TW202502303A - Lipid particles for delivering a payload - Google Patents

Abstract

Provided herein are lipid particles, pharmaceutical compositions comprising the lipid particles described herein, and methods of use comprising administering the lipid particles described herein.

Description

Lipid particles for delivery of payloads

The present disclosure relates to lipid particles, pharmaceutical compositions comprising the lipid particles described herein, and methods of use including administering the lipid particles described herein.

Although lipid nanoparticles have been developed to deliver payloads to cells, there remains a need in the art for improved lipid particle compositions suitable for therapeutic use. Disclosed herein are lipid particles designed to deliver payloads (e.g., nucleic acids) that are well tolerated and provide an adequate therapeutic response.

The present disclosure is based, in part, on lipid particles comprising one or more ionizable lipids, one or more neutral lipids, one or more sterols, one or more charged lipids, and one or more stealth lipids.

Contemplated herein are lipid particles comprising cholesterol hemisuccinate ("CHEMS") and a payload and, optionally, one or more of the following: a. one or more ionizable lipids; b. one or more neutral lipids; c. one or more sterols; and d. one or more stealth lipids.

Also contemplated herein are methods for targeting a payload to the spleen of an individual in vivo, comprising combining a lipid particle or a composition comprising a payload encapsulated within a lipid particle or a composition comprising at least 11% CHEMS in a molar amount. In some aspects, the lipid particle or composition further comprises one or more ionizable lipids, one or more neutral lipids, one or more sterols, one or more stealth lipids, or any combination thereof. In some aspects, the lipid particle delivers the payload to the spleen.

In some aspects, one or more ionizable lipids in the lipid particles described herein are selected from one or more of the group consisting of: DLin-MC3-DMA ("MC3"), DLin-KC2-DMA ("KC2"), ssPalmO-Phe ("SS-OP"), C12-200, SM-102, α-D-tocopheryl succinyl ("SS-EC"), ALC-0315 ("ALC"), 3-(ethyl(methyl)amino)propionic acid tri-N-tridecyl ester ("304-O13"), 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionic acid tetrakis(2-(octyldisulfanyl)ethyl) ester ("306-O12B"), 9-[4-(dimethylamino)-1-oxobutyloxy]-heptadecanedioic acid 1,17-di-(2Z)-2-nonene-1 -yl ester ("L319"), 9,12-octadecadienoic acid (9Z,12Z)-1,1',1'',1'''-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1-butanediylaminobis-4,1-butanediyl)] ester ("OF-C4-Deg-Lin"), 2-(dioctylamino)ethyl nonyl hydrogen phosphate ("9A1P9"), (9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoic acid) 5-(((3-(Dibutylamino)propyl)amino)methyl)-6-hydroxyundecane-1,11-diyl ester ("IR-117-17"), 3-[4,4-bis(octyloxy)-1-oxobutyloxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl 9Z,12Z-octadecadienoate ("LP-01") and 4A3-SC8. In some aspects, one or more ionizable lipids in the lipid particles described herein are selected from one or more of the group consisting of: DLin-MC3-DMA ("MC3"), DLin-KC2-DMA ("KC2"), ssPalmO-Phe ("SS-OP"), C12-200, SM-102, α-D-tocopheryl succinyl ("SS-EC"), and ALC-0315 ("ALC"). In some aspects, only one ionizable lipid (e.g., SS-OP) is in the lipid particles.

In some aspects, the lipid particles described herein include one or more neutral lipids. In some aspects, the molar amount of one or more neutral lipids in the lipid particles is at least 0.5%. In some aspects, the one or more neutral lipids in the lipid particles are one or more phosphatidylcholine ("PC"), phosphatidylethanolamine ("PE") and combinations thereof. In some aspects, one or more neutral lipids in the lipid particles described herein are selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine ("DSPC"), 1,2-dioleoyl-sn-glycero-3-phosphocholine ("DOPC"), 1,2-dioctadecanoyl-sn-glycerol ("DSDG"), 1-2-dioleoyl-sn-glycerol, 1-2-di-(9Z-octadecenoyl)-sn-glycerol ("DODG"), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine ("DOPE"), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, Cerebrosides, diacylglycerol dipalmitoylphosphatidylcholine ("DPPC"), palmitoyloleyl-phosphatidylcholine ("POPC"), palmitoyloleyl-phosphatidylethanolamine ("POPE"), palmitoyloleyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine ("DPPE"), dimyristan In some embodiments, only one neutral lipid (e.g., DOPE, DODG) is present in the lipid particles described herein.

In some aspects, the lipid particles described herein include one or more sterols. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is at least 5%. In some aspects, one or more sterols in the lipid particles described herein are cholesterol, β-sterol, stigmasterol, campesterol, fucosterol, ergosterol, 9,11-dehydroergosterol, carotene, β-sterol acetate and other C-24 alkyl derivatives and combinations thereof. In some aspects, one or more sterols in the lipid particles described herein are cholesterol, β-sterol and combinations thereof. In some aspects of the lipid particles described herein, only one sterol (e.g., cholesterol) is in the lipid particles.

In some aspects, the lipid particles described herein include one or more stealth lipids. In some aspects, the molar amount of one or more stealth lipids in the lipid particles described herein is at least 0.1%. In some aspects, one or more stealth lipids in the lipid particles described herein are one or more PEG-terminated lipids, one or more poly(saccharide) derivatives or combinations thereof. In some aspects, only one polyethylene glycol ("PEG")-terminated lipid (DMG-PEG2000, DMPE-PEG2000, PEG2000-PE) is in the lipid particles described herein.

Also contemplated herein are lipid particles comprising: a. about 20% to about 70% molar amount of one or more ionizable lipids; b. about 2.5% to about 25% molar amount of one or more neutral lipids; c. about 20% to about 50% molar amount of one or more sterols; d. about 10% or less molar amount of one or more charged lipids; and e. about 0.25% to about 3% molar amount of one or more stealth lipids.

Methods for targeting a payload to a tissue other than the spleen of an individual in vivo, the methods comprising combining a lipid particle or a composition comprising a payload encapsulated within a lipid particle or a composition comprising one or more ionizable lipids, one or more neutral lipids, one or more sterols, one or more stealth lipids and/or one or more charged lipids, wherein the lipid particle or the composition (a) does not comprise cholesterol hemisuccinate ("CHEMS") or (b) comprises a molar amount of about 10% or less than 10% CHEMS.

In some aspects, one or more of the charged lipids in the lipid particles described herein is selected from the group consisting of cholesterol hemisuccinate ("CHEMS"), 1,2-distearoyl-3-trimethylammonium-propane ("DSTAP"), 18:1 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP"), 1,2-dioleoyl-sn-glycero-3-phosphate ("18:1 PA"), 18:1 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) ("18:1 PG”), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-butyrylphosphatidylethanolamine, N-pentylphosphatidylethanolamine, phosphatidylglycerol, palmitoyloleylphosphatidylglycerol (“POPG”), 1,2-dimyristoyl-3-trimethylammonium-propane (“DMTAP”), 1,2-dipalmitoyl-3-trimethylammonium-propane (“DPTAP”), palmitoyloleyl-3-trimethylammonium-propane (“PO TAP”), 1,2-dioleoyl-3-dimethylammonium-propane (“DODAP”), palmitoyloleyl-3-dimethylammonium-propane (“PODAP”), 1,2-dimyristoyl-3-dimethylammonium-propane (“DMDAP”), 1,2-dipalmitoyl-3-dimethylammonium-propane (“DPDAP”), 1,2-distearoyl-3-dimethylammonium-propane (“DSDAP”), 1,2-dioleoyl-3-dimethylhydroxyethyl-ammonium-propane (“DODMHEAP”) (also known as DORI), palmitoyloleyl-3-dimethylhydroxyethyl-ammonium-propane (“PODMHEAP”) (also known as PORI), 1,2-dimyristoyl-3-dimethylhydroxyethyl-ammonium-propane (“DMDMHEAP”) (also known as DMRI), 1,2-dipalmitoyl-3-dimethylhydroxyethyl-ammonium-propane (“DPDMHEAP”) (also known as DPRI), 1,2-distearoyl-3-dimethylhydroxyethyl-ammonium-propane (“DSDMHEAP”) (also known as DSRI), 1,2-dioleyl-3-methylhydroxyethylammonium-propane ("DOMDHEAP"), palmitoyloleyl-3-methylhydroxyethylammonium-propane ("POMDHEAP"), 1,2-dimyristoyl-3-methyldihydroxyethylammonium-propane ("DMMDHEAP"), 1,2-dipalmitoyl-3-methyldihydroxyethylammonium-propane ("DPMDHEAP"), 1,2-distearoyl-3-methyldihydroxyethylammonium-propane ("DPMDHEAP"), hydroxyethylammonium propane ("DSMDHEAP"), 1,2-dioleoyl-3-methyldihydroxyethylammonium propane ("DOMHEAP"), palmitoyloleyl-3-methylhydroxyethylammonium propane ("POMHEAP"), 1,2-dimyristoyl-3-methylhydroxyethylammonium propane ("DMMHEAP"), 1,2-dipalmitoyl-3-methylhydroxyethylammonium propane ("DPMHEAP"), 1,2-distearyl-3 -methylhydroxyethylammonium-propane ("DSMHEAP"), 1,2-dioleoyl-3-dihydroxyethylammonium-propane ("DODHEAP"), palmityloleyl-3-dihydroxyethylammonium-propane ("PODHEAP"), 1,2-dimyristoyl-3-dihydroxyethylammonium-propane ("DMDHEAP"), 1,2-dipalmitoyl-3-dihydroxyethylammonium-propane ("DPDHEAP"), 1,2-distearoyl-3-dihydroxyethylammonium-propane ("DSDHEAP"), dimethylbisoctadecylammonium bromide ("DDAB"), dioleyldimethylammonium chloride ("DODAC"), 1,2-di Oleyl-sn-glycero-3-ethylphosphocholine ("DOEPC"), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine ("DMEPC"), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine ("DPEPC"), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine ("DSEPC"), palmitoyloleyl-sn-glycero-3-ethylphosphocholine ("POEPC"), 1,2-dioleyl-3-dimethyl-hydroxyethylammonium propane ("DORIE"), 1,2-dimyristoyl-3-dimethyl-hydroxyethylammonium propane ("DMRIE") 、1,2-dioleyl-3-methyl-(methoxycarbonyl-ethyl)ammonium-propane ("DOMCAP")、1,2-dioleyl-3-methyl-(methoxycarbonylmethyl)ammonium-propane ("DOMGME")、1,2-dioleyl-3-N-pyrrolidinium-propane ("DOP5P")、1,2-dioleyl-3-N-pyridinium-propane bromide salt ("DOP6P")、3b-[N-(N9,N9-dimethylaminoethane)aminomethyl]cholesterol ("DC-Chol")、3b-[N-(N9,N9-trimethylaminoethane)aminomethyl]cholesterol ("TC-Chol")、3b (N-(N,N'-dimethylaminoethane)-aminoformyl) cholesterol ("DAC-Chol"), cholesterol-oxycarbonyl-methyl-trimethylammonium chloride ("Chol-betaine"), N-methyl{4-N-amino[(3'-β-cholesterol)aminoformyl]}piperazine ("N-methyl-PipChol"), hexadecyltrimethylammonium bromide ("CTAB"), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"), 4-(2-aminoethyl)-morpholinyl-cholesterol hemisuccinate ("MoChol"), histamino-cholesterol hemisuccinate Cholesterol ("HisChol"), (3-imidazol-1-ylpropyl) carbamate ("Chim"), (N-2-propylamino[(3'-β-cholesterol)carbamate]}morpholine ("MoC3Chol"), [(3-morpholine-4-yl-propylcarbamate)-methyl]-cholesterol carbamate ("Chol-C3N-Mo3"), ("Chol-C3N-Mo2"), [(2-morpholine-4-yl-ethylcarbamate)methyl]-cholesterol carbamate ("Chol-C4N-Mo2"), [1-methyl-2-(2-morpholine-4-yl-ethylcarbamate)-propylcarbamate [Chol-DMC3N-Mo2], 2-(2-Phenyl-4-yl-ethylaminoformyl)-cyclohexanecarboxylic acid cholesterol ester ("CholC4Hex-Mo2"), 4-(2-aminoethyl)-phenyl-cholesterol-2,3-dimethyl hemi-succinate ("DmC4Mo2"), 4-(2-aminoethyl)-phenyl-cholesterol-2,2-dimethyl hemi-malonate ("DmC3Mo2"), 4-(2-aminoethyl)-phenyl-cholesterol-hemi-malonate ("C3Mo2"), 4-(2-aminopropyl)-phenyl-cholesterol-hemi-malonate ("C 3Mo3”), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemigatanoate (“C5Mo2”), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemigatanoate (“C6Mo2”), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemigatanoate (“C8Mo2”), 4-(2-aminobutyl)-morpholinyl-cholesterol-hemisuccinate (“C4Mo4”), 4{N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}piperazine (“PipC2Chol”), {N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}morpholinyl (“MoC2Ch ol”), {N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}pyrrolidine (“PyrroC2Chol”), {N-2-propylamino[(3'-β-cholesterol)aminoformyl]}imidazole (“ImC3Chol”), {N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}pyridine (“PyC2Chol”), 1,2-dioleoyl-3-N-pyroline-propane (“MoDO”), 1,2-dipalmitoyl-3-N-pyroline-propane (“MoDP”), 4,(2,3-bis-acyloxy-propyl)-1-methyl-1H-imidazole (“DOIM”) (also known as DPIM); diacylglycerol hemisuccinates, such as dioctadecylamido-glycidylspermine ("DOGS"), dimyristoylglycerol hemisuccinate ("DMGS") (also known as DMG-Succ), 1-palmitoyl-2-oleylglycerol hemisuccinate ("POGS") (also known as POG-Succ), dipalmitoylglycerol hemisuccinate ("DPGS") (also known as POG-Succ), distearylglycerol hemisuccinate ("DSGS") (also known as DSG-Succ); diacylglycerol hemimalonates, such as dioleylglycerol hemimalonate ("DOGM"), dimyristoylglycerol hemimalonate ("DMGM"); diacylglycerol hemiglutarate, such as dioleylglycerol hemiglutarate ("DOGG"), dimyristoylglycerol hemiglutarate ("DMGG"); diacylglycerol hemiadipate, such as dioleylglycerol hemiadipate ("DOGA"), dimyristoylglycerol hemiadipate ("D MGA”), diacylglycerol hemicyclohexane-1,4-dicarboxylic acid, such as dioleoylglycerol hemicyclohexane-1,4-dicarboxylic acid (“DO-cHA”), dimyristoylglycerol hemicyclohexane-1,4-dicarboxylic acid (“DM-cHA”), (2,3-diacyl-propyl)amino}-oxoalkanoic acid, such as 4-{(2,3-dioleoyl-propyl)amino}-4-oxobutanoic acid (“DOAS”), 3-{(2,3-dioleoyl-propyl)amino}-3-oxopropionic acid ( DOAM), 5-{(2,3-dioleyl-propyl)amino}-5-oxopentanoic acid (“DOAG”), 6-{(2,3-dioleyl-propyl)amino}-6-oxopentanoic acid (“DOAA”), 4-{(2,3-dimyristyl-propyl)amino}-4-oxopentanoic acid (“DMAS”), 3-{(2,3-dimyristyl-propyl)amino}-3-oxopentanoic acid (“DMAM”), 5-{(2,3-dimyristyl-propyl)amino}-6-oxopentanoic acid (“DOAG”), 6-{(2,3-dioleyl-propyl)amino}-6-oxopentanoic acid (“DOAA”), 4-{(2,3-dimyristyl-propyl)amino}-4-oxopentanoic acid (“DMAS”), 3-{(2,3-dimyristyl-propyl)amino}-3-oxopentanoic acid (“DMAM”), )amino}-5-hydroxypentanoic acid ("DMAG"), 6-{(2,3-dimyristyl-propyl)amino}-6-hydroxyhexanoic acid ("DMAA"); diacyl-alkanoic acids, such as 2,3-dioleyl-propionic acid ("DOP"), 3,4-dioleyl-butyric acid ("DOB"), 5,6-dioleyl-hexanoic acid ("DOS"), 4,5-dioleyl-pentanoic acid ("DOM"), 6,7-dioleyl-heptanoic acid ("DOG"), 7,8-dioleyl-octanoic acid ("DOB"), 1,2-dioleyl-1,2-dioleyl-2,3-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-2,3-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-3,4-dioleyl-4,5-dioleyl- (“DOA”), 2,3-dimyristyl-propionic acid (“DMP”), 3,4-dioleyl-butyric acid (“DOB”), 5,6-dimyristyl-hexanoic acid (“DMS”), 4,5-dimyristyl-pentanoic acid (“DMM”), 6,7-dimyristyl-heptanoic acid (“DMG”), 7,8-dimyristyl-octanoic acid (“DMA”); cholesterol oxycarbonylcarbamates, such as cholesterol hemidodecanedicarboxylic acid (“Chol-C12”), 12-cholesterol Steroloxycarbonylaminododecanoic acid ("CholC13N"); fatty acids, such as oleic acid, myristic acid, palmitic acid, stearic acid, cervical acid, behenic acid; dioleylphosphatidic acid ("DOPA"), 1,2-dimyristyl-sn-glycero-3-phosphate ("DMPA"), 1,2-dipalmitoyl-sn-glycero-3-phosphate ("DPPA"), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate ("POPA"), 1,2-distearyl sn-glycerol-3-phosphate (DSPA), cholesterol sulfate (Chol-SO4), dioleoylphosphatidylglycerol (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-racemic-glycerol), (DMPG), dimalmitoylphosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-distearyl-sn-glycero-3-phosphoglycerol (POPG), Phospho-rac-glycerol ("DSPG"), dioleylphosphatidylserine ("DOPS"), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine ("DMPS"), dimalmitoylphosphatidylserine ("DPPS"), 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-L-serine ("POPS"), 1,2-distearyl-sn-glycero-3-phospho-L-serine ("DSPS") or hexadecyl phosphate. In some aspects, one or more of the charged lipids in the lipid particles described herein is selected from the group consisting of cholesterol hemisuccinate ("CHEMS"), 1,2-distearoyl-3-trimethylammonium-propane ("DSTAP"), 18:1 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP"), 1,2-dioleoyl-sn-glycero-3-phosphate ("18:1 PA"), 18:1 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) ("18:1 PG"), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-succinylatedphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, phosphatidylglycerol and palmitoyloleylphosphatidylglycerol ("POPG"). In some aspects, only one charged lipid (e.g., CHEMS) is in the lipid particles described herein. In some aspects, the molar concentration of one or more charged lipids (e.g., CHEMS) in the lipid particles described herein is about 10% or less. In some aspects, the lipid particles described herein do not include a charged lipid (e.g., CHEMS).

The lipid particles described herein may include a payload. In some aspects, the payload includes a bioactive molecule. In some aspects, the bioactive molecule is a small molecule, a nucleic acid, an aptamer, or any combination thereof. In some aspects, the bioactive molecule includes a nucleic acid. In some aspects, the nucleic acid comprises a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a micro RNA (miRNA), a primary micro RNA (pri-miRNA), a long noncoding RNA (lncRNA), a messenger RNA (mRNA), a cluster of regularly interspaced repeats (CRISPR)-related nucleic acid, a CRISPR-RNA (crRNA), a single guide RNA (sgRNA), a trans-activating CRISPR RNA (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer RNA (tRNA), an antisense oligonucleotide (ASO), an antisense RNA (RNA), a guide RNA, a deoxyribonucleic acid (DNA), a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded RNA (ssRNA), a double-stranded RNA (dsRNA), a CRISPR-associated (Cas) protein, or a combination thereof. In some aspects, the nucleic acid in the lipid particles described herein comprises mRNA. In some aspects, the nucleic acid in the lipid particles described herein encodes a peptide having therapeutic activity. In some aspects, the peptide comprises an antigenic determinant amino acid sequence. In some aspects, the peptide induces immune tolerance to cells.

In some aspects, when the lipid particles described herein are administered to a subject in need thereof, the subject exhibits one or more of the following: (i) an increase in Treg cells, (ii) a decrease in effector T cells, (iii) a decrease in B cells, (iv) a decrease in interleukin production, (v) a decrease in activated B cells, (vi) a decrease in activated effector T cells; or (vii) any combination thereof.

In some aspects, the lipid particles described herein comprise one or more ionizable lipids comprising α-D-tocopheryl succinyl ("SS-EC"). In some aspects, subjects administered lipid particles comprising SS-EC described herein exhibit (i) higher levels of B cells, (ii) higher levels of T cells, and (iii) higher levels of INFα compared to subjects administered lipid particles containing MC-3 as an ionizable lipid.

Pharmaceutical compositions comprising lipid particles described herein and one or more pharmaceutically acceptable carriers are contemplated. In some embodiments, one of the pharmaceutically acceptable carriers is sucrose. In some embodiments, one of the pharmaceutically acceptable carriers is saline. In some embodiments, the saline is buffered with tris.

Contemplated are methods of targeting a payload to the pancreas of an individual in need thereof, the methods comprising intraperitoneally administering to the individual a lipid particle described herein.

Also contemplated are methods of targeting a payload to lymph nodes in a subject in need thereof, the methods comprising subcutaneously administering to the subject a lipid particle described herein.

Also contemplated are methods for treating a disease or condition in an individual in need thereof, the methods comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising the lipid particles described herein. In some aspects, the disease is an autoimmune disease. In some aspects, the autoimmune disease is selected from the group consisting of autoimmune hepatitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), nonalcoholic fatty pancreatitis, scleroderma, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, chylous diarrhea, type 1 diabetes, Guillain-Barré syndrome, Hashimoto's thyroiditis, thyroiditis), polymyalgia rheumatica, alopecia areata, fibrosis, psoriasis, pemphigus vulgaris, vitiligo, ankylosing spondylitis, juvenile idiopathic arthritis, psoriasis arthritis, mixed connective tissue disease, neuromyelitis optica, latent autoimmune diabetes in adults ("LADA"), autoimmune thyroid disease, Grave's disease, Addison's disease, autoimmune atrophic gastritis, pernicious anemia, atopic dermatitis, bullous pemphigoid, myasthenia gravis, polymyositis/dermatomyositis, rheumatic fever, primary sclerosing cholangitis, autoimmune uveitis, and Behcet's disease. In some embodiments, the disease or disorder is an autoimmune disease, such as autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, Goodpasture's syndrome, autoimmune nephritis, glomerulonephritis, Wegener's granulomatosis, chronic inflammatory demyelinating polyradiculoneuropathy, Sjogren's syndrome, primary biliary cholangitis, Parkinson's disorder, and antiphospholipid syndrome. In some embodiments, the autoimmune disease is autoimmune hepatitis, type 1 diabetes, or multiple sclerosis. In some embodiments, the disease or disorder is an infectious disorder or cancer.

Further contemplated are methods of preventing or delaying the onset or recurrence of a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the lipid particles described herein.

Contemplated are methods for restoring immune homeostasis in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the lipid particles described herein.

Also contemplated are methods for limiting or reducing an immunogenic response in a subject in need thereof, the methods comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the lipid particles described herein.

Also contemplated are methods for limiting or reducing an inflammatory response in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a lipid particle composition described herein.

Cross-references to related applications

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/619,395 filed on January 10, 2024 and U.S. Provisional Patent Application No. 63/511,130 filed on June 29, 2023, the disclosures of each of which are incorporated herein by reference in their entirety.

The present disclosure is based in part on a method of targeting a payload to a tissue (e.g., spleen, pancreas, lymph node, or liver) of an individual in need thereof, the method comprising combining the payload with one or more neutral lipids, one or more ionizable lipids (e.g., cholesterol hemisuccinate (e.g., cholesterol hemisuccinate ("CHEMS")), one or more sterols, one or more stealth lipids, or any combination thereof. Lipid particles or compositions comprising a payload encapsulated within a lipid particle as described herein can be used to treat a disease or condition in an individual, such as an autoimmune disease, an infectious disease, or cancer. I. Definitions

To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

As used herein, the terms "a" or "an" mean "one or more" and include the plural unless the context is inappropriate.

Where the term "about" is used before a quantitative value, unless expressly stated otherwise, the disclosure also includes the specific quantitative value itself. Unless otherwise indicated or inferred, as used herein, the term "about" refers to a variation of ±10% from the nominal value.

Unless otherwise understood from the context and usage, as used herein, the term "and/or" in connection with two or more listed items includes each of the listed items individually and various combinations of two or more of the listed items.

As used herein, the term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and includes DNA and RNA. Examples of nucleic acids include small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), primary micro RNA (pri-miRNA), long non-coding RNA (lncRNA), messenger RNA (mRNA), clustered regularly interspaced repeats (CRISPR)-related nucleic acid, CRISPR-RNA (crRNA), single guide RNA (sgRNA), trans-activating CRISPR RNA (tracrRNA), plasmid deoxyribonucleic acid (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), antisense RNA (RNA), guide RNA, deoxyribonucleic acid (DNA), double-stranded deoxyribonucleic acid (dsDNA), single-stranded deoxyribonucleic acid (ssDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), CRISPR-associated (Cas) protein or a combination thereof.

Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have binding properties similar to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, heterologs, SNPs, and complementary sequences as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res ., 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem ., 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes , 8: 91-98 (1994)).

As used herein, the term "effective amount" refers to an amount of a payload (e.g., a compound of the present disclosure) sufficient to achieve beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration.

As used herein, the percentage of "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence after the sequences are aligned and, if necessary, spaces are introduced to achieve the maximum percentage of sequence identity. Similarly, the percentage of "identity" between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence after the sequences are aligned and, if necessary, spaces are introduced to achieve the maximum percentage of sequence identity. For the purpose of determining the percentage of sequence identity (e.g., amino acid sequence identity or nucleic acid sequence identity), alignment can be achieved in various ways within the skill of the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

Unless the context clearly states or understands otherwise, the use of the terms "include/includes/including", "have/has/having" or "contain/contains/containing" (including their grammatical equivalents) should generally be understood as open and non-restrictive, for example, not excluding additional unlisted elements or steps.

As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water, emulsions (e.g., oil/water or water/oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Adejare, Remington , The Science and Practice of Pharmacy (23rd ed. 2020).

As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., rodents, monkeys, horses, cows, pigs, dogs, cats, and the like), and more preferably include humans.

As used herein, the term "treating" includes any action that ameliorates a disorder, disease, condition, and the like or improves symptoms thereof, such as by alleviating, reducing, modulating, improving, or eliminating.

As used herein, the term "lipids" refers to a group of organic compounds that include but are not limited to fatty acid esters and are characterized by being insoluble in water but soluble in many organic solvents.

As used herein, the term "lipid particle" refers to a lipid formulation that can be used to deliver active agents or therapeutic agents, such as nucleic acids (e.g., interfering RNA), to a target site of interest, such as the spleen.

As used herein, the term "cholesterol hemisuccinate"("CHEMS") refers to an acidic cholesterol ester having the following structure: .

CHEMS is also known as 3β-hydroxy-5-cholesterol 3-hemisuccinate, 5-cholesterol-3β-ol 3-hemisuccinate and cholesterol hydrogen succinate.

As used herein, the term "SS-OP" refers to an ionizable lipid having the following structure: .

SS-OP is available from NOF America Corporation (Coatsome SS-OP). SS-OP is also known as ssPalmO-Phe.

As used herein, the term "SS-EC" refers to an ionizable lipid having the following structure: .

SS-EC is available from NOF America Corporation (Coatsome SS-EC). SS-EC was formerly known as SS-33/4PE-15. SS-EC is also known as α-D-tocopheryl succinyl.

As used herein, the term "ALC-0315" refers to an ionizable lipid having the following structure: .

ALC-0315 is also known as [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

As used herein, the term "SM-102" refers to an ionizable lipid having the following structure: .

SM-102 is also known as 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate 9-heptadecyl ester and 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate 1-octylnonyl ester.

As used herein, the term "DLin-KC2-DMA"("KC2") refers to an ionizable lipid having the following structure: .

KC2 is also known as N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxacyclopentane-4-ethylamine.

As used herein, the term "DLin-MC3-DMA"("MC3") refers to an ionizable lipid having the following structure: .

MC3 is also known as 4-(dimethylamino)-butyric acid (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester.

As used herein, the term "DODG" refers to a neutral lipid having the following structure: .

DODG is also known as 1-2-di-(9Z-octadecenyl)-sn-glycerol, (Z)-octadec-9-enoic acid [(2S)-3-hydroxy-2-[(Z)-octadec-9-enoyl]oxypropyl] ester.

As used herein, the term "DOPE" refers to a neutral lipid having the following structure:

DOPE is also known as 1,2-dioleyl-sn-glycero-3-phosphoethanolamine.

As used herein, the term "stealth lipid" refers to a lipid that, when used in a lipid particle, reduces recognition by the mononuclear phagocyte system, prevents protein adsorption, and allows for prolonged circulation time. An example of a stealth lipid is a PEG-terminated lipid.

As used herein, the term "DMG-PEG2000" refers to a PEG-terminated lipid having the following structure:

DMG-PEG2000 is also known as 1,2-dimyristyl-racemic-glycerol-3-methoxypolyethylene glycol-2000.

As used herein, the term "DMPE-PEG2000" refers to a PEG-terminated lipid having the following structure:

DMPE-PEG is also known as 3-[hydroxy-[2-[2-(2-methoxyethoxy)ethylamino]ethoxy]phosphoyl]oxy-2-tetradecanoyloxypropyl]tetradecanoate.

As used herein, the term "18:1 PEG2000-PE" refers to a PEG-terminated lipid having the following structure:

18:1 PEG2000-PE is also known as 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. II. Lipid particles

The present disclosure provides lipid particles comprising one or more ionizable lipids, one or more neutral lipids, one or more sterols, one or more charged lipids, and/or one or more polyethylene glycol ("PEG")-capped lipids.

Contemplated herein are lipid particles comprising cholesterol hemisuccinate ("CHEMS") and a payload and, optionally, one or more of the following: a. one or more ionizable lipids; b. one or more neutral lipids; c. one or more sterols; and d. one or more stealth lipids.

Also contemplated herein are methods of targeting a payload to the spleen of an individual in vivo comprising combining a lipid particle, or a composition comprising a payload encapsulated within a lipid particle, or a composition comprising a molar amount of at least 11% CHEMS.

Also contemplated herein are lipid particles comprising: a. about 20% to about 70% molar amount of one or more ionizable lipids; b. about 2.5% to about 25% molar amount of one or more neutral lipids; c. about 20% to about 50% molar amount of one or more sterols; d. about 10% or less molar amount of one or more charged lipids; and e. about 0.25% to about 3% molar amount of one or more stealth lipids.

Also contemplated herein are methods of targeting a payload to a tissue other than the spleen in an individual in vivo, the methods comprising combining a lipid particle or a composition comprising a payload encapsulated within a lipid particle or a composition comprising one or more ionizable lipids, one or more neutral lipids, one or more sterols, one or more stealth lipids, and/or one or more charged lipids, wherein the lipid particle or the composition does not comprise cholesterol hemisuccinate ("CHEMS") or comprises a molar amount of about 10% or less than 10% CHEMS.

In some embodiments, the desired tissue of the subject is the spleen. In some embodiments, the desired tissue of the subject is the liver. In some embodiments, the desired tissue of the subject is a lymph node. In some embodiments, the desired tissue of the subject is the pancreas. In some embodiments, the desired tissue of the subject is the brain, muscle, or lung. In some embodiments, the desired tissue contains one or more tumors. In some embodiments, the one or more tumors are malignant. a. Components of lipid particles i. Ionizable lipids

The lipid particles described herein may comprise one or more ionizable lipids. Ionizable lipids are lipids that can be easily protonated, such as amine-containing lipids.

The molar amount of one or more ionizable lipids in the lipid particles described herein is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. The molar amount of one or more ionizable lipids in the lipid particles described herein is about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 70%, about 25 ... % to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 3 0% to about 40%, about 30% to about 35%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about From about 40% to about 45%, from about 45% to about 70%, from about 45% to about 65%, from about 45% to about 60%, from about 45% to about 55%, from about 45% to about 50%, from about 50% to about 70%, from about 50% to about 65%, from about 50% to about 60%, from about 55% to about 70%, from about 55% to about 65%, from about 60% to about 70%, from about 60% to about 65%, or from about 65% to about 70%. In some aspects, the molar amount of one or more ionizable lipids in the lipid particles described herein is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%. In some aspects, the molar amount of one or more ionizable lipids in the lipid particles described herein is about 20%. In some aspects, one or more ionizable lipids in the lipid particles described herein are present in a molar amount of about 30%. In some aspects, one or more ionizable lipids in the lipid particles described herein are present in a molar amount of about 40%. In some aspects, one or more ionizable lipids in the lipid particles described herein are present in a molar amount of about 50%. In some aspects, one or more ionizable lipids in the lipid particles described herein are present in a molar amount of about 60%. In some aspects, one or more ionizable lipids in the lipid particles described herein are present in a molar amount of about 65%. In some aspects, one or more ionizable lipids in the lipid particles described herein are present in a molar amount of about 70%.

One or more ionizable lipids in the lipid particles described herein are selected from one or more of the group consisting of: DLin-MC3-DMA ("MC3"), DLin-KC2-DMA ("KC2"), ssPalmO-Phe ("SS-OP"), C12-200, SM-102, α-D-tocopheryl succinyl ("SS-EC"), ALC-0315 ("ALC"), 3-(ethyl(methyl)amino)propionic acid tri-N-tridecyl ester ("304-O13"), 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionic acid tetrakis(2-(octyldisulfanyl)ethyl) ester ("306-O12B"), 9-[4-(dimethylamino)-1-oxobutyloxy]-heptadecanedioic acid 1,17-di-(2Z)-2-nonene-1 -yl ester ("L319"), 9,12-octadecadienoic acid (9Z,12Z)-1,1',1'',1'''-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1-butanediylaminobis-4,1-butanediyl)] ester ("OF-C4-Deg-Lin"), 2-(dioctylamino)ethyl nonyl hydrogen phosphate ("9A1P9"), (9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoic acid) 5-(((3-(Dibutylamino)propyl)amino)methyl)-6-hydroxyundecane-1,11-diyl ester ("IR-117-17"), 3-[4,4-bis(octyloxy)-1-oxobutyloxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl 9Z,12Z-octadecadienoate ("LP-01") and 4A3-SC8. In some aspects, one or more ionizable lipids in the lipid particles described herein are selected from one or more of the group consisting of: DLin-MC3-DMA ("MC3"), DLin-KC2-DMA ("KC2"), ssPalmO-Phe ("SS-OP"), C12-200, SM-102, α-D-tocopheryl succinyl ("SS-EC"), and ALC-0315 ("ALC"). In some aspects, the lipid particles described herein contain only one ionizable lipid.

