TW202321271A - Compositions comprising hydroxyethyl-capped cationic peptoids - Google Patents

Abstract

The present disclosure provides delivery vehicle compositions comprising hydroxyethyl-capped tertiary amino lipidated cationic peptoids, and complexes of the delivery vehicles with polyanionic compounds, such as nucleic acids. The disclosure further provides methods of making and using the delivery vehicle compositions and complexes, such as for the delivery polyanionic compounds (e.g., nucleic acids) to cells. The disclosure also provides methods of eliciting an immune response with the delivery vehicle complexes of the disclosure.

Description

Compositions comprising hydroxyethyl-terminated cationic peptoids

Therapeutic nucleic acids, such as mRNA, small interfering RNA (siRNA), small activating RNA (saRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plastids, and immunostimulatory Prevention and treatment of diseases hold great promise. However, nucleic acids typically undergo rapid degradation in blood, renal clearance, poor cellular uptake, and inefficient endosomal escape. Therefore, for nucleic acids to be therapeutically useful, safe and efficient systems for delivering nucleic acids to the nucleus or cytosol are needed. Traditional methods for the delivery of polyanionic compounds such as oligonucleotides in cells and in vivo include viral vectors, cationic lipid nanoparticles (LNPs), and polycationic polymers. These delivery systems can be plagued by limitations such as poor stability, rapid clearance, adverse toxicology, immune response issues and the sub-optimal performance of their polyanionic cargos.

There is a need for stable, safe and efficient systems for the delivery of nucleic acids to cells. Accordingly, the present disclosure is directed to delivery vehicle compositions comprising hydroxyethyl-terminated tertiary aminolipidated cationic peptoids, and complexes of such delivery vehicle compositions with polyanionic compounds, such as nucleic acids. The disclosure further relates to methods of making and using delivery vehicle compositions and complexes for the intracellular delivery of polyanionic compounds, such as mRNA, and methods of eliciting an immune response using the complexes of the disclosure.

，其中n為1、2、3、4、5或6；R ¹為H、C _1-3烷基或羥乙基；且各R ²獨立地為C _8-24烷基或C _8-24烯基。在一些情況下，n為3。在各種情況下，n為4。在一些實現方式中，R ¹為H。在一些情況下，R ¹為乙基或羥乙基。在各種情況下，R ²獨立地為C _8-18烷基或C _8-18烯基。在一些實現方式中，各R ²係選自由以下組成之群：

。在各種情況下，各R ²獨立地選自由以下組成之群：

。在一些實現方式中，R ²獨立地選自由以下組成之群：

。在各種實現方式中，各R ²為

。在一些情況下，式（I）化合物具有選自由以下組成之群的結構：

。在各種情況下，式（I）化合物具有結構：

。本文進一步揭示式（I）化合物之醫藥學上可接受之鹽。 In one aspect, the disclosure provides a compound having the following structure of formula (I):

, wherein n is 1, 2, 3, 4, 5 or 6; R ¹ is H, C _1-3 alkyl or hydroxyethyl; and each R ² is independently C _8-24 alkyl or C _8-24 Alkenyl. In some cases, n is 3. n was 4 in each case. In some implementations, R ¹ is H. In some instances, R ¹ is ethyl or hydroxyethyl. In each instance, R ² is independently C _8-18 alkyl or C _8-18 alkenyl. In some implementations, each ^R is selected from the group consisting of:

. In each instance, ^each R is independently selected from the group consisting of:

. In some implementations, R ^is independently selected from the group consisting of:

. In various implementations, each ^R2 is

. In some cases, the compound of Formula (I) has a structure selected from the group consisting of:

. In each case, the compound of formula (I) has the structure:

. Further disclosed herein are pharmaceutically acceptable salts of compounds of formula (I).

Another aspect of the disclosure provides a delivery vehicle composition comprising a compound disclosed herein, or a pharmaceutically acceptable salt thereof. In some implementations, the composition further comprises one or more of phospholipids, sterols, and pegylated lipids. In some implementations, the compound or salt of formula (I) is present in the delivery vehicle composition in an amount from about 30 mol % to about 60 mol %. In some implementations, the compound or salt of formula (I) is present in the delivery vehicle composition in an amount from about 35 mol % to about 55 mol %. In various implementations, the compound or salt of formula (I) is present in the delivery vehicle composition in an amount from about 30 mol % to about 45 mol %. In various implementations, the compound or salt of formula (I) is present in the delivery vehicle composition in an amount from about 35 mol % to about 39 mol %. In some instances, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount from about 39 mol % to about 52 mol %. In various implementations, the compound or salt of formula (I) is present in the delivery vehicle composition in an amount from about 30 mol % to about 35 mol %. In various implementations, the compound or salt of formula (I) is present in the delivery vehicle composition in an amount from about 40 mol % to about 45 mol %. In each instance, the compound or salt of formula (I) is present in an amount from about 42 mol % to about 49 mol %. In some implementations, the compound or salt of formula (I) is present in an amount of about 50 mol% to about 52 mol%.

