Lipid nanoparticles comprising polymer-conjugated lipids and uses thereof

Abstract

Compounds are provided having the following structure: or a pharmaceutically acceptable salt, or stereoisomer thereof, wherein R1, R2, R3, R4, R5, Y, m, n, w and x are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a payload, compositions comprising the compounds and methods for their use and preparation are also provided.

Description

[0001] LIPID NANOPARTICLES COMPRISING POLYMER-CONJUGATED LIPIDS

[0002] AND USES THEREOF

[0003] RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 514,405 filed July 19, 2023 and U.S. Provisional Application No. 63 / 666,914 filed July 2, 2024. The entire content of each of the foregoing applications is hereby incorporated by reference herein in its entirety.

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to novel polymer-conjugated lipid compounds. The invention also relates to the preparation of the polymer-conjugated lipid compounds and intermediates used in their preparation, compositions containing the polymer-conjugated lipid compounds, and uses of the polymer-conjugated lipid compounds including in combination with other lipid components, such as neutral lipids, steroids and ionizable cationic lipids, to form lipid nanoparticles which may be combined with a payload (e.g. therapeutic nucleic acids including but not limited to oligonucleotides and messenger RNA, proteins, hydrophobic small molecular drugs (e.g. anti-cancer drugs), or imaging agents) to facilitate the intracellular delivery of said payload, both in vitro and in vivo.

[0007] REFERENCE TO SEQUENCE LISTING

[0008] This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .xml format. The .xml file contains a sequence listing entitled "PC072923A Sequence Listing.xml” created on July 3, 2024 and having a size of 25 KB. The sequence listing contained in this .xml file is part of the specification and is incorporated herein by reference in its entirety.

[0009] BACKGROUND OF THE INVENTION

[0010] There are many challenges associated with the delivery of a payload (e.g. a nucleic acid) to affect a desired response in a biological system. For example, nucleic acid-based therapeutics have enormous potential but there remains a need for more effective delivery of nucleic acids to appropriate sites within a cell or organism in order to realize this potential. Therapeutic nucleic acids include, e.g., messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products as would be useful in the treatment of, for example, diseases related to a deficiency of a protein or enzyme, or as a vaccine. The therapeutic applications of translatable nucleotide delivery are extremely broad as constructs can be synthesized to produce any chosen protein sequence, whether or not indigenous to the system. The expression products of the nucleic acid can augment existing levels of protein, replace missing or non-functional versions of a protein, or introduce new protein and associated functionality in a cell or organism.

[0011] RNA may be delivered to a subject using different delivery vehicles, mostly based on lipids or conjugates thereof which together with the RNA form nanoparticles, e.g. Lipid Nanoparticles (LNPs). The nanoparticles are intended to protect the RNA from degradation, enable delivery of the RNA to the target site and facilitate cellular uptake and processing by the target cells. For delivery efficacy, in addition to the molecular composition, parameters like particle size, charge, or grafting with molecular moieties, such as polyethylene glycol (PEG) or other polymers or ligands, play a role. Grafting with PEG or other polymer is considered to reduce serum interactions, to increase serum stability and to increase circulation time, which can be helpful for certain targeting approaches. Ligands which bind to receptors at the target site can help to improve targeting efficacy.

[0012] Furthermore, PEGylation (the covalent grafting of PEG) or other polymer conjugation can be used for particle engineering. For example, if Lipid Nanoparticles (LNPs) are manufactured by mixing an aqueous phase of the RNA with an organic phase of the lipids a certain fraction of PEG-conjugated lipid in the lipid mixture is required, otherwise the particles aggregate during the mixing step. It has been shown that by variation of the molar fraction of PEG-lipids comprising PEG at different molar masses the size of the particles can be adjusted. In addition, the particle size may be adjusted by variation of the molar mass of the PEG moiety of the PEGylated lipids. Typical sizes which are accessible are in the range between 30 and 200 nm (Belliveau et al, 2012, Molecular Therapy-Nucleic Acids 1 , e37). So-formed particles have the additional advantage that, due to the PEG fraction, they interact less with serum components, and have a longer circulation half-life, which is desirable in many drug delivery approaches. Without PEG- lipids, no particles with discrete size can be formed; the particles form large aggregates and precipitate.

[0013] So, for techniques where LNPs are formed from an ethanolic and an aqueous phase, one of the primary roles of PEG-lipids is to facilitate particle self-assembly by providing a steric barrier at the surface of nascent particles formed when nucleic acids are rapidly mixed in ethanol solutions containing lipids to bind the RNA. PEG steric hindrance prevents inter-particle fusion and promotes the formation of a homogeneous population of LNPs with diameters <100 nm.

[0014] PEG is the most widely used polymer in drug delivery. PEG-lipids are typically incorporated into systems to prepare a homogenous and colloidally stable nanoparticle population due to its hydrophilic steric hindrance property (PEG shell prevents electrostatic or Van der Waals attraction that leads to aggregation). PEGylation attracts a water shell around the polymer shielding the RNA complex from opsonization with serum proteins, increasing serum half-life as well as reducing rapid renal clearance which results in an improvement of the pharmacokinetic behavior. Variation of the length of the acyl chains (C18, C16 or C14) of the lipids modifies the stability of the incorporation of the PEG-lipid in the particles which leads to a modulation of the pharmacokinetics. The use of a PEG-lipid containing short (C14) acyl chains that dissociates from LNPs in vivo with a halftime <30 min results in optimum hepatocyte genesilencing potency (Chen et al, 2014, J Control Release 196:106-12; Ambegia et al., 2005, Biochimica et Biophysica Acta 1669:155-163). In addition, tight control of particle size can be obtained by varying the PEG-lipid parameter: higher PEG MW or higher molar fraction of PEG- lipids in the particles lead to smaller particles.

[0015] Despite these advantages, PEGylation of nanoparticles may lead as well to several effects which are detrimental to the intended use for drug delivery. PEGylation of liposomes and LNPs is known to reduce cellular uptake and endosomal escape, thus reducing the overall transfection efficiency. Indeed, the PEG shell provides a steric barrier to efficient binding of particles to the cell and also hinders endosomal release by preventing membrane fusion between the liposome and the endosomal membrane. This is the reason the type of PEG-lipid and the amount of PEG-lipid used must always be carefully adjusted. It should provide sufficient stealth effect for in vivo and stabilization aspects on the one hand, while not hindering transfection on the other. This phenomenon is known as the “PEG Dilemma”.

[0016] Besides lowering transfection efficiency, PEGylation has been associated with accelerated blood clearance (ABC) phenomenon induced by anti-PEG antibodies and / or complement activation as well as storage diseases (Bendele A et al., 1998, Toxicological Sciences 42, 152-157; Young M A et al., 2007, Translational Research 149(6), 333-342; S. M. Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478). Ishida et al. and Laverman et al. reported that intravenous injection in rats of PEG-grafted liposomes may significantly alter the pharmacokinetic behavior of a second dose when this second dose is administered after an interval of several days (Laverman P et al., 2001 , J Pharmacol Exp Ther. 298(2), 607-12; Ishida et al., 2006, J Control Release 115(3), 251-8). The phenomenon of “accelerated blood clearance” (ABC) appears to be inversely related to the PEG content of liposomes. The presence of anti- PEG antibodies in the plasma induces a higher clearance of the particles by the Monophagocyte System (MPS) which at the end reduces the efficacy of the drug.

[0017] PEG is also known to induce complement activation, which can lead to hypersensitivity reaction, also known as Complement-Activation Related Pseudo-Allergy (CARPA). It is still not clear from the literature if the activation of complement is due to the nanoparticle in general or to the presence of PEG in particular.

[0018] The presence of PEG in lipidic nanoparticles may also induce a specific immune response. Semple et al. reported that liposomes containing PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotide or plasmid DNA elicit a strong immune response that results in the rapid blood clearance of subsequent doses in mice. The magnitude of this response was sufficient to induce significant morbidity and, in some instances, mortality. Rapid elimination of liposome-encapsulated oligodeoxynucleotides (ODN) from blood depended on the presence of PEG-lipid in the membrane because the use of non-pegylated liposomes or liposomes containing rapidly exchangeable PEG-lipid abrogated the response. The generation of anti-PEG antibody and the putative complement activation were a likely explanation for the rapid elimination of the vesicles from the blood. (Semple et al., 2005, J Pharmacol Exp Ther. 312(3), 1020-6).

[0019] Since PEG may induce undesirable immune responses there is a need to avoid its use for certain applications where multiple injections are needed. Examples are therapies using mRNA, for example (but not limited to) protein replacement therapy. Here, the risk can be particularly high due to the potential intrinsic immunogenicity of RNA.

[0020] Thus, there remains a need in the art for efficient methods and compositions for introducing RNA or other payloads (e.g. hydrophobic small moleculardrugs) into cells which avoid the disadvantages accompanied by use of PEG-lipids or derivatives thereof.

[0021] SUMMARY OF THE INVENTION

[0022] The present disclosure demonstrates that the RNA particle formulations described herein comprising the novel polymer-conjugated lipid compounds having Formula (I) fulfill the abovementioned need. In particular it is demonstrated that the polymer-conjugated lipid compounds having Formula (I) described herein are suitable components for assembly of lipid nanoparticles comprising a payload including, but not limited to, RNA. The polymer-conjugated lipid compounds having Formula (I) described herein may comprise the polymer poly-2- methacryloyloxyethylphosphorylcholine (PMPC) which is composed of repeated units of methacryloyloxyethylphosphorylcholine (MPC) which is biocompatible. PMPC-lipid conjugated compounds enable manufacturing of lipid nanoparticles (e.g. RNA lipid nanoparticles) with different techniques, resulting in defined surface properties and controlled size ranges. Manufacturing can be done by robust processes, compliant with the requirements for pharmaceutical manufacturing. The compounds of the invention may also be end-group functionalized with different moieties to modulate charge or to introduce specific molecular moieties like ligands.

[0023] The present invention provides, in part, compounds of Formula (I) and pharmaceutically acceptable salts thereof. Also provided are pharmaceutical compositions, comprising the compounds or salts of the invention, alone or in combination with additional therapeutic agents or excipients. The present invention also provides, in part, methods for preparing such compounds, pharmaceutically acceptable salts and compositions of the invention, and methods of using the foregoing. This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used in isolation as an aid in determining the scope of the claimed subject matter. According to an embodiment of the invention there is provided a polymer-conjugated lipid compound of Formula or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0024] R1and R2are each independently hydrogen or methyl;

[0025] R3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor; R5is hydrogen or halogen; m is an integer from 1 to 6;

[0026] Y is a polymer having Formula (II):

[0027] (ID n is an integer from 1 to 1000; x is an integer from 2 to 6; and w is an integer from 2 to 4. In one aspect, the present disclosure relates to a polymer-conjugated lipid compound having Formula (la) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0028] R3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor;

[0029] R5is hydrogen or halogen; and n is an integer from 1 to 1000.

[0030] In another aspect, the present disclosure relates to a polymer-conjugated lipid compound having Formula (lb) or a pharmaceutically acceptable salt or stereoisomer thereof. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows the1H NMR spectrum of DiTDA-polymerization initiator.

[0033] FIG. 2A and 2B show the dose responses of different molar ratios of LNPs comprising DiTDA- PMPC8k in HEK293F cells in vitro expression assay. The EC50 (ng) of each LNP formulation as measured by %Positive cells is described in Table 2. “PEG lipid” as described in this figure is ALC-0159.

[0034] FIG. 3 shows in vitro expression assay in HEK293F cells. Different molar ratios of LNPs comprising DiTDA-PMPC1 1 k show dose dependent increase. The EC50 (ng) of each LNP formulation as measured by %Positive cells is shown in Table 3. “PEG lipid” as described in this figure is ALC-0159.

[0035] FIG. 4A & 4B show in vitro cell potency and protein expression levels after long term 4°C storage; FIG. 4A) Stability of cell potency up to 16 weeks; FIG. 4B) MFI (Mean fluorescence intensity) from 293 cells treated with LNPs that were stored at 4°C for 16 weeks.

[0036] FIG. 5 shows results of immunogenicity testing of the DiTDA-PMPC8k polymer lipid nanoparticles. LNP with modRNA encoding a representative antigen was dosed to mice IM, and neutralizing antibody titer was measured. DiTDA-PMPC8k polymer LNPs induced significantly higher titers than the ALC-0159 (PEG) control. Statistics were determined by ANOVA with Tukey’s post-hoc analysis and with p < 0.05. "Indicates significant difference compared to each other.

[0037] FIG.6A - 6C show that in vitro cell potency and protein expression levels were stable at -80 °C (FIG. 6A), 4 °C (FIG. 6B) and 25 °C (FIG. 6C) after long term storage for up to 16 weeks.

[0038] FIG. 7 shows immunogenicity testing of the polymer lipid nanoparticles. LNPs with modRNA encoding a representative antigen were dosed to mice IM, and neutralizing antibody titers were measured.

[0039] DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the Examples included herein. It is to be understood that this invention is not limited to specific synthetic methods of making that may of course vary. It is to be also understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Described below are embodiments of the invention, where for convenience Embodiment 1 (E1) is identical to the embodiment of Formula (I) provided above. Exemplary embodiments (E) of the invention provided herein include: E1 . A polymer conjugated lipid compound of Formula (I): or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1and R2are each independently hydrogen or methyl;

[0041] R3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor;

[0042] R5is hydrogen or halogen; m is an integer from 1 to 6; Y is a polymer having Formula (II): n is an integer from 1 to 1000; x is an integer from 2 to 6; and w is an integer from 2 to 4.

[0043] In a preferred embodiment, m is 3, x is 2 and w is 2. In another embodiment, R5is -Br.

[0044] E2. The polymer conjugated lipid compound of embodiment E1 having Formula (la): or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0045] R3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor;

[0046] R5is hydrogen or halogen; and n is an integer from 1 to 1000.

[0047] E3. The polymer conjugated lipid compound of embodiment E1 having Formula (lb): (lb) or a pharmaceutically acceptable salt or stereoisomer thereof.

[0048] E4. The polymer conjugated lipid compound of embodiment E1 , wherein (i) n ranges from 15 to 19, (ii) n ranges from 20 to 24, (iii) n ranges from 25 to 29, (iv) n ranges from 30 to 33, or (v) n ranges from 34 to 38.

[0049] E5. The polymer conjugated lipid compound of embodiment E1 , wherein the polymer conjugated lipid compound has a dispersity (£>) of about 1 .0 to about 2.0.

[0050] E6. The polymer conjugated lipid compound of embodiment E5, wherein the polymer conjugated lipid compound has a dispersity (£>) of between 1 .0 to 1 .4. In a preferred embodiment, the dispersity (£>) is 1 .2.

[0051] E7. The polymer conjugated lipid compound of embodiment E1 , wherein the polymer has a molecular weight of between 100 Daltons and 100,000 Daltons.

[0052] E8. The polymer conjugated lipid compound of embodiment E7, wherein the polymer has a molecular weight of between 4,500 Daltons and 12,000 Daltons.

[0053] E9. The polymer conjugated lipid compound of embodiment E7, wherein the polymer has a molecular weight of between about 4,800 Daltons and about 5,300 Daltons.

[0054] E10. The polymer conjugated lipid compound of embodiment E7, wherein the polymer has a molecular weight of between about 8,300 Daltons and about 8,900 Daltons.

[0055] E11 . The polymer conjugated lipid compound of embodiment E7, wherein the polymer has a molecular weight of between about 11 ,200 Daltons and about 11 ,800 Daltons.

[0056] E12. The polymer conjugated lipid compound of embodiment E1 selected from the group consisting of:

[0057] (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-8k]; and

[0058] (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-11 k], or a pharmaceutically acceptable salt or stereoisomer thereof.

[0059] E13. A composition comprising the polymer conjugated lipid compound according to any one of embodiments E1-E12, or a pharmaceutically acceptable salt thereof, at least one payload, and at least one pharmaceutically acceptable excipient. E14. The composition of embodiment E13, wherein the payload comprises a small molecule, a nucleic acid, an adjuvant, or a combination thereof.

[0060] E15. The composition of embodiment E14, wherein the pharmaceutically acceptable excipient is selected from the group consisting of neutral lipids, steroids and ionizable cationic lipids.

[0061] E16. The composition of embodiment E15, wherein the composition comprises one or more neutral lipids selected from 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1-stearoyl-2- oleoylphosphatidyethanolamine (SOPE), and / or 1 ,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (transDOPE).

[0062] E17. The composition of embodiment E15, wherein the steroid is cholesterol.

[0063] E18. The composition of any one of embodiments E13 to E17, wherein the nucleic acid is RNA.

[0064] E19. The composition of embodiment E18, wherein the RNA is mRNA.

[0065] E20. The composition of embodiment E19, wherein the mRNA is modRNA or saRNA.

[0066] E21 . The composition of any one of embodiments E13 to E17, wherein the nucleic acid is circular DNA (cDNA).

[0067] E22. The composition of any one of embodiments E13 to E21 , wherein the ionizable cationic lipid comprises N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2-dilinoleyloxy- N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), (4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecane-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl) amino) octanoate (SM- 102), or a mixture thereof. E23. The composition of any one of embodiments E13 to E22, wherein the composition is a lipid nanoparticle comprising a neutral lipid, a steroid, an ionizable cationic lipid and a polymer conjugated lipid compound having a mol% ratio of ALC- 0315:cholesterol:DSPC:polymer conjugated lipid compound of any one of embodiments E1 -E12 selected from the group consisting of: a) 46.3:44.1 :9.4:0.2; b) 46.3:43.9:9.4:0.4; c) 46.3:43.8:9.4:0.5; d) 46.3:43.5:9.4:0.8; e) 46.3:42.7:9.4:1.6 and f) 46.3:41.1 :9.4:3.2.

[0068] E24. The composition of embodiment E23, wherein the polymer conjugated lipid is

[0069] (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-8k]; or

[0070] (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-11 k], or a pharmaceutically acceptable salt or stereoisomer thereof.

[0071] E25. A method for delivering a payload to a subject in need thereof comprising administering to the subject the composition of any one of embodiments E13 to E24.

[0072] E26. The method of embodiment E25, wherein the payload comprises a small molecule, a nucleic acid, an adjuvant, or a combination thereof.

[0073] E27. A method for delivering a nucleic acid to a subject in need thereof comprising administering to the subject a composition of any one of embodiments E13 to E24 comprising a nucleic acid.

[0074] E28. A method for delivering a therapeutic peptide or protein to a subject in need thereof, the method comprising administering to the subject a composition of any one of embodiments E13 to E24 comprising a nucleic acid, wherein the nucleic acid encodes the therapeutic peptide or protein.

[0075] E29. A method for treating or preventing a disease or disorder in a subject comprising administering to the subject a composition of any one of embodiments E13 to E24 comprising a nucleic acid, wherein administering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder.

[0076] E30. A method for treating or preventing a disease or disorder in a subject comprising administering to the subject a composition of any one of embodiments E13 to E24 comprising a nucleic acid, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.

[0077] E31. The method of any one of embodiments E25 to E30, wherein the subject is a mammal.

[0078] E32. The method of embodiment E31 , wherein the mammal is a human.

[0079] E33. A method for producing a polymer conjugated lipid compound of any one of embodiments E1 to E12, wherein the method is a Controlled Radical Polymerization.

[0080] E34. The method of embodiment E33, wherein the Controlled Radical Polymerization is Atom Transfer Radical Polymerization (ATRP) or Reversible Addition / Fragmentation Chain Transfer Polymerization (RAFT).

[0081] Each of the embodiments described herein may be combined with any other embodiment(s) described herein not inconsistent with the embodiments) with which it is combined. In addition, any of the compounds described in the Examples, or pharmaceutically acceptable salts thereof, may be claimed individually or grouped together with one or more other compounds of the Examples, or pharmaceutically acceptable salts thereof, for any of the embodiment(s) described herein.

[0082] Furthermore, each of the embodiments described herein envisions within its scope pharmaceutically acceptable salts of the compounds described herein.

[0083] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0084] All references cited herein, including patent applications, patent publications, UniProtKB accession numbers are herein incorporated by reference, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety.

[0085] Definitions

[0086] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by those of ordinary skill in the art.

[0087] The invention described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. “Compounds of the invention” include compounds of Formula (I), (la), (lb), (II) and the novel intermediates used in the preparation thereof. One of ordinary skill in the art will appreciate that compounds of the invention include conformational isomers (e.g., cis and trans isomers) and all optical isomers (e.g., enantiomers and diastereomers), racemic, diastereomeric and other mixtures of such isomers thereof, where they may exist. One of ordinary skill in the art will also appreciate that compounds of the invention include solvates, hydrates, isomorphs, polymorphs, esters, salt forms, and isotopically labelled versions thereof, where they may be formed.

[0088] As used herein, the singular form "a", "an", and "the" include plural references unless indicated otherwise. For example, "a" substituent includes one or more substituents.

[0089] As used herein, the term “about” when used to modify a numerically defined parameter (e.g., the dose of XXX) means that the parameter may vary by as much as 10% below or above the stated numerical value for that parameter. For example, a dose of about 5 mg means 5 mg ± 10%, e.g., it may vary between 4.5 mg and 5.5 mg.

[0090] As used herein, the term “aqueous solution” refers to a composition comprising water.

[0091] If substituents are described as being “independently selected” from a group, each substituent is selected independent of the other. Each substituent therefore may be identical to or different from the other substituent(s).

[0092] The term “halo” or “halogen” refers to fluorine (which may be depicted as -F), chlorine (which may be depicted as -Cl), bromine (which may be depicted as -Br), or iodine (which may be depicted as -I).

[0093] “Optional" or "optionally" means that the subsequently described event or circumstance may, but need not occur, and the description includes instances where the event or circumstance occurs and instances in which it does not.

[0094] The terms “optionally substituted” and “substituted or unsubstituted” are used interchangeably to indicate that the particular group being described may have no non-hydrogen substituents (e.g., unsubstituted), or the group may have one or more non-hydrogen substituents (e.g., substituted). If not otherwise specified, the total number of substituents that may be present is equal to the number of H atoms present on the unsubstituted form of the group being described. Where an optional substituent is attached via a double bond, such as an oxo (=O) substituent, the group occupies two available valences, so the total number of other substituents that are included is reduced by two. In the case where optional substituents are selected independently from a list of alternatives, the selected groups may be the same or different. Throughout the disclosure, it will be understood that the number and nature of optional substituent groups will be limited to the extent that such substitutions make chemical sense to one of ordinary skill in the art.

[0095] Salts

[0096] Salts encompassed within the term “pharmaceutically acceptable salts” refer to the compounds of this invention which are generally prepared by reacting the free base or free acid with a suitable organic or inorganic acid, or a suitable organic or inorganic base, respectively, to provide a salt of the compound of the invention that is suitable for administration to a subject or patient.

[0097] In addition, the compounds of Formula I may also include other salts of such compounds which are not necessarily pharmaceutically acceptable salts, which may be useful as intermediates for one or more of the following: 1) preparing compounds of Formula I; 2) purifying compounds of Formula I; 3) separating enantiomers of compounds of Formula I; or 4) separating diastereomers of compounds of Formula I.

[0098] Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include, but are not limited to, acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, 1 ,5- naphathalenedisulfonic acid and xinofoate salts.

[0099] Hemisalts of acids and bases may also be formed, for example, hemisulfate and hemicalcium salts.

[0100] For a review on suitable salts, see Paulekun, G. S. et al., Trends in Active Pharmaceutical Ingredient Salt Selection Based on Analysis of the Orange Book Database, J. Med. Chem. 2007; 50(26), 6665-6672.

[0101] Pharmaceutically acceptable salts of compounds of the invention may be prepared by methods well known to one skilled in the art, including but not limited to the following procedures

[0102] (i) by reacting a compound of the invention with the desired acid;

[0103] (ii) by removing an acid- or base-labile protecting group from a suitable precursor of a compound of the invention or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid; or

[0104] (iii) by converting one salt of a compound of the invention to another. This may be accomplished by reaction with an appropriate acid or by means of a suitable ion exchange procedure.

[0105] These procedures are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent.

[0106] Solvates

[0107] The compounds of the invention, and pharmaceutically acceptable salts thereof, may exist in unsolvated and solvated forms. The term ‘solvate’ is used herein to describe a molecular complex comprising the compound of the invention, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ is employed when said solvent is water.

[0108] In addition, the compounds of Formula I may also include other solvates of such compounds which are not necessarily pharmaceutically acceptable solvates, which may be useful as intermediates for one or more of the following: 1) preparing compounds of Formula I; 2) purifying compounds of Formula I; 3) separating enantiomers of compounds of Formula I; or 4) separating diastereomers of compounds of Formula I.

[0109] A currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal-ion coordinated hydrates - see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995). Isolated site hydrates are ones in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules lie in lattice channels where they are next to other water molecules. In metal-ion coordinated hydrates, the water molecules are bonded to the metal ion.

[0110] When the solvent or water is tightly bound, the complex may have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water / solvent content may be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.

[0111] Solid form

[0112] The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term ‘amorphous’ refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically, such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterized by a change of state, typically second order (‘glass transition’). The term ‘crystalline’ refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterized by a phase change, typically first order (‘melting point’).

[0113] The compounds of the invention may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution) and consists of two dimensional order on the molecular level. Mesomorphism arising as the result of a change in temperature is described as ‘thermotropic’ and that resulting from the addition of a second component, such as water or another solvent, is described as ‘lyotropic’. Compounds that have the potential to form lyotropic mesophases are described as ‘amphiphilic’ and consist of molecules which possess an ionic (such as -COO Na+, -COO K+, or -S03Na+) or non-ionic (such as -N N+(CH3)3) polar head group. For more information, see Crystals and the Polarizing Microscope by N. H. Hartshorne and A. Stuart, 4thEdition (Edward Arnold, 1970).

[0114] Stereoisomers

[0115] Some compounds of the invention may exist as two or more stereoisomers. Stereoisomers of the compounds may include cis and trans isomers (geometric isomers), optical isomers such as R and S enantiomers, diastereomers, rotational isomers, atropisomers, and conformational isomers. For example, compounds of the invention containing one or more asymmetric carbon atoms may exist as two or more stereoisomers. Where a compound of the invention contains an alkenyl or alkenylene group, geometric cis / trans (or Z / E) isomers are possible. Cis / trans isomers may also exist for saturated rings.

[0116] The pharmaceutically acceptable salts of compounds of the invention may also contain a counterion which is optically active (e.g., d-lactate or l-lysine) or racemic (e.g. dl-tartrate or dl- arginine).

[0117] Cis / trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallization.

[0118] Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where a compound of the invention contains an acidic or basic moiety, a base or acid such as 1 -phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography, fractional crystallization, or by using both of said techniques, and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person. Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC Concentration of the eluate affords the enriched mixture. Chiral chromatography using sub-and supercritical fluids may be employed. Methods for chiral chromatography useful in some embodiments of the present invention are known in the art (see, for example, Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998), 75 (Supercritical Fluid Chromatography with Packed Columns), pp. 223-249 and references cited therein).

[0119] When any racemate crystallizes, crystals of two different types are possible. The first type is the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate wherein two crystal forms are produced in equimolar amounts each comprising a single enantiomer. While both of the crystal forms present in a racemic mixture have identical physical properties, they may have different physical properties compared to the true racemate. Racemic mixtures may be separated by conventional techniques known to those skilled in the art - see, for example, Stereochemistry of Organic Compounds by E. L. Eliel and S. H. Wilen (Wiley, 1994).

[0120] Isotopes

[0121] The present invention includes all pharmaceutically acceptable isotopically-labeled compounds of the invention wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature.

[0122] Examples of isotopes suitable for inclusion in the compounds of the invention may include isotopes of hydrogen, such as2H and3H, carbon, such as11C,13C and14C, chlorine, such as36CI, fluorine, such as18F, iodine, such as123l and125l, nitrogen, such as13N and15N, oxygen, such as150,17O and18O, phosphorus, such as32P, and sulfur, such as35S.

[0123] Certain isotopically-labelled compounds of the invention, for example those incorporating a radioactive isotope, are useful in one or both of drug or substrate tissue distribution studies. The radioactive isotopes tritium, e.g.,3H, and carbon-14, e.g.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0124] Substitution with deuterium, e.g.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability.

[0125] Substitution with positron emitting isotopes, such as11C,18F,15O and13N, may be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

[0126] Isotopically-labeled compounds of the invention may generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically- labeled reagent in place of the non-labeled reagent previously employed.

[0127] Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g., D2O, d6-acetone, d6- DMSO.

[0128] Metabolites

[0129] Also included within the scope of the invention are active metabolites of compounds of the invention, that is, compounds formed in vivo upon administration of the drug, often by oxidation or dealkylation. Some examples of metabolites in accordance with the invention include, but are not limited to,

[0130] (i) where the compound of the invention contains an alkyl group, a hydroxyalkyl derivative thereof (-CH > -COH): (ii) where the compound of the invention contains an alkoxy group, a hydroxy derivative thereof (-OR -> -OH);

[0131] (iii) where the compound of the invention contains a tertiary amino group, a secondary amino derivative thereof (-NRR -> -NHR or -NHR);

[0132] (iv) where the compound of the invention contains a tertiary amino group, an N-oxide derivative thereof (-NRR -> -N(O)RR’);

[0133] (v) where the compound of the invention contains a secondary amino group, a primary derivative thereof (-NHR -> -NH2);

[0134] (vi) where the compound of the invention contains a phenyl moiety, a phenol derivative thereof (-Ph -> -PhOH);

[0135] (vii) where the compound of the invention contains an amide group, a carboxylic acid derivative thereof (-CONH2-> COOH); and

[0136] (viii) where the compound contains a hydroxy or carboxylic acid group, the compound may be metabolized by conjugation, for example with glucuronic acid to form a glucuronide. Other routes of conjugative metabolism exist. These pathways are frequently known as Phase 2 metabolism and include, for example, sulfation or acetylation. Other functional groups, such as NH groups, may also be subject to conjugation.

[0137] Lipid Nanoparticles (LNPs)

[0138] The present invention is based, in part, upon the discovery of novel polymer-conjugated lipid compounds as exemplified by the compounds of Formula (I), (la) and (lb) that provide advantages when used in lipid nanoparticles for the in vivo delivery of an active or therapeutic agent such as a nucleic acid into a cell of a mammal. In particular embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel polymer-conjugated lipid compounds described herein that provide increased activity of the nucleic acid, increased in vitro stability of the nanoparticle and improved tolerability of the compositions in vivo, resulting in an increase in the therapeutic index as compared to nucleic acid-lipid nanoparticle compositions previously described.

[0139] In particular embodiments, the present invention provides novel polymer-conjugated lipid compounds as exemplified by the compounds of Formula (I), (la) and (lb) that enable the formulation of improved compositions for the in vitro and in vivo delivery of a payload including, but not limited to, mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for expression of protein encoded by mRNA. In other embodiments, these improved lipid nanoparticles compositions are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNAs regulating one target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for down-regulating (e.g., silencing) the protein levels and / or mRNA levels of target genes. In some other embodiments, the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes. In yet other embodiments, the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antigen or antibody. The present disclosure further provides for RNA molecules that are messenger-RNA (mRNA), which can be either nucleoside-modified RNA (modRNA), self-amplifying RNA (saRNA) or circular RNA (cRNA). In some aspects, the RNA is an mRNA. In some aspects, the RNA is a modRNA. In other aspects, the RNA is an saRNA. In a further aspect, the RNA is cRNA.

[0140] The lipid nanoparticles and compositions of the present invention may be used for a variety of purposes including, but not limited to, the delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells, both in vitro and in vivo. Accordingly, embodiments of the present invention provide methods of treating or preventing diseases or disorders in a subject in need thereof by contacting the subject with a lipid nanoparticle that encapsulates or is associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more of the novel polymer-conjugated lipid compounds described herein.

[0141] As described herein, embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, the lipid nanoparticles and compositions of the present invention may be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel polymer-conjugated lipid compounds described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA or plasmid encoding the desired protein) or inhibit processes that terminate expression of mRNA (e.g., miRNA inhibitors). Alternatively, the lipid nanoparticles and compositions of the present invention may be used to decrease the expression of target genes and proteins both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel polymer-conjugated lipid compounds described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that reduces target gene expression (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)). The lipid nanoparticles and compositions of the present invention may also be used for co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination, such as may be useful to provide an effect requiring colocalization of different nucleic acids (e.g., mRNA encoding for a suitable gene modifying enzyme and DNA segment(s) for incorporation into the host genome). Nucleic acids for use with this invention may be prepared according to any available technique. For mRNA, the primary methodology of preparation is, but not limited to, enzymatic synthesis (also termed in vitro transcription) which currently represents the most efficient method to produce long sequence-specific mRNA. In vitro transcription describes a process of template- directed synthesis of RNA molecules from an engineered DNA template comprised of an upstream bacteriophage promoter sequence (e.g., including but not limited to that from the T7, T3 and SP6 coliphage) linked to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J. L and Conn, G. L., General protocols for preparation of plasmid DNA template and Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed), New York, N.Y. Humana Press, 2012).

[0142] Transcription of the RNA occurs in vitro using the linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant 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 kits (Life Technologies) as well as with commercially available reagents including RNA polymerases and rNTPs. The methodology for in vitro transcription of mRNA is well known in the art. (see, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v. 41 409-46; Kamakaka, R. T. and Kraus, W. L. 2001 . In Vitro Transcription. Current Protocols in Cell Biology. 2:1 1 .6:11 .6.1-11 .6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J. L. and Green, R., 2013, Chapter Five — In vitro transcription from plasmid or PCR- amplified DNA, Methods in Enzymology v. 530, 101 -1 14; all of which are incorporated herein by reference).

[0143] The desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or associated reactions (including unincorporated rNTPs, protein enzyme, salts, short RNA oligos, etc.). Techniques for the isolation of the mRNA transcripts are well known in the art. Well known procedures include phenol / chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional, non-limiting examples of purification procedures which can be used include size exclusion chromatography (Lukavsky, P. J. and Puglisi, J. D., 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v. 10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed), New York, N.Y. 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).

[0144] Furthermore, while reverse transcription can yield large quantities of mRNA, the products can contain a number of aberrant RNA impurities associated with undesired polymerase activity which may need to be removed from the full-length mRNA preparation. These include short RNAs that result from abortive transcription initiation as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates and self-complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses. This in turn, can dramatically reduce mRNA translation since protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques to remove 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, (Weissman, D., Pardi, N., Muramatsu, H., Kariko, K. (2013). HPLC Purification of In Vitro Transcribed Long RNA. In: Rabinovich, P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology, vol 969. Humana Press, Totowa, NJ. https: / / doi.org / 10.1007 / 978-1 -62703- 260-5_3). HPLC-purified mRNA has been reported to be translated at much greater levels, particularly in primary cells and in vivo.

[0145] A significant variety of modifications have been described in the art which are used to alter specific properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications to the 5' and 3' termini of the mRNA. Endogenous eukaryotic mRNA typically contains a cap structure on the 5'-end of a mature molecule which plays an important role in mediating binding of the mRNA Cap Binding Protein (CBP), which is in turn responsible for enhancing mRNA stability in the cell and efficiency of mRNA translation. Therefore, highest levels of protein expression are achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate linkage between the 5'-most nucleotide and guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the ultimate and penultimate most 5'-nucleotides on the 2'- hydroxyl group.

[0146] Multiple distinct cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co-transcriptionally with chemical cap analogs (e.g. capping during in vitro transcription). For example, the Anti-Reverse Cap Analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine linkage where one guanine contains an N7 methyl group as well as a 3'-O-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcriptional process and the synthetic cap analog is not identical to the 5'-cap structure of an authentic cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5'-cap structure that more closely mimics, either structurally or functionally, the endogenous 5'-cap which have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5' decapping. Numerous synthetic 5'-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see eg. Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, R. E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v. 969 (Rabinovich, P. H. Ed), 2013).

[0147] On the 3'-terminus, a long chain of adenine nucleotides (poly-A tail) is normally added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl to which poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly-A tail has been extensively shown to enhance both translational efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A), poly (A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001 , Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11 -23; Dreyfus, M. And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. 11 1 , 61 1 -613).

[0148] Poly (A) tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post- transcriptional addition using Poly (A) polymerase. The first case allows in vitro transcription of mRNA 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 the enzymatic addition of a poly (A) tail to in vitro transcribed mRNA using poly (A) polymerase which catalyzes the incorporation of adenine residues onto the 3’ termini of RNA, requiring no additional manipulation of the DNA template, but results in mRNA 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 with commercially available reagents, various ARCA caps, Poly (A) polymerase, etc.

[0149] In addition to 5' cap and 3' poly adenylation, other modifications of the in vitro transcripts have been reported to provide benefits as related to efficiency oftranslation and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by a variety of sensors within eukaryotes and trigger potent innate immune responses. The ability to discriminate between pathogenic and self-DNA and -RNA has been shown to be based, at least in part, on structure and nucleoside modifications since most nucleic acids from natural sources contain modified nucleosides In contrast, in vitro synthesized RNA lacks these modifications, thus rendering it immunostimulatory which in turn can inhibit effective mRNA translation as outlined above. The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translation capacity (see e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v. 10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v. 969 (Rabinovich, P. H. Ed), 2013); Kariko, K., Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v. 16, 1833-1840. The modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared monitored and utilized using general methods and procedures known in the art. A large variety of nucleoside modifications are available that may be incorporated alone or in combination with other modified nucleosides to some extent into the in vitro transcribed mRNA (see e.g., US2012 / 0251618). In vitro synthesis of nucleoside- modified mRNA have been reported to have reduced ability to activate immune sensors with a concomitant enhanced translational capacity.

[0150] Other components of mRNA which can be modified (modRNA) to provide benefit in terms of translatability and stability include the 5' and 3' untranslated regions (UTR). Optimization of the UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNAs), either both or independently, have been shown to increase mRNA stability and translational efficiency of in vitro transcribed mRNA (see e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v. 969 (Rabinovich, P. H. Ed), 2013).

[0151] A “modified RNA” or “modRNA” refers to an RNA molecule having at least one addition, deletion, substitution, and / or alteration of one or more nucleotides as compared to naturally occurring RNA. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides, or to the 5' and / or 3' end(s) of RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as an alteration to the base of the nucleotide. For example, a modified nucleotide may replace one or more uridine and / or cytidine nucleotides. For example, these replacements may occur for every instance of uridine and / or cytidine in the RNA sequence, or may occur for only select uridine and / or cytidine nucleotides. Such alterations to the standard nucleotides in RNA may include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide may be replaced with N1 -methylpseudouridine in an RNA sequence. Other such altered nucleotides are known to those of skill in the art. Such altered RNA molecules are considered analogs of naturally-occurring RNA. In some aspects, the RNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleotides.

[0152] In some aspects, the RNA molecule may be an saRNA. “Self-amplifying RNA,” “selfamplifying RNA,” and “replicon” refer to RNA with the ability to replicate itself. Self-amplifying RNA molecules may be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNA molecules. These daughter RNA molecules, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNA molecules and so the encoded gene of interest, e.g., a viral antigen, becomes a major polypeptide product of the cells.

[0153] In some aspects, the self-amplifying RNA includes at least one or more genes including any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins, or combination thereof. In some aspects, the self-amplifying RNA may also include 5’- and 3 end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0154] In addition to mRNA, other nucleic acid payloads may be used for this invention. For oligonucleotides, methods of preparation include but are not limited to chemical synthesis and enzymatic, chemical cleavage of a longer precursor, in vitro transcription as described above, etc. Methods of synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference). For plasmid DNA, preparation for use with this invention commonly utilizes but is not limited to expansion and isolation of the plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows those bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures. Methods of isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, K. L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41 :11:1.7:1.7.1-1 .7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R. and Schmidt, S. R. (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566; and U.S. Pat. No. 6,197,553B1). 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 with commercially available reagents.

[0155] The term “nucleic acid” as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O- methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

[0156] The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0157] “Gene product,” as used herein, refers to a product of a gene such as an RNA transcript or a polypeptide.

[0158] The term “hydrophobic anchor” shall mean any hydrophobic moiety including but not limited to a lipoid, cholesterol, etc.

[0159] The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0160] A “steroid” is a compound comprising the following carbon skeleton:

[0161] Non-limiting examples of steroids include cholesterol, and the like.

[0162] An “ionizable lipid” refers to a lipid capable of being positively charged. Exemplary ionizable lipids include one or more amine group(s) which bear the positive charge. Preferred ionizable lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the ionizable lipid affects the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S. C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic non-bilayer structures (Hafez, I. M., et al., Gene Ther 8:1188-1196 (2001)) critical to the intracellular delivery of nucleic acids. As used herein, an “ionizable lipid” may also include, but is not limited to, a “cationic lipid”.

[0163] The term “polymer-conjugated lipid” compound refers to a compound comprising both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid compound is a compound comprising a lipid and a polymer having the structure set forth in Formula I, (la) or (lb).

[0164] The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like. The term “neutral lipid” refers to any of a number of lipid species that exist either 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), phophatidylethanolamines such as 1 ,2-Dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0165] The term “charged lipid” refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range e.g., pH ~3 to pH ~9. Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates, dialkyl trimethylammonium-propanes, (e.g., DOTAP, DOTMA), dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g. DC-Chol).

[0166] The term “lipid nanoparticle” refers to particles having at least one dimension on the order of nanometers (e.g., 1 -1 ,000 nm) which include one or more of the compounds of of the invention having Formula (I), (la) or (lb). In some embodiments, 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., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles of the invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a payload and one or more excipients selected from neutral lipids, charged lipids, steroids and polymer-conjugated lipid compounds having Formula (I), (la) or (lb). In some embodiments, the payload is an active agent or therapeutic agent, such as a nucleic acid, which may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g., an adverse immune response.

[0167] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 1 10 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 1 15 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0168] As used herein, the “polydispersity index," or "PDI" is a ratio that describes the homogeneity of the particle size distribution of a system. Calculations used for the determination of size and PDI parameters may be found in the ISO standard documents 13321 :1996 E and ISO 22412:2008 (Worldwide M.l. Dynamic Light Scattering, Common Terms Defined. Malvern Instruments Limited; Malvern, UK: 201 1 . pp. 1 -6. Inform White Paper). A value of less than 0.3 indicates a narrow particle size distribution. For polymers, the term “dispersity” or has been recommended by IUPAC in place of the term polydispersity index (PDI), and is a measure of either molecular mass or degree of polymerization in a given polymer sample. D of a polymer is calculated using the equation DM= MJMn, where Mwis the weight-average molar mass and Mnis the number-average molar mass. It can also be calculated according to degree of polymerization, where £>x = Xw / Xn, where Xwis the weight-average degree of polymerization and XJs the number-average degree of polymerization. In certain cases where DM= Dx, it is simply referred to as D. The dispersity indicates the distribution of individual molecular masses in a batch of polymers. D has a value equal to orgreaterthan 1 , but as the polymer chains approach uniform chain length, D approaches unity (e.g., D approaches 1). Dispersity is a measure of the dispersion (or spread) of a molar-mass, relative-molecular-mass, molecular-weight, or degree- of-polymerization distribution. For a uniform polymer, D = 1 ; for a polymer of sufficiently high Xnhaving a Poisson distribution of molar masses, relative molecular masses, or molecular weights, D = 1 ; and for a polymer of sufficiently high X„ having a most-probable distribution of molar masses, relative molecular masses, or molecular weights, D = 2. (Stepto, R. F. T.; Gilbert, R. G.; Hess, M.; Jenkins, A. D.; Jones, R. G.; Kratochvil P. (2009) "Dispersity in Polymer Science" Pure Appl. Chem. 81 (2): 351-353. DGI:10.1351 / PAC-REC-08-05-02).

[0169] As used herein, “lipid encapsulated” refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), with full encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticle

[0170] As used herein, "encapsulation efficiency” refers to the percentage of a therapeutic agent that becomes part of a nanoparticle composition, relative to the initial total amount of therapeutic agent used in the preparation. Encapsulation efficiency (EE%) is calculated by (total therapeutic agent added - free non-entrapped therapeutic agent) divided by the total therapeutic agent added.

[0171] As used herein, "size" or "mean size" in the context of lipid nanoparticles refers to the mean diameter of a nanoparticle composition.

[0172] “Serum-stable” in relation to nucleic acid-lipid nanoparticles means that the nucleotide is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.

[0173] “Stable compound” and “stable structure” are meant to indicate a compound (orstructure) that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0174] In an aspect, the compositions disclosed herein comprise lipids. For example, compositions can include lipids and mRNA (e.g., modRNA or saRNA), and the lipids and mRNA (e.g., modRNA or saRNA) can together form nanoparticles, thereby producing mRNA- containing nanoparticles comprising lipids. The lipids can encapsulate or associate with the mRNA in the form of a lipid nanoparticle (LNP) to aid stability, cell entry, and intracellular release of the RNA / lipid nanoparticles.

[0175] In some instances, a LNP comprises a micelle, a solid lipid nanoparticle, a nanoemulsion, a liposome, etc., or a combination thereof.

[0176] The lipid component of a LNP may include, for example, an ionizable lipid, a phospholipid , a polymer-conjugated lipid compound of the invention (e.g., conjugate comprising a lipid and a polymer of Formula (I), (la) or (lb)), a structural lipid, a neutral lipid, or any combination thereof. The elements of the lipid component may be provided in specific fractions. Suitable phospholipids, polymer-conjugated lipids, structural lipids, ionizable lipids, and neutral lipids for the methods of the present disclosure are further disclosed herein.

[0177] In some aspects, the lipid component of a LNP includes any one or more of a phospholipid, a polymer-conjugated lipid compound having Formula (I), (a) or (b), a structural lipid, an ionizable lipid, and / or a neutral lipid. In certain aspects, the lipid component of the lipid nanoparticle includes about 0 mol % to about 60 mol % ionizable lipid (e.g., at least about, at most about, between any two of, or exactly 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % ionizable lipid); about 0 mol % to about 60 mol % phospholipid (e.g., at least about, at most about, between any two of, or exactly 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % phospholipid); about 0 mol % to about 60 mol % structural lipid (e.g., at least about, at most about, between any two of, or exactly 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 mol % structural lipid); about 0 mol % to about 60 mol % of the polymer- conjugated lipid compound of Formula (I), (la) or (lb) (e.g., at least about, at most about, between any two of, or exactly 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% polymer-conjugated lipid compound of Formula (I), (la) or (lb)); and / or about 0 mol % to about 60 mol % neutral lipid (e.g., at least about, at most about, between any two of, or exactly 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % neutral lipid). The LNP can have any amount of the foregoing lipid components, provided that the total mol % does not exceed 100%. As used herein, “mol percent” refers to a component’s molar percentage relative to total moles of all lipid components in the LNP (e.g., total moles of ionizable lipid(s), the neutral lipid, the steroid and the polymer-conjugated lipid compound of Formula (I), (la) or (lb)).

[0178] In some aspects, the lipid component of the lipid nanoparticle includes about 35 mol % to about 55 mol % compound of ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 50 mol % structural lipid, and about 0.2 mol % to about 10 mol % of the polymer-conjugated lipid compound of Formula (I), (la) or (lb). In a particular aspect, the lipid component includes about 50 mol % said ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of the polymer-conjugated lipid compound of Formula (I), (la) or (lb). In another particular aspect, the lipid component includes about 40 mol % said ionizable lipid, about 20 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of the polymer-conjugated lipid compound of Formula (I), (la) or (lb).

[0179] In some aspects, the phospholipid may be DOPE or DSPC. In other aspects, the polymer-conjugated lipid compound may be a lipid conjugated to a polymer having Formula (I), (la) or (lb) and / or the structural lipid may be cholesterol.

[0180] In some aspects, the lipid nanoparticle includes: i) between 40 and 50 mol percent of an ionizable lipid; ii) between 9 and 10 mol percent of a phospholipid and / or a neutral lipid; iii) between 40 and 44 mol percent of a structural lipid; iv) between 0.2 and 3.2 mol percent of a polymer-conjugated lipid compound comprising a lipid conjugated to a polymer having Formula (I), (la) or (lb); and v) a therapeutic agent (e.g., RNA) encapsulated within or associated with the lipid nanoparticle.

[0181] In some aspects, the lipid nanoparticle comprises from 41 to 50 mol percent, from 42 to 50 mol percent, from 43 to 50 mol percent, from 44 to 50 mol percent, from 45 to 50 mol percent, from 46 to 50 mol percent, or from 47 to 50 mol percent of the ionizable lipid. In certain specific aspects, the lipid nanoparticle comprises at least about, at most about, between any two of, or exactly 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 mol percent of the ionizable lipid. In certain specific aspects, the lipid nanoparticle comprises at least about, at most about, between any two of, or exactly 41 .0, 41.1 , 41 .2, 41 .3, 41 .4, 41 .5, 41 .6, 41 .7, 41 .8, 41 .9, 42.0, 42.1 , 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1 , 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1 , 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1 , 45.2, 45.3, 45.4, 45.5, 45.6,

[0182] 45.7, 45.8, 45.9, 46.0, 46.1 , 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1 , 47.2, 47.3,

[0183] 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1 , 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49.0,

[0184] 49.1 . 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, or 50 mol percent of the ionizable lipid.

[0185] In some aspects, the phospholipid and / or neutral lipid is present in a concentration ranging from 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent. In certain aspects, the phospholipid and / or neutral lipid is present in a concentration of at least about, at most about, in between any two of, or exactly 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 mol percent. In certain aspects, the phospholipid and / or neutral lipid is present in a concentration of at least about, at most about, in between any two of, or exactly 5, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 ,

[0186] 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1 , 10.2,

[0187] 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11 , 11.1 , 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1 , 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1 , 13.2, 13.3, 13.4, 13.5, 13.6,

[0188] 13.7, 13.8, 13.9, 14, 14.1 , 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15 mol percent. In certain specific aspects, the phospholipid and / or neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent.

[0189] In some aspects, the molar ratio of the ionizable lipid to the phospholipid and / or neutral lipid ranges from about 4.1 :1 .0 to about 4.9:1 .0, from about 4.5:1 .0 to about 4.8:1 .0, or from about 4.7:1 .0 to 4.8:1 .0. In other aspects, the molar ratio of the phospholipid and / or neutral lipid to the ionizable lipid is 1 :4.1 , 1 :4.2, 1 :4.3, 1 :4.4, 1 :4.5, 1 :4.6, 1 :4.7, 1 :4.8, or 1 :4.9.

[0190] In some aspects, the structural lipid is a steroid. In some aspects, the steroid is cholesterol. In some aspects, the structural lipid is present in a concentration ranging from about 39 to 49 mol percent, 40 to 46 molar percent, from 40 to 44 mol percent, from 41 to 44 molar percent, from 40 to 42 mol percent, from 42 to 44 mol percent, or from 44 to 46 mol percent. . In certain specific aspects, the structural lipid is present in a concentration of 40, 41 , 42, 43, 44, 45, or 46 mol percent. In certain specific aspects, the structural lipid is present in a concentration of at least about, at most about, in between any two of, or exactly 39, 39.1 , 39.2,

[0191] 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, 40, 40.1 , 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9,

[0192] 41 . 41.1. 41 .2, 41 .3, 41 .4, 41 .5, 41 .6, 41 .7, 41 .8, 41 .9, 42, 42.1 , 42.2, 42.3, 42.4, 42.5, 42.6,

[0193] 42.7, 42.8, 42.9, 43, 43.1 , 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.1 , 44.2, 44.3,

[0194] 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45, 45.1 , 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46,

[0195] 46.1 . 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47, 47.1 , 47.2, 47.3, 47.4, 47.5, 47.6, 47.7,

[0196] 47.8, 47.9, 48, 48.1 , 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, or 49 mol percent

[0197] In other aspects, the structural lipid is present in a concentration ranging from 40 to 60 mol %. In certain aspects, the structural lipid is present in a concentration of at least about, at most about, in between any two of, or exactly 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol percent. In certain specific aspects, the structural lipid is present in a concentration of about 48, 49, or 50 mol percent.

[0198] In certain aspects, the molar ratio of ionizable lipid to the structural lipid ranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1. O to 1.0:1.2, e.g., 1 :0.9, 1 :1 , 1 :1.1 , or 1 :1.2.

[0199] In a particular aspect, the lipid component of the lipid nanoparticle includes an ionizable lipid, phospholipid, structural lipid, and polymer-conjugated lipid compound of Formula I at a molar ratio of about 47.5: 10: 40.7: 1.8. In another aspect, the lipid component of the lipid nanoparticle includes an ionizable lipid, phospholipid, structural lipid, and polymer-conjugated lipid compound of Formula I at a molar ratio of about 46.3: 9.4: 44.1 : 0.2. In another aspect, the lipid component of the lipid nanoparticle includes an ionizable lipid, phospholipid, structural lipid, and polymer-conjugated lipid compound of Formula I at a molar ratio of about 46.3: 9.4: 43.9: 0.4. In another aspect, the lipid component of the lipid nanoparticle includes an ionizable lipid, phospholipid, structural lipid, and polymer-conjugated lipid compound of Formula I at a molar ratio of about 46.3: 9.4: 43.8: 0.5. In another aspect, the lipid component of the lipid nanoparticle includes an ionizable lipid, phospholipid, structural lipid, and polymer-conjugated lipid compound of Formula I at a molar ratio of about 46.3: 9.4: 43.5: 0.8. In another aspect, the lipid component of the lipid nanoparticle includes an ionizable lipid, phospholipid, structural lipid, and polymer-conjugated lipid compound of Formula I at a molar ratio of about 46.3: 9.4: 42.7: 1 .6. In another particular aspect, the lipid component of the lipid nanoparticle includes an ionizable lipid, phospholipid, structural lipid, and polymer-conjugated lipid compound of Formula I at a molar ratio of about 46.3: 9.4: 41 .1 : 3.2.

[0200] POLYPHOSPHORYLCHOLINE POLYMER-CONJUGATED LIPIDS

[0201] The lipid nanoparticles described herein such as the RNA particles described herein comprising an ionizable lipid and an additional lipid further include a polymer lipid such as a polyphosphorylcholine-conjugated lipid. The polyphosphorylcholine may be conjugated, in particular covalently bound to or linked to, any particle forming component such as a lipid or lipid-like material. The polyphosphorylcholine-lipid conjugate is a molecule wherein polyphosphorylcholine is conjugated to a lipid. In certain embodiments, the polyphosphorylcholine-lipid conjugate or a conjugate of polyphosphorylcholine and a lipid-like material comprises the following general Formula (lb): The symbol “n” in Formula (lb) refers to the number of polymer units and may be a number as defined herein. in certain embodiments, the polyphosphorylcholine-Hpid conjugate or a conjugate of polyphosphorylcholine and a lipid-like material is a poly-2- methacryloyloxyethylphosphorylcholine (PMPC) conjugate.

[0202] In certain instances, the polyphosphorylcholine-lipid conjugate compound may comprise from about 0.1 mol % to about 50 mol %, from about 0.25 mol % to about 30 mol %, from about 0.5 mol % to about 25 mol %, from about 0.75 mol % to about 25 mol %, from about 1 mol % to about 25 mol %, from about 1 mol % to about 20 mol %, from about 1 mol % to about 15 mol %, from about 1 mol % to about 10 mol %, from about 1 mol % to about 5 mol %, from about 1 .5 mol % to about 25 mol %, from about 1 .5 mol % to about 20 mol %, from about 1 .5 mol % to about 15 mol %, from about 1.5 mol % to about 10 mol %, from about 1.5 mol % to about 5 mol %, from about 2 mol % to about 25 mol %, from about 2 mol % to about 20 mol %, from about 2 mol % to about 15 mol %, from about 2 mol % to about 10 mol %, or from about 2 mol % to about 5 mol % of the total lipid present in the lipid nanoparticle.

[0203] In one embodiment, the polyphosphorylcholine-lipid conjugate compound is (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-8k] (“DiTDA-PMPC8k”); or (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-11 k] (“DiTDA-PMPC11 k”).

[0204] In one aspect, DiTDA-PMPC8k may comprise from about 0.1 mol% to 3.2 mol% of the total lipid present in the lipid nanoparticle. In one aspect, DiTDA-PMPC8k may comprise from about 0.1 mol% to 1.6 mol% of the total lipid present in the lipid nanoparticle. In one aspect, DiTDA-PMPC8k may comprise from about 0.1 mol% to 0.8 mol% of the total lipid present in the lipid nanoparticle. In another aspect, DiTDA-PMPC8k may comprise from about 0.4 mol% to 0.8 mol% of the total lipid present in the lipid nanoparticle. In one aspect, DiTDA-PMPC8k may comprise from about 0.1 mol% to 0.5 mol% of the total lipid present in the lipid nanoparticle. In one aspect, DiTDA-PMPC8k may comprise from about 0.1 mol% to 0.2 mol% of the total lipid present in the lipid nanoparticle. In another aspect, DiTDA-PMPC8k may comprise from about 0.1 mol%, 0.2 mol%, 0.3 mol%, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%,

[0205] 1.0 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%,

[0206] 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%,

[0207] 2.8 mol%, 2.9 mol%, 3.0 mol%, 3 / 1 mol% or 3.2 mol% of the total lipid present in the lipid nanoparticle. In a particular aspect, DiTDA-PMPC8k may comprise 0.2 mol% of the total lipid present in the lipid nanoparticle. In another particular aspect, DiTDA-PMPC8k may comprise 0.4 mol% of the total lipid present in the lipid nanoparticle. In another particular aspect, DiTDA- PMPC8k may comprise 0.5 mol% of the total lipid present in the lipid nanoparticle. In another particular aspect, DiTDA-PMPC8k may comprise 0.8 mol% of the total lipid present in the lipid nanoparticle. In another particular aspect, DiTDA-PMPC8k may comprise 1 .6 mol% of the total lipid present in the lipid nanoparticle. In another particular aspect, DiTDA-PMPC8k may comprise 3.2 mol% of the total lipid present in the lipid nanoparticle.

[0208] In one aspect, DiTDA-PMPC11 k may comprise 0.8 + / - 0.7 mol% of the total lipid present in the lipid nanoparticle. In one aspect, DiTDA-PMPC11 k may comprise from about 0.1 mol% to

[0209] 1.5 mol% of the total lipid present in the lipid nanoparticle. . In one aspect, DiTDA-PMPC11 k may comprise from about 0.1 mol% to 3.2 mol% of the total lipid present in the lipid nanoparticle. In another aspect, DiTDA-PMPC11 k may comprise from about 0.4 mol% to 0.8 mol% of the total lipid present in the lipid nanoparticle. In another aspect, DiTDA-PMPC11 k may comprise from about 0.1 mol%, 0.2 mol%, 0.3 mol%, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%,

[0210] 0.8 mol%, 0.9 mol%, 1 .0 mol%, 1 .1 mol%, 1 .2 mol%, 1 .3 mol%, 1 .4 mol%, 1 .5 mol%, 1 .6 mol%,

[0211] 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%,

[0212] 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3.0 mol%, 3 / 1 mol% or 3.2 mol % of the total lipid present in the lipid nanoparticle. In a particular aspect, DiTDA-PMPC11 k may comprise 0.2 mol% of the total lipid present in the lipid nanoparticle. In a preferred aspect, DiTDA-PMPC11 k may comprise 0.4 mol% of the total lipid present in the lipid nanoparticle. In another preferred aspect, DiTDA-PMPC11 k may comprise 0.5 mol% of the total lipid present in the lipid nanoparticle. In another preferred aspect, DiTDA-PMPC11 k may comprise 0.6 mol% of the total lipid present in the lipid nanoparticle. In another preferred aspect, DiTDA-PMPC11 k may comprise 0.7 mol% of the total lipid present in the lipid nanoparticle. In another preferred aspect, DiTDA-PMPC11 k may comprise 0.8 mol% of the total lipid present in the lipid nanoparticle. In another particular aspect, DiTDA-PMPC11 k may comprise 1 .6 mol% of the total lipid present in the lipid nanoparticle. In another particular aspect, DiTDA-PMPC11 k may comprise 3.2 mol% of the total lipid present in the lipid nanoparticle.

[0213] Typically, the polyphosphorylcholine polymer moiety has between 1 and 1 ,000, between 1 and 500, between 2 and 200, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, or between 10 and 80, or between 10 and 40 monomer units as set forth in Formula (II). In a preferred embodiment, the polyphosphorylcholine polymer moiety has 14, 16, 27, 38, 28 or 36 monomer units as set forth in Formula (II). In a further preferred embodiment, the monomer unit is 2-methacryloyloxyethylphosphorylcholine (MPC).

[0214] In embodiments of the polymer-conjugated lipid compounds of the present invention, the polymer moiety of the compound is also defined by its molecular weight in kiloDaltons. For example, one embodiment of the invention is (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl- 1 -oxopropan-2-yl)[(poly(2-((oxido(2-(trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2- methacrylate)-8k], wherein the “8k” at the end of the chemical name signifies that the PMPC polymer moiety of the polymer-conjugated lipid compound has a molecular weight of about 8000 Daltons (or 8 kiloDaltons or “8k”). Another embodiment of the invention is (1-(4- (ditetradecylamino)-4-oxobutoxy)-2-methyl-1 -oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-1 1 k], wherein the “11 k” at the end of the chemical name signifies that the PMPC polymer moiety of the polymer-conjugated lipid compound has a molecular weight of about 11000 Daltons (or 11 kiloDaltons or “1 1 k”).

[0215] The polymer conjugated lipid compound of embodiments E1 to E12 may be produced by a Controlled Radical Polymerization method. Controlled Radical Polymerization is known in the art (“Controlled Radical Polymerization Guide” by MilliporeSigma (2019) https: / / www.google.com / url?sa=i&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=0 CDgQw7AJahcKEwjgxZbR9pqAAxUAAAAAHQAAAAAQAw&url=https%3A%2F%2Fwww.sigm aaldrich.com%2Fdeepweb%2Fassets%2Fsigmaaldrich%2Fmarketing%2Fglobal%2Fdocument s%2F716%2F722%2Fcrp-guide-br5077en- mk.pdf&psig=AOvVaw3YkLDsyEurzrlSAfh2EsyM&ust=1689861435292190&opi=89978449) and includes Atom Transfer Radical Polymerization (ATRP) or Reversible Addition / Fragmentation Chain Transfer Polymerization (RAFT).

[0216] IONIZABLE CATIONIC LIPIDS

[0217] Cationic or cationically ionizable lipids or lipid-like materials refer to a lipid or lipid-like material capable of being positively charged and able to electrostatically bind nucleic acid. As used herein, a “cationic lipid” or “cationic lipid-like material” refers to a lipid or lipid like material having a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Cationic lipids may encapsulate negatively charged RNA.

[0218] In some aspects, cationic lipids are ionizable such that they may exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. Without wishing to be bound by theory, this ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH. For purposes of the present disclosure, such “cationically ionizable” lipids or lipid-like materials are comprised by the term “cationic lipid” or “cationic lipid-like material” unless contradicted by the circumstances. In some aspects, a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the particle. In some aspects, a cationic lipid may be at least, at most, exactly, or between any two of 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, 90 mol %, or 100 mol %, or any range or value derivable therein, of the total lipid present in the particle.

[0219] Examples of cationic lipids include, but are not limited to: ((4- hydroxybutyl)azanediyl)bis(hexane-6,1 -diyl)bis(2-hexyldecanoate); 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1 ,2- di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N — (N’,N’- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1 ,2-diacyloxy-3- dimethylammonium propanes; 1 ,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1 ,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE),

[0220] 1 .2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1 ,2-dimyristoyl-3- trimethylammonium propane (DMTAP), 1 ,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l- propanamium trifluoroacetate (DOSPA), 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc- tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l- (cis,cis-9’,12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1 ,2-N,N’-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy- N,N-dimethylpropylamine (DLinDAP), 1 ,2-N,N’-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1 ,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3] - dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3] -dioxolane (DLin- KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3 -DM A) , N-(2-Hydroxyethyl)-N,N-dimethyl-2,3 -bis(tetradecyloxy )-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1 propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-1 -propanaminium bromide (bAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-

[0221] 2.3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3b)-cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1 -amine (Octyl-CLinDMA), 1 ,2- dimyristoyl-3-dimethylammonium-propane (DMDAP), 1 ,2-dipalmitoyl-3-dimethylammonium- propane (DPDAP), N1 -[2-((1 S)-1 -[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1 ,2-dioleoyl-sn- glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N- dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8’- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan-1 -amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1 -amine (DM DMA), Di((Z)-non-2-en-l-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2- dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}- ethylamino)propionamide (lipidoid 98N12-5), 1 -[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2- [bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid 02-200); or heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl) amino) octanoate (SM-102).

[0222] In some aspects, the lipid nanoparticles comprise one or more cationic lipids. In one aspect, the lipid nanoparticles comprise (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate) (ALC-0315), having the formula:

[0223] Cationic lipids are disclosed in, e.g., U.S. 10,166,298, the full disclosures of which are herein incorporated by reference in their entirety for all purposes. Representative cationic lipids include:

[0224]

[0225]

[0226] In some aspects, the RNA-LNPs comprise a cationic lipid, a RNA molecule as described herein and one or more of neutral lipids, steroids, pegylated lipids, or combinations thereof. If more than one cationic lipid is incorporated within the LNP, such percentages apply to the combined cationic lipids. In one aspect, the cationic lipid is present in the LNP in an amount such as at least, at most, exactly, or between any two of about 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60 mole percent, respectively.

[0227] In some aspects of the disclosure the LNP comprises a combination or mixture of any the lipids described above.

[0228] ADDITIONAL LIPIDS

[0229] In certain aspects, the LNP comprises one or more additional lipids or lipid-like materials that stabilize the formation of particles during their formation. Suitable stabilizing or structural lipids include non-cationic lipids, e.g., neutral lipids and anionic lipids. Without being bound by any theory, optimizing the formulation of LNPs by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable / cationic lipid or lipid-like material may enhance particle stability and efficacy of nucleic acid delivery.

[0230] As used herein, an “anionic lipid” refers to any lipid that is negatively charged at a selected pH. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. In some aspects, additional lipids comprise one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.

[0231] Representative neutral lipids include phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines, ceramides, sphingomyelins, dihydro-sphingomyelins, cephalins, and cerebrosides. Exemplary phospholipids include, for example, phosphatidylcholines, e.g., diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPO), 1 ,2-di-G-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and 1 - hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC); and phosphatidylethanolamines, e.g., diacylphosphatidylethanolamines, such as dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), distearoyl-phosphatidylethanolamine (DSPE), iphytanoyl-phosphatidylethanolamine (DpyPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and 1 ,2-dielaidoyl-sn- glycero-3-phophoethanolamine (transDOPE). In one aspect, the neutral lipid is 1 ,2-distearoyl-sn- glycero-3phosphocholine (DSPC), having the formula:

[0232] In some aspects, the LNPs comprise a neutral lipid, and the neutral lipid comprises one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, or SM.

[0233] In various aspects, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

[0234] In certain aspects, the steroid or steroid analogue is cholesterol. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4’-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof. In one aspect, the cholesterol has the formula:

[0235] Without being bound by any theory, the amount of the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some aspects, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2:1 to about 8:1 , or from about 10:0 to about 1 :9, about 4:1 to about 1 :2, or about 3:1 to about 1 :1.

[0236] In some aspects, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and / or cholesterol), may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in the particle. In some aspects, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and / or cholesterol), may be at least, at most, exactly, or between any two of 0 mol %, 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, or 90 mol % of the total lipid present in the particle.

[0237] In preferred aspects, the composition further includes a nucleic acid. In preferred aspects, the nucleic acid comprises messenger RNA. In some aspects, the composition further includes one or more excipients selected from neutral lipids and steroids. In some aspects, the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. Preferably, in some aspects, the neutral lipid is DSPC. Preferably, in some aspects, the steroid is cholesterol.

[0238] A LNP may include one or more components described herein. In some aspects, the LNP formulation of the disclosure includes at least one lipid nanoparticle component. Lipid nanoparticles may include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic, such as a nucleic acid. A LNP may be designed for one or more specific applications or targets. The elements of a LNP may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a LNP may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combinations of elements. The efficacy and tolerability of a LNP formulation may be affected by the stability of the formulation.

[0239] Lipid nanoparticles may be designed for one or more specific applications or targets. For example, a LNP may be designed to deliver a therapeutic and / or prophylactic such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal’s body. Physiochemical properties of lipid nanoparticles may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs. The therapeutic and / or prophylactic included in a LNP may also be selected based on the desired delivery target or targets. For example, a therapeutic and / or prophylactic may be selected for a particular indication, condition, disease, or disorder and / or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery). In certain aspects, a LNP may include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest. Such a composition may be designed to be specifically delivered to a particular organ. In some aspects, a composition may be designed to be specifically delivered to a mammalian liver. In some aspects, a composition may be designed to be specifically delivered to a lymph node. In some aspects, a composition may be designed to be specifically delivered to a mammalian spleen.

[0240] In some aspects, a surface altering agent may be included in and / or used to encapsulate or partially encapsulate a LNP. Surface altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., ionizable surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin p4, dornase alfa, neltenexine, and erdosteine), and DNases (e.g., rhDNase). A surface altering agent may be disposed within a nanoparticle and / or on the surface of a LNP (e.g., by coating, adsorption, covalent linkage, or other process).

[0241] A LNP may also comprise one or more functionalized lipids. For example, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction. In particular, a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition, or imaging. The surface of a LNP may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0242] In addition to these components, lipid nanoparticles may include any substance useful in pharmaceutical compositions. For example, the lipid nanoparticle may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, surface active agents, buffering agents, preservatives, and other species.

[0243] Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxy ethylene sorbitan monolaurate [TWEENO20], polyoxy ethylene sorbitan [TWEEN® 60], polyoxy ethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0244] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, free radical scavengers, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™, KATHON™, and / or EUXYL®. An exemplary free radical scavenger includes butylated hydroxytoluene (BHT or butylhydroxytoluene) or deferoxamine. In some preferred aspects, the composition does not include a preservative.

[0245] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d- gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, and / or combinations thereof. In some aspects, the concentration of the buffer in the composition is about 10 mM. For example, the buffer concentration can be equal to any one of, at least any one of, at most any one of, or between any two of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or any range or value derivable therein. In specific aspects, the buffer concentration is 10 mM. The buffer can be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the buffer can be at pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In specific aspects, the buffer is at pH 7.4.

[0246] In some aspects, the formulation including an LNP may further include a salt, such as a chloride salt. In some aspects, the formulation including a LNP may further includes a sugar such as a disaccharide. In some aspects, the formulation further includes a sugar but not a salt, such as a chloride salt. In some aspects, a LNP may further include one or more small hydrophobic molecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof). The characteristics of a LNP may depend on the components thereof. For example, an LNP including cholesterol as a structural lipid may have different characteristics than an LNP that includes a different structural lipid. As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In some aspects, the structural lipid is a steroid. In some aspects, the structural lipid is cholesterol. In some aspects, the structural lipid is an analog of cholesterol. In some aspects, the structural lipid is alpha-tocopherol.

[0247] In some aspects, the characteristics of an LNP may depend on the absolute or relative amounts of its components. For instance, an LNP including a higher molar fraction of a phospholipid may have different characteristics than an LNP including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the lipid nanoparticle. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0248] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. In some aspects, a ionizable phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. In some aspects, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidyl-ethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. In some aspects, a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC. Formulations comprising amphiphilic polymers and lipid nanoparticles may be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may include one or more amphiphilic polymers and one or more lipid nanoparticles. For example, a pharmaceutical composition may include one or more amphiphilic polymers and one or more lipid nanoparticles including one or more different therapeutics and / or prophylactics. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington’s The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a LNP or the one or more amphiphilic polymers in the formulation of the disclosure. An excipient or accessory ingredient may be incompatible with a component of a LNP or the amphiphilic polymer of the formulation if its combination with the component or amphiphilic polymer may result in any undesirable biological effect or otherwise deleterious effect.

[0249] In some aspects, the composition may comprise a pharmaceutically acceptable carrier and / or vehicle. In some aspects, the composition may further include pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g., phosphate, citrate etc. buffered solutions. In some aspects, the composition may include water and / or a buffer containing a sodium salt, such as at least 50 mM of a sodium salt, a calcium salt, in some aspects at least 0.01 mM of a calcium salt, and optionally a potassium salt, in some aspects at least 3 mM of a potassium salt. In some aspects the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g., chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Examples of sodium salts include e.g., NaCI, Nal, NaBr, Na2CO3, NaHCO3, Na2SO4, examples of the potassium salts include e.g., KCI, KI, KBr, K2CO3, KHCO3, K2SO4, and examples of calcium salts include e.g., CaCI2, Cal2, CaBr2, CaCO3, CaSO4, Ca(OH)2. In some aspects, organic anions of the aforementioned cations may be contained in the buffer. In some aspects, the composition may include salts selected from sodium chloride (NaCI), calcium chloride (CaCI2) and potassium chloride (KCI), wherein further anions may be present additional to the chlorides. CaCI2can also be replaced by another salt like KCI. In some aspects, the injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, e.g. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein such concentrations of the afore mentioned salts may be used, which minimizes damage of cells due to osmosis or other concentration effects. The concentration of the salts in the composition can be about 70 mM to about 140 mM. For example, the salt concentration can be equal to any one of, at least any one of, at most any one of, or between any two of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM, or any range or value derivable therein. The salt can be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the salt can be at a pH equal to any one of, at least any one of, at most any one of, or between any two of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein.

[0250] In some aspects, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a LNP. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some aspects, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. Examples of excipients, which refer to ingredients in the compositions that are not active ingredients, include but are not limited to carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compression agents, wet granulation agents, or colorants. Preservatives for use in the compositions disclosed herein include but are not limited to benzalkonium chloride, chlorobutanol, paraben and thimerosal. As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer’s dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Diluents, or diluting or thinning agents, include but are not limited to ethanol, glycerol, water, sugars such as lactose, sucrose, mannitol, and sorbitol, and starches derived from wheat, corn rice, and potato; and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 10% to about 90% by weight of the total composition, about 25% to about 75%, about 30% to about 60% by weight, or about 12% to about 60%.

[0251] In some aspects, an excipient is approved for use in humans and for veterinary use. In some aspects, an excipient is approved by United States Food and Drug Administration. In some aspects, an excipient is pharmaceutical grade. In some aspects, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia. Relative amounts of the one or more amphiphilic polymers, the one or more lipid nanoparticles, the one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more lipid nanoparticles. As another example, a pharmaceutical composition may comprise between 0.1 % and 15% (wt / vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% w / v).

[0252] The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.

[0253] In certain aspects, the lipid nanoparticles and / or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and / or shipment (e.g., being stored at a temperature of 10 °C or lower, such as a temperature at about 4 °, a temperature between about -150 °C and about 10 °C (e.g., about 10 °C, 9 °C, 8 °C, 7 °C, 6 °C, 5 °C, 4 °C, 3 °C, 2 °C, 1 °C, 0 °C, -1 °C, -2 °C, -3 °C, -4 °C, -5 °C, -6 °C, -7 °C, -8 °C, -9 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C) or a temperature between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C). For example, the pharmaceutical composition comprising one or more amphiphilic polymers and one or more lipid nanoparticles is a solution or solid (e.g., via lyophilization) that is refrigerated for storage and / or shipment at, for example, about -20 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80°C or - 90 °C.

[0254] In certain aspects, the disclosure also relates to a method of increasing stability of the lipid nanoparticles by adding an effective amount of an amphiphilic polymer and by storing the lipid nanoparticles and / or pharmaceutical compositions thereof at a temperature of 10 °C or lower, such as a temperature at about 4 °C, a temperature between about -150 °C and about 10 °C (e.g., about 10 °C, 9 °C, 8 °C, 7 °C, 6 °C, 5 °C, 4 °C, 3 °C, 2 °C, 1 °C, 0 °C, -1 °C, -2 °C, - 3°C, -4 °C, -5 °C, -6 °C, -7 °C, -8 °C, -9 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C) or a temperature between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C).

[0255] The chemical properties of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of an LNP. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of an LNP, such as particle size, polydispersity index, and zeta potential.

[0256] The mean size of an LNP may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 150 nm, such as about 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. In some aspects, the mean size of a LNP may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain aspects, the mean size of a LNP may be from about 70 nm to about 100 nm. In a particular aspect, the mean size may be about 80 nm. In other aspects, the mean size may be about 100 nm.

[0257] An LNP may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index from about 0 to about 0.3, such as 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 or 0.30. In some aspects, the polydispersity index of an LNP may be from about 0.10 to about 0.20.

[0258] The zeta potential of a LNP may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively neutral charges are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some aspects, the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about - 10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0259] The efficiency of encapsulation of a therapeutic and / or prophylactic describes the amount of therapeutic and / or prophylactic that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and / or prophylactic in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and / or prophylactic (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a therapeutic and / or prophylactic may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some aspects, the encapsulation efficiency may be at least 80%. In certain aspects, the encapsulation efficiency may be at least 90%. In some aspects, the LNP encapsulation efficiency of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of blank LNPs (e.g., lipid nanoparticles comprising the lipids listed in herein but not encapsulating any nucleic acid) is about 50% or higher, about 55% or higher, about 60% or higher, about 65% or higher, about 70% or higher, about 75% or higher, about 80% or higher, about 8% or higher, about 90% or higher, about 91% or higher, about 92% or higher, about 93% or higher, about 94% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, or about 99% or higher than the LNP encapsulation efficiency of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lesser concentration of blank LNPs or in the absence of blank LNPs.

[0260] In some aspects, electrophoresis (e.g., capillary electrophoresis) or chromatography (e.g., reverse phase liquid chromatography) may be used to examine the mRNA integrity.

[0261] In some aspects, the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of blank LNPs (e.g., lipid nanoparticles comprising the lipids listed in herein but not encapsulating any nucleic acid) is about 20% or higher, about 25% or higher, about 30% or higher, about 35% or higher, about 40% or higher, about 45% or higher, about 50% or higher, about 55% or higher, about 60% or higher, about 65% or higher, about 70% or higher, about 75% or higher, about 80% or higher, about 85% or higher, about 90% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, or about 99% or higher than the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lesser concentration of blank LNPs or in the absence of blank LNPs.

[0262] In some aspects, the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of blank LNPs (e.g., lipid nanoparticles comprising the lipids listed in herein but not encapsulating any nucleic acid) is higher than the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lesser concentration of blank LNPs or in the absence of blank LNPs by about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 folds or more, about 2 folds or more, about 3 folds or more, about 4 folds or more, about 5 folds or more, about 10 folds or more, about 20 folds or more, about 30 folds or more, about 40 folds or more, about 50 folds or more, about 100 folds or more, about 200 folds or more, about 300 folds or more, about 400 folds or more, about 500 folds or more, about 1000 folds or more, about 2000 folds or more, about 3000 folds or more, about 4000 folds or more, about 5000 folds or more, or about 10000 folds or more.

[0263] As used herein, “Tx” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to degrade to about X of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For example, ”T80” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to degrade to about 80% of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For another example, ”T1 / 2” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to degrade to about 1 / 2 of the initial concentration of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation.

[0264] In some aspects, the Txo% of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of blank LNPs (e.g., lipid nanoparticles comprising the lipids listed in herein but not encapsulating any nucleic acid) is about 12 months or longer, about 15 months or longer, about 18 months or longer, about 21 months or longer, about 24 months or longer, about 27 months or longer, about 30 months or longer, about 33 months or longer, about 36 months or longer, about 48 months or longer, about 60 months or longer, about 72 months or longer, about 84 months or longer, about 96 months or longer, about 108 months or longer, about 120 months or longer than the Txo% of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lesser concentration of blank LNPs or in the absence of blank LNPs. In some aspects, the Txo% of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of blank LNPs (e.g., lipid nanoparticles comprising the lipids listed in herein but not encapsulating any nucleic acid) is longer than the Txo% of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lesser concentration of blank LNPs or in the absence of blank LNPs by about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 fold or more, about 2 folds or more, about 3 folds or more, about 4 folds or more, about 5 folds or more.

[0265] In some aspects, the T1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of blank LNPs (e.g., lipid nanoparticles comprising the lipids listed in herein but not encapsulating any nucleic acid) is about 12 months or longer, about 15 months or longer, about 18 months or longer, about 21 months or longer, about 24 months or longer, about 27 months or longer, about 30 months or longer, about 33 months or longer, about 36 months or longer, about 48 months or longer, about 60 months or longer, about 72 months or longer, about 84 months or longer, about 96 months or longer, about 108 months or longer, about 120 months or longerthan the T1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lesser concentration of blank LNPs or in the absence of blank LNPs.

[0266] In some aspects, the T1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of blank LNPs (e.g., lipid nanoparticles comprising the lipids listed in herein but not encapsulating any nucleic acid) is longerthan the T1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lesser concentration of blank LNPs or in the absence of blank LNPs by about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 fold or more, about 2 folds or more, about 3 folds or more, about 4 folds or more, about 5 folds or more.

[0267] The amount of a therapeutic and / or prophylactic in a LNP may depend on the size, composition, desired target and / or application, or other properties of the lipid nanoparticle as well as on the properties of the therapeutic and / or prophylactic. For example, the amount of an RNA useful in a LNP may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic and / or prophylactic (e.g., pharmaceutical substance) and other elements (e.g., lipids) in a LNP may also vary. In some aspects, the wt / wt ratio of the lipid component to a therapeutic and / or prophylactic in a LNP may be from about 5:1 to about 60:1 , such as 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 , 11 :1 , 12:1 , 13:1 , 14:1 , 15:1 , 16:1 , 17:1 , 18:1 , 19:1 , 20:1 , 25:1 , 30:1 , 35:1 , 40: 1 , 45: 1 , 50: 1 , and 60: 1 . For example, the wt / wt ratio of the lipid component to a therapeutic and / or prophylactic may be from about 10: 1 to about 40:1 . In certain aspects, the wt / wt ratio is about 20:1 . The amount of a therapeutic and / or prophylactic in a LNP may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy). In some aspects, the mRNA to lipid ratio in the LNP (e.g., N:P, where N represents the moles of ionizable lipid and P represents the moles of phosphate present as part of the nucleic acid backbone) range from 2:1 to 30:1 , for example 3:1 to 22:1 . In other aspects, N:P ranges from 6:1 to 20:1 or 2:1 to 12:1 . Exemplary N:P ranges include about 3:1 , about 6:1 , about 12:1 and about 22:1 .

[0268] Various exemplary embodiments of the lipid nanoparticles and compositions comprising the same, and their use to deliver active (e.g., therapeutic agents), such as nucleic acids, to modulate gene and protein expression, are described in further detail below.

[0269] Pharmaceutical Compositions

[0270] In another embodiment, the invention comprises pharmaceutical compositions. For pharmaceutical composition purposes, the compound per se or pharmaceutically acceptable salt thereof will simply be referred to as the compounds of the invention.

[0271] A "pharmaceutical composition" refers to a mixture of one or more of the compounds of the invention, or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof as an active ingredient, and at least one pharmaceutically acceptable excipient.

[0272] The term ’excipient’ is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0273] As used herein, "excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, carriers, diluents and the like that are physiologically compatible. Examples of excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof, and may include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol, or sorbitol in the composition. Examples of excipients also include various organic solvents (such as hydrates and solvates). The pharmaceutical compositions may, if desired, contain additional excipients such as flavorings, binders / binding agents, lubricating agents, disintegrants, sweetening or flavoring agents, coloring matters or dyes, and the like. For example, for oral administration, tablets containing various excipients, such as citric acid may be employed togetherwith various disintegrants such as starch, alginic acid and certain complex silicates and with binding agents such as sucrose, gelatin and acacia. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules. Non-limiting examples of excipients, therefore, also include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with additional excipients such as water, ethanol, propylene glycol, glycerin, or combinations thereof.

[0274] Examples of excipients also include pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the compound.

[0275] The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, capsules, pills, powders, liposomes and suppositories. The form depends on the intended mode of administration and therapeutic application.

[0276] Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with antibodies in general. One mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In another embodiment, the compound is administered by intravenous infusion or injection. In yet another embodiment, the compound is administered by intramuscular or subcutaneous injection.

[0277] Oral administration of a solid dosage form may be, for example, presented in discrete units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the invention. In another embodiment, the oral administration may be in a powder or granule form. In another embodiment, the oral dosage form is sub-lingual, such as, for example, a lozenge. In such solid dosage forms, the compounds of the invention are ordinarily combined with one or more adjuvants. Such capsules or tablets may comprise a controlled release formulation. In the case of capsules, tablets, and pills, the dosage forms also may comprise buffering agents or may be prepared with enteric coatings.

[0278] In another embodiment, oral administration may be in a liquid dosage form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art (e.g., water). Such compositions also may comprise adjuvants, such as one or more of wetting, emulsifying, suspending, flavoring (e.g., sweetening), or perfuming agents.

[0279] In another embodiment, the invention comprises a parenteral dosage form. "Parenteral administration" includes, for example, subcutaneous injections, intravenous injections, intraperitoneally, intramuscular injections, intrasternal injections, and infusion. Injectable preparations (e.g., sterile injectable aqueous or oleaginous suspensions) may be formulated according to the known art using one or more of suitable dispersing, wetting agents, or suspending agents. In another embodiment, the invention comprises a topical dosage form. "Topical administration" includes, for example, dermal and transdermal administration, such as via transdermal patches or iontophoresis devices, intraocular administration, or intranasal or inhalation administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments, and creams. A topical formulation may include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. When the compounds of this invention are administered by a transdermal device, administration will be accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical excipients include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated - see, for example, B. C. Finnin and T. M. Morgan, J. Pharm. Sci., vol. 88, pp. 955-958, 1999.

[0280] Formulations suitable for topical administration to the eye include, for example, eye drops wherein the compound of this invention is dissolved or suspended in a suitable excipient. Atypical formulation suitable for ocular or aural administration may be in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or methylcellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

[0281] For intranasal administration, the compounds of the invention are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant. Formulations suitable for intranasal administration are typically administered in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1 , 1 ,1 ,2- tetrafluoroethane or 1 ,1 ,1 ,2,3,3, 3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin. In another embodiment, the invention comprises a rectal dosage form. Such rectal dosage form may be in the form of, for example, a suppository. Other excipients and modes of administration known in the pharmaceutical art may also be used. Pharmaceutical compositions of the invention may be prepared by any of the well-known techniques of pharmacy, such as effective formulation and administration procedures. The above considerations in regard to effective formulations and administration procedures are well known in the art and are described in standard textbooks. Formulation of drugs is discussed in, for example, Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

[0282] Acceptable excipients are nontoxic to subjects at the dosages and concentrations employed, and may comprise one or more of the following: 1) buffers such as phosphate, citrate, or other organic acids; 2) salts such as sodium chloride; 3) antioxidants such as ascorbic acid or methionine; 4) preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol; 5) alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol; 6) low molecular weight (less than about 10 residues) polypeptides; 7) proteins such as serum albumin, gelatin, or immunoglobulins; 8) hydrophilic polymers such as polyvinylpyrrolidone; 9) amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; 10) monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrins; 1 1) chelating agents such as EDTA; 12) sugars such as sucrose, mannitol, trehalose or sorbitol; 13) salt-forming counter-ions such as sodium, metal complexes (e.g., Zn- protein complexes), or 14) non-ionic surfactants such as polysorbates (e.g., polysorbate 20 or polysorbate 80), poloxamers or polyethylene glycol (PEG).

[0283] For oral administration, the compositions may be provided in the form of tablets or capsules containing 0.01 , 0.05, 0.1 , 0.5, 1 .0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 75.0, 100, 125, 150, 175, 200, 250 or 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, or in another embodiment, from about 1 mg to about 100 mg of active ingredient. Intravenously, doses may range from about 0.01 to about 10 mg / kg / minute during a constant rate infusion.

[0284] Liposome containing compounds of the invention may be prepared by methods known in the art (See, for example, Chang, H.I.; Yeh, M.K.; Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy; Int J Nanomedicine 2012; 7; 49-60). Particularly useful liposomes may be generated by the reverse phase evaporation method . Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. The formulations to be used for intravenous administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Compounds of the invention are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0285] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

[0286] “Systemic delivery,” as used herein, refers to delivery of a therapeutic product that can result in a broad exposure of an active agent within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts 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.

[0287] “Local delivery,” as used herein, refers to delivery of an active agent directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor, other target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like. Local delivery can also include topical applications or localized injection techniques such as intramuscular, subcutaneous or intradermal injection. Local delivery does not preclude a systemic pharmacological effect.

[0288] Administration and Dosing

[0289] The term "treating", "treat" or "treatment" as used herein embraces both preventative, e.g., prophylactic, and palliative treatment, e.g., relieve, alleviate, or slow the progression of the patient’s disease (or condition) or any tissue damage associated with the disease.

[0290] As used herein, the terms, “subject, “individual” or “patient,” used interchangeably, refer to any animal, including mammals. Mammals according to the invention include canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, humans and the like, and encompass mammals in utero. In an embodiment, humans are suitable subjects. Human subjects may be of any gender and at any stage of development. As used herein, the phrase “therapeutically effective amount” or “effective amount” refers to the amount of active compound or pharmaceutical agent, such as a nucleic acid, that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include one or more of the following:

[0291] (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease;

[0292] (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (e.g., arresting (or slowing) further development of the pathology or symptomatology or both); and

[0293] (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (e.g., reversing the pathology or symptomatology or both).

[0294] An “effective amount” or “therapeutically effective amount” of nucleic acid is an amount sufficient to produce the desired effect, e.g., an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid. An increase in 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. In the case where the expression product is present at some level prior to contact with the nucleic acid, an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control 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 greater. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art.

[0295] The phrase “induce expression of a desired protein” refers to the ability of a nucleic acid to increase expression of the desired protein. To examine the extent of protein expression, a test sample (e.g., a sample of cells in culture expressing the desired protein) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) is contacted with a nucleic acid (e.g., nucleic acid in combination with a lipid of the present invention). Expression of the desired protein in the test sample or test animal is compared to expression of the desired protein in a control sample (e.g., a sample of cells in culture expressing the desired protein) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that is not contacted with or administered the nucleic acid. When the desired protein is present in a control sample or a control mammal, the expression of a desired protein in a control sample or a control mammal may be assigned a value of 1 .0. In particular embodiments, inducing expression of a desired protein is achieved when the ratio of desired protein expression in the test sample or the test mammal to the level of desired protein expression in the control sample or the control mammal is greater than 1 , for example, about 1 .1 , 1 .5, 2.0. 5.0 or 10.0. When a desired protein is not present in a control sample or a control mammal, inducing expression of a desired protein is achieved when any measurable level of the desired protein in the test sample or the test mammal is detected. One of ordinary skill in the art will understand appropriate assays to determine the level of protein expression in a sample, for example dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0296] The phrase “inhibiting expression of a target gene” refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To examine the extent of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) is contacted with a nucleic acid that silences, reduces, or inhibits expression of the target gene. Expression ofthe target gene in the test sample or test animal is compared to expression of the target gene in a control sample (e.g., a sample of cells in culture expressing the target gene) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that is not contacted with or administered the nucleic acid. The expression of the target gene in a control sample or a control mammal may be assigned a value of 100%. In particular embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in the test sample or the test mammal relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, the nucleic acids are capable of silencing, reducing, or inhibiting the expression of a target gene by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a test mammal relative to the level of target gene expression in a control sample or a control mammal not contacted with or administered the nucleic acid. Suitable assays for determining the level of target gene expression include, without limitation, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[0297] Typically, a compound of the invention is administered in an amount effective to treat a condition as described herein. The compounds of the invention may be administered as compound per se, or alternatively, as a pharmaceutically acceptable salt. For administration and dosing purposes, the compound per se or pharmaceutically acceptable salt thereof will simply be referred to as the compounds of the invention.

[0298] The compounds of the invention are administered by any suitable route in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The compounds of the invention may be administered orally, rectally, vaginally, parenterally, topically, intranasally, or by inhalation.

[0299] The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the bloodstream directly from the mouth.

[0300] In another embodiment, the compounds of the invention may also be administered parenterally, for example directly into the bloodstream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

[0301] In another embodiment, the compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. In another embodiment, the compounds of the invention may also be administered intranasally or by inhalation. In another embodiment, the compounds of the invention may be administered rectally or vaginally. In another embodiment, the compounds of the invention may also be administered directly to the eye or ear.

[0302] The dosage regimen for the compounds of the invention or compositions containing said compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Thus, the dosage regimen may vary widely. In one embodiment, the total daily dose of a compound of the invention is typically from about 0.01 to about 100 mg / kg (e.g., mg compound of the invention per kg body weight) for the treatment of the indicated conditions discussed herein. In another embodiment, total daily dose of the compound of the invention is from about 0.00001 to about 50 mg / kg, and in another embodiment, from about 0.0001 to about 30 mg / kg. It is not uncommon that the administration of the compounds of the invention will be repeated a plurality of times in a day (typically no greater than 4 times). Multiple doses per day typically may be used to increase the total daily dose, if desired. Therapeutic Methods and Uses

[0303] The compounds of the invention may be useful for treating or preventing a disease, disorder, or condition. In particular, such compositions may be useful in treating or preventing a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity. Diseases, disorders, and / or conditions characterized by dysfunctional or aberrant protein or polypeptide activity for which a composition may be administered include, but are not limited to rare diseases, infectious diseases (as both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases.

[0304] Co-administration

[0305] The compounds of the invention may be used alone, or in combination with one or more other therapeutic agents. The invention provides any of the uses, methods or compositions as defined herein wherein the compound of the invention, or pharmaceutically acceptable salt thereof, is used in combination with one or more other therapeutic agent discussed herein.

[0306] The administration of two or more compounds “in combination” means that all of the compounds are administered closely enough in time to affect treatment of the subject. The two or more compounds may be administered simultaneously or sequentially, via the same or different routes of administration, on same or different administration schedules and with or without specific time limits depending on the treatment regimen. Additionally, simultaneous administration may be carried out by mixing the compounds prior to administration or by administering the compounds at the same point in time but as separate dosage forms at the same or different site of administration. Examples of “in combination” include, but are not limited to, “concurrent administration,” “co-administration,” “simultaneous administration,” “sequential administration” and “administered simultaneously”.

[0307] A compound of the invention and the one or more other therapeutic agents may be administered as a fixed or non-fixed combination of the active ingredients. The term "fixed combination" means a compound of the invention, or a pharmaceutically acceptable salt thereof, and the one or more therapeutic agents, are both administered to a subject simultaneously in a single composition or dosage. The term "non-fixed combination" means that a compound of the invention, or a pharmaceutically acceptable salt thereof, and the one or more therapeutic agents are formulated as separate compositions or dosages such that they may be administered to a subject in need thereof simultaneously or at different times with variable intervening time limits, wherein such administration provides effective levels of the two or more compounds in the body of the subject.

[0308] These agents and compounds of the invention may be combined with pharmaceutically acceptable vehicles such as saline, Ringer’s solution, dextrose solution, and the like. The particular dosage regimen, e.g., dose, timing and repetition, will depend on the particular individual and that individual’s medical history.

[0309] Kits

[0310] Another aspect of the invention provides kits comprising the compound of the invention or pharmaceutical compositions comprising the compound of the invention. A kit may include, in addition to the compound of the invention or pharmaceutical composition thereof, diagnostic or therapeutic agents. A kit may also include instructions for use in a diagnostic or therapeutic method. In some embodiments, the kit includes the compound or a pharmaceutical composition thereof and a diagnostic agent.

[0311] In yet another embodiment, the invention comprises kits that are suitable for use in performing the methods of treatment described herein. In one embodiment, the kit contains a first dosage form comprising one or more of the compounds of the invention in quantities sufficient to carry out the methods of the invention. In another embodiment, the kit comprises one or more compounds of the invention in quantities sufficient to carry out the methods of the invention and a container for the dosage.

[0312] Synthetic Methods

[0313] Compounds of the present invention may be synthesized by synthetic routes that include processes analogous to those well-known in the chemical arts, particularly in light of the description contained herein. The starting materials are generally available from commercial sources or may be prepared using methods well known to those skilled in the art. Many of the compounds used herein, are related to, or may be derived from compounds in which one or more of the scientific interest or commercial need has occurred. Accordingly, such compounds may be one or more of 1) commercially available; 2) reported in the literature or 3) prepared from other commonly available substances by one skilled in the art using materials which have been reported in the literature.

[0314] For illustrative purposes, the reaction schemes depicted below provide potential routes for synthesizing the compounds of the present invention as well as key intermediates. For a more detailed description of the individual reaction steps, see the Examples section below. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the inventive compounds. Although specific starting materials and reagents are discussed below, other starting materials and reagents may be substituted to provide one or more of a variety of derivatives or reaction conditions. In addition, many of the compounds prepared by the methods described below may be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

[0315] The skilled person will appreciate that the experimental conditions set forth in the schemes that follow are illustrative of suitable conditions for effecting the transformations shown, and that it may be necessary or desirable to vary the precise conditions employed for the preparation of compounds of the invention. It will be further appreciated that it may be necessary or desirable to carry out the transformations in a different order from that described in the schemes, or to modify one or more of the transformations, to provide the desired compound of the invention.

[0316] In the preparation of compounds of the invention it is noted that some of the preparation methods useful for the preparation of the compounds described herein may require protection of remote functionality (e.g., a primary amine, secondary amine, carboxyl, etc. in a precursor of a compound of the invention). The need for such protection will vary depending on the nature of the remote functionality and the conditions of the preparation methods. The need for such protection is readily determined by one skilled in the art. The use of such protection / deprotection methods is also within the skill in the art. For a general description of protecting groups and their use, see March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure 8th Edition; or Green’s Protective Groups in Organic Synthesis, 5th Edition, Wuts, P.G.M. Ed.

[0317] For example, if a compound contains an amine or carboxylic acid functionality, such functionality may interfere with reactions at other sites of the molecule if left unprotected. Accordingly, such functionalities may be protected by an appropriate protecting group (PG) which may be removed in a subsequent step. Suitable protecting groups for amine and carboxylic acid protection include those protecting groups commonly used in peptide synthesis (such as / V-t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and 9- fluorenylmethylenoxycarbonyl (Fmoc) for amines and lower alkyl or benzyl esters for carboxylic acids) which are generally not chemically reactive under the reaction conditions described and may typically be removed without chemically altering other functionality in a compound of the invention.

[0318] General Experimental Details

[0319] In the non-limiting Examples and Preparations that illustrate the invention and that are set out in the description, and in the following Schemes, the following abbreviations, definitions and analytical procedures may be referred to:

[0320] 1H NMR spectra were recorded on a Bruker 400 MHz spectrometer. The chemical shifts are reported in parts per million (ppm) and all spectra are referenced to their residual nondeuterated solvent peaks as follows: CHCI3(7.26 ppm), CD3OD (3.31 ppm), DMSO-c / 6 (2.50 ppm), D2O (4.75 ppm). Coupling constants (J) are reported to the nearest 0.1 Hz. Multiplicities are reported as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad singlet (br s). Exchangeable protons are not always observed.

[0321] LCMS data were acquired on an Agilent Prime-6125B instrument, Agilent Poroshell 120 EC-C18 2.7um 3.0*30mm column, acetonitrile / water gradients with TFA modifiers, using evaporative light scattering detector (ELSD) (see method A and B). Preparative chiral supercritical fluid chromatography (prep-SFC) was performed using DAICEL ChiralPAK-AD, -AS, -IC, DAICEL ChiralCEL-OJ, or -OD columns; gradient eluting with CO2mixtures with 0.1 % NH3H2O in EtOH and UV detection was used to trigger fraction collection. The LCMS-ELSD purity was verified by the following analytical methods and noted as area %: instrument = Agilent 1260 Infinity with 6150 MSD; Column = Waters XBridge C8 100*2.1 mm, 3.5pm; Mobile phase A, 0.05% DFA in water; Mobile phase B, 0.05% DFA in acetonitrile; Gradient = 50%-100% (solvent B) over 5 minutes and holding at 100% for 2 minutes at a flow rate of 1 .0 mL / min, total time of 7.0 min, 40 °C. The LC-CAD purity was verified by the following analytical methods and noted as area %: instrument = Thermo Vanquish F; Column = Waters XBridge C8 4.6*150mm, 3.5pm; Mobile phase A, 1 L water + 0.05% TFA; Mobile phase B, 1 L acetonitrile + 0.05% TFA; Gradient = 50%- 100% (solvent B) over 10 minutes and holding at 100% for 5 minutes at a flow rate of 1 .0 mL / min, total time of 15.0 min, 40 °C. The charged aerosol detection (CAD) data collection rate: 10 Hz; evaporator temperature: 35 °C.

[0322] The LCMS-ELSD methods used to monitor the reactions:

[0323] Method A: Analytical LCMS data collected on instrument = Agilent Prime-6125B; Column = Agilent Poroshell 120 EC-C18 2.7um 3.0*30mm; Mobile phase A, water(4L) + TFA(1.5mL); Mobile phase B, acetonitrile(4L) + TFA(0.75mL); Gradient = 5%-95% (solvent B) over 0.4 minutes and holding at 100% for 0.3minutes at a flow rate of 2.0 mL / min, total time of 1 .0 min, 50 °C.

[0324] Method B: Analytical LCMS data collected on instrument = Agilent Prime-6125B; Column = Agilent Poroshell 120 EC-C18 2.7um 3.0*30mm; Mobile phase A, water(4L) + TFA(1.5mL); Mobile phase B, acetonitrile(4L) + TFA(0.75mL); Gradient = 95%-100% (solvent B) over 0.4 minutes and holding at 100% for 0.3minutes at a flow rate of 2.0 mL / min, total time of 1 .0 min, 50 °C.

[0325] Abbreviations

[0326] °20 is degrees 2-theta;

[0327] AcCI is acetyl chloride;

[0328] AcOH is acetic acid;

[0329] APCI is atmospheric pressure chemical ionization; aq is aqueous;

[0330] ATRP is Atom-Transfer Radical Polymerization;

[0331] Bn is benzyl;

[0332] Boc is te / Y-butoxycarbonyl;

[0333] BOC20 is di-te / Y-butyl dicarbonate;

[0334] Bpy is 2,2'-bipyridine; br is broad; tBu is te / Y-butyl; tBuOH is te / Y-butanol; tBuOK is potassium te / Y-butoxide;

[0335] °C is degrees Celsius;

[0336] CDCh is deutero-chloroform;

[0337] CDI is 1 ,1 ’-carbonyldiimidazole;

[0338] D is dispersity for polymers;

[0339] 6 is chemical shift in ppm; d is doublet; dd is doublet of doublets; ddd is doublet of doublet of doublets; dt is doublet of triplets;

[0340] DCE is 1 ,2-dichloroethane;

[0341] DCM is dichloromethane; methylene chloride;

[0342] DI is deionized;

[0343] DIAD is diisopropyl azodicarboxylate;

[0344] DIPEA is N-ethyldiisopropylamine, also known as N,N-diisopropylethylamine;

[0345] DiTDA is ditetradecylamine or ditetradecylamino-;

[0346] DMA is N,N-dimethylacetamide;

[0347] DME is 1 ,2-dimethoxyethane;

[0348] DMAP is 4-dimethylaminopyridine;

[0349] DMF is N,N-dimethylformamide;

[0350] DMSO is dimethyl sulfoxide;

[0351] DMSO-de is deuterodimethylsulfoxide;

[0352] DP is the degree of polymerization;

[0353] EDC is N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide;

[0354] EDC.HCI is N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride; equiv or eq is molar equivalents;

[0355] ESI is electrospray ionization;

[0356] Et2O is diethyl ether;

[0357] EtOAc is ethyl acetate;

[0358] EtOH is ethanol;

[0359] Et3N is triethylamine; g is gram;

[0360] HATU is 1-[bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate;

[0361] HPLC is high pressure liquid chromatography;

[0362] HOBt is 1 -hydroxybenzotriazole hydrate; hr(s) is hour(s); IPA is isopropyl alcohol; iPrOAc is isopropyl acetate;

[0363] L is liter;

[0364] LCMS is liquid chromatography with mass spectrometry detection; m is multiplet;

[0365] M is molar; m-CPBA is 3-chloroperbenzoic acid;

[0366] MeCN is acetonitrile;

[0367] MeMgBr is methylmagnesium bromide;

[0368] MeNHOMe HCI is N.O-dimethylhydroxylamine hydrochloride;

[0369] MeOD-d4is deuterated methanol;

[0370] MeOH is methanol;

[0371] 2-MeTHF is 2-methyl tetrahydrofuran; mg is milligram;

[0372] MHz is mega Hertz; min(s) is minute(s); mL is milliliter; mmol is millimole; mol is mole;

[0373] MPC is methacryloyloxyethyl phosphorylcholine;

[0374] MS (m / z) is mass-spectrometry signal with mass-to-charge ratio;

[0375] MsCI is mesyl chloride;

[0376] MTBE is te / Y-butyl methyl ether;

[0377] MWCO is molecular weight cut-off;

[0378] NEt3 or TEA is triethylamine

[0379] NMR is nuclear magnetic resonance;

[0380] Pd / C is palladium on carbon;

[0381] Pd2(dba)3is palladium tris(dibenzylideneacetone)dipalladium(0);

[0382] Pd(dppf)Ch is [1 ,1 ’-bis(diphenylphophino)ferrocene]dichloropalladium(ll);

[0383] Pd(PPh3)4is tetrakis(triphenylphosphine)palladium(0);

[0384] Pet. ether is the petroleum fraction consisting of aliphatic hydrocarbons and boiling in the range 35-60 °C;

[0385] PMB is para-methoxybenzyl;

[0386] PMB-NH2 is para-methoxybenzylamine;

[0387] PPh3is triphenylphosphine; pH is power of hydrogen; ppm is parts per million;

[0388] PSD is position sensitive detector; psi is pounds per square inch; PXRD is powder X-ray diffraction; q is quartet; rotovap is rotary evaporation technique; rt is room temperature; RT is retention time; s is singlet; SEM-Cl is 2-(trimethylsilyl)ethoxymethyl chloride; SFC is supercritical fluid chromatography; t is triplet; TBAF is tert-butyl ammonium fluoride; TBDMSCl is tert-butyldimethylsilyl chloride; TFA is trifluoroacetic acid; THF is tetrahydrofuran; TLC is thin layer chromatography; TMEDA is N,N,N’N’-tetramethylethylenediamine; TMSCl is trimethylsilyl chloride; TMSCN is trimethylsilyl cyanide; TMSCHN2is (diazomethyl)trimethylsilane; TsCl is p-toluenesulfonyl chloride; Ts2O is p-toluenesulfonic anhydride; µL is microliter; µmol is micromole The Schemes / Examples described below are intended to provide a general description of the methodology employed in the preparation of the compounds of the present invention. Some of the compounds of the present invention contain one or more chiral centers. In the following Schemes / Examples, the general methods for the preparation of the compounds are shown either in racemic or enantioenriched form. It will be apparent to one skilled in the art that all of the synthetic transformations may be conducted in a precisely similar manner whether the materials are enantioenriched or racemic. Moreover, the resolution to the desired optically active material may take place at any desired point in the sequence using well known methods such as described herein and in the chemistry literature. General Methods: In some cases, compounds described having Formula (I), (Ia), (Ib) or (II) may contain protecting groups, which may be appended or removed by additional steps in the synthetic sequence using conditions known in the art (March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure 8th Edition or Green’s Protective Groups in Organic Synthesis, 5th Edition, Wuts, P.G.M. Ed.). Compounds at every step may be purified by standard techniques, such as column chromatography, crystallization, or reverse phase SFC or HPLC. Variables m, n, o, p, s or t are as defined in the embodiments, schemes, examples, and claims herein.

[0389] EXAMPLES

[0390] In orderthat this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

[0391] The compounds and intermediates described below were named using the naming convention provided with ChemDraw, Version 20.1 .1 .125 (Perkin Elmer). The naming convention provided with ChemDraw, Version 20.1 .1.125 is well known by those skilled in the art and it is believed that the naming convention provided with ChemDraw, Version 20.1 .1.125 generally comports with the IUPAC (International Union for Pure and Applied Chemistry) recommendations on Nomenclature of Organic Chemistry or the CAS Index rules. Unless noted otherwise, all reactants were obtained commercially without further purifications or were prepared using methods known in the literature.

[0392] Example 1 Preparation of DiTDA Polymerization Initiator

[0393] Scheme 1. Synthetic Route to DiTDA Polymerization Initiator

[0394] In a sealed tube equipped with magnetic stir bar, ditetradecylamine (CI4H29)2NH (DiTDA, 1 , 10 g, 24 mmol) and gammabutyrolactone (2, 75 mL, 985 mmol) and triethylamine (8.5 mL, 61 mmol) were mixed and stirred for 3 days at 130°C. The crude mixture was cooled to room temperature and diluted with heptane. After mixing, two layers were formed, and the top layer (heptane layer) was collected, and the excess volatile was removed by rotavap. The product was further purified by a flash column using heptane / ethyl acetate = 3: 1 as mobile phase. The volatile was removed by rotavap. Compound 3 was collected as a brown oil (10.8 g, 22 mmol, 92% yield).

[0395] In a one-neck RB flask equipped with magnetic stir bar, 3 (5.3 g, 11 mmol), DIPEA (2.9 mL, 16.5 mmol), and anhydrous DCM (50 mL) were added respectively. 2-bromoisobutyl bromide (4, 2.5 g, 11 mmol) was dropwise added into the mixture. The reaction mixture was stirred at room temperature overnight. The crude mixture was washed with brine and the DCM layers were collected and dried with sodium sulfate. The product was further purified by flash column using heptane / ethyl acetate = 10 : 1 as mobile phase. After removing volatiles under reduced pressure, DiTDA-polymerization initiator was obtained as a transparent oil (5.6 g, 79% yield).1H NMR spectrum of DiTDA-polymerization initiator is shown in FIG 1 .

[0396] Example 2

[0397] (DiTDA-PMPC8k) (1)

[0398] (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1 -oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-8k]

[0399] 1. Polymerization of MPC (DiTDA-PMPC 8k-Br / H)

[0400] In a Schlenk flask equipped with magnetic stir bar, DiTDA-Polymerization initiator (0.602 g, 0.93 mmol, 1 equiv), MPC monomer (1 .65 g, 5.6 mmol, 6 equiv), CuE3r2 (21 .4 mg, 0.096 mmol, 0.1 equiv), and 2 ,2'-bipyridine (91 .7 mg, 0.587 mmol, 0.63 equiv) were added and dissolved in a mixture of DMF (27 mL) and MeOH (9 mL). The Schlenk flask was sealed with a rubber septum and degassed by purging with nitrogen for 30 min. Copper wire (length: 90 mm, diameter: 0.25 mm, surface area: 71 mm2) was coiled around a glass pipette and added under a strong nitrogen counterflow. The system was further purged with nitrogen for five minutes and under a strong nitrogen counterflow, the septum was replaced by a glass stopper. The mixture was stirred at 25 °C for 16 h. The reaction was stopped by opening the flask to air under vigorous stirring and the color of the crude solution turned green after exposure to air within 5 min. The solution was stirred for 30 min. Silica gel and basic alumina were added into a column successively (Note: both silica gel and basic alumina were washed with MeOH and dried in the oven before use). The crude polymer solution was diluted with MeOH and passed through the column mentioned above. The column was further washed with MeOH (200 mL). The volatile was removed by rotary evaporation and the excess DMF was decanted to obtain the amorphous white polymer solid. The solid was dissolved with minimum amount of MeOH and precipitated with THF. The resulting white precipitate was collected by centrifuge sedimentation at 3,000 rpm for 3 min. The product was redissolved in MeOH and precipitated with THF one more time and the white solids were collected by centrifugation and dried under high vacuum. After drying, white solid (710 mg, 43% yield) was collected. The DP of polymer product was determined by 1H NMR spectrum collected in CD3OD. The DP of the polymer was used to estimate a molecular weight of approximately 8kD. 1H NMR (300 MHz, CD3OD) δ 4.51-3.94 (br, 159H), δ 3.85-3.62 (br, 54H), δ 2.50-2.38 (t, J= 7.1 Hz, 2H), δ2.12-1.44 (m, 54H), δ 1.39-1.22 (br, 44H), δ 1.21-0.79 (br, 84H). Đ (GPC, [solvent]): 1.2. Example 3 (DiTDA-PMPC11k) (2) (1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-11k] 1. Polymerization of MPC (DiTDA-PMPC11k-Br / H) g, 0.72 mmol, 1 equiv), MPC monomer (2.068 g, 7.0 mmol, 9.7 equiv), CuBr2 (17 mg, 0.076 mmol, 0.11 equiv), and 2,2′-bipyridine (68 mg, 0.44 mmol, 0.6 equiv) were added and dissolved in a mixture of DMF (24 mL) and MeOH (8 mL). The Schlenk flask was sealed with a rubber septum and degassed by purging with nitrogen for 30 min. Copper wire (length: 69 mm, diameter: 0.25 mm, surface area: 54 mm2) was coiled around a glass pipette and added under a strong nitrogen counterflow. The system was further purged with nitrogen for five minutes and under a strong nitrogen counterflow, the septum was replaced by a glass stopper. The mixture was stirred at 25 °C for 15 h. The reaction was stopped by open the flask to air under vigorous stirring and the color of the crude solution turned green / blue after exposure to air. The solution was stirred for 30 min. Silica gel and basic alumina were added (silica gel : basic alumina = 1 : 3 (v / v)) into a column respectively (Note: both silica gel and basic alumina were washed with MeOH and dried in the oven before use). The crude polymer solution was diluted with MeOH and passed through the column mentioned above. The column was further washed with MeOH (200 mL). The volatile was removed by rotary evaporation and the excess DMF was decanted out to obtain the amorphous white polymer solid. The product was further purified by two precipitation cycles, using minimum amount of MeOH as a solvent and THF as a counter- solvent. The volume ratio of THF to MeOH was 10: 1. The white precipitate was collected by centrifuge sedimentation at 3,000 rpm for 3 min and dried under high vacuum. The product was collected as a white solid (1.604 g, 78% yield). The DP of polymer product was determined by 1H NMR spectrum collected in CD3OD. The DP of the polymer was used to estimate a molecular weight of approximately 11kD. 1H NMR (300 MHz, CD3OD) δ 4.47-3.96 (br, 210H), δ 3.84-3.65 (br, 71H), δ 2.50-2.37 (t, J= 7.1 Hz, 2H), δ 2.10-1.46 (m, 68H), δ 1.37-1.25 (br, 44H), δ 1.25-0.82 (br, 105H). Reference: Magenau, A. J. D.; Kwak, Y.; Matyjaszewski, K. Macromolecules 2010, 43, 9682–9689. 2. Dehalogenation of DiTDA-PMPC11k-Br / H DiTDA-PMPC11k-Br / H (1.563 g) was dissolved by MeOH (20 mL). Palladium on carbon (100 mg) was weighted into a vial containing water (10 mL) and transferred to the polymer solution by a glass pipette. The oxygen in the system was removed by three evacuation / nitrogen backfill cycles. Under vacuum, the vessel was pressurized by hydrogen (50 psi) and the reaction was allowed to shake for 12 h. The catalyst was removed by passing the crude material through a celite column and excess MeOH was used to elute all the residual product out. (The process can be repeated if any black suspension is observed.) The excess volatile was removed on a rotary evaporator. The product was dissolved in a minimum amount of water and dialyzed (Spectra / Por 6 Dialysis Tubing, MWCO: 3.5 k) against DI water. The excess water is removed by rotary evaporator and the solid was redissolved with a minimum amount of MeOH and precipitated by THF. The solid was collected by centrifuge sedimentation at 3000 rpm for 3 min. The white amorphous polymer was dried under vacuum until constant weight observed (1.35 g). The DP of the polymer product was determined by 1H NMR spectrum collected in CD3OD and estimated to give a MW of approximately 11kD.1H NMR (300 MHz, CD3OD) δ 4.49-3.95 (br, 204H), δ 3.83-3.65 (br, 68H), δ 2.49-2.40 (t, J= 7.1 Hz, 2H), δ 2.15-1.44 (m, 63H), δ 1.38-1.23 (br, 44H), δ 1.23-0.75 (br, 103H). MS (ESI): Calc’d for C410H819N35O207P34 [M+H]: 10600.62; found: 10610.09 Đ calculated from MS (ESI): 1.2 Reference: Gutekunst, W. R.; Anastasaki, A.; Lunn, D. J.; Truong, N. P.; Whitfield, R.; Jones, G. R.; Treat, N. J.; Abdilla, A.; Barton, B. E.; Clark, P. G.; Haddleton, D. M.; Davis, T. P.; Hawker, C. J. Macromol. Chem. Phys.2017, 218, 1700107. Example 4 Preparation, characterization, and determination of efficacy for lipid nanoparticle formulations containing polyphosphocholine (PC) polymer lipids and mRNA (WISC HA modFlu) The Examples are based on the influenza modRNA, unless specified otherwise. The influenza modRNA immunogenic composition is comprised of one or more nucleoside modified mRNAs that encode the full-length HA glycoprotein derived from seasonal human influenza strains. This Example describes the use of the specific construct (Wisconsin HA modRNA) as the active ingredient in the immunogenic composition. In addition to the codon-optimized sequence encoding the antigen, the RNA contains common structural elements optimized for mediating high RNA stability and translational efficiency (5′-cap, 5′UTR, 3′-UTR, poly(A)-tail; see table and sequences below). Furthermore, an intrinsic signal peptide (sec) is part of the open reading frame and is translated as an N-terminal peptide. The RNA does not contain any uridines; instead of uridine, the modified N1-methylpseudouridine is used in RNA synthesis. The specific constructs each comprise the elements shown below in Table 1Table 1: Table 1: Construct Elements Element Description Position

[0401] Sequences of Elements:

[0402] Cap and 5'-UTR: GAGAATAAAC MJAGMJAMJMJCMJMJ CMJGGMJCCCCA CAGACMJCAGA GAGAACCCGC CACC (SEQ ID NO:1), where the bolded and underlined text corresponds to the cap and the unmodified text corresponds to the 5'-UTR.

[0403] 3'-UTR: CMJCGAGCMJGGMJ ACMJGCAMJGCA CGCAAMJGCMJA GC^GCCCCM^

[0404] MJCCCGMJCCMJG GGMJACCCCGA GMJCMJCCCCCG ACCMJCGGGMJC CCAGGMJAMJGC

[0405] MJCCCACCMJCC ACCMJGCCCCA CMJCACCACCMJ CMJGCMJAGMJMJC CAGACACCMJC

[0406] CCAAGCACGC AGCAAMJGCAG CMJCAAAACGC 4H+’AGCC’+’AGC CACACCCCCA CGGGAAACAG CAG’+’GA’+”+’AA CC^M^AGCAA ^AAACGAAAGl4JI4JI4JAACl4JAAG

[0407] CMJAMJACMJAAC CCCAGGGMJMJG GMJCAAMJMJMJCG ^GCCAGCCAC ACCCMJGGAGC I’AGC (SEQ ID NO:2)

[0408] Poly(A) tail: AAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGCATAT GACTAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAA (SEQ ID NO:3) As used herein, T represents 1 -methyl-3'-pseudouridylyl.

[0409] The 5'-cap analog (m27,3'OMeGppp(mi2' °)ApG) for production of RNA containing a cap1 structure is shown below.

[0410]

[0411] The above structure corresponds to Trilink’s CleanCap AG (3’OMe) - m27,3’-OGppp (m12’-O)ApG. This molecule is identical to the natural RNA cap structure in that it starts with a guanosine methylated at N7, and is linked by a 5’ to 5’ triphosphate linkage to the first coded nucleotide of the transcribed RNA (in this case, an adenosine). This guanosine is also methylated at the 3’ hydroxyl of the ribose to mitigate possible reverse incorporation of the cap molecule. Finally, the 2’ hydroxyl of the ribose on the adenosine is methylated, conferring a Cap1 structure - by contrast, leaving this as a 2’ hydroxyl would give this a CapO structure. Cap1 structures should provide superior transcription to RNA’s with a CapO structure in eukaryotes.

[0412] The influenza modRNA vaccine candidates may encode the HA protein derived from A / Wisconsin / 588 / 2019 (H1 N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 02 / 2019 (B / Victoria-lineage) and B / Phuket / 3073 / 2013 (B / Yamagata lineage), which are the recommended vaccine strains for the cell culture-based influenza vaccines for the Northern Hemisphere 2021 - 2022 season. The number of A nucleotides present in the poly(A)-tail in the sequences preferably reflect how it would be in the final RNA after linearization with BspQ1 (or its isoschizomer): 30A- linker-70A. In the transcribed RNA with Cap 1 structure, the first two nucleotides in the mRNA sequence (AG) are actually provided by the CLEANCAP reagent and the 2’ hydroxyl of the ribose on the first adenosine is methylated.

[0413] The cap1 structure (e.g., containing a 2'-O-methyl group on the penultimate nucleoside of the 5'-end of the RNA chain) is incorporated into the drug substance by using a respective cap analog during in vitro transcription. For RNAs with modified uridine nucleotides, the cap1 structure is superior to other cap structures, since cap1 is not recognized by cellular factors such as IFIT1 and, thus, cap1 -dependent translation is not inhibited by competition with eukaryotic translation initiation factor 4E. In the context of IFIT1 expression, mRNAs with a cap1 structure give higher protein expression levels.