The selection of one or more ionizable lipids depends on the tissue to which the payload is to be delivered or the use of the lipid particles described herein. For example, if the lipid particles deliver the payload to the spleen of an individual (e.g., for a tolerogenic vaccine), one or more ionizable lipids in the lipid particles described herein may include SS-OP, KC2, and combinations thereof. In addition, if the lipid particles deliver the payload to the liver of an individual (e.g., for a cancer or infectious agent vaccine), one or more ionizable lipids in the lipid particles described herein may include MC3, KC2, and combinations thereof.

In addition, in some aspects, when administered to an individual in need, the lipid particles described herein reduce the immunogenicity of the individual (e.g., increase Treg cells, reduce effector T cells, reduce B cells, reduce interleukin production, reduce activated B cells and/or reduce activated effector T cells). In some aspects, the reduction in immunogenicity is compared to the reduction in immunogenicity of a reference lipid particle. In some aspects, the reduction in immunogenicity of an individual is compared to an individual before administration of the lipid particles described herein. In some aspects, the reference lipid particles contain MC-3 and one or more ionizable lipids in the lipid particles include SS-OP. In some aspects, the reference lipid particles contain KC-2 and one or more ionizable lipids in the lipid particles include SS-OP. In some aspects, the reference lipid particle contains SM-102 and one or more ionizable lipids in the lipid particle comprises SS-OP.

In some aspects, when administered to an individual in need thereof, the lipid particles increase the immunogenicity of the individual (e.g., decrease Treg cells, increase effector T cells, increase B cells, increase interleukin production, increase activated B cells, and/or increase activated effector T cells). In some aspects, the increase in immunogenicity is compared to the increase in immunogenicity of a reference lipid particle or individual before administration of the lipid particles described herein. In some aspects, the reference lipid particle contains MC-3 and one or more ionizable lipids in the lipid particle include SS-EC. In some aspects, the reference lipid particle contains KC-2 and one or more ionizable lipids in the lipid particle include SS-EC. In some aspects, the reference lipid particle contains SM-102 and one or more ionizable lipids in the lipid particle include SS-EC.

In some embodiments, the only ionizable lipid in the lipid particles described herein is SS-OP. In some embodiments, the only ionizable lipid in the lipid particles described herein is KC2. In some embodiments, the only ionizable lipid in the lipid particles described herein is MC3. In some embodiments, the only ionizable lipid in the lipid particles described herein is SM-102.

In some aspects, the molar amount of SS-OP in the lipid particles described herein is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. In some aspects, the molar amount of SS-OP in the lipid particles described herein is about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 70%, about 25 ... to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45 %, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 70%, about 60% to about 65%, or about 65% to about 70%. In some aspects, the molar amount of SS-OP in the lipid particles described herein is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%. In some aspects, the SS-OP in the lipid particles described herein is present in a molar amount of about 20%. In some aspects, the SS-OP in the lipid particles described herein is present in a molar amount of about 30%. In some aspects, the SS-OP in the lipid particles described herein is present in a molar amount of about 40%. In some aspects, the SS-OP in the lipid particles described herein is present in a molar amount of about 50%. In some aspects, the SS-OP in the lipid particles described herein is present in a molar amount of about 60%. In some aspects, the SS-OP in the lipid particles described herein is present in a molar amount of about 65%. In some aspects, the SS-OP in the lipid particles described herein is present in a molar amount of about 70%.

In some aspects, the molar amount of KC2 in the lipid particles described herein is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. In some aspects, the molar amount of KC2 in the lipid particles described herein is about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 70%, about 25% to about 50%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 70%, about 25% to about about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 4 0%, about 30% to about 35%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45 %, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 70%, about 60% to about 65%, or about 65% to about 70%. In some aspects, the molar amount of KC2 in the lipid particles described herein is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%. In some aspects, the KC2 in the lipid particles described herein is present in a molar amount of about 20%. In some aspects, the KC2 in the lipid particles described herein is present in a molar amount of about 30%. In some aspects, the KC2 in the lipid particles described herein is present in a molar amount of about 40%. In some aspects, the KC2 in the lipid particles described herein is present in a molar amount of about 50%. In some aspects, the KC2 in the lipid particles described herein is present in a molar amount of about 60%. In some aspects, the KC2 in the lipid particles described herein is present in a molar amount of about 65%. In some aspects, the KC2 in the lipid particles described herein is present in a molar amount of about 70%.

In some aspects, the molar amount of MC3 in the lipid particles described herein is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. In some aspects, the molar amount of MC3 in the lipid particles described herein is about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 70%, about 25% to about 3 ... about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 4 0%, about 30% to about 35%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45 In some aspects, the molar amount of MC3 in the lipid particles described herein is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%. In some aspects, the molar amount of MC3 in the lipid particles described herein is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%. In some aspects, the molar amount of MC3 in the lipid particles described herein is about 20%. In some aspects, the MC3 in the lipid particles described herein is present in a molar amount of about 30%. In some aspects, the molar amount of MC3 in the lipid particles described herein is about 40%. In some aspects, the molar amount of MC3 in the lipid particles described herein is about 50%. In some aspects, the molar amount of MC3 in the lipid particles described herein is about 60%. In some aspects, the molar amount of MC3 in the lipid particles described herein is about 65%. In some aspects, the molar amount of MC3 in the lipid particles described herein is about 70%.

In some aspects, the molar amount of SM-102 in the lipid particles described herein is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. In some aspects, the molar amount of SM-102 in the lipid particles described herein is about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 70%, about 25 ... % to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about about 40%, about 30% to about 35%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 35% to about 40%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 40% 5%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 70%, about 60% to about 65%, or about 65% to about 70%. In some aspects, the molar amount of SM-102 in the lipid particles described herein is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%. In some aspects, the SM-102 in the lipid particles described herein is present in a molar amount of about 20%. In some aspects, the SM-102 in the lipid particles described herein is present in a molar amount of about 30%. In some aspects, the SM-102 in the lipid particles described herein is present in a molar amount of about 40%. In some aspects, the SM-102 in the lipid particles described herein is present in a molar amount of about 50%. In some aspects, the SM-102 in the lipid particles described herein is present in a molar amount of about 60%. In some aspects, the SM-102 in the lipid particles described herein is present in a molar amount of about 65%. In some aspects, the SM-102 in the lipid particles described herein is present in a molar amount of about 70%. ii. Neutral lipids

The lipid particles disclosed herein may comprise one or more neutral lipids. Neutral lipids are uncharged lipids or zwitterionic molecules, i.e., molecules with positive and negative charges, resulting in overall neutral molecules.

In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 5%, at least 7.5%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, or at least 22.5%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 2.5% to about 25%, about 2.5% to about 20%, about 2.5% to about 17.5%, about 2.5% to about 15%, about 2.5% to about 12.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 25%, about 5% to about 20%, about 5% to about 17.5%, about 5% to about 15%, about 5% to about 12.5%, about 5% to about 10%, about %, about 7.5% to about 25%, about 7.5% to about 20%, about 7.5% to about 17.5%, about 7.5% to about 15%, about 7.5% to about 12.5%, about 7.5% to about 10%, about 10% to about 25%, about 10% to about 20%, about 10% to about 17.5%, about 10% to about 15%, about 10% to about 12.5%, about 12.5% to about 25%, about 12.5% to about 20%, about 12.5% to about 17.5%, or about 12.5% to about 15%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about 22.5%, or about 25%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 5% to about 20%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 7.5% to about 17.5%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 2.5%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 5%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 7.5. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 10%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 12.5%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 15%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 17.5%. In some aspects, the molar amount of one or more neutral lipids in the particles described herein is about 20%. In some aspects, the molar amount of one or more neutral lipids in the lipid particles described herein is about 25%.

In some aspects, the lipid particles described herein do not contain neutral lipids.

In some aspects, the lipid particles described herein include one or more neutral lipids. In some aspects, one or more neutral lipids in the lipid particles are one or more phosphatidylcholine ("PC"), phosphatidylethanolamine ("PE") and combinations thereof. One or more neutral lipids in the lipid particles described herein may be selected from one or more of the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine ("DSPC"), 1,2-dioleoyl-sn-glycero-3-phosphocholine ("DOPC"), 1,2-dioctadecanoyl-sn-glycerol ("DSDG"), 1-2-dioleoyl-sn-glycerol, 1-2-di-(9Z-octadecenoyl)-sn-glycerol ("DODG"), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine ("DOPE"), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cerebroside, diacylglycerol Dipalmitoylphosphatidylcholine (“DPPC”), palmitoyloleyl-phosphatidylcholine (“POPC”), palmitoyloleyl-phosphatidylethanolamine (“POPE”), palmitoyloleyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (“DPPE”), dimyristoyl-phosphatidylethanolamine (“ In some embodiments, the lipid particles described herein contain only one neutral lipid.

In some aspects, the only neutral lipid in the lipid particles described herein is DOPE. In some aspects, the molar amount of DOPE is at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 5%, at least 7.5%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, or at least 22.5%. In some aspects, the molar amount of DOPE is about 2.5% to about 25%, about 2.5% to about 20%, about 2.5% to about 17.5%, about 2.5% to about 15%, about 2.5% to about 12.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 25%, about 5% to about 20%, about 5% to about 17.5%, about 5% to about 15%, about 5% to about 12.5%, about 5% to about 10%, about 5% to about 7.5%. %, about 7.5% to about 25%, about 7.5% to about 20%, about 7.5% to about 17.5%, about 7.5% to about 15%, about 7.5% to about 12.5%, about 7.5% to about 10%, about 10% to about 25%, about 10% to about 20%, about 10% to about 17.5%, about 10% to about 15%, about 10% to about 12.5%, about 12.5% to about 25%, about 12.5% to about 20%, about 12.5% to about 17.5%, or about 12.5% to about 15%. In some aspects, the molar amount of DOPE is about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about 22.5%, or about 25%. In some aspects, the molar amount of DOPE is about 5% to about 20%. In some aspects, the molar amount of DOPE is about 7.5% to about 17.5%. In some aspects, the molar amount of DOPE is about 2.5%. In some aspects, the molar amount of DOPE is about 5%. In some aspects, the molar amount of DOPE is about 7.5. In some aspects, the molar amount of DOPE is about 10%. In some aspects, the molar amount of DOPE is about 12.5%. In some aspects, the molar amount of DOPE is about 15%. In some aspects, the molar amount of DOPE is about 17.5%. In some aspects, the molar amount of DOPE is about 20%. In some aspects, the molar amount of DOPE is about 25%.

In some aspects, the only neutral lipid in the lipid particles described herein is DODG. In some aspects, the molar amount of DODG is at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 5%, at least 7.5%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, or at least 22.5%. In some aspects, the molar amount of DODG is about 2.5% to about 25%, about 2.5% to about 20%, about 2.5% to about 17.5%, about 2.5% to about 15%, about 2.5% to about 12.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 25%, about 5% to about 20%, about 5% to about 17.5%, about 5% to about 15%, about 5% to about 12.5%, about 5% to about 10%, about 5% to about 7.5%. %, about 7.5% to about 25%, about 7.5% to about 20%, about 7.5% to about 17.5%, about 7.5% to about 15%, about 7.5% to about 12.5%, about 7.5% to about 10%, about 10% to about 25%, about 10% to about 20%, about 10% to about 17.5%, about 10% to about 15%, about 10% to about 12.5%, about 12.5% to about 25%, about 12.5% to about 20%, about 12.5% to about 17.5%, or about 12.5% to about 15%. In some aspects, the molar amount of DODG is about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about 22.5%, or about 25%. In some aspects, the molar amount of DODG is about 5% to about 20%. In some aspects, the molar amount of DODG is about 7.5% to about 17.5%. In some aspects, the molar amount of DODG is about 2.5%. In some aspects, the molar amount of DODG is about 5%. In some aspects, the molar amount of DODG is about 7.5. In some aspects, the molar amount of DODG is about 10%. In some aspects, the molar amount of DODG is about 12.5%. In some aspects, the molar amount of DODG is about 15%. In some aspects, the molar amount of DODG is about 17.5%. In some aspects, the molar amount of DODG is about 20%. In some aspects, the molar amount of DODG is about 25%. iii. Sterols

The lipid particles disclosed herein may include one or more sterols. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 20% to about 50%. In some aspects, the molar amount of one or more sterols in the lipid particle is about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 50%, or about 40% to about 45%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 20% to about 40%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 20% to about 30%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 30% to about 40%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 20%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 28.5%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 30%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 38.5%. In some aspects, the molar amount of one or more sterols in the lipid particles described herein is about 40%.

In some embodiments, one or more sterols in the lipid particles described herein are cholesterol, β-myristol, stigmasterol, campesterol, fucosterol, ergosterol, 9,11-dehydroergosterol, carotene, β-myristol acetate and other C-24 alkyl derivatives or combinations thereof. In some embodiments, the sterol is cholesterol, β-myristol and combinations thereof. In some embodiments, there is only one sterol in the lipid particles. In some embodiments, one of the sterols in the lipid particles comprises cholesterol.

In some aspects, the molar amount of cholesterol in the lipid particles described herein is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 20% to about 50%. In some aspects, the molar amount of cholesterol in the lipid particles is about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 50%, or about 40% to about 45%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 20% to about 40%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 20% to about 30%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 30% to about 40%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 20%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 28.5%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 30%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 38.5%. In some aspects, the molar amount of cholesterol in the lipid particles described herein is about 40%. iv. Charged lipids

The lipid particles disclosed herein may include one or more charged lipids. In some aspects, the molar amount of one or more charged lipids is about 10% or less. The charged lipids are anionic lipids, i.e., lipids that have more negative charges than positive charges in aqueous media, or cationic lipids, i.e., lipids that have more positive charges than negative charges in aqueous media.

In some aspects, the molar amount of one or more charged lipids in the lipid particles disclosed herein is 10%. In some aspects, the molar amount of one or more charged lipids in the lipid particles disclosed herein is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.1%. In some aspects, the molar amount of one or more charged lipids in the lipid particles disclosed herein is about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about From about 4%, from about 2% to about 3%, from about 3% to about 10%, from about 3% to about 9%, from about 3% to about 8%, from about 3% to about 7%, from about 3% to about 6%, from about 3% to about 5%, from about 3% to about 4%, from about 4% to about 10%, from about 4% to about 9%, from about 4% to about 8%, from about 4% to about 7%, from about 4% to about 6%, from about 4% to about 5%, from about 5% to about 10%, from about 5% to about 9%, from about 5% to about 8%, from about 5% to about 7%, or from about 5% to about 6%. In some aspects, the molar amount of one or more charged lipids in the lipid particles disclosed herein is about 0.1%, about 0.25%, about 0.5%, about 0.7%, about 1%, about 1.25%, about 1.5%, about 1.75%, about 2%, about 2.25%, about 2.5%, about 2.75%, about 3%, about 3.25%, about 3.5%, about 3.75%, about 4%, about 4.25%, about 4.5%, about 4.75%, about 5%, about 5.25%, about 5.5%, about 5.75%, about 6%, about 6.25%, about 6.5%, about 6.75%, about 7%, about 7.25%, about 7.5%, about 7.75%, about 8%, about 8.25%, about 8.5%, about 8.75%, about 9%, about 9.25%, about 9.5%, about 9.75%, or about 10%.

In some aspects, one or more of the charged lipids in the lipid particle is selected from the group consisting of cholesterol hemisuccinate ("CHEMS"), 1,2-distearoyl-3-trimethylammonium-propane ("DSTAP"), 18:1 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP"), 1,2-dioleoyl-sn-glycero-3-phosphate ("18:1 PA"), 18:1 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) ("18:1 PG”), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-butyrylphosphatidylethanolamine, N-pentylphosphatidylethanolamine, phosphatidylglycerol, palmitoyloleylphosphatidylglycerol (“POPG”), 1,2-dimyristoyl-3-trimethylammonium-propane (“DMTAP”), 1,2-dipalmitoyl-3-trimethylammonium-propane (“DPTAP”), palmitoyloleyl-3-trimethylammonium-propane (“PO TAP”), 1,2-dioleoyl-3-dimethylammonium-propane (“DODAP”), palmitoyloleyl-3-dimethylammonium-propane (“PODAP”), 1,2-dimyristoyl-3-dimethylammonium-propane (“DMDAP”), 1,2-dipalmitoyl-3-dimethylammonium-propane (“DPDAP”), 1,2-distearoyl-3-dimethylammonium-propane (“DSDAP”), 1,2-dioleoyl-3-dimethylhydroxyethyl-ammonium-propane (“DODMHEAP”) (also known as DORI), palmitoyloleyl-3-dimethylhydroxyethyl-ammonium-propane ("PODMHEAP") (also known as PORI), 1,2-dimyristoyl-3-dimethylhydroxyethyl-ammonium-propane ("DMDMHEAP") (also known as DMRI), 1,2-dipalmitoyl-3-dimethylhydroxyethyl-ammonium-propane ("DPDMHEAP") (also known as DPRI), 1,2-distearyl-3-dimethylhydroxyethyl-ammonium-propane ("DSDMHEAP") (also known as DSRI), 1,2-dioleyl-3-methylhydroxyethylammonium-propane ("DOMDHEAP"), palmitoyloleyl-3-methylhydroxyethylammonium-propane ("POMDHEAP"), 1,2-dimyristoyl-3-methyldihydroxyethylammonium-propane ("DMMDHEAP"), 1,2-dipalmitoyl-3-methyldihydroxyethylammonium-propane 1,2-dioleyl-3-methyldihydroxyethylammonium-propane ("DOMHEAP"), palmityloleyl-3-methylhydroxyethylammonium-propane ("POMHEAP"), 1,2-dimyristoyl-3-methylhydroxyethylammonium-propane ("DPMDHEAP"), 1,2-distearyl-3-methyldihydroxyethylammonium-propane ("DSMDHEAP"), 1,2-dioleyl-3-methyldihydroxyethylammonium-propane ("DOMHEAP"), palmityloleyl-3-methylhydroxyethylammonium-propane ("POMHEAP"), 1,2-dimyristoyl-3-methylhydroxyethylammonium -propane ("DMMHEAP"), 1,2-dipalmitoyl-3-methylhydroxyethylammonium-propane ("DPMHEAP"), 1,2-distearoyl-3-methylhydroxyethylammonium-propane ("DSMHEAP"), 1,2-dioleoyl-3-dihydroxyethylammonium-propane ("DODHEAP"), palmitoyloleoyl-3-dihydroxyethylammonium-propane (" PODHEAP”), 1,2-dimyristoyl-3-dihydroxyethylammonium-propane (“DMDHEAP”), 1,2-dipalmitoyl-3-dihydroxyethylammonium-propane (“DPDHEAP”), 1,2-distearoyl-3-dihydroxyethylammonium-propane (“DSDHEAP”), dimethylbisoctadecylammonium bromide (“DDAB”), dioleyl dimethyl ammonium chloride ("DODAC"), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine ("DOEPC"), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine ("DMEPC"), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine ("DPEPC"), 1,2-distearyl-sn-glycero-3 -ethylphosphocholine ("DSEPC"), palmitoyloleyl-sn-glycero-3-ethylphosphocholine ("POEPC"), 1,2-dioleyl-3-dimethyl-hydroxyethylammonium propane ("DORIE"), 1,2-dimyristyl-3-dimethyl-hydroxyethylammonium propane ("DMRIE"), 1,2-dioleyl-3-methyl-(methoxycarbonyl- 1,2-dioleyl-3-methyl-(methoxycarbonylmethyl)ammonium-propane ("DOMGME"), 1,2-dioleyl-3-N-pyrrolidinium-propane ("DOP5P"), 1,2-dioleyl-3-N-pyridinium-propane bromide salt ("DOP6P"), 3b-[N-(N9,N9-dioleyl)-3-methyl-(methoxycarbonylmethyl)ammonium-propane ("DOMGME"), 1,2-dioleyl-3-N-pyrrolidinium-propane ("DOP5P"), 1,2-dioleyl-3-N-pyridinium-propane bromide salt ("DOP6P"), Cholesterol ("3-( ... -betaine"), N-methyl{4-N-amino[(3'-β-cholesterol)aminomethyl]}piperazine ("N-methyl-PipChol"), hexadecyltrimethylammonium bromide ("CTAB"), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"), 4-(2-aminoethyl)-morpholinyl-cholesterol Cholesterol hemisuccinate ("MoChol"), histamine-cholesterol hemisuccinate ("HisChol"), cholesterol-(3-imidazol-1-ylpropyl) carbamate ("Chim"), (N-2-propylamino[(3'-β-cholesterol)carbamyl]}morpholine ("MoC3Chol"), [(3-morpholine-4-yl-propylcarbamyl) -methyl]-cholesterol carbamate ("Chol-C3N-Mo3"), ("Chol-C3N-Mo2"), [(2-oxolin-4-yl-ethylcarbamoyl)methyl]-cholesterol carbamate ("Chol-C4N-Mo2"), [1-methyl-2-(2-oxolin-4-yl-ethylcarbamoyl)-propyl]-cholesterol carbamate ("Chol-DMC3N-Mo2"), 2-(2-morpholin-4-yl-ethylaminoformyl)-cyclohexanecarboxylic acid cholesterol ester ("CholC4Hex-Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-2,3-dimethyl hemisuccinate ("DmC4Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-2,2-dimethyl Methyl hemimalonate ("DmC3Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemimalonate ("C3Mo2"), 4-(2-aminopropyl)-morpholinyl-cholesterol-hemimalonate ("C3Mo3"), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemiglutarate ("C5Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemiglutarate ("C5Mo2"), morpholinyl-cholesterol-hemisuccinate ("C6Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemisuccinate ("C8Mo2"), 4-(2-aminobutyl)-morpholinyl-cholesterol-hemisuccinate ("C4Mo4"), 4{N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}piperazine ("PipC2Chol ”), {N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}morpholine (“MoC2Chol”), {N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}pyrrolidine (“PyrroC2Chol”), {N-2-propylamino[(3'-β-cholesterol)aminoformyl]}imidazole (“ImC3Chol”) 、{N-2-ethylamino[(3'-β-cholesterol)aminomethyl]}pyridine ("PyC2Chol")、1,2-dioleoyl-3-N-pyroline-propane ("MoDO")、1,2-dipalmitoyl-3-N-pyroline-propane ("MoDP")、4,(2,3-bis-acyloxy-propyl)-1-methyl-1H-imidazole ("DOIM") (also known as DPIM); diacylglycerol hemisuccinates, such as dioctadecylamido-glycidylspermine ("DOGS"), dimyristoylglycerol hemisuccinate ("DMGS") (also known as DMG-Succ), 1-palmitoyl-2-oleylglycerol hemisuccinate ("POGS") (also known as POG-Succ), dipalmitoylglycerol hemisuccinate ("DPGS") (also known as POG-Succ), distearylglycerol hemisuccinate ("DSGS") (also known as DSG-Succ); diacylglycerol hemimalonates, such as dioleylglycerol hemimalonate ("DOGM"), dimyristoylglycerol hemimalonate ("DMGM"); diacylglycerol hemiglutarate, such as dioleylglycerol hemiglutarate ("DOGG"), dimyristoylglycerol hemiglutarate ("DMGG"); diacylglycerol hemiadipate, such as dioleylglycerol hemiadipate ("DOGA"), dimyristoylglycerol hemiadipate ("D MGA”), diacylglycerol hemicyclohexane-1,4-dicarboxylic acid, such as dioleoylglycerol hemicyclohexane-1,4-dicarboxylic acid (“DO-cHA”), dimyristoylglycerol hemicyclohexane-1,4-dicarboxylic acid (“DM-cHA”), (2,3-diacyl-propyl)amino}-oxoalkanoic acid, such as 4-{(2,3-dioleoyl-propyl)amino}-4-oxobutanoic acid (“DOAS”), 3-{(2,3-dioleoyl-propyl)amino}-3-oxopropionic acid ( DOAM), 5-{(2,3-dioleyl-propyl)amino}-5-oxopentanoic acid (“DOAG”), 6-{(2,3-dioleyl-propyl)amino}-6-oxopentanoic acid (“DOAA”), 4-{(2,3-dimyristyl-propyl)amino}-4-oxopentanoic acid (“DMAS”), 3-{(2,3-dimyristyl-propyl)amino}-3-oxopentanoic acid (“DMAM”), 5-{(2,3-dimyristyl-propyl)amino}-6-oxopentanoic acid (“DOAG”), 6-{(2,3-dioleyl-propyl)amino}-6-oxopentanoic acid (“DOAA”), 4-{(2,3-dimyristyl-propyl)amino}-4-oxopentanoic acid (“DMAS”), 3-{(2,3-dimyristyl-propyl)amino}-3-oxopentanoic acid (“DMAM”), )amino}-5-hydroxypentanoic acid ("DMAG"), 6-{(2,3-dimyristyl-propyl)amino}-6-hydroxyhexanoic acid ("DMAA"); diacyl-alkanoic acids, such as 2,3-dioleyl-propionic acid ("DOP"), 3,4-dioleyl-butyric acid ("DOB"), 5,6-dioleyl-hexanoic acid ("DOS"), 4,5-dioleyl-pentanoic acid ("DOM"), 6,7-dioleyl-heptanoic acid ("DOG"), 7,8-dioleyl-octanoic acid ("DOB"), 1,2-dioleyl-1,2-dioleyl-2,3-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-2,3-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-3,4-dioleyl-4,5-dioleyl- (“DOA”), 2,3-dimyristyl-propionic acid (“DMP”), 3,4-dioleyl-butyric acid (“DOB”), 5,6-dimyristyl-hexanoic acid (“DMS”), 4,5-dimyristyl-pentanoic acid (“DMM”), 6,7-dimyristyl-heptanoic acid (“DMG”), 7,8-dimyristyl-octanoic acid (“DMA”); cholesterol oxycarbonylcarbamates, such as cholesterol hemidodecanedicarboxylic acid (“Chol-C12”), 12-cholesterol Steroloxycarbonylaminododecanoic acid ("CholC13N"); fatty acids, such as oleic acid, myristic acid, palmitic acid, stearic acid, cervical acid, behenic acid; dioleylphosphatidic acid ("DOPA"), 1,2-dimyristyl-sn-glycero-3-phosphate ("DMPA"), 1,2-dipalmitoyl-sn-glycero-3-phosphate ("DPPA"), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate ("POPA"), 1,2-distearyl sn-glycerol-3-phosphate (DSPA), cholesterol sulfate (Chol-SO4), dioleoylphosphatidylglycerol (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-racemic-glycerol), (DMPG), dimalmitoylphosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-distearyl-sn-glycero-3-phosphoglycerol (POPG), Phospho-rac-glycerol ("DSPG"), dioleylphosphatidylserine ("DOPS"), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine ("DMPS"), dimalmitoylphosphatidylserine ("DPPS"), 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-L-serine ("POPS"), 1,2-distearyl-sn-glycero-3-phospho-L-serine ("DSPS") or hexadecyl phosphate. In some aspects, one or more of the charged lipids in the lipid particle is selected from the group consisting of cholesterol hemisuccinate ("CHEMS"), 1,2-distearoyl-3-trimethylammonium-propane ("DSTAP"), 18:1 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP"), 1,2-dioleoyl-sn-glycero-3-phosphate ("18:1 PA"), 18:1 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) ("18:1 PG"), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-butyrylphosphatidylethanolamine, N-pentylphosphatidylethanolamine, phosphatidylglycerol and palmitoyloleylphosphatidylglycerol ("POPG"). In some aspects, the lipid particles described herein contain only one charged lipid. In some aspects, the only charged lipid is CHEMS.

In some aspects, the lipid particles disclosed herein contain CHEMS. In some aspects, the molar amount of CHEMS in the lipid particles disclosed herein is 10%. In some aspects, the molar amount of CHEMS in the lipid particles disclosed herein is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.1%. In some aspects, the molar amount of CHEMS in the lipid particles disclosed herein is about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4 %, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, or about 5% to about 6%. In some aspects, the molar amount of CHEMS in the lipid particles disclosed herein is about 0.1%, about 0.25%, about 0.5%, about 0.7%, about 1%, about 1.25%, about 1.5%, about 1.75%, about 2%, about 2.25%, about 2.5%, about 2.75%, about 3%, about 3.25%, about 3.5%, about 3.75%, about 4%, about 4.25%, about 4.5%, about 4.75%, about 5%, about 5.25%, about 5.5%, about 5.75%, about 6%, about 6.25%, about 6.5%, about 6.75%, about 7%, about 7.25%, about 7.5%, about 7.75%, about 8%, about 8.25%, about 8.5%, about 8.75%, about 9%, about 9.25%, about 9.5%, about 9.75% or about 10%. In some aspects, the lipid particles described herein do not contain charged lipids. In some embodiments, the lipid particles disclosed herein do not contain CHEMS.

In some aspects, CHEMS is included in the lipid particles disclosed herein in a molar amount sufficient to deliver the effective load delivered by the lipid particles to the spleen, such as at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%. In some aspects, the molar amount of CHEMS in the lipid particles disclosed herein that delivers the effective load to the spleen is about 11% to about 45%, about 11% to about 40%, about 11% to about 35%, about 11% to about 30%, about 11% to about 25%, about 11% to about 20%, about 11% to about 15%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15 ... From about 15% to about 20%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%, from about 20% to about 25%, from about 25% to about 45%, from about 25% to about 40%, from about 25% to about 35%, from about 25% to about 30%, from about 30% to about 45%, from about 30% to about 40%, from about 30% to about 35%, from about 35% to about 45%, from about 35% to about 40%, or from about 40% to about 45%. In some aspects, the molar amount of CHEMS in the lipid particles disclosed herein that delivers a payload to the spleen is about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some embodiments, the lipid particles that deliver the payload to the spleen contain about 11% to about 20%, about 20% to about 30%, or about 11% to about 30% CHEMS in a molar amount. In some embodiments, the lipid particles contain about 11% to about 20% CHEMS in a molar amount. In some embodiments, the lipid particles contain about 11% CHEMS in a molar amount. In some embodiments, the lipid particles contain about 15% CHEMS in a molar amount. In some embodiments, the lipid particles contain about 20% CHEMS in a molar amount. In some embodiments, the lipid particles contain about 25% CHEMS in a molar amount. v. Stealth Lipids

The lipid particles disclosed herein may include one or more stealth lipids. In some aspects, the molar amount of one or more stealth lipids in the lipid particles described herein is at least 0.1%, at least 0.15%, at least 0.2% or at least 0.25%. In some aspects, the molar amount of one or more stealth lipids in the lipid particles described herein is about 0.1% to about 3%, about 0.1% to 2.75%, about 0.1% to about 2.5%, about 0.1% to about 2.25%, about 0.1% to about 2%, about 0.1% to about 1.75%, about 0.1% to about 1.5%, about 0.1% to about 1.25%, about 0.1% to about 1%, about 0.1% to about 0.75%, about 0.1% to about 0.5%, about 0.1% to about 0.25%, about 0.25% to about 3%, about 0.25% to about 2.75%, about 0.25% to about 2.5%, about 0.25% to about 2.25%, about 0.25% to about 2%, about 0. 25% to about 1.75%, about 0.25% to about 1.5%, about 0.25% to about 1.25%, about 0.25% to about 1%, about 0.25% to about 0.75%, about 0.25% to about 0.5%, about 0.5% to about 3%, about 0.5% to about 2.75%, about 0.5% to about 2.5%, about 0.5% to about 2.25%, about 0.5% to about 2%, about 0.5% to about 1.75%, about 0.5% to about 1.5%, about 0.5% to about 1.25%, about 0.5% to about 1%, about 0.5% to about 0.75%, about 0.75% to about 3%, about 0.75% to about 2.75%, about 0.75% to about 2.5%, about 0.75% to about 2%, about 0.75% to about 1.75%, about 0.75% to about 1.5%, about 0.75% to about 1.25%, about 0.75% to about 1%, about 1% to about 3%, about 1% to about 2.75%, about 1% to about 2.5%, about 1% to about 2.25%, about 1% to about 2%, about 1% to about 1.75%, about 1% to about 1.5%, about 1% to about 1.25%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1.5% to about 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 1.75% to about 3%, about 1.75% to about 2.75%, about 1.75% to about 2.5%, about 1.75% to about 2.25%, about 1.75% to about 2%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25%, about 2.25% to about 3%, about 2.25% to about 2.75%, about 2.25% to about 2.5%, about 2.5% to about 3%, about 2.5% to about 2.75%, about 2.75% to about 3%. In some aspects, the molar amount of one or more stealth lipids in the lipid particles described herein is about 1% to about 3%. In some aspects, the molar amount of one or more stealth lipids in the lipid particles described herein is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, or about 3%. In some aspects, the molar amount of one or more stealth lipids in the lipid particles described herein is about 1%. In some aspects, the molar amount of one or more stealth lipids in the lipid particles described herein is about 1.5%. In some aspects, the molar amount of one or more stealth lipids in the lipid particles described herein is about 2%. In some aspects, the molar amount of one or more stealth lipids in the lipid particles described herein is about 2.5%.

In some aspects, one or more stealth lipids in the lipid particles disclosed herein include one or more PEG-terminated lipids, one or more poly(saccharoline) derivatives, or a combination thereof. In some aspects, only one or more PEG-terminated lipids are present. PEG-terminated lipids are a class of polyethylene ("PEG") derivatives attached to a lipid portion.

In some aspects, the molar amount of one or more PEG-capped lipids in the lipid particles described herein is at least 0.1%, at least 0.15%, at least 0.2%, or at least 0.25%. In some aspects, the molar amount of one or more PEG-capped lipids in the lipid particles described herein is about 0.1% to about 3%, about 0.1% to 2.75%, about 0.1% to about 2.5%, about 0.1% to about 2.25%, about 0.1% to about 2%, about 0.1% to about 1.75%, about 0.1% to about 1.5%, about 0.1% to about 1.25%, about 0.1% to about 1%, about 0.1% to about 0.75%, about 0.1% to about 0.5%, about 0.1% to about 0.25%, about 0.25% to about 3%, about 0.25% to about 2.75%, about 0.25% to about 2.5%, about 0.25% to about 2.25%, about 0.25% to about 2%, about 0.25% to about 1.75%, about 0.25% to about 1.5%, about 0.25% to about 1.25%, about 0.25% to about 1%, about 0.25% to about 0.75%, about 0.25% to about 0.5%, about 0.5% to about 3%, about 0.5% to about 2.75%, about 0.5% to about 2.5%, about 0.5% to about 2.25%, about 0.5% to about 2%, about 0.5% to about 1.75%, about 0.5% to about 1.5%, about 0.5% to about 1.25%, about 0.5% to about 1%, about 0.5% to about 0.75%, about 0.75% to about 3%, about 0.75% to about 2.75%, about 0.75% to about 2.5%, about 0.75% to about 2.5%, about 0.75% to about about 2%, about 0.75% to about 1.75%, about 0.75% to about 1.5%, about 0.75% to about 1.25%, about 0.75% to about 1%, about 1% to about 3%, about 1% to about 2.75%, about 1% to about 2.5%, about 1% to about 2.25%, about 1% to about 2%, about 1% to about 1.75%, about 1% to about 1.5%, about 1% to about 1.25%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1.5 % to about 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 1.75% to about 3%, about 1.75% to about 2.75%, about 1.75% to about 2.5%, about 1.75% to about 2.25%, about 1.75% to about 2%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25%, about 2.25% to about 3%, about 2.25% to about 2.75%, about 2.25% to about 2.5%, about 2.5% to about 3%, about 2.5% to about 2.75%, about 2.75% to about 3%. In some aspects, the molar amount of one or more PEG-capped lipids in the lipid particles described herein is about 1% to about 3%. In some aspects, the molar amount of one or more PEG-capped lipids in the lipid particles described herein is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5% or about 3%. In some aspects, the molar amount of one or more PEG-capped lipids in the lipid particles described herein is about 1%. In some aspects, the molar amount of one or more PEG-capped lipids in the lipid particles described herein is about 1.5%. In some aspects, the molar amount of one or more PEG-capped lipids in the lipid particles described herein is about 2%. In some aspects, the molar amount of one or more PEG-capped lipids in the lipid particles described herein is about 2.5%.

One or more PEG-terminated lipids in the lipid particles may be one or more selected from the group consisting of: 1,2-dimyristyl-rac-glycero-3-methoxypolyethylene glycol-2000 ("DMG-PEG2000"), distearyl-rac-glycero-PEG2K ("DSG-PEG2k"), 2-(2-methoxyethoxycarbonylamino)ethyl phosphate [(2R)-2,3-dioctadecanoyloxy)propyl] ester ("C18-mPEG2000"), tetradecanoic acid [3-[3-(2-methoxyethoxy)propylaminomethyloxy]-2-tetradecanoyloxypropyl] ester ("PEG2000-c-DMG"), tetradecanoic acid 3-[hydroxy-[2-[2-(2-methoxyethoxy)ethylamino]ethoxy]phosphoyl]oxy-2-tetradecanoyloxypropyl] ester ("DMPE-PEG2000"), 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ("18:1 In some embodiments, the lipid particles described herein contain only one PEG-terminated lipid.

In some aspects, the only PEG-terminated lipid in the lipid particles described herein is DMG-PEG 2000. In some aspects, the molar amount of DMG-PEG 2000 is at least 0.1%, at least 0.15%, at least 0.2%, or at least 0.25%. In some aspects, the molar amount of DMG-PEG2000 is about 0.1% to about 3%, about 0.1% to 2.75%, about 0.1% to about 2.5%, about 0.1% to about 2.25%, about 0.1% to about 2%, about 0.1% to about 1.75%, about 0.1% to about 1.5%, about 0.1% to about 1.25%, about 0.1% to about 1%, about 0.1% to about 0.75%, about 0.1% to about 0.5%, about 0.1% to about 0.25%, about 0.25% to about 3%, about 0.25% to about 2.75%, about 0.25% to about 2.5%, about 0.25% to about 2.25%, about 0.25% to about 2%, about 0.25% to about 1 .75%, about 0.25% to about 1.5%, about 0.25% to about 1.25%, about 0.25% to about 1%, about 0.25% to about 0.75%, about 0.25% to about 0.5%, about 0.5% to about 3%, about 0.5% to about 2.75%, about 0.5% to about 2.5%, about 0.5% to about 2.25%, about 0.5% to about 2%, about 0.5% to about 1.75%, about 0.5% to about 1.5%, about 0.5% to about 1.25%, about 0.5% to about 1%, about 0.5% to about 0.75%, about 0.75% to about 3%, about 0.75% to about 2.75%, about 0.75% to about 2.5%, about 0.75% to about 2%, about 0.75% to about 1.75%, about 0.75% to about 1.5%, about 0.75% to about 1.25%, about 0.75% to about 1%, about 1% to about 3%, about 1% to about 2.75%, about 1% to about 2.5%, about 1% to about 2.25%, about 1% to about 2%, about 1% to about 1.75%, about 1% to about 1.5%, about 1% to about 1.25%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1.5% to about In some aspects, the molar amount of DMG-PEG2000 is about 1% to about 3%. In some aspects, the molar amount of DMG-PEG2000 is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5% or about 3%. In some aspects, the molar amount of DMG-PEG2000 is about 1%. In some aspects, the molar amount of DMG-PEG2000 is about 1.5%. In some aspects, the molar amount of DMG-PEG2000 is about 2%. In some aspects, the molar amount of DMG-PEG2000 is about 2.5%.

In some aspects, the only PEG-terminated lipid in the lipid particles described herein is DMPE-PEG 2000. In some aspects, the molar amount of DMPE-PEG 2000 is at least 0.1%, at least 0.15%, at least 0.2%, or at least 0.25%. In some aspects, the molar amount of DMPE-PEG2000 is about 0.1% to about 3%, about 0.1% to 2.75%, about 0.1% to about 2.5%, about 0.1% to about 2.25%, about 0.1% to about 2%, about 0.1% to about 1.75%, about 0.1% to about 1.5%, about 0.1% to about 1.25%, about 0.1% to about 1%, about 0.1% to about 0.75%, about 0.1% to about 0.5%, about 0.1% to about 0.25%, about 0.25% to about 3%, about 0.25% to about 2.75%, about 0.25% to about 2.5%, about 0.25% to about 2.25%, about 0.25% to about 2%, about 0.25% to about 1 .75%, about 0.25% to about 1.5%, about 0.25% to about 1.25%, about 0.25% to about 1%, about 0.25% to about 0.75%, about 0.25% to about 0.5%, about 0.5% to about 3%, about 0.5% to about 2.75%, about 0.5% to about 2.5%, about 0.5% to about 2.25%, about 0.5% to about 2%, about 0.5% to about 1.75%, about 0.5% to about 1.5%, about 0.5% to about 1.25%, about 0.5% to about 1%, about 0.5% to about 0.75%, about 0.75% to about 3%, about 0.75% to about 2.75%, about 0.75% to about 2.5%, about 0.75% to about 2%, about 0.75% to about 1.75%, about 0.75% to about 1.5%, about 0.75% to about 1.25%, about 0.75% to about 1%, about 1% to about 3%, about 1% to about 2.75%, about 1% to about 2.5%, about 1% to about 2.25%, about 1% to about 2%, about 1% to about 1.75%, about 1% to about 1.5%, about 1% to about 1.25%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1.5% to about 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 1.75% to about 3%, about 1.75% to about 2.75%, about 1.75% to about 2.5%, about 1.75% to about 2.25%, about 1.75% to about 2%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25%, about 2.25% to about 3%, about 2.25% to about 2.75%, about 2.25% to about 2.5%, about 2.5% to about 3%, about 2.5% to about 2.75%, about 2.75% to about 3%. In some aspects, the molar amount of DMPE-PEG2000 is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5% or about 3%. In some aspects, the molar amount of DMPE-PEG2000 is about 1%. In some aspects, the molar amount of DMPE-PEG2000 is about 1.5%. In some aspects, the molar amount of DMPE-PEG2000 is about 2%. In some aspects, the molar amount of DMPE-PEG2000 is about 2.5%.

In some aspects, the only PEG-terminated lipid in the lipid particles described herein is 18:1 PEG2000-PE. In some aspects, the molar amount of 18:1 PEG2000-PE is at least 0.1%, at least 0.15%, at least 0.2%, or at least 0.25%. In some aspects, the molar amount of 18:1 PEG2000-PE is about 0.25% to about 3%, about 0.5% to about 3%, about 1% to about 3%, about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 0.25% to about 2.5%, about 0.5% to about 2.5%, about 1% to about 2.5%, about 1.5% to about 2.5%, about 2% to about 2.5%, about 0.25% to about 2%, about 0.5% to about 2%, about 1% to about 2%, about 1.5% to about 2%, about 0.25% to about 1.5%, about 0.5% to about 1.5%, or about 1% to about 1.5%. In some aspects, the molar amount of 18:1 PEG2000-PE is about 1% to about 3%. In some aspects, the molar amount of 18:1 PEG2000-PE is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, or about 3%. In some aspects, the molar amount of 18:1 PEG2000-PE is about 1%. In some aspects, the molar amount of 18:1 PEG2000-PE is about 1.5%. In some aspects, the molar amount of 18:1 PEG2000-PE is about 2%. In some aspects, the molar amount of 18:1 PEG2000-PE is about The molar amount of PEG2000-PE is about 2.5%, the molar amount is about 0.1% to about 3%, about 0.1% to 2.75%, about 0.1% to about 2.5%, about 0.1% to about 2.25%, about 0.1% to about 2%, about 0.1% to about 1.75%, about 0.1% to about 1.5%, about 0.1% to about 1.25%, about 0.1% to about 1%, about 0.1% to about 0.75%, about 0.1% to about 0.5%, about 0.1% to about 0.25%, about 0.25% to about 3%, about 0.25% to about 2.75%, about 0.25% to about 2.5%, about 0.25% to about 2.25%, about 0.25% to about 2%, about 0.25% to about 1.75%, about 0.25% to about 1.5%, about 0.25% to about 1.25%, about 0.25% to about 1%, about 0.25% to about 0.75%, about 0.25% to about 0.5%, about 0.5% to about 3%, about 0.5% to about 2.75%, about 0.5% to about 2.5%, about 0.5% to about 2.25%, about 0.5% to about 2%, about 0.5% to about 1.75%, about 0.5% to about 1.5%, about 0.5% to about 1.25%, about 0.5% to about 1%, about 0.5% to about 0.75%, about 0.75% to about 3%, about 0.75% to about 2.75%, about 0.75% to about 2.5%, about 0.75% to about 2%, about 0.75% to about 1.75%, about 0.75% to about 1.5%, about 0.75% to about 1.25%, about 0.75% to about 1%, about 1% to about 3%, about 1% to about 2.75%, about 1% to about 2.5%, about 1% to about 2.25%, about 1% to about 2%, about 1% to about 1.75%, about 1% to about 1.5%, about 1% to about 1.25%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1% to about 1.25%, about 1.25% to about 3%, about 1.25% to about 2.75%, about 1.25% to about 2.5%, about 1.25% to about 2.25%, about 1.25% to about 2%, about 1.25% to about 1.75%, about 1.25% to about 1.5%, about 1.5% to about About 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 1.75% to about 3%, about 1.75% to about 2.75%, about 1.75% to about 2.5%, about 1.75% to about 2.25%, about 1.75% to about 2%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25%, about 2.25% to about 3%, about 2.25% to about 2.75%, about 2.25% to about 2.5%, about 2.5% to about 3%, about 2.5% to about 2.75%, about 2.75% to about 3%.

In some aspects, one or more stealth lipids in the lipid particles disclosed herein comprise one or more poly(isosinoic acid) derivatives or combinations thereof. In some aspects, one or more polysarcosine derivatives in the lipid particle are one or more selected from the group consisting of: N-tetradecyl-polysarcosine-25 ("N-tetradecyl-pSar25"), N-hexadecyl-polysarcosine-25 ("N-hexadecyl-pSar25"), N-octadecyl-polysarcosine-25, N-dodecyl-polysarcosine-25 ("N-octadecyl-pSar25"), 1,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 ("DMG-pSar25"), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-polysarcosine-25 (ammonium salt) ("18:1 PE (DOPE), pSar25”), N,N-ditetradecylamine-N-succinyl[methyl(poly(saccharin)45] (“N-TETAMINE-pSar45”), N,N-ditetradecylamine-N-succinyl[methyl(poly(saccharin)35] (“N-TETAMINE-pSar35”), N,N-ditetradecyl-poly(saccharin)-25 (“N-TETAMINE-pSar25”), N-TETAMINE-pSar45-butene diimide or N-TETAMINE-PEOZ-40. In some embodiments, only one poly(saccharin) derivative is in the lipid particle. b. Exemplary lipid particles

In some aspects, the lipid particles described herein comprise about 25% to about 45% SS-OP, about 5% to about 25% DOPE, about 15% to about 35% cholesterol, about 10% to about 30% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 35% SS-OP, about 15% DOPE, about 28.5% cholesterol, about 20% CHEMS, and about 1.5% DMG-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 25% to about 45% SS-OP, about 5% to about 25% DODG, about 15% to about 35% cholesterol, about 10% to about 30% CHEMS, and about 0.5% to about 2.5% DMPE-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 35% SS-OP, about 15% DODG, about 28.5% cholesterol, about 20% CHEMS, and about 1.5% DMPE-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 25% to about 45% SS-OP, about 5% to about 25% DOPE, about 15% to about 35% cholesterol, about 10% to about 30% CHEMS, and about 0.5% to about 2.5% DMPE-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 35% SS-OP, about 15% DOPE, about 28.5% cholesterol, about 20% CHEMS, and about 1.5% DMPE-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 25% to about 45% SS-OP, about 5% to about 25% DODG, about 15% to about 35% cholesterol, about 10% to about 30% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles comprise about 35% SS-OP, about 15% DODG, about 28.5% cholesterol, about 20% CHEMS, and about 1.5% DMG-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 25% to about 45% SS-OP, about 5% to about 25% DODG, about 15% to about 35% cholesterol, about 10% to about 30% CHEMS, and about 0.5% to about 2.5% 18:1 PEG2000-PE (in molar amounts). In some aspects, the lipid particles described herein comprise about 35% SS-OP, about 15% DODG, about 28.5% cholesterol, about 20% CHEMS, and about 1.5% 18:1 PEG2000-PE (in molar amounts).

In some aspects, the lipid particles described herein comprise about 25% to about 45% SS-OP, about 5% to about 25% DOPE, about 15% to about 35% cholesterol, about 10% to about 30% CHEMS, and about 0.5% to about 2.5% 18:1 PEG2000-PE (in molar amounts). In some aspects, the lipid particles described herein comprise about 35% SS-OP, about 15% DOPE, about 28.5% cholesterol, about 20% CHEMS, and about 1.5% 18:1 PEG2000-PE (in molar amounts).

In some aspects, the lipid particles described herein comprise about 30% to about 50% SS-OP, about 15% to about 55% cholesterol, about 10% to about 30% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles comprise about 40% SS-OP, about 38.5% cholesterol, about 20% CHEMS, and about 1.5% DMG-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 20% to about 45% SS-OP, about 15% to about 55% cholesterol, about 15% to about 35% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 35% SS-OP, about 38.5% cholesterol, about 25% CHEMS, and about 1.5% DMG-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 20% to about 40% SS-OP, about 15% to about 55% cholesterol, about 20% to about 40% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 30% SS-OP, about 38.5% cholesterol, about 30% CHEMS, and about 1.5% DMG-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 15% to about 35% SS-OP, about 15% to about 55% cholesterol, about 25% to about 45% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 25% SS-OP, about 38.5% cholesterol, about 35% CHEMS, and about 1.5% DMG-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 30% to about 50% SS-OP, about 15% to about 55% cholesterol, about 15% to about 35% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 40% SS-OP, about 33.5% cholesterol, about 25% CHEMS, and about 1.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 30% to about 50% SS-OP, about 15% to about 55% cholesterol, about 20% to about 40% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 40% SS-OP, about 28.5% cholesterol, about 30% CHEMS, and about 1.5% DMG-PEG2000 (by molar amount).

In some aspects, the lipid particles described herein comprise about 40% to about 60% SS-OP, about 1% to about 20% DODG, about 25% to about 45% cholesterol, about 1% to about 10% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 50% SS-OP, about 10% DODG, about 38.5% cholesterol, about 10% CHEMS, and about 1.5% DMG-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 40% to about 60% SS-OP, about 1% to about 20% DODG, about 25% to about 45% cholesterol, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 50% SS-OP, about 10% DODG, about 38.5% cholesterol, and about 1.5% DMG-PEG2000 (in molar amounts).

In some aspects, the lipid particles described herein comprise about 40% to about 60% SS-OP, about 1% to about 20% DODG, about 25% to about 45% cholesterol, and about 0.5% to about 2.5% DMG-PEG2000 (in molar amounts). In some aspects, the lipid particles described herein comprise about 50% SS-OP, about 10% DODG, about 38.5% cholesterol, and about 1.5% DMG-PEG2000 (in molar amounts).

The lipid particles described herein can be contained in a composition. The composition comprises lipid particles having an average particle size of less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 85 nm, less than 80 nm, or less than 75 nm. In some aspects, the composition contains an average particle size of about 50 nm to about 150 nm, about 50 nm to about 140 nm, about 50 nm to about 140 nm, about 50 nm to about 130 nm, about 50 nm to about 120 nm, about 50 nm to about 110 nm, about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 60 nm to about 150 nm, about 60 nm to about 140 nm, about 60 nm to about 140 nm, about 60 nm to about 130 nm, about 60 nm to about 120 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 70 nm to about 150 nm, about 70 nm to about 140 nm, about 70 nm to about 140 nm, about 70 nm to about 130 nm, about 70 nm to about 120 nm, In some embodiments, the size of the lipid particles is determined by dynamic light scattering using a NanoBrook Omni instrument from Brookhaven Instruments.

The composition comprises lipid particles having an encapsulation efficiency of less than 99.9%, less than 99.5%, less than 99%, less than 98.5%, less than 98%, less than 97.5%, less than 97%, less than 96.5%, less than 96%, less than 95.5%, or less than 95%. In some aspects, the encapsulation efficiency is about 75% to about 99.9%, about 80% to about 99.9%, about 85% to about 99.9%, about 90% to about 99.9%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, or about 75% to about 90%. In some aspects, the encapsulation efficiency is about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In some aspects, the encapsulation efficiency is determined by QuantiT Ribogreen assay. In some aspects, the encapsulation efficiency is determined by LC-MS. III. Payload

The lipid particles described herein may include a payload. The payload may be a biologically active molecule. In some aspects, the biologically active molecule includes a nucleic acid, a small molecule, a peptide, a protein, or any combination thereof.

In some aspects, the lipid particles described herein comprise a nucleic acid. Examples of nucleic acids include, but are not limited to, small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), primary micro RNA (pri-miRNA), long noncoding RNA (lncRNA), messenger RNA (mRNA), clustered regularly interspaced repeats (CRISPR)-related nucleic acid, CRISPR-RNA (crRNA), single guide RNA (sgRNA), trans-activating CRISPR RNA (tracrRNA), plasmid deoxyribonucleic acid (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), antisense RNA (RNA), guide RNA, deoxyribonucleic acid (DNA), double-stranded deoxyribonucleic acid (dsDNA), single-stranded deoxyribonucleic acid (ssDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), CRISPR-associated (Cas) protein, or a combination thereof.

In some aspects, the lipid particles described herein include nucleic acids as mRNA. In some aspects, the nucleic acid encodes a peptide with therapeutic activity. The peptide with therapeutic activity may include an antigenic determinant amino acid sequence. The peptide may also induce immune tolerance of an individual (e.g., a human) to a cell. The lipid particles or compositions described herein deliver a payload (e.g., a nucleic acid) in vivo to a target organ of interest of an individual. In some aspects, the lipid particles or compositions described herein deliver a payload (e.g., a nucleic acid) in vivo to a target organ of interest of an individual, and the target organ is the spleen. In some aspects, the ratio of payload delivered to the spleen to the liver is greater than 1.1. In some aspects, the ratio of the effective load delivered to the spleen to the liver is about 1.1 to about 12, about 1.1 to about 11, about 1.1 to about 10, about 1.1 to about 9, about 1.1 to about 8, about 1.1 to about 7, about 1.1 to about 6, about 1.1 to about 5, about 1.1 to about 4, about 1.5 to about 12, about 1.5 to about 11, about 1.5 to about 10, about 1.5 to about 9, about 1.5 to about 8, about 1.5 to about 7, about 1.5 to about 6, about 1.5 to about 5 , about 1.5 to about 4, about 2 to about 12, about 2 to about 11, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2.5 to about 12, about 2.5 to about 11, about 2.5 to about 10, about 2.5 to about 9, about 2.5 to about 8, about 2.5 to about 7, about 2.5 to about 6, about 2.5 to about 5, about 2.5 to about 4, about 3 to about 12, about 3 to about 11, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 3.5 to about 12, about 3.5 to about 11, about 3.5 to about 10, about 3.5 to about 9, about 3.5 to about 8, about 3.5 to about 7, about 3.5 to about 6, about 3.5 to about 5, about 4 to about 12, about 4 to about 11, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 5 to about 12, about 5 to about 11, From about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5 to about 6, about 6 to about 12, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 7 to about 12, about 7 to about 11, about 7 to about 10, about 7 to about 9, about 7 to about 8, about 8 to about 12, about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 to about 12, about 9 to about 11, about 9 to about 10, about 10 to about 12, or about 10 to about 11.

The ratio of the effective load of the spleen and the liver is determined by comparing the BLI value of the effective load of luciferase mRNA in the spleen with the BLI value in the liver. In some aspects, the BLI values in the spleen and the liver are determined by imaging the organ of interest. In some aspects, the BLI values in the spleen and the liver are determined by imaging the organ of interest using an imaging system. In some aspects, software is used to determine the BLI value based on the image generated by the imaging system. In some aspects, the BLI values in the spleen and the liver are determined by imaging the organ of interest using a LICOR Pearl imaging system. In some aspects, the software used to determine the BLI value is software provided by LICOR. In some aspects, the software used to determine the BLI value is Image Studio software from LICOR. In some aspects, the software used to determine the BLI value is Image Studio software from LICOR version 5.2.5.

In some aspects, the ratio of spleen to liver payload is determined by homogenizing organs from individuals (e.g., mice) and quantifying the luciferase signal using a plate-based luciferase assay.

In some aspects, the lipid particles or compositions described herein deliver a payload (e.g., a nucleic acid) intravitally to a target organ of interest of an individual that is not the spleen. In some aspects, the lipid particles or compositions described herein deliver a payload (e.g., a nucleic acid) intravitally to the liver of an individual. In some aspects, the lipid particles or compositions described herein deliver a payload (e.g., a nucleic acid) intravitally to the pancreas of an individual. In some aspects, the lipid particles or compositions described herein deliver a payload (e.g., a nucleic acid) intravitally to the lymph nodes of an individual. In some aspects, the lipid particles or compositions described herein deliver a payload (e.g., a nucleic acid) intravitally to the brain of an individual. In some aspects, the lipid particles or compositions described herein deliver a payload (e.g., a nucleic acid) intravitally to the muscle cells of an individual. In some aspects, the lipid particles or compositions described herein deliver a payload (e.g., a nucleic acid) to the lungs of a subject in vivo.

In some aspects, the ratio of the effective load delivered to the liver to the spleen is greater than 1.1. In some aspects, the ratio of the effective load delivered to the liver to the spleen is about 1.1 to about 12, about 1.1 to about 11, about 1.1 to about 10, about 1.1 to about 9, about 1.1 to about 8, about 1.1 to about 7, about 1.1 to about 6, about 1.1 to about 5, about 1.1 to about 4, about 1.5 to about 12, about 1.5 to about 11, about 1.5 to about 10, about 1.5 to about 9, about 1.5 to about 8, about 1.5 to about 7, about 1.5 to about 6, about 1.5 to about 5 , about 1.5 to about 4, about 2 to about 12, about 2 to about 11, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2.5 to about 12, about 2.5 to about 11, about 2.5 to about 10, about 2.5 to about 9, about 2.5 to about 8, about 2.5 to about 7, about 2.5 to about 6, about 2.5 to about 5, about 2.5 to about 4, about 3 to about 12, about 3 to about 11, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 3.5 to about 12, about 3.5 to about 11, about 3.5 to about 10, about 3.5 to about 9, about 3.5 to about 8, about 3.5 to about 7, about 3.5 to about 6, about 3.5 to about 5, about 4 to about 12, about 4 to about 11, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 5 to about 12, about 5 to about 11, From about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5 to about 6, about 6 to about 12, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 7 to about 12, about 7 to about 11, about 7 to about 10, about 7 to about 9, about 7 to about 8, about 8 to about 12, about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 to about 12, about 9 to about 11, about 9 to about 10, about 10 to about 12, or about 10 to about 11.

The ratio of the effective load of the liver to the spleen is determined by comparing the BLI value in the spleen to the BLI value in the liver. In some aspects, the BLI values in the spleen and liver are determined by imaging the organ of interest.

In some embodiments, the BLI values in the spleen and liver are determined by imaging the organs of interest using an imaging system. In some embodiments, software is used to determine the BLI values based on images generated by the imaging system. In some embodiments, the BLI values in the spleen and liver are determined by imaging the organs of interest using a LICOR Pearl imaging system. In some embodiments, the software used to determine the BLI values is software provided by LICOR. In some embodiments, the software used to determine the BLI values is Image Studio software from LICOR. In some embodiments, the software used to determine the BLI values is Image Studio software from LICOR version 5.2.5.

In some embodiments, the ratio of spleen to liver payload is determined by homogenizing organs from an individual (e.g., mouse) and quantifying the luciferase signal using a plate-based luciferase assay. IV. Pharmaceutical Compositions

The present disclosure provides pharmaceutical compositions (e.g., tolerogenic compositions) comprising one or more lipid particles described herein, the one or more lipid particles comprising one or more payloads (e.g., nucleic acids encoding one or more peptides or antigenic determinants). In some aspects, the peptides or antigenic determinants constitute fragments of complete antigenic proteins. Such pharmaceutical compositions are also referred to as minigene vaccines.

In some aspects, at least one nucleic acid included in the lipid particles disclosed herein encodes a plurality of different peptides. The nucleic acid can be engineered to insert a protease cleavage site and/or a ribosome skipping element between the peptides so that the encoded peptides are processed to produce independent peptides. Such polynucleotide constructs (also referred to as "strings") can be used as pharmaceutical compositions (see, e.g., Velders et al., J. Immunol . (2001) 166:5366-73; Schubert et al., Genome Medicine (2016) 8:9). In some aspects, the pharmaceutical composition comprises the lipid particles described herein, the lipid particles comprising one or more polynucleotide constructs, the one or more polynucleotide constructs encoding one or more peptides having one or more protease cleavage sites and/or ribosome skipping elements between the peptides. Both coding strands and non-coding strands can be incorporated into the nucleic acid included in the lipid particles disclosed herein. In some aspects, the string refers to a polynucleotide chain that tandemly encodes multiple peptides. In some aspects, the string encodes 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 50 to 100, 50 to 90, 50 to 80, 50 to 70, or 50 to 60 peptides. In some embodiments, the peptides are arranged on a string to maximize recognition by a certain HLA. In some embodiments, the antigenic determinant coding sequence in the string construct is flanked by one or more sequences selected for better cleavage of peptide presentation to MHC, better expression and/or improved translation in the cells of an individual. The flanking sequences may comprise ribosomal skipping elements such as T2A, P2A, F2A or E2A sequences. In some embodiments, one or more cleavage sequences are selected from the group consisting of FRAC, KRCF, KKRY, ARMA, RRSG, MRAC, KMCG, ARCA, KKQG, YRSY, SFMN, FKAA, KRNG, YNSF, KKNG, RRRG, KRYS and ARYA. In some aspects, the amino acid sequence encoded by the string construct further comprises a signal protein sequence. In some aspects, the string construct can be mRNA. In some aspects, the lipid particles described herein can include one or more mRNA string constructs.

In some embodiments, the pharmaceutical composition comprises a lipid particle containing a nucleic acid, the nucleic acid comprising RNA encoding one or more antigenic peptides and/or T cell antigenic determinants. In some embodiments, the RNA is a messenger RNA (mRNA). The RNA may further comprise one or more chemical modifications as appropriate. For example, the RNA may comprise a 5' and/or 3' non-translated region (UTR) and may be further stabilized by a 7-methylguanosine (m7G) 5' cap and a 3' poly (A) tail and/or a suitable poly (A) sequence, respectively. In some embodiments, the mRNA comprises modified nucleosides, such as pseudouridine (Ψ), 1-methyl pseudouridine (m1Ψ) and 5-methylcytidine (m5C). In some aspects, the RNA comprises a self-amplifying RNA (saRNA), which further encodes an RNA-dependent RNA polymerase that can be translated in situ, thereby amplifying a peptide-encoding sequence or an antigen-encoding sequence in situ (see, e.g., Bloom et al., Gene Therapy (2021) 28: 117-29). An overview of mRNA compositions (such as mRNA cancer vaccines) is provided, e.g., in Miao, L. et al., Molecular Cancer (2021) 20: 41.

In some aspects, the pharmaceutical composition comprises lipid particles as described herein, wherein the lipid particles comprise an antigenic peptide and a nucleic acid encoding the peptide. In some aspects, the antigenic peptide is linked to the nucleic acid.

In some aspects, the lipid particles deliver a payload (e.g., a nucleic acid) to a target cell (e.g., a tolerogenic target cell). After the target cell internalizes the peptide, it can present the peptide on the cell surface with a cognate MHC, thereby stimulating a tolerogenic T cell response. The lipid particles described herein deliver nucleic acids to a target cell (e.g., a tolerogenic target cell).

In some embodiments, the nucleic acid in the lipid particles described herein further comprises an immunomodulator. An immunomodulator can be an immunosuppressant that reduces the activity of effector cells (e.g., cytotoxic T cells), or an immunostimulator that promotes T cell anergy or exhaustion (e.g., Kwong et al., Immunology & Cell Biology (2021) 99: 486-495, and Linsley et al., Curr Opin Endocrinol Diabetes Obes (2019) 26: 213-218). When delivered to or expressed by target cells, immunomodulators can induce or stabilize the tolerance, anergy, or exhaustion of target cells or nearby cells. In some aspects, the immunomodulator comprises an immunomodulatory interleukin, such as an immunomodulatory interleukin selected from IL-2, IL-10, TGF-β, IL-37, IL-27, IL-35, vasoactive intestinal peptide (VIP) and variants thereof. In some aspects, the immunomodulatory interleukin is a mutant IL-2 that preferentially activates the IL-2Rαβγ receptor complex relative to the IL-2Rβγ receptor complex (see, e.g., Ghelani et al., Front. Immunol . (2020) 11:1106; Khoryati et al., Sci Immunol . (2020) 5(50):eaba5264). In some aspects, the immunomodulator comprises a nucleic acid encoding an immunomodulatory interleukin. In some aspects, the immunomodulator comprises a nucleic acid encoding an intracellular or transmembrane immunomodulatory protein, such as an immunomodulatory protein selected from PD-L1, PD-L2, ICOS ligand, ILT3, ILT4, BTLA, Fas, CD39 and indoleamine 2,3-dioxygenase 1 (IDO1), heme oxygenase 1, HLA-G, CD95L, galectin-1 and DC-SIGN. In some embodiments, the immunomodulator comprises an immunomodulatory compound, such as an immunomodulatory compound selected from vitamin A, vitamin D (e.g., 1α, 25-dihydroxyvitamin D3, also known as calcitriol), adenosine, kynurenine, retinoic acid, rapamycin, dexamethasone, corticosteroids, and AhR ligands (e.g., 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE)). Depending on the form of the immunomodulator and delivery agent, the immunomodulator can be encapsulated or otherwise contained in the lipid particle, or covalently or non-covalently linked to the outer or inner surface of the lipid particle.

Pharmaceutical compositions comprising lipid particles described herein and one or more pharmaceutically acceptable carriers are contemplated. In some aspects, a pharmaceutically acceptable carrier is one that does not have harmful side effects or toxicity under the conditions of use. In some aspects, the pharmaceutical composition is sterile and produced according to GMP guidelines. One or more pharmaceutically acceptable carriers contained in the pharmaceutical composition described herein include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, ubber arable, potassium phosphate, alginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition described herein may further include lubricants, moisturizers, sweeteners, flavoring agents, emulsifiers, suspending agents and preservatives.

In some embodiments, the pharmaceutical composition is in the form of a lyophilized formulation or an aqueous solution. The pharmaceutical composition may be in the form of a dose suspended in any appropriate pharmaceutical vehicle or carrier in a volume sufficient to carry the dose.

Pharmaceutical compositions containing such adjuvants, carriers and/or excipients can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to an individual according to an appropriate dosage.

In some embodiments, the pharmaceutical composition is in the form of a lyophilized formulation or aqueous solution. The pharmaceutical composition may be in a dosage suspended in any appropriate pharmaceutical carrier in a volume sufficient to carry the dosage. In general, the final volume including carriers, adjuvants, and the like will typically be at least 0.5 mL. The upper limit is determined by the practicality of the amount to be administered, generally in the range of 0.5 mL to about 4.0 mL, such as 0.5 mL to about 2.0 mL. In some embodiments, the pharmaceutical composition is administered to the patient enterally or parenterally. In some embodiments, the pharmaceutical composition is administered to the patient by intravenous, intramuscular, intratumoral, intradermal, intrajejunal, intraileal, intracolonic, or intrarectal administration.

In some aspects, the lipid particles and pharmaceutical compositions disclosed herein are stable after storage at -80° C. In some aspects, the lipid particles and pharmaceutical compositions disclosed herein are stable after storage at -80° C. for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, the lipid particles and pharmaceutical compositions disclosed herein are stable after storage at 4° C. In some aspects, the lipid particles and pharmaceutical compositions disclosed herein are stable after storage at 4° C. for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, the lipid particles and pharmaceutical compositions disclosed herein are stable after storage at room temperature. In some aspects, the lipid particles and pharmaceutical compositions disclosed herein are stable after storage at room temperature for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, the lipid particles and pharmaceutical compositions disclosed herein are stored as lyophilized formulations at -80°C, 4°C, or room temperature.

In some embodiments, the lyophilized formulation is reconstituted with an aqueous solution (e.g., saline) or a buffered formulation (e.g., Tris buffer) to prepare a reconstituted formulation. In some embodiments, the reconstituted formulation is stored at 4° C. or at room temperature. In some aspects, the reconstituted formulation is stable for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours. , at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, the reconstituted formulation is stable after storage at 4°C for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, the reconstituted formulation is stable after storage at room temperature for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, the lipid particles and pharmaceutical compositions disclosed herein are stored as aqueous solution formulations (e.g., saline) or buffer formulations (e.g., Tris buffer) at -80°C, 4°C, or room temperature. In some aspects, the aqueous solution formulation is stable for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours , at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, the aqueous solution formulation is stable after storage at -80°C for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours, at least 31 hours, at least 32 hours, at least 33 hours, at least 34 hours, at least 35 hours, at least 36 hours 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, the aqueous formulation is stable after storage at 4°C for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours, at least 31 hours, at least 32 hours, at least 33 hours, at least 34 hours 6 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, the aqueous formulation is stable after storage at room temperature for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours, at least 31 hours, at least 32 hours, at least 33 hours, at least 34 hours 6 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

In some aspects, lipid particles and pharmaceutical compositions are administered intranasally, intracranially, intrathecally, intradermally, intratracheally, percutaneously, intravenously, intraperitoneally, by injection, by infusion, intramuscularly or subcutaneously to an individual (e.g., a human being). In some aspects, lipid particles and pharmaceutical compositions disclosed herein are administered intravenously, intraperitoneally, by injection, by infusion, intramuscularly or subcutaneously to an individual (e.g., a human being).

Contemplated are methods of targeting a payload to the pancreas of an individual in need thereof, the methods comprising intraperitoneally administering to the individual a lipid particle or pharmaceutical composition disclosed herein.

Also contemplated are methods of targeting a payload to lymph nodes of a subject in need thereof, the methods comprising subcutaneously administering to the subject a lipid particle or pharmaceutical composition disclosed herein.

Methods of targeting a payload to one or more tumors in a subject in need thereof are contemplated, the methods comprising injecting into the subject a lipid particle or pharmaceutical composition disclosed herein.

Also contemplated are methods of preventing or delaying the onset or recurrence of a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a lipid particle or pharmaceutical composition disclosed herein.

Methods of restoring immune homeostasis in an individual in need thereof are contemplated, comprising administering to the individual a therapeutically effective amount of a lipid particle or pharmaceutical composition disclosed herein. In some aspects, the individual exhibits an increased percentage of MOG-specific CD4+ splenocytes.

Also contemplated are methods of limiting or reducing an immunogenic response in an individual in need thereof, the methods comprising administering a therapeutically effective amount of a lipid particle or pharmaceutical composition disclosed herein. In some aspects, the individual exhibits increased FR4+CD73+ anergic T cells, as expressed as a percentage of learned CD4+ splenocytes.

Further contemplated are methods of limiting or reducing an inflammatory response in a subject in need thereof, comprising administering a therapeutically effective amount of a lipid particle or pharmaceutical composition disclosed herein. In some aspects, the subject exhibits one or more of the following: (i) an increase in Treg cells, (ii) a decrease in effector T cells, (iii) a decrease in B cells, (iv) a decrease in interleukin production, (v) a decrease in activated B cells, (vi) a decrease in activated effector T cells; or (vii) any combination thereof.

The lipid particles and pharmaceutical compositions disclosed herein can be used to treat autoimmune diseases. Therefore, the present disclosure provides methods for treating autoimmune diseases, which methods include administering a therapeutically effective amount of the lipid particles or pharmaceutical compositions disclosed herein to a subject in need thereof. In some aspects, the autoimmune disorder is selected from the group consisting of autoimmune hepatitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), nonalcoholic fatty pancreatitis, scleroderma, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, chylous diarrhea, type 1 diabetes, Guillain-Barré syndrome, Hashimoto's thyroiditis, polymyalgia rheumatica, alopecia areata, fibrosis, psoriasis, pemphigus vulgaris, vitiligo, ankylosing spondylitis, juvenile idiopathic arthritis, psoriasis arthritis, mixed connective tissue disease, neuromyelitis optica, latent autoimmune diabetes in adults ("LADA"), autoimmune thyroid disease, Grave's disease, Addison's disease, autoimmune atrophic gastritis, pernicious anemia, atopic dermatitis, bullous pemphigoid, myasthenia gravis, polymyositis/dermatomyositis), rheumatic fever, primary sclerosing cholangitis, autoimmune uveitis, and Behcet's disease), diseases affecting the blood or bone marrow (e.g., autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, pale DePasture's syndrome, autoimmune nephritis, glomerulonephritis, Wegener's granulomatosis, chronic inflammatory demyelinating polyradiculoneuropathy, Sjögren's syndrome, primary biliary cholangitis, Parkinson's disease, and antiphospholipid syndrome. In some aspects, the autoimmune disorder is autoimmune hepatitis, type 1 diabetes, or multiple sclerosis.

The lipid particles and pharmaceutical compositions disclosed herein can also be used to treat cancer. Therefore, the present disclosure provides methods for treating cancer, which methods include administering a therapeutically effective amount of the lipid particles or pharmaceutical compositions disclosed herein to an individual in need thereof. In some aspects, the cancer is selected from the group consisting of: breast cancer, pancreatic cancer, colorectal cancer, endometrial cancer, pancreatic cancer, lung cancer, bladder cancer, kidney cancer, and thyroid cancer.

The lipid particles and pharmaceutical compositions disclosed herein can also be used to treat infectious diseases. Therefore, the present disclosure provides methods for treating infectious diseases, which methods include administering a therapeutically effective amount of the lipid particles or pharmaceutical compositions disclosed herein to an individual in need. In some aspects, the present disclosure provides methods for preventive treatment of infectious diseases, which methods include administering a therapeutically effective amount of the lipid particles or pharmaceutical compositions disclosed herein to an individual in need. In some aspects, the infectious disease is COVID, influenza, monkeypox, Middle East Respiratory Syndrome ("MERS"), severe acute respiratory syndrome ("SARS"), human immunodeficiency virus ("HIV"), cytomegalovirus ("CMV"), Epstein-Barr virus ("EBV"), human papillomavirus ("HPV"), hepatitis B virus ("HBV"), herpes simplex virus ("HSV"), Ebola, Lyme disease, avian influenza, or rabies.

In some aspects, the individual is a mammal. In some aspects, the mammal is a human.

In some embodiments, the pharmaceutical composition comprising the lipid particles described herein can be administered in a single injection in a desired dose depending on the intended use. The pharmaceutical composition comprising the lipid particles described herein can be delivered according to a desired dosing regimen, such as once, weekly, monthly, or yearly. In some embodiments, the pharmaceutical composition comprising the lipid particles described herein can be administered as a booster vaccination. V. Manufacturing Methods

Methods for making the lipid particles and pharmaceutical compositions described herein are contemplated. Specifically, the methods include (a) dissolving one or more lipids (e.g., CHEMS, one or more ionizable lipids, one or more neutral lipids, cholesterol, and/or one or more PEG-terminated lipids) in a solvent; (b) adding a payload to the lipid mixture; and (c) mixing.

In some embodiments, the solvent used to dissolve lipids is an organic acid. In some embodiments, the solvent used to dissolve lipids is a mixture of organic solvents. In addition, the solvent can be a mixture of one or more organic solvents and water. In some embodiments, the organic solvent is ethanol or butanol (e.g., tertiary butanol). In some embodiments, anhydrous ethanol (100%), 95% ethanol/water mixture, or 90% tertiary butanol/water mixture is used.

In some embodiments, the effective load is a nucleic acid as described above. In some embodiments, the nucleic acid is included in a buffer. Examples of suitable buffers include citrate, acetate, phosphate, and 2-(N-morpholinyl)ethane sulfonate (MES). In some embodiments, the pH of the buffer is less than the pKa of the protonatable lipid in the lipid mixture. In some aspects, the pH of the buffer is about 2 to about 8, about 2 to about 7.5, about 2 to about 7, about 2 to about 6.5, about 2 to about 6, about 2 to about 5.5, about 2 to about 5, about 2 to about 4.5, about 2 to about 4, about 2 to about 3.5, about 2 to about 3, about 2 to about 2.5, about 2.5 to about 8, about 2.5 to about 7.5, about 2.5 to about 7, about 2.5 to about 6.5, about 2.5 to about 6, about 2.5 to about 5.5, about 2.5 to about 5, about 2.5 to about 4.5, about 2.5 to about 4, about 2.5 to about 3.5, about 2.5 to about 3, about 3 to about 8, about 3 to about 7.5, about 3 to about 7, about 3 to about 6.5, about 3 to about 6, about 3 to about 5.5, about 3 to about 5, about 3 to about 4.5, about 3 to about 4, about 3 to about 3.5, about 4 to about 8, about 4 to about 7.5, about 4 to about 7, about 4 to about 6.5, about 4 to about 6, about 4 to about 5.5, about 4 to about 5, about 4 to about 4.5, about 4.5 to about 8, about 4.5 to about 7.5, about 4.5 to about 7, about 4.5 to about 6.5, about 4.5 to about 6, about 4.5 to about 5.5, about 4.5 to about 5, about 5 to about 8, about 5 to about 7.5, about 5 to about 7 , about 5 to about 6.5, about 5 to about 6, about 5 to about 5.5, about 5.5 to about 8, about 5.5 to about 7.5, about 5.5 to about 7, about 5.5 to about 6.5, about 5.5 to about 6, about 6 to about 8, about 6 to about 7.5, about 6 to about 7, about 6 to about 6.5, about 6.5 to about 8, about 6.5 to about 7.5, about 6.5 to about 7, about 7 to about 8, or about 7.5 to about 8. In some aspects, the pH of the buffer is about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8 , about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8.

In some embodiments, the buffer is citrate. In some embodiments, the pH of the citrate buffer is about 2 to about 8, about 2 to about 7.5, about 2 to about 7, about 2 to about 6.5, about 2 to about 6, about 2 to about 5.5, about 2 to about 5, about 2 to about 4.5, about 2 to about 4, about 2 to about 3.5, about 2 to about 3, about 2 to about 2.5, about 2.5 to about 8, about 2.5 to about 7.5, about 2.5 to about 7, about 2.5 to about 6 .5, about 2.5 to about 6, about 2.5 to about 5.5, about 2.5 to about 5, about 2.5 to about 4.5, about 2.5 to about 4, about 2.5 to about 3.5, about 2.5 to about 3, about 3 to about 8, about 3 to about 7.5, about 3 to about 7, about 3 to about 6.5, about 3 to about 6, about 3 to about 5.5, about 3 to about 5, about 3 to about 4.5, about 3 to about 4, about 3 to about 3.5, about 4 to about 8, about 4 to about 7.5, about 4 to about 7, about 4 to about 6.5, about 4 to about 6, about 4 to about 5.5, about 4 to about 5, about 4 to about 4.5, about 4.5 to about 8, about 4.5 to about 7.5, about 4.5 to about 7, about 4.5 to about 6.5, about 4.5 to about 6, about 4.5 to about 5.5, about 4.5 to about 5, about 5 to about 8, about 5 to about 7.5, about 5 to about 7, about 5 to about 6.5, about 5 to about 6, about 5 to about 5.5, about 5.5 to about 8, about 5.5 to about 7.5, about 5.5 to about 7, about 5.5 to about 6.5, about 5.5 to about 6, about 6 to about 8, about 6 to about 7.5, about 6 to about 7, about 6 to about 6.5, about 6.5 to about 8, about 6.5 to about 7.5, about 6.5 to about 7, about 7 to about 8, or about 7.5 to about 8. In some aspects, the pH of the citrate buffer is about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.10, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6. .8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8. In some embodiments, the buffer is a citrate having a pH of about 4. Example

The present invention, which is now generally described, will be more easily understood by referring to the following examples, including those which are only used for the purpose of illustrating certain aspects and embodiments of the present invention and are not intended to limit the present invention. Example 1. Process flow of lipid nanoparticles

A general process that can be used to make lipid particles described herein is provided in FIG1 . Specifically, the desired lipids (e.g., CHEMS, one or more ionizable lipids, one or more neutral lipids, cholesterol, and/or one or more stealth lipids (e.g., PEG-terminated lipids)) are dissolved in a solvent (e.g., ethanol). Citrate buffer (pH 4.0) containing mRNA can be added to the lipid solution and mixed (e.g., using a T-junction mixer or microfluidics) to produce lipid particles. The particles can then be stabilized by dilution with an in-line buffer and a two-step pH adjustment (e.g., 20 mM citrate, pH 6.0, followed by 1 M Tris HCl, pH 8.0). The lipid particles can then be dialyzed against Tris-buffered saline (pH 7.4), PBS, or HEPES (pH 7.4). The lipid particles can then be concentrated (e.g., using a 100 kDa concentrator). The lipid particles can then be sterile filtered through a 0.22 µm polyethersulfone (PES) syringe filter to produce the final lipid particle formulation.

The particle size and size distribution of the lipid particles can then be characterized using, for example, dynamic light scattering (DLS), the surface charge (zeta potential) using, for example, phase analysis light scattering (PALS), the mRNA encapsulation efficiency using, for example, the Ribogreen assay or LC-MS, the integrity of the mRNA in the LNP using capillary electrophoresis, and the endotoxin load using, for example, a LAL kit-based test (Endosafe® LAL kit from Charles River labs). Example 2. Lipid particles

Lipid particles having the compositions listed in Tables 1 and 2 were consistently prepared using the method provided in Example 1. The formulations listed in Table 3 were prepared on a small scale. Table 1. Lipid particles Lipid particle formulation number Composition (molar ratio) molar % Molar ratio (molar % of each lipid in the formulation) Anionic lipids Mole % of anionic lipids CHEMS% ( if used) Organ harvesting Size(nm) Encapsulation% 1 SS-OP/DODG/CHEMS/CHOL/DMG-PEG2k 35/15/20/28.5/1.5 CHEMS 20 20 Spleen 85.45 ± 3.66 88.21 ± 3.59 2 SS-OP/DOPE/CHEMS/CHOL/DMG-PEG 2k 35/15/20/28.5/1.5 CHEMS 20 20 Spleen 82.82 ± 1.4 91.99 3 SS-OP/DODG/Chol/DMPE-PEG2k/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Spleen 84.75 ± 2.82 87.49 4 SS-OP/DOPE/Chol/DMPE-PEG2k/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Spleen 80.86 ± 2.17 92.51 5 SS-OP/DODG/Chol/18:1 PEG-PE/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Spleen 78.38 ± 0.89 84.12 6 SS-OP/DOPE/Chol/18:1 PEG-PE/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Spleen 75.42 ± 2.08 92.38 7 SS-OP/Chol/DMG-PEG2k/CHEMS 40/38.5/1.5/20 CHEMS 20 20 Spleen 95.3 ± 1.1 85.3 8 SS-OP/Chol/DMG-PEG2k/CHEMS 35/38.5/1.5/25 CHEMS 25 25 Spleen 89.93 ± 2.05 89.35 9 SS-OP/Chol/DMG-PEG2k/CHEMS 30/38.5/1.5/30 CHEMS 30 30 Spleen 89.50 ± 1.90 84 10 SS-OP/Chol/DMG-PEG2k/CHEMS 25/38.5/1.5/35 CHEMS 35 35 Spleen 97.92 ± 0.63 73.07 11 SS-OP/Chol/DMG-PEG2k/CHEMS 40/33.5/1.5/25 CHEMS 25 25 Spleen 93.41 ± 0.96 85.42 12 SS-OP/Chol/DMG-PEG2k/CHEMS 40/28.5/1.5/30 CHEMS 30 30 Spleen 88.46 ± 1.64 85.39 13 SS-OP/DODG/Chol/DMG-PEG2k 50/10/38.5/1.5 Liver 122.24 ± 4.28 89.04 14 SS-OP/DODG/CHEMS/CHOL/DMG-PEG2k 50/10/10/28.5/1.5 CHEMS 10 10 Spleen and liver 98.55 ± 1.74 85.10 15 SS-OP/DODG/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/27/1.5/30 18:1 PA 0 Liver and spleen 100.24 ± 1.70 76.10 16 SS-OP/DODG/Chol/CHEMS/DMG-PEG2k 34.6/6.9/27/30/1.5 CHEMS 30 30 Spleen 98.69 ± 2.89 87.80 17 SS-OP/DODG/Chol/18:1PG/DMG-PEG2k 34.6/6.9/27/30/1.5 18:1 PG 30 0 Liver and spleen 97.12 ± 4.14 74.60 18 SS-OP/DOPE/Chol/CHEMS/DMG-PEG2k 45/15/28.5/10/1.5 CHEMS 28.5 28.5 Liver and spleen 95.56 ± 1.74 75.40 19 MC3/DODG/Chol/DMG-PEK2k 50/10/38.5/1.5 Liver 113.40 ± 2.32 76.30 20 SS-OP/DODG/Chol/DMG-PEG2k 40/20/38.5/1.5 Liver 112.93 ± 2.66 80.16 twenty one SS-OP/DODG/Chol/DMG-PEG2k 45/15/38.5/1.5 Liver 121.15 ± 2.25 77.04 twenty two SS-OP/DODG/Chol/DMG-PEG2k 48/12/38.5/1.5 Liver 122.50 ± 2.73 86.71 twenty three SS-OP/DOPE/Chol/DMG-PEG2k/CHEMS 34.6/6.9/27/1.5/30 CHEMS 30 30 Spleen 91.06 ± 3.09 89.63 twenty four SS-OP/DSPC/Chol/DMG-PEG2k/CHEMS 34.6/6.9/27/1.5/30 CHEMS 30 30 Spleen 82.83 ± 1.58 91.18 25 SS-OP/DODG/CHEMS/CHOL/DMG-PEG2k 30/10/40/18.5/1.5 CHEMS 40 40 Spleen 87.08 ± 2.23 70.69 26 SS-OP/DODG/CHEMS/CHOL/DMG-PEG2k 50/8/25/15.5/1.5 CHEMS 25 25 Spleen 90.86 ± 3.49 84.12 27 SS-OP/DODG/CHEMS/CHOL/DMG-PEG2k 45/7/15/31.5/1.5 CHEMS 15 15 Spleen mainly and some liver renewal 90.58 ± 2.63 84.19 28 SS-OP/DODG/CHEMS/CHOL/DMG-PEG2k 40/10/10/38.5/1.5 CHEMS 10 10 Spleen and liver 95.17 ± 1.3 89.63 29 SS-OP/DODG/CHEMS/CHOL/DMG-PEG2k 45/15/10/28.5/1.5 CHEMS 10 10 Spleen and liver 102.89 ± 1.0 87.29 30 SS-OP/DODG/CHEMS/CHOL/DMG-PEG2k 35/15/25/23.5/1.5 CHEMS 25 25 Spleen 86.36 ± 1.26 84.65 31 SS-OP/DODG/CHEMS/CHOL/DMG-PEG2k 35/20/20/23.5/1.5 CHEMS 20 20 Overall poor capture 89.76 ± 2.35 86.32 32 SS-OP/DOPE/CHEMS/CHOL/DMG-PEG 2k 40/20/20/18.5/1.5 CHEMS 20 20 Spleen 91.78 ± 1.91 84.44 33 SS-OP/DOPE/CHEMS/CHOL/DMG-PEG 2k 34.5/17/21.5/25.5/1.5 CHEMS 21.5 21.5 Spleen 85.97 ± 2.32 89.46 34 MC3/DODG/Chol/DMG-PEG2k/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Spleen 88.58 ± 1.52 70.28 35 SM-102/DODG/Chol/DMG-PEG2k/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Spleen 93.78 ± 2.72 72.21 36 ALC-0315/DODG/Chol/DMG-PEG2k/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Unencapsulated 89.48 ± 3.42 N/A 37 SS-OP/DOPC/Chol/DMG-PEG2k/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Very low spleen uptake 83.04 ± 1.77 85.14 38 SS-OP/DOCP/Chol/DMG-PEG2k/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Very low spleen uptake 85.24 ± 2.58 79.26 39 SS-OP/DOCPe/Chol/DMG-PEG2k/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Low spleen uptake 81.13 ± 1.52 87.38 40 SS-OP/DOPC/Chol/DMG-PEG2k 50/10/38.5/1.5 Spleen and liver 96.29 ± 1.11 84.25 41 SS-OP/DOCP/Chol/DMG-PEG2k 50/10/38.5/1.5 Spleen and low liver uptake 99.84 ± 1.60 83.28 42 SS-OP/DOCPe/Chol/DMG-PEG2k 50/10/38.5/1.5 Spleen and low liver uptake 91.58 ± 2.06 90.88 43 SS-OP/DODG/DMG-PEG2k/CHEMS 50/10/1.5/38.5 CHEMS 38.5 38.5 Low spleen uptake 89.98 ± 1.86 75.63 44 SS-OP/DODG/Chol/DMPE-PEG2k 50/10/1.5/38.5 Liver 120.18 ± 1.97 77.32 45 SS-OP/DODG/Chol/DSPE-PEG-COOH 50/10/38.5/1.5 Liver 127.83 ± 3.28 82.03 46 SS-OP/DODG/Chol/DMG-PEG2k/CHEMS 55/10/23.5/1.5/10 CHEMS 10 10 Liver and spleen 116.19 ± 2.95 74.16 47 SS-OP/DODG/Chol/DMG-PEG2k 55/10/33.5/1.5 Liver 140.06 ± 1.94 56.64 48 SS-OP/DODG/Chol/DMG-PEG2k 60/10/28/2 Liver 137.45 ± 2.21 48.17 49 SS-OP/DODG/Chol/DMPE-PEG2k/CHEMS 50/10/28.5/1.5/10 CHEMS 10 10 Liver and spleen 104.77 ± 2.15 78.32 50 SS-OP/DODG/Chol/18:1 PEG2k-PE 50/10/38.5/1.5 Liver 118.26 ± 2.42 81.63 51 SS-OP/DODG/Chol/18:1 PEG2k-PE/CHEMS 50/10/28.5/1.5/10 CHEMS 10 10 Spleen and low liver uptake 95.96 ± 2.10 89.79 52 SS-OP/DODG/Chol/DMG-PEG2k 60/10/28.5/1.5 Liver 155.86 ± 2.41 56.32 53 SS-OP/DODG/Chol/DMG-PEG2k/CHEMS 35/15/29/1/20 CHEMS 20 20 Spleen 103.81 ± 1.47 90.03 54 SS-OP/DODG/Chol/DMG-PEG2k/CHEMS 35/10/33.5/1.5/20 CHEMS 20 20 Spleen 88.35 ± 2.37 91.15 55 SS-OP/DODG/Chol/DMG-PEG2k/CHEMS 35/10/28.5/1.5/25 CHEMS 25 25 Spleen 84.65 ± 1.82 83.86 56 SS-OP/DOPE/Chol/DMG-PEG2k/CHEMS 35/10/33.5/1.5/20 CHEMS 20 20 Spleen 90.25 ± 1.70 90.07 57 SS-OP/DOPE/Chol/DMPE-PEG2k/CHEMS 35/10/33.5/1.5/20 CHEMS 20 20 Spleen 86.05 ± 2.15 92.67 58 SS-OP/DODG/Chol/DMPE-PEG2k/CHEMS 45/10/18.5/1.5/25 CHEMS 25 25 Spleen 89.24 ± 2.10 77.59 59 SS-OP/DOPE/Chol/DMPE-PEG2k/CHEMS 45/10/18.5/1.5/25 CHEMS 25 25 Spleen and liver 95.48 ± 1.95 86.33 60 SS-OP/DODG/Chol/DMG-PEG2k/CHEMS 45/10/18.5/1.5/25 CHEMS 25 25 Spleen and liver 102.69 ± 1.74 82.36 61 SS-OP/DODG/Chol/DMG-PEG2k/CHEMS 35/8/30.5/1.5/25 CHEMS 25 25 Spleen 96.71 ± 2.20 87.48 62 SS-OP/DOPE/Chol/DMPE-PEG2k/CHEMS 35/10/28.5/1.5/25 CHEMS 25 25 Spleen 98.89 ± 2.26 66.21 63 SS-OP/DODG/β-Mycosterol/CHEMS/DMG-PEG2k 35/15/28.5/20/1.5 CHEMS 20 20 Spleen 84.23 ± 0.66 85.59 64 SS-OP/DOPE/Chol/DMG-PEG2k/CHEMS 33.5/15/30/1.5/20 CHEMS 20 20 Spleen 84.95 ± 2.34 90.38 65 SS-OP/DOPE/β-Mycosterol/DMG-PEG2k/CHEMS 33.5/15/30/1.5/20 CHEMS 20 20 Spleen 79.47 ± 0.50 93.24 66 SS-OP/DODG/β-Mycosterol/DMGPEG2k/CHEMS 33.5/15/30/1.5/20 CHEMS 20 20 Spleen 97.56 ± 1.66 74.13 67 SS-OP/DODG/Chol/DMPE-PEG/CHEMS 33.5/15/30/1.5/20 CHEMS 20 20 Spleen 86.42 ± 1.91 84.08 68 SS-OP/DODG/Chol/DMG-PEG2k/CHEMS 33.5/15/30/1.5/20 CHEMS 20 20 Spleen 89.55 ± 1.44 87.43 69 SS-OP/DSPC/Chol/DMG-PEG2k/CHEMS 35/15/28.5/1.5/20 CHEMS 20 20 Spleen 79.86 ± 1.10 93.41 70 SS-OP/DODG/Chol/DMG-PEG2k/CHEMS 30/10/38.5/1.5/20 CHEMS 20 20 Spleen 92.60 ± 1.29 87.03 71 SS-OP/DODG/Chol/DMG-PEG2k/CHEMS 35/5/38.5/1.5/20 CHEMS 20 20 Spleen 96.62 ± 2.84 89.93 72 SS-OP/DOPE/Chol/DMG-PEG2k/CHEMS 30/10/38.5/1.5/20 CHEMS 20 20 Spleen 87.25 ± 3.03 92.69 73 SS-OP/DOPE/Chol/DMG-PEG2k/CHEMS 35/5/38.5/1.5/20 CHEMS 20 20 Spleen 112.88 ± 2.60 81.48 74 SS-EC/DOPC/Chol/DSG-PEG2k 60/10/30/3 Spleen negligible 104.5 ± 2.89 44.52 75 SS-OP/Chol/DMG-PEG2k/CHEMS 50/38.5/1.5/10 CHEMS 10 10 Spleen and liver 92.86 ± 1.95 82.56 76 SS-OP/Chol/DMG-PEG2k/CHEMS 45/38.5/1.5/15 CHEMS 10 10 Spleen and liver 94.08 ± 0.86 77.72 77 SS-OP/Chol/DMG-PEG2k/CHEMS 55/38.5/1.5/5 CHEMS 5 5 Spleen and liver 94.21 ± 1.27 60.12 78 SS-OP/Chol/DMG-PEG2k/CHEMS 40/43.5/1.5/15 CHEMS 15 15 Spleen and liver 90.94 ± 1.72 85.40 Table 2. Lipid particle formulation number Composition (molar ratio) molar % Molar ratio (molar % of each lipid in the formulation) Anionic lipids Mole % of anionic lipids CHEMS% ( if used) Organ harvesting Size(nm) Encapsulation% 79 MC3/DSPC/Chol/DMG-PEG2k 50/10/38/2 Liver 76.1 ± 0.5 88.5 80 SS-OP/DSPC/Chol/DMG-PEG2k 50/10/38/2 Liver 93.94 ± 3.44 85.97 81 SS-OP/DSPC/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 18:1 PA 30 Liver and spleen 98.07 ± 2.10 77.7 82 MC3/DSDG/Chol/DMG-PEG2k 50/10/38/2 Liver 141.65 ± 3.81 80.5 83 SS-OP/DSDG/Chol/DMG-PEG2k 50/10/38/2 Liver 119.80 ± 3.07 76.6 84 MC3/DSPC/Chol/DMG-PEG2k/CHEMS 34.6/6.9/26.5/2/30 CHEMS 30 30 78.64 ± 0.68 79.5 85 SS-OP/DSDG/Chol/DMG-PEG2k/CHEMS 34.6/6.9/26.5/2/30 CHEMS 30 30 148.43 ± 1.38 70.1 86 SS-OP/DSPC/Chol/DSG-PEG2k 50/10/35/5 80.74 ± 1.87 65.86 87 SS-OP/DSPC/Chol/MPEG2k-C18 50/10/38/2 111.72 ± 1.08 88.39 88 SS-OP/DSDG/Chol/DMG-PEG2k 50/10/38/2 119.80 ± 3.07 76.62 89 SS-OP/DSDG/Chol/MPEG2k-C18 50/10/38/2 137.23 ± 4.82 66.47 90 SS-OP/DSPC/Chol/DMG-PEG2k/18:1 PA low PEG 34.6/6.9/26.5/1.1/30 18:1 PA 30 140.02 ± 3.21 76.20 91 SS-OP/DSDG/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 18:1 PA 30 112.96 ± 2.40 79.08 92 SS-OP/DSPC/Chol/DSG-PEG2k 50/10/35/5 82.9 ± 1.32 64.3 93 SS-OP/DSDG/Chol/MPEG2k-C18 50/10/38.5/1.5 123.01 ± 1.43 65 94 SS-OP/DSDG/Chol/MPEG2k-C18/CHEMS 34.6/6.9/27/1.5/30 CHEMS 30 30 132.40 ± 3.13 73.6 95 SS-OP/DSDG/Chol/MPEG2k-C18/18:1 PA 34.6/6.9/27/1.5/30 18:1 PA 30 147.62 ± 3.27 70.9 96 SM-102/DSDG/Chol/MPEG2k-C18 50/10/38.5/1.5 123.17 ± 2.25 74.3 97 SM-102/DSDG/Chol/MPEG2k-C18 (10:1)Lipid:mRNA 50/10/38.5/1.5 126.60 ± 2.24 57.4 98 SM-102/DSDG/Chol/MPEG2k-C18 50/10/38.5/1.5 124.19 ± 2.36 89.2 99 SM-102/DODG/Chol/MPEG2k-C18 50/10/38.5/1.5 128.24 ± 3.44 67.3 100 SM-102/DSDG/Chol/MPEG2k-C18 50/10/38.5/1.5 125.60 ± 4.37 86.7 101 SM-102/DSDG/Chol/MPEG2k-C16 50/10/38.5/1.5 105.76 ± 1.86 69.9 102 SS-OP/DSPC/Chol/MPEG2k-C18 50/10/38.5/1.5 117.28 ± 2.42 93.06 103 SS-OP/DODG/Chol/MPEG2k-C18 50/10/38.5/1.5 121.49 ± 4.52 79.9 104 SS-OP/DODG/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/27/1.5/30 18:1 PA 30 Liver and spleen 102.93 ± 1.37 80.7 105 SS-OP/DODG/Chol/DMG-PEG2k 50/10/38/2 Liver 116.65 ± 1.35 76.7 106 SS-OP/DODG/Chol/MPEG2k-C18 50/10/38/2 Liver 107.55 ± 1.70 85.6 Table 3 Lipid particle formulation number Composition Molar ratio (mol%) size PDI Zeta potential Encapsulation% 80 SS-OP/DSPC/Chol/DMG-PEG2k 50/10/38/2 89.8 ± 3.9 0.157 ± 0.033 -3.5 ± 7.8 95.2 107 SS-OP/DOPE/Chol/DMG-PEG2k 50/10/38/2 80.1 ± 3.5 0.160 ± 0.049 1.2 ± 6.5 94.2 83 SS-OP/DSDG/Chol/DMG-PEG2k 50/10/38/2 85.5 ± 1.7 0.173 ± 0.021 -3.3 ± 5.3 93.2 108 SS-OP/DSPC/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 77.0 ± 2.4 0.204 ± 0.012 -10.8 ± 0.5 94 109 SS-OP/DOPE/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 74.6 ± 2.2 0189 ± 0.021 -4.7 ± 1.1 92.6 110 SS-OP/DSDG/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 81.3 ± 4.3 0.183 ± 0.036 -8.3 ± 1.5 93.4 111 SS-OP/DOPC/CHOL/DMG-PEG2k 50/10/38/2 95.8 ± 2.3 0.159 ± 0.033 -7.5 ± 3.12 92.4 112 SSOP/DOPC/CHOL/DMGPEG2k/CHEMS 30/10/38/2/20 84.7 ± 1.1 0.193 ± 0.024 -8.9 ± 3.2 91.1 81 SS-OP/DSPC/CHOL/18:1 PA/DMG-PEG2k 34.6/6.9/26.5/30/2 117.4 ± 2.5 0.125 ± 0.064 -8.5 ± 2.2 88.3 82 SS-OP/DOPE/CHOL/18:1 PA/DMG-PEG2k 34.6/6.9/26.5/30/2 108.7 ± 2.5 0.162 ± 0.043 -7.3 ± 1.9 84.8 91 SS-OP/DSDG/CHOL/18:1 PA/DMG-PEG2k 34.6/6.9/26.5/30/2 102.6 ± 1.8 0.193 ± 0.035 -8.8 ± 2.4 94.3 113 SS-OP/DOPC/CHOL/18:1 PA/DMG-PEG2k 34.6/6.9/26.5/30/2 99.6 ± 4.2 0.211 ± 0.027 -10.9 ± 2.6 85.4 114 SS-OP/DSPC/CHOL/CHEMS/DMG-PEG2k 34.6/6.9/26.5/30/2 81.8 ± 2.1 0.202 ± 0.025 0.5 ± 1.54 89.9 115 SS-OP/DOPE/CHOL/CHEMS/DMG-PEG2k 34.6/6.9/26.5/30/2 74.7 ± 0.70 0.148 ± 0.068 0.6 ± 1.40 94 85 SS-OP/DSDG/CHOL/CHEMS/DMG-PEG2k 34.6/6.9/26.5/30/2 75.8 ± 1.4 0.153 ± 0.055 0.1 ± 0.9 96.2 116 SS-OP/DOPC/CHOL/CHEMS/DMG-PEG2k 34.6/6.9/26.5/30/2 82.8 ± 2.6 0.148 ± 0.032 -1.9 ± 1.3 92.6 117 SS-OP/DSPC/CHOL/DMG-PEG2k/18:1 PA 30/8/30/2/30 97.95 ± 2.43 0.117 ± 0.055 -0.42 ± 0.61 89.5 118 SS-OP/DSPC/CHOL/DMG-PEG2k/18:1 PA 25/8/35/2/30 97.29 ± 1.91 0.104 ± 0.052 -0.77 ± 2.28 78.7 119 SS-OP/DSPC/CHOL/DMG-PEG2k/18:1 PA 20/10/38/2/30 79.31 ± 0.91 0.139 ± 0.043 0.01 ± 0.75 70.1 120 SS-OP/DSPC/CHOL/DMG-PEG2k/18:1 PA 25/8/30/2/35 105.63 ± 1.49 0.150 ± 0.025 0.38 ± 9.63 40.3 121 SS-OP/DSPC/CHOL/DMG-PEG2k/18:1 PA 30/7/26/2/35 93.27 ± 7.39 0.196 ± 0.120 -1.49 ± 2.64 39.7 122 SS-OP/DSPC/CHOL/DMG-PEG2k/18:1 PA 28/7/30/2/33 72.16 ± 0.59 0.158 ± 0.039 -0.08 ± 0.95 <1% 123 SS-OP/DSPC/CHOL/DMG-PEG2k/18:1 PA 30/10/38/2/20 78.24 ± 4.10 0.154 ± 0.070 -1.57 ± 1.51 95.1 124 SS-OP/DSPC/CHOL/DMG-PEG2k/18:1 PA 27/8/30/2/33 85.08 ± 3.98 0.241 ± 0.022 0.06 ± 1.78 <1% 89 SS-OP/DSDG/Chol/MPEG-C18 50/10/38/2 108.75 ± 2.49 0.168 ± 0.048 0.81 ± 0.49 82.8 105 SS-OP/DODG/Chol/DMG-PEG2k 50/10/38/2 100.65 ± 1.04 0.102 ± 0.049 -0.12 ± 0.29 89.8 87 SS-OP/DSPC/Chol/DSG-PEG2k 50/10/35/5 63.37 ± 0.87 0.164 ± 0.026 0.21 ± 1.51 93.3 125 SS-OP/DSPC/Chol/DMG-PEG2k/18:1 PG 30/10/38/2/20 93.68 ± 1.98 0.110 ± 0.039 -0.26 ± 0.35 88.8 91 SS-OP/DSPC/Chol/DMG-PEG2k/18:1 PG 34.6/6.9/26.5/2/30 105.54 ± 1.90 0.095 ± 0.036 0.46 ± 2.33 83.5 41 SS-OP/DOCP/Chol/DMG-PEG2k 50/10/38.5/1.5 126.93 ± 1.95 0.269 ± 0.017 -1.35 ± 3.30 85.5 42 SS-OP/DOCPe/Chol/DMG-PEG2k 50/10/38.5/1.5 140.05 ± 6.22 0.239 ± 0.027 1.02 ± 0.54 92.7 126 SS-OP/DODG/Chol/Bis DSPE PEG2k 50/10/38.5/1.5 125.86 ± 4.88 0.162 ± 0.046 1.68 ± 6.99 85.3 50 SS-OP/DODG/Chol/18:1 PEG2k PE 50/10/38.5/1.5 123.08 ± 3.08 0.131 ± 0.043 0.08 ± 0.75 89.6 127 SS-OP/DODG/Chol/DMPE-PEG2k 50/10/38.5/1.5 116.95 ± 3.82 0.104 ± 0.063 0.57 ± 0.89 89.2 45 SS-OP/DODG/Chol/DSPE-PEG2k-COOH 50/10/38.5/1.5 120.13 ± 7.52 0.117 ± 0.057 -0.22 ± 0.64 86.5 128 KC2/DSPC/Chol/DMG-PEG2k 50/10/38/2 84.3 ± 1.5 0.132 ± 0.040 -0.20 ± 0.88 87.72 129 KC2/DOPE/Chol/DMG-PEG2k 50/10/38/2 85.7 ± 1.8 0.142 ± 0.020 0.01 ± 0.46 92.15 130 KC2/DSDG/Chol/DMG-PEG2k 50/10/38/2 87.6 ± 2.1 0.115 ± 0.024 -1.37 ± 4.84 90.52 131 KC2/DOPC/Chol/DMG-PEG2k 50/10/38/2 93.6 ± 1.9 0.092 ± 0.032 -0.62 ± 1.60 93.13 132 KC2/DSPC/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 80.3 ± 4.2 0.223 ± 0.014 -1.1 ± 1.1.75 84.36 133 KC2/DOPE/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 86.8 ± 3.7 0.170 ± 0.029 -0.99 ± 1.12 84.04 134 KC2/DSDG/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 86.2 ± 5.2 0.207 ± 0.019 1.56 ± 0.46 92.09 135 KC2/DOPC/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 90.9 ± 1.4 0.138 ± 0.066 0.24 ± 0.72 88.09 136 KC2/DSPC/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 135.3 ± 3.4 0.189 ± 0.020 -0.56 ± 0.60 11.97 137 KC2/DOPE/Chol/DMG-PEG2k/18:1PA 34.6/6.9/26.5/2/30 135.1 ± 4.2 0.123 ± 0.057 0.54 ± 1.04 9.23 138 KC2/DSDG/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 136.5 ± 2.8 0.135 ± 0.017 1.24 ± 1.29 8.98 139 KC2/DOPC/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 135.2 ± 5.9 0.134 ± 0.058 -0.17 ± 1.65 6.23 140 MC3/DSPC/Chol/CHEMS/DMG-PEG2k 30/10/37/21/2 73.9 ± 3.1 0.225 ± 0.019 -11.5 ± 2.4 86 84 MC3/DSPC/Chol/DMG-PEG2k/CHEMS 34.6/6.9/26.5/2/30 80.3 ± 1.0 0.199 ± 0.030 0.73 ± 0.84 86.5 141 MC3/DOPE/Chol/DMG-PEG2k/CHEMS 34.6/6.9/26.5/2/30 78.8 ± 1.4 0.124 ± 0.042 -0.51 ± 0.29 84.7 142 MC3/DSDG/Chol/DMG-PEG2k/CHEMS 34.6/6.9/26.5/2/30 77.0 ± 2.7 0.155 ± 0.043 -0.25 ± 0.75 91.3 143 MC3/DOPE/Chol/DMG-PEG2k/CHEMS 34.6/6.9/26.5/2/30 79.9 ± 1.9 0.157 ± 0.035 0.13 ± 0.75 85.9 144 MC3/DSPC/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 81.1 ± 2.2 0.225 ± 0.013 0.18 ± 0.45 86.2 145 MC3/DOPE/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 87.3 ± 1.2 0.147 ± 0.056 0.33 ± 2.41 86.9 146 MC3/DSDG/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 79.4 ± 2.2 0.197 ± 0.031 -0.04 ± 1.30 86.5 147 MC3/DOPC/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 87.5 ± 3.0 0.187 ± 0.020 -0.23 ± 0.97 88.7 82 MC3/DSDG/Chol/DMG-PEG2k 50/10/38/2 94.04 ± 4.69 0.106 ± 0.034 -1.50 ± 1.44 89.48 148 MC3/DSPC/Chol/DSG-PEG2k 50/10/35/5 60.59 ± 1.97 0.173 ± 0.036 0.64 ± 1.03 89.25 149 MC3/DSPC/Chol/DMG-PEG2k/18:1 PG 30/10/38/2/20 98.53 ± 1.87 0.167 ± 0.024 -1.30 ± 1.96 84.56 150 MC3/DSPC/Chol/DMG-PEG2k/18:1 PG 34.6/6.9/26.5/2/30 118.37 ± 2.67 0.124 ± 0.027 1.27 ± 0.51 72.00 151 MC3/DOPE/Chol/DMG-PEG2k (50/10/38/2) 50/10/38/2 73.35 ± 1.36 0.133 ± 0.014 0.08 ± 1.32 93.3 152 MC3/DSDG/Chol/C18-MPEG (50/10/38/2) 50/10/38/2 119.30 ± 1.11 0.169 ± 0.021 0.74 ± 0.42 89.48 153 MC3/DODG/Chol/DMG-PEG2k (50/10/38/2) 50/10/38/2 85.84 ± 1.99 0.129 ± 0.041 -0.32 ± 1.15 89.82 79 MC3/DSPC/Chol/DMG-PEG2k (50/10/38/2) 50/10/38/2 78.27 ± 2.11 0.143 ± 0.041 0.92 ± 1.33 91.48 154 SM-102/DSPC/Chol/DMG-PEG2k 50/10/38/2 84.3 ± 1.5 0.211 ± 0.030 -0.2 ± 0.9 92.7 155 SM-102/DSDG/Chol/DMG-PEG2k 50/10/38/2 83.4 ± 1.2 0.116 ± 0.023 -1.1 ± 0.6 90.6 156 SM-102/DOPE/Chol/DMG-PEG2k 50/10/38/2 75.8 ± 2.1 0.104 ± 0.056 -1.1 ± 2.3 98 157 SM-102/DODG/Chol/DMG-PEG2k 50/10/38/2 96.0 ± 1.6 0.154 ± 0.038 1.4 ± 1.5 89.7 158 SM-102/DSPC/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 145.4 ± 5.7 0.241 ± 0.020 0.4 ± 1.9 <1% 159 SM-102/DSDG/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 126.3 ± 2.7 0.232 ± 0.022 0.4 ± 1.9 <1% 160 SM-102/DSDG/Chol/sc PEG 50/10/38/2 132.9 ± 2.5 0.204 ± 0.025 -0.1 ± 1.5 92.39 161 SM-102/DSPC/Chol/sc PEG 50/10/38/2 100.2 ± 2.8 0.195 ± 0.034 -0.7 ± 3.6 90.57 162 SM-102/DOPE/Chol/sc PEG 50/10/38.5/1.5 136.8 ± 2.8 0.184 ± 0.027 -0.01 ± 1.3 96.4 163 SM-102/DODGChol/sc PEG 50/10/38.5/1.5 132.4 ± 1.9 0.100 ± 0.063 0.12 ± 1.0 92.4 164 SM-102/DSDG/Chol/sc PEG 50/10/38.5/1.5 123.4 ± 3.6 0.083 ± 0.045 0.9 ± 0.9 97.3 165 SM-102/DODG/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/27/1.5/30 134.0 ± 1.6 0.180 ± 0.018 -0.03 ± 0.5 <1% 166 SM-102/DSDG/Chol/sc PEG/CHEMS 34.6/6.9/27/1.5/30 117.3 ± 2.5 0.124 ± 9.031 -0.3 ± 0.5 69.27 167 SM-102/DSDG/Chol/C16 sc PEG 50/10/38.5/1.5 131.0 ± 3.9 0.186 ± 0.029 -0.8 ± 0.83 94 168 ALC/DSPC/Chol/DMG-PEG2k 50/10/38/2 81.26 ± 1.72 0.229 ± 0.014 -0.09 ± 1.38 89.8 169 ALC/DSDG/Chol/DMG-PEG2k 50/10/38/2 115.11 ± 2.21 0.204 ± 0.022 1.33 ± 1.28 79.3 170 ALC/DODG/Chol/DMG-PEG2k 50/10/38/2 81.82 ± 2.51 0.087 ± 0.065 0.69 ± 2.67 81 171 ALC/DSPC/Chol/MPEG2k-C18 50/10/38/2 86.28 ± 2.24 0.169 ± 0.036 0.79 ± 5.59 92.24 172 ALC/DSDG/Chol/MPEG2k-C18 50/10/38/2 167.25 ± 4.09 0.139 ± 0.051 0.21 ± 0.96 <1% 173 ALC/DSPC/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 152.32 ± 4.02 0.283 ± 0.029 1.96 ± 2.72 <1% 174 ALC/DSDG/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 153.02 ± 4.28 0.237 ± 0.024 0.85 ± 0.96 <1% 175 C12-200/DSPC/Chol/DMG-PEG2k 50/10/38/2 123.75 ± 5.50 0.157 ± 0.038 0.70 ± 2.39 87.79 176 C12-200/DOPE/Chol/DMG-PEG2k 50/10/38/3 85.77 ± 1.18 0.063 ± 0.020 0.88 ± 2.14 73.27 177 C12-200/DSDG/Chol/DMG-PEG2k 50/10/38/4 91.00 ± 3.30 0.114 ± 0.038 -0.15 ± 1.52 <1% 178 C12-200/DOPC/Chol/DMG-PEG2k 50/10/38/5 94.63 ± 2.75 0.116 ± 0.033 -0.09 ± 2.52 90.06 179 C12-200/DSPC/Chol/DMG-PEG2k/CHEMS 30/10/38/2/20 74.49 ± 5.81 0.165 ± 0.052 -0.66 ± 1.14 74.45 180 C12-200/DOPE/Chol/DMG-PEG2k/CHEMS 30/10/38/2/21 79.20 ± 1.35 0.113 ± 0.027 0.31 ± 1.32 62.77 181 C12-200/DSDG/Chol/DMG-PEG2k/CHEMS 30/10/38/2/22 84.58 ± 2.29 0.147 ± 0.028 0.58 ± 1.74 30.9 182 C12-200/DOPC/Chol/DMG-PEG2k/CHEMS 30/10/38/2/23 92.08 ± 3.89 0.161 ± 0.030 -0.81 ± 1.44 75.11 183 C12-200/DSPC/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 75.47 ± 0.81 0.124 ± 0.049 0.42 ± 1.05 <1% 184 C12-200/DOPE/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 69.80 ± 1.02 0.122 ± 0.023 0.78 ± 0.93 <1% 185 C12-200/DSDG/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 70.96 ± 1.02 0.080 ± 0.048 -0.42 ± 0.73 <1% 186 C12-200/DOPC/Chol/DMG-PEG2k/18:1 PA 34.6/6.9/26.5/2/30 75.69 ± 1.76 0.086 ± 0.033 1.32 ± 1.32 <1% Example 3. BLI solution

C57BL/6J mice aged 6-8 weeks were used for this study. Prior to injection of the specific lipid particle formulation to be tested, the fur of the mice was cut open along the left side of the abdomen up to the spine to highlight the spleen and liver expression field. 100 μL of the lipid particle formulation to be tested was injected intravenously into the mice via the tail vein. The lipid particle formulation to be tested contained the equivalent of 20 μg of firefly luciferase mRNA. Six hours later, the mice were administered 200 μL of D-luciferin (3 mg/mouse) via intraperitoneal injection (ip), anesthetized with isoflurane and imaged within 10 minutes using a LICOR Pearl imager. Images were obtained in the right flank position with 3% isoflurane used to maintain anesthesia. LICOR settings: BLI and white channels only, 85um resolution, focus at 0. BLI values were determined using Image Studio software version 5.2.5 from LICOR. Regions of interest were drawn around organs (e.g., spleen or liver) using software tools, and the corresponding BLI signal was calculated by the software after background subtraction. Data were reported as spleen BLI value, liver BLI value, and spleen to liver ratio (i.e., the ratio obtained by dividing the BLI value in the spleen by the BLI value in the liver of the same animal). Example 4. DOPE vs. DODG in lipid particles

The spleen to liver ratio of each of lipid particle formulations No. 1, No. 3-6, and No. 53-55 (in PBS) and PBS control was determined using the BLI protocol described in Example 3. The results are provided in Figures 2A-2C . Example 5. Effect of CHEMS on spleen targeting

The spleen to liver ratio of each of lipid particle formulations No. 1, No. 2, and No. 30-33 (in PBS) and the PBS control was determined using the BLI protocol described in Example 3. The results are provided in Figures 3A-3C .

The spleen to liver ratio of each of lipid particle formulations No. 1, No. 13, and No. 43-48 (in PBS) and the PBS control was determined using the BLI protocol described in Example 3. The results are provided in Figures 4A-4C .

In addition, the spleen to liver ratio of each of lipid particle formulations No. 13, No. 15, No. 16, and No. 20-24 (in PBS) and the PBS control was determined using the BLI protocol described in Example 3. The results are provided in Figures 5A-5C .

The BLI values of lipid particle formulations No. 1, No. 14, No. 16, and No. 25-29 (in PBS) and each in PBS in the spleen of mice are provided in Figure 6. The spleen to liver ratio of lipid particle formulations No. 1, No. 16, and No. 23-26 (in PBS) in mice is provided in Table 4. The BLI values were determined using the protocol described in Example 3. Table 4. Spleen to liver ratio Preparation Auxiliary lipids Spleen BLI S/L Ratio ATP-18* DODG 16.62 7.49 ATP-21 DOPE 14.36 5.78 ATP-27 DOPC 1.87 1.44 ATP-28 DOCP 0.78 0.08 ATP-29 DOCPe 4.54 4.96 ATP-63 DSPC 0.42 2.37 Lipid particle formulation number Auxiliary lipids Spleen BLI S/L Ratio 1* DODG 16.62 7.49 twenty one DOPE 14.36 5.78 37 DOPC 1.87 1.44 38 DOCP 0.78 0.08 39 DOCPe 4.54 4.96 69 DSPC 0.42 2.37 Example 6. Effect of neutral lipids

The spleen to liver ratios for each of lipid particle formulations No. 1, No. 13, and No. 37-42 (in PBS) and PBS controls were determined using the protocol described in Example 3. The results are provided in Figures 7A-7C . Using the same lipid molar ratio, the choice of neutral lipids affects the spleen signal. DODG and DOPE target the payload to the spleen better than DOPC, DSPC, or DOCP. Example 7. Dual Targeted Formulations (Spleen and Liver)

The spleen to liver ratios for each of lipid particle formulations No. 1, No. 14, No. 16, and No. 25-29 (in PBS) and the PBS control were determined using the protocol described in Example 3. The results are provided in Figures 8A-8C . Example 8. Four-component system targeting the spleen

The BLI values of lipid particle formulation No. 7 (four components) and lipid particle formulation No. 14 (intravenous administration), No. 1 (Tris containing drug composition administered intravenously) and No. 1 (frozen lipid particles) each containing five components in the mouse spleen were determined and reported in Figures 9A-9C . BLI values were determined using the imaging protocol and software described in Example 3. Example 9. Intraperitoneal delivery

C57BL/6J mice aged 6-8 weeks were injected intravenously or intraperitoneally with 100 μl of lipid particle formulation No. 1 or No. 14 (in TBS) containing the equivalent of 20 μg of firefly luciferase mRNA. Six hours after injection, mice were injected with 200 μl of D-luciferin (3 mg/mouse) administered intraperitoneally. After allowing luciferin to distribute for 2 minutes, mice were placed in a CO ₂ chamber and euthanized. After necropsy, the organs of interest (spleen, liver, and pancreas) were excised and placed individually in wells of a 24-well plate. Sufficient luciferin (300 μg/mL) was added to cover the tissue (approximately 0.5 mL/well). The organs were transferred to a solution basin, with compartments separated to avoid spillover of luminescence from larger organs. If necessary, additional luciferin solution was added to the chamber during imaging. Organs were imaged using a LICOR Pearl imager. Images of individual organs were analyzed using Image studio software from LICOR (version 5.2.5). Areas of interest were drawn around individual organs using software tools. Organs isolated from PBS-injected mice were used as background controls. The resulting images and BLI values of the organs from the software are reported. The results are provided in Figures 15A-15E .

As shown in FIG. 15B and FIG. 15E , luciferase was detected in the pancreas after intraperitoneal administration of each lipid particle formulation No. 1 and No. 14. However, as shown in FIG. 15A , FIG. 15C , and FIG. 15D , when the same lipid particle formulation was administered intravenously, luciferase was detected in the liver and/or spleen. Example 10. Subcutaneous delivery of lipid particle formulation No. 1, No. 13, and No. 14

C57BL/6J mice aged 6-8 weeks were used for this study. Prior to injection of lipid particle formulations No. 1, 13, or 14 (in PBS), the fur of mice was cut open along the left side of the abdomen up to the spine to highlight the spleen and liver expression field. 100 μL of lipid particle formulations No. 1, 13, or 14 (in PBS) containing 20 μg of firefly luciferase mRNA equivalent was injected subcutaneously into the left flank of the mice. 16 hours, 24 hours, and 48 hours later, mice were administered 200 μL of D-luciferin (3 mg/mouse) by intraperitoneal injection (ip), anesthetized with isoflurane, and imaged within 10 minutes using a LICOR Pearl imager. Images were obtained in the right flank position under 3% isoflurane for maintenance of anesthesia. LICOR settings: BLI and white channel only, 85um resolution, focus at 0. Lymph nodes ("LN") were imaged only at the 24 hour time point. For LN imaging, mice were euthanized using _CO2 , inguinal lymph nodes were isolated, placed in weigh boats and imaged using the LICOR Pearl imaging system. BLI values were determined using Image Studio software version 5.2.5 from LICOR. For whole body images of the animal or for isolated inguinal LNs, a region of interest ("ROI") was drawn around an organ (e.g., liver, injection site, or LN) using the software tool and the corresponding BLI signal was calculated by the software after background subtraction. Data are reported as spleen BLI values, liver BLI values, and spleen to liver ratio (i.e., the ratio obtained by dividing the BLI value in the spleen by the BLI value in the liver of the same animal) for whole body imaging. For LN imaging, BLI values of dissected LNs and underlying subcutaneous tissue are reported for each formulation. Images are provided in Figure 16 .

As shown in Figure 16 , after subcutaneous administration to mice, luciferase from lipid particle formulation No. 13 was shown to accumulate in the liver for at least 48 hours. In contrast, luciferase from lipid particle formulation No. 14 had a low liver signal for up to 24 hours. Most of the luciferase from lipid particle formulations No. 1, No. 13, and No. 14 remained at the injection site and was taken up into the lymph nodes. Luciferase from lipid particle formulation No. 1 accumulated to a higher extent in the lymph nodes, and the subcutaneous signal was negligible. Example 11. Spleen targeting using different ionizable lipids

The BLI values of luciferase delivered by lipid particle formulations 1, 34, and 35 in the spleen and liver of mice were determined using the protocol of Example 3. The results are provided in Figures 10A-10B . Example 12. Four-component lipid particles

The mean spleen BLI values, mean liver BLI values, and spleen to liver ratios of lipid particle formulations Nos. 7-10 and 76-77 (in PBS) in mice were determined using the protocol provided in Example 3. The results are reported in Table 5 below. Each of the formulations contained 38.5% (molar weight percentage) cholesterol and 1.5% (molar weight percentage) DMG-PEG2000. Table 5 Lipid Particle Formulation No. 77 Lipid Particle Formulation No. 75 Lipid Particle Formulation No. 76 Lipid Particle Formulation No. 7 Lipid Particle Formulation No. 8 Lipid Particle Formulation No. 9 Lipid Particle Formulation No. 10 SS-OP/CHEMS ratio (mol%) 55/5 50/10 45/15 40/20 35/25 30/30 25/35 Mean spleen BLI 15.3 14.2 20.35 9.9 6.88 2.97 2.68 Mean liver BLI 130 70.11 41.13 4.35 1.18 0.37 0.38 Spleen to liver ratio 0.117 0.202 0.4985 2.28 5.83 8.02 7.05 Example 13. Four-component lipid particles

The mean spleen BLI values, mean liver BLI values, and spleen to liver ratios of luciferase delivered by lipid particle formulations No. 7, No. 11, No. 12, and No. 78 (in PBS) were determined using the protocol described in Example 3. The results are reported in Table 6 below. Each of the formulations contained 40% (molar ratio) of SS-OP and 1.5% (molar ratio) of DMG-PEG2000. Table 6 Lipid Particle Formulation No. 78 Lipid Particle Formulation No. 7 Lipid Particle Formulation No. 11 Lipid Particle Formulation No. 12 Cholesterol/CHEMS ratio (mol %) 43.5/15 38.5/20 33.25/25 28.5/30 Mean spleen BLI 8.17 9.9 5.03 4.16 Mean liver BLI 11.33 4.35 0.78 0.59 Spleen to liver ratio 0.78 2.28 5.61 6.14 Example 14. Stability

The spleen BLI values ( FIG. 11A ), liver BLI values ( FIG. 11B ), and spleen to liver ratio ( FIG. 11C ) of lipid particle formulation No. 1 in mice were determined (at different time points after manufacture: fresh at 4°C, after 1 month of storage at 4°C, freshly frozen at -80°C (frozen for two weeks), after two freeze-thaw cycles at -80°C, after 1 month of storage at -80°C, or freshly prepared). "Fresh" formulation means that it is typically prepared one day before injection and stored at 4°C overnight. BLI values were determined using the protocol described in Example 3. Example 15. Immunogenicity Assay

The goal of the study was to determine the immunogenicity of mRNA encapsulated in the lipid particle formulation of interest. mRNA was purified by oligo-dT column or RP-HPLC to remove dsRNA.

C57BL/6 mice aged 6-8 weeks were injected intravenously with lipid particle formulations No. 79 (containing MC3) or No. 80 (containing SS-OP) via the tail vein. R848 (Resimod, Invivogen) was injected as a control for B cell and T cell activation. Six hours after injection, mice were sacrificed and blood was collected by cardiac puncture into tubes containing anticoagulant. Plasma was separated by centrifugation of the blood at 3000 rpm for 10 minutes at 4°C. Plasma was analyzed for INF-α using an ELISA kit (Mouse Quantikine IFN-α ELISA, R&D Systems). Spleens were treated with GentleMACS in RPMI medium to obtain splenocytes. Splenocytes (1 × 10 ⁶ cells/well) were then blocked with FcBlock (BioLegend) and stained for CD45 (all immune cells), CD3 (T cells), CD19 (B cells), and CD69 (activation markers) (all 1:100) from BioLegend in MACS buffer (PBS, 0.05% BSA, 2mM EDTA) for 1 hour at 4°C. Cells were washed with MACS buffer and flow cytometry was performed on a BD FACSCelesta. Activated B cells were gated as CD45+ CD19+ CD69+ CD19-, and activated T cells were gated as CD45+ CD3+ CD69+. The percentages in the graphs indicate the percentage of CD69+ cells of the parental gate (B cells or T cells).

IFN-α is a marker of innate immune cell activation by dsRNA to TLR3. CD69 is a marker of activated B cells and T cells. R848 is a small molecule TLR7/8 agonist that induces inflammation and activates B cells and T cells in C57BL/6 mice, but does not induce IFN-α production. The results are provided in Figures 12A-12C .

Neither MC3 nor SS-OP ionizable lipids alone (without luciferase mRNA) induced any IFN-α or resulted in activation of B or T cells. Liposome formulations encapsulating OdT purified fLuc mRNA (which was shown to contain some residual dsRNA) potently induced IFN-α and activation of CD69+ B and T cells. Liposome formulation No. 80 (containing SS-OP), which contained the same amount of fLuc mRNA as Liposome formulation No. 79 (containing MC3), induced significantly less IFN-α and B and T cell activation. HPLC purification of mRNA did not activate B or T cells and did not produce detectable IFN-α, indicating that HPLC is an excellent method for removing dsRNA. Example 16. Induction of Treg and anergic T cells

C57BL/6J female mice were injected intravenously via the tail vein with 100 μL of Formulation 1. The following groups were injected on days 0, 3, 7, and 10: MOG (20 μg), MOG+IL-10 (20+20) μg, MOG+TGFβ (20+20) μg, MOG+PD-L1 (20+20) μg, MOG+IL-10+PD-L1 (20+20+20) μg, or MOG+TGFβ+PD-L1 (20+20+20) μg.

Three days after the final dose, C57BL/6J female mice were euthanized using _CO2 on day 13. Spleens were harvested and weighed and placed in gentleMACS tubes containing 5 mL RPMI on ice. Spleens were homogenized in a gentleMACS dissociator for 1 min, filtered through a 40 μm filter cap into a 50 mL conical tube and washed with MACS buffer. Cells were pelleted after spinning at 500 × g for 5 min and RBC lysis was performed using ACK lysis buffer at room temperature for 2-3 min. Cells were washed with PBS (10 mL) and pelleted again. Finally, cells were resuspended in 5 mL MACS and counted using a Cellaca MX high-throughput cell counter after diluting 1:1 with 25 μL AOPI. For flow cytometry staining, 4 million cells were placed in a 1.5 mL eppendorf tube and made up to 1 mL with MACS buffer; then, cells were spun down and resuspended in 0.4 mL MACS buffer, followed by plating 2e6 cells/well in a 96-well VB plate by transferring 0.2 mL. For FMO, 0.2 mL from each group was pooled and dispensed into 9 separate wells at 0.2 mL. Flow staining was performed for tetramer and Treg groups. Briefly, cells were viability stained with Zombie Aqua and Fc Blocker in 200 μL PBS at room temperature for 15 minutes. Then, after pelleting and removal of supernatant, cells were stained in 50 μL MACS containing 10 μL MOG: IA-b Tetramer-PE (MBL International) for 1 hour at room temperature to obtain 2e6 total cells. After 1 hour, surface staining antibodies were added to each well at 50 μL/well to make up to 100 μL, and cells were stained for another 20 minutes at room temperature. After pelleting and removal of supernatant, cells were fixed with Foxp3 transcription factor fixation/permeabilization buffer at room temperature for 20 minutes and finally stored in 200 μL MACS buffer overnight. The next day, cells were pelleted and resuspended in 80 μL permeabilization/wash (P/W) buffer containing 1 μL Fc blocking agent and 1 μL mouse serum per well for 30 minutes. Then, Foxp3 antibody was added directly to the wells at 20 μL and stained in a total of 100 μL for 30 minutes at room temperature. Cells were washed with 100 μL P/W buffer and resuspended in 100 μL MACS buffer. Cells then received 20 μL MACS buffer containing 10k counting beads per well. Cells were run on a BD Celesta and programmed to record 1 million viable cells per sample, however, the viable cells collected ranged from 100-300k cells. Final analysis was performed using FlowJo and excel.

The results of the assay are provided in Figures 13 and 14. The results of a similar study are provided in Figures 17A-17C . These Figures show that lipid particle formulation No. 1 is able to deliver and express mRNA encoding the antigenic determinant or the antigenic determinant together with IL-10 in the targeted tissue (i.e., spleen). Example 17. In vivo efficacy in the EAE mouse model of multiple sclerosis

C57BL/6 mice (10-13 weeks of age) were anesthetized with isoflurane on day 0. EAE was induced by subcutaneous (sc) injection of a total of 200 µL of MOG _35-55 emulsion in complete Freund's adjuvant (MOG _{35 -55} /CFA) (#EK-2110, Hooke Laboratory) (containing approximately 200 ug MOG _35-55 ). Two injections of 100 µL each were administered into the flank just posterior to the spine. The animals were returned to their cages for 3 hours and each mouse was injected intraperitoneally (ip) with 100 µL PBS containing 150 ng pertussis toxin (PTX).

On day 1, all animals were again injected intraperitoneally (ip) with 100 μL PBS containing 150 ng PTX/mouse. On days 7 and 10 (preventive studies) or days 12 and 15 (therapeutic studies), lipid particle formulation No. 15 containing 20 μg MOG _35-55 or irrelevant mRNA was administered intravenously via tail vein injection. Control mice were injected with MOG _35-55 /CFA and PTX, but not lipid particle formulation No. 15. Animals were monitored for body weight and scored daily for signs of EAE induction starting on day 10 using the EAE scoring scale in Table 7 below. The study was terminated on day 20, and spleens and spinal cords were harvested from mice on day 21. Table 7. EAE Scoring

The results of the study are provided in Figures 18A-18B . These figures show that the formulation is able to deliver and express mRNA encoding MOG antigen to the spleen and spinal cord of target mice.

In addition, serum IL-17 levels were measured in inducible EAE mice after treatment with lipid particle formulation No. 15 encoding MOG peptide or lipid particle formulation No. 15 encoding irrelevant mRNA. A significant decrease in IL-17 levels was observed in EAE mice treated with formulations containing mRNA encoding MOG peptide compared to EAE mice treated with formulations containing mRNA encoding irrelevant mRNA ( Figure 18A ). Re-stimulation assay (interleukin analysis):

After termination of the study, C57BL/6J female mice were euthanized using CO _2. Spleens were harvested, homogenized, and after removal of RBCs using ACK lysis, splenocytes were counted and resuspended in RPMI medium containing 10% FBS. Large numbers of splenocytes were plated in duplicate at 200,000 cells per 96-well plate. Cells were stimulated with 10 µg/ml MOG peptide or left unstimulated (no peptide) for 48 hours. Supernatants were collected from the plates and run on Magi using a custom 7-plex (IL-2, IL-4, IL-6, IL-10, IFNy, TNFa, and IL-17a) Luminex kit from R&D systems. Data were analyzed using two-way ANOVA with Tukey post hoc test for multiple comparisons. Example 18. In vivo efficacy in the NOD mouse model of type 1 diabetes

NOD mice were immunized intravenously once a week for 5 weeks starting when the mice were 6 weeks old. The tolerogenic vaccine used in this experiment used the following lipid particle formulation 1: • Formulation 1: Lipid particle formulation 15 with tol105 mRNA and KIF1a mRNA (20 µg each, 40 µg total) • Formulation 2: Lipid particle formulation 15 with tol105 mRNA, KIF1a mRNA, and IL-10HA mRNA (20 µg each, 60 µg total) • Formulation 3: Lipid particle formulation 15 without mRNA, administered at a lipid equivalent to the 60 µg dose of formulation 2.

Tol105 is a construct made using the antigenic determinants described in Podojil, JR et al., "Tolerogenic Immune-Modifying Nanoparticles Encapsulating Multiple Recombinant Pancreatic β Cell Proteins Prevent Onset and Progression of Type 1 Diabetes in Nonobese Diabetic Mice", J Immunol. (Aug 1, 2022):209(3):465-75. doi: 10.4049/jimmunol.2200208. Epub 2022 Jun 20. PMID: 35725270; PMCID: PMC9339508.

KIF1a mRNA consists of two overlapping DECODE class I antigenic determinants with flanking regions. The sequences of all antigenic determinants in tol105 and Kif1a sequences are listed in Table 8 below. Table 8 Mouse peptide antigen sequence Insulin A14-20 YQLENYC IGRP (206-214) VYLKTNVFL Chromogranin A (359-372) SRMDQLAKELTAEK Insulin B9-23 SHLVEALYLVCGERG GAD65 (286-300) KGAAALGIGTDSVI GAD65 (514-524) RTLEDNEERMS HSP60 (437-460) VLGGGCALLRCIPALDSLKPANED ZnT8 (345-359) LTIQIESAADQDPSC IGRP(123-145) WYVMVTAALSYTISRMEESSVTL Kif1a (decoding) SLVGRAFVYLSNLLYPVPLVHRVAIVSE

After all five immunizations, the blood glucose levels of the mice were monitored once a week. Hyperglycemia is indicated when the blood glucose level exceeds 200 mg/dL glucose, and diabetes is indicated when the blood glucose level exceeds 400 mg/dL. The diabetic mice were euthanized and their spleens and pancreases were collected. All remaining mice in the study were euthanized when the mice were 30 weeks of age and the spleens and pancreases were collected. The results of the study are provided in Figures 19A-19B . The results again show that the formulation is able to deliver and express mRNA encoding T1D antigenic determinants to the target tissue, i.e., the spleen. Example 19. Determination of the pKa of lipid particle formulations using (2-(p-toluidinyl)-6-naphthalenesulfonic acid) ("TNS") assay

TNS is a non-fluorescent dye in aqueous solution, which exhibits strong fluorescence by binding to cationic lipids or polymers. At low pH values, the interaction between TNS and cationic lipids increases and leads to a steady increase in fluorescence until it reaches a plateau. The pH value at the half-maximum of fluorescence represents the pKa of lipid particle formulations No. 1 ( FIG. 20A ), No. 13 ( FIG. 20B ), and No. 14 ( FIG. 20C ).

Prepare a master buffer stock solution using 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM ammonium acetate, and 150 mM sodium chloride. Prepare pH buffers from 2.5 to 11.0 by adjusting the master buffer with concentrated NaOH and HCl in increments of 0.5 units. First, add 250 μL of buffer at each pH in triplicate to a 96-well black plate. Next, add 5 μL of the lipid particle formulation diluted to 0.1 mg/mL to each of the wells containing buffer. Finally, add TNS from a 0.16 mM stock solution in DMSO (10 μL) at a final concentration of 6 uM, mix the wells thoroughly, and let the plate sit for 5 minutes. TNS fluorescence was read using a Spectramax ID3 plate reader at 322 _Ex /431 _Em . Plots of normalized fluorescence of TNS versus pH were prepared. The pKa of a particular lipid particle formulation was determined from the plots, which was defined as the pH at half-maximum fluorescence of TNS ( Figures 20A-20C ).

The pKa of Formulations 1, 13, and 14 are each within the range reported for commercially approved lipid nanoparticles. Example 20. mRNA Integrity

To determine the integrity of mRNA, mRNA was first released from the mRNA-lipid particle formulation. In this study, lipid particle formulations 1, 13, and 14 were used.

For capillary gel electrophoresis ("CE") analysis under denaturing conditions, 10 μL of each mRNA-lipid particle formulation sample was mixed with 20 μL of CE-grade water containing 0.3% Triton X-100, 50 μL of formamide solution (SLS), and 10 μL of CE-grade water. The samples were incubated at room temperature for 20 minutes and then heated at 70°C for 10 minutes using a thermocycler. Immediately thereafter, the samples were cooled on ice for at least 5 minutes and transferred to a polycapillary sample plate pre-filled with RNA dilution buffer. An RNA ladder was included on the plate. The plate was run on an Agilent 5200 fragment analyzer. Relative concentrations (%) were determined from the RFU values. The decrease in relative mRNA concentration (%) of each sample at time T was compared to the original sample at time T = 0, and data were obtained for up to 16 weeks. A cutoff value of 85% was used as the minimum value for the integrity measure.

The results are provided in Figures 21, 22A and 22B . For lipid particle formulation No. 1, mRNA integrity was maintained for up to 4 months at 2-8°C; for lipid particle formulations Nos. 13 and 14, the relative concentration % values decreased after 6 weeks and 11 weeks, respectively. Example 21. Multiple mRNAs can be encapsulated into lipid particle formulations

Lipid particle formulation No. 1 was used as a representative example. Three mRNAs (MOG _35-55 , OV, A and IL-10) were used to formulate lipid particle formulation No. 1. 20 μg of each of the three mRNAs was added to the mRNA mixture, and the final combined concentration was 0.5 mg/mL. After formulation, the lipid particles were dissolved and analyzed using capillary gel electrophoresis to observe the integrity of the mRNA encapsulated by the lipid particles (the method is described in detail in Example 20 ). The relative concentration (%) of each mRNA was determined by CE electrophoresis.

As shown in Figures 23A and 23B , it is apparent that the lipid particle formulations described herein (eg, lipid particle formulation No. 1) can encapsulate at least three different mRNA payloads at one time. Example 22. Liposome formulations do not produce IL-1β in human PBMCs in vitro.

The mRNA lipid particle formulations were prepared using the T-mix method as described in Figure 1. Lipid particle formulation No. 1 with different ionizable lipids (i.e., SS-OP, MC-3, and SM-102) and the base formulation ( ionizable lipid /DSPC/cholesterol/DMG-PEG2000, 50/10/38.5/1.5) were prepared using HPLC purified mRNA. All formulations were tested for endotoxin before processing cells.

Fresh human PBMCs were seeded at 2e ⁵ cells/well in U-bottom 96-well plates in RPMI with 2% human AB serum. Cells were pre-sensitized with LPS (1 μg/ml) or R848 (0.5 μg/ml) for 2 hours. After pre-sensitization, cells were treated with various mRNA liposome formulations at a dose of 30 μg/mL mRNA for 17 hours or with nigericin (positive control) at a dose of 10 μg/mL for 1 hour. After 17 hours, plates were centrifuged at 300 × g for 5 minutes, supernatants were transferred to fresh plates, and IL-1β levels were analyzed using the human IL-1β/IL-1F2 quantikine ELISA kit from R&D systems.

As shown in Figure 24 , none of the lipid particle formulations produced any significant levels of IL-1β relative to the untreated (medium alone) group. This shows that the production and cleaning process used for the mRNA and lipid particle formulations produces a drug product that does not induce the production of pro-inflammatory interleukins (such as IL-1β) and does not stimulate the innate immune system. The positive control nigericin produced significantly higher levels of IL-1β after treatment. Example 23. Delivery efficacy of lipid particle formulations in human PBMC subsets

Fresh human PBMCs were used to isolate PBMC subsets (DCs, monocytes, CD4 T cells, CD8 T cells, and B cells). Individual cell subsets were isolated using the StemCell kit (positive or negative selection) according to the manufacturer's instructions. For each of these cell types, approximately 2e ⁵ cells/well/100 μL were seeded in the respective cell culture medium. Cells were treated with 100 μL of the specific lipid particle formulation (3 wells/treatment) for 17 hours, equivalent to an mRNA concentration of 20 μg/ml (firefly luciferase mRNA, 4 μg/well). Lipid particle formulations No. 13, No. 14, and No. 1 were compared with a comparator 4-component formulation ( ionizable lipid /DSPC/cholesterol/DMG-PEG2k, 50/10/38.5/1.5) using DLin-MC3-DMA, SM-102, or SS-OP as ionizable lipids. At the end of the incubation, the plate was spun at 300 × g for 5 minutes and the supernatant was removed. The cells were then resuspended in 100 μL of fresh medium, and 100 μL of steady-glo luciferin substrate was added to the cells and mixed thoroughly. After lysing the cells for 5 minutes, 150 μL of the lysate was transferred to a white opaque plate, and the luciferase signal was read using the luminescence protocol in a Spectramax iD3 plate reader. The relative luminescence units ("RLU") for each treatment are plotted. Higher signals indicate greater delivery efficacy.

As shown in Figures 25A-25E , lipid particle formulation No. 1 showed significantly higher delivery efficacy in all PBMC subsets compared to the other lipid particle formulations tested. Example 24. Delivery efficacy of lipid particle formulations in human PBMCs

Human PBMCs were seeded at 2e ⁵ cells/well/100 μL in RPMI containing 2% human AB serum. Cells were treated with 100 μL of liposome formulations (3 wells/treatment) for 17 hours, equivalent to an mRNA concentration of 20 μg/mL (firefly luciferase mRNA, 4 μg/well). Liposome formulations No. 13, No. 14, and No. 1 were compared to a comparator 4-component formulation ( ionizable lipid /DSPC/cholesterol/DMG-PEG2k, 50/10/38.5/1.5) using DLin-MC3-DMA or SM-102 as ionizable lipids. At the end of the incubation, the plates were spun at 300 × g for 5 minutes and the supernatant was removed. The cells were then resuspended in 100 μL of fresh RPMI containing 2% human AB serum. 100 μL of steady-glo luciferin substrate was added to the cells and mixed thoroughly. After 5 minutes of cell lysis, 150 μL of the lysate was transferred to a white opaque plate and the luciferase signal was read in a Spectramax iD3 plate reader using the luminescence protocol. The relative luminescence units ("RLU") of each treatment were plotted. Higher signals indicate greater delivery efficacy.

As shown in FIG. 26 , lipid particle formulation No. 1 showed significantly higher delivery efficacy compared to the other lipid particle formulations tested Example 25. mRNA-lipid particle formulation dose response in human PBMCs in vitro

Human PBMCs were seeded at 2e ⁵ cells/well/100 μL in RPMI containing 2% human AB serum. Cells were treated with 100 μL of Liposome Formulation No. 1 (3 wells/treatment) containing various mRNA concentrations (firefly luciferase mRNA) for 17 hours. The final mRNA concentration range of 0.25 to 30 μg/mL (0.05 to 6 μg mRNA/well) was evaluated. At the end of the incubation, the plates were spun at 300 × g for 5 minutes and the supernatant was removed. The cells were then resuspended in 100 μL of fresh RPMI containing 2% human AB serum, and 100 μL of steady-glo luciferin substrate was added to the cells and mixed well. After 5 minutes of cell lysis, 150 μL of the lysate was transferred to a white opaque plate and the luciferase signal was read in a Spectramax iD3 plate reader using a luminescence protocol. The relative luminescence units ("RLU") for each treatment were plotted. Higher signals indicate greater delivery efficiency.

As shown in Figure 27 , a clear dose response was observed with increasing mRNA concentrations. It is also evident that lipid particle formulation No. 1 improved delivery efficacy. Example 26. Induction of antigen-specific Tregs in vivo by lipid particle formulations co-encapsulating antigenic determinants and immunomodulators

C57BL/6J female mice were immunized subcutaneously with 100 μg MOG _35-55 /CFA emulsion. The mice were treated with lipid particle formulation No. 1 encapsulating an irrelevant mRNA (mouse serum albumin ("MSA")), MOG _35-55 , IL-10, or both MOG _35-55 and IL-10. The mice were treated with four doses of lipid particle formulation No. 1 twice a week (Day 7, Day 10, Day 14, and Day 17) starting from Day 7 after immunization.

Three days after the final dose, mice were euthanized using CO ₂ on day 17. Spleens were harvested, weighed, and placed in gentleMACS tubes containing 5 mL RPMI on ice. Spleens were homogenized in a gentleMACS dissociator for 1 min, filtered through a 40 μm filter cap into a 50 mL conical tube and washed with MACS buffer. Cells were pelleted after spinning at 500 × g for 5 min, and RBC lysis was performed using ACK lysis buffer at room temperature for 2-3 min. Cells were washed with PBS (10 mL) and pelleted again. Finally, cells were resuspended in 5 mL MACS and counted using a Cellaca MX high-throughput cell counter after diluting 1:1 with 25 μL AOPI. For flow cytometry staining, 4 million cells were placed in a 1.5 mL eppendorf tube and made up to 1 mL with MACS buffer. Cells were then spun down and resuspended in 0.4 mL MACS buffer, followed by plating 2e ⁶ cells/well in a 96-well VB plate by transferring 0.2 mL. For FMO, 0.2 mL from each group was pooled and dispensed into 9 separate wells at 0.2 mL.

Flow staining was performed for tetramer and Treg groups. Briefly, cells were stained for viability with Zombie Aqua and Fc blocker in 200 μL PBS at room temperature for 15 minutes. Then, after pelleting and removal of supernatant, cells were stained for 1 hour at room temperature in 50 μL MACS containing 10 μL MOG:IA-b tetramer-PE (MBL International) to obtain 2e6 total cells. After 1 hour, surface staining antibodies were added to each well at 50 μL/well to make up to 100 μL, and cells were stained for another 20 minutes at room temperature. After pelleting and removal of supernatant, cells were fixed with Foxp3 transcript fixation/permeabilization buffer for 20 min at room temperature and finally stored in 200 μL MACS buffer overnight.

The next day, cells were pelleted and resuspended in 80 μL permeabilization/wash (P/W) buffer containing 1 μL Fc blocking agent and 1 μL mouse serum per well for 30 minutes. Foxp3 antibody was then added directly to the wells at 20 μL and stained in a total of 100 μL for 30 minutes at room temperature. Cells were washed with 100 μL P/W buffer and resuspended in 100 μL MACS buffer. Cells then received 20 μL MACS buffer containing 10k counting beads per well. Cells were run on a BD Celesta and the program was set to record 1 million live cells per sample. The number of live cells collected ranged from 100-300k cells. FlowJo and Excel were used for final analysis.

As shown in Figures 28A-28B , co-encapsulation of the antigenic determinant (MOG _35-55 ) and the immunomodulator (IL-10) induced significantly higher levels of MOG-specific Tregs compared to either the antigenic determinant or the immunomodulator alone. The data clearly show the synergistic effect of administering both mRNAs together. Example 27. IL-10 was detected in mouse plasma on days 8 and 18, i.e., 24 hours after injection.

C57BL/6J female mice were immunized subcutaneously with 100 μg MOG _35-55 /CFA emulsion. The mice were treated with lipid particle formulation No. 1 encapsulating an irrelevant mRNA (mouse serum albumin ("MSA")), MOG _35-55 , IL-10, or both MOG _35-55 and IL-10.

Mice were treated with four doses of Liposome Formulation No. 1 twice a week (Day 7, Day 10, Day 14, and Day 17) starting from Day 7 after immunization. On Day 8 and Day 18, 24 hours after the 1st and 4th doses, mice were bled via the retro-orbital sinus and the blood was collected into tubes containing EDTA. Plasma was separated from the blood and stored at -80°C until analysis. To detect IL-10 levels, plasma samples were diluted 2-fold and analyzed using the Mouse IL-10 Quantikine ELISA Kit from R&D systems according to the manufacturer's instructions.

As shown in Figure 29 , a clear dose response was observed for IL-10 concentrations in plasma, with higher IL-10 mRNA doses producing significantly higher plasma IL-10. Lipid particle formulation No. 1 maintained mRNA delivery efficacy throughout the duration of the study, with no difference in IL-10 levels at day 8 and day 18. Example 28. Lipid particle formulation stock solutions stored at -80°C for up to 6 months retain original key quality attributes and functional mRNA delivery in vivo

For long-term storage stability studies, multiple batches of Liposome Formulation No. 1 containing Firefly luciferase mRNA were prepared. Prior to filtration, sucrose from an 80% w/v sucrose stock solution in Tris-buffered saline was incorporated into the formulations, resulting in a final sucrose concentration of 7% w/v for each batch. The formulations were then stored at -80°C until the indicated time points.

At designated time points of 1 month, 3 months, and 6 months, each lipoparticle formulation No. 1 in the batch was analyzed for particle size, PDI, and mRNA encapsulation. Functional delivery of mRNA was assessed by injecting the formulation into 6-8 week old C57BL/6J mice (20 μg) and comparing the BLI signal of the freshly prepared batch of lipoparticle formulation No. 1 with that of the formulation stored at 80°C.

Prior to injection, the fur of mice was cut open along the left side of the abdomen up to the spine to highlight the spleen and liver expression field. Mice were injected intravenously via the tail vein with 100 μL of lipid particle formulation No. 1 containing the equivalent of 20 μg of firefly luciferase mRNA. Six hours later, mice were administered 200 μL of D-luciferin (3 mg/mouse) via intraperitoneal injection (i.p.), anesthetized with isoflurane and imaged within 10 minutes using a LICOR Pearl imager. Images were acquired in the right flank position under 3% isoflurane for maintenance of anesthesia. LICOR settings: BLI and white channel only, 85um resolution, focus at 0. Bioluminescence intensity (BLI) values were determined using Image Studio software version 5.2.5 from LICOR. A region of interest was drawn around an organ (e.g., spleen or liver) using a software tool, and the corresponding bioluminescence intensity (BLI) signal was calculated by the software after background subtraction. Data were reported as spleen BLI value, liver BLI value, and spleen to liver ratio (i.e., the ratio obtained by dividing the BLI value in the spleen by the BLI value in the liver of the same animal).

As shown in Figures 30A-30D , lipid particle formulation stock No. 1 retained its original key quality attributes (size, PDI and mRNA encapsulation) and functional delivery after long-term storage at -80°C for 6 months. Spleen specificity was also maintained. Example 29. EAE Method

Under isoflurane anesthesia, C57BL/6J mice were injected subcutaneously with 200 μL total MOG/CFA containing approximately 200 μg of MOG35-55. For this, two 100 μL injections were made in the flank just behind the spine. The animals were returned to their cages for 3 hours and then injected with 100 μL PBS containing 150 ng pertussis toxin ("PTX")/mouse via the intraperitoneal route (Day 0). One day later, they were injected again with 100 μL PBS containing 150 ng PTX/mouse via the intraperitoneal route (Day 1). Animals were injected with lipid particle formulation No. 1 containing MOG, MOG/IL-10, or control mRNA (eGFP). Untreated mice did not receive any treatment.

MOG/IL-10 was administered subcutaneously (back) to a group of mice. Two doses were administered to mice (day 7 and day 10). Starting from day 10, the disease signs of mice were monitored and scored daily until day 16 (EAE peak). On day 16, mice were killed by CO ₂ euthanasia and intracardiac perfusion with 10-15 mL PBS. Spleens were harvested, followed by 2× inguinal lymph nodes (LN) and 2× brachial/axillary LN, and kept on ice in PBS. Complete spinal column dissection was performed with decapitation just below the cerebellum, and the spinal column was flushed distally with a 19G needle containing PBS. All spinal columns were also kept on ice in PBS until all animals were killed. Spleens were weighed and subjected to gentleMACS lysis in 5 mL MACS buffer. LNs were also kept on ice in a 48-well plate.

Next, spinal cords were weighed and digested in 2.5 mL of Multitissue Dissociation Kit I per spinal cord for 20 min at +37°C and kept on ice. All tissues were crushed with the blunt end of a syringe plunger and filtered through a 70 μm filter with MACS buffer, followed by washing and spinning at 500 × g for 5 min. LN were resuspended in 2 mL of MACS buffer. Spleens were subjected to RBC lysis with ACK lysis buffer for 2 min, followed by making up to 10 mL with MACS buffer, washed, and resuspended in 5 mL of MACS buffer. Resuspend the spinal cord in 4 mL of 30% Percoll/PBS mixture using a P1000 pipette tip in a 15 mL conical tube. Next, place a borosilicate glass Pasteur pipette in each 15 mL conical tube. To serve as the basal layer for the Percoll gradient, slowly pipette the 70% Percoll/PBS mixture into the borosilicate glass Pasteur pipette (starting with 2 mL), being careful not to disrupt the cell suspension during the basal layer. Next, pipette the last 1 mL into the borosilicate glass Pasteur pipette for a total of 3 mL. Carefully remove each glass pipette from the Percoll gradient suspension, ensuring that the basal layer reaches 3 mL in the 15 mL conical tube. Spin the suspension at 690 × g for 20 minutes at room temperature to separate the mononuclear cell layer in the center of the conical tube from the neuronal debris. Aspirate the top of the Percoll suspension until the volume above the mononuclear cell layer is approximately 1 mL. Next, pipette the mononuclear cells containing the leukocyte fraction from the CNS into a separate 15 mL conical tube and make up to a total of approximately 13-14 mL with PBS. Invert all tubes to mix the spinal cord samples, then pellet the cells by spinning at 520 × g for 6 minutes. Resuspend the spinal cord mononuclear cells in MACS buffer at 150 μL per tube, and place all samples in separate 96-well RB plates for flow staining.

Splenocytes and LN cells were all pipetted into Nexcelom 96-well plates (20 μL per well) and cells were counted by resuspending 1:1 with AOPI viability dye and counting via Cellaca.

A volume associated with 6-8 million cells was pipetted into a 1.5 mL microcentrifuge tube and the cells were pelleted into SP and LN cells. The cell pellets were then resuspended in MACS buffer at 600-800 μL per sample at a concentration of 1 million cells/0.1 mL. Two million cells corresponding to 0.2 mL from each tube were then dispensed into 96-well RB plates for staining for flow cytometry analysis. FMOs were set up accordingly by pooling cells from the MOG/IL10HA group and then redistributed at 2 million cells per well.

All cells were washed and then first stained for viability by adding 100 μL PBS containing 1:500 Zombie Aqua and 1:100 FcX Plus and incubated for 15 minutes at room temperature (RT). Cells were then washed and resuspended in 50 μL MACS buffer containing a 1:10 dilution of MOG 35-55 I-Ab Tetramer PE (MBL International) and incubated for 45 minutes at room temperature in the dark. At the 45 minute mark, the remaining 50 μL of the MACS buffer mixture containing cell surface marker antibodies was added to each well and the samples were incubated for 20 minutes at room temperature in the dark. After washing with MACS buffer, cells were fixed with eBioscience Foxp3 Transcription Factor Buffer Set Fixation/Permeabilization Reagent at room temperature in the dark for 30 minutes. Then, cells were washed and resuspended in MACS buffer overnight and stored in the refrigerator overnight. Cells were washed and resuspended in 80 μL 1× Permeabilization Buffer and allowed to stand at room temperature for 30 minutes to re-permeabilize after overnight incubation. For Foxp3 staining, 20 μL containing Foxp3 antibody (diluted 1:200 in 100 μL) was added to a total of 100 μL of 1× Permeabilization Buffer, and cells were stained at room temperature for 30 minutes. The cells were then run on a BD Celesta after fresh replenishment with beads.

All publications and patents cited throughout this specification (including all patents, patent applications, scientific publications, manufacturer's instructions, guides, etc.) are hereby incorporated by reference in their entirety for all purposes. If the material incorporated by reference contradicts or is inconsistent with this specification, this specification will supersede any such material. Equivalents

Without departing from the spirit or essential features of the present invention, the present invention may be embodied in other specific forms. Therefore, the foregoing embodiments should be considered in all respects as illustrative and not limiting of the invention described herein. Therefore, the scope of the present invention is indicated by the appended patent application rather than the foregoing description, and all changes within the equivalent meaning and scope of the patent application are intended to be included therein.

FIG. 1 shows an exemplary process for preparing lipid particles provided herein. Exemplary components and reagents are non-limiting and representative only. Alternative components or reagents or additional or fewer components or reagents may be used in the process. FIG. 2A-C shows bioluminescence imaging ("BLI") values of luciferase in mice administered lipid particle formulations No. 1, No. 3-6, and No. 53-55 (in phosphate buffered saline ("PBS")) and PBS controls (each containing mRNA encoding luciferase). FIG. 2A shows the BLI values of luciferase in the spleen of mice. FIG. 2B shows the BLI values of luciferase in the liver of mice. FIG. 2C is a graph of the spleen and liver ratio of the BLI values of luciferase. Figures 3A-3C show the BLI values of luciferase in mice administered lipid particle formulations No. 1, No. 30, No. 31, No. 2, No. 32 and No. 33 (in PBS) and PBS controls (each containing mRNA encoding luciferase). Figure 3A shows the BLI values of luciferase in the spleen of mice. Figure 3B shows the BLI values of luciferase in the liver of mice. Figure 3C is a graph of the spleen and liver ratio of the BLI values of luciferase. Figures 4A-4C show the BLI values of luciferase in mice administered lipid particle formulations No. 13, No. 1 and No. 43-48 (in PBS) and PBS controls. Figure 4A shows the BLI values of luciferase in the liver of mice. FIG. 4B shows the BLI values of luciferase in the spleen of mice. FIG. 4C is a graph of the ratio of the BLI values of luciferase in the spleen to the liver. FIG. 5A-5C show the BLI values of luciferase in mice administered with lipid particle formulations No. 15, No. 16, No. 20-22, No. 13, No. 23, and No. 24 (in PBS) and PBS control (each including mRNA encoding luciferase). FIG. 5A shows the BLI values of luciferase in the spleen of mice. FIG. 5B shows the BLI values of luciferase in the liver of mice. FIG. 5C is a graph of the ratio of the BLI values of luciferase in the spleen to the liver. FIG6 shows the BLI values of luciferase in the spleen of mice administered lipid particle formulations No. 16, No. 25-29, No. 14 and No. 1 (in PBS) and PBS control (each including mRNA encoding luciferase). FIG7A-7C shows the BLI values of luciferase in mice administered lipid particle formulations No. 13, No. 1 and No. 37-42 and PBS control (each including mRNA encoding luciferase). FIG7A shows the BLI values of luciferase in the liver of mice. FIG7B shows the BLI values of luciferase in the spleen of mice. FIG7C is a graph of the spleen and liver ratio of the BLI values of luciferase. Figures 8A-8C show the BLI values of luciferase in mice administered lipid particle formulations No. 16, No. 25-29, No. 14 and No. 1 (in PBS) and PBS control (each including mRNA encoding luciferase). Figure 8A shows the BLI values of luciferase in the liver of mice. Figure 8B shows the BLI values of luciferase in the spleen of mice. Figure 8C is a graph of the spleen to liver ratio of the BLI values of luciferase. Figures 9A-9C show the BLI values of luciferase in mice administered lipid particle formulations No. 14 (in PBS) (administered intravenously), No. 1 (administered intravenously as a Tris buffer solution), No. 1 (in PBS) (frozen for 1 month), and No. 7 (in PBS) (each including mRNA encoding luciferase). Figure 9A shows the BLI values of luciferase in the spleen of mice. Figure 9B shows the BLI values of luciferase in the liver of mice. Figure 9C is a graph of the spleen and liver ratio of the BLI values of luciferase. FIG. 10A and FIG. 10B show the spleen BLI values ( FIG. 10A ) and spleen to liver BLI values ( FIG. 10B ) of luciferase in mice after intravenous administration of lipid particle formulations No. 34, No. 35 and No. 1 (in PBS) (each including mRNA encoding luciferase). Figures 11A-11C show spleen BLI values ( Figure 11A ), liver BLI values ( Figure 11B ), and spleen to liver ratio (Figure 11C ) in mice after administration of lipid particle formulation No. 1 including mRNA encoding luciferase at different time points after production: fresh at 4°C, after 1 month of storage at 4°C, freshly frozen at -80°C (frozen for two weeks), after two freeze-thaw cycles at -80°C, after 1 month of storage at -80°C, or freshly prepared. "Fresh" formulation means that it is typically prepared the day before injection and stored at 4°C overnight. Figures 12A-12C show the levels of INFα ( Figure 12A), CD69+ B cells (Figure 12B), and CD69+ T cells ( Figure 12C ) in mice after administration of PBS control, R848 (resiquimod), lipid particle formulation No. 79 (in PBS), and lipid particle formulation No. 80 (in PBS). Figure 13 shows the levels of T regulatory cells as a percentage of MOG-specific CD4+ spleen cells in mice after administration of lipid particle formulation No. 1 (in PBS) containing or not encoding mRNA for myelin oligodendrocyte glycoprotein ("MOG") epitopes, IL-10, TGF-β, and/or PD-L1. Figure 14 shows FR4+ CD73+ anergic T cells as a percentage of learned CD4+ spleen cells in mice following administration of lipid particle formulation No. 1 (in PBS) with or without mRNA encoding MOG epitope, IL-10, TGF-β and/or PD-L1. Figures 15A-15E show images of tissues in mice where the lipid particle formulation delivered mRNA encoding luciferase to the liver, spleen and/or pancreas. Figure 15A shows lipid particle formulation No. 14 (in Tris buffered saline ("TBS")) delivering mRNA encoding luciferase to the liver and spleen when administered intravenously to mice. Figure 15B shows lipid particle formulation No. 14 (in TBS) that delivers mRNA encoding luciferase to the pancreas and spleen when administered intraperitoneally to mice. Figure 15C shows lipid particle formulation No. 1 (in TBS) that delivers mRNA encoding luciferase to the spleen after two weeks of freezing when administered intravenously to mice. Figure 15D shows lipid particle formulation No. 1 (in TBS) that delivers mRNA to the spleen when administered intravenously to mice. Figure 15E shows lipid particle formulation No. 1 (in TBS) that delivers mRNA to the pancreas and spleen when administered intraperitoneally to mice. Figure 16 shows whole body images of mice 16 hours, 24 hours, or 48 hours after subcutaneous administration of lipid particle formulations No. 13, No. 14, and No. 1 (in PBS) delivering mRNA encoding luciferase. The region of interest ("ROI") on the image shows the subcutaneous injection site and the liver. Figures 17A-17C show the effects of administering lipid particle formulation No. 1 delivering mRNA encoding the MOG epitope with and without IL-10 on T cells in mice. Figure 17A provides an overview of the experiment. Figure 17B shows the number of epitope-specific Foxp3+ regulatory T cells after administration of lipid particle formulation No. 1. Figure 17C shows the number of antigen-specific CD4+, CD44hi, Foxp3-learned T cells after administration of lipid particle formulation No. 1. Figures 18A-18B show the in vivo efficacy of mRNA tolerogenic vaccines in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Figure 18A provides EAE scores and serum IL-17 levels after administration of lipid particle formulation No. 15 in the preventive study. Figure 18B provides EAE scores after administration of lipid particle formulation No. 15 in the therapeutic study. Figures 19A-19B show the in vivo efficacy of mRNA tolerogenic vaccines in the non-obese diabetic ("NOD") mouse model of type 1 diabetes. Figure 19A provides blood glucose levels (mg/dL) in mice after administration of lipid particle formulation No. 1 containing KIF1a, tol105 (9-epitope) and IL-10 mRNA. Figure 19B provides normal glucose percentage in NOD mice after administration of lipid particle formulation No. 1 containing KIF1a mRNA, tol105 (9-epitope) and IL-10. Figures 20A-20C show pKa determinations for lipid particle formulations No. 1 ( Figure 20A ), No. 13 ( Figure 20B ) and No. 14 ( Figure 20C ). Figure 21 shows that for lipid particle formulation No. 1, the relative concentration % (fLUC mRNA) remained above 85% for 4 months. For lipid particle formulations No. 13 and No. 14, the relative concentration (%) dropped below 85% after storage at 2°C-8°C for 6 weeks and 11 weeks, respectively. Figures 22A - 22B show capillary gel electrophoresis ("CE") analysis of lipid particle formulations No. 1, No. 13, and No. 14 and fLuc mRNA at T=0 and T=16 weeks of storage at 2-8°C. Figures 23A - 23B provide results from CE analysis. Figure 23A provides CE electropherograms of three different mRNA constructs encoding MOG epitopes, IL-10, and ovalbumin ("OVA") epitopes loaded in lipid particle formulation No. 1. Figure 23B provides CE gel images of three different mRNA constructs loaded in Formulation 1. Figure 24 provides IL-1β levels for lipid particle formulation No. 1 and lipid particle formulations containing ionizable lipids (SS-OP, MC-3 or SM-102), DSPC, cholesterol and DMG-PEG2000 at a molar ratio of 50/10/38.5/1.5 (molar % of each lipid in the formulation). None of the formulations produced any significant level of IL-1β relative to the medium alone (no LNP) group. This shows that the generation and cleaning process used for mRNA and formulations produces a drug product that does not induce the production of pro-inflammatory cytokines (such as IL-1β) and does not stimulate the innate immune system. The positive control nigericin produced significantly higher levels of IL-1β after treatment. Figures 25A-25E show the delivery efficacy of lipid particle formulations No. 13, No. 14, and No. 1 and a four-component formulation containing ionizable lipids (DLin-MC3-DMA, SM-102, or SS-OP), DSPC, cholesterol, and DMG-PEG2k at a molar ratio of 50/10/38.5/1.5 (molar % of each lipid in the formulation) in human peripheral blood mononuclear cells (PBMCs) and PBMC subsets (dendritic cells ("DC") ( Figure 25A ); monocytes ( Figure 25B ); B cells ( Figure 25C ); CD8 ( Figure 25D ); and CD4 ( Figure 25E )) in vitro. Compared to the other lipid particle formulations tested, lipid particle formulation No. 1 showed significantly higher delivery efficacy in all human PBMC subsets. Figure 26 provides a comparison of lipid particle formulations No. 13, No. 14 and No. 1 with a four-component formulation containing ionizable lipids (DLin-MC3-DMA or SM-102), DSPC, cholesterol and DMG-PEG2k in a molar ratio of 50/10/38.5/1.5 (molar % of each lipid in the formulation). Compared to the other lipid particle formulations tested, lipid particle formulation No. 1 showed significantly higher delivery efficacy in human PBMC. FIG. 27 provides the relative luminescence units ("RLU") observed in human PBMCs after treatment with lipid particle formulation No. 1 at various mRNA concentrations (firefly luciferase mRNA). Dose responses were observed at increasing concentrations of mRNA. FIG. 28A-28B show the levels of T regulatory cells in mice after administration of lipid particle formulation No. 1 encapsulating mouse serum albumin (untreated), MOG _35-55 , IL-10, or both MOG _35-55 and IL-10. FIG . 28A reports the number of MOG-specific FoxP3-positive Tregs observed. FIG. 28B reports the percentage of FoxP3-positive Tregs. Co-encapsulation of an antigenic determinant (MOG35-55) and an immunomodulator (IL-10) induced significantly higher levels of MOG-specific Tregs compared to either the antigenic determinant or the immunomodulator alone. The data clearly show an additive effect of co-administering both mRNAs. Figure 29 reports IL-10 concentrations in mice treated with lipid particle formulation No. 1 encapsulating mouse serum albumin ("MSA") MOG _35-55 , IL-10, or both MSA or MOG _35-55 and IL-10. Untreated mice were not treated with lipid particle formulation No. 1. A clear dose response of IL-10 concentrations in plasma was observed, with higher IL-10 mRNA doses producing significantly higher plasma IL-10. Lipid Formulation No. 1 maintained mRNA delivery efficacy throughout the duration of the study, with no differences in IL-10 levels at days 8 and 18. Figures 30A-30D provide particle size ( Figure 30A ), polydispersity index ("PDI") ( Figure 30B ), mRNA encapsulation ( Figure 30C ), BLI ( Figure 30D ) of Lipid Particle Formulation No. 1 containing firefly luciferase mRNA after storage at -80°C for the indicated time points. Lipid Particle Formulation No. 1 stock retained its original key quality attributes (size, PDI, and mRNA encapsulation) and functional delivery after long-term storage at -80°C for 6 months. Spleen specificity was also maintained. Figures 31A-31C show the liver BLI values ( Figure 11A ), spleen BLI values ( Figure 11B), and spleen to liver ratio (Figure 11C ) in mice after administration of lipid particle formulation No. 1 including mRNA encoding luciferase at different time points after production: fresh at 4°C, frozen at -80°C (frozen for two weeks), after storage at -80°C for 1 month, after storage at -80°C for 3 months, and after storage at -80°C for 6 months. Figures 31A-31C show liver BLI values ( Figure 31A ), spleen BLI values ( Figure 31B) and spleen to liver ratio (Figure 31C ) in mice after administration of lipid particle formulation No. 1 including mRNA encoding luciferase at different time points after production: fresh at 4°C, frozen at -80°C (frozen for two weeks), after 1 month of storage at -80°C, after 3 months of storage at -80°C, and after 6 months of storage at -80°C. Figures 32A-32E show the characteristics of lipid particle formulation No. 1 at 4°C at T=0 and after 1 month at 4°C compared to the second freshly prepared lipid particle formulation No. 1. These characteristics are particle size ( FIG. 32A ), polydispersity index ( FIG. 32B ), % mRNA encapsulation ( FIG. 32C ), spleen BLI ( FIG. 32D ), liver BLI ( FIG. 32E ), and spleen to liver ratio ( FIG. 32F ). FIGs. 33A-33C provide whole body images of mice administered lipid particle formulation No. 1 at T=0 at 4°C and 1 month later at 4°C compared to a second freshly prepared lipid particle formulation No. 1. FIGs. 34A-34B provide whole body images of mice administered lipid particle formulations Nos. 78, 7, 11, 12 ( FIG. 34A ), 77, 75, 76, and 7-10 ( FIG. 34B ) as well as mean spleen BLI, mean liver BLI, and spleen to liver ratio. Figures 35A-35C show that lipid formulation No. 1 inhibits disease activity and reduces monocyte infiltration when administered intravenously or subcutaneously in mice. Figure 35A shows that both intravenous and subcutaneous administration inhibit paralysis in the EAE mouse model. Figure 35B compares the number of monocytes in mice administered mouse serum albumin (untreated), control mRNA, and MOG _35-55 and IL-10 in lipid particle formulation No. 1 administered intravenously or subcutaneously. FIG. 35C reports the percentage of MOG-specific CD4+ spleen cells that serve as Tregs in mice following administration to mice of mouse serum albumin (untreated), control mRNA, and MOG _35-55 and IL-10 in lipid particle formulation No. 1 administered intravenously or subcutaneously.

Claims (302)

A lipid particle comprising cholesterol hemisuccinate ("CHEMS") and a payload and, as appropriate, one or more of the following: a. one or more ionizable lipids; b. one or more neutral lipids; c. one or more sterols; and d. one or more stealth lipids. A method of targeting a payload to the spleen of an individual in vivo, the method comprising combining a lipid particle or a composition comprising the payload encapsulated within a lipid particle or a composition comprising a molar amount of at least 11% CHEMS. The method of claim 2, wherein the lipid particle or the composition further comprises one or more ionizable lipids, one or more neutral lipids, one or more sterols, one or more stealth lipids or any combination thereof. The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle delivers the payload to the spleen. The lipid particle of claim 1 or 4 or the method of claim 2 or 3, wherein the ratio of the effective load delivered to the spleen and the liver is greater than 1.1. The lipid particle of any one of claims 1, 4 and 5 or the method of any one of claims 2 to 5, wherein the ratio of the payload delivered to the spleen to the liver is about 1.1 to about 12. The lipid particle of any one of claims 1 and 4 to 6 or the method of any one of claims 2 to 6, wherein the ratio of the effective load delivered to the spleen to the liver is about 4 to about 11. The lipid particle of any one of claims 1 and 4 to 7 or the method of any one of claims 2 to 7, wherein the ratio of the payload delivered to the spleen and the liver is determined using an imaging system. The lipid particle of claim 8 or the method of claim 8, wherein the imaging system is a LICOR Pearl Trilogy imaging system using Image Studio software from LICOR version 5.2.5. The lipid particle of any one of claims 1 and 4 to 9 or the method of any one of claims 2 to 9, wherein the molar amount of the CHEMS in the lipid particle is at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19% or at least 20%. The lipid particle of any one of claims 1 and 4 to 10 or the method of any one of claims 2 to 10, wherein the molar amount of the CHEMS in the lipid particle is about 11% to about 45%, about 15% to about 45%, about 20% to about 45%, about 5% to about 40%, about 7.5% to about 40%, about 10% to about 40%, about 15% to about 40%, about 20% to about 40%, about 5% to about 35 %, about 7.5% to about 35%, about 10% to about 35%, about 15% to about 35%, about 20% to about 35%, about 5% to about 30%, about 7.5% to about 30%, about 10% to about 30%, about 15% to about 30%, about 20% to about 30%, about 5% to about 25%, about 7.5% to about 25%, about 10% to about 25%, about 15% to about 25%, or about 20% to about 25%. The lipid particle of any one of claims 1 and 4 to 11 or the method of any one of claims 2 to 11, wherein the molar amount of the CHEMS in the lipid particle is about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40% or about 45%. The lipid particle of any one of claims 1 and 4 to 11 or the method of any one of claims 2 to 11, wherein the molar amount of the CHEMS in the lipid particle is about 11% to about 20%, about 20% to about 30%, or about 11% to about 30%. The lipid particle of any one of claims 1 and 4 to 11 or the method of any one of claims 2 to 11, wherein the molar amount of the CHEMS in the lipid particle is about 11% to about 20%. The lipid particle of any one of claims 1 and 4 to 11 or the method of any one of claims 2 to 11, wherein the molar amount of the CHEMS in the lipid particle is about 11%. The lipid particle of any one of claims 1 and 4 to 11 or the method of any one of claims 2 to 11, wherein the molar amount of the CHEMS in the lipid particle is about 15%. The lipid particle of any one of claims 1 and 4 to 11 or the method of any one of claims 2 to 11, wherein the molar amount of the CHEMS in the lipid particle is about 20%. The lipid particle of any one of claims 1 and 4 to 11 or the method of any one of claims 2 to 11, wherein the molar amount of the CHEMS in the lipid particle is about 25%. The lipid particle of any one of claims 1 and 4 to 18 or the method of any one of claims 2 to 18, wherein the lipid particle comprises one or more ionizable lipids. A lipid particle as in any one of claims 1 and 4 to 19 or a method as in any one of claims 2 to 19, wherein the molar amount of the one or more ionizable lipids in the lipid particle is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19% or at least 20%. The lipid particle of any one of claims 1 and 4 to 20 or the method of any one of claims 2 to 20, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 20% to about 70%. The lipid particle of any one of claims 1 and 4 to 21 or the method of any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 25% to about 65%, about 30% to about 65%, about 35% to about 65%, about 45% to about 65%, about 50% to about 65%, about 25% to about 60%, about 30% to about 60%, about 35% to about 60%, about 45% to about 60%, about 50% to about 60%, about 25% to about 55%, about 30% to about 55%, about 35% to about 55%, about 45% to about 55%, about 50% to about 55%, about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, or about 45% to about 50%. The lipid particle of any one of claims 1 and 4 to 21 or the method of any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% or about 65%. The lipid particle of any one of claims 1 and 4 to 21 or the method of any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 30% to about 60%. The lipid particle of any one of claims 1 and 4 to 21 or the method of any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 40% to about 60%. The lipid particle of any one of claims 1 and 4 to 21 or the method of any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 40% to about 50%. The lipid particle of any one of claims 1 and 4 to 21 or the method of any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 50% to about 60%. A lipid particle as claimed in any one of claims 1 and 4 to 21 or a method as claimed in any one of claims 2 to 21, wherein the one or more ionizable lipids in the lipid particle are present in a molar amount of about 30%. A lipid particle as in any one of claims 1 and 4 to 21 or a method as in any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 40%. A lipid particle as in any one of claims 1 and 4 to 21 or a method as in any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 50%. A lipid particle as in any one of claims 1 and 4 to 21 or a method as in any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 55%. A lipid particle as in any one of claims 1 and 4 to 21 or a method as in any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 60%. A lipid particle as in any one of claims 1 and 4 to 21 or a method as in any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 65%. A lipid particle as in any one of claims 1 and 4 to 21 or a method as in any one of claims 2 to 21, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 70%. The lipid particle of any one of claims 1 and 4 to 34 or the method of any one of claims 2 to 34, wherein the one or more ionizable lipids in the lipid particles are one or more selected from the group consisting of: DLin-MC3-DMA ("MC3"), DLin-KC2-DMA ("KC2"), ssPalmO-Phe ("SS-OP"), C12-200, SM-102, α-D-tocopheryl succinyl ("SS-EC"), ALC-0315 ("ALC"), 3-(ethyl(methyl)amino)propionic acid tri-N-tridecyl ester ("304-O13"), 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionic acid tetrakis(2-(octyldisulfanyl)ethyl) ester ("306-O12B"), 9-[4-(dimethylamino)-1-oxobutyloxy]-heptadecanedioic acid 1,17-di-(2Z)-2-nonene-1 -yl ester ("L319"), 9,12-octadecadienoic acid (9Z,12Z)-1,1',1'',1'''-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1-butanediylaminobis-4,1-butanediyl)] ester ("OF-C4-Deg-Lin"), 2-(dioctylamino)ethyl nonyl hydrogen phosphate ("9A1P9"), (9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoic acid) 5-(((3-(Dibutylamino)propyl)amino)methyl)-6-hydroxyundecane-1,11-diyl ester ("IR-117-17"), 3-[4,4-bis(octyloxy)-1-oxobutyloxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl 9Z,12Z-octadecadienoate ("LP-01") and 4A3-SC8. A lipid particle as in any one of claims 1 and 4 to 35 or a method as in any one of claims 2 to 35, wherein the one or more ionizable lipids in the lipid particle are one or more selected from the group consisting of: DLin-MC3-DMA ("MC3"), DLin-KC2-DMA ("KC2"), ssPalmO-Phe ("SS-OP"), C12-200, SM-102, α-D-tocopheryl succinyl ("SS-EC") and ALC-0315 ("ALC"). A lipid particle as claimed in claim 36 or a method as claimed in claim 36, wherein the one or more ionizable lipids in the lipid particle are one or more selected from the group consisting of: ssPalmO-Phe ("SS-OP"), C12-200, SM-102 and ALC-0315 ("ALC"). A lipid particle as claimed in any one of claims 1 and 4 to 37 or a method as claimed in any one of claims 2 to 36, wherein only one ionizable lipid is present in the lipid particle. A lipid particle as claimed in claim 37 or a method as claimed in claim 38, wherein the only ionizable lipid in the lipid particle is SS-OP. A lipid particle as claimed in claim 39 or a method as claimed in claim 39, wherein when administered to an individual in need thereof, the individual exhibits one or more of the following: (i) an increase in Treg cells, (ii) a decrease in effector T cells, (iii) a decrease in B cells, (iv) a decrease in interleukin production, (v) a decrease in activated B cells, (vi) a decrease in activated effector T cells; or (vii) any combination thereof. The lipid particle of claim 39 or 40 or the method of claim 39 or 40, when administered to a subject in need thereof, reduces the level of B cells in the subject. The lipid particle of claim 39 or 40 or the method of claim 39 or 40, when administered to a subject in need thereof, reduces B cell levels in the subject, compared to a reference lipid particle comprising an ionizable lipid other than SS-OP. The lipid particle of claim 39 or 40 or the method of claim 39 or 40, when administered to a subject in need thereof, reduces the level of B cells in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 39 to 43 or the method of any one of claims 39 to 43, when administered to a subject in need thereof, reduces T cell levels in the subject. The lipid particle of any one of claims 39 to 43 or the method of any one of claims 39 to 43, when administered to a subject in need thereof, reduces T cell levels in the subject, compared to a reference lipid particle containing an ionizable lipid other than SS-OP. The lipid particle of any one of claims 39 to 43 or the method of any one of claims 39 to 43, when administered to a subject in need thereof, reduces T cell levels in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 39 to 46 or the method of any one of claims 39 to 46, when administered to a subject in need thereof, reduces the level of IFN-α in the subject. The lipid particle of any one of claims 39 to 46 or the method of any one of claims 39 to 46, when administered to a subject in need thereof, reduces IFN-α levels in the subject compared to a reference lipid particle containing an ionizable lipid other than SS-OP. The lipid particle of any one of claims 39 to 46 or the method of any one of claims 39 to 46, when administered to a subject in need thereof, reduces the level of IFN-α in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 1 and 4 to 49 or the method of any one of claims 2 to 49, wherein the one or more ionizable lipids in the lipid particle comprises α-D-tocopheryl succinyl ("SS-EC"). The lipid particle of claim 50 or the method of claim 50, when administered to a subject in need thereof, increases the level of B cells in the subject. The lipid particle of claim 50 or the method of claim 50, when administered to a subject in need thereof, increases B cell levels in the subject, compared to a reference lipid particle comprising an ionizable lipid other than SS-EC. The lipid particle of claim 50 or the method of claim 50, when administered to a subject in need thereof, increases the level of B cells in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 50 to 53 or the method of any one of claims 50 to 53, when administered to a subject in need thereof, increases T cell levels in the subject. The lipid particle of any one of claims 50 to 53 or the method of any one of claims 50 to 53, when administered to a subject in need thereof, increases T cell levels in the subject, compared to a reference lipid particle comprising an ionizable lipid other than SS-EC. The lipid particle of any one of claims 50 to 53 or the method of any one of claims 50 to 53, when administered to a subject in need thereof, increases T cell levels in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 50 to 53 or the method of any one of claims 50 to 53, when administered to a subject in need thereof, increases the level of IFN-α in the subject. The lipid particle of any one of claims 50 to 53 or the method of any one of claims 50 to 53, when administered to a subject in need thereof, increases IFN-α levels in the subject compared to a reference lipid particle comprising an ionizable lipid other than SS-EC. The lipid particle of any one of claims 50 to 53 or the method of any one of claims 49 to 52, when administered to a subject in need thereof, increases the level of IFN-α in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 1 and 4 to 59 or the method of any one of claims 2 to 59, wherein the lipid particle comprises one or more neutral lipids. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4% or at least 2.5%. The lipid particle of claim 60 or the method of claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 2.5% to about 25%, about 2.5% to about 20%, about 2.5% to about 17.5%, about 2.5% to about 15%, about 2.5% to about 12.5%, about 2.5% to about 10%, about 5% to about 25%, about 5% to about 20%, about 5% to about 17.5%, about 5% to about 15%, about 5% to about 12.5%, about 5% to about 10%, From about 7.5% to about 25%, from about 7.5% to about 20%, from about 7.5% to about 17.5%, from about 7.5% to about 15%, from about 7.5% to about 12.5%, from about 7.5% to about 10%, from about 10% to about 25%, from about 10% to about 20%, from about 10% to about 17.5%, from about 10% to about 15%, from about 10% to about 12.5%, from about 12.5% to about 25%, from about 12.5% to about 20%, from about 12.5% to about 17.5%, or from about 12.5% to about 15%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about 22.5% or about 25%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 5% to about 20%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 7.5% to about 17.5%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 2.5%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 5%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 7.5%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 10%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 12.5%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 15%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 17.5%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 20%. A lipid particle as claimed in claim 60 or a method as claimed in claim 60, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 25%. A lipid particle as in any one of claims 60 to 74 or a method as in any one of claims 60 to 74, wherein the one or more neutral lipids in the lipid particles are one or more phosphatidylcholine ("PC"), phosphatidylethanolamine ("PE") and combinations thereof. The lipid particle of any one of claims 60 to 74 or the method of any one of claims 60 to 74, wherein the one or more neutral lipids in the lipid particle are one or more selected from the group consisting of: 1,2-distearyl-sn-glycero-3-phosphocholine ("DSPC"), 1,2-dioleoyl-sn-glycero-3-phosphocholine ("DOPC"), ”), 1,2-dioctadecanoyl-sn-glycerol (“DSDG”), 1-2-dioleoyl-sn-glycerol, 1-2-di-(9Z-octadecenoyl)-sn-glycerol (“DODG”), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (“DOPE”), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, Sphingomyelin, cephalin, cerebroside, diacylglycerol dipalmitoylphosphatidylcholine ("DPPC"), palmitoyloleyl-phosphatidylcholine ("POPC"), palmitoyloleyl-phosphatidylethanolamine ("POPE"), palmitoyloleyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine ("DPPE"), dimyristoyl -phosphatidylethanolamine (“DMPE”), distearyl-phosphatidylethanolamine (“DSPE”), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (“DEPE”), stearyloleyl-phosphatidylethanolamine (“SOPE”) and phosphatidylcholine (“EPC”). The lipid particle of claim 76 or the method of claim 76, wherein the one or more neutral lipids in the lipid particle are DOPE, DODG or a combination thereof. A lipid particle as claimed in claim 76 or 77 or a method as claimed in claim 76 or 77, wherein only one neutral lipid is present in the lipid particle. A lipid particle as claimed in claim 78 or a method as claimed in claim 78, wherein the only neutral lipid in the lipid particle is DOPE. A lipid particle as claimed in claim 78 or a method as claimed in claim 78, wherein the only neutral lipid in the lipid particle is DODG. The lipid particle of any one of claims 1 and 4 to 80 or the method of any one of claims 2 to 80, wherein the lipid particle comprises one or more sterols. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19% or at least 20%. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 20% to about 50%, about 20% to about 45%, about 25% to about 45%, about 30% to about 45%, about 35% to about 45%, about 40% to about 45%, about 20% to about 40%, about 25% to about 40%, about 30% to about 40%, about 35% to about 40%, about 20% to about 30%, about 25% to about 30% or about 20% to about 25%. The lipid particle of claim 81 or the method of claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50%. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 20% to about 40%. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 20% to about 30%. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 30% to about 40%. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 20%. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 28.5%. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 30%. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 38.5%. A lipid particle as claimed in claim 81 or a method as claimed in claim 81, wherein the molar amount of the one or more sterols in the lipid particle is about 40%. A lipid particle as in any one of claims 81 to 92 or a method as in any one of claims 81 to 92, wherein the one or more sterols in the lipid particle are cholesterol, β-ergosterol, stigmasterol, campesterol, fucosterol, ergosterol, 9,11-dehydroergosterol, carotene, β-ergosterol acetate or other C-24 alkyl derivatives and combinations thereof. A lipid particle as in any one of claims 81 to 92 or a method as in any one of claims 81 to 92, wherein the one or more sterols in the lipid particle are cholesterol, β-sterol and a combination thereof. A lipid particle as claimed in any one of claims 81 to 93 or a method as claimed in any one of claims 81 to 94, wherein only one sterol is present in the lipid particle. A lipid particle as claimed in claim 95 or a method as claimed in claim 95, wherein the only sterol in the lipid particle is cholesterol. The lipid particle of any one of claims 1 and 4 to 96 or the method of any one of claims 2 to 96, wherein the lipid particle comprises one or more stealth lipids. A lipid particle as claimed in claim 97 or a method as claimed in claim 97, wherein the molar amount of the one or more stealth lipids in the lipid particle is at least 0.1%, at least 0.15%, at least 0.2% or at least 0.25%. A lipid particle as claimed in claim 97 or a method as claimed in claim 97, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 0.25% to about 3%, about 0.5% to about 3%, about 1% to about 3%, about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 0.25% to about 2.5%, about 0.5% to about 2.5%, about 1% to about 2.5%, about 1.5% to about 2.5%, about 2% to about 2.5%, about 0.25% to about 2%, about 0.5% to about 2%, about 1% to about 2%, about 1.5% to about 2%, about 0.25% to about 1.5%, about 0.5% to about 1.5% or about 1% to about 1.5%. A lipid particle as claimed in claim 97 or a method as claimed in claim 97, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 1% to about 3%. The lipid particle of claim 97 or the method of claim 97, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5% or about 3%. A lipid particle as claimed in claim 97 or a method as claimed in claim 97, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 1%. The lipid particle of claim 974 or the method of claim 97, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 1.5%. A lipid particle as claimed in claim 97 or a method as claimed in claim 97, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 2%. A lipid particle as claimed in claim 97 or a method as claimed in claim 97, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 2.5%. A lipid particle as claimed in claim 97 or a method as claimed in claim 97, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 3%. A lipid particle as in any one of claims 97 to 106 or a method as in any one of claims 94 to 103, wherein the one or more stealth lipids are one or more PEG-capped lipids, one or more poly(isosinoic acid) derivatives, or a combination thereof. A lipid particle as claimed in claim 107 or a method as claimed in claim 107, wherein the one or more stealth lipids are one or more PEG-terminated lipids. The lipid particle of claim 108 or the method of claim 108, wherein the one or more PEG-terminated lipids in the lipid particle are one or more selected from the group consisting of: 1,2-dimyristyl-rac-glycero-3-methoxypolyethylene glycol-2000 ("DMG-PEG2000"), distearyl-rac-glycero-PEG2K ("DSG-PEG2k"), 2-(2-methoxyethoxycarbonylamino)ethyl phosphate [(2R)-2,3-dioctadecanoyloxy)propyl] ester ("C18-mPEG2000"), tetradecanoic acid [3-[3-(2-methoxyethoxy)propylaminomethyloxy]-2-tetradecanoyloxypropyl] ester ("PEG2000-c-DMG"), tetradecanoic acid 3-[hydroxy-[2-[2-(2-methoxyethoxy)ethylamino]ethoxy]phosphoyl]oxy-2-tetradecanoyloxypropyl] ester ("DMPE-PEG2000"), 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ("18:1 PEG2000-PE"), 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] ("DSPE-PEG2000-COOH)" and bis(1,2-distearyl-sn-glycero-3-phosphoethanolamine)-N-[(polyethylene glycol)-2000] ("Bis-DSPE-PEG2000"). The lipid particle of claim 109 or the method of claim 109, wherein the one or more PEG-terminated lipids in the lipid particle are DMG-PEG2000, DMPE-PEG2000 or a combination thereof. A lipid particle as claimed in any one of claims 108 to 110 or a method as claimed in any one of claims 105 to 107, wherein only one PEG-capped lipid is in the lipid particle. The lipid particle of claim 108 or the method of claim 111, wherein the only PEG-terminated lipid in the lipid particle is DMG-PEG2000. The lipid particle of claim 108 or the method of claim 111, wherein the only PEG-terminated lipid in the lipid particle is DMPE-PEG2000. The lipid particle of claim 108 or the method of claim 111, wherein the only PEG-terminated lipid in the lipid particle is PEG2000-PE. The lipid particle of claim 104 or the method of claim 107, wherein the one or more stealth lipids are one or more poly(mycosine) derivatives. The lipid particle of claim 112 or the method of claim 115, wherein the one or more polysarcosine derivatives in the lipid particle are one or more selected from the group consisting of: N-tetradecyl-polysarcosine-25 ("N-tetradecyl-pSar25"), N-hexadecyl-polysarcosine-25 ("N-hexadecyl-pSar25"), N-octadecyl-polysarcosine-25, N-dodecyl-polysarcosine-25 ("N-octadecyl-pSar25"), 1,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 ("DMG-pSar25"), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-polysarcosine-25 (ammonium salt) ("18:1 PE (DOPE)"), N,N-ditetradecylamine-N-succinyl[methyl(polysarcosine)45] ("N-TETAMINE-pSar45"), N,N-ditetradecylamine-N-succinyl[methyl(polysarcosine)35] ("N-TETAMINE-pSar35"), N,N-ditetradecyl-polysarcosine-25 ("N-TETAMINE-pSar25"), N-TETAMINE-pSar45-butenediimide or N-TETAMINE-PEOZ-40. A lipid particle as claimed in claim 115 or 116 or a method as claimed in claim 115 or 116, wherein only one poly(sarcosine) derivative is present in the lipid particle. The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 25% to about 45% SS-OP, about 5% to about 25% DODG, about 15% to about 35% cholesterol, about 11% to about 30% CHEMS and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 118 or the method of claim 118, wherein the lipid particle comprises about 35% SS-OP, about 15% DODG, about 28.5% cholesterol, about 20% CHEMS and about 1.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 25% to about 45% SS-OP, about 5% to about 25% DOPE, about 15% to about 35% cholesterol, about 11% to about 30% CHEMS and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 121 or the method of claim 121, wherein the lipid particle comprises about 35% SS-OP, about 15% DOPE, about 28.5% cholesterol, about 20% CHEMS and about 1.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 25% to about 45% SS-OP, about 5% to about 25% DODG, about 15% to about 35% cholesterol, about 11% to about 30% CHEMS and about 0.5% to about 2.5% DMPE-PEG2000 (by molar amount). The lipid particle of claim 122 or the method of claim 122, wherein the lipid particle comprises about 35% SS-OP, about 15% DODG, about 28.5% cholesterol, about 20% CHEMS and about 1.5% DMPE-PEG2000 (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 25% to about 45% SS-OP, about 5% to about 25% DOPE, about 15% to about 35% cholesterol, about 11% to about 30% CHEMS and about 0.5% to about 2.5% DMPE-PEG2000 (by molar amount). The lipid particle of claim 124 or the method of claim 124, wherein the lipid particle comprises about 35% SS-OP, about 15% DOPE, about 28.5% cholesterol, about 20% CHEMS and about 1.5% DMPE-PEG2000 (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 25% to about 45% SS-OP, about 5% to about 25% DODG, about 15% to about 35% cholesterol, about 11% to about 30% CHEMS, and about 0.5% to about 2.5% 18:1 PEG2000-PE (by molar amount). The lipid particle of claim 126 or the method of claim 126, wherein the lipid particle comprises about 35% SS-OP, about 15% DODG, about 28.5% cholesterol, about 20% CHEMS and about 1.5% 18:1 PEG2000-PE (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 25% to about 45% SS-OP, about 5% to about 25% DOPE, about 15% to about 35% cholesterol, about 11% to about 30% CHEMS and about 0.5% to about 2.5% 18:1 PEG2000-PE (by molar amount). The lipid particle of claim 128 or the method of claim 128, wherein the lipid particle comprises about 35% SS-OP, about 15% DOPE, about 28.5% cholesterol, about 20% CHEMS and about 1.5% 18:1 PEG2000-PE (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 30% to about 50% SS-OP, about 15% to about 55% cholesterol, about 11% to about 30% CHEMS and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 130 or the method of claim 130, wherein the lipid particle comprises about 40% SS-OP, about 38.5% cholesterol, about 20% CHEMS and about 1.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 20% to about 45% SS-OP, about 15% to about 55% cholesterol, about 15% to about 35% CHEMS and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 132 or the method of claim 132, wherein the lipid particle comprises about 35% SS-OP, about 38.5% cholesterol, about 25% CHEMS and about 1.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 20% to about 40% SS-OP, about 15% to about 55% cholesterol, about 20% to about 40% CHEMS and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 134 or the method of claim 134, wherein the lipid particle comprises about 30% SS-OP, about 38.5% cholesterol, about 30% CHEMS and about 1.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 15% to about 35% SS-OP, about 15% to about 55% cholesterol, about 25% to about 45% CHEMS and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 136 or the method of claim 136, wherein the lipid particle comprises about 25% SS-OP, about 38.5% cholesterol, about 35% CHEMS and about 1.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 30% to about 50% SS-OP, about 15% to about 55% cholesterol, about 15% to about 35% CHEMS and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 138 or the method of claim 138, wherein the lipid particle comprises about 40% SS-OP, about 33.5% cholesterol, about 25% CHEMS and about 1.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 1 or the method of claim 2 or 3, wherein the lipid particle comprises about 30% to about 50% SS-OP, about 15% to about 55% cholesterol, about 20% to about 40% CHEMS and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 140 or the method of claim 140, wherein the lipid particle comprises about 40% SS-OP, about 28.5% cholesterol, about 30% CHEMS and about 1.5% DMG-PEG2000 (by molar amount). A lipid particle comprising: a. about 20% to about 70% molar amount of one or more ionizable lipids; b. about 2.5% to about 25% molar amount of one or more neutral lipids; c. about 20% to about 50% molar amount of one or more sterols; d. about 10% or less molar amount of one or more charged lipids; and e. about 0.25% to about 3% molar amount of one or more stealth lipids. The method of claim 142, wherein the one or more charged lipids in the lipid particle are selected from the group consisting of: cholesterol hemisuccinate ("CHEMS"), 1,2-distearoyl-3-trimethylammonium-propane ("DSTAP"), 18:1 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP"), 1,2-dioleoyl-sn-glycero-3-phosphate ("18:1 PA"), 18:1 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) ("18:1 PG”), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-butyrylphosphatidylethanolamine, N-pentylphosphatidylethanolamine, phosphatidylglycerol, palmitoyloleylphosphatidylglycerol (“POPG”), 1,2-dimyristoyl-3-trimethylammonium-propane (“DMTAP”), 1,2-dipalmitoyl-3-trimethylammonium-propane (“DPTAP”), palmitoyloleyl-3-trimethylammonium-propane (“PO TAP”), 1,2-dioleoyl-3-dimethylammonium-propane (“DODAP”), palmitoyloleyl-3-dimethylammonium-propane (“PODAP”), 1,2-dimyristoyl-3-dimethylammonium-propane (“DMDAP”), 1,2-dipalmitoyl-3-dimethylammonium-propane (“DPDAP”), 1,2-distearoyl-3-dimethylammonium-propane (“DSDAP”), 1,2-dioleoyl-3-dimethylhydroxyethyl-ammonium-propane (“DODMHEAP”) (also known as DORI), palmitoyloleyl-3-dimethylhydroxyethyl-ammonium-propane ("PODMHEAP") (also known as PORI), 1,2-dimyristoyl-3-dimethylhydroxyethyl-ammonium-propane ("DMDMHEAP") (also known as DMRI), 1,2-dipalmitoyl-3-dimethylhydroxyethyl-ammonium-propane ("DPDMHEAP") (also known as DPRI), 1,2-distearyl-3-dimethylhydroxyethyl-ammonium-propane ("DSDMHEAP") (also known as DSRI), 1,2-dioleyl-3-methylhydroxyethylammonium-propane ("DOMDHEAP"), palmitoyloleyl-3-methylhydroxyethylammonium-propane ("POMDHEAP"), 1,2-dimyristoyl-3-methyldihydroxyethylammonium-propane ("DMMDHEAP"), 1,2-dipalmitoyl-3-methyldihydroxyethylammonium-propane 1,2-dioleyl-3-methyldihydroxyethylammonium-propane ("DOMHEAP"), palmityloleyl-3-methylhydroxyethylammonium-propane ("POMHEAP"), 1,2-dimyristoyl-3-methylhydroxyethylammonium-propane ("DPMDHEAP"), 1,2-distearyl-3-methyldihydroxyethylammonium-propane ("DSMDHEAP"), 1,2-dioleyl-3-methyldihydroxyethylammonium-propane ("DOMHEAP"), palmityloleyl-3-methylhydroxyethylammonium-propane ("POMHEAP"), 1,2-dimyristoyl-3-methylhydroxyethylammonium -propane ("DMMHEAP"), 1,2-dipalmitoyl-3-methylhydroxyethylammonium-propane ("DPMHEAP"), 1,2-distearoyl-3-methylhydroxyethylammonium-propane ("DSMHEAP"), 1,2-dioleoyl-3-dihydroxyethylammonium-propane ("DODHEAP"), palmitoyloleoyl-3-dihydroxyethylammonium-propane (" PODHEAP”), 1,2-dimyristoyl-3-dihydroxyethylammonium-propane (“DMDHEAP”), 1,2-dipalmitoyl-3-dihydroxyethylammonium-propane (“DPDHEAP”), 1,2-distearoyl-3-dihydroxyethylammonium-propane (“DSDHEAP”), dimethylbisoctadecylammonium bromide (“DDAB”), dioleyl dimethyl ammonium chloride ("DODAC"), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine ("DOEPC"), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine ("DMEPC"), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine ("DPEPC"), 1,2-distearyl-sn-glycero-3 -ethylphosphocholine ("DSEPC"), palmitoyloleyl-sn-glycero-3-ethylphosphocholine ("POEPC"), 1,2-dioleyl-3-dimethyl-hydroxyethylammonium propane ("DORIE"), 1,2-dimyristyl-3-dimethyl-hydroxyethylammonium propane ("DMRIE"), 1,2-dioleyl-3-methyl-(methoxycarbonyl- 1,2-dioleyl-3-methyl-(methoxycarbonylmethyl)ammonium-propane ("DOMGME"), 1,2-dioleyl-3-N-pyrrolidinium-propane ("DOP5P"), 1,2-dioleyl-3-N-pyridinium-propane bromide salt ("DOP6P"), 3b-[N-(N9,N9-dioleyl)-3-methyl-(methoxycarbonylmethyl)ammonium-propane ("DOMGME"), 1,2-dioleyl-3-N-pyrrolidinium-propane ("DOP5P"), 1,2-dioleyl-3-N-pyridinium-propane bromide salt ("DOP6P"), Cholesterol ("3-( ... -betaine"), N-methyl{4-N-amino[(3'-β-cholesterol)aminomethyl]}piperazine ("N-methyl-PipChol"), hexadecyltrimethylammonium bromide ("CTAB"), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"), 4-(2-aminoethyl)-morpholinyl-cholesterol Cholesterol hemisuccinate ("MoChol"), histamine-cholesterol hemisuccinate ("HisChol"), cholesterol-(3-imidazol-1-ylpropyl) carbamate ("Chim"), (N-2-propylamino[(3'-β-cholesterol)carbamyl]}morpholine ("MoC3Chol"), [(3-morpholine-4-yl-propylcarbamyl) -methyl]-cholesterol carbamate ("Chol-C3N-Mo3"), ("Chol-C3N-Mo2"), [(2-oxolin-4-yl-ethylcarbamoyl)methyl]-cholesterol carbamate ("Chol-C4N-Mo2"), [1-methyl-2-(2-oxolin-4-yl-ethylcarbamoyl)-propyl]-cholesterol carbamate ("Chol-DMC3N-Mo2"), 2-(2-morpholin-4-yl-ethylaminoformyl)-cyclohexanecarboxylic acid cholesterol ester ("CholC4Hex-Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-2,3-dimethyl hemisuccinate ("DmC4Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-2,2-dimethyl Methyl hemimalonate ("DmC3Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemimalonate ("C3Mo2"), 4-(2-aminopropyl)-morpholinyl-cholesterol-hemimalonate ("C3Mo3"), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemiglutarate ("C5Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemiglutarate ("C5Mo2"), morpholinyl-cholesterol-hemisuccinate ("C6Mo2"), 4-(2-aminoethyl)-morpholinyl-cholesterol-hemisuccinate ("C8Mo2"), 4-(2-aminobutyl)-morpholinyl-cholesterol-hemisuccinate ("C4Mo4"), 4{N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}piperazine ("PipC2Chol ”), {N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}morpholine (“MoC2Chol”), {N-2-ethylamino[(3'-β-cholesterol)aminoformyl]}pyrrolidine (“PyrroC2Chol”), {N-2-propylamino[(3'-β-cholesterol)aminoformyl]}imidazole (“ImC3Chol”) 、{N-2-ethylamino[(3'-β-cholesterol)aminomethyl]}pyridine ("PyC2Chol")、1,2-dioleoyl-3-N-pyroline-propane ("MoDO")、1,2-dipalmitoyl-3-N-pyroline-propane ("MoDP")、4,(2,3-bis-acyloxy-propyl)-1-methyl-1H-imidazole ("DOIM") (also known as DPIM); diacylglycerol hemisuccinates, such as dioctadecylamido-glycidylspermine ("DOGS"), dimyristoylglycerol hemisuccinate ("DMGS") (also known as DMG-Succ), 1-palmitoyl-2-oleylglycerol hemisuccinate ("POGS") (also known as POG-Succ), dipalmitoylglycerol hemisuccinate ("DPGS") (also known as POG-Succ), distearylglycerol hemisuccinate ("DSGS") (also known as DSG-Succ); diacylglycerol hemimalonates, such as dioleylglycerol hemimalonate ("DOGM"), dimyristoylglycerol hemimalonate ("DMGM"); diacylglycerol hemiglutarate, such as dioleylglycerol hemiglutarate ("DOGG"), dimyristoylglycerol hemiglutarate ("DMGG"); diacylglycerol hemiadipate, such as dioleylglycerol hemiadipate ("DOGA"), dimyristoylglycerol hemiadipate ("D MGA”), diacylglycerol hemicyclohexane-1,4-dicarboxylic acid, such as dioleoylglycerol hemicyclohexane-1,4-dicarboxylic acid (“DO-cHA”), dimyristoylglycerol hemicyclohexane-1,4-dicarboxylic acid (“DM-cHA”), (2,3-diacyl-propyl)amino}-oxoalkanoic acid, such as 4-{(2,3-dioleoyl-propyl)amino}-4-oxobutanoic acid (“DOAS”), 3-{(2,3-dioleoyl-propyl)amino}-3-oxopropionic acid ( DOAM), 5-{(2,3-dioleyl-propyl)amino}-5-oxopentanoic acid (“DOAG”), 6-{(2,3-dioleyl-propyl)amino}-6-oxopentanoic acid (“DOAA”), 4-{(2,3-dimyristyl-propyl)amino}-4-oxopentanoic acid (“DMAS”), 3-{(2,3-dimyristyl-propyl)amino}-3-oxopentanoic acid (“DMAM”), 5-{(2,3-dimyristyl-propyl)amino}-6-oxopentanoic acid (“DOAG”), 6-{(2,3-dioleyl-propyl)amino}-6-oxopentanoic acid (“DOAA”), 4-{(2,3-dimyristyl-propyl)amino}-4-oxopentanoic acid (“DMAS”), 3-{(2,3-dimyristyl-propyl)amino}-3-oxopentanoic acid (“DMAM”), )amino}-5-hydroxypentanoic acid ("DMAG"), 6-{(2,3-dimyristyl-propyl)amino}-6-hydroxyhexanoic acid ("DMAA"); diacyl-alkanoic acids, such as 2,3-dioleyl-propionic acid ("DOP"), 3,4-dioleyl-butyric acid ("DOB"), 5,6-dioleyl-hexanoic acid ("DOS"), 4,5-dioleyl-pentanoic acid ("DOM"), 6,7-dioleyl-heptanoic acid ("DOG"), 7,8-dioleyl-octanoic acid ("DOB"), 1,2-dioleyl-1,2-dioleyl-2,3-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-2,3-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-3,4-dioleyl-4,5-dioleyl-5,6-dioleyl-3,4-dioleyl-4,5-dioleyl- (“DOA”), 2,3-dimyristyl-propionic acid (“DMP”), 3,4-dioleyl-butyric acid (“DOB”), 5,6-dimyristyl-hexanoic acid (“DMS”), 4,5-dimyristyl-pentanoic acid (“DMM”), 6,7-dimyristyl-heptanoic acid (“DMG”), 7,8-dimyristyl-octanoic acid (“DMA”); cholesterol oxycarbonylcarbamates, such as cholesterol hemidodecanedicarboxylic acid (“Chol-C12”), 12-cholesterol Steroloxycarbonylaminododecanoic acid ("CholC13N"); fatty acids, such as oleic acid, myristic acid, palmitic acid, stearic acid, cervical acid, behenic acid; dioleylphosphatidic acid ("DOPA"), 1,2-dimyristyl-sn-glycero-3-phosphate ("DMPA"), 1,2-dipalmitoyl-sn-glycero-3-phosphate ("DPPA"), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate ("POPA"), 1,2-distearyl sn-glycerol-3-phosphate (DSPA), cholesterol sulfate (Chol-SO4), dioleoylphosphatidylglycerol (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-racemic-glycerol), (DMPG), dimalmitoylphosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-distearyl-sn-glycero-3-phosphoglycerol (POPG), Phospho-rac-glycerol ("DSPG"), dioleylphosphatidylserine ("DOPS"), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine ("DMPS"), dimalmitoylphosphatidylserine ("DPPS"), 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-L-serine ("POPS"), 1,2-distearyl-sn-glycero-3-phospho-L-serine ("DSPS") or hexadecyl phosphate. The lipid particle of claim 142, wherein the one or more charged lipids in the lipid particle are selected from the group consisting of: cholesterol hemisuccinate ("CHEMS"), 1,2-distearoyl-3-trimethylammonium-propane ("DSTAP"), 18:1 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP"), 1,2-dioleoyl-sn-glycero-3-phosphate ("18:1 PA"), 18:1 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) ("18:1 PG”), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, phosphatidylglycerol and palmitoyloleylphosphatidylglycerol (“POPG”). A lipid particle as in any one of claims 142 to 144, wherein only one charged lipid is present in the lipid particle. The lipid particle of claim 145, wherein the only charged lipid in the lipid particle is CHEMS. A lipid particle as in any of claims 142 to 146, wherein the molar concentration of the one or more charged lipids in the lipid particle is about 10%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.1%. The lipid particle of any one of claims 142 to 146, wherein the molar amount of the one or more charged lipids in the lipid particle is about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5 %, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, or about 5% to about 6%. The lipid particle of claim 142 to 146, wherein the molar amount of the one or more charged lipids in the lipid particle is about 10%. The lipid particle of claim 142, wherein the lipid particle does not contain charged lipids. The lipid particle of any one of claims 142 to 150, further comprising a payload. A method for targeting a payload to a tissue other than the spleen of an individual in vivo, the method comprising combining a lipid particle or a composition comprising the payload encapsulated within a lipid particle or a composition comprising one or more ionizable lipids, one or more neutral lipids, one or more sterols, one or more stealth lipids and/or one or more charged lipids, wherein the lipid particle or the composition does not contain CHEMS or contains a molar amount of about 10% or less than 10% CHEMS. The method of claim 152, wherein the molar amount of CHEMS is about 10%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.1%. The method of claim 152, wherein the molar amount of the CHEMS is about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, From about 2% to about 3%, from about 3% to about 10%, from about 3% to about 9%, from about 3% to about 8%, from about 3% to about 7%, from about 3% to about 6%, from about 3% to about 5%, from about 3% to about 4%, from about 4% to about 10%, from about 4% to about 9%, from about 4% to about 8%, from about 4% to about 7%, from about 4% to about 6%, from about 4% to about 5%, from about 5% to about 10%, from about 5% to about 9%, from about 5% to about 8%, from about 5% to about 7%, or from about 5% to about 6%. The method of claim 152, wherein the molar amount of CHEMS is about 10%. The method of claim 152, which does not include CHEMS. The method of any one of claims 152 to 156, wherein the molar amount of the one or more ionizable lipids is about 20% to about 70%. The method of any one of claims 152 to 157, wherein the molar amount of the one or more neutral lipids is about 2.5% to about 25%. The method of any one of claims 152 to 158, wherein the molar amount of the one or more sterols is about 20% to about 50%. The method of any one of claims 152 to 159, wherein the one or more stealth lipids are present in a molar amount of about 0.25% to about 3%. The lipid particle of any one of claims 1142 to 151 or the method of any one of claims 148 to 156, wherein the lipid particle delivers the payload to the liver. The lipid particle of claim 161 or the method of claim 161, wherein the ratio of the payload delivered to the liver and spleen is greater than 1.1. The lipid particle of claim 161 or the method of claim 161, wherein the ratio of the payload delivered to the liver and spleen is about 1.1 to about 12. The lipid particle of any one of claims 142 to 151 and 161 to 163 or the method of any one of claims 152 to 163, wherein the ratio of the payload delivered to the spleen and the liver is determined using an imaging system. The lipid particle of claim 164 or the method of claim 164, wherein the imaging system is a LICOR Pearl Trilogy imaging system using Image Studio software from LICOR version 5.2.5. The lipid particle of any one of claims 142 to 151 and 161 to 165 or the method of any one of claims 152 to 166, wherein the lipid particles comprise one or more ionizable lipids. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 25% to about 65%, about 30% to about 65%, about 35% to about 65%, about 45% to about 65%, about 50% to about 65%, about 25% to about 60%, about 30% to about 60%, about 35% to about 60%, about 45% to about 60%, about 50% to about 60%, about 25% to about 55%, about 30% to about 55%, about 35% to about 55%, about 45% to about 55%, about 50% to about 55%, about 25% to about 50%, about 30% to about 50%, about 35% to about 50% or about 45% to about 50%. The lipid particle of claim 166 or the method of claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% or about 65%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 30% to about 60%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 40% to about 60%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 40% to about 50%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 50% to about 60%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 30%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 40%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 50%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 55%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 60%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 65%. A lipid particle as claimed in claim 166 or a method as claimed in claim 166, wherein the molar amount of the one or more ionizable lipids in the lipid particle is about 70%. The lipid particle of any one of claims 166 to 179 or the method of any one of claims 166 to 179, wherein the one or more ionizable lipids in the lipid particles are one or more selected from the group consisting of: DLin-MC3-DMA ("MC3"), DLin-KC2-DMA ("KC2"), ssPalmO-Phe ("SS-OP"), C12-200, SM-102, α-D-tocopheryl succinyl ("SS-EC"), ALC-0315 ("ALC"), 3-(ethyl(methyl)amino)propionic acid tri-N-tridecyl ester ("304-O13"), 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionic acid tetrakis(2-(octyldisulfanyl)ethyl) ester ("306-O12B"), 9-[4-(dimethylamino)-1-oxobutyloxy]-heptadecanedioic acid 1,17-di-(2Z)-2-nonene-1 -yl ester ("L319"), 9,12-octadecadienoic acid (9Z,12Z)-1,1',1'',1'''-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1-butanediylaminobis-4,1-butanediyl)] ester ("OF-C4-Deg-Lin"), 2-(dioctylamino)ethyl nonyl hydrogen phosphate ("9A1P9"), (9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoic acid) 5-(((3-(Dibutylamino)propyl)amino)methyl)-6-hydroxyundecane-1,11-diyl ester ("IR-117-17"), 3-[4,4-bis(octyloxy)-1-oxobutyloxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl 9Z,12Z-octadecadienoate ("LP-01") and 4A3-SC8. A lipid particle as claimed in any one of claims 166 to 179 or a method as claimed in any one of claims 166 to 179, wherein the one or more ionizable lipids in the lipid particle are one or more selected from the group consisting of: DLin-MC3-DMA ("MC3"), DLin-KC2-DMA ("KC2"), ssPalmO-Phe ("SS-OP"), C12-200, SM-102, α-D-tocopheryl succinyl ("SS-EC") and ALC-0315 ("ALC"). A lipid particle as claimed in claim 181 or a method as claimed in claim 181, wherein the one or more ionizable lipids in the lipid particle are one or more selected from the group consisting of: ssPalmO-Phe ("SS-OP"), C12-200, SM-102 and ALC-0315 ("ALC"). A lipid particle as claimed in any one of claims 166 to 182 or a method as claimed in any one of claims 166 to 182, wherein the only ionizable lipid is in the lipid particle. A lipid particle as claimed in claim 183 or a method as claimed in claim 183, wherein the only ionizable lipid in the lipid particle is SS-OP. A lipid particle as claimed in claim 183 or 184 or a method as claimed in claim 183 or 184, wherein when administered to an individual in need thereof, the individual exhibits one or more of the following: (i) an increase in Treg cells, (ii) a decrease in effector T cells, (iii) a decrease in B cells, (iv) a decrease in interleukin production, (v) a decrease in activated B cells, (vi) a decrease in activated effector T cells; or (vii) any combination thereof. The lipid particle of claim 183 or 184 or the method of claim 183 or 184, when administered to a subject in need thereof, the lipid particle or the method reduces the level of B cells in the subject. The lipid particle of claim 183 or 184 or the method of claim 183 or 184, when administered to a subject in need thereof, reduces B cell levels in the subject, compared to a reference lipid particle comprising an ionizable lipid other than SS-OP. The lipid particle of claim 183 or 184 or the method of claim 183 or 184, when administered to a subject in need thereof, reduces the level of B cells in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 183 to 188 or the method of any one of claims 183 to 188, when administered to a subject in need thereof, reduces T cell levels in the subject. The lipid particle of any one of claims 183 to 188 or the method of any one of claims 183 to 188, when administered to a subject in need thereof, reduces T cell levels in the subject, compared to a reference lipid particle containing an ionizable lipid other than SS-OP. The lipid particle of any one of claims 183 to 188 or the method of any one of claims 183 to 188, when administered to a subject in need thereof, reduces T cell levels in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 183 to 191 or the method of any one of claims 183 to 191, when administered to a subject in need thereof, reduces the level of IFN-α in the subject. The lipid particle of any one of claims 183 to 191 or the method of any one of claims 183 to 191, when administered to a subject in need thereof, reduces IFN-α levels in the subject compared to a reference lipid particle containing an ionizable lipid other than SS-OP. The lipid particle of any one of claims 183 to 191 or the method of any one of claims 183 to 191, when administered to a subject in need thereof, reduces the level of IFN-α in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 166 to 179 or the method of any one of claims 166 to 179, wherein the one or more ionizable lipids in the lipid particle comprises α-D-tocopheryl succinyl ("SS-EC"). The lipid particle of claim 195 or the method of claim 195, when administered to a subject in need thereof, increases the level of B cells in the subject. The lipid particle of claim 195 or the method of claim 195, when administered to a subject in need thereof, increases B cell levels in the subject compared to a reference lipid particle comprising an ionizable lipid other than SS-EC. The lipid particle of claim 195 or the method of claim 195, when administered to a subject in need thereof, increases the level of B cells in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 195 to 198 or the method of any one of claims 190 to 193, when administered to a subject in need thereof, increases T cell levels in the subject. The lipid particle of any one of claims 195 to 198 or the method of any one of claims 190 to 193, when administered to a subject in need thereof, increases T cell levels in the subject, compared to a reference lipid particle comprising an ionizable lipid other than SS-EC. The lipid particle of any one of claims 195 to 198 or the method of any one of claims 190 to 193, when administered to a subject in need thereof, increases T cell levels in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 195 to 201 or the method of any one of claims 190 to 196, when administered to a subject in need thereof, increases the level of IFN-α in the subject. The lipid particle of any one of claims 195 to 201 or the method of any one of claims 190 to 196, when administered to a subject in need thereof, increases IFN-α levels in the subject compared to a reference lipid particle containing an ionizable lipid other than SS-EC. The lipid particle of any one of claims 195 to 201 or the method of any one of claims 190 to 196, when administered to a subject in need thereof, increases the level of IFN-α in the subject, compared to a reference lipid particle containing MC-3 as the ionizable lipid. The lipid particle of any one of claims 142 to 151 and 161 to 203 or the method of any one of claims 148 to 199, wherein the lipid particle comprises one or more neutral lipids. The lipid particle of claim 205 or the method of claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 2.5% to about 20%, about 2.5% to about 17.5%, about 2.5% to about 15%, about 2.5% to about 12.5%, about 2.5% to about 10%, about 5% to about 25%, about 5% to about 20%, about 5% to about 17.5%, about 5% to about 15%, about 5% to about 12.5%, about 5% to about 10%, about 7.5% to about 25%, about 7.5% to about 20%, about 7.5% to about 17.5%, about 7.5% to about 15%, about 7.5% to about 12.5%, about 7.5% to about 10%, about 10% to about 25%, about 10% to about 20%, about 10% to about 17.5%, about 10% to about 15%, about 10% to about 12.5%, about 12.5% to about 25%, about 12.5% to about 20%, about 12.5% to about 17.5%, or about 12.5% to about 15%. The lipid particle of claim 205 or the method of claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about 22.5% or about 25%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 5% to about 20%. The lipid particle of claim 205 or the method of claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 7.5% to about 17.5%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 2.5%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 5%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 7.5%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 10%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 12.5%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 15%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 17.5%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 20%. A lipid particle as claimed in claim 205 or a method as claimed in claim 205, wherein the molar amount of the one or more neutral lipids in the lipid particle is about 25%. A lipid particle as in any one of claims 205 to 218 or a method as in any one of claims 205 to 218, wherein the one or more neutral lipids in the lipid particles are one or more phosphatidylcholine ("PC"), phosphatidylethanolamine ("PE") and combinations thereof. The lipid particle of any one of claims 205 to 218 or the method of any one of claims 205 to 218, wherein the one or more neutral lipids in the lipid particle are one or more selected from the group consisting of: wherein the one or more neutral lipids in the lipid particle are one or more selected from the group consisting of: 1,2-distearyl-sn-glycero-3-phosphocholine ("DSP C”), 1,2-dioleoyl-sn-glycero-3-phosphocholine (“DOPC”), 1,2-dioctadecanoyl-sn-glycerol (“DSDG”), 1-2-dioleoyl-sn-glycerol, 1-2-di-(9Z-octadecenoyl)-sn-glycerol (“DODG”), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (“DOPE”) , diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cerebroside, diacylphosphatidylcholine ("DPPC"), palmitoyloleyl-phosphatidylcholine ("POPC"), palmitoyloleyl-phosphatidylethanolamine ("POPE"), palmitoyloleyl-phosphatidylglycerol (POPG), diacylphosphatidylcholine ("DPPC"), palmitoyloleyl-phosphatidylethanolamine ("POPE"), palmitoyloleyl-phosphatidylglycerol (POPG), ethanolamine ("DPPE"), dimyristoyl-phosphatidylethanolamine ("DMPE"), distearyl-phosphatidylethanolamine ("DSPE"), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dioleyl-phosphatidylethanolamine ("DEPE"), stearyloleyl-phosphatidylethanolamine ("SOPE") and phosphatidylcholine ("EPC"). The lipid particle of any one of claims 205 to 220 or the method of any one of claims 205 to 220, wherein the one or more neutral lipids in the lipid particle are DOPE, DODG or a combination thereof. A lipid particle as claimed in any one of claims 205 to 2221 or a method as claimed in any one of claims 205 to 2221, wherein only one neutral lipid is present in the lipid particle. The lipid particle of claim 222 or the method of claim 222, wherein the only neutral lipid in the lipid particle is DOPE. The lipid particle of claim 222 or the method of claim 222, wherein the only neutral lipid in the lipid particle is DODG. The lipid particle of any one of claims 142 to 151 and 161 to 224 or the method of any one of claims 152 to 2224, wherein the lipid particle comprises one or more sterols. The lipid particle of claim 225 or the method of claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 20% to about 45%, about 25% to about 45%, about 30% to about 45%, about 35% to about 45%, about 40% to about 45%, about 20% to about 40%, about 25% to about 40%, about 30% to about 40%, about 35% to about 40%, about 20% to about 30%, about 25% to about 30% or about 20% to about 25%. The lipid particle of claim 2225 or the method of claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50%. The lipid particle of claim 225 or the method of claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 20% to about 40%. The lipid particle of claim 225 or the method of claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 20% to about 30%. The lipid particle of claim 225 or the method of claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 30% to about 40%. A lipid particle as claimed in claim 225 or a method as claimed in claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 20%. The lipid particle of claim 225 or the method of claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 28.5%. A lipid particle as claimed in claim 225 or a method as claimed in claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 30%. The lipid particle of claim 225 or the method of claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 38.5%. A lipid particle as claimed in claim 225 or a method as claimed in claim 225, wherein the molar amount of the one or more sterols in the lipid particle is about 40%. A lipid particle as in any one of claims 225 to 235 or a method as in any one of claims 225 to 235, wherein the one or more sterols are cholesterol, β-ergosterol, stigmasterol, campesterol, fucosterol, ergosterol, 9,11-dehydroergosterol, carotene, β-ergosterol acetate and other C-24 alkyl derivatives or combinations thereof. The lipid particle of any one of claims 225 to 236 or the method of any one of claims 225 to 236, wherein the one or more sterols are cholesterol, β-sterol or a combination thereof. The lipid particle of any one of claims 225 to 236 or the method of any one of claims 219 to 230, wherein the only sterol is in the lipid particle. The lipid particle of claim 238 or the method of claim 238, wherein the only sterol in the lipid particle is cholesterol. The lipid particle of any one of claims 225 to 239 or the method of any one of claims 193 to 206, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 0.5% to about 3%, about 1% to about 3%, about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 0.25% to about 2.5%, about 0.5% to about 2.5%, about 1% to about 2.5%, about 1.5% to about 2.5%, about 2% to about 2.5%, about 0.25% to about 2%, about 0.5% to about 2%, about 1% to about 2%, about 1.5% to about 2%, about 0.25% to about 1.5%, about 0.5% to about 1.5%, or about 1% to about 1.5%. The lipid particle of any one of claims 225 to 239 or the method of any one of claims 193 to 206, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 1% to about 3%. The lipid particle of any one of claims 225 to 239 or the method of any one of claims 193 to 206, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 0.5%, about 1%, about 1.5%, about 2%, about 2.5% or about 3%. A lipid particle as claimed in any one of claims 225 to 239 or a method as claimed in any one of claims 193 to 206, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 1%. The lipid particle of any one of claims 225 to 239 or the method of any one of claims 193 to 206, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 1.5%. The lipid particle of any one of claims 225 to 239 or the method of any one of claims 193 to 206, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 2%. The lipid particle of any one of claims 225 to 239 or the method of any one of claims 193 to 206, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 2.5%. The lipid particle of any one of claims 225 to 239 or the method of any one of claims 193 to 206, wherein the molar amount of the one or more stealth lipids in the lipid particle is about 3%. A lipid particle as in any one of claims 225 to 247 or a method as in any one of claims 219 to 240, wherein the one or more stealth lipids are one or more PEG-capped lipids, one or more poly(sarcosine) derivatives, or a combination thereof. A lipid particle as claimed in claim 248 or a method as claimed in claim 248, wherein the one or more stealth lipids are one or more PEG-terminated lipids. The lipid particle of claim 248 or 249 or the method of claim 248 or 249, wherein the one or more PEG-terminated lipids in the lipid particle are one or more selected from the group consisting of: 1,2-dimyristyl-rac-glycero-3-methoxypolyethylene glycol-2000 ("DMG-PEG2000"), distearyl-rac-glycero-PEG2K ("DSG-PEG2k"), 2-(2-methoxyethoxycarbonylamino)ethyl phosphate [(2R)-2,3-dioctadecanoyloxy)propyl] ester ("C18-mPEG2000"), tetradecanoic acid [3-[3-(2-methoxyethoxy)propylaminomethyloxy]-2-tetradecanoyloxypropyl] ester ("PEG2000-c-DMG"), tetradecanoic acid 3-[hydroxy-[2-[2-(2-methoxyethoxy)ethylamino]ethoxy]phosphoyl]oxy-2-tetradecanoyloxypropyl] ester ("DMPE-PEG2000"), 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ("18:1 PEG2000-PE"), 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] ("DSPE-PEG2000-COOH)" and bis(1,2-distearyl-sn-glycero-3-phosphoethanolamine)-N-[(polyethylene glycol)-2000] ("Bis-DSPE-PEG2000"). The lipid particle of any one of claims 248 to 250 or the method of any one of claims 241 to 243, wherein the one or more PEG-terminated lipids in the lipid particle are DMG-PEG2000, DMPE-PEG2000 or a combination thereof. A lipid particle as claimed in any one of claims 248 to 251 or a method as claimed in any one of claims 241 to 244, wherein only one PEG-capped lipid is in the lipid particle. The lipid particle of claim 245 or the method of claim 252, wherein the only PEG-terminated lipid in the lipid particle is DMG-PEG2000. The lipid particle of claim 245 or the method of claim 252, wherein the only PEG-terminated lipid in the lipid particle is DMPE-PEG2000. The lipid particle of claim 245 or the method of claim 252, wherein the only PEG-terminated lipid in the lipid particle is PEG2000-PE. The lipid particle of claim 241 or the method of claim 248, wherein the one or more stealth lipids are one or more poly(mycosine) derivatives. The lipid particle of claim 249 or the method of claim 256, wherein the one or more polysarcosine derivatives in the lipid particle are one or more selected from the group consisting of: N-tetradecyl-polysarcosine-25 ("N-tetradecyl-pSar25"), N-hexadecyl-polysarcosine-25 ("N-hexadecyl-pSar25"), N-octadecyl-polysarcosine-25, N-dodecyl-polysarcosine-25 ("N-octadecyl-pSar25"), 1,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 ("DMG-pSar25"), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-polysarcosine-25 (ammonium salt) ("18:1 PE (DOPE)"), N,N-ditetradecylamine-N-succinyl[methyl(polysarcosine)45] ("N-TETAMINE-pSar45"), N,N-ditetradecylamine-N-succinyl[methyl(polysarcosine)35] ("N-TETAMINE-pSar35"), N,N-ditetradecyl-polysarcosine-25 ("N-TETAMINE-pSar25"), N-TETAMINE-pSar45-butenediimide or N-TETAMINE-PEOZ-40. A lipid particle as claimed in claim 256 or 257 or a method as claimed in claim 256 or 257, wherein only one poly(mycosine) derivative is present in the lipid particle. The lipid particle of claim 142 or the method of claim 152, wherein the lipid particle comprises about 40% to about 60% SS-OP, about 1% to about 20% DODG, about 25% to about 45% cholesterol, about 1% to about 10% CHEMS, and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 259 or the method of claim 259, wherein the lipid particle comprises about 50% SS-OP, about 10% DODG, about 38.5% cholesterol, about 10% CHEMS and about 1.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 142 or the method of claim 152, comprising about 40% to about 60% SS-OP, about 1% to about 20% DODG, about 25% to about 45% cholesterol and about 0.5% to about 2.5% DMG-PEG2000 (by molar amount). The lipid particle of claim 261 or the method of claim 261, wherein the lipid particle comprises about 50% SS-OP, about 10% DODG, about 38.5% cholesterol and about 1.5% DMG-PEG2000 (by molar amount). A method of targeting a payload to the pancreas of an individual in need thereof, the method comprising intraperitoneally administering to the individual the lipid particle of any one of claims 1, 4 to 151, and 162 to 262. A method for targeting a payload to lymph nodes of an individual in need thereof, the method comprising subcutaneously administering to the individual the lipid particle of any one of claims 1, 4 to 151, and 162 to 262. The lipid particle of any one of claims 1, 4 to 151 and 162 to 262 or the method of any one of claims 2 to 138 and 148 to 255, wherein the payload comprises a biologically active molecule. The lipid particle of claim 265 or the method of claim 265, wherein the biologically active molecule is a small molecule, a nucleic acid, an aptamer or any combination thereof. A lipid particle as in claim 265 or a method as in claim 265, wherein the biologically active molecule comprises a nucleic acid. The lipid particle of claim 267 or the method of claim 267, wherein the nucleic acid comprises small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), primary micro RNA (pri-miRNA), long noncoding RNA (lncRNA), messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, CRISPR-RNA (crRNA), single guide RNA (sgRNA), trans-activating CRISPR RNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), antisense RNA (RNA), guide RNA, deoxyribonucleic acid (DNA), double-stranded deoxyribonucleic acid (dsDNA), single-stranded deoxyribonucleic acid (ssDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), CRISPR-associated (Cas) protein or a combination thereof. The lipid particle of claim 268 or the method of claim 268, wherein the nucleic acid in the lipid particle comprises mRNA. The lipid particle of claim 268 or the method of claim 268, wherein the nucleic acid in the lipid particle encodes a peptide having therapeutic activity. The lipid particle of claim 270 or the method of claim 270, wherein the peptide comprises an antigen-determining amino acid sequence. The lipid particle of claim 270 or the method of claim 270, wherein the peptide induces immune tolerance to cells. A nucleic acid-lipid particle comprising the lipid particle of any one of claims 1, 4 to 151 and 162 to 262 and a nucleic acid. A composition comprising the lipid particle of any one of claims 1, 4 to 151 and 162 to 262. A pharmaceutical composition comprising the lipid particles of any one of claims 1, 4 to 151 and 162 to 262 and one or more pharmaceutically acceptable carriers. The pharmaceutical composition of claim 275, wherein one of the pharmaceutically acceptable carriers is sucrose. The pharmaceutical composition of claim 275 or 276, wherein one of the pharmaceutically acceptable carriers is saline. The pharmaceutical composition of claim 277, wherein the saline solution is buffered with tris. The pharmaceutical composition of any one of claims 275 to 278, wherein the pharmaceutical composition is stored at -80°C. The pharmaceutical composition of any one of claims 275 to 278, wherein the pharmaceutical composition is stored at 4°C. A method for producing a nucleic acid-lipid particle, the method comprising mixing a nucleic acid with the lipid particle of any one of claims 1, 4 to 151, and 162 to 262. The method of claim 281, wherein the nucleic acid comprises small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), primary micro RNA (pri-miRNA), long noncoding RNA (lncRNA), messenger RNA (mRNA), clusters of regularly interspaced repeats (CRISPR)-related nucleic acid, CRISPR-RNA (crRNA), single guide RNA (sgRNA), trans-activating CRISPR RNA (tracrRNA), plasmid deoxyribonucleic acid (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), antisense RNA (RNA), guide RNA, deoxyribonucleic acid (DNA), double-stranded deoxyribonucleic acid (dsDNA), single-stranded deoxyribonucleic acid (ssDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), CRISPR-related (Cas) protein or a combination thereof. The method of claim 282, wherein the nucleic acid comprises mRNA. The method of claim 282, wherein the nucleic acid encodes a peptide having therapeutic activity. The method of claim 284, wherein the peptide comprises an antigen-determining amino acid sequence. The method of claim 284, wherein the peptide induces immune tolerance to cells. A method for introducing a nucleic acid into a cell, the method comprising contacting the cell with the lipid particle of any one of claims 1, 4 to 151, and 162 to 262. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 275 to 280. A method for preventing or delaying the onset or recurrence of a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 275 to 280. A method for restoring immune homeostasis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 275 to 280. A method for limiting or reducing an immunogenic response in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 275 to 280. The method of any one of claims 288 to 291, wherein after administration to a subject in need thereof, the subject exhibits one or more of the following: (i) an increase in Treg cells, (ii) a decrease in effector T cells, (iii) a decrease in B cells, (iv) a decrease in interleukin production, (v) a decrease in activated B cells, (vi) a decrease in activated effector T cells; or (vii) any combination thereof. A method for limiting or reducing an inflammatory response in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 275 to 280. The method of any one of claims 288 to 293, wherein the pharmaceutical composition is administered intranasally, intracranialy, intrathecally, intradermally, intratracheally, transdermally, intravenously, intraperitoneally, by injection, by infusion, intramuscularly or subcutaneously. The method of any one of claims 288 to 293, wherein the pharmaceutical composition is administered to the subject intravenously, intraperitoneally, by injection, by infusion, intramuscularly, or subcutaneously. The method of any of claims 288 to 295, wherein the individual is a mammal. The method of claim 296, wherein the mammal is a human. The method of any of claim 288, wherein the disease is an autoimmune disease. The method of claim 298, wherein the autoimmune disease is selected from the group consisting of autoimmune hepatitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), nonalcoholic fatty pancreatitis, scleroderma, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, chylous diarrhea, type 1 diabetes, Guillain-Barré syndrome, Hashimoto's thyroiditis, thyroiditis), rheumatic polymyalgia, alopecia areata, fibrosis, psoriasis, pemphigus vulgaris, vitiligo, ankylosing spondylitis, juvenile idiopathic arthritis, psoriasis arthritis, mixed connective tissue disease, neuromyelitis optica, latent autoimmune diabetes in adults ("LADA"), autoimmune thyroid disease, Grave's disease, Addison's disease, autoimmune atrophic gastritis, pernicious anemia, atopic dermatitis, bullous pemphigoid, myasthenia gravis, polymyositis/dermatomyositis), rheumatic fever, primary sclerosing cholangitis, autoimmune uveitis, and Behcet's disease. disease), diseases that affect the blood or bone marrow (e.g., autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, Goodpasture's syndrome, autoimmune nephritis, glomerulonephritis, Wegener's granulomatosis, chronic inflammatory demyelinating polyradiculoneuropathy, Sjogren's syndrome, primary biliary cholangitis, Parkinson's disorder, and antiphospholipid syndrome. The method of claim 299, wherein the autoimmune disease is autoimmune hepatitis, type 1 diabetes, or multiple sclerosis. The method of claim 288, wherein the disease is an infectious disease. The method of claim 288, wherein the disease is cancer.