（tomatidine）、熊果酸、α-生育酚及其混合物。在一些情況下，固醇為膽固醇。在一些實現方式中，聚乙二醇化脂質係選自由以下組成之群：經PEG改質之磷脂醯乙醇胺、經PEG改質之磷脂酸、經PEG改質之神經醯胺、經PEG改質之二烷基胺、經PEG改質之二醯甘油、經PEG改質之二烷基甘油、經PEG改質之固醇及經PEG改質之磷脂。在各種實現方式中，經PEG改質之脂質係選自由以下組成之群：經PEG改質之膽固醇、N-辛醯基-神經鞘胺醇-1-{丁二醯基[甲氧基(聚乙二醇)]}、N-軟脂醯基-神經鞘胺醇-1-{丁二醯基[甲氧基(聚乙二醇)]}、經PEG改質之DMPE（DMPE-PEG）、經PEG改質之DSPE（DSPE-PEG）、經PEG改質之DPPE（DPPE-PEG）、經PEG改質之DOPE（DOPE-PEG）、二肉豆蔻醯基甘油-聚乙二醇（DMG-PEG）、二硬脂醯基甘油-聚乙二醇（DSG-PEG）、二軟脂醯基甘油-聚乙二醇（DPG-PEG）、二油醯基甘油-聚乙二醇（DOG-PEG）及其組合。在一些情況下，經PEG改質之脂質為二肉豆蔻醯基甘油-聚乙二醇2000（DMG-PEG 2000）。在各種情況下，組合物包含約38.2 mol%之化合物140、約11.8 mol%之DSPC、約48.2 mol%之膽固醇及約1.9 mol%之DMG-PEG 2000。在一些實現方式中，組合物包含約42.6 mol%之化合物140、約10.9 mol%之DSPC、約44.7 mol%之膽固醇及約1.7 mol%之DMG-PEG 2000。在一些實現方式中，組合物包含約48.2 mol%之化合物140、約9.9 mol%之DSPC、約40.4 mol%之膽固醇及約1.6 mol%之DMG-PEG 2000。在各種情況下，組合物包含約51.3 mol%之化合物140、約9.3 mol%之DSPC、約38 mol%之膽固醇及約1.5 mol%之DMG-PEG 2000。在各種情況下，組合物包含約44.4 mol%之化合物140、約10.6 mol%之DSPC、約43.3 mol%之膽固醇及約1.7 mol%之DMG-PEG 2000。在各種情況下，組合物包含約44.4 mol%之化合物140、約10.6 mol%之DSPC、約43.4 mol%之膽固醇及約1.7 mol%之DMG-PEG 2000。在各種情況下，組合物包含約33.1 mol%之化合物140、約10.6 mol%之DSPC、約53.8 mol%之膽固醇及約2.5 mol%之DMG-PEG 2000。 In various implementations, the composition comprises phospholipids, sterols, and pegylated lipids. In some instances, the compositions consist essentially of a compound disclosed herein, or a salt thereof, a phospholipid, a sterol, and a pegylated lipid. In some cases, the composition comprises about 30 mol% to about 60 mol% of the compound of formula (I); about 3 mol% to about 20 mol% of phospholipids, about 25 mol% to about 60 mol% of sterols and about 1 mol% to about 5 mol% of pegylated lipids. In each case, the composition comprises from about 35 mol% to about 55 mol% of a compound or salt of formula (I); from about 5 mol% to about 15 mol% of phospholipids, from about 30 mol% to about 55 mol% of solid Alcohol and about 1 mol% to about 3 mol% of pegylated lipids. In some implementations, the composition comprises about 38 mol% to about 52 mol% of the compound or salt of formula (I); about 9 mol% to about 12 mol% of phospholipids, about 35 mol% to about 50 mol% of Sterols and about 1 mol% to about 2 mol% of pegylated lipids. In various implementations, the composition comprises about 30 mol% to about 49 mol% of the compound of formula (I); about 5 mol% to about 15 mol% of phospholipids, about 30 mol% to about 55 mol% of sterols and About 1 mol% to about 3 mol% pegylated lipid. In some cases, the composition comprises about 35 mol% to about 49 mol% of a compound or salt of formula (I); about 7 mol% to about 12 mol% of phospholipids, about 35 mol% to about 50 mol% of solid Alcohol and about 1 mol% to about 2 mol% of pegylated lipids. In some cases, the composition comprises about 30 mol% to about 45 mol% of a compound or salt of formula (I); about 7 mol% to about 12 mol% of phospholipids, about 40 mol% to about 55 mol% of solid Alcohol and about 1 mol% to about 3 mol% of pegylated lipids. In some cases, the composition comprises about 30 mol% to about 35 mol% of a compound or salt of formula (I); about 7 mol% to about 12 mol% of phospholipids, about 50 mol% to about 55 mol% of solid Alcohol and about 2 mol% to about 3 mol% of pegylated lipids. In some cases, the composition comprises about 40 mol% to about 45 mol% of a compound or salt of formula (I); about 7 mol% to about 12 mol% of phospholipids, about 40 mol% to about 45 mol% of solid Alcohol and about 1 mol% to about 2 mol% of pegylated lipids. In some cases, the phospholipid is selected from the group consisting of 1,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristyl-sn-glycerol - Phosphocholine (DMPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine ( DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecyl-sn-glycero-phosphocholine (DUPC), 1-soft Fattyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 di ether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16 Lyso PC), 1,2-dilinoleyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-di (Docosyl)hexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-disalmitoyl Diphytyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearyl-sn- Glycerol-3-phosphoethanolamine, 1,2-Dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, Arachidonyl-sn-glycero-3-phosphoethanolamine, 1,2-bis(docos)hexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleyl-sn - Glycerol-3-dioxaphosphoryl-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and combinations thereof. In some instances, the phospholipid is DOPE, DSPC, or a combination thereof. In each case, the phospholipid is DSPC. In some implementations, the sterol is selected from the group consisting of cholesterol, coprosterol, phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomato

(tomatidine), ursolic acid, alpha-tocopherol and mixtures thereof. In some instances, the sterol is cholesterol. In some implementations, the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified Dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, PEG-modified sterols, and PEG-modified phospholipids. In various implementations, the PEG-modified lipid is selected from the group consisting of PEG-modified cholesterol, N-octyl-sphingosine-1-{butadiyl[methoxy(polyethylene Diol)]}, N-palmitoyl-sphingosine-1-{butanediyl[methoxy(polyethylene glycol)]}, DMPE modified by PEG (DMPE-PEG), PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), PEG-modified DOPE (DOPE-PEG), dimyristyl glycerol-polyethylene glycol (DMG- PEG), distearoylglycerol-polyethylene glycol (DSG-PEG), disalmitylglycerol-polyethylene glycol (DPG-PEG), dioleylglycerol-polyethylene glycol (DOG- PEG) and combinations thereof. In some instances, the PEG-modified lipid is dimyristylglycerol-polyethylene glycol 2000 (DMG-PEG 2000). In each case, the composition comprised about 38.2 mol% of Compound 140, about 11.8 mol% of DSPC, about 48.2 mol% of cholesterol, and about 1.9 mol% of DMG-PEG 2000. In some implementations, the composition comprises about 42.6 mol% Compound 140, about 10.9 mol% DSPC, about 44.7 mol% cholesterol, and about 1.7 mol% DMG-PEG 2000. In some implementations, the composition comprises about 48.2 mol% Compound 140, about 9.9 mol% DSPC, about 40.4 mol% cholesterol, and about 1.6 mol% DMG-PEG 2000. In each case, the composition comprised about 51.3 mol% of Compound 140, about 9.3 mol% of DSPC, about 38 mol% of cholesterol, and about 1.5 mol% of DMG-PEG 2000. In each case, the composition comprised about 44.4 mol% of Compound 140, about 10.6 mol% of DSPC, about 43.3 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000. In each case, the composition comprised about 44.4 mol% of Compound 140, about 10.6 mol% of DSPC, about 43.4 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000. In each case, the composition comprised about 33.1 mol% of Compound 140, about 10.6 mol% of DSPC, about 53.8 mol% of cholesterol, and about 2.5 mol% of DMG-PEG 2000.

Further disclosed herein is a delivery vehicle complex comprising the delivery vehicle composition described herein and a polyanionic compound. In some cases, a compound of formula (I) or a salt thereof is complexed with a polyanionic compound. In each case, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of from about 5:1 to about 25:1. In some implementations, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 7:1 to about 20:1. In each case, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of from about 10:1 to about 17:1. In some cases, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 19:1. In some cases, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 20:1. In some cases, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 10:1. In each case, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 12:1. The compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 13:1. In some implementations, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 15:1. In various implementations, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 17:1. In some cases, the phospholipid and polyanionic compound are present in a mass ratio of about 2:1 to about 10:1. In some cases, the phospholipid and polyanionic compound are present in a mass ratio of about 2:1 to about 4:1. In each case, the phospholipid and polyanionic compound are present in a mass ratio of about 2:1 to about 3:1. In each case, the phospholipid and polyanionic compound are present in a mass ratio of about 4.0:1. In each case, the phospholipid and polyanionic compound were present in a mass ratio of about 2.7:1. In some implementations, the sterol to polyanionic compound is present in a mass ratio of about 5:1 to about 8:1. In some implementations, the sterol and polyanionic compound are present in a mass ratio of about 5:1 to about 6:1. In various implementations, the sterol to polyanionic compound is present in a mass ratio of about 5.4:1. In some cases, the sterol to polyanionic compound is present in a mass ratio of about 8.1:1. In some cases, the sterol to polyanionic compound is present in a mass ratio of about 6.7:1. In some instances, the pegylated lipid and polyanionic compound are present in a mass ratio of about 0.5:1 to about 2.5:1. In each case, the pegylated lipid and polyanionic compound are present in a mass ratio of about 1:1 to about 2:1. In some cases, the phospholipid and polyanionic compound are present in a mass ratio of about 2.1:1. In some cases, the phospholipid and polyanionic compound are present in a mass ratio of about 1.4:1. In each case, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 10:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, Cholesterol at about 5.4:1 and DMG-PEG 2000 at a mass ratio of about 1.4:1 to the polyanionic compound. In each case, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 12:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, Cholesterol at about 5.4:1 and DMG-PEG 2000 at a mass ratio of about 1.4:1 to the polyanionic compound. In some cases, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 15:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, and in a mass ratio to the polyanionic compound of Cholesterol at about 5.4:1 and DMG-PEG 2000 at a mass ratio of about 1.4:1 to the polyanionic compound. In each case, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 17:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, Cholesterol at about 5.4:1 and DMG-PEG 2000 at a mass ratio of about 1.4:1 to the polyanionic compound. In each case, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 13:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, Cholesterol at about 5.4:1 and DMG-PEG 2000 at a mass ratio of about 1.4:1 to the polyanionic compound. In each case, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 19:1 to the polyanionic compound, DSPC in a mass ratio of about 4.0:1 to the polyanionic compound, Cholesterol at about 5.4:1 and DMG-PEG 2000 at a mass ratio of about 2.1:1 to the polyanionic compound. In each case, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 9.7:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, Cholesterol at about 6.7:1 and DMG-PEG 2000 at a mass ratio of about 2.1:1 to the polyanionic compound.

In some cases, the composite exhibits a particle size of about 50 nm to about 200 nm and/or a polydispersity index (PDI) of less than 0.25. In each case, the complex exhibits a particle size ranging from about 60 nm to about 100 nm. In some implementations, the complex exhibits a particle size of about 60 nm to about 90 nm. In various implementations, the complex exhibits a particle size of about 105 nm to about 200 nm. In each case, the complex exhibits a particle size of about 150 nm to about 200 nm. In some instances, the delivery vehicle complex exhibits a particle size of about 105 nm to about 200 nm. In some cases, the delivery vehicle complex exhibits a particle size of about 40 nm to about 115 nm, or about 55 nm to about 95 nm, or about 70 to about 80 nm, or about 75 nm. In each case, the delivery vehicle complex exhibits a particle size of about 135 nm to about 225 nm, or about 155 nm to about 195 nm, or about 170 to about 180 nm, or about 175 nm. In each case, at least 80% of the polyanionic compound remains after storage at 4°C for 48 days, or the delivery vehicle complex retains at least 80% of its original size after storage at 4°C for 48 days, or both.

In some cases, the polyanionic compound comprises at least one nucleic acid. In each case, at least one nucleic acid comprises RNA, DNA, or a combination thereof. In each case, at least one nucleic acid comprises RNA. In some implementations, the RNA is mRNA encoding a peptide, protein or functional fragment thereof. In various implementations, the mRNA encodes a viral peptide, a viral protein, or a functional fragment of any of the foregoing. In some cases, the mRNA encodes a human papillomavirus (HPV) protein or a functional fragment thereof. In each case, the mRNA encodes an HPV E6 protein and/or an HPV E7 protein, a variant thereof, or a functional fragment of any of the foregoing. In some instances, the HPV protein is from HPV subtypes HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and/or 68. In each case, the HPV protein is from HPV subtypes HPV 16 and/or HPV 18. In some cases, the mRNA encodes a viral spike protein or a functional fragment thereof. In each case, the mRNA encodes a SARS-CoV Spike (S) protein, a variant thereof, or a functional fragment of any of the foregoing. In some cases, the RNA encodes a SARS-associated coronavirus (e.g. severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV ), human coronavirus 229E (HCoV-229E), human coronavirus 0C43 (HCoV-0C43), human coronavirus HKU1 (HCoV-HKU1) or human coronavirus NL63 (HCoV-NL63)). In some instances, the mRNA encodes influenza hemagglutinin (HA), a variant thereof, or a functional fragment of any of the foregoing. In some implementations, the influenza A virus has a subtype HA selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. In various implementations, the influenza subtype is HA strain H1, H2, H3 or H5. In each case, one mRNA encodes the SARS-CoV Spike (S) protein and one mRNA encodes influenza hemagglutinin (HA), a variant thereof, or a functional fragment of any of the foregoing.

Further disclosed herein is a pharmaceutical composition comprising the delivery vehicle complex of the disclosure and a pharmaceutically acceptable excipient. In some instances, the pharmaceutical composition is an intratumoral (IT) or intramuscular (IM) composition.

Also disclosed herein is a method of inducing an immune response in an individual in need thereof, comprising administering to the individual an effective amount of a delivery vehicle complex described herein or a pharmaceutical formulation comprising the delivery vehicle complex, whereby in An immune response is induced in the individual. Further disclosed herein is a method of treating a viral infection in an individual in need thereof comprising administering to the individual an effective amount of a delivery vehicle complex described herein or a pharmaceutical formulation comprising the delivery vehicle complex, thereby treating A viral infection in the individual. Also disclosed herein is a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a delivery vehicle complex described herein or a pharmaceutical formulation comprising the delivery vehicle complex, thereby treating the subject of cancer. In some instances, the cancer is cervical cancer, head and neck cancer, B cell lymphoma, T cell lymphoma, prostate cancer, lung cancer, or a combination thereof. In each case, administration is by intramuscular, intratumoral, intravenous, intraperitoneal or subcutaneous delivery.

Also disclosed herein is a method of delivering a polyanionic compound to a cell comprising contacting the cell with a delivery vehicle complex described herein or a pharmaceutical formulation comprising the delivery vehicle complex. In some instances, the cells are muscle cells, tumor cells, or combinations thereof. In some cases, the polyanionic compound is mRNA encoding a peptide, protein, or fragment of any of the foregoing, and the cell expresses the peptide, protein, or fragment following contact with the delivery vehicle complex.

Also disclosed herein is a method of forming a delivery vehicle complex disclosed herein comprising contacting a compound or salt of formula (I) with a polyanionic compound. In some cases, the method comprises admixing a solution comprising a compound or salt of formula (I) with a solution comprising a polyanionic compound.

Also disclosed herein are vaccines comprising the delivery vehicle complexes disclosed herein or the pharmaceutical compositions disclosed herein. Vaccines comprising the delivery vehicle complexes disclosed herein or the pharmaceutical compositions disclosed herein are also disclosed for use in the treatment of cancer. Also disclosed are methods of treating or preventing cancer in a patient comprising administering to the patient a delivery vehicle complex disclosed herein or a pharmaceutical composition disclosed herein. In various instances, the cancer is cervical cancer, head and neck cancer, B cell lymphoma, T cell lymphoma, prostate cancer, lung cancer, or combinations thereof.

It is to be appreciated that all combinations of the foregoing concepts and implementations, as well as additional concepts and implementations discussed in greater detail below, are contemplated as part of the subject matter of the disclosure disclosed herein and may be employed in any suitable combination to achieve a goal as described herein. benefit. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are intended to be a part of the subject matter of this disclosure disