Nanoparticle formulations for treatment of inflammations

Abstract

The present invention provides lipid nanoparticles and formulations comprising cationic lipids and helper phospholipid. These lipid nanoparticles may be formulated with therapeutic agents to facilitate their intracellular delivery for both in vitro and in vivo therapeutic applications. The present invention is specifically directed to nanoparticles and formulations that can target inflamed tissues and treat inflammatory diseases.

Description

[0001] NANOPARTICLE FORMULATIONS FOR TREATMENT OF INFLAMMATIONS FIELD OF THE INVENTION The present invention provides lipid nanoparticle formulations and lipid nanoparticles 5 (LNPs) comprising cationic lipids and helper phospholipid. These lipid nanoparticles may be formulated with therapeutic agents to facilitate their intracellular delivery for both in vitro and in vivo applications. The present invention is specifically directed to nanoparticles formulations and LNPs that can target inflamed tissues BACKGROUND OF THE INVENTION 10 Therapeutic nucleic acids including small interfering RNA (siRNA), micro RNA (miRNA), antisense oligonucleotides, messenger RNA (mRNA), ribozymes, pDNA and immune stimulating nucleic acids act via a variety of mechanisms. Specific proteins can be downregulated by siRNA or miRNA through RNA interference (RNAi). Hematopoietic cells, such as leukocytes in general, and primary T lymphocytes and B-cells in particular, 15 are notoriously hard to transfect with small interfering RNAs (siRNAs). Modulating immune cell function, such as T cells and B cells, by downregulating specific genes using RNA interference (RNAi) holds tremendous potential in advancing targeted therapies in many immune-related disorders including cancer, inflammation, autoimmunity and viral infections. The therapeutic applications of RNAi are extremely broad, since siRNA and 20 miRNA constructs can be synthesized with any nucleotide sequence directed against a target protein. To date, siRNA constructs have shown the ability to specially silence target proteins in both in vitro and in vivo models. These are currently being evaluated in clinical studies. Messenger RNA (mRNA) is the family of large RNA molecules which transport the genetic 25 information from DNA to ribosome. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products. Such nucleic acids would be useful in the treatment to the of diseases related deficiency of a protein or enzyme. However, there are many problems associated with nucleic acids in therapeutic contexts. 1

[0002] One of the major problems with therapeutic nucleic acids is the stability of the phosphodiester inter nucleotide link and its susceptibility to nucleases. Apart from that these nucleic acids have limited ability to cross the cell membrane. Various lipids, e.g., cationic lipids, have proved to be excellent carriers of nucleic acids to 5 treat different diseases in gene therapy applications. Lipid nanoparticles formed from cationic lipids and other co-lipids such as cholesterol, DSPC and PEGylated lipids encapsulated oligonucleotides which protect them from degradation and facilitate the cellular uptake. WO 2018 / 087753 discloses a cationic lipid comprising a functional group represented by 10 the structure: -W-(T=O)m-X-(CH2)z-Y, wherein X and Y are each independently O, N or NH, wherein X and Y cannot both be O; W is a bond, O, NH or S; T is C or S; m is 0 or 1; and z is 0 or 2, wherein said functional group is linked to at least one saturated or unsaturated fatty acid residue. Tanaka et al. (Heliyon. 2018 Dec 3;4(12):e00959. doi: 10.1016 / j.heliyon.2018.e00959. 15 PMID: 30555953; PMCID: PMC6280607) disclose delivery of mRNA to colon inflammatory lesions by lipid-nano-particles containing environmentally-sensitive lipid- like materials with oleic acid scaffolds. Mei Yang et al. (J Bio-X Res. 2020;03:157-173.DOI:10.1097 / JBR.0000000000000078) review the use of nanoparticle-based therapeutics for treatment of inflammatory bowel 20 diseases. WO 2022 / 201167 discloses lipids and lipid nanoparticle formulations comprising these lipids, alone or in combination with other lipids. These lipid nanoparticles may be formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications. 25 Nevertheless, there remains a need in the art for suitable and efficient delivery platforms for targeted delivery of nucleic acid and therapeutic agents to specifically to inflamed tissues, in order to treat inflammations, such as inflammatory bowel diseases, rheumatoid arthritis and others.

[0003] 2

[0004] SUMMARY OF THE INVENTION The present invention relates to novel lipid nanoparticle (LNP) formulations and LNPs. These lipid nanoparticles protect nucleic acids from degradation, clearance from circulation and intracellular release. In addition, the nucleic acid encapsulated lipid nanoparticles 5 advantageously are well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The LNPs and formulations of the present invention are suitable for nucleic acid (e.g., mRNA) delivery to different organs, and, in particular, to inflamed tissue(s). Targeted organs include, but are not limited to, the colon, the intestines, 10 the kidneys, the lung and / or the spleen. Surprisingly LNPs distributed specifically in areas where inflammation is present and significantly higher protein expression was observed compared to the expression levels in the healthy counterpart organs; and in the liver (of both healthy and inflamed subjects). The results provided herein indicate that given similar exposure, the formulations in 15 accordance with the principles of the invention is unexpectedly more effective in delivering biological agents specifically to inflamed tissues than hitherto known formulations. The high specificity of the present lipid nanoparticle formulations and LNPs in inflamed tissue targeting is highly advantageous, as it reduces the side effects that accompany the delivery of active agents to undesired healthy organs. Advantageously, this high specificity 20 also enables use of lower dosages, as the therapeutic is made locally and therefore less active agent is lost, which, in its turn, also contributes to side effect reduction. The present lipid nanoparticle formulations and LNPs comprise a specific combination of lipid ingredients, which include a neutral phospholipid, at a specific molar concentration as specified herein. 25 According to some embodiments, there is provided a lipid nanoparticle formulation for use in treating an inflammation. According to some embodiments, the lipid nanoparticle formulation comprises at least one cationic lipid; at least one neutral phospholipid; at least one sterol; and at least one PEGylated lipid. According to some embodiments, a lipid

[0005] 3 portion of the lipid nanoparticle formulation consists of the at least one cationic lipid; the at least one neutral phospholipid; the at least one sterol; and the at least one PEGylated lipid. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation is in the range of 20% mol / mol to 40% mol / mol. 5 According to some embodiments, the lipid nanoparticle formulation comprises a plurality of lipid nanoparticles, wherein the LNPs have a phase transition temperature peak in the range of 50℃ to 63℃, as measured by Differential Scanning Calorimetry (DSC), including each value and sub-range within the specified range. According to some embodiments, the phase transition temperature peak is in the range of 55°C to 63°C. 10 According to some embodiments, there is provided a lipid nanoparticle comprising: at least one cationic lipid; at least one neutral phospholipid; at least one sterol; and at least one PEGylated lipid, wherein a total neutral phospholipid concentration within the lipid nanoparticle is in the range of 20% mol / mol to 40% mol / mol, wherein the lipid nanoparticle has a phase transition temperature peak in the range of 50℃ to 63℃, as measured by 15 Differential Scanning Calorimetry (DSC). According to some embodiments, the lipid nanoparticle is for use in treating an inflammation. According to some embodiments, the cationic lipid is represented by the structure of Formula (IA), Formula (IB) or Formula (II), or salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof, wherein the structures 20 of Formula (IA), Formula (IB) and Formula (II) are represented below; Formula (IA): ,

[0006] R1Ais selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNARNA’, wherein each one of RNAand RNA’is individually C1-4 alkyl or RNAand RNA’together with the nitrogen to which they are bound, form a ring; 5 R2Ais selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2- C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5-15alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; nAis selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 nAis 10 selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 nAis selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; XAis selected from the group consisting of: -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH; jAis selected from the group consisting of: 0, 1, 2, 3 and 4; 15 YAis selected from the group consisting of: absent, -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH; mAis selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; R3Ais selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNA’’RNA’’’, wherein each one of RNA’’and RNA’’’is 20 individually C1-4alkyl or RNA’’and RNA’’’together with the nitrogen to which they are bound, form a ring; and R4Ais selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2- C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5-1525 alkenyl, C5-15alkenylene-O2C-C5-15alkyl, C5-15alkenylene-O2C-C5-15alkenyl; Formula (IB):

[0007] 5

[0008] , wherein R1Bis selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNBRNB’, wherein each one of RNAand RNA’is 5 individually C1-4alkyl or RNAand RNA’together with the nitrogen to which they are bound, form a ring; R2Bis selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2- C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5-1510 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; WBis a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; YBis selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene; 15 R3Bis selected from the group consisting of: C5-25alkyl, C5-25alkenyl, C5-15alkylene-CO2- C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15alkyl, C5-15alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; and R4Bis selected from the group consisting of: C5-25alkyl, C5-25alkenyl, C5-15alkylene-CO2- 20 C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15alkyl, C5-15alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15alkenylene-O2C-C5-15alkyl, C5-15alkenylene-O2C-C5-15alkenyl;

[0009] 6

[0010] Formula (II): , wherein R1Cis selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2- 5 C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5-15alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; R2Cis selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2- C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15alkyl, C5-1510 alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5-15alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; Y1Cis selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6alkylene; W1Cis a C4-12 alkylene, optionally substituted with at least one substituent selected from 15 the group consisting of hydroxy and halogen; R5Cis selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNIIRNII’, wherein each one of RNIIand RNII’is individually C1-4 alkyl or RNIIand RNII’together with the nitrogen to which they are bound, form a ring; 20 W2Cis a C4-12alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen;

[0011] 7

[0012] Y2Cis selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene; R3Cis selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2- C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15alkyl, C5-155 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; and R4Cis selected from the group consisting of: C4-18alkyl and C12-24alkenyl. According to some embodiments, the cationic lipid is selected from the group consisting of: lipid II-1, lipid IA-10, lipid ALC-0315, lipid EA-502 (lipid 15), lipid SM-102, lipid II- 10 25 (NV1-022) and a combination thereof;

[0013] 8

[0014]

[0015] The term “at least one” means a single element or object or a plurality of elements and / or objects. The term “plurality” means two or more elements or objects. It is to be understood that “one cationic lipid” relates to a single species of a cationic lipid, which may include a plurality of molecules of the same kind. 5 It is to be understood that specifying that the lipid nanoparticle formulation or LNP comprises at least one cationic lipid, wherein the cationic lipid is represented by the structure of Formula (IA), Formula (IB) or Formula (II), or salts, etc., thereof broadly covers both the option that the lipid nanoparticle formulation or LNP comprises only cationic lipid(s) of Formula (IA) and / or (II) and / or (IB), and the option that the lipid nanoparticle formulation 10 or LNP comprises cationic or other ionizable lipids, as long as the lipid nanoparticle formulation or LNP comprises at least one cationic lipid of Formula (IA), at least one cationic lipid of Formula (IB) and / or at least one of Formula (II). According to some embodiments, the lipid nanoparticle formulation comprises 25% mol / mol to 35% mol / mol total neutral phospholipid concentration. According to some 15 embodiments, the lipid nanoparticle formulation comprises about 30% mol / mol total neutral phospholipid concentration. According to some embodiments, the lipid nanoparticle comprises 25% mol / mol to 35% mol / mol total neutral phospholipid concentration. According to some embodiments, the lipid nanoparticle comprises about 30% mol / mol total neutral phospholipid concentration 20 According to some embodiments, the neutral phospholipid comprises a permanently charged chemical group. According to some embodiments, the neutral phospholipid comprises a phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), or a combination thereof. Each possibility 25 represents a separate embodiment of the invention. According to some embodiments, the neutral phospholipid comprises a phosphatidylcholine (PC). According to some embodiments, the phosphatidylcholine (PC) is selected from the group consisting of: dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline

[0016] 10

[0017] (DPPC) palmitoyloleoylphosphatidylcholine (POPC) and a combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the neutral phospholipid comprises a phosphatidylglycerol (PG). According to some embodiments, the phosphatidylglycerol (PG) is selected from the 5 group consisting of: dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG) and a combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the neutral phospholipid comprises a phosphatidylserine 10 (PS). According to some embodiments, the phosphatidylserine (PS) is selected from the group consisting of: dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS), dipalmitoyl phosphatidyl serine (DPPS) and a combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the neutral phospholipid comprises a 15 phosphatidylethanolamine (PE). According to some embodiments, the phosphatidylethanolamine (PE) is selected from the group consisting of: dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), 20 dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), 1,2-Diphytanoyl-sn- glycero-3-phosphoethanolamine (DPhPE), Biotin-Phosphatidylethanolamine, and a 25 combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the neutral phospholipid comprises l,2-distearoyl-sn- glycero-3-phosphocholine (DSPC). According to some embodiments, the cationic lipid is not a phospholipid.

[0018] 11

[0019] According to some embodiments, the lipid nanoparticle formulation comprises 20% mol / mol to 60% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation comprises 30% mol / mol to 50% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation comprises 33% 5 mol / mol to 43% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation comprises about 38% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle comprises 20% mol / mol to 60% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle comprises 30% mol / mol to 50% mol / mol of the cationic lipid. According to some embodiments, the lipid 10 nanoparticle comprises 33% mol / mol to 43% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle comprises about 38% mol / mol of the cationic lipid. According to some embodiments, the sterol comprises cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 20% 15 mol / mol to 40% mol / mol of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises 25% mol / mol to 35% mol / mol of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises about 30% mol / mol of the sterol. According to some embodiments, the lipid nanoparticle comprises 20% mol / mol to 40% mol / mol of the sterol. According to some embodiments, the lipid nanoparticle 20 comprises 25% mol / mol to 35% mol / mol of the sterol. According to some embodiments, the lipid nanoparticle comprises about 30% mol / mol of the sterol. According to some embodiments, the PEGylated lipid comprises 1,2-Dimyristoyl-sn- glycero-3-methoxypolyethylene glycol (DMG-PEG). According to some embodiments, the DMG-PEG comprises 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 25 2000 (DMG-PEG 2000). According to some embodiments, the lipid nanoparticle formulation comprises 1% to 5% mol / mol DMG-PEG. According to some embodiments, the lipid nanoparticle formulation comprises 1% to 3% mol / mol DMG-PEG. According to some embodiments, the lipid nanoparticle formulation comprises about 2% mol / mol DMG- PEG. According to some embodiments, the lipid nanoparticle comprises 1% to 5% mol / mol

[0020] 12

[0021] DMG-PEG. According to some embodiments, the lipid nanoparticle comprises 1% to 3% mol / mol DMG-PEG. According to some embodiments, the lipid nanoparticle comprises about 2% mol / mol DMG-PEG. According to some embodiments, the lipid nanoparticle formulation comprises: at least one 5 cationic lipid, 30% to 50% mol / mol; at least one neutral phospholipid, 20% to 40% mol / mol; at least one sterol 20% to 40% mol / mol; and at least one PEGylated lipid, 1% to 5% mol / mol. According to some embodiments, the lipid nanoparticle formulation comprises at least one cationic lipid, 33% to 43% mol / mol; at least one neutral phospholipid, 25% to 35% mol / mol; at least one sterol 25% to 35% mol / mol; and at least one PEGylated lipid, 1% to 3% mol / mol. 10 According to some embodiments, the lipid nanoparticle formulation comprises at least one cationic lipid, about 38% mol / mol; at least one neutral phospholipid, about 30% mol / mol; at least one sterol, about 30% mol / mol; and at least one PEGylated lipid, about 2% mol / mol. According to some embodiments, the lipid nanoparticle comprises: at least one cationic lipid, 30% to 50% mol / mol; at least one neutral phospholipid, 20% to 40% mol / mol; at least 15 one sterol 20% to 40% mol / mol; and at least one PEGylated lipid, 1% to 5% mol / mol. According to some embodiments, the lipid nanoparticle comprises at least one cationic lipid, 33% to 43% mol / mol; at least one neutral phospholipid, 25% to 35% mol / mol; at least one sterol 25% to 35% mol / mol; and at least one PEGylated lipid, 1% to 3% mol / mol. According to some embodiments, the lipid nanoparticle comprises at least one cationic lipid, about 38% 20 mol / mol; at least one neutral phospholipid, about 30% mol / mol; at least one sterol, about 30% mol / mol; and at least one PEGylated lipid, about 2% mol / mol. According to some embodiments, the lipid nanoparticle formulation is selected from the group consisting of: Formulation 1, Formulation 2, Formulation 3, Formulation 7, Formulation 9 and Formulation 10: 25 Formulation 1: lipid II-1: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35 mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol;

[0022] 13

[0023] Formulation 2: IA-10: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 3: ALC-0315: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 5 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 7: EA-502 (lipid 15): 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; 10 Formulation 9: SM-102: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 10: II-25 (NV1-022): 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 15 1% to 3% mol / mol. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle has a lipid ratio as in Formulation 1, Formulation 2, Formulation 3, Formulation 7, Formulation 9 or Formulation 10. According to some embodiments, the lipid nanoparticle formulation is devoid of a targeting moiety. According to some embodiments, the lipid nanoparticle is devoid of a targeting 20 moiety. According to some embodiments, the lipid nanoparticle further comprises a nucleic acid encapsulated therein. According to some embodiments, the lipid nanoparticle formulation further comprises a nucleic acid encapsulated within at least one particle thereof. According to some embodiments, the nucleic acid is selected from the group consisting of small 25 interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. Each possibility represents a separate embodiment of the invention.

[0024] 14

[0025] According to some embodiments, the lipid nanoparticle formulation further comprises a therapeutic agent encapsulated within at least one particle thereof. According to some embodiments, the lipid nanoparticle further comprises a therapeutic agent encapsulated therein. 5 According to some embodiments, the lipid nanoparticle formulation further comprises an anti-inflammatory therapeutic agent encapsulated within at least one particle thereof. According to some embodiments, the lipid nanoparticle further comprises an anti- inflammatory therapeutic agent encapsulated therein. According to some embodiments, the anti-inflammatory therapeutic agent comprises an 10 anti-inflammatory nucleic acid. According to some embodiments, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. According to some embodiments, the nucleic acid is an 15 mRNA. According to some embodiments, the mRNA encodes an anti-inflammatory polypeptide. According to some embodiments, the anti-inflammatory polypeptide comprises an anti-inflammatory peptide or an anti-inflammatory protein. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulation further comprises a 20 pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the lipid nanoparticle formulation is formulated for oral, intramuscular (IM), intravenous (IV), intraperitoneal (IP), subcutaneous (SC), topical, intradermal (ID) or intrathecal administration. Each possibility represents a separate embodiment of the invention. According to some embodiments, the administration is IV or 25 SC. According to some embodiments, the lipid nanoparticle formulation is selectively targeting an organ selected from the group consisting of: gut, lymphoid organs, lungs, heart, brain, spleen. Each possibility represents a separate embodiment of the invention. According to

[0026] 15

[0027] some embodiments, the lipid nanoparticle is selectively targeting an organ selected from the group consisting of: gut, lymphoid organs, lungs, heart, brain, spleen. Each possibility represents a separate embodiment of the invention According to some embodiments, the lipid nanoparticle formulation is selectively targeting 5 the gut. According to some embodiments, the lipid nanoparticle formulation is selectively targeting the colon. According to some embodiments, the lipid nanoparticle formulation is selectively targeting an inflamed tissue. According to some embodiments, the lipid nanoparticle is selectively targeting the gut. According to some embodiments, the lipid nanoparticle is selectively targeting the colon. 10 According to some embodiments, the lipid nanoparticle is selectively targeting an inflamed tissue. According to some embodiments, the inflammation is an inflammatory bowel disease (IBD). According to some embodiments, the lipid nanoparticle formulation is for treatment of 15 colitis. According to some embodiments, the lipid nanoparticle is for treatment of colitis. According to some embodiments, the lipid nanoparticle formulation is for treatment of rheumatoid arthritis. According to some embodiments, the lipid nanoparticle is for treatment of rheumatoid arthritis. According to some embodiments, there is provided a method of treating an inflammatory 20 disease or disorder. According to some embodiments, the method comprises the step of administering to a subject in need thereof the lipid nanoparticle formulation or LNP disclosed herein, and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the method is for treating an inflammatory bowel disease (IBD). 25 According to some embodiments, the method is for treating colitis. According to some embodiments, the method is for treating rheumatoid arthritis.

[0028] 16

[0029] According to some embodiments, the method comprises the step of orally, intramuscularly (IM), intravenously (IV), intraperitoneally (IP), subcutaneously (SC), topically, intradermally (ID) or Intrathecally administering to a subject in need thereof the lipid nanoparticle formulation or LNP and the pharmaceutically acceptable carrier, diluent or 5 excipient. Each possibility represents a separate embodiment of the invention. According to some embodiments, the administration is IV or SC. According to some embodiments, there is provided a method of delivery of a therapeutic agent or a nucleic acid to an organ selected from the group consisting of: lymphoid organs, lungs, heart, brain, and skin. According to some embodiments, the method comprises the 10 step of administering to a subject in need thereof the lipid nanoparticle formulation or LNP disclosed herein, and a pharmaceutically acceptable carrier, diluent or excipient. Each possibility represents a separate embodiment of the invention. According to some embodiments, the method is for delivery of a therapeutic agent or a nucleic acid to the gut. 15 According to some embodiments, the method is for delivery of a therapeutic agent or a nucleic acid to the colon. According to some embodiments, the method comprises the step of orally intramuscularly (IM), intravenously (IV), intraperitoneally (IP), subcutaneously (SC), topically, intradermally (ID) or Intrathecally administering to a subject in need thereof the lipid 20 nanoparticle formulation or LNP and the pharmaceutically acceptable carrier, diluent or excipient. Each possibility represents a separate embodiment of the invention. According to some embodiments, the administration is IV or SC. According to some embodiments, there is provided a method of delivery of a therapeutic agent or a nucleic acid to an inflamed tissue. According to some embodiments, the method 25 comprises the step contacting the lipid nanoparticle formulation disclosed herein with an inflamed tissue. According to some embodiments, the method comprises the step contacting the lipid nanoparticle disclosed herein with an inflamed tissue. According to some embodiments, the contacting is performed in vitro.

[0030] 17

[0031] According to some embodiments, the method comprises the step of administering to a subject in need thereof the lipid nanoparticle formulation or LNP and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the method comprises the step of orally, intramuscularly 5 (IM), intravenously (IV), intraperitoneally (IP), subcutaneously (SC), topically, intradermally (ID) or Intrathecally administering to a subject in need thereof the lipid nanoparticle formulation or LNP and the pharmaceutically acceptable carrier, diluent or excipient. Each possibility represents a separate embodiment of the invention. According to some embodiments, the administration is IV or SC. 10 Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those 15 skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a1H NMR spectrum of lipid IA-12 (NV3-006). Figure 1B is an ESI-MS spectrum of lipid IA-12 (NV3-006). Figure 1C is a1H NMR spectrum of lipid IA-11 (NV3-004). 20 Figure 1D is an ESI-MS spectrum of lipid IA-11 (NV3-004). Figure 1E is a1H NMR spectrum of lipid IA-10 (NV3-002). Figure 1F is an ESI-MS spectrum of lipid IA-10 (NV3-002). Figures 2A-E are bar charts showing expression of fLuc in healthy (empty bars) and in DSS colitis (full bars) mice administered with Formulation 1 (third to the right), 25 Formulation 2 (second to the right) and Formulation 3 (right), which target inflamed tissues (right group) and of Formulation 4 (left), Formulation 5 (second to the left) and Formulation 6 (third to the left), having standard ratio of components (left group). Figure

[0032] 18

[0033] 2A: colon / liver ratio; Figure 2B: intestines / liver ratio; Figure 2C: kidneys / liver ratio; Figure 2D: lungs / liver ratio; and Figure 2E: spleen / liver ratio. Figure 3A is a1H NMR spectrum of lipid IB-4 (NV2-004). Figure 3B is an ESI-MS spectrum of lipid IB-4 (NV2-004). 5 Figure 4A is a1H NMR spectrum of lipid IB-13 (NV2-015). Figure 4B is an ESI-MS spectrum of lipid IB-13 (NV2-015). Figure 5A is a1H NMR spectrum of lipid IB-9 (NV2-009). Figure 5B is an ESI-MS spectrum of lipid IB-9 (NV2-009). Figure 6A is a1H NMR spectrum of lipid IB-24 (NV2-027). 10 Figure 6B is an ESI-MS spectrum of lipid IB-24 (NV2-027). Figure 7A is a1H NMR spectrum of lipid IA-13 (NV3-013). Figure 7B is an ESI-MS spectrum of lipid IA-13 (NV3-013). Figure 8A is a1H NMR spectrum of lipid IA-14 (NV3-014). Figure 8B is an ESI-MS spectrum of lipid IA-14 (NV3-014). 15 Figure 9A is a1H NMR spectrum of lipid IA-15 (NV3-016). Figure 9B is an ESI-MS spectrum of lipid IA-15 (NV3-016). Figures 10A-L are bar charts showingLNPs interaction with different cells populations isolated from mice-induced with DSS. Colonic lamina propria and blood leukocytes were stained. Mice were i.v. injected with Cy-5-labelled RNA-loaded LNPs at a dose of 10ug of 20 RNA per mouse. After 2 hours or 24 hours from administration, the mice colons and blood were harvested to assess the LNPs interaction with different cell lines via FACS cells staining according to the antibodies panels. These populations include: colonic B cells (Figure 10A), colonic myeloid cells (Figure 10B), colon neutrophils (Figure 10C), colon monocytes (Figure 10D), general colon immune cells (Figure 10E), colon endothelial cells 25 (Figure 10F), colonic dendritic cells (Figure 10G), blood B cells (Figure 10I) blood myeloid cells (Figure 10J), blood neutrophils (Figure 10K) and blood monocytes (Figure 10L). The

[0034] 19

[0035] experiment was performed in triplicate with 3 mice for each experimental group, graphs represent average values ± SEM. Figures 11A-C are bar charts showing payload expression in DSS colitis mice administered with Formulation 1 (middle), Formulation 9 (left) and Formulation 10 (right). 5 Figure 11A: colon expression; Figure 11B: liver expression; and Figure 2C: colon / liver ratio. Figure 12A is a bar chart showing payload expression in sera in healthy mice administered with Formulation 1: after 24 hours (second to the left), after 48 hours (third to the right) and after 72 hours (right); and with Formulation 11: after 24 hours (left), after 48 hours 10 (third to the left) and after 72 hours (second to the right). Figure 12B is a bar chart showing payload expression in the liver in healthy mice administered with Formulation 1: after 24 hours (second to the left), after 48 hours (third to the right) and after 72 hours (right); and with Formulation 11: after 24 hours (left), after 48 hours (third to the left) and after 72 hours (second to the right). 15 Figures 13A-B are small angle X-ray scattering (SAXS) overlay graphs representing the measurements of DSPC formulations 1, 12-18 (Figure 13A) and DOPC formulations 19- 26 (Figure 13B). Figures 14A-I are DSC curves from DSPC LNPs for Formulation 12 (Figure 14A),Formulation 13 (Figure 14B), Formulation 14 (Figure 14C), Formulation 15 (Figure 14D), 20 Formulation 16 (Figure 14E), Formulation 1 (Figure 14F), Formulation 17 (Figure 14G) and Formulation 18 (Figure 14H). Figure 15 is a graph showing the phase transition temperature of Formulation 12 (5% mol / mol DSPC), Formulation 13 (10% mol / mol DSPC), Formulation 14 (15% mol / mol DSPC), Formulation 15 (20% mol / mol DSPC), Formulation 16 (25% mol / mol DSPC), 25 Formulation 1 (30% mol / mol DSPC), Formulation 17 (35% mol / mol DSPC) and Formulation 18 (40% mol / mol DSPC). Figure 16 is a graph showing the integrated enthalpy change (30℃-70℃) of Formulation 12 (5% mol / mol DSPC), Formulation 13 (10% mol / mol DSPC), Formulation 14 (15%

[0036] 20

[0037] mol / mol DSPC), Formulation 15 (20% mol / mol DSPC), Formulation 16 (25% mol / mol DSPC), Formulation 1 (30% mol / mol DSPC), Formulation 17 (35% mol / mol DSPC) and Formulation 18 (40% mol / mol DSPC). DETAILED DESCRIPTION OF THE PRESENT INVENTION 5 The present invention is based on the discovery of lipid nanoparticle (LNP) compositions useful in selectively delivering active agents to inflamed tissues. Specifically, the present lipid nanoparticle formulations and LNPs include a specific ratio between the different lipids contained therein. According to some embodiments, the lipid nanoparticle compositions of the present invention can include different cationic lipids as the at least 10 one cationic lipid component. The at least one cationic lipid component may include the lipids ALC-0315, EA-502, SM-102, II-25 (NV1-022) or other cationic lipid(s), such as those represented by Formula (IA), Formula (II), and / Formula (IB). According to some embodiments, the cationic lipid is not a phospholipid. Specifically, according to some embodiments, the phospholipid portion of the present lipid nanoparticle 15 formulations and LNPs is detailed herein separately. Cationic lipids - Formula (IA) As contemplated herein, the present invention relates to a cationic lipid(s) represented by the structure of Formula (IA): 20 nc u ng sa s, y raes, so va es, po ymorp s, op ca somers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.

[0038] 21

[0039] According to some embodiments, R1Ais selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNARNA’, wherein each one of RNAand RNA’is individually C1-4 alkyl or RNAand RNA’together with the nitrogen to which they are bound, form a ring. Each possibility represents a separate 5 embodiment of the invention. According to some embodiments, the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl. According to some embodiments, R1Ais selected from the group consisting of: OH, -(CH2CH2O)2-6H and C1-6 hydroxyalkyl. According to some embodiments, R1Ais 10 selected from the group consisting of: OH, -(CH2CH2O)2-4H and C1-4hydroxyalkyl. According to some embodiments, R1Ais -(CH2CH2O)1-4H or OH. According to some embodiments, R1Ais selected from the group consisting of: -CH2CH2OH, -CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH and OH. According to some embodiments, R1Ais OH. According to some embodiments, R1Ais -CH2CH2OH. According to some 15 embodiments, R1Ais, -CH2CH2OCH2CH2OH. According to some embodiments, R1Ais the same as R3A. It is the be understood that the term “the same” in the previous paragraph means that the two specified R substituents have the same chemical definition. For example, lipid 1A-2 as shown below has the same R1Aand R3A, each of which is-CH2CH2OH. This lipid also has 20 the same R2Aand R4A, each of which is -(CH2)8CH=CHCH2CH=CHC5H11. According to some embodiments, R2Ais selected from the group consisting of: C5-25alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15alkenylene-CO2-C5-15alkenyl, C5-15alkenylene-O2C-C5-15alkyl, C5-1525 alkenylene-O2C-C5-15alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2Ais selected from the group consisting of: C6-18alkyl and C12-24alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some

[0040] 22

[0041] embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R2Ais selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, -C12H25, -(CH2)5CH=CHCH2CH=CHC8H17, -(CH2)8CH=CHC8H17and 5 -(CH2)7CH=CHCH2CH=CHC4H9. According to some embodiments, R2Ais selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11 and -C12H25. According to some embodiments, R2Ais -(CH2)8CH=CHCH2CH=CHC5H11. According to some embodiments, R2Ais -C12H25. According to some embodiments, R2Ais the same as R4A. 10 According to some embodiments, nAis selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. According to some embodiments, nAis selected from the group consisting of: 8, 9 and 10. According to some embodiments, nAis 9 or 10. According to some embodiments, nAis 9. According to some embodiments, nAis 10. According to some embodiments, XAis selected from the group consisting of:-COO-, -OOC, 15 -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-. Each possibility represents a separate embodiment of the invention. According to some embodiments, XAis selected from the group consisting of: -COO-, -OOC-, -NHCO- and -CONH. According to some embodiments, XAis -COO- or -OOC-. According to some embodiments, XAis -COO- and the lipid is represented by Formula 20 (IA1) . According to some embodiments, j is selected from the group consisting of: 0, 1, 2, 3 and 4. Each possibility represents a separate embodiment of the invention. According to some

[0042] 23

[0043] embodiments, jAis selected from the group consisting of: 0, 1 and 2. According to some embodiments, jAis 0 or 2. According to some embodiments, jAis 0. According to some embodiments, YAis selected from the group consisting of: absent, -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-. Each 5 possibility represents a separate embodiment of the invention. According to some embodiments, YAis absent, -COO- or -OOC-. Each possibility represents a separate embodiment of the invention. According to some embodiments, YAis absent or -OOC-. According to some embodiments, YAis absent. According to some embodiments, YAis absent, jAis 0 and the lipid of Formula (IA) is 10 represented by Formula (IA2): . According to some embodiments, Y is absent, jAis 0, XAis -COO- and the lipid of Formula (IA) is represented by Formula (IA3) . 15 According to some embodiments, m is selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. Each possibility represents a separate embodiment of the invention. According to some embodiments, mAis selected from the group consisting of: 6, 7, 8, 9, 10, 11, 12 and 13. According to some embodiments, mAis selected from the

[0044] 24

[0045] group consisting of: 8, 9, and 10. According to some embodiments, mAis 9 or 10. According to some embodiments, mAis 9. According to some embodiments, mAis 10. According to some embodiments, R3Ais selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6hydroxyalkyl and C1-6haloalkyl and C1-3alkylene-NRNA’’RNA’’’, 5 wherein each one of RNA’’and RNA’’’is individually C1-4 alkyl or RNA’’and RNA’’’together with the nitrogen to which they are bound, form a ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl. 10 According to some embodiments, R3Ais selected from the group consisting of: OH, -(CH2CH2O)2-6H and C1-6hydroxyalkyl. According to some embodiments, R3Ais selected from the group consisting of: OH, -(CH2CH2O)2-4H and C1-4 hydroxyalkyl. According to some embodiments, R3Ais -(CH2CH2O)1-4H or OH. According to some embodiments, R3Ais selected from the group consisting of: -CH2CH2OH, 15 -CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH, and OH. According to some embodiments, R3Ais OH. According to some embodiments, R3Ais -CH2CH2OH. According to some embodiments, R1Ais, -CH2CH2OCH2CH2OH. According to some embodiments, R4Ais selected from the group consisting of: C5-25alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 20 alkylene-O2C-C5-15alkyl, C5-15alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5-15alkenyl, C5-15alkenylene-O2C-C5-15alkyl, C5-15alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R4Ais selected from the group consisting of: C6-18alkyl 25 and C12-24alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R4Ais selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, -C12H25,

[0046] 25

[0047] -(CH2)5CH=CHCH2CH=CHC8H17, -(CH2)8CH=CHC8H17and -(CH2)7CH=CHCH2CH=CHC4H9. According to some embodiments, R4Ais selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11 and -C12H25. According to some embodiments, R4Ais -(CH2)8CH=CHCH2CH=CHC5H11. According to some 5 embodiments, R4Ais -C12H25. According to some embodiments, the cationic lipid of Formula (IA) is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14, and IA-15 including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each 10 possibility represents a separate embodiment of the invention. According to some embodiments, the lipid of Formula (IA) is selected from the group consisting of: lipid IA- 4, lipid IA-5, lipid IA-8, lipid IA-10, lipid IA-11, lipid IA-12, lipid IA-13, lipid IA-14, and lipid IA-15. According to some embodiments, the lipid of Formula (IA) is selected from the group consisting of: IA-10, IA-11 and a combination thereof. According to some 15 embodiments, the lipid of Formula (IA) is IA-5. According to some embodiments, the lipid of Formula (IA) is IA-10. According to some embodiments, the lipid of Formula (IA) is IA-11. According to some embodiments, the lipid of Formula (IA) is IA-12. Formula (IB) As contemplated herein, the present invention relates to a cationic lipid(s) represented by 20 the structure of Formula (IB): c u g sa s, y aes, so va es, po y o phs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Specifically, a cationic lipid(s)

[0048] 26

[0049] represented by the structure of Formula (IB) is optionally included in the lipid nanoparticle formulation or LNP of the present invention. According to some embodiments, R1Bis selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6hydroxyalkyl and C1-6haloalkyl and C1-3alkylene-NRNBRNB’, 5 wherein each one of RNAand RNA’is individually C1-4 alkyl or RNAand RNA’together with the nitrogen to which they are bound, form a ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl. 10 According to some embodiments, R1Bis selected from the group consisting of: -CH2CH2OCH2CH2OH, -CH2CH2Cl, -CH2CH2OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2CH2OH and -CH2CH2NMe2. Each possibility represents a separate embodiment of the invention. According to some embodiments, R1Bis selected from the group consisting of: CH2CH2OCH2CH2OH, -CH2CH2Cl and -CH2CH2OH. According to 15 some embodiments, R1Bis -CH2CH2OCH2CH2OH or -CH2CH2Cl. According to some embodiments, R1Bis -CH2CH2OCH2CH2OH. According to some embodiments, R1Bis - CH2CH2Cl. It is to be understood that the group -CH2CH2CH2N(CH2)4, which is specified herein refers to propyl pyrrolidine, i.e., the group drawn below: 20 Similarly, the group -CH2CH2CH2N(CH2)5 refers to the structure drawn below:

[0050] 27

[0051] w: g , . 5 According to some embodiments, R2Bis selected from the group consisting of: C5-25alkyl, C5-25alkenyl, C5-15alkylene-CO2-C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15alkenyl. Each possibility represents a separate embodiment of the 10 invention. According to some embodiments, R2Bis selected from the group consisting of: C6-18 alkyl and C12-24alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the 15 alkenyl is an unsubstituted alkenyl. According to some embodiments, R2Bis selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, - (CH2)7CH=CHCH2CH=CHC6H13, -(CH2)8CH=CHC8H17 and -C12H25. According to some embodiments, R2Bis -(CH2)8CH=CHCH2CH=CHC5H11 or -C12H25. According to some embodiments, R2Bis -(CH2)8CH=CHCH2CH=CHC5H11. According to some embodiments, 20 R2Bis -C12H25.

[0052] 28

[0053] According to some embodiments, R2Ais the same as R4A. According to some embodiments, R2Ais the same as R3A. According to some embodiments, WBis a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen. According to 5 some embodiments, WBis a C4-12 alkylene. According to some embodiments, WBis a straight chain alkylene. According to some embodiments, WBis a C5-9 alkylene. According to some embodiments, WBis selected from the group consisting of: -(CH2)9-, -(CHMe)- (CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, WBis -(CH2)9-. 10 According to some embodiments, YBis selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6alkylene. According to some embodiments, YBis selected from the groups consisting of: absent, -CH2CH2OCH2CH2- and -CH2CH2-. It is to be understood by the person having ordinary skill in the art that when a variable is said to be “absent”, no chemical group will appear in the specified place, and the atoms 15 drawn as bonded to the variable, will be bonded to each other. For example, Formula (IB1) is an embodiment of Formula (IB), wherein the variable YBis absent. In Formula (IB), YBis drawn as bonded to an oxygen atom and to a nitrogen atom. Thus, according to some embodiments, if YBis absent the oxygen will be directly bonded to the nitrogen via a single (signa) bond. 20 According to some embodiments, YBis absent and the lipid of Formula (IB) is represented by Formula (IB1): . According to some embodiments, R3Bis selected from the group consisting of: C5-25 alkyl, C5-25alkenyl, C5-15alkylene-CO2-C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15

[0054] 29

[0055] alkylene-O2C-C5-15alkyl, C5-15alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. 5 According to some embodiments, R3Bis selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R3Bis a C4-16 alkyl. 10 According to some embodiments, R3Bis selected from the group consisting of: -C12H25, - C6H13and -C8H17. According to some embodiments, R3Bis -C6H13or -C8H17. According to some embodiments, R3Bis -C6H13. According to some embodiments, R3Bis -C8H17. According to some embodiments, R3Bis the same as R4B. According to some embodiments, R4Bis selected from the group consisting of: C5-25alkyl, 15 C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15alkenylene-CO2-C5-15alkenyl, C5-15alkenylene-O2C-C5-15alkyl, C5-15alkenylene-O2C-C5-15alkenyl. Each possibility represents a separate embodiment of the invention. 20 According to some embodiments, R4Bis selected from the group consisting of: C6-18alkyl and C12-24alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R4Bis a C4-16alkyl. 25 According to some embodiments, R4Bis selected from the group consisting of: -C12H25, - C6H13 and -C8H17. According to some embodiments, R4Bis -C6H13 or -C8H17. According to some embodiments, R4Bis -C6H13. According to some embodiments, R4Bis -C8H17. According to some embodiments, the cationic lipid of Formula (IB) is selected from the group consisting of: IB-1, IB-2, IB-3, IB-4, IB-5, IB-6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-

[0056] 30

[0057] 12, IB-13, IB-14, IB-15, IB-16, IB-17, IB-18, IB-19, IB-20, IB-21, IB-22, IB-23, and IB- 24, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the cationic lipid of 5 Formula (IB) is selected from the group consisting of: IB-4, IB-6, IB-10 and IB-24. According to some embodiments, the lipid of Formula (IB) is IB-4. According to some embodiments, the lipid of Formula (IB) is IB-6. According to some embodiments, the lipid of Formula (IB) is IB-10. Formula (II) 10 As contemplated herein, the present invention relates to a cationic lipid(s) represented by the structure of Formula (II): g , y , , p y p , p , geometrical isomers, enantiomers, diastereomers, and mixtures thereof. 15 According to some embodiments, R1Cis selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15alkyl, C5-15alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of 20 the invention. According to some embodiments, R1Cis selected from the group consisting of: C4-18alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some

[0058] 31

[0059] embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R1Cis a C4-16 alkyl. According to some embodiments, R1Cis a C4-10 alkyl. According to some embodiments, R1Cis selected from the group consisting of: -C12H25, -C6H13and -C8H17. According to 5 some embodiments, R1Cis -C6H13 or -C8H17. According to some embodiments, R1Cis - C6H13. According to some embodiments, R1Cis -C8H17. According to some embodiments, R1Cis the same as R2C. According to some embodiments, R1Cis the same as R3C. According to some embodiments, R1Cis the same as R4C. According to some embodiments, R1C, R2C, R3C, and R4Care the same. 10 According to some embodiments, R2Cis selected from the group consisting of: C5-25alkyl, C5-25alkenyl, C5-15alkylene-CO2-C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15alkenyl. Each possibility represents a separate embodiment of the 15 invention. According to some embodiments, R2Cis selected from the group consisting of: C4-18 alkyl and C12-24alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the 20 alkenyl is an unsubstituted alkenyl. According to some embodiments, R2Cis a C4-16alkyl. According to some embodiments, R2Cis a C4-10alkyl. According to some embodiments, R2Cis selected from the group consisting of: -C12H25, -C6H13 and -C8H17. According to some embodiments, R2Cis -C6H13 or -C8H17. According to some embodiments, R2Cis - C6H13. According to some embodiments, R2Cis -C8H17. 25 According to some embodiments, Y1Cis selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y1Cis selected from the groups consisting of: absent, -CH2CH2OCH2CH2- and -CH2CH2-. According to some embodiments, Y1Cis absent.

[0060] 32

[0061] According to some embodiments, Y1Cis the same as Y2C. According to some embodiments, each one of Y1Cand Y2Cis absent and the lipid of Formula (II) is represented by Formula (II1): . 5 According to some embodiments, W is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen. According to some embodiments, W1Cis a C4-12 alkylene. According to some embodiments, W1Cis a straight chain alkylene. According to some embodiments, W1Cis a C5-9 alkylene. According to some embodiments, W1Cis selected from the group consisting of: -(CH2)9-, -(CHMe)- 10 (CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, W1Cis -(CH2)9-. According to some embodiments, W1Cand W2Care the same. According to some embodiments, each one of W1Cand W2Cis a C4-12straight chain alkylene and the lipid of Formula (II) is represented by Formula (II2): 15 . According to some embodiments, each one of Y and Y is absent, each one of W1Cand W2Cis a C4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II3):

[0062] 33

[0063] . According to some embodiments, R is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNIIRNII’, wherein each one of RNIIand RNII’is individually C1-4alkyl or RNIIand RNII’together with 5 the nitrogen to which they are bound, form a ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl. According to some embodiments, R5Cis selected from the group consisting of: 10 OH, -(CH2CH2O)2-3H, C1-4 hydroxyalkyl and C1-4 haloalkyl and C1-3 alkylene-NRNIIRNII’, wherein each one of RNIIand RNII’is individually C1-4alkyl or RNIIand RNII’together with the nitrogen to which they are bound, form a 5-6 membered ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, R5Cis selected from the group consisting of: 15 -CH2CH2OH, -CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2N(CH2)5, -CH2CH2N(CH2)5, -CH2CH2Cl, -OH and -CH2CH2NMe2. According to some embodiments, R5Cis -CH2CH2OH or -CH2CH2OCH2CH2OH. According to some embodiments, W2Cis a C4-12alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen. According 20 to some embodiments, W2Cis a C4-12 alkylene. According to some embodiments, W2Cis a straight chain alkylene. According to some embodiments, W2Cis a C5-9alkylene. According to some embodiments, W2Cis selected from the group consisting of: -(CH2)9-, -(CHMe)- (CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, W2Cis -(CH2)9-.

[0064] 34

[0065] According to some embodiments, Y2Cis selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y2Cis selected from the groups consisting of: absent, -CH2CH2OCH2CH2- and -CH2CH2-. According to some 5 embodiments, Y2Cis absent. According to some embodiments, R3Cis selected from the group consisting of: C5-25 alkyl, C5-25alkenyl, C5-15alkylene-CO2-C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 10 alkenylene-O2C-C5-15alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R3Cis selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some 15 embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R3Cis a C4-16 alkyl. According to some embodiments, R3Cis a C4-10alkyl. According to some embodiments, R3Cis selected from the group consisting of: -C12H25, -C6H13and -C8H17. According to some embodiments, R3Cis -C6H13 or -C8H17. According to some embodiments, R3Cis - 20 C6H13. According to some embodiments, R3Bis -C8H17. According to some embodiments, R4Cis selected from the group consisting of: C4-18alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the 25 alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R4Cis a C4-16 alkyl. According to some embodiments, R4Cis a C4-10alkyl. According to some embodiments, R4Cis selected from the group consisting of: -C12H25, -C6H13and -C8H17. According to some

[0066] 35

[0067] embodiments, R4Cis -C6H13or -C8H17. According to some embodiments, R4Cis -C6H13. According to some embodiments, R4Cis -C8H17. According to some embodiments, the cationic lipid of Formula (II) is selected from the group consisting of: II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, 5 II-11, II-12, II-13, II-14, II-15, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-23, II-24 and II-25, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: II-1, II-5, II-25 and a 10 combination thereof. According to some embodiments, the lipid of Formula (II) is II-1. According to some embodiments, the lipid of Formula (II) is II-5. According to some embodiments, the lipid of Formula (II) is II-25. The chemical structures of each of the specific lipids are detailed below in the “Exemplary Cationic Lipids” Section and in the claims. 15 Exemplary Cationic Lipids Exemplary lipids according to Formula (IA), Formula (IB) and Formula (II) of the present invention are shown below. It is to be understood that, according to some embodiments, the invention is not limited to any one or more of the following exemplary lipids. The following exemplary lipids are portrayed as non-limiting examples of the cationic 20 lipids of the present invention (designation below the chemical structure). -

[0068] 36

[0069] 5 . 10 IA-5 Lipid IA-5 is also referred herein as NV3-005.

[0070] 37

[0071] 5 10 IA-10 Lipid IA-10 is also referred herein as NV3-002. 5 IA-11 Lipid IA-11 is also referred herein as NV3-004. - Lipid IA-12 is also referred herein as NV3-006. 10 Lipid IA-13 is also referred herein as NV3-013. IA-14 Lipid IA-14 is also referred herein as NV3-014. 5 10

[0072] 5 10 p - - .

[0073] 41

[0074] 5 IB-9 Lipid IB-9 is also referred herein as NV2-009. 10 Li p - - .

[0075] 42

[0076] 5 10

[0077] 5 IB-24 Lipid IB-24 is also referred herein as NV2-027.

[0078] 45 Lipid II-1 is also referred herein as NV1-001. 5 II-5 Lipid II-5 is also referred herein as NV1-005. 5

[0079]

[0080] -20

[0081] II-25 Lipid II-25 is also referred herein as NV1-022. 5 - ;

[0082] 52

[0083] . ALC-0315 is a commercial cationic lipid, its systematic name is [(4- Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate). 5 SM-102 is a commercial cationic lipid, its systematic name is 9-Heptadecanyl 8-{(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate. According to some embodiments, the cationic lipid comprises ALC-0315, EA-502, SM- 102, II-25, lipid II-1, lipid IA-10 or any combination thereof. According to some embodiments, there is provided the lipid II-25 (NV1-022) 10 Chemical Definitions The term “ionizable lipid”, as used herein refers to lipid species that carries a charge at a selected pH. Selected pH values include, but not limited to physiological pH, pH=7 and the like. It is to be understood by the person having ordinary skill in the art that the lipids of Formulae (IA), (IB) and (II) may be considered as ionizable lipids. 15 The term “cationic lipid”, as used herein refers to lipid species that carries a net positive charge at a selected pH. Selected pH values include, but not limited to physiological pH (e.g., blood pH), pH=7 and the like. It is to be understood by the person having ordinary skill in the art that the lipids of Formulae (IA), (IB) and (II) may be considered as cationic lipids, since they bear 2 or more nitrogen atoms, where these atoms are typically basic and 20 protonizable at the selected pH, so that the lipids may carry a net positive charge. According to some embodiments, the lipid of the present invention is a cationic lipid. The term “permanently charged lipid”, as used herein refers to lipid species that carries a charge at on one or more of its atoms and that has a total net (positive or negative) charge.

[0084] 53

[0085] The charged atom may not be de-charged in the permanently charged lipid. Specifically, the charged atom may not be de-charged at different pH environments. According to some embodiments, the charge of the charged atom may not be altered at different pH environments. The term “permanently charged lipid” includes both charged lipids coupled 5 to a counterion and zwitterionic lipids. Zwitterionic permanently charged lipids may require unequal number of positively charged atoms and negatively charged atom in the lipid, and also include a counterion not bonded to the lipid. For example distearoylphosphatidylcholine (DSPC) has permanently charged phosphate and ammonium groups, and has a net zero charge so it in not permanently charged. Charged 10 atoms of the permanently charged lipid include, but are not limited to nitrogen (e.g., ammonium groups) and / or oxygen (e.g., sulfate groups, carboxylate groups, phosphate groups, etc.). According to some embodiments, the permanently charged lipid comprises a quaternary ammonium group. Specifically, quaternary ammonium groups are considered to have permanently charged nitrogen atom, due to the three-valent nitrogen bonded to 4 15 groups (e.g., alkyl groups). According to some embodiments, the permanently charged lipid have a net charge. It is to be understood that a permanently charged lipid which has a net charge has a permanently charged atom as part of or chemically bonded to the lipid backbone, and a counterion, which is not covalently bonded to the lipid backbone. The term “permanently charged chemical group” means a chemical group that has a net 20 charge, which is not affected by pH environment. Notable permanently charged chemical groups include, but are not limited to, quaternary ammonium groups. The term “PEG” refers to polyethylene glycol. The term “PEGylated lipid” means a lipid that is bonded to PEG. The term “quaternary ammonium group” as used herein refers to a chemical functional 25 group containing at least one quaternized nitrogen wherein the nitrogen atom is attached to four organic groups. A permanently charged lipid according to the present invention may comprise one or more quaternized nitrogen atoms. According to some embodiments, the quaternary ammonium group is a tetra-alkyl ammonium.

[0086] 54

[0087] The term “tetra-alkyl ammonium” refers to a group or compound (e.g., lipid) which contain such group, that has a nitrogen atom bonded to four alkyl groups. The term “alkyl” is defined below. An “alkyl” group refers to any saturated aliphatic hydrocarbon, including straight-chain 5 and branched-chain alkyl groups. The alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl. The term "Cn-m alkyl", refers to an alkyl group having n to m carbon atoms. An alkyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment 10 of the alkyl group to the remainder of the compound. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, 3-pentyl, hexyl, 1,2,2-trimethylpropyl and the like. The term "alkylene," employed alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene group formally 15 corresponds to an alkane with two C-H bonds replaced by points of attachment of the alkylene group to the remainder of the compound. The term "Cn-m alkylene" refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethan-l,2-diyl, ethan-l,l-diyl, propan-l,3-diyl, propan-l,2-diyl, propan-l,l- diyl, butan-l,4-diyl, butan-l,3-diyl, butan-1,2- diyl, 2-methyl-propan-l,3-diyl, -(CHMe)- 20 (CH2)4-, -(CHMe)-(CH2)4- and the like. It is to be understood that for branched alkylene groups the total number of carbon atoms is counted. For example, the substituent -(CHMe)- (CH2)4-, is a C6 alkylene. It is to be understood that C0-alkylene means that the specified substituent is absent. In various section of the present application ranges of alkyl chains are presented, e.g., C4-16alkyl, C6-18alkyl etc. It is to be understood that such ranges include 25 any sub range thereof. for example, C6-18 alkyl may include and / or be directed to: C6-12 alkyl, C8-14 alkyl, C12-18 alkyl, C8 alkyl etc. An "alkenyl" group refers to an aliphatic hydrocarbon group containing at least one carbon- carbon double bond including straight-chain, branched-chain and cyclic alkenyl groups. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-

[0088] 55

[0089] enyl, n-pentenyl, heptenyl, octenyl, cyclohexyl-butenyl and decenyl. The alkenyl group can be unsubstituted or substituted through available carbon atoms with one or more groups defined hereinabove for alkyl. Alkenyls according to the present invention may include more than one carbon-carbon double bond. Thus, dienes (see e.g., lipid IA-10, NV3-002, 5 substituent R4A) and trienes are within the definition of alkenyl. According to some embodiments, the alkenyl is a dienyl. The term "Cn-m alkenyl", refers to an alkyl group having n to m carbon atoms. An alkenyl group formally corresponds to an alkene with one C-H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound. Examples of alkenyl moieties include, but are not limited to, chemical groups 10 such as ethenyl, propenyl, isopropenyl, n- butenyl, sec-butenyl the like. The term "alkenylene," employed alone or in combination with other terms, refers to a divalent alkenyl linking group. An alkenylene group formally corresponds to an alkane with two C- H bonds replaced by points of attachment of the alkenylene group to the remainder of the compound. The term "Cn-malkenylene" refers to an alkenylene group having n to m carbon 15 atoms. In various section of the present application ranges of alkenyl chains are presented, e.g., C2-8 alkenyl, C4-20 alkenyl etc. It is to be understood that such ranges include any sub range thereof. for example, C4-14alkenyl may include and / or be directed to: C4-8alkenyl, C8-14alkenyl, C6-12alkenyl, C9alkenyl etc. According to some embodiments, each one of the alkenyl double bond has a cis configuration. 20 One or more of the lipids of the invention, may be present as a salt. The term "salt" encompasses both basic and acid addition salts, including but not limited to, carboxylate salts or salts with amine nitrogen atoms, and include salts formed with the organic and inorganic anions and cations discussed below. Furthermore, the term includes salts that form by standard acid-base reactions with basic groups (such as amino groups) and organic 25 or inorganic acids. Such acids include hydrochloric, hydrofluoric, trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids. Each possibility represents a separate embodiment of the invention.

[0090] 56

[0091] The term "organic or inorganic cation" refers to counter-ions for the anion of a salt. The counter-ions include, but are not limited to, alkali and alkaline earth metals (such as lithium, sodium, potassium, barium, aluminum and calcium); ammonium and mono-, di- and tri- alkyl amines such as trimethylamine, cyclohexylamine; and the organic cations, such as 5 dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2- hydroxyethyl)ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations. See, for example, Berge et al., J. Pharm. Sci. (1977), 66:1-19, which is incorporated herein by reference. Particles, Formulations Compositions and Uses 10 According to some embodiments, the present invention provides a particle comprising the present at least one cationic lipid, at least one neutral phospholipid, at least one sterol, and at least one PEGylated lipid. According to some embodiments, there is provided a composition comprising a plurality of particles as discloses herein and a pharmaceutically acceptable carrier, diluent or 15 excipient. According to some embodiments, the composition is a liposomal composition. According to some embodiments, the particles of the present invention are in the form of liposomes. According to some embodiments, the composition further comprises one or more components selected from the group consisting of a neutral lipid, a charged lipid, a steroid, and a polymer-conjugated lipid. Each possibility represents a separate embodiment 20 of the present invention. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least one cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least one neutral phospholipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least one sterol 25 According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least one PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least one cationic lipid; at least one neutral phospholipid; at least one sterol; and at least one PEGylated lipid.

[0092] 57

[0093] According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises a phospholipid. According to some embodiments, the phospholipid is a neutral phospholipid. According to some embodiments, the neutral phospholipid comprises a phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), 5 phosphatidylserine (PS), phosphatidylinositol (PI), or a combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the neutral phospholipid comprises a phosphatidylcholine (PC). The term “phosphatidylcholine” is defined herein. According to some embodiments, the phosphatidylcholine (PC) is selected from the group 10 consisting of: dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC) palmitoyloleoylphosphatidylcholine (POPC) and a combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the phosphatidylcholine comprises DOPC, DSPC or both. 15 According to some embodiments, the phosphatidylcholine comprises DOPC. According to some embodiments, the phosphatidylcholine comprises DSPC. According to some embodiments, the neutral phospholipid comprises a phosphatidylglycerol (PG). The term “phosphatidylglycerol” is defined herein. According to some embodiments, the phosphatidylglycerol (PG) is selected from the group 20 consisting of: dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG) and a combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the neutral phospholipid comprises a phosphatidylserine 25 (PS). The term “phosphatidylserine” is defined herein. According to some embodiments, the phosphatidylserine (PS) is selected from the group consisting of: dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine

[0094] 58

[0095] (DSPS), dioleoyl phosphatidyl serine (DOPS), dipalmitoyl phosphatidyl serine (DPPS) and a combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the neutral phospholipid comprises a phosphatidylethanolamine (PE). The term “phosphatidylethanolamine” is defined herein. 5 According to some embodiments, the phosphatidylethanolamine (PE) is selected from the group consisting of: dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyloleoyl- phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-10 ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl- 2-oleoyl-phosphatidyethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE), 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), Biotin- Phosphatidylethanolamine, and a combination thereof. Each possibility represents a 15 separate embodiment of the invention. According to some embodiments, the neutral phospholipid DSPC, DOPE or both. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is the phospholipid shown below: 20 . According to some embodiments, the neutral phospholipid comprises DSPC. l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is the phospholipid shown below:

[0096] 59

[0097] According to some embodiments, the neutral phospholipid comprises a permanently charged chemical group. According to some embodiments, the neutral phospholipid has 5 neutral net charge at physiological pH, while having a permanently charged chemical group. According to some embodiments, the neutral phospholipid comprises a permanently positively charged chemical group. According to some embodiments, the neutral phospholipid has neutral net charge at physiological pH, while having a permanently positively charged chemical group. This is possible, according to some embodiments, in 10 case that the neutral phospholipid is zwitterionic, bearing, e.g, a positively permanently charged chemical group, such as tetraalkyl ammonium. According to some embodiments, the phospholipid is a zwitterionic phospholipid. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is in the range of 11% mol / mol to 49% mol / mol, 15 including each value and sub-range within the specified range. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is in the range of 15% mol / mol to 45% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is in the range of 20% mol / mol to 40% mol / mol. According to some 20 embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is in the range of 25% mol / mol to 35% mol / mol. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises about 30% mol / mol total neutral phospholipid concentration. According to some embodiments, a total neutral phospholipid concentration within the lipid 25 nanoparticle formulation and / or LNP is at least 11% mol / mol. According to some

[0098] 60

[0099] embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is at least 12% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is at least 14% mol / mol. According to some embodiments, a total neutral phospholipid 5 concentration within the lipid nanoparticle formulation and / or LNP is at least 16% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is at least 18% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is at least 20% mol / mol. According to some embodiments, a total 10 neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is at least 21% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is at least 22% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is at least 24% mol / mol. According to some 15 embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is at least 26% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is at least 28% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid 20 nanoparticle formulation and / or LNP is no more than 49% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is no more than 48% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is no more than 46% mol / mol. According to some embodiments, a total neutral 25 phospholipid concentration within the lipid nanoparticle formulation and / or LNP is no more than 44% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is no more than 42% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is no more than 40% mol / mol. According to 30 some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle

[0100] 61

[0101] formulation and / or LNP is no more than 39% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is no more than 38% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is no more 5 than 36% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is no more than 34% mol / mol. According to some embodiments, a total neutral phospholipid concentration within the lipid nanoparticle formulation and / or LNP is no more than 32% mol / mol. Reference is made to the cationic lipid of the present invention and to its mole proportion 10 within the LNPs. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises 20% mol / mol to 60% mol / mol of the cationic lipid, including each value and sub-range within the specified range. According to some embodiments, the lipid nanoparticle formulation comprises 25% mol / mol to 55% mol / mol of the cationic lipid. According to 15 some embodiments, the lipid nanoparticle formulation and / or LNP comprises 30% mol / mol to 50% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises 33% mol / mol to 43% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises about 38% mol / mol of the cationic lipid. 20 According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least 15% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least 20% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least 25% mol / mol of the cationic lipid. According to some embodiments, the lipid 25 nanoparticle formulation and / or LNP comprises at least 30% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least 33% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least 35% mol / mol of the cationic lipid.

[0102] 62

[0103] According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises no more than 65% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises no more than 60% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP 5 comprises no more than 55% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises no more than 50% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises no more than 45% mol / mol of the cationic lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises 10 no more than 40% mol / mol of the cationic lipid. Reference is made to the sterol of the present invention and to its mole proportion therein. The term “sterol” is defined herein According to some embodiments, the sterol comprises cholesterol. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises cholesterol. 15 According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is in the range of 11% mol / mol to 49% mol / mol, including each value and sub-range within the specified range. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is in the range of 15% mol / mol to 45% mol / mol. According to some embodiments, a total sterol concentration 20 within the lipid nanoparticle formulation and / or LNP is in the range of 20% mol / mol to 40% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is in the range of 25% mol / mol to 35% mol / mol. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises about 30% mol / mol total sterol concentration. 25 According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 11% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 12% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 14% mol / mol. According to some

[0104] 63

[0105] embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 16% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 18% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation 5 and / or LNP is at least 20% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 21% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 22% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 24% 10 mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 26% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is at least 28% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle 15 formulation and / or LNP is no more than 49% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 48% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 46% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation 20 and / or LNP is no more than 44% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 42% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 40% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or 25 LNP is no more than 39% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 38% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 36% mol / mol. According to some embodiments, a total sterol concentration within the lipid nanoparticle formulation and / or 30 LNP is no more than 34% mol / mol. According to some embodiments, a total sterol

[0106] 64

[0107] concentration within the lipid nanoparticle formulation and / or LNP is no more than 32% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is in the range of 11% mol / mol to 49% mol / mol, 5 including each value and sub-range within the specified range. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is in the range of 15% mol / mol to 45% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is in the range of 20% mol / mol to 40% mol / mol. According to some 10 embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is in the range of 25% mol / mol to 35% mol / mol. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises about 30% mol / mol total cholesterol concentration. According to some embodiments, a total cholesterol concentration within the lipid 15 nanoparticle formulation and / or LNP is at least 11% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is at least 12% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is at least 14% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid 20 nanoparticle formulation and / or LNP is at least 16% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is at least 18% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is at least 20% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid 25 nanoparticle formulation and / or LNP is at least 21% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is at least 22% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is at least 24% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid

[0108] 65

[0109] nanoparticle formulation and / or LNP is at least 26% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is at least 28% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid 5 nanoparticle formulation and / or LNP is no more than 49% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 48% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 46% mol / mol. According to some embodiments, a total cholesterol concentration 10 within the lipid nanoparticle formulation and / or LNP is no more than 44% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 42% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 40% mol / mol. According to some embodiments, a total 15 cholesterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 39% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 38% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 36% mol / mol. According to some 20 embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 34% mol / mol. According to some embodiments, a total cholesterol concentration within the lipid nanoparticle formulation and / or LNP is no more than 32% mol / mol. Reference is made to the PEGylated lipid of the present invention and to its mole proportion 25 therein. The terms “PEG” and “PEGylated lipid” are defined herein According to some embodiments, the sterol comprises a PEGylated lipid. According to some embodiments, the PEGylated lipid comprises a PEG moiety having a molecular weight in the range of 1000 gr / mol to 3000 gr / mol, including each value and sub-range within the specified range. According to some embodiments, the PEGylated lipid

[0110] 66

[0111] comprises a PEG moiety having a molecular weight in the range of 1000 gr / mol to 2000 gr / mol. According to some embodiments, the PEG moiety has a molecular weight of about 2000 gr / mol. According to some embodiments, the PEGylated lipid comprises DMG-PEG. According 5 to some embodiments, the PEGylated lipid comprises DMG-PEG-2000. The term “DMG-PEG” means 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol, or ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol. The term “DMG-PEG- 2000” means 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol, or ,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol, wherein the polyethylene glycol 10 has a molecular weight of about 2000 gr / mol. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises 0.5% to 5 mol% PEGylated lipid, including each value and sub-range within the specified range. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises 1 to 3.5 mol% PEGylated lipid. According to some embodiments, the lipid 15 nanoparticle formulation and / or LNP comprises about 2 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least 1 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least 0.5 mol% PEG. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises no more than 10 20 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises no more than 5 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises no more than 3 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises no more than 2.5 mol% PEGylated lipid. 25 It is to be understood that by the phrase “the molar percentage of the component is at least x mol% of the particle” it is meant that at least x% of the particle molecules are of the component. Similarly, the phrase “the molar percentage of the component is no more than x mol% of the particle” it is meant that no more than x% of the particle molecules are of the component. The unit “mol%” is also sometimes referred as “mol:mol” or “% mol:mol”.

[0112] 67

[0113] According to some embodiments, a lipid portion of the lipid nanoparticle formulation and / or LNP consists of the at least one cationic lipid; the at least one neutral phospholipid; the at least one sterol; and the at least one PEGylated lipid. It is to be understood that the phrase “lipid portion of the lipid nanoparticle formulation 5 and / or LNP” relates to the total of all the lipids within the lipid nanoparticle formulation and / or LNP (e.g., the cationic lipid(s), the neutral phospholipid(s), the sterol(s), and the PEGylated lipid(s)), while excluding any non-lipid component (e.g., therapeutic agent(s), nucleic acid(s), carriers, excipients, etc.) According to some embodiments, the lipid nanoparticle formulation and / or LNP 10 comprises: at least one cationic lipid, 20% to 60% mol / mol; at least one neutral phospholipid, 11% to 49% mol / mol; at least one sterol 11% to 49% mol / mol; and at least one PEGylated lipid, 0.5% to 10% mol / mol. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises: at least one cationic lipid, 30% to 50% mol / mol; at least one neutral phospholipid, 20% to 40% mol / mol; at least one sterol 20% to 40% mol / mol; and 15 at least one PEGylated lipid, 1% to 5% mol / mol. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least one cationic lipid, 33% to 43% mol / mol; at least one neutral phospholipid, 25% to 35% mol / mol; at least one sterol 25% to 35% mol / mol; and at least one PEGylated lipid, 1% to 3% mol / mol. According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises at least one cationic 20 lipid, about 38% mol / mol; at least one neutral phospholipid, about 30% mol / mol; at least one sterol, about 30% mol / mol; and at least one PEGylated lipid, about 2% mol / mol. According to some embodiments, the lipid nanoparticle formulation is selected from the group consisting of: Formulation 1, Formulation 2, Formulation 3, Formulation 7, Formulation 9 and Formulation 10: 25 Formulation 1: lipid II-1: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25 mol% to 35 mol%; and DMG-PEG 2000: 1 mol% to 3 mol%;

[0114] 68

[0115] Formulation 2: IA-10: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25 mol% to 35 mol%; and DMG-PEG 2000: 1 mol% to 3 mol%; Formulation 3: ALC-0315: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 5 25% to 35% mol / mol; cholesterol: 25 mol% to 35 mol%; and DMG-PEG 2000: 1 mol% to 3 mol%; Formulation 7: EA-502: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25 mol% to 35 mol%; and DMG-PEG 2000: 1 mol% to 3 mol%; 10 Formulation 9: SM-102: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25 mol% to 35 mol%; and DMG-PEG 2000: 1 mol% to 3 mol%; Formulation 10: II-25: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25 mol% to 35 mol%; and DMG-PEG 2000: 1 mol% to 3 15 mol%. According to some embodiments, the lipid nanoparticle has a lipid ration as in Formulation 1, Formulation 2, Formulation 3, Formulation 7, Formulation 9 or Formulation 10. According to some embodiments, Formulation 1 comprises lipid II-1: about 38% mol / mol; distearoyl phosphatidyl choline (DSPC): about 30% mol / mol; cholesterol: about 30% 20 mol / mol mol%; and DMG-PEG 2000: about 2% mol / mol. According to some embodiments, Formulation 2 comprises lipid IA-10: about 38% mol / mol; distearoyl phosphatidyl choline (DSPC): about 30% mol / mol; cholesterol: about 30% mol / mol mol%; and DMG-PEG 2000: about 2% mol / mol. According to some embodiments, Formulation 3 comprises ALC-0315: about 38% 25 mol / mol; distearoyl phosphatidyl choline (DSPC): about 30% mol / mol; cholesterol: about 30% mol / mol mol%; and DMG-PEG 2000: about 2% mol / mol.

[0116] 69

[0117] According to some embodiments, Formulation 7 comprises EA-502: about 38% mol / mol; distearoyl phosphatidyl choline (DSPC): about 30% mol / mol; cholesterol: about 30% mol / mol mol%; and DMG-PEG 2000: about 2% mol / mol. According to some embodiments, Formulation 9 comprises SM-102: about 38% mol / mol; 5 distearoyl phosphatidyl choline (DSPC): about 30% mol / mol; cholesterol: about 30% mol / mol mol%; and DMG-PEG 2000: about 2% mol / mol. According to some embodiments, Formulation 10 comprises II-25: about 38% mol / mol; distearoyl phosphatidyl choline (DSPC): about 30% mol / mol; cholesterol: about 30% mol / mol mol%; and DMG-PEG 2000: about 2% mol / mol. 10 According to some embodiments, the lipid nanoparticle formulation and / or LNP comprises a targeting moiety. According to some embodiments, the lipid nanoparticle formulation is conjugated to a targeting moiety. According to some embodiments, the lipid nanoparticle formulation comprises at least one nanoparticle, which is conjugated to a targeting moiety. According to some embodiments, the targeting moiety is selected from the group consisting 15 of: colon targeting moiety, intestines targeting moiety, kidneys targeting moiety, lung targeting moiety and spleen targeting moiety. Each possibility represents a separate embodiment of the invention. According to some embodiments, the targeting moiety is a gut targeting moiety. According to some embodiments, the targeting moiety is a colon targeting moiety. 20 According to some embodiments, the lipid nanoparticle formulation and / or LNP is devoid of targeting moieties. According to some embodiments, the lipid nanoparticle formulation and / or LNP is devoid of targeting moieties selected from the group consisting of: colon targeting moiety, intestines targeting moiety, kidneys targeting moiety, lung targeting moiety and spleen targeting moiety. Each possibility represents a separate embodiment of 25 the invention. According to some embodiments, the lipid nanoparticle formulation and / or LNP is devoid of gut targeting moieties. According to some embodiments, the lipid nanoparticle formulation and / or LNP is devoid of colon targeting moieties.

[0118] 70

[0119] Specifically, it was found that the present lipid nanoparticles are highly effective in targeting inflamed tissues, e.g., inflammatory cells in the colon of a DSS-induced colitis mouse model. Accordingly, the incorporation of such moieties, which are typically employed for targeting purposes, may be avoided. This enables a simplified formulation 5 process at significantly reduced costs. According to some embodiments, the lipid nanoparticle formulation further comprises a nucleic acid encapsulated within at least one particle thereof. According to some embodiments, the lipid nanoparticle further comprises a nucleic acid encapsulated therein. According to some embodiments, the nucleic acid is selected from the group consisting of 10 small interfering RNA (siRNA), micro-RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. According to some embodiments, the particle further comprises a nucleic acid. According to some embodiments, the nucleic acid is encapsulated within the LNP. According to some 15 embodiments, the nucleic acid is encapsulated within a particle of the lipid nanoparticle formulation. According to some embodiments, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. In some embodiments, the 20 composition may further comprise a nucleic acid. Examples of nucleic acids include small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. Each possibility represents a separate embodiment of the present invention. 25 According to some embodiments, the nucleic acid comprises an anti-inflammatory nucleic acid. According to some embodiments, the anti-inflammatory nucleic acid is selected from the group consisting of anti-inflammatory small interfering RNA (siRNA), anti-inflammatory micro RNA (miRNA), anti-inflammatory antisense oligo nucleotides, anti-inflammatory

[0120] 71

[0121] messenger RNA (mRNA), anti-inflammatory ribozymes, anti-inflammatory pDNA, anti- inflammatory CRISPR anti-inflammatory mRNA, anti-inflammatory gRNA, anti- inflammatory circular RNA and anti-inflammatory immune stimulating nucleic acids. According to some embodiments, the anti-inflammatory nucleic acid is an anti- 5 inflammatory mRNA. According to some embodiments, the mRNA encodes an anti- inflammatory polypeptide. According to some embodiments, the anti-inflammatory polypeptide comprises an anti-inflammatory peptide or an anti-inflammatory protein. Each possibility represents a separate embodiment of the invention. According to some embodiments, the anti-inflammatory polypeptide comprises an anti-inflammatory protein. 10 According to some embodiments, the mol ratio between the nucleic acid and the lipid mixture may be adjusted so as to achieve maximal biological effect by the nucleic acid on the target site. According to some embodiments, weight ratio between the nucleic acid and the lipid phase is in the range of 1:1 to 1:20., including each value and sub-range within the specified range. According to some embodiments, weight ratio between the nucleic acid 15 and the lipid phase is in the range of 1:2 to 1:10. According to some embodiments, weight ratio between the nucleic acid and the lipid phase is in the range of 1:3 to 1:9. According to some embodiments, weight ratio between the nucleic acid and the lipid phase is in the range of 1:4 to 1:8. According to some embodiments, weight ratio between the nucleic acid and the lipid phase is in the range of 1:5 to 1:6. According to some embodiments, weight 20 ratio between the nucleic acid and the lipid phase is about 1:6. According to some embodiments, the particle further comprises a therapeutic agent. According to some embodiments, the therapeutic agent is encapsulated within a particle comprising the lipid. According to some embodiments, the lipid nanoparticle formulation comprises a therapeutic agent encapsulated within at least one particle thereof. According 25 to some embodiments, the lipid nanoparticle comprises a therapeutic agent encapsulated therein. As detailed herein the present lipid nanoparticle formulation may include a plurality of nanoparticles. As detailed herein the present lipid nanoparticle formulation may include a

[0122] 72

[0123] plurality of the nanoparticles as disclosed herein. The following section related to the diameter of the nanoparticles. According to some embodiments, the lipid nanoparticle formulation has average nanoparticle size (Zaverage) in the range of 10 to 500 nanometers. According to some 5 embodiments, the lipid nanoparticle formulation has average nanoparticle size in the range of 25 to 400 nanometers. According to some embodiments, the lipid nanoparticle formulation has nanoparticle size (Zaverage) in the range of 50 to 200 nanometers. According to some embodiments, the lipid nanoparticle formulation has nanoparticle size (Z average) in the range of 60 to 150 nanometers. According to some embodiments, the lipid nanoparticle 10 formulation has nanoparticle size (Zaverage) in the range of 110 to 120 nanometers. In some embodiments, the particles (including any nucleic acid, therapeutic agent and the like encapsulated within and any targeting moiety conjugated thereto) have a particle size (diameter) in the range of about 10 to about 500 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 10 to about 350 nm. In some 15 embodiments, the particles have a particle size (diameter) in the range of about 40 to about 270 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 75 to about 200 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 90 to about 160 nm. In some embodiments, the particles have a particle size (diameter) in the range of over about 10 nm. In some embodiments, the 20 particles have a particle size (diameter) of over about 20 nm. In some embodiments, the particles have a particle size (diameter) of over about 30 nm. In some embodiments, the particles have a particle size (diameter) of over about 40 nm. In some embodiments, the particles have a particle size (diameter) of over about 50 nm. In some embodiments, the particles have a particle size (diameter) of over about 60 nm. In some embodiments, the 25 particles have a particle size (diameter) of over about 70 nm. In some embodiments, the particles have a particle size (diameter) of over about 80 nm. In some embodiments, the particles have a particle size (diameter) of over about 90 nm. In some embodiments, the particles have a particle size (diameter) of over about 100 nm. In some embodiments, the particles have a particle size (diameter) of over about 110 nm. In some embodiments, the

[0124] 73

[0125] particles have a particle size (diameter) of not more than about 500 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 400 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 300 nm. In some embodiments, the particles have a particle size (diameter) of not more 5 than about 200 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 150 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 125 nm. In some embodiments, the size is a hydrodynamic diameter. According to some embodiments, the lipid nanoparticle formulation and / or LNP has Zeta 10 potential in the range of -5 to 20 mV. According to some embodiments, the lipid nanoparticle formulation and / or LNP has Zeta potential in the range of-4 to 15 mV. According to some embodiments, the lipid nanoparticle formulation and / or LNP has Zeta potential in the range of -3 to 6 mV. The term “zeta potential” refers to a physical measurement of a colloidal system by 15 electrophoresis. It gives the value of the potential (in mV) of a colloid in a suspension at the boundary between the Stern layer and the diffuse layer. In other words, the zeta potential in a colloidal system is the difference in potential between the immovable layer attached to the surface of the dispersed phase and the dispersion medium. The zeta potential is related to stability of suspensions of particles. Zeta potential may be adjusted, in part, for 20 example, by adjusting the concentration of an electrolyte in the buffer system. According to some embodiments, the lipid nanoparticle formulation has polydispersity index (PDI) of no more than 0.75. According to some embodiments, the lipid nanoparticle formulation has PDI of no more than 0.5. According to some embodiments, the lipid nanoparticle formulation has PDI of no more than 0.25. According to some embodiments, 25 the lipid nanoparticle formulation has PDI of no more than 0.2. According to some aspects and embodiments, the pharmaceutical formulations of the invention are provided in a form suitable for parenteral administration, for example by injection, implantation or infusion. Each possibility represents a separate embodiment. The term “parenteral” as used herein refers to routes of administration selected from

[0126] 74

[0127] subcutaneous (SC), intravenous (IV), intramuscular (IM), intradermal (ID), intraperitoneal (IP), topical, intrathecal (IT) and the like. Each possibility represents a separate embodiment. Intradermal delivery can be performed through nanoneedles or microneedles for example using a patch as described in Larraneta et al. (Mater. Sci. Eng. R 104: 1-32, 5 2016). According to some embodiments, there is provided a composition suitable for administration orally intramuscularly (IM), intravenously (IV), intraperitoneally (IP), topical, subcutaneously (SC), intradermally (ID) and / or Intrathecally. According to some embodiments, the administration is IV or SC. 10 According to some embodiments, the composition is a liquid composition. According to some embodiments, the lipid nanoparticle formulation and / or LNP is selectively targeting an organ selected from the group consisting of: gut, lymphoid tissues, lungs, heart, brain, spleen, kidneys. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulation and / or 15 LNP is selectively targeting an organ selected from the group consisting of: colon, intestines, kidneys, lung and spleen. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulation and / or LNP is selectively targeting the gut. According to some embodiments, the lipid nanoparticle 20 formulation and / or LNP is selectively targeting the colon. According to some embodiments, the lipid nanoparticle formulation and / or LNP is selectively targeting an inflamed cell. According to some embodiments, the lipid nanoparticle formulation and / or LNP is selectively targeting an inflamed tissue. According to some embodiments, the present lipid nanoparticle formulation and / or LNP 25 exhibits higher targeting to inflamed tissues compared to healthy counterparts. Each possibility represents a separate embodiment of the invention. According to some embodiments, the present lipid nanoparticle formulation and / or LNP exhibits higher targeting to the colon compared to the liver.

[0128] 75

[0129] According to some embodiments, the present lipid nanoparticle formulation comprises a nucleic acid and / or protein encapsulated within at least one particle thereof, wherein administration in mammals of the formulation results in higher delivery to the colon of the mammal compared to the mammal’s liver. According to some embodiments, the present 5 lipid nanoparticle formulation comprises a nucleic acid and / or protein encapsulated within at least one particle thereof, wherein administration in mammals, who suffers from inflammation, of the formulation results in higher expression in inflamed tissues of the mammal compared to the mammal’s healthy tissues. According to some embodiments, the present lipid nanoparticle comprises a nucleic acid and / or protein encapsulated therein, 10 wherein administration in mammals of the formulation results in higher delivery to the colon of the mammal compared to the mammal’s liver. According to some embodiments, the present lipid nanoparticle comprises a nucleic acid and / or protein encapsulated therein, wherein administration in mammals, who suffers from inflammation, of the formulation results in higher expression in inflamed tissues of the mammal compared to the mammal’s 15 healthy tissues. According to some embodiments, the mammal is human. According to some embodiments, the lipid nanoparticle formulation and / or LNP is for use in treating an inflammation. According to some embodiments, the lipid nanoparticle formulation and / or LNP is for use in treating an inflammatory disease or disorder. Inflammation is part of the complex biological response of body tissues to harmful stimuli, 20 such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and initiate tissue repair. The classical signs of inflammation are heat, pain, redness, swelling, and loss of function. 25 Inflammation is a generic response, and therefore it is considered as a mechanism of innate immunity. Inflammatory diseases include a vast array of disorders and conditions that are characterized by chronic inflammation. Examples include allergy, asthma, transplant rejection, autoimmune diseases, and ocular inflammation, to name just a few.

[0130] 76

[0131] According to some embodiments, the inflammatory disease or disorder is selected from the group consisting of: an eye inflammatory disease or disorder, an ear inflammatory disease or disorder, a lung inflammatory disease or disorder, a bowel inflammatory disease or disorder, or an inflammatory autoimmune disease or disorder. Each possibility represents 5 a separate embodiment of the invention. According to some embodiments, the disease or disorder is an inflammatory bowel disease (IBD). According to some embodiments, the IBD is colitis. According to some embodiments, the inflammatory bowel disease is selected from the group consisting of Crohn's disease, ulcerative colitis and celiac disease. 10 The term inflammatory bowel disease: is defined herein. According to some embodiments, the inflammatory disease or disorder in an autoimmune disease or disorder. According to still further embodiments the autoimmune disease or disorder is selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, lupus, and Sjogren 15 syndrome. According to some embodiments, the autoimmune disease or disorder is rheumatoid arthritis. The phrase “rheumatoid arthritis” as used herein refers to a chronic, autoimmune disorder in which multiple joints are inflamed. Inflammation of the synovial joint lining is accompanied by joint pain and stiffness and usually leads to bone and joint destruction, 20 deformity, disability, and even death. According to some embodiments, the lipid nanoparticle formulation and / or LNP is for treatment of inflammatory arthritis. According to some embodiments, the lipid nanoparticle formulation and / or LNP is for treatment of rheumatoid arthritis. According to some embodiments, the autoimmune disease or disorder is lupus. The term 25 "lupus" as used herein refers to a chronic, inflammatory autoimmune disorder called lupus erythematosus that may affect many organ systems including the skin, joints and internal organs. Lupus is a general term which includes a number of specific types of lupus, including systemic lupus, lupus nephritis, and lupus cerebritis. In systemic lupus (SLE),

[0132] 77

[0133] the body's natural defenses are turned against the body and rogue immune cells attack the body's tissues. Antibodies may be produced that can react against the body's blood cells, organs, and tissues. This reaction leads to immune cells attacking the affected systems, producing a chronic disease. Lupus nephritis, also referred to as lupus glomerular disease, 5 is kidney disorder that is usually a complication of SLE, and is characterized by damage to the glomerulus and progressive loss of kidney function. Lupus cerebritis refers to another complication of SLE, which is inflammation of the brain and / or central nervous system. According to some embodiments, the inflammatory disease or disorder is osteoarthritis (OA). OA is also referred to as hypertrophic osteoarthritis, osteoarthrosis, and degenerative 10 joint disease. OA is a chronic degenerative disease of skeletal joints, which affects specific joints, commonly knees, hips, hand joints and spine, in adults of all ages. OA is characterized by a number of the following manifestations including degeneration and thinning of the articular cartilage with associated development of "ulcers" or craters, osteophyte formation, hypertrophy of bone at the margins, and changes in the synovial 15 membrane and enlargement of affected joints. Furthermore, osteoarthritis is accompanied by pain and stiffness, particularly after prolonged activity. Characteristic radiographic features of osteoarthritis include joint space narrowing, subchondral sclerosis, osteophytosis, subchondral cyst formation, loose osseous body (or "joint mouse"). According to some embodiments, the inflammatory disease or disorder is an inflammatory 20 disease or disorder of the lung. According to some embodiments, the inflammatory disease or disorder of the lung is selected from the group consisting of asthma, bronchitis, pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis and cystic fibrosis. 25 According to some embodiments, the inflammatory disease or disorder is an inflammatory disease or disorder of the ear. According to some embodiments, the inflammatory disease or disorder of the ear is selected from the group consisting of inflammatory symptoms associated with ear infection, otitis media, otitis externa, mastoiditis and otomastoiditis.

[0134] 78

[0135] According to some embodiments, the compositions of the present invention may be used as a delivery system to administer a therapeutic agent to its target location in the body. According to some embodiments, the location in the body is selected from the group consisting of: colon, intestines, kidneys, lung and spleen. Each possibility represents a 5 separate embodiment of the invention. According to some embodiments, the compositions of the present invention may be used as a delivery system to administer a therapeutic agent specifically to an inflamed tissue in any location in the body. According to some embodiments, the compositions of the present invention may be used as a delivery system to administer a therapeutic agent specifically 10 to an inflamed tissue in a location in the body selected from the group consisting of: colon, intestines, kidneys, lung and spleen. Each possibility represents a separate embodiment of the invention Thus, according to some embodiments, the present invention relates to a method for administering a therapeutic agent, by preparing a lipid nanoparticle formulation and / or 15 LNP as described herein and a biologically active agent (e.g., a therapeutic agent, a protein, a nucleic acid and the like), and administering the composition to a subject in need thereof. According to some embodiments, the present invention relates to a method for administering a therapeutic agent, by preparing a particle as described herein comprising an active agent, and administering the composition to a subject in need thereof. According 20 to some embodiments, the method further comprises encapsulating the active agent within a particle comprising the lipid. According to some embodiments, there is provided a method of delivery of a therapeutic agent or a nucleic acid to an organ selected from the group consisting of: colon, intestines, kidneys, lung and spleen. According to some embodiments, the method comprises the step 25 of administering to a subject in need thereof the lipid nanoparticle formulation and / or LNP disclosed herein, and a pharmaceutically acceptable carrier, diluent or excipient. Each possibility represents a separate embodiment of the invention.

[0136] 79

[0137] According to some embodiments, the method is for delivery of a therapeutic agent or a nucleic acid to the gut. According to some embodiments, the method is for delivery of a therapeutic agent or a nucleic acid to the colon. According to some embodiments, there is provided a method of delivery of a therapeutic 5 agent or a nucleic acid to an inflamed tissue. According to some embodiments, the method comprises the step contacting the lipid nanoparticle formulation and / or LNP disclosed herein with an inflamed tissue. According to some embodiments, there is provided a method of delivery of a therapeutic agent or a nucleic acid to an inflamed cell. According to some embodiments, the method comprises the step contacting the lipid nanoparticle 10 formulation and / or LNP disclosed herein with an inflamed cell. According to some embodiments, the method comprises the step of orally, intramuscularly (IM), intravenously (IV), intraperitoneally (IP), subcutaneously (SC), topically, intradermally (ID) or Intrathecally administering to a subject in need thereof the lipid nanoparticle formulation and / or LNP and the pharmaceutically acceptable carrier, diluent 15 or excipient. Each possibility represents a separate embodiment of the invention. According to some embodiments, the administration is IV or SC. According to some embodiments, the contacting is performed in vitro. According to some embodiments, these lipid nanoparticle formulations and / or LNPs are useful for expression of protein encoded by mRNA. 20 According to some embodiments, these improved lipid nanoparticles formulations and LNPs are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA to an inflamed tissue. According to some embodiments, these improved lipid nanoparticle formulations and 25 LNPs are useful for down-regulating (e.g., silencing) the protein levels and / or mRNA levels of target genes within inflamed tissues. According to some embodiments, the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes within inflamed tissues.

[0138] 80

[0139] According to some 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 antibody 5 within inflamed cells and / or tissues. According to some embodiments, the active agent is selected from the group consisting of: a protein, a therapeutic agent and a nucleic acid. Each possibility represents a separate embodiment of the invention. According to some embodiments, the active agent is a therapeutic agent. According to some embodiments, the active agent is a nucleic acid. 10 Exemplary nucleic acids are discussed above. According to some embodiments, there is provided a method of treating an inflammatory disease or disorder. According to some embodiments, the method comprises the step of administering to a subject in need thereof the lipid nanoparticle formulation and / or LNP disclosed herein, and a pharmaceutically acceptable carrier, diluent or excipient. 15 According to some embodiments, the method is for treating an inflammatory bowel disease (IBD). According to some embodiments, the method is for treating colitis. According to some embodiments, the method is for treating rheumatoid arthritis. According to some embodiments, the method comprises the step of orally, intramuscularly (IM), intravenously (IV), intraperitoneally (IP), subcutaneously (SC), topically, intradermally (ID) or 20 Intrathecally administering to a subject in need thereof the lipid nanoparticle formulation and / or LNP and the pharmaceutically acceptable carrier, diluent or excipient. Each possibility represents a separate embodiment of the invention. According to some embodiments, the administration is IV or SC. Definitions 25 To facilitate an understanding of the present invention, a number of terms and phrases are defined below. It is to be understood that these terms and phrases are for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0140] 81

[0141] The term “about” means ±20%, ±15%, ±10% or ±5% or a specified value. Each possibility represents a separate embodiment of the invention. The phrase "inflammatory disease or disorder" used herein refers to a disease, condition or disorder associated with inflammation. The term "inflammation" as used herein refers the 5 process by which a subject's immune system coordinates a response to tissue damage, infection, antigenic challenge, etc. Inflammation may be associated with an increased blood supply to the tissue, increased capillary permeability in the tissue and / or increased leukocyte migration to the tissue. The term “inflammatory bowel disease” or “IBD” as used herein refers to diseases or 10 disorders of the gastrointestinal tract. Non-limiting examples of IBD include, Crohn's disease (CD), ulcerative colitis (UC), indeterminate colitis (IC), microscopic colitis, diversion colitis, Behcet's disease, and other inconclusive forms of IBD. According to some embodiments, IBD comprises fibrosis, fibrostenosis, stricturing and / or penetrating disease, obstructive disease, or a disease that is medically refractory (e.g., mrUC, refractory CD), 15 perianal CD, or other complicated forms of IBD. As used herein, the term “nanoparticle” refers to a nanostructure that is generally or substantially spherical or spheroidal. Typically, each dimension of a nanoparticle is in a range of about 1 nm to about 1000 nm, According to some embodiments, “neutral lipid” refers to any of a number of lipid species 20 that exist either in an uncharged or neutral zwitterionic form at physiological pH, such lipids include, but are not limited to, sterols, such as cholesterol, phosphotidylcholines such as l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn-glyccro-3- phosphocholine (DPPC), 1,2-Dimyristoyl-sn-glyccro-3-phosphocholine (DMPC), 1- Palmitoyl-2-olcoyl-sn-glyccro-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-25 phosphocholine (DOPC), phophatidyl ethanolamines such as 1,2-Diolcoyl-sn-glyccro-3- phosphoethanolamine (DOPE), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived. The term “polypeptide“, as used herein, refers to a linear polymer composed of at least two amino acid residues linked by peptide bonds. The term includes peptides (i.e., relatively

[0142] 82

[0143] short chains, typically fewer than about 50 amino acids), oligopeptides, and proteins (i.e., longer chains which may adopt a defined three-dimensional structure and / or exhibit biological activity). Polypeptides may be naturally occurring, synthetic, or recombinant, and may include non-naturally occurring amino acids, modified amino acids, or other 5 chemical modifications, such as glycosylation, pegylation, or incorporation of labels or protecting groups. The term encompasses both full-length proteins and fragments thereof, including functional domains, fusion proteins, and analogs, provided that the essential peptide backbone is retained. The term “anti-inflammatory therapeutic agent”, as used herein, refers to any compound, 10 composition, or biologic species that reduces, suppresses, or modulates inflammatory responses in a subject. The term includes, but is not limited to, small molecules, nucleic acids (e.g., anti-inflammatory nucleic acids), polypeptides (e.g., anti-inflammatory polypeptides), antibodies, cytokine antagonists, or other agents with demonstrated or intended anti-inflammatory activity. The anti-inflammatory effect may be direct or indirect 15 and may target specific inflammatory pathways, mediators, or cells. The term “anti-inflammatory nucleic acid”, as used herein, refers to a nucleic acid molecule that exerts or encodes an effect that reduces, suppresses, or modulates inflammation. The term includes, but is not limited to, DNA, RNA, or chemically modified nucleic acids, whether single-stranded or double-stranded, linear or circular. Non-limiting examples 20 include antisense oligonucleotides, small interfering RNAs (siRNA), microRNAs (miRNA), aptamers, plasmids, and messenger RNAs (mRNA) that encode an anti- inflammatory polypeptide. The anti-inflammatory nucleic acid may act through gene expression modulation or through encoding a therapeutic product. The term “anti-inflammatory polypeptide”, as used herein, refers to any polypeptide that 25 exhibits, induces, or contributes to an anti-inflammatory effect. The term encompasses peptides and proteins that act by suppressing, regulating, or modulating one or more components of the inflammatory response, including cytokine production, immune cell recruitment or activation, or inflammatory signaling pathways. The polypeptide may be of natural, recombinant, or synthetic origin and includes full-length proteins, fragments,

[0144] 83

[0145] variants, analogs, fusion proteins, and engineered sequences with anti-inflammatory function. The term “anti-inflammatory”, as used herein, refers to an activity, property, or effect that reduces, suppresses, mitigates, or modulates inflammation in a subject. The term 5 encompasses therapeutic effects on acute or chronic inflammation, and includes, without limitation, activity directed against inflammatory diseases or conditions such as inflammatory bowel disease (IBD), including colitis and Crohn’s disease, and autoimmune inflammatory disorders such as rheumatoid arthritis. The anti-inflammatory effect may be exerted through modulation of immune cells, inhibition of pro-inflammatory cytokines, 10 induction of anti-inflammatory mediators, or interference with inflammatory signaling pathways. The term “phospholipid” refers any lipid or fatty acid having a covalently attached a phosphate group in the molecular structure. Phospholipids include, but are not limited to phosphatidylcholines (PCs), phosphatidic acids (PAs), phosphatidylethanolamines (PEs), 15 phosphatidylglycerols (PGs), phosphatidylserines (PSs), and phosphatidylinositols (PIs) and any lipid which includes a phosphatidyl moiety. The term “zwitterionic phospholipid” means a phospholipid having a proton acceptor in the molecular structure so that the phosphate group can bear a negative charge and the proton acceptor can be a positive charge due to an intra-molecular acid-base reaction; or 20 that the phospholipid has a positively charged group (e.g., a permanently positively charged group) that counter ionizes the negative phosphate group. The term “phosphatidylethanolamine” refers to a phospholipid comprising a phosphoethanolamine group and two fatty acyl groups. Phosphatidylethanolamines include, but are not limited to, dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyloleoyl-25 phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl- 2-oleoyl-phosphatidyethanolamine (SOPE), 1,2-dielaidoyl-sn-glycero-3-

[0146] 84

[0147] phophoethanolamine (transDOPE), 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), and biotin- Phosphatidylethanolamine. A notable phosphatidylethanolamine is DOPE. 5 phosphoethanolamine The term “phosphatidylcholine” refers to a phospholipid comprising a phosphocholine group and two fatty acyl groups. Phosphatidylcholines include, but are not limited to, dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC) 10 palmitoyloleoylphosphatidylcholine (POPC). A notable phosphatidylethanolamine is DSPC. e The term “phosphatidylglycerol” refers to a phospholipid comprising a phosphoglycerol 15 group and two fatty acyl groups. Phosphatidylglycerols include, but are not limited to, dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG) and dipalmitoyl phosphatidyl glycerol (DPPG). 20 The term “phosphatidylserine” refers to a phospholipid comprising a phosphoserine group and two fatty acyl groups. Phosphatidylserines include, but are not limited to, dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS) and dipalmitoyl phosphatidyl serine (DPPS).

[0148] 85

[0149] The term “sterol” as used herein, includes, but is not limited to any sterol, any stanol, and mixtures thereof; also included, but not limited to, are esters of sterols, esters of stanols, and 5 mixtures thereof. The esters can be carboxylic acid esters such as fatty acid esters. The sterol can be any natural or synthetic sterol. SteroIs / Stanols The term "sterol" includes all sterols without limitation, for example: sitosterol, campesterol, stigmasterol, brassicasterol (including dihydrobrassicasterol), desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, 10 nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollinastasterol, cholesterol and all natural or synthesized forms and derivatives thereof, including isomers. As referred to herein, the terms "nucleic acid", "nucleic acid molecules" “oligonucleotide”, "polynucleotide", and "nucleotide" may interchangeably be used herein. The terms are 15 directed to polymers of deoxy ribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded, double stranded, triple stranded, or hybrids thereof. The term also encompasses RNA / DNA hybrids. The polynucleotides may include sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA. The 20 DNA or RNA molecules may be, for example, but not limited to: complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, Antisense RNA, CRISPR / Cas and the like. Each possibility represents a separate embodiment of the present invention. The terms further include oligonucleotides composed of naturally occurring 25 bases, sugars, and covalent inter nucleoside linkages, as well as oligonucleotides having non-naturally occurring portions, which function similarly to respective naturally occurring portions.

[0150] 86

[0151] The term "construct", as used herein, refers to an artificially assembled or isolated nucleic acid molecule which may include one or more nucleic acid sequences, wherein the nucleic acid sequences may include coding sequences (that is, sequence which encodes an end product), regulatory sequences, non-coding sequences, or any combination thereof. The 5 term construct includes, for example, vector but should not be seen as being limited thereto. "Expression vector" refers to constructs that have the ability to incorporate and express heterologous nucleic acid fragments (such as, for example, DNA), in a foreign cell. In other words, an expression vector comprises nucleic acid sequences / fragments (such as DNA, mRNA, tRNA, rRNA), capable of being transcribed. Many prokaryotic and eukaryotic 10 expression vectors are known and / or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art. In some representative embodiments, the expression vector may encode for a double stranded RNA molecule in the target site. The term "expression", as used herein, refers to the production of a desired end-product 15 molecule in a target cell. The end-product molecule may include, for example an RNA molecule; a peptide or a protein; and the like; or combinations thereof. As used herein, the terms "introducing" and "transfection" may interchangeably be used and refer to the transfer of molecules, such as, for example, nucleic acids, polynucleotide molecules, vectors, and the like into a target cell(s), and more specifically into the interior 20 of a membrane-enclosed space of a target cell(s). The molecules can be "introduced" into the target cell(s) by any means known to those of skill in the art, for example as taught by Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001), the contents of which are incorporated by reference herein. Means of "introducing" molecules into a cell include, for example, but are not limited to: heat 25 shock, calcium phosphate transfection, PEI transfection, electroporation, lipofection, transfection reagent(s), viral-mediated transfer, and the like, or combinations thereof. The transfection of the cell may be performed on any type of cell, of any origin, such as, for example, human cells, animal cells, plant cells, virus cell, and the like. The cells may be

[0152] 87 selected from isolated cells, tissue cultured cells, cell lines, cells present within an organism body, and the like. The term “treating” and "treatment" as used herein refers to abrogating, inhibiting, slowing or reversing the progression of a disease or condition, ameliorating clinical symptoms of a 5 disease or condition or preventing the appearance of clinical symptoms of a disease or condition. The term “preventing” is defined herein as barring a subject from acquiring a disorder or disease or condition. Examples EXAMPLE 1: Synthesis of cationic lipids 10 Example 1A: Synthesis of lipid IA-12:

[0153] 2-(((9Z,12Z)-Octadeca-9,12-dien-1-yl)amino)ethan-1-ol o a so u on o no e c a co o ( . g, . mmol, 1 equiv.) in dry CH2Cl2(80 mL), molecular sieves (4Å MS) were added under argon atmosphere. Then PCC (6.4 g, 30.0 5 mmol, 2 equiv.) was added portion wise over a period of 10 min and stirred for 2 hr at room temperature. After completing the reaction filtered it through a silica gel pad using CH2Cl2to remove PCC. The solvent was evaporated under reduced pressure to obtain the linoleic aldehyde 3.9 g (99%) as a colorless liquid. Linoleic aldehyde (3.20 g, 12.12 mmol, 1 equiv.) and ethanolamine (0.89 ml, 14.54 mmol, 10 1.2 equiv.) were dissolved in dry CH2Cl2 (60 mL) under nitrogen atmosphere and stirred for 1 hr. at room temperature. Then sodium triacetoxyborohydride (5.10 g, 24.24 mmol, 2 equiv.) was added portion wise over a period of 15 min, and stirred it for 16 hr at the same temperature. Later, the reaction mixture was quenched with sat.NaHCO3solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and 15 dried over anhydrous Na2SO4. The solvent was evaporated and the residue was purified by column chromatography using 0-10% MeOH in CHCl3to get 1 (2.0 g, 54%) as a pale yellowish liquid. 1H NMR (400 MHz, CDCl3): δ 5.40-5.30 (4 H, m), 3.64 (2 H, t, J = 5.2 Hz), 2.83-2.73 (4 H, m), 2.62 (2 H, t, J = 7.2 Hz), 2.04 (4 H, q, J = 6.8 Hz), 1.54-1.43 (2 H, m), 1.41-1.21 20 (16 H, m), 0.88 (3 H, t, J = 6.8 HZ). ESI-MS: m / z 310.5 [M+1]+(9Z,12Z)-N-(2-((Tert-butyldiphenylsilyl)oxy)ethyl)octadeca-9,12-dien-1-amine o a s rre so u on o ( . g, . mmo , equv.) and imidazole (628 mg, 9.24 mmol, 25 2.2 equiv.) in dry CH2Cl2 (40 mL), TBDPS-Cl (1.31 mL, 5.04 mmol, 1.2 equiv.) was added drop-wise under argon atmosphere and stirred it for overnight at room temperature. The

[0154] 89 reaction mixture was then poured in brain solution and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-3% MeOH in CHCl3 to afford TBDPS protected compound 2 in quantitative yield as a yellow color liquid. 51H NMR (400 MHz, CDCl3): δ 7.69-7.63 (4 H, m), 7.45-7.33 (6 H, m), 5.44-5.28 (4 H, m), 3.78 (2 H, t, J = 5.6 Hz), 2.77 (2 H, t, J = 6.4 Hz), 2.74 (2 H, t, J = 5.2 Hz), 2.59 (2 H, t, J = 7.2 Hz), 2.05 (4 H, q, J = 6.8 Hz), 1.53-1.42 (2 H, m), 1.41-1.21 (16 H, m), 1.05 (9 H, s), 0.89 (3 H, t, J = 6.8 HZ) ESI-MS: m / z 548.7 [M+1]+10 10-Oxodecanoic acid To a solution of IBX (11.9 g, 45 wt.%, 19.15 mmol, 1.2 equiv.) in DMSO (40 mL), a solution of 10-Hydroxydecanoic acid (3.0 g, 15.96 mmol, 1.0 equiv.) in THF (20 mL) was added and stirred for 6 hr at room temperature. After that, the reaction was quenched with 15 water (20 mL) and the precipitated solid was removed by filtration. The filtrate was diluted with water and extracted with diethyl ether (4 x 100 mL). The organic layer dried over anhydrous Na2SO4 and the solvent was removed on rotary evaporator. The crude product was purified by column chromatography using 0-20% ethyl acetate in hexane to obtain 10- oxodecanioc acid 3 (2.60 g, 87%) as a white solid. 201H NMR (400 MHz, CDCl3): δ 9.75 (1 H, t, J = 2.0 Hz), 2.41 (2 H, dt, J = 7.2, 2.0 Hz), 2.33 (2 H, t, J = 7.6 Hz), 1.61 (4 H, quint, J = 7.2 Hz), 1.39-1.23 (8 H, m). 10-((2-((tert-Butyldiphenylsilyl)oxy)ethyl)((9Z,12Z)-octadeca-9,12-dien-1- yl)amino)decanoic acid

[0155] The compound 2 (700 mg, 1.27 mmol, 1.0 equiv.) and 10-oxodecanioc acid 3 (285 mg, 1.53 mmol, 1.2 equiv.) were dissolved in dry CH2Cl2 (30 mL) under argon atmosphere and stirred for 1 hr at room temperature. Then sodium triacetoxyborohydride (404 mg, 1.91 mmol, 1.5 equiv.) was added and stirred it for 24 hr at the same temperature. Later, the 5 reaction was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated rotary evaporator and the residue was purified by column chromatography using 0-3% MeOH in CHCl3 to provide 5 (778 mg, 85%) as a pale yellowish viscus liquid. 101H NMR (400 MHz, CDCl3): δ 7.67-7.61 (4 H, m), 7.46-7.34 (6 H, m), 5.43-5.27 (4 H, m), 3.86 (2 H, t, J = 5.6 Hz), 2.96 (2 H, t, J = 5.6 Hz), 2.85-2.66 (6 H, m), 2.21 (2 H, t, J = 7.2 Hz), 2.11-1.96 (4 H, m), 1.65-1.44 (6 H, m), 1.42-1.13 (26 H, m), 1.04 (9 H, s), 0.88 (3 H, t, J = 6.8 Hz). ESI-MS: m / z 718.9 [M+1]+; 716.9 [M-1]- 10-Hydroxydecanal 15 , 0- ecanedo ( .0 g, 5.7 mmol, 1 equiv.) was dissolved in dry THF (40 mL) under argon atmosphere and molecular sieves (4Å MS) were added. Then PCC (1.5 g, 6.9 mmol, 1.2 equiv.) was added portion wise to the reaction mixture over a period of 5 min and stirred for 2 hr at room temperature. After that, the reaction mixture was filtered through silica gel 20 pad to remove PCC followed by wash with 30% ethyl acetate in hexane (2 × 100 mL). The solvent was evaporated under reduced pressure and the residue was purified by column chromatography using 5-15% ethyl acetate in hexane to obtain the 10-hydroxydecanal 4 (0.49 g, 49%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 9.77 (1 H, t, J = 2.0 Hz), 3.64 (2 H, t, J = 6.8 Hz), 2.43 (2 25 H, dt, J = 7.6, 2.0 Hz), 1.69-1.52 (4 H, m), 1.41-1.25 (10 H, m). 10-((2-((Tert-butyldiphenylsilyl)oxy)ethyl)((9Z,12Z)-octadeca-9,12-dien-1- yl)amino)decan-1-ol

[0156] 91

[0157] e compoun ( . g, . mmo , equ v.) and 10-hydroxydecanal 4 (678 mg, 3.94 mmol, 1.2 equiv.) were dissolved in dry CH2Cl2 (50 mL) under argon atmosphere and stirred for 1 hr. at room temperature. Then sodium triacetoxyborohydride (1.38 g, 6.56 5 mmol, 2 equiv.) was added and stirred it for 24 hr at the same temperature. After that, the reaction was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 × 30 mL). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated on rotary evaporator and the residue was purified by column chromatography using 0-2% MeOH in CHCl3 to obtain 6 (1.9 g, 82%) as a yellowish viscus 10 liquid. 1H NMR (400 MHz, CDCl3): δ 7.70-7.63 (4 H, m), 7.45-7.33 (6 H, m), 5.43-5.27 (4 H, m), 3.85-3.68 (2 H, br), 3.63 (2 H, dt, J = 6.8, 2.0 Hz), 2.77 (2 H, t, J = 6.4 Hz), 2.72-2.57 (2 H, br), 2.53-2.32 (4 H, br), 2.04 (4 H, q, J= 8.0 Hz) 1.65-1.47 (4 H, m), 1.45-1.12 (30 H, m), 1.05 (9 H, s), 0.89 (3 H, t, J = 7.2 Hz). 15 ESI-MS: m / z 704.9 [M+1]+10-((2-Hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decyl 10-((2- hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decanoate The alcohol 6 (303 mg, 0.43 mmol, 1.0 equiv.), acid 5 (370 mg, 0.51 mmol, 1.2 equiv.), 20 EDC.HCl (165 mg, 0.86 mmol, 2.0 equiv.) and DMAP (11 mg, 0.09 mmol, 0.2 equiv.) were dissolved in dry CH2Cl2 (15 mL) under nitrogen atmosphere and stirred for 24 hr at room temperature. Later, the reaction was quenched with sat. NaHCO3and extracted with CH2Cl2 (3 times). The organic portion was washed with brine solution and dried over

[0158] 92

[0159] anhydrous Na2SO4. The solvent was evaporated and the residue purified with a short silica gel column (2% IPA / CHCl3) to get the desired product (417 mg, 69%). The obtained product (417 mg, 0.3 mmol, 1 equiv.) was dissolved in THF (5 mL), and TBAF (1.2 mL, 1.0 M in THF, 1.19 mmol, 4.0 equiv.) was added. The reaction was stirred for 3 hr at room 5 temperature and quenched with sat. NH4Cl, and extracted with 20% ethyl acetate in diethyl ether. Then the combined organic portion was washed with sat. NH4Cl solution (3 times) to remove TBAF completely. The solvent was evaporated under reduced pressure and the residue purified by column chromatography using 0-15% isopropanol in chloroform to obtain the IA-12 (NV3-006) (220 mg, 80%) as a yellow color viscus liquid. 101H NMR (400 MHz, CDCl3): δ 5.45-5.26 (8 H, m), 4.05 (2 H, t, J = 6.8 Hz), 3.60 (4 H, t, J = 4.8 Hz), 2.77 (4 H, t, J = 6.4 Hz), 2.71-2.62 (4 H, br), 2.59-2.47 (8 H, br), 2.28 (4 H, t, J = 7.6), 2.05 (8 H, q, J = 6.8 Hz), 1.60 (6 H, quint, J = 6.8 Hz), 1.55-1.42 (8 H, m), 1.40- 1.20 (50 H, m), 0.89 (6 H, t, J = 7.2 Hz). ESI-MS: m / z 928.3 [M+1]+; 464.7 [M / 2+1]+15 Figure 1A is a1H NMR spectrum of lipid IA-12 (NV3-006). Figure 1B is an ESI-MS spectrum of lipid IA-12 (NV3-006). Example 1B: Synthesis of lipid IA-11:

[0160] 93

[0161] 2-(Dodecylamino)ethan-1-ol o a so ut on o o ecana ( . m , 10.87 mmol, 1.0 equiv.) in dry CH2Cl2 (60 mL), 5 ethanolamine (0.79 ml, 13.04 mmol, 1.2 equiv.) was added under nitrogen atmosphere and stirred for 1 hr. at room temperature. Then sodium triacetoxyborohydride (4.6 g, 21.74 mmol, 2.0 equiv.) was added portion wise over a period of 15 min and stirred for 24 hr at

[0162] 94

[0163] the same temperature. The reaction was quenched with sat.NaHCO3solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue was purified by column chromatography using 0-10% MeOH in CHCl3to obtain 7 (0.97 g, 40%) and 8 5 (0.45 g, 19%) as white solid and pale yellowish liquid respectively. 1H NMR (400 MHz, CDCl3): δ 3.64 (2 H, t, J = 5.2 Hz), 3.08 (2 H, br), 2.75 (2 H, t, J = 5.2 Hz), 2.60 (2 H, t, J = 7.2 Hz), 1.48 (2 H, quint, J = 7.2 Hz), 1.35-1.17 (18 H, m), 0.86 (3 H, t, J = 7.2 Hz). ESI-MS: m / z 230.4 [M+1]+10 2-(Didodecylamino)ethan-1-ol ( z, ): . ( H, t, J = 5.6 Hz), 2.60 (2 H, t, J = 5.2 Hz), 2.46 (4 H, t, J = 7.6 Hz), 1.44 (4 H, quint, J = 6.8 Hz), 1.34-1.19 (36 H, m), 0.88 (6 H, t, J = 7.2 Hz). 15 ESI-MS: m / z 398.7 [M+1]+N-(2-((Tert-butyldiphenylsilyl)oxy)ethyl)dodecan-1-amine To a solution of 7 (4.0 g, 17.47 mmol, 1.0 equiv.) and imidazole (2.38 g, 34.94 mmol, 2.0 equiv.) in dry CH2Cl2(100 mL), TBDPS-Cl (5.0 mL, 19.22 mmol, 1.1 equiv.) was added 20 drop-wise over a period of 5 min under argon atmosphere and stirred for overnight at room temperature. Then the reaction mixture was poured in brain solution and extracted with CH2Cl2(3 times). The organic portion was dried over anhydrous Na2SO4and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-2% MeOH in CHCl3 to afford TBDPS protected compound 9 (7.73 g, 95%) as a pale 25 yellowish liquid.

[0164] 951H NMR (400 MHz, CDCl3): δ 7.69-7.63 (4 H, m), 7.45-7.34 (6 H, m), 3.78 (2 H, t, J = 5.2 Hz), 2.74 (2 H, t, J = 5.2 Hz), 2.60 (2 H, t, J = 7.2 Hz), 1.49 (2 H, quint, J = 6.8 Hz), 1.36-1.18 (18 H, m), 1.05 (9 H, s), 0.88 (3 H, t, J = 6.8 Hz). ESI-MS: m / z 468.7 [M+1]+5 10-((2-((Tert-butyldiphenylsilyl)oxy)ethyl)(dodecyl)amino)decanoic acid e co pou . g, . o , 1.0 equiv.) and 10-oxodecanioc acid 3 (2.4 g, 12.90 mmol, 1.2 equiv.) were dissolved in dry CH2Cl2(180 mL) under argon atmosphere and stirred for 1 hr. at room temperature. Then sodium triacetoxyborohydride (3.4 g, 16.12 10 mmol, 1.5 equiv.) was added, and the reaction mixture was stirred for 24 hr at the same temperature. Later, the reaction was quenched with sat. NaHCO3solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated rotary evaporator and the residue was purified by column chromatography using 0-5% IPA in CHCl3to provide 10 (6.6 g, 97%) 15 as a pale yellowish viscus liquid. 1H NMR (400 MHz, CDCl3): δ 7.69-7.60 (4 H, m), 7.46-7.33 (6 H, m), 3.86 (2 H, t, J = 5.6 Hz), 2.95 (2 H, t, J = 5.6 Hz), 2.81-2.66 (4 H, m), 2.21 (2 H, t, J = 7.2 Hz), 1.64-1.43 (6 H, m), 1.36-1.13 (28 H, m), 1.04 (9 H, s), 0.87 (3 H, t, J = 6.8 Hz). ESI-MS: m / z 638.9 [M+1]+; 636.8 [M-1]-20 10-((2-((Tert-butyldiphenylsilyl)oxy)ethyl)((9Z,12Z)-octadeca-9,12-dien-1- yl)amino)decyl 10-((2-((tert-butyldiphenylsilyl)oxy)ethyl)(dodecyl)amino)decanoate

[0165] The alcohol 6 (338 mg, 0.48 mmol, 1.0 equiv.), acid 10 (460 mg, 0.72 mmol, 1.5 equiv.), EDC.HCl (183 mg, 0.96 mmol, 2.0 equiv.) and DMAP (12 mg, 0.01 mmol, 0.2 equiv.) were dissolved in dry CH2Cl2 (10 mL) under argon atmosphere and stirred for 24 hr at room temperature. Then the reaction was quenched with sat. NaHCO3and extracted with 5 CH2Cl2 (3 times). The organic portion was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue purified by column chromatography using 0-2% IPA / CHCl3to obtain the desired product 11 (590 mg, 93%) as pale yellowish liquid. 1H NMR (400 MHz, CDCl3): δ 7.71-7.64 (8 H, m), 7.45-7.33 (12 H, m), 5.44-5.23 (4 H, 10 m), 4.05 (2 H, t, J = 6.8 Hz), 3.70 (4 H, t, J = 6.8 Hz), 2.77 (2 H, t, J = 6.4 Hz), 2.61 (4 H, t, J = 6.8 Hz), 2.37 (8 H, t, J = 7.2 Hz), 2.28 (2 H, t, J = 7.6 Hz), 2.05 (4 H, q, J = 7.2 Hz), 1.72-1.52 (6 H, m), 1.44-1.11 (62 H, m), 1.05 (18 H, s), 0.93-0.84 (6 H, m). ESI-MS: m / z 663.3 [M / 2+1]+10-((2-Hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decyl 10-(dodecyl(2- 15 hydroxyethyl)amino)decanoate g, . , . y , TBAF (1.6 mL, 1.57 mmol, 4.0 equiv.) was added. The reaction was stirred for 3 hrs at room temperature and quenched with sat. NH4Cl and extracted with the mixture of ethyl acetate and diethyl 20 ether (20:80). Then the combined organic portion was washed with sat. NH4Cl solution (3 times) to remove TBAF completely. The solvent was evaporated, and the residue purified by column chromatography using 0-15% isopropanol in chloroform to obtain the IA-11 (NV3-004; 244 mg, 74%) as a yellow color viscus liquid. 1H NMR (400 MHz, CDCl3): δ 5.44-5.26 (4 H, m), 4.05 (2 H, t, J = 6.4 Hz), 3.55 (4 H, t, 25 J = 5.2 Hz), 2.77 (2 H, t, J = 6.4 Hz), 2.60 (4 H, t, J = 5.2 Hz), 2.47 (8 H, t, J = 7.2 Hz),

[0166] 97

[0167] 2.28 (2 H, t, J = 7.2 Hz), 2.05 (4 H, q, J = 6.8 Hz), 1.61 (6 H, quint, J = 6.8 Hz),1.50-1.39 (8 H, m), 1.38-1.13 (54 H, m), 0.93-0.82 (6 H, m). ESI-MS: m / z 848.3 [M+1]+; 424.7 [M / 2+1]+Figure 1C is a1H NMR spectrum of lipid IA-11 (NV3-004). Figure 1D is an ESI-MS 5 spectrum of lipid IA-11 (NV3-004). Example 1C: Synthesis of lipid IA-10: 10-((2-((Tert-butyldiphenylsilyl)oxy)ethyl)((9Z,12Z)-octadeca-9,12-dien-1- yl)amino)decyl 10-oxodecanoate 10 e aco o 6 (5 mg, 0.73 mmo, .0 equ v.), 0-oxodecan oc ac d 3 (202 mg, 1.09 mmol, 1.5 equiv.), EDC.HCl (277 mg, 1.45 mmol, 2.0 equiv.) and DMAP (18 mg, 0.14

[0168] 98

[0169] mmol, 0.2 equiv.) were dissolved in dry CH2Cl2(10 mL) under argon atmosphere and stirred for 24 hr at room temperature. The reaction mixture was quenched with sat. NaHCO3 and extracted with CH2Cl2 (3 times). The organic portion was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated on rotary 5 evaporator and the crude product was purified by column chromatography using 0-5% isopropanol in chloroform to obtain the 13 (564 mg, 89%) as a pale yellow color viscus liquid. 10-((2-Hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decyl 10- (dodecyl(hydroxy)amino)decanoate 10 y y y g, . , 1.0 equiv.) in dry CH2Cl2(5 mL), trimethylamine (67 µL, 0.48 mmol, 1.0 equiv.) was added under argon atmosphere and stirred for 5 min at room temperature. Later, a solution of aldehyde 13 (420 mg, 0.48 mmol, 1.0 equiv.) in dry CH2Cl2 (10 mL) was added drop wisely and stirred it for 15 2 hr. After that, the reaction mixture was diluted with another 10 mL of dry CH2Cl2, and sodium triacetoxyborohydride (150 mg, 0.72 mmol, 1.5 equiv.) was added portion wise over a period of 10 min and stirred for another 10 min. Then a solution of dodecanal (160 µl, 0.721 mmol, 1.5 equiv.) in dry CH2Cl2 (10 mL) was added drop wisely to the reaction mixture and stirred for another 10 min. Later the remaining amount of sodium 20 triacetoxyborohydride (150 mg, 0.72 mmol, 1.5 equiv.) was added portion wise over a period of 15 min and left for the overnight stirring at room temperature under argon atmosphere. The reaction was quenched with sat.NaHCO3solution and extracted with CH2Cl2 (3 times). The solvent was evaporated on rotary evaporator and the crude product was dissolved in THF (10 mL) and TBAF (1.0 mL, 1.0 M in THF, 0.96 mmol, 2.0 equiv.) 25 was added. The reaction was stirred for 3 hr at room temperature and quenched with sat. NH4Cl, and extracted with 20% ethyl acetate in diethyl ether (3 times). The combined organic portion was washed with sat. NH4Cl solution (3 times) to remove TBAF

[0170] 99

[0171] completely. The solvent was evaporated under reduced pressure and the residue purified by column chromatography using 0-5% isopropanol in chloroform to bestow the IA-10 (NV3-002; 208 mg, 53%) as a pale yellowish gum. 1H NMR (400 MHz, CDCl3): δ 5.44-5.27 (4 H, m), 4.06 (2 H, t, J = 6.8 Hz), 3.81-3.58 (2 5 H, m), 2.77 (4 H, t, J = 6.4 Hz), 2.64 (8 H, t, J = 7.6 Hz), 2.29 (2 H, t, J = 7.6 Hz), 2.05 (4 H, q, J = 6.8 Hz), 1.69-1.48 (14 H, m), 1.38-1.13 (54 H, m), 0.89 (3 H, t, J = 6.8 Hz), 0.88 (3 H, t, J = 6.8 Hz). ESI-MS: m / z 820.3 [M+1]+; 410.7 [M / 2+1]+1H NMR (400 MHz, CDCl3): δ 5.44-5.27 (4 H, m), 4.06 (2 H, t, J = 6.8 Hz), 3.69-3.59 (2 H, br), 2.77 (2 H, t, J = 6.4 Hz), 2.74-2.67 (2 10 H, br), 2.63 (4 H, t, J = 7.6 Hz), 2.67-2.51 (4 H, br), 2.28 (2 H, t, J = 7.6 Hz), 2.05 (4 H, q, J = 6.8 Hz), 1.66-1.45 (14 H, m), 1.38-1.13 (54 H, m), 0.89 (3 H, t, J = 6.8 Hz), 0.88 (3 H, t, J = 6.8 Hz). ESI-MS: m / z 820.28 [M+1]+; 410.68 [M / 2+1]+Figure 1E is a1H NMR spectrum of lipid IA-10 (NV3-002). Figure 1F is an ESI-MS 15 spectrum of lipid IA-10 (NV3-002). Examples 1D and 1E: Syntheses of lipids II-1 and II-5:

[0172] 100

[0173] To a solution of hexanol (4.0 g, 39.2 mmol, 1 equiv.) in dry CH2Cl2 (100 mL), molecular 5 sieves (4Å MS) were added under argon atmosphere. Then PCC (12.64 g, 58.8 mmol, 1.5 equiv.) was added portion wise over a period of 10 min and stirred for 2 hr at room temperature. After completing the reaction filtered it through a silica gel pad using CH2Cl2

[0174] 101

[0175] to remove PCC. The solvent was evaporated on rotary evaporator with low vacuum to get a solution of hexanal (20 mL). The aldehyde solution was dried over anhydrous sodium sulfate and subjected to the next step. To a suspension of hydroxylamine hydrochloride (871 mg, 11.76 mmol, 0.3 equiv.) in dry 5 CH2Cl2 (20 mL), dry trimethylamine (1.6 mL, 11.76 mmol, 0.3 equiv.) was added under argon atmosphere and stirred for 10 min at room temperature. Then, a solution of hexanal in dry CH2Cl2(30 mL) was added drop wisely and stirred for 2 hr. After that, the reaction mixture was diluted with another 50 mL of dry CH2Cl2 and sodium triacetoxyborohydride (7.4 g, 35.3 mmol, 0.9 equiv.) was added portion wise and left for the overnight stirring at 10 room temperature. Later, the reaction was quenched with sat. NaHCO3solution followed by extract with CH2Cl2(3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue was purified by column chromatography using 0-10% EtOAc in Hexane to obtain N,N- dihexylhydroxylamine 18 (2.2 g, 91%). 151H NMR (400 MHz, CDCl3): δ 2.64 (4 H, t, J = 7.6 Hz), 1.58 (4 H, quint, J = 7.2 Hz), 1.40- 1.18 (12 H, m), 0.87 (6 H, t, J = 6.8 Hz). ESI-MS: m / z 202.4 [M+1]+10-((Dihexylamino)oxy)-10-oxodecan-1-ol 20 The above hydroxylamine 18 (500 mg, 2.49 mmol, 1.0 equiv.), 10-((tert- butyldiphenylsilyl)oxy)decanoic acid 19 (1.27 g, 2.98 mmol, 1.2 equiv.), EDC.HCl (950 mg, 4.97 mmol, 2.0 equiv.) and DMAP (60 mg, 0.50 mmol, 0.2 equiv.) were dissolved in dry CH2Cl2 (20 mL) under argon atmosphere and left for the overnight stirring at room temperature. Then the reaction was quenched with sat. NaHCO3followed by extract with 25 CH2Cl2 (3 times) and washed with brine solution and dried over with anhydrous Na2SO4.

[0176] 102

[0177] The solvent was evaporated on rotary evaporator and the crude product was dissolved in THF (10 mL) and TBAF (5.0 mL, 1.0 M in THF, 4.97 mmol, 2.0 equiv.) was added. The reaction was stirred for 3 hr at room temperature and quenched with sat. NH4Cl, and extracted with ethyl acetate (3 times). The solvent was evaporated and the residue purified 5 by column chromatography using 0-5% EtOAc in hexane to obtain the desired alcohol 20 (721 mg, 78%) as a colorless liquid. 1H NMR (400 MHz, CDCl3): δ 3.63 (2 H, q, J = 4.4 Hz), 2.80 (4 H, t, J = 7.6 Hz), 2.27 (2 H, t, J = 7.6 Hz), 1.71-1.44 (8 H, m), 1.41-1.17 (22 H, m), 0.87 (6 H, t, J = 6.8 Hz). ESI-MS: m / z 394.5 [M+Na]+10 2-(Bis(10-((dihexylamino)oxy)-10-oxodecyl)amino)ethanol o a sout on o (70 mg, 5 wt.%, . 3 mmo , . equiv.) in DMSO (6 mL), a solution of a solution of alcohol 20 (350 mg, 0.94 mmol, 1.0 equiv.) in THF (6 mL) was added and stirred for 5 hr at room temperature. After that, the reaction was quenched with water (10 15 mL) and the precipitated solid was removed by filtration. The filtrate was diluted with water and extracted with diethyl ether (4 x 10 mL). The organic layer dried over anhydrous Na2SO4 and the solvent was removed on rotary evaporator. The crude product was dried and subject to the next step. The crude product was dissolved in dry CH2Cl2 (20 mL) and ethanolamine (27 µL, 0.45 mmol, 0.475 equiv.) was added under argon atmosphere and 20 stirred for 2 hr. at room temperature. Then sodium triacetoxyborohydride (298 mg, 1.41 mmol, 1.5 equiv.) was added portion wise and stirred for 24 hr at the same temperature. The reaction was quenched with sat. NaHCO3solution followed by extract with CH2Cl2(3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue was purified by column

[0178] 103

[0179] chromatography using 0-6% IPA in CHCl3to obtain the lipid II-1 (NV1-001) (195 mg, 57%) as colorless liquid. 1H NMR (400 MHz, CDCl3): δ 3.54 (2 H, t, J = 5.2 Hz), 2.80 (8 H, t, J = 7.6 Hz), 2.60 (2 H, t, J = 5.2 Hz), 2.46 (4 H, t, J = 7.2 Hz), 2.27 (4 H, t, J = 7.7.2 Hz), 1.64 (6 H, quint, J = 5 7.6 Hz), 1.56-1.38 (10 H, m), 1.41-1.17 (44 H, m), 0.87 (12 H, t, J = 6.8 Hz). ESI-MS: m / z 769.01 [M+1]+6-(10-((dihexylamino)oxy)-10-oxodecyl)-18-hexyl-16-oxo-3,17-dioxa-6,18- diazatetracosan-1-ol: 10 The crude product of 20 (460.0 mg, 1.00 wt) was dried and subject to the next step. The crude product (1.00 wt, 1.00 equiv) was dissolved in dry DCM (20 mL, 40 vol) and 2- (aminoethoxy)ethanol (56.2 mg, 0.12 wt, 0.45 equiv.) was added under a nitrogen atmosphere. The reaction was stirred for 2 h at room temperature then sodium triacetoxyborohydride (754.6 mg, 3.0 equiv.) was added portion wise and stirred for at least 15 5 h at the same temperature. The reaction was quenched with saturated NaHCO3solution (40 vol) followed by extract with DCM (3 x 10 vol). The organic layer was washed with brine solution (10 vol) and dried over anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-1% (v / v) MeOH in CHCl3to obtain II-5 (NV1-005) as colorless oil in 3% yield. 201H NMR (400 MHz, CDCl3): δ 3.71-3.63 (3 H, m), 3.61 (2 H, t), 2.80 (7 H, t), 2.74 (2 H, t), 2.57 (5 H, t), 2.27 (6 H, t), 1.69-1.59 (9 H, m), 1.54-1.43 (10 H, m), 1.41-1.22 (48 H, m), 0.87 (12 H, t).

[0180] 104

[0181] ESI-MS: m / z 812.0 [M+H]+Example 1F: Synthesis of lipid IB-10: 4-((10-((dihexylamino)oxy)-10-oxodecyl)(dodecyl)amino)butan-1-ol 5 To a clean flask were charged 10-((dihexylamino)oxy)-10-oxodecan-1-ol (1 wt, 1.0 equiv) and PCC, 98% (2 wt, 2.0 equiv). The reagents were suspended in anhydrous DCM (5 vol) and celite (3 wt) was added to the mixture. The reaction mixture was stirred at 18 to 25 °C for at least 3 h or until complete by TLC analysis in 20% (v / v) EtOAc in hexane. On reaction completion, the crude reaction mixture was filtered over silica gel in hexane. The 10 silica was washed with 20% (v / v) EtOAc in hexane (2 x 25 vol). The filtrates were combined, and solvents were removed using rotavapor to yield orange colored oil. The crude intermediate aldehyde (0.34g, 0.9mol) was dissolved in dry CH2Cl2(20 mL) and 4-aminobutanol (0.08g, 0.9mol) was added under a nitrogen atmosphere. The reaction was stirred for 2 h at room temperature then sodium triacetoxyborohydride (0.3g, 1.35mol) was 15 added portion-wise and stirred for 5 h at the same temperature. On detection of the single chain intermediate charged dodecanal (1.10 equiv). The reaction was quenched with saturated NaHCO3 solution followed by extraction with CH2Cl2 (3 x 10 vol). The organic layer was washed with brine solution (10 vol) and dried over anhydrous Na2SO4. The

[0182] 105

[0183] solvent was evaporated under vacuum at 40 °C and the residue was purified by column chromatography. The chromatography was performed in 0 - 3% v / v MeOH in chloroform to remove the impurities and the lipid desired lipid IB10 (NV2-011) was isolated as a pale-yellow oil 5 (0.05g). 1H NMR (400 MHz, CDCl3): δ 3.6 (2 H, s), 2.77 (3 H, t), 2.56 (5 H, s), 2.45 (3 H, t), 2.11 (2 H, 2), 1.66-1.60 (8 H, m), 1.55-1.37 (8 H, m), 1.36-1.17 (42 H, m), 0.88 (9 H, t). ESI-MS: m / z 612.0 [M+H]+Example 1G: Synthesis of lipid IB-4 (NV2-004): 10 Synthesis of 10-((dihexylamino)oxy)-10-oxodecanal :

[0184] 106

[0185] no)oxy)-10-oxodecan-1-ol (1 wt, 1.0 equiv) and PCC, 98% (2 wt, 2.0 equiv). The reagents were suspended in anhydrous DCM (5 vol) and celite (3 wt) was added to the mixture. The reaction mixture was stirred at 18 to 25 °C 5 for at least 3 h or until complete by TLC analysis in 20% (v / v) EtOAc in hexane. On reaction completion, the crude reaction mixture was filtered over silica gel in hexane. The silica was washed with 20% (v / v) EtOAc in hexane (2 x 25 vol). The filtrates were combined, and solvents were removed using rotavapor to yield orange colored oil. 1H NMR (400 MHz, CDCl3): δ 9.76 (t, J = 1.8 Hz, 1H), 2.84 - 2.76 (m, 4H), 2.42 (td, J = 10 7.3, 1.8 Hz, 2H), 2.27 (t, J = 7.5 Hz, 2H), 1.68 - 1.56 (m, 4H), 1.55 (s, 8H), 1.37 - 1.19 (m, 23H), 0.87 (t, J = 6.7 Hz, 9H). ESI-MS: m / z 370.8 [M+H]+Synthesis of (6Z,9Z)-18-bromooctadeca-6,9-diene To a stirred solution of linoleyl alcohol (3.0g, 11.300 mmol, 1.0 equiv.) in DCM (100 mL) 15 was added triphenyl phosphine (3.56g, 13.600mmol, 1.2 equiv.), stirred for 10 min then carbon tetra bromide (4.510g, 13.600 mmol, 1.2 equiv.) added in one portion. Then the reaction mixture stirred at room temperature for overnight. The progress of the reaction monitored by TLC analysis (5% EtOAc in Hexane). After completion of the reaction, the solvent was removed and purified by column chromatography using 0-5% EtOAc in 20 Hexane to afford 3.7g of (6Z,9Z)-18-bromooctadeca-6,9-diene (2) as a colourless liquid. SM = starting material; co= SM and RM and RM= reaction mixture; Blue spot indicates product (It applies where these terms appear). 1H NMR (400 MHz, CDCl3): δ 5.44 - 5.28 (m, 4H), 3.41 (t, J = 6.9 Hz, 2H), 2.78 (t, J = 6.5 Hz, 2H), 2.05 (q, J = 6.9 Hz, 4H), 1.91 - 1.80 (m, 2H), 1.46 - 1.35 (m, 4H), 1.35 - 1.22 25 (m, 12H), 0.93 - 0.85 (m, 3H).

[0186] 107

[0187] Synthesis of 2-(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl) amino) ethoxy) ethan-1-ol (8) To a stirred solution of (6Z,9Z)-18-bromooctadeca-6,9-diene (2) (4.0 g, 12.144 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added 2-(2-aminoethoxy) ethan-1-ol (12.16mL, 5 121.43 mmol, 10.0 equiv.) at room temperature and then stirred for 24h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in 10 CHCl3 to give the 3.2 g of 2-(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl) amino) ethoxy) ethan-1-ol (8) as pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 5.44 - 5.27 (m, 4H), 3.76 - 3.69 (m, 2H),3.67-3.57 (m,4H), 2.85 - 2.73 (m, 4H), 2.66 - 2.58 (m, 2H), 2.10-2.00 (m,4H), 1.50 (q, J = 7.2 Hz, 2H), 1.42 - 1.28 (m, 18H), 0.94-0.84(m, 3H). ESI-MS: m / z 354.7 [M+H]+. 15 Synthesis of 18-hexyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)-16-oxo-3,17-dioxa-6,18- diazatetracosan-1-ol (NV2-004) To a stirred solution of 2-(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl) amino) ethoxy) ethan-1- ol (8) (0.2g, 0.565 mmol, 1.0 equiv.) in DCM (15 mL) was added 10-((dihexylamino)oxy)- 20 10-oxodecanal (0.25 g, 0.678 mmol, 1.2 equiv.) at room temperature, stirred for 1 h. Then STAB (0.239 g, 1.131 mmol, 2.0 equiv.) added at room temperature, stirred for 3h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion

[0188] 108 of aldehyde, the reaction mixture was quenched with sat. NaHCO3 solution (pH=7-8) and extracted with DCM (3x20 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-12% IPA in CHCl3to give the 270mg g of 18-hexyl-6- 5 ((9Z,12Z)-octadeca-9,12-dien-1-yl)-16-oxo-3,17-dioxa-6,18-diazatetracosan-1-ol (NV2-004; lipid IB-4) as yellow colour liquid. 1H NMR (400 MHz, CDCl3): δ 5.44 - 5.27 (m, 4H), 3.75 - 3.69 (m, 3H), 3.62 (dd, J = 5.3, 3.3 Hz, 2H), 2.84 - 2.73 (m, 8H), 2.27 (t, J = 7.5 Hz, 2H), 2.05 (q, J = 6.9 Hz, 5H), 1.63 (p, J = 7.5 Hz, 5H), 1.51 (s, 6H), 1.42 - 1.34 (m, 3H), 1.34 - 1.22 (m, 36H), 0.88 (t, J = 6.6 Hz, 10 9H). ESI-MS: m / z 708 [M+H]+Figure 3A is a1H NMR spectrum of lipid IB-4 (NV2-004). Figure 3B is an ESI-MS spectrum of lipid IB-4 (NV2-004). Example 1H: Synthesis of lipid IB-13 (NV2-015):

[0189] Synthesis of 6-oxohexanoic acid of 6-hydroxyhexanoic acid (1.5g, 11.349 mmol, 1.0 equiv.) in DMSO (12 mL) was added IBX (4.76g, 17.024mmol, 1.5equiv.) at rt, stirred for overnight. The 5 progress of the reaction monitored by TLC analysis (60% EtOAc in Hexane). After completion of the reaction, the reaction mixture quenched with the addition of H2O (20ml), solids were precipitated out which were collected by filtration. Collect the filtrate and extracted into diethyl ether (2x50mL). Then combined organic layers were dried over anhydrous Na2SO4, filtered and distilled off to give the crude material. The crude was 10 purified by Buchi flash using0-30% EtOAc in hexane to afford 608 mg of 6-oxohexanoic acid as a semi solid. 1H NMR (400 MHz, CDCl3): δ 9.78 (t, J = 1.6 Hz, 1H), 2.55- 2.27 (m, 4H), 1.77-1.60 (m, 4H). ESI-MS: N / A Synthesis of 2-(didodecylamino) ethan-1-ol : 15 . g, 16.276 mmol, 1.0 equiv.) in dry DCM (30 mL) was added ethanol amine (0.447g, 7.324 mmol, 0.45 equiv.) at rt, stirred for 2h. Then reaction mixture was further diluted with dry DCM (30 mL). Then STAB (6.9 g, 32.552 mmol, 2.0 equiv.) was charged to the reaction mixture at rt, stirred for 18 h. The progress 20 of the reaction was monitored by TLC (5% MeOH in CHCl3). The reaction mixture was quenched with sat. NaHCO3 solution and extracted with DCM (2x100 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4and evaporated the solvent. The crude was purified by Buchi flash pure system using 0-5% MeOH in CHCl3 to afford 1.75 g of 2-(didodecylamino) ethan-1-ol (2) as yellow liquid.

[0190] 110

[0191] 1H NMR (400 MHz, CDCl3): δ 3.53 (t, J = 5.4 Hz, 2H), 2.58 (t, J = 5.4 Hz, 2H), 2.49 - 2.41 (m, 4H), 1.43 (q, J = 6.6 Hz, 4H), 1.26 (d, J = 3.6 Hz, 40H), 0.92 - 0.85 (m, 7H). ESI- MS: N / A Synthesis of 2-(didodecylamino) ethyl 6-oxohexanoate : 5 . g, .842 mmol, 1.0 equiv.) and of 2- (didodecylamino) ethan-1-ol (2) (1.22 g, 3.073 mmol, 0.80 equiv.) in dry DCM (25 mL) were added EDC.HCl (1.32 g, 6.915 mmol, 1.8 equiv.) and DMAP (0.093 g, 0.768 mmol, 0.2 equiv. ) at room temperature, stirred for 3-4 h.The progress of the reaction was 10 monitored by TLC (5% MeOH in CHCl3). After completion of acid, the reaction mixture was quenched with water and extracted into 100%EtOAc (3x50 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% IPA in CHCl3 to give 0.495 g of 2-(didodecylamino) ethyl 6-oxohexanoate (4) as yellow liquid. 151H NMR (400 MHz, CDCl3): δ 9.77 (t, J = 1.6 Hz, 1H), 4.12 (td, J = 6.3, 2.5 Hz, 2H), 2.67 (t, J = 6.3 Hz, 2H), 2.45 (ddd, J = 9.5, 7.2, 5.3 Hz, 5H), 2.33 (td, J = 5.8, 2.5 Hz, 2H), 1.66 (h, J = 3.2 Hz, 4H), 1.40 (q, J = 6.8 Hz, 4H), 1.26 (s, 35H), 0.92 - 0.84 (m, 6H). ESI- MS: m / z 542.8 [M+MeOH]+and 510.8 [M+H]+Synthesis of 2-(2-(dodecylamino) ethoxy) ethan-1-ol (6) 20 . g, 16.048 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added 2-(2-aminoethoxy) ethan-1-ol (16 mL, 160.48 mmol, 10.0 equiv.) at room temperature and then stirred for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was 25 removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4and evaporated the

[0192] 111

[0193] solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3to give the 3.1 g of 2-(2-(dodecylamino) ethoxy) ethan-1-ol (6) as an off-white solid. 1H NMR (400 MHz, CDCl3): δ 3.88 (t, J = 4.9 Hz, 2H), 3.80 - 3.73 (m, 2H), 3.65 (dd, J = 5.0, 3.3 Hz, 2H), 3.19 (t, J = 4.9 Hz, 2H), 3.05 - 2.96 (m, 2H), 1.95 - 1.83 (m, 2H), 1.31- 5 1.25 (m, 18H), 0.88 (t, J = 6.8 Hz, 3H). ESI-MS: m / z 274.8 [M+H]+Synthesis of 2-(didodecylamino) ethyl 6-(dodecyl(2-(2-hydroxyethoxy) ethyl) amino) hexanoate (NV2-015; IB-13): To a stirred solution of 2-(2-(dodecylamino) ethoxy) ethan-1-ol (0.2 g, 0.731 mmol, 1.0 10 equiv.) in dry DCM (15 mL) was added 2-(didodecylamino) ethyl 6-oxohexanoate (4) (0.447 g, 0.877 mmol, 1.2 equiv.) at room temperature, stirred for 1 h. Then STAB (0.310 g, 1.462 mmol, 2.0 equiv.) added at room temperature, stirred for 3h at rt. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of aldehyde, the reaction mixture was quenched with saturated Sat.NaHCO3 solution and extracted with 15 DCM (3x20 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% IPA in CHCl3 to give the 0.280 g of 2-(didodecylamino) ethyl 6-(dodecyl(2- (2-hydroxyethoxy) ethyl) amino)hexanoate (NV2-015) as a pale-yellow liquid in pure and 0.20g of NV2-015 as yellow liquid in less pure. 201H NMR (400 MHz, CDCl3): δ 4.11 (t, J = 6.3 Hz, 2H), 3.72 - 3.64 (m, 2H), 3.64 - 3.57 (m, 4H), 2.64 (dt, J = 20.5, 5.9 Hz, 4H), 2.44 (s, 8H), 2.30 (t, J = 7.5 Hz, 3H), 1.64 (p, J = 7.5 Hz, 2H), 1.55 - 1.42 (m, 4H), 1.42 - 1.38 (m, 2H), 1.26 (s, 57H), 0.92 - 0.84 (m, 9H). ESI-MS: m / z 768.2 ; 384.8 [M+H]+

[0194] 112

[0195] Figure 4A is a1H NMR spectrum of lipid IB-13 (NV2-015). Figure 4B is an ESI-MS spectrum of lipid IB-13 (NV2-015). Example 1I: Synthesis of lipid IB-9 (NV2-009): 5 Experimental procedure for NV2-009 Synthesis of 10-oxodecanoic acid (2) o a st rred so ut on o ( .602g, 9.295mmol, 1.75equiv.) in DMSO (13 mL) was added a solution of 10-hydroxydecanoic acid (1.0g, 5.311 mmol, 1.0 equiv.) in THF (10 mL) at 10 rt, stirred for 4-5h. The progress of the reaction monitored by TLC analysis (60% EtOAc in Hexane). After completion of the reaction, the reaction mixture quenched with the addition of H2O (20ml), solids were precipitated out which were collected by filtration.

[0196] 113

[0197] Collect the filtrate and extracted into diethyl ether (2x50mL). Then combined organic layers were dried over anhydrous Na2SO4, filtered and distilled off to give the crude material. The crude was purified by Buchi flash using0-30% EtOAc in hexane to afford 620mg of 10-oxodecanoic acid as a white solid. 51H NMR (400 MHz, CDCl3): δ 9.77 (t, J = 1.8 Hz, 1H), 2.42 (td, J = 7.3, 1.8 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.63 - 1.59(m, 4H), 1.35 - 1.32 (m, 8H). ESI-MS: m / z 186.6 [M+H]+Synthesis of N, N-dioctylhydroxylamine (4): o a st rred so ut on o Octanal (3.03g, 23.632 mmol, 1.0 equiv.) in dry DCM (30 mL) was 10 added NH2OH.HCl (0.821g, 11.8160mmol, 0.5 equiv.) and TEA (1.19g, 11.816 mmol, 0.5 equiv.) at rt, stirred for 2h. Then reaction mixture was further diluted with dry DCM (20 mL). Then STAB (15.03g, 70.899mmol, 3.0 equiv.) was charged to the reaction mixture at rt, stirred for 4h. The progress of the reaction was monitored by TLC (20% EtOAc in hexane). The reaction mixture was quenched with sat. NaHCO3 solution and extracted with 15 DCM (2x100 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4and evaporated the solvent. The crude was triturated with acetonitrile and collected the solids by filtration to afford 2.78 g of N, N- dioctylhydroxylamine (4) as an off-white solid. 1H NMR (400 MHz, CDCl3): δ 2.68 - 2.60 (m, 4H), 1.58 (p, J = 7.8 Hz, 4H), 1.35 - 1.25 20 (m, 20H), 0.92 - 0.84 (m, 6H). ESI-MS: m / z 258.8 [M+H]+Synthesis of 10-((dioctylamino)oxy)-10-oxodecanal (5): o a st rre so ut on o -oxo ecano c ac ( ) ( .22 g, 6.583 mmol, 1.13 equiv.) and N, N-dioctylhydroxylamine (4) (1.5 g, 5.826 mmol, 1.0 equiv.) in dry DCM (40 mL) was

[0198] 114

[0199] added EDC.HCl (2.23 g, 11.65 mmol, 2.0 equiv.) and DMAP (0.142 g, 1.165 mmol, 0.2 equiv. ) at room temperature, stirred for 3-4 h.The progress of the reaction was monitored by TLC (30% EtOAc in hexane). After completion of acid, the reaction mixture was quenched with water and extracted into 100%EtOAc (3x50 mL). The combined organic 5 layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-20% EtOAc in hexane to give 0.66 g of 10-((dioctylamino)oxy)-10-oxodecanal (5) as pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 9.76 (t, J = 1.8 Hz, 1H), 2.84 - 2.76 (m, 4H), 2.42 (td, J = 7.3, 1.8 Hz, 2H), 2.28 (t, J = 7.5 Hz, 2H), 1.63 (h, J = 6.9 Hz, 4H), 1.50 (s, 4H), 1.37 - 1.12 10 (m, 31H), 0.88 (q, J = 8.1 Hz, 6H). ESI-MS: m / z 426.6 [M+H]+Synthesis of 2-(dodecylamino) ethan-1-ol (7) o a st rred sout on o - romo dodecane (4.0 g, 16.049 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added ethanolamine (9.7 mL, 160.494 mmol, 10.0 equiv.) at room 15 temperature and then stirred for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3to give the 3.1 g of 2- 20 (dodecylamino) ethan-1-ol (7) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 3.67 - 3.60 (m, 2H), 2.81 - 2.74 (m, 2H), 2.65 - 2.57 (m, 2H), 1.47 (q, J = 7.0 Hz, 2H), 1.32-1.26 (m, 18H), 0.88 (t, J = 8.0 Hz, 3H). ESI-MS: m / z 230.5 [M+H]+. Synthesis 2-((10-((dioctylamino)oxy)-10-oxodecyl) (dodecyl)amino) ethan-1-ol (NV2- 25 009):

[0200] 115

[0201] To a stirred solution of 2-(dodecylamino) ethan-1-ol (7) (0.2 g, 0.871 mmol, 1.0 equiv.) in dry DCM (10 mL) was added 10-((dioctylamino)oxy)-10-oxodecanal (5) (0.408 g, 0.958 mmol, 1.1 equiv.) at room temperature, stirred for 1 h. Then STAB (0.369 g, 1.743 mmol, 5 2.0 equiv.) added at room temperature, stirred for 3h at rt. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of aldehyde, the reaction mixture was quenched with saturated Sat.NaHCO3 solution and extracted with DCM (3x20 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% 10 IPA in DCM to give the 0.383 g of 2-((10-((dioctylamino)oxy)-10-oxodecyl) (dodecyl)amino) ethan-1-ol (NV2-009; lipid IB-9) as a yellow gummy liquid. 1H NMR (400 MHz, CDCl3): δ 3.52 (t, J = 5.4 Hz, 2H), 2.84 - 2.76 (m, 4H), 2.57 (t, J = 5.4 Hz, 2H), 2.44 (t, J = 7.4 Hz, 4H), 2.27 (t, J = 7.5 Hz, 2H), 1.64 (s, 3H), 1.46 (dp, J = 29.3, 6.8 Hz, 9H), 1.27 (d, J = 6.3 Hz, 47H), 0.88 (td, J = 6.9, 2.9 Hz, 9H). ESI-MS: m / z 15 640.0 [M+H]+Figure 5A is a1H NMR spectrum of lipid IB-9 (NV2-009). Figure 5B is an ESI-MS spectrum of lipid IB-9 (NV2-009). Example 1J: Synthesis of lipid IB-24 (NV2-027):

[0202] 116

[0203] Experimental procedure for NV2-027 Synthesis of 2-(dioctyl amino) ethan-1-ol (2)

[0204] 117

[0205] o a st rred so ut on o Octanal (3.0 g, 23.397 mmol, 1.0 equiv.) in dry DCM (50 mL) was added ethanolamine (0.7 mL, 11.698mmol, 0.5 equiv.) at room temperature and then stirred for 2h. Then STAB (9.920g, 46.794mmol, 2.0 equiv.) was charged at room temperature, 5 stirred for 16h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the reaction was quenched with sat. NaHCO3 solution and extracted into DCM (2x60ml). The combined organic layers washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3to give the 1.797 g of 2-(dioctyl amino) ethan- 10 1-ol (2) as a pale-yellow liquid. 1H NMR (400 MHz, CDCl3) δ 3.76 (t, J = 5.0 Hz, 2H), 2.86 (t, J = 5.0 Hz, 2H), 2.79 - 2.71 (m, 4H), 1.62 (dq, J = 13.1, 6.5 Hz, 4H), 1.29 (dt, J = 10.6, 4.5 Hz, 20H), 0.92 - 0.83 (m, 6H). ESI-MS: m / z 286.8 [M+H]+. Synthesis of 2-(dioctyl amino) ethyl 6-oxoheptanoate (4) 15 To a stirred solution of 2-(dioctyl amino) ethan-1-ol (2) (1.73 g, 6.059 mmol, 1.0 equiv.) and 6-oxoheptanoic acid (1.048g, 7.270 mmol, 1.2 equiv.) in DCM (40 mL) was added DMAP (0.148g, 1.218 mmol, 0.2 equiv.) at room temperature, stirred for 10 min. Then EDC.HCl (2.32 g, 12.118 mmol, 2.0 equiv.) was added at room temperature, stirred for 16 20 h. The progress of the reaction was monitored by TLC (60% EtOAc in hexane). The reaction mixture was quenched with water and extracted with DCM (3x25 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by column using 0-30% EtOAc in

[0206] 118

[0207] hexane to give the 1.98 g of 2-(dioctyl amino) ethyl 6-oxoheptanoate (4) as pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 4.11 (t, J = 6.3 Hz, 2H), 2.67 (t, J = 6.3 Hz, 2H), 2.49 - 2.39 (m, 6H), 2.36 - 2.28 (m, 2H), 2.14 (s, 3H), 1.56 (s, 7H), 1.41 (h, J = 6.7 Hz, 4H), 1.27 5 (d, J = 2.3 Hz, 20H), 0.88 (t, J = 6.7 Hz, 6H). ESI-MS: m / z 412.38 [M+H]+. Synthesis of 2-(dioctyl amino) ethyl 6-((2-hydroxyethyl) amino) heptanoate (5): To a stirred solution of 2-(dioctyl amino) ethyl 6-oxoheptanoate (4) (1.97g, 4.785 mmol, 1.0 equiv.) in dry DCM (30 mL) was added ethanolamine (2.3 mL, 38.283 mmol, 8.0 10 equiv.) at room temperature and then stirred for 2h. Then STAB (5.07 g, 23.926 mmol, 5.0 equiv.) was charged at room temperature, stirred for 16h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the reaction was quenched with sat. NaHCO3 solution and extracted into DCM (2x60ml). The combined organic layers washed with brine solution and dried over anhydrous Na2SO4 and 15 evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3to give the 1.90 g of 2-(dioctyl amino) ethyl 6-((2-hydroxyethyl) amino) heptanoate (5) as a pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 4.12 (t, J = 6.3 Hz, 2H), 3.71 (td, J = 4.5, 2.3 Hz, 2H), 2.95 - 2.73 (m, 3H), 2.68 (t, J = 6.3 Hz, 2H), 2.49 - 2.40 (m, 4H), 2.32 (t, J = 7.4 Hz, 2H), 20 1.64 (p, J = 7.5 Hz, 3H), 1.41 (dq, J = 11.8, 7.5 Hz, 7H), 1.35 - 1.20 (m, 23H), 1.15 (d, J = 6.3 Hz, 3H), 0.92 - 0.84 (m, 7H). ESI-MS: m / z 458.0 [M+H]+Synthesis of 2-(dioctyl amino) ethyl 6-((2-hydroxyethyl) ((9Z,12Z)-octadeca-9,12- dien-1-yl) amino) heptanoate (NV2-027):

[0208] 119

[0209] To a stirred solution of 2-(dioctyl amino) ethyl 6-((2-hydroxyethyl) amino) heptanoate (5) (0.442 g, 0.968 mmol, 0.8 equiv.) in dry DCM (10 mL) was added linoleyl aldehyde (0.32 g, 1.210 mmol, 1.0 equiv.) at room temperature, stirred for 1 h. Then STAB (0.513 g, 2.420 5 mmol, 2.0 equiv.) added at room temperature, stirred for 24h at rt. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of aldehyde, the reaction mixture was quenched with saturated Sat.NaHCO3 solution and extracted with DCM (3x20 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash10 using 0-10% IPA in CHCl3 to give the 0.405 g of 2-(dioctyl amino) ethyl 6-((2- hydroxyethyl) ((9Z,12Z)-octadeca-9,12-dien-1-yl) amino) heptanoate (NV2-027; IB- 24) as a yellow liquid. 1H NMR (400 MHz, CDCl3): δ 5.44 - 5.27 (m, 4H), 4.12 (t, J = 6.3 Hz, 2H), 3.48 (tt, J = 10.6, 4.7 Hz, 2H), 2.81 - 2.62 (m, 5H), 2.53 - 2.42 (m, 4H), 2.42 - 2.26 (m, 5H), 2.05 (q, J 15 = 6.8 Hz, 4H), 1.62 (p, J = 7.5 Hz, 4H), 1.50 - 1.21 (m, 47H), 0.94 (d, J = 6.5 Hz, 3H), 0.89 (td, J = 6.8, 4.1 Hz, 9H). ESI-MS: m / z 706.0; 353.8 [M+H]+Figure 6A is a1H NMR spectrum of lipid IB-24 (NV2-027). Figure 6B is an ESI-MS spectrum of lipid IB-24 (NV2-027). Example 1K: Synthesis of lipid IA-13 (NV3-013):

[0210] 120

[0211] Experimental procedure for NV3-013 Synthesis of 4-(dodecylamino) butan-1-ol (2) 5 To a stirred solution of 1-Bromo dodecane (4.0 g, 16.049 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added 4-amino butanol (9.7 mL, 160.494 mmol, 10.0 equiv.) at room temperature and then stirred for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with 10 brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was triturated with acetonitrile to give the 3.1 g of 4-(dodecylamino) butan-1-ol (2) as an white solid.

[0212] 121

[0213] 1H NMR (400 MHz, CDCl3) δ 3.60 - 3.53 (m, 2H), 2.68 - 2.56 (m, 4H), 1.63 (s, 5H), 1.49 (p, J = 7.3 Hz, 2H), 1.27 (d, J = 9.3 Hz, 18H), 0.88 (t, J = 6.8 Hz, 3H). ESI-MS: m / z 258.28 [M+H]+. Synthesis of 10-bromodecyl 10-bromodecanoate (5) 5 To a stirred solution of 10-bromodecan-1-ol (0.5 g, 2.100 mmol, 1.0 equiv.) and 10- bromodecanoic acid (0.627g, 2.500mmol,1.2 equiv.) in DCM (20 mL) was added DMAP (0.04g, 0.400 mmol, 0.2 equiv.) at room temperature, stirred for 10 min. Then EDC.HCl (0.805 g, 4.200 mmol, 2.0 equiv.) was added at room temperature, stirred for 2-3 h. The 10 progress of the reaction was monitored by TLC (30% EtOAc in hexane). The reaction mixture was quenched with water and extracted with DCM (3x15 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4and evaporated the solvent. The crude was purified by column using 0-5% EtOAc in hexane to give the 0.87 g of 10-bromodecyl 10-bromodecanoate (3) as color-less liquid. 151H NMR (400 MHz, CDCl3): δ 4.06 (t, J = 6.7 Hz, 2H), 3.41 (td, J = 6.8, 1.3 Hz, 4H), 2.29 (t, J = 7.5 Hz, 2H), 1.91 - 1.79 (m, 4H), 1.61 (q, J = 7.1 Hz, 4H), 1.42 (t, J = 7.2 Hz, 4H), 1.30-1.25 (m, 18H). Synthesis of 10-(dodecyl(4-hydroxybutyl) amino) decyl 10-(dodecyl(4-hydroxybutyl) amino) decanoate (NV3-013, IA-13): 20 To a stirred solution of 4-(dodecylamino) butan-1-ol (2) (0.421g, 1.637mmol, 2.2 equiv.) and 10-bromodecyl 10-bromodecanoate (0.350 g, 0.744 mmol, 1.0 equiv.) in DMF (12 mL)

[0214] 122

[0215] was added KI (0.296 g, 1.785 mmol, 2.4 equiv.) and K2CO3(0.514g, 3.720 mmol, 5.0 equiv. ) at room temperature, stirred at 70°C for 36h.The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of amino alcohol, the reaction mixture was quenched with water and extracted into EtOAc (3x15 mL). The combined 5 organic layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-22% IPA in CHCl3 to give the 0.302 g of 10-(dodecyl(4-hydroxybutyl) amino) decyl 10-(dodecyl(4- hydroxybutyl) amino) decanoate (NV3-013) as pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 4.05 (t, J = 6.8 Hz, 2H), 3.55 (t, J = 4.6 Hz, 4H), 2.49 - 10 2.37 (m, 11H), 2.28 (t, J = 7.6 Hz, 2H), 1.71 - 1.55 (m, 12H), 1.53 - 1.41 (m, 8H), 1.27 (d, J = 7.0 Hz, 57H), 0.88 (t, J = 6.8 Hz, 6H). ESI-MS: m / z 823.71; 412.41 [M+H]+Figure 7A is a1H NMR spectrum of lipid IA-13 (NV3-013). Figure 7B is an ESI-MS spectrum of lipid IA-13 (NV3-013). Example 1L: Synthesis of lipid IA-14 (NV3-014): 15 pe e a p oce u e o - Synthesis of ethane-1,2-diyl bis(10-bromodecanoate) (2):

[0216] 123

[0217] o a st rred so ut on o et y ene g yco (0.3 g, .8 mmo , .0 equ v.) and 10-bromodecanoic acid (3.61 g, 14.4 mmol,1.2 equiv.) in DCM (60 mL) was added DMAP (0.238g, 1.900 mmol, 0.4 equiv.) at room temperature, stirred for 10 min. Then EDC.HCl (3.68 g, 19.200 5 mmol, 2.0 equiv.) was added at room temperature, stirred for 16 h. The progress of the reaction was monitored by TLC (20% EtOAc in hexane). The reaction mixture was quenched with water and extracted with DCM (3x50 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by column using 0-10% EtOAc in hexane to give the 2.3 g of 10 ethane-1,2-diyl bis(10-bromodecanoate) (2) as color-less liquid. 1H NMR (400 MHz, CDCl3): δ 4.27 (s, 4H), 3.41 (t, J = 6.8 Hz, 4H), 2.32 (t, J = 7.5 Hz, 4H), 1.85 (dq, J = 8.7, 6.9 Hz, 4H), 1.64 (s, 4H), 1.55 (s, 2H), 1.41 (q, J = 7.0 Hz, 4H), 1.36 - 1.26 (m, 16H). Synthesis of 4-(dodecylamino) butan-1-ol (4): 15 o a st rred sout on o - romo dodecane (3.0 g, 12.036 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added butanol amine (10.71 g, 120.365 mmol, 10.0 equiv.) at room temperature and then stirred for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted 20 with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4and evaporated the solvent. The crude was triturated with acetonitrile to afford 2.36g of 4-(dodecylamino) butan-1-ol (4) as an off- white solid.

[0218] 124

[0219] 1H NMR (400 MHz, CDCl3) δ 3.60 - 3.53 (m, 2H), 2.68 - 2.56 (m, 4H), 1.63 (s, 5H), 1.49 (p, J = 7.3 Hz, 2H), 1.27 (d, J = 9.3 Hz, 18H), 0.88 (t, J = 6.8 Hz, 3H). ESI-MS: m / z 258.28 [M+H]+. Synthesis of ethane-1,2-diyl bis(10-(dodecyl(4-hydroxybutyl) amino) decanoate (NV3- 5 014. Lipid IA-14): To a stirred solution of 4-(dodecylamino) butan-1-ol (4) (0.428 g, 1.665 mmol, 2.2 equiv.) and ethane-1,2-diyl bis(10-bromodecanoate) (2) (0.40 g, 0.757 mmol, 1.0 equiv.) in DMF (12 mL) was added KI (0.289 g, 1.741 mmol, 2.3 equiv.) and K2CO3(0.522 g, 3.785 mmol, 10 5.0 equiv. ) at room temperature, stirred at 70°C for 48 h.The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of amino alcohol, the reaction mixture was quenched with water and extracted into EtOAc (3x20 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in 15 CHCl3 to give the 0.35 g of ethane-1,2-diyl bis(10-(dodecyl(4-hydroxybutyl) amino) decanoate (NV3-014) as yellow gummy liquid. 1H NMR (400 MHz, CDCl3): δ 4.27 (s, 4H), 3.59 - 3.52 (m, 4H), 2.47 (t, J = 7.5 Hz, 10H), 2.32 (t, J = 7.6 Hz, 4H), 1.70 - 1.62 (m, 10H), 1.59 (d, J = 7.3 Hz, 2H), 1.49 (s, 8H), 1.27 (d, J = 10.9 Hz, 60H), 0.88 (t, J = 6.8 Hz, 6H). ESI-MS: m / z 882.4 [M+H]+; 441.9 20 [M+H]+. Figure 8A is a1H NMR spectrum of lipid IA-14 (NV3-014). Figure 8B is an ESI-MS spectrum of lipid IA-14 (NV3-014). Example 1M: Synthesis of lipid IA-15 (NV3-016): 25

[0220] Experimental procedure for NV3-016 Synthesis of (Z)-1-bromooctadec-9-ene (1) 5 To a stirred solution of Oleyl alcohol (5.0g, 18.622 mmol, 1.0 equiv.) in DCM (100 mL) was added triphenyl phosphine (5.85g, 22.347 mmol, 1.2 equiv.), stirred for 10 min then carbon tetra bromide (7.41g, 22.347 mmol, 1.2 equiv.) added in one portion. Then the reaction mixture stirred at room temperature for overnight. The progress of the reaction monitored by TLC analysis (5% EtOAc in Hexane). After completion of the reaction, the 10 solvent was removed and purified by column chromatography using 0-5% EtOAc in Hexane to afford 6.14g of (Z)-1-bromooctadec-9-ene (1) as a pale-brown liquid. 1H NMR (400 MHz, CDCl3): δ 5.35 (td, J = 7.0, 4.0 Hz, 2H), 3.40 (t, J = 6.9 Hz, 2H), 2.01 (q, J = 6.3 Hz, 4H), 1.85 (p, J = 7.0 Hz, 2H), 1.48 - 1.37 (m, 2H), 1.30 (t, J = 9.8 Hz, 20H), 0.88 (t, J = 6.7 Hz, 3H).

[0221] 126

[0222] Synthesis of (Z)-2-(octadec-9-en-1-ylamino) ethan-1-ol (2) To a stirred solution of (Z)-1-bromooctadec-9-ene (4.0 g, 12.070 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added ethanolamine (14.74g, 241.41 mmol, 20.0 equiv.) at 5 room temperature and then stirred for 24h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3to give the 2.08 g of (Z)-2-(octadec- 10 9-en-1-ylamino) ethan-1-ol (2) as pale-brown liquid. 1H NMR (400 MHz, CDCl3): δ 5.41 - 5.28 (m, 2H), 4.12 (q, J = 7.2 Hz, 1H), 3.66 (dd, J = 6.3, 4.0 Hz, 2H), 2.85 - 2.74 (m, 3H), 2.62 (q, J = 5.7 Hz, 2H), 2.07 - 1.92 (m, 4H), 1.50 (q, J = 7.4 Hz, 2H), 1.35 - 1.22 (m, 22H), 0.92 - 0.83 (m, 3H). ESI-MS: m / z 312.9 [M+H] +. 15 Synthesis of 10-bromodecyl 10-bromodecanoate (5) To a stirred solution of 10-bromodecan-1-ol (0.5 g, 2.100 mmol, 1.0 equiv.) and 10- bromodecanoic acid (0.627g, 2.500mmol,1.2 equiv.) in DCM (20 mL) was added DMAP (0.04g, 0.400 mmol, 0.2 equiv.) at room temperature, stirred for 10 min. Then EDC.HCl 20 (0.805 g, 4.200 mmol, 2.0 equiv.) was added at room temperature, stirred for 2-3 h. The progress of the reaction was monitored by TLC (30% EtOAc in hexane). The reaction mixture was quenched with water and extracted with DCM (3x15 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by column using 0-5% EtOAc in hexane to 25 give the 0.87 g of 10-bromodecyl 10-bromodecanoate (5) as color-less liquid.

[0223] 127

[0224] 1H NMR (400 MHz, CDCl3): δ 5.40 - 5.30 (m, 2H), 3.68 - 3.61 (m, 2H), 2.82 - 2.75 (m, 2H), 2.67 - 2.58 (m, 2H), 2.07 - 1.94 (m, 7H), 1.49 (s, 2H), 1.37 - 1.25 (m, 22H), 0.92 - 0.84 (m, 3H). Synthesis of 10-((2-hydroxyethyl) ((Z)-octadec-9-en-1-yl) amino) decyl 10-((2- 5 hydroxyethyl) ((Z)-octadec-9-en-1-yl) amino) decanoate (NV3-016, lipid IA-15): To a stirred solution of (Z)-2-(octadec-9-en-1-ylamino) ethan-1-ol (2) (0.582g, 1.871mmol, 2.2equiv.) and 10-bromodecyl 10-bromodecanoate (0.400 g, 0.850 mmol, 1.0 equiv.) in 10 DMF(12 mL) was added KI (0.324 g, 1.956 mmol, 2.3 equiv.) and K2CO3(0.586g, 4.252 mmol, 5.0 equiv. )at room temperature, stirred at 65-70°C for 32 h.The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of amino alcohol, the reaction mixture was quenched with water and extracted into EtOAc (3x15 mL). The combined organic layers were washed with brine solution and dried over 15 anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3 to give the 0.498 and 0.14g of 10-((2-hydroxyethyl) ((Z)- octadec-9-en-1-yl) amino) decyl 10-((2-hydroxyethyl) ((Z)-octadec-9-en-1-yl) amino) decanoate (NV3-016) as yellow liquid. 1H NMR (400 MHz, CDCl3) δ 5.40 - 5.28 (m, 4H), 4.05 (t, J = 6.8 Hz, 2H), 3.53 (t, J = 20 5.4 Hz, 4H), 2.58 (t, J = 5.4 Hz, 4H), 2.45 (dd, J = 8.5, 6.4 Hz, 8H), 2.29 (t, J = 7.5 Hz, 2H), 2.06 - 1.97 (m, 8H), 1.61 (s, 4H), 1.44 (p, J = 6.8 Hz, 9H), 1.37 - 1.24 (m, 65H), 0.92 - 0.82 (m, 6H). ESI-MS: m / z 932.4 [M+H]+;466.9[M+H]+Figure 9A is a1H NMR spectrum of lipid IA-15 (NV3-016). Figure 9B is an ESI-MS spectrum of lipid IA-15 (NV3-016).

[0225] 128

[0226] EXAMPLE 2: Preparation and characterization of LNPs comprising cationic lipids Cationic lipids were synthesized as detailed in Example 1. Other lipids: ALC-315 was purchased from BroadPharm, DSPC, DOTAP, Cholesterol and DMG-PEG were purchased from Avanti polar lipids. 5 Example 2A: LNPs using inflamed tissue-targeting formulation: Lipid nanoparticles were synthesized by microfluidic mixing device. Briefly, one volume of lipid mix (composition & mol ratios were mentioned in Table 1) in ethanol solution and three volumes of mRNA (total lipid to mRNA ratio at 40:1 w / w) in citrate buffer (pH 4.5) were mixed through a microfluidic mixing device Ignite (Precision Nanosystems Inc) at10 total flow rate of 12 ml / min. The resultant mRNA-LNPs were dialyzed against phosphate- buffered saline (PBS, pH 7.4) for 24hr. The details of Formulations 1-3 are presented in Table 1: Table 1: Formulation details Formulation Cationic lipid DSPC Chol DMG- b PEG 15 Example 2B: LNPs using standard formulation: Lipid nanoparticles were synthesized by microfluidic mixing device. Briefly, one volume of lipid mix (composition & mol ratios were mentioned in Table 1) in ethanol solution and three volumes of mRNA (cationic lipid to mRNA ratio at 6:1 mol / mol) in citrate buffer (pH 4.5) were mixed through a microfluidic mixing device Ignite (Precision Nanosystems 20 Inc) at total flow rate of 12 ml / min. The resultant mRNA-LNPs were dialyzed against phosphate-buffered saline (PBS, pH 7.4) for 24hr. fLuc mRNA was used. The details of Formulations 4-6 are presented in Table 2:

[0227] 129

[0228] Table 2: Formulation details Formulation Cationic lipid DSPC Chol DMG- b PEG EXAMPLE 3: RNA encapsulation and quantification The Quant-iT RiboGreen RNA assay kit (Life Technologies) was used to measure the 5 mRNA encapsulation in LNPs. In brief, 0.5 µL of LNP was diluted in a final volume of 100 µL of TE buffer (20 mм EDTA, 10 mм Tris-HCL) with or without 1% Triton X-100 (Sigma-Aldrich). Samples were loaded in a 96-well black plate (Costar, Corning). The plate was incubated for 5 min at 37 °C before adding 100 µL of RiboGreen in TE buffer (1:200 v / v) to each well. The fluorescence was detected using GloMax plate reader (Promega) 10 according to the manufacturer’s protocol. EXAMPLE 4: Size and ζ-potential analysis of LNPs Nano size and ζ-potential of mRNA-LNPs were analyzed by dynamic light scattering (DLS) using a Malvern nano ZS ζ-sizer (Malvern Instruments). Briefly, mRNA-LNPs were diluted in double-distilled water (1:50, volume ratio) and PBS (1:50, volume ratio) for ζ 15 potential and size measurements, respectively. The physico-chemical properties - particle size, polydispersity index (PDI), Zeta potential and encapsulation efficiency (EE%) - of Formulations 1, 2, 3, 4, 5 and 6 are presented in Table 3. Table 3: Physico-chemical properties of formulations 1-6 Formulation Size (d.nm) PDI Zeta EE (%) 1 1104 0032 276 8959

[0229] 130

[0230] 5 111.3 0.025 -0.05 97.80 6 7503 004 017 9905 EXAMPLE 5: In vivo luciferase assay & organ distribution study Example 5A: fLuc expression in healthy mice: 5 Healthy mice injected i.v with 200µl of LNPs (10ug fLuc-mRNA-LNP per mouse) to the retro orbital sinus. After 6hr mice were injected with 3mg of D-Luciferin and evaluated by IVIS imaging system for fLuc reporter expression. Ex vivo imaging of organs (colon, small intestine, heart, lung, spleen, liver, kidney) was also performed and images quantified. Example 5B: fLuc expression in DSS-colitis mice: 10 Mice received 2% DSS (dextran sodium sulphate) in drinking water. Mice were monitored daily for signs of colitis. 7 days post DSS treatment, mice were injected with LNPs and tested for fLuc reporter expression as described above. LNPs were administered to actual mice weight estimated to be 17-18gr (~9ug fLuc-mRNA-LNP per mouse). Figures 2A-E are bar charts showing expression of fLuc in healthy (empty bars) and in 15 DSS colitis (full bars) mice administered with Formulations 1-3, which target inflamed tissues (right group) and of Formulations 4-6, having standard ratio of components (left group). As shown in Figures 2A-E, Formulations 1-3 showed higher relative expression (to liver) of the fLuc reporter in inflamed organs of DSS-colitis mice, in comparison with 20 Formulations 4-6. These results show the potential for local production of therapeutics delivered systemically to treat inflammatory diseases. EXAMPLE 6: Biodistribution of Cy5-LNPs in the blood and colon of DSS mice over time Rationale: We wanted to get some insights in which cell populations might be responsible 25 to n-LNPs preferential tropism towards inflamed tissues. Since the inflammatory setting is time sensitive, we did not select tdTtomato mice which require at least 48h from treatment before imaging. Furthermore, mCherry of EGFP mRNA-loaded LNPs do not work very

[0231] 131 well in vivo, showing very low signal. Thus, we opted for Cy5 labelled particles to check for LNPs association with cells, with the important caveat that this model does not technically show transfection. Experimental setup: 5 Mouse strain: Wild type female C57BL6 mice, 7-8 weeks old. Induction of colitis: DSS 2% (w / v) in drinking water ad libitum for 7 days. DSS was changed on day 3. Assessment of Colitis: monitoring mice body weights on day 0, 3, 4, 5, 6 and 7 and compared to healthy mice; assessment of mice colon lengths on day 7 compared to healthy 10 animals. LNPs formulations: we employed LNPs formulated using the Helix Custom System in our Lab using the following conditions TFR:12ml / min, FRR (Acq / EtOH) 3:1, N / P ratios of 12, 0.25mL pre waste, 0.9mL sample collected, 0.05mL post-waste. Aqueous phase (same for all formulations): Citrate buffer 25mM (pH=4.5), RNA cargo: 15 Cy5-labelled noncoding siRNA (Cy5-NC5) and modified Luciferase-encoding mRNA (mLuc) 50:50 ratio (w / w). The LNPs formulation had the following lipid compositions (in molar ratios): Benchmark LNPs (b-LNPs): 10% DSPC, 1,5% PEG-DMG (in house); 38.5% cholesterol; 50% Lipid 15 (EA-502) - referred herein as Formulation 8. 20 New LNPs (n-LNPs): 30% DSPC, 2% PEG-DMG (in house); 30% cholesterol; 38% Lipid 15 (EA-502) - referred herein as Formulation 7.

[0232] After formulation, LNPs were dialyzed against 0.5X PBS (1L) for 3 hours and then against PBS 1X (1L) overnight. Timing of treatment: b-LNPs or n-LNPs were injected retro-orbitally at a dose of 10ug of RNA per mouse on day 6 (24 hours before sacrifice) or on day 7 (2 hours before sacrifice). 5 Mice processing: on day 7, mice were anesthetized using isoflurane and terminally bled via cheek incision. Blood was collected in EDTA tubes to prevent coagulation. Then, mice were sacrificed via cervical dislocation. Their colons were harvested and measured before proceeding to tissue dissociation. Blood processing: Mice blood was treated to induce RBC lysis. 10 Colon processing to extract immune cells from the lamina propria: Mice colons were processed. Cells staining: after performing tissue dissociation, immune cells isolated from the blood or from the colonic lamina propria were counted and divided in FACS tubes to have approximately 1 million cells per tube. Cells were then incubated using FcR Blocking 15 Reagent for mouse (Miltenyi biotech) and then stained using the antibody panels summarized in Table 4 (blood) and Table 5 (Colon lamina propria). Each panel was previously optimized and validated by creating an ad hoc compensation matrixes. Finally, cells were stained using DAPI to assess cells viability. Table 4: Summary of the antibodies panel used for Blood leukocytes and colonic immune 20 cells profiling. All antibodies were purchased from Biolegend. Markers Selected fluorofores Laser Filter

[0233] 133

[0234] Table 5: Summary of the antibodies panels used to characterize immune, endothelial and epithelial cells in the colonic lamina propria and lungs. All antibodies were purchased from Biolegend. Markers Selected fluorofores Laser Filter 5 FACS reading: Samples were read using a CytoFlex Flow Cytometer and Data were gated and analyzed using the CytExpert Software. Gates for different cell populations were defined against unstained samples from each experimental group. For LNPs Uptake (Cy5 + cells), gates were defined by comparison with stained but untreated mice. 50,000 live singlet cells were recoded for each sample. 10 Results: As displayed in Figures 10A-L, n-LNPs show higher interactions with all the investigated cell populations in the colonic lamina propria and in the blood compared to b- LNPs, and especially with endothelial cells (CD45- CD31+), neutrophils (CD11b+, Ly6C low, Ly6G+), monocytes (CD11b+, Ly6C+, Ly6G-), and macrophages (CD31- CD45+ CD11b+ F4 / 80+). Interestingly, the n-LNPs + cells in the blood sharply decreased in 15 between 2 hours and 24 hours, suggesting how n-LNPs could be transported by these cells to other tissues. However, these tissues does not seem to significantly include the colon since there is no clear trend over time for LNPs accumulation in the blood. EXAMPLE 7: In vivo study: expression of a therapeutic protein encoded by mRNA- LNPs in a model of acute colitis 20 The experiment was performed in an established model of acute colitis: mice received 2% DSS (dextran sodium sulphate) in drinking water for 8 days, and they were monitored daily

[0235] 134

[0236] for signs colitis. On day 3 and 5 from the start of DSS treatment, mice were intravenously administered with mRNA-LNPs (dose: 1mg / kg) encoding a clinically validated full-length monoclonal antibody. On day 8, mice were culled: colon and livers were snap-frozen for tissue ELISA analysis. The Formulations details are presented in Table 6 and their physico- 5 chemical properties are detailed in Table 7. Table 6: Formulation details Formulation Cationic lipid DSPC Chol DMG-PEG b l% Table 7: Physico-chemical properties of formulations 1, 9-10 Formulation Size (d.nm) PDI RNA integrity (%) EE (%) 1 1014 ± 09 0027 ± 003 81 92 gures an s ow e pay oa express on n co on an ver ssues respectively (each dot represents a mouse); and Figure 11C the colon to liver expression ratio. Results indicate that there is high expression of the protein (monoclonal antibody, whose 15 half-life is higher than 5 days) encoded by the systemically injected mRNA-LNPs at site of inflammation (colon), to the same extent (or higher) as found in the liver. 3 different ionizable lipids were tested, with the detailed formulation. A relevant therapeutic protein can be made at site of inflammation (at an effective concentration, as per published data), when mRNA-LNPs are systemically injected.

[0237] 135

[0238] EXAMPLE 8: In vivo study: expression kinetic of a therapeutic protein encoded by mRNA-LNPs in a model of spontaneous chronic colitis The experiment was performed in healthy mice. Animals were intravenously administered with mRNA-LNPs (dose: 0.75mg / kg) encoding an exogenous cytokine. At 24h, 48h, 72h 5 post-administration mice were culled: organs were snap-frozen, and sera collected for ELISA analysis. Figures 12A and 12B show the payload expression in sera (Figure 12A) and liver (Figure 12B) over time (each dot represents a mouse). The Formulations details are presented in Table 8 and their physico-chemical properties are detailed in Table 9. 10 Table 8: Formulation details Formulation Cationic lipid DSPC Chol DMG-PEG b l% Table 9: Physico-chemical properties of formulations 1, 11 Formulation Size (d.nm) PDI RNA integrity (%) EE (%) 1 942 0042 93 90 15 Results in Figures 12A-B indicate that there is higher systemic expression of the cytokine when this is encoded in mRNA-LNPs formulated with standard formulation (Formulation 11), compared to Formulation 1 (Figure 12A). Likewise, the expression in liver is significantly higher in groups treated with standard formulation 11 (Figure 12B). The same 20 ionizable lipid is used in both formulations. The expression decreases with time, as expected – the cytokine half-life is <5 hours.

[0239] 136

[0240] EXAMPLE 9: small angle X-ray scattering (SAXS) and scanning differerential calorimetry (DSC) measurements LNPs with increasing molar fractions of 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were manufactured with 5 standard protocols using a commercial microfluidic device. The mol fraction of the PC was varied in 5 mol% steps between 5% and 40%, with compositions and manufacturing conditions as indicated in Tables 10 and 11. Table 10: Formulation details (DSPC) Formulation Cationic lipid DSPC Chol DMG-PEG b l% 10 Table 11: Formulation details (DOPC) Formulation Cationic lipid DOPC Chol DMG-PEG b l%

[0241] 137

[0242] The LNPs were prepared at a total volume of 1.2 ml, with total mRNA (fLuc) of 0.155- 0.163 with a 10mM Tris HCl buffer (pH 7.4) at 2℃-8℃. Results from physicochemical characterization are shown in Table 12 for the DSPC LNPs and in Table 13 for the DOPC LNPs. 5 Table 12: Physico-chemical properties of formulations 12-18 (DSPC) Formulation Size PDI RNA integri Total mRNA conc (dnm) ty (%) (u / ml) EE (%) Table 13: Physico-chemical properties of formulations 19-26 (DOPC) Formulation Size (d.nm) PDI Total mRNA conc (ug / ml) EE (%) 19 954 005 209 98 mall angle X-ray scattering (SAXS) and scanning differerential calorimetry (DSC) measurements in order 15 to derive the dependence of physicochemical characteristics on the -PC mol fraction. SAXS measurements allow to obtain structural information at different length scales. The scattering curves for both, DSPC and DOPC show the characteristics as expected for LNPs, having a widely unstructured shape with a broad maximum around 1 nm-1. The maximum derives from Bragg peak where the peak position is indicative for a low number

[0243] 138

[0244] of repeating lipid and LNP multilayers (Cited as: Nogueira et al., (2020) Polysarcosine- Functionalized Lipid Nanoparticles for Therapeutic mRNA Delivery. ACS Appl Nano Mater 3(11): 10634-1064; Wilhelmy et al., (2025) Direct structural investigation of pH responsiveness in mRNA lipid nanoparticles: Refining paradigms. J Control Release 384: 5 113848). From the position of the peak maximum, the repeat distance, d, can be calculated using Bragg’s law (d= 2*p / q, with q the momentum transfer 4*pi / l*sin(q), l the X-ray wavelength, q the scattering angle). For both, DSPC and DOPC, we observe a systematic evolution of the curve characteristics as a function of the mol fraction of -PC: As already obvious by visual inspection (also 10 highlighted by the black lines), there is a shift of the peak position as a function of -PC fraction. In the low molar range the peak shifts to lower q, indicative for increase of the repeat distance. At a certain fraction, around 30 mol% this trend inverts and the peak shifts back to higher q and therefore indicating decrease of repeat distance. This indicates that different types of internal molecular organization, are present above and 15 below this inflection point. Possible reasons can be related to phase separation, phase transition, or similar effects. The critical mol fraction where this change of structure occurs is around 30 mol% in both cases. Figure 16 shows results from DSC measurements, which have been performed from the same formulations. Here only data for DSPC are shown, because DOPC has a phase 20 transition temperature below 0°C, which makes measurements more complex. In the calorimetry traces for the DSPC samples, depending on the mol fraction, a first order phase transition between 40°C and 70 °C is clearly visible. Notably, at 35 and 40% of DSPC, the pattern changes drastically and substantial additional enthalpy changes at higher temperature are determined. Analysis of the phase transition temperature as a function of 25 the DSPC mol fraction, Figure 15, reveals a systematic decrease of the phase transition temperature up to 30-35 °C, where the effect level off. Accordingly (Figure 16), the integrated enthalpy shows a discontinuous behavior in the same range. While the melting enthalpy is roughly the same up to 30 mol%, it increases drastically at higher fractions of DSPC.

[0245] 139

[0246] Taking together results from SAXS and DCS measurements, both indicate, that the 30 mol% of -PC in the LNPs reflect a critical composition with the properties above and below being characterized by a different nature. While in the low and in the high range the properties as a function of the PC- fraction change continuously (linearly), the 30% reflect 5 a critical transition point with very strong changes of different physicochemical properties. The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the figures. While the present invention has been particularly described, persons skilled in the art will 10 appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.

[0247] 140

Claims

CLAIMS What is claimed is:

1. A lipid nanoparticle formulation for use in treating an inflammation, the lipid nanoparticle formulation comprising: 5 at least one cationic lipid; at least one neutral phospholipid; at least one sterol; and at least one PEGylated lipid, wherein a total neutral phospholipid concentration within the lipid nanoparticle 10 formulation is in the range of 20% mol / mol to 40% mol / mol.

2. The lipid nanoparticle formulation for use of claim 1, comprising a plurality of lipid nanoparticles, wherein the LNPs have a phase transition temperature peak in the range of 50℃ to 63℃, as measured by Differential Scanning Calorimetry (DSC).

3. The lipid nanoparticle formulation for use of claim 2, wherein the phase transition 15 temperature peak is in the range of 55℃ to 63℃.

4. The lipid nanoparticle formulation for use of any one of claims 1 to 3, further comprising an anti-inflammatory therapeutic agent encapsulated within at least one particle thereof.

5. The lipid nanoparticle formulation for use of claim 4, wherein the anti-inflammatory 20 therapeutic agent comprises an anti-inflammatory nucleic acid.

6. The lipid nanoparticle formulation for use of claim 5, wherein the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. 25 7. The lipid nanoparticle formulation for use of claim 6, wherein the nucleic acid is an mRNA.1418. The lipid nanoparticle formulation for use of claim 7, wherein the mRNA encodes an anti-inflammatory polypeptide.

9. The lipid nanoparticle formulation for use of any one of claims 1 to 8, wherein the cationic lipid is represented by the structure of Formula (IA), Formula (IB) or Formula 5 (II), or salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof, wherein the structures of Formula (IA), Formula (IB) and Formula (II) are represented below; Formula (IA): , 10wherein R1Ais selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNARNA’, wherein each one of RNAand RNA’is individually C1-4alkyl or RNAand RNA’together with the nitrogen to which they are bound, form a ring; 15 R2Ais selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene- CO2-C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15alkyl, C5-15alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5- 15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; nAis selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; 20 XAis selected from the group consisting of: -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH; jAis selected from the group consisting of: 0, 1, 2, 3 and 4;142YAis selected from the group consisting of: absent, -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH; mAis selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; R3Ais selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6hydroxyalkyl and 5 C1-6 haloalkyl and C1-3 alkylene-NRNA’’RNA’’’, wherein each one of RNA’’and RNA’’’is individually C1-4 alkyl or RNA’’and RNA’’’together with the nitrogen to which they are bound, form a ring; and R4Ais selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene- CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-1510 alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5- 15alkenyl, C5-15alkenylene-O2C-C5-15alkyl, C5-15alkenylene-O2C-C5-15alkenyl; Formula (IB): ,wherein 15 R1Bis selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6hydroxyalkyl and C1-6haloalkyl and C1-3alkylene-NRNBRNB’, wherein each one of RNAand RNA’is individually C1-4 alkyl or RNAand RNA’together with the nitrogen to which they are bound, form a ring; R2Bis selected from the group consisting of: C5-25alkyl, C5-25alkenyl, C5-15alkylene- 20 CO2-C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15alkyl, C5-15alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5- 15alkenyl, C5-15alkenylene-O2C-C5-15alkyl, C5-15alkenylene-O2C-C5-15alkenyl;143WBis a C4-12alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; YBis selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6alkylene; 5 R3Bis selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene- CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5- 15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; and R4Bis selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene- 10 CO2-C5-15alkyl, C5-15alkylene-CO2-C5-15alkenyl, C5-15alkylene-O2C-C5-15alkyl, C5-15alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5- 15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; Formula (II): ,15 wherein R1Cis selected from the group consisting of: C5-25alkyl, C5-25alkenyl, C5-15alkylene- CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5- 15alkenyl, C5-15alkenylene-O2C-C5-15alkyl, C5-15alkenylene-O2C-C5-15alkenyl; 20 R2Cis selected from the group consisting of: C5-25alkyl, C5-25alkenyl, C5-15alkylene- CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15144alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5- 15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; Y1Cis selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6alkylene; 5 W1Cis a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; R5Cis selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNIIRNII’, wherein each one of RNIIand RNII’is individually C1-4 alkyl or RNIIand RNII’together with the nitrogen to which they are bound, 10 form a ring; W2Cis a C4-12alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; Y2Cis selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6alkylene; 15 R3Cis selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene- CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15alkenyl, C5-15alkenylene-CO2-C5-15alkyl, C5-15alkenylene-CO2-C5- 15alkenyl, C5-15alkenylene-O2C-C5-15alkyl, C5-15alkenylene-O2C-C5-15alkenyl; and R4Cis selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. 20 10. The lipid nanoparticle formulation for use of any one of claims 1 to 8, wherein the cationic lipid is selected from the group consisting of: lipid II-1, Lipid IA-10, EA-502, lipid ALC-0315, lipid SM-102, lipid II-25 and a combination thereof;II- 111. The lipid nanoparticle formulation for use of any one of claims 1 to 10, comprising 25% mol / mol to 35% mol / mol total neutral phospholipid concentration. 5 12. The lipid nanoparticle formulation of any one of claims 1 to 11, wherein the neutral phospholipid comprises a permanently charged chemical group.

13. The lipid nanoparticle formulation for use of any one of claims 1 to 12, wherein the neutral phospholipid comprises a phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), 10 phosphatidylinositol (PI), or a combination thereof.

14. The lipid nanoparticle formulation for use of claim 13, wherein the neutral phospholipid comprises a phosphatidylcholine (PC).

15. The lipid nanoparticle formulation for use of any one of claims 1 to 14, wherein the neutral phospholipid comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 15 16. The lipid nanoparticle formulation for use of any one of claims 1 to 15, wherein the cationic lipid is not a phospholipid.

17. The lipid nanoparticle formulation for use of any one of claims 1 to 17, comprising 30% mol / mol to 50% mol / mol of the cationic lipid.

18. The lipid nanoparticle formulation for use of any one of claims 1 to 17, wherein the 20 sterol comprises cholesterol.

19. The lipid nanoparticle formulation for use of any one of claims 1 to 18, comprising 25% mol / mol to 35% mol / mol of the sterol.

20. The lipid nanoparticle formulation for use of any one of claims 1 to 19, comprising:147at least one cationic lipid, 30% to 50% mol / mol; at least one neutral phospholipid, 20% to 40% mol / mol; at least one sterol 20% to 40% mol / mol; and at least one PEGylated lipid, 1% to 5% mol / mol. 5 21. The lipid nanoparticle formulation for use of any one of claims 1 to 20, comprising: at least one cationic lipid, 33% to 43% mol / mol; at least one neutral phospholipid, 25% to 35% mol / mol; at least one sterol 25% to 35% mol / mol; and at least one PEGylated lipid, 1% to 3% mol / mol. 10 22. The lipid nanoparticle formulation for use of any one of claims 1 to 21, comprising: at least one cationic lipid, about 38% mol / mol; at least one neutral phospholipid, about 30% mol / mol; at least one sterol, about 30% mol / mol; and at least one PEGylated lipid, about 2% mol / mol. 15 23. The lipid nanoparticle formulation for use of any one of claims 1 to 22, which is selected from the group consisting of: Formulation 1, Formulation 2, Formulation 3, Formulation 7, Formulation 9 and Formulation 10: Formulation 1: lipid II-1: 33% to 43% mol / mol; 20 distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 2: IA-10: 33% to 43% mol / mol;148distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 3: 5 ALC-0315: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 7: 10 EA-502: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 9: 15 SM-102: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; II-25: 20 EA-502: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol;149wherein the structures of lipid II-1, lipid IA-10, EA-502, lipid ALC-0315, lipid SM- 102 and lipid II-25 are as shown in claim 10.

24. The lipid nanoparticle formulation for use of any one of claims 1 to 23, which is selectively targeting the gut. 5 25. The lipid nanoparticle formulation for use of claim 24, which is selectively targeting the colon.

26. The lipid nanoparticle formulation for use of any one of claims 1 to 25, which is selectively targeting an inflamed tissue.

27. The lipid nanoparticle formulation for use of any one of claims 1 to 26, wherein the 10 inflammation is an inflammatory bowel disease (IBD).

28. The lipid nanoparticle formulation for use of claim 27, for treatment of colitis.

29. The lipid nanoparticle formulation for use of any one of claims 1 to 27, for treatment of rheumatoid arthritis.

30. A lipid nanoparticle comprising: 15 at least one cationic lipid; at least one neutral phospholipid; at least one sterol; and at least one PEGylated lipid, wherein a total neutral phospholipid concentration within the lipid nanoparticle 20 formulation is in the range of 20% mol / mol to 40% mol / mol, wherein the lipid nanoparticle has a phase transition temperature peak in the range of 50°C to 63°C, as measured by Differential Scanning Calorimetry (DSC).

31. The lipid nanoparticle of claim 30, wherein the phase transition temperature peak is in the range of 55°C to 63°C. 25 32. The lipid nanoparticle of any one of claims 30 to 31, further comprising an anti- inflammatory therapeutic agent encapsulated within at least one particle thereof.15033. The lipid nanoparticle of claim 32, wherein the anti-inflammatory therapeutic agent comprises an anti-inflammatory nucleic acid.

34. The lipid nanoparticle of claim 33, wherein the nucleic acid is an mRNA that encodes an anti-inflammatory polypeptide. 5 35. The lipid nanoparticle of any one of claims 30 to 34, wherein the cationic lipid is selected from the group consisting of: lipid II-1, Lipid IA-10, EA-502, lipid ALC- 0315, lipid SM-102, lipid II-25 and a combination thereof; 10536. The lipid nanoparticle of any one of claims 30 to 35, comprising 25% mol / mol to 35% mol / mol total neutral phospholipid concentration.

37. The lipid nanoparticle of any one of claims 30 to 36, wherein the neutral phospholipid 10 comprises a phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), or a combination thereof.

38. The lipid nanoparticle of any one of claims 30 to 37, wherein the neutral phospholipid comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).15239. The lipid nanoparticle of any one of claims 30 to 38, wherein the cationic lipid is not a phospholipid.

40. The lipid nanoparticle of any one of claims 30 to 39, comprising 30% mol / mol to 50% mol / mol of the cationic lipid. 5 41. The lipid nanoparticle of any one of claims 30 to 40, wherein the sterol comprises cholesterol.

42. The lipid nanoparticle of any one of claims 30 to 41, comprising 25% mol / mol to 35% mol / mol of the sterol.

43. The lipid nanoparticle f of any one of claims 30 to 42, comprising: 10 at least one cationic lipid, 33% to 43% mol / mol; at least one neutral phospholipid, 25% to 35% mol / mol; at least one sterol 25% to 35% mol / mol; and at least one PEGylated lipid, 1% to 3% mol / mol.

44. The lipid nanoparticle of any one of claims 1 to 32, comprising: 15 at least one cationic lipid, about 38% mol / mol; at least one neutral phospholipid, about 30% mol / mol; at least one sterol, about 30% mol / mol; and at least one PEGylated lipid, about 2% mol / mol.

45. The lipid nanoparticle of any one of claims 30 to 44, which has a lipid ratio as in 20 Formulation 1, Formulation 2, Formulation 3, Formulation 7, Formulation 9 or Formulation 10: Formulation 1: lipid II-1: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; 25 cholesterol: 25% to 35% mol / mol; and153DMG-PEG 2000: 1% to 3% mol / mol; Formulation 2: IA-10: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; 5 cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 3: ALC-0315: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; 10 cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 7: EA-502: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; 15 cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; Formulation 9: SM-102: 33% to 43% mol / mol; distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; 20 cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; II-25: EA-502: 33% to 43% mol / mol;154distearoyl phosphatidyl choline (DSPC): 25% to 35% mol / mol; cholesterol: 25% to 35% mol / mol; and DMG-PEG 2000: 1% to 3% mol / mol; wherein the structures of lipid II-1, lipid IA-10, EA-502, lipid ALC-0315, lipid SM- 5 102 and lipid II-25 are as shown in claim 35.

46. The lipid nanoparticle of any one of claims 30 to 45, which is selectively targeting the gut.

47. The lipid nanoparticle of claim 46, which is selectively targeting the colon.

48. The lipid nanoparticle of any one of claims 30 to 47, which is selectively targeting an 10 inflamed tissue.

49. The lipid nanoparticle of any one of claims 30 to 48, for use in treating an inflammation.

50. The lipid nanoparticle of claim 49, wherein the inflammation is an inflammatory bowel disease (IBD). 15 51. The lipid nanoparticle of claim 50, for treatment of colitis.

52. The lipid nanoparticle of any one of claims 30 to 51, for treatment of rheumatoid arthritis.

53. A method of treating an inflammatory disease or disorder, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation of 20 any one of claims 1 to 29 or the nanoparticle of any one of claims 30 to 52 and a pharmaceutically acceptable carrier, diluent or excipient.

54. The method of claim 53, for treating an inflammatory bowel disease (IBD).

55. The method of any one of claims 53 to 54, for treating colitis.

56. The method of any one of claims 53 to 54, for treating inflammatory arthritis. 25 57. The method of any one of claims 53 to 56, comprising the step of orally, intramuscularly (IM), intravenously (IV), intraperitoneally (IP), subcutaneously (SC),155topically, intradermally (ID) or Intrathecally administering to a subject in need thereof the lipid nanoparticle formulation or lipid nanoparticle, and the pharmaceutically acceptable carrier, diluent or excipient.

58. A method of delivery of a therapeutic agent or a nucleic acid to an organ selected from 5 the group consisting of: lymphoid organs, lungs, heart, brain, spleen and skin, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation of any one of claims 1 to 29 or the lipid nanoparticle of any one of claims 30 to 52 and a pharmaceutically acceptable carrier, diluent or excipient.

59. The method of claim 58, for delivery of a therapeutic agent or a nucleic acid to the gut. 10 60. The method of any one of claims 58 to 59, for delivery of a therapeutic agent or a nucleic acid to the colon.

61. The method of any one of claims 58 to 60, comprising the step of orally, intramuscularly (IM), intravenously (IV), intraperitoneally (IP), subcutaneously (SC), topically, intradermally (ID) or intrathecally administering to a subject in need thereof 15 the lipid nanoparticle formulation or lipid nanoparticle and the pharmaceutically acceptable carrier, diluent or excipient.

62. A method of delivery of a therapeutic agent or a nucleic acid to an inflamed tissue, the method comprising the step contacting the lipid nanoparticle formulation of any one of claims 1 to 29 or the lipid nanoparticle of any one of claims 30 to 52 with the 20 inflamed tissue.

63. The method of claim 62, wherein the contacting is performed in vitro.

64. The method of any one of claim 63, comprising the step of orally, intramuscularly (IM), intravenously (IV), intraperitoneally (IP), subcutaneously (SC), topically, intradermally (ID) or intrathecally administering to a subject in need thereof the lipid 25 nanoparticle formulation or lipid nanoparticle and the pharmaceutically acceptable carrier, diluent or excipient.156

Images