[0414] In some preferred embodiments, the Influenza vaccine drug substance is a singlestranded, 5'-capped mRNA that is translated into the respective protein (the encoded antigen) which corresponds to the Hemagglutinin (HA) protein from Influenza virus. The general structure of the antigen-encoding RNA is determined by the respective nucleotide sequence of the DNA used as template for in vitro RNA transcription. In addition to the codon-optimized sequence encoding the antigen, the RNA contains common structural elements optimized for mediating high RNA stability and translational efficiency (5'-cap, 5’UTR, 3'-UTR, poly(A) - tail; see below).

[0415] The manufacturing process comprises RNA synthesis via an in vitro transcription (IVT) step followed by DNase I and proteinase K digestion steps, purification by ultrafiltration / diafiltration (UFDF), final filtration, dispense into an appropriate container, and storage at -20°C. A platform approach to the IVT, digestion, and purification process steps is used in the production of the four modRNAs. The mRNA clinical batches are prepared at a scale of 37.6 L starting volume for IVT. All the material is purified by a single 2-stage UFDF to produce mRNA drug substance.

[0416] Lipid nanoparticles are prepared and tested according to the general procedures described in US Patent 9737619 (PCT Pub. No. WO2015 / 199952) and US Patent 10166298 (PCT Pub. No. WO 2017 / 075531) and PCT Pub. No. W02020 / 146805. The novel polymer conjugated lipid of the invention, cholesterol, DSPC and ionizable lipid are solubilized in Ethanol at a molar ratio of about 46.3:42.7:9.4:1 .6. Lipid nanoparticles (LNP) are prepared at a total lipid to mRNA ratio of about 23:1 . In short, the mRNA (WISC HA modFlu) is diluted in buffer. Syringe pumps are used to mix the lipid solution with the mRNA solution. The ethanol is removed and external buffer replaced with another buffer (e.g., Tris) by dialysis. Finally, the lipid nanoparticle size and size distribution is determined by dynamic light scattering using an Unchained Labs Stunner (Unchained Labs, USA). RNA encapsulation efficiency is determined using the Quant-iT RiboGreen RNA assay (Life Technologies, USA). Briefly, LNPs are incubated with RiboGreen dye (200-fold dilution per manufactures’ instruction) in the presence and absence of 1% Triton-X 100 and fluorescence intensities (Excitation / Emission: 485 / 528nM) are measured for unencapsulated RNA and total RNA after release from LNPs by Triton-X 100.

[0417] Plated Hek293T or Hela cells are dosed with lipid nanoparticles in a total volume of 40 pl cell culture media and incubated overnight at 37 °C and 5% CO2. After fixation with 4% paraformaldehyde in PBS cells are washed in PBS containing 0.3% Triton-X100 and 3% BSA (w / v), followed by nuclear staining with Hoechst and antibody staining for the encoded gene of interest. Nuclear count and identification of cells stained positive for gene of interest is done on an Opera Phenix high content imager (PerkinElmer, USA).

[0418] An alternate method comprises the use of plated Hek293F cells dosed with lipid nanoparticles in a total volume of 250 pl cell culture media and incubated 18-20hr at 37 °C , 5% CO2, 80% humidity with 300rpm shaking. After live / dead staining, cells are fixed with BD Cytofix / Cytoperm solution, followed by antibody staining for the encoded gene of interest. Count viable cells and identification of cells stained positive for gene of interest is done on an BD LSRFortessa. Example 5

[0419] Preparation, characterization, and determination of efficacy for lipid nanoparticle formulations comprising polyphosphocholine (DiTDA-PMPC) polymer lipids and mRNA (CA HA modFlu mRNA)

[0420] The specific construct (California HA modRNA) is the only active ingredient in the immunogenic composition. In addition to the codon-optimized sequence encoding the antigen, the RNA contains common structural elements optimized for mediating high RNA stability and translational efficiency (5'-cap, 5'UTR, 3'-UTR, poly(A)-tail; see table and sequences described in Example 4). Furthermore, an intrinsic signal peptide (sec) is part of the open reading frame and is translated as an N-terminal peptide. The RNA does not contain any uridines; instead of uridine, the modified N1 -methylpseudouridine is used in RNA synthesis.

[0421] Lipid nanoparticles were prepared and tested according to the general procedures described in US Patent 9737619 (PCT Pub. No. WO2015 / 199952), US Patent 10166298 (PCT Pub. No. WO 2017 / 075531) and PCT Pub. No. W02020 / 146805 and further set forth in Example 4.

[0422] 1. LNPs comprising DiTDA-PMPC8k at various concentrations

[0423] To test the novel polymer-conjugated lipid of the invention (DiTDA-PMPC8k) LNPs comprising four different lipids: ionizable lipid ALC-0315 (4-hydroxy butyl) azanediyl)bis (hexane- 6,1-diyl)bis(2-hexyldecanoate), cholesterol, distearoylphosphatidylcholine (DSPC) and the novel polymer-conjugated lipid of the invention (DiTDA-PMPC8k) were solubilized in ethanol at five molar ratios (46.3:44.1 :9.4:0.2; 46.3:43.8:9.4:0.5; 46.3:43.5:9.4:0.8, 46.3:42.7:9.4:1.6 and 46.3:41 .1 :9.4:3.2) with N:P ratio of about 6:1 .

[0424] Total RNA concentration and encapsulation efficiency were assessed using the RiboGreen RNA quantitation assay. RNA integrity pre- and post-formulation was monitored by Agilent Fragment Analyzer capillary gel electrophoresis. Malvern Zetasizer dynamic light scattering (DLS) was used to determine LNP size and polydispersity index (PDI). The in vitro expression levels of the lipid nanoparticles were evaluated in HEK293F cells using flow cytometry, LNPs having a mol ratio of ALC-0315:cho!esterol:DSPC:ALC-0159 of 46.3:42.7:9.4:1.6 were used as a positive control. Results:

[0425] Table 2: Biophysics characterizations for DiTDA-PMPC8k containing LNPs

[0426] * This assay was done in a separate experiment, ALC-0315 / cholesterol / DSPC / ALC-0159 control EC50=7.52 ng / well

[0427] %EE=Encapsulation Efficiency; PDI= Polydispersity; POI = Peak of Interest; IVE = In Vitro Expression

[0428] As shown in Table 2 and FIG. 2, ILNPs with different ratios of DiTDA-PMPC8k polymers show good size and RNA integrity. Nanoparticles can be formed with the incorporation of as low as 0.2 mol% DiTDA-PMPC8k. The encapsulation efficiency and transfection efficiency of LNP comprising DiTDA-PMPC8k polymer at 0.8% is higher than the DiTDA-PMPC8k polymer at 1 .6% or 3.2%.

[0429] 2. LNPs comprising DiTDA-PMPC11 k at various concentrations To test the novel polymer-conjugated lipid of the invention (DiTDA-PMPC11 k) LNPs comprising four different ratios of lipids (ALC-0315:cholesterol:DSPC:DiTDA-PMPC11 k) were optimized, including 1) 46.3:43.9:9.4:0.4; 2) 46.3:43.5:9.4:0.8; 3) 46.3:42.7:9.4:1.6 and 4) 46.3:41 .1 :9.4:3.2 with N:P ratio of about 6:1 .

[0430] RNA concentration and encapsulation efficiency (EE%) were measured using RiboGreen RNA quantitation assay. RNA integrity was monitored by Agilent Fragment Analyzer capillary gel electrophoresis. Malvern Zetasizer dynamic light scattering (DLS) was used to determine LNP size and polydispersity index (PDI). The in vitro expression levels of the lipid nanoparticles were evaluated in HEK293F cells using flow cytometry, standard LNPs having mol ratio of ALC- 0315 / cholesterol / DSPC / ALC-0159 equal to 46.3:42.7:9.4:1.6 LNPs were used as control. Table 3: Biophysics characterizations for DiTDA-PMPC11k containing LNPs

[0431] %EE=Encapsulation Efficiency; PDI= Polydispersity; POI = Peak of Interest; IVE=ln Vitro

[0432] Expression As shown in Table 3 and FIG. 3, LNPs with several ratios of DiTDA-PMPC polymers show good size and RNA integrity. The encapsulation efficiency and transfection efficiency of LNPs comprising DiTDA-PMPC11 k are: 0.4% > 0.8% > 1 .6% > 3.2%.

[0433] 3. LNP cell potency stability study of DiTDA-PMPC8k polymer lipid-containing CA HA modRNA LNPs

[0434] FIG. 4A and Table 4 show the EC50 value for LNPs comprising DiTDA-PMPC8k polymer lipid encapsulating CA HA modRNA over the course of 16 weeks stored at 4°C. FIG. 4B shows the Mean Fluorescence Intensity (MFI) for LNPs comprising DiTDA-PMPC8k polymer lipid encapsulating CA HA modRNA at 16 weeks stored at 4°C. The LNPs comprising DiTDA-PMPC8k stored at 4°C remained stable and demonstrated comparable potency as PEG- lipid (ALC-0159) control LNPs.

[0435] Table 4. EC50 values in the cell potency stability study up to 16 weeks.

[0436] 4. In vivo immunogenicity study using DiTDA-PMPC8k polymer lipid-containing LNPs

[0437] LNPs manufactured using DiTDA-PMPC8k (ALC-0315 / Cholesterol / DSPC / DiTDA- PMPC8k 0.8 mol%) encapsulating CA HA modFlu mRNA were used in an animal study to demonstrate its in vivo efficacy. Mice were immunized intramuscularly (I.M.) with saline, ALC- 0159 (PEG) LNP control, and DiTDA-PMPC8k 0.8 mol% LNPs at 0.2 micrograms mRNA dose on Day 0 and boosted on Day 21 with the same dose. Postdose 2 bleed was taken on Day 42 and neutralizing antibody titer was determined. As shown in FIG. 5 and Table 5, modRNA LNPs made using DiTDA-PMPC8k polymer lipid induced titers significantly higher than the ALC-0159 LNP control. Table 5. In Vivo Mouse Study (2 Weeks Post-dose 2)

[0438] 5. LNP cell potency stability study of DiTDA-PMPC11 k polymer lipid-containing LNPs FIG. 6A to 6C and Table 6 show that the EC50 value for LNPs comprising DiTDA-

[0439] PMPC1 1 k polymer lipid over the course of 16 weeks at -80°C, 4°C and 25°C. DiTDA-PMPC1 1 k polymer lipid LNPs at 0.4 mol% demonstrated good stability up to 16 weeks at -80°C and 4°C and had a lower EC50 than the PEG lipid control LNPs at 25°C. DiTDA-PMPC1 1 k polymer lipid LNPs at 0.8 mol% demonstrated similar cell potency up to 16 weeks at 25°C but was not as potent at 4°C compared to the 0.4 mol% LNPs.

[0440] Table 6. EC50 values in the cell potency stability study up to 16 weeks. 6. In vivo immunogenicity study using DiTDA-PMPC11 k polymer lipid-containing

[0441] LNPs

[0442] ALC-0315 / Cholesterol / DSPC / DiTDA-PMPC11 k 0.4 mol% LNPs encapsulating California HA modRNA were tested in an animal study. Mice were immunized intramuscularly (I.M.) with saline, ALC-0159 LNP control, or DiTDA-PMPC1 1 k polymer LNPs at a dose of 0.2 micrograms mRNA on Day 0 and boosted on Day 21 with the same dose. Postdose 2 bleed was taken on Day 42 and neutralizing antibody titer was determined. modRNA LNPs made using DiTDA- PMPC11 k polymer lipid induced higher titers than ALC-0315 / cholesterol / DSPC / ALC-0159 control LNPs as shown in FIG. 7 and Table 7.

[0443] Table 7. In Vivo Mouse Study (2 Weeks Post-dose 2)

[0444] It will be apparent to those skilled in the art that various modifications and variations may be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0445] All references cited herein, including patents, patent applications, papers, textbooks, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entireties. In the event that one or more of the incorporated literature and similar materials differ from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

Claims

What is claimed is:1 . A polymer conjugated lipid compound of Formula (I):or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR1and R2are each independently hydrogen or methyl;R3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor;R5is hydrogen or halogen; m is an integer from 1 to 6;Y is a polymer having Formula (II):(ID n is an integer from 1 to 1000;x is an integer from 2 to 6; and w is an integer from 2 to 4.

2. The polymer conjugated lipid compound of claim 1 having Formula (la):or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR3and R4are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 14 to 18 carbon atoms, an alkene, a lipid or a hydrophobic anchor; R5is hydrogen or halogen; and n is an integer from 1 to 1000.

3. The polymer conjugated lipid compound of claim 1 having Formula (lb):or a pharmaceutically acceptable salt or stereoisomer thereof.

4. The polymer conjugated lipid compound of claim 1 , wherein (i) n ranges from 15 to 19, (ii) n ranges from 20 to 24, (iii) n ranges from 25 to 29, (iv) n ranges from 30 to 33, or (v) n ranges from 34 to 38.

5. The polymer conjugated lipid compound of claim 1 , wherein the polymer conjugated lipid compound has a Dispersity of about 1 .0 to about 2.0.

6. The polymer conjugated lipid compound of claim 5, wherein the polymer conjugated lipid compound has a Dispersity of between 1 .0 to 1 .4.

7. The polymer conjugated lipid compound of claim 1 , wherein the polymer has a molecular weight of between 100 Daltons and 100,000 Daltons.

8. The polymer conjugated lipid compound of claim 7, wherein the polymer has a molecular weight of between 4,500 Daltons and 12,000 Daltons.

9. The polymer conjugated lipid compound of claim 7, wherein the polymer has a molecular weight of between about 4,800 Daltons and about 5,300 Daltons.

10. The polymer conjugated lipid compound of claim 7, wherein the polymer has a molecular weight of between about 8,300 Daltons and about 8,900 Daltons.11 . The polymer conjugated lipid compound of claim 7, wherein the polymer has a molecular weight of between about 11 ,200 Daltons and about 11 ,800 Daltons.

12. The polymer conjugated lipid compound of claim 1 selected from the group consisting of:(1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-8k]; and(1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-11 k], or a pharmaceutically acceptable salt or stereoisomer thereof.

13. A composition comprising the polymer conjugated lipid compound according to any one of claims 1-12, or a pharmaceutically acceptable salt thereof, at least one payload, and at least one pharmaceutically acceptable excipient.

14. The composition of claim 13, wherein the payload comprises a small molecule, a nucleic acid, an adjuvant, or a combination thereof.

15. The composition of claim 14, wherein the pharmaceutically acceptable excipient is selected from the group consisting of neutral lipids, steroids and ionizable cationic lipids.

16. The composition of claim 15, wherein the composition comprises one or more neutral lipids selected from 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1-stearoyl-2- oleoylphosphatidyethanolamine (SOPE), and / or 1 ,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (transDOPE).

17. The composition of claim 15, wherein the steroid is cholesterol.

18. The composition of any one of claims 13 to 17, wherein the nucleic acid is RNA.

19. The composition of claim 18, wherein the RNA is mRNA.

20. The composition of claim 19, wherein the mRNA is modRNA or saRNA.21 . The composition of any one of claims 13 to 17, wherein the nucleic acid is circular DNA (cDNA).

22. The composition of any one of claims 13 to 21 , wherein the ionizable cationic lipid comprises N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2-dilinoleyloxy- N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), (4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecane-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl) amino) octanoate (SM- 102), or a mixture thereof.

23. The composition of any one of claim 13 to 22, wherein the composition is a lipid nanoparticle comprising a neutral lipid, a steroid, an ionizable cationic lipid and a polymer conjugated lipid compound having a mol% ratio of ALC-0315:cholesterol:DSPC:polymer conjugated lipid compound of any one of claims 1-12 selected from the group consisting of: a) 46.3:44.1 :9.4:0.2;b) 46.3:43.9:9.4:0.4; c) 46.3:43.8:9.4:0.5; d) 46.3:43.5:9.4:0.8; e) 46.3:42.7:9.4:1.6 and f) 46.3:41.1 :9.4:3.2.

24. The composition of claim 23, wherein the polymer conjugated lipid is(1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-8k]; or(1-(4-(ditetradecylamino)-4-oxobutoxy)-2-methyl-1-oxopropan-2-yl)[(poly(2-((oxido(2- (trimethylammonio)ethoxy)phosphoryl)oxy)ethyl)-2-methacrylate)-11 k], or a pharmaceutically acceptable salt or stereoisomer thereof.

25. A method for delivering a payload to a subject in need thereof comprising administering to the subject the composition of any one of claims 13 to 24.

26. The method of claim 25, wherein the payload comprises a small molecule, a nucleic acid, an adjuvant, or a combination thereof.

27. A method for delivering a nucleic acid to a subject in need thereof comprising administering to the subject a composition of any one of claims 13 to 24 comprising a nucleic acid.

28. A method for delivering a therapeutic peptide or protein to a subject in need thereof, the method comprising administering to the subject a composition of any one of claims13 to 24 comprising a nucleic acid, wherein the nucleic acid encodes the therapeutic peptide or protein.

29. A method for treating or preventing a disease or disorder in a subject comprising administering to the subject a composition of any one of claims 13 to 24 comprising a nucleic acid, wherein administering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder.

30. A method for treating or preventing a disease or disorder in a subject comprising administering to the subject a composition of any one of claims 13 to 24 comprising a nucleic acid, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.31 . The method of any one of claims 25 to 30, wherein the subject is a mammal.

32. The method of claim 31 , wherein the mammal is a human.

33. A method for producing a polymer conjugated lipid compound of any one of claims 1 to 12, wherein the method is a Controlled Radical Polymerization.

34. The method of claim 33, wherein the Controlled Radical Polymerization is Atom TransferRadical Polymerization (ATRP) or Reversible Addition / Fragmentation Chain Transfer Polymerization (RAFT).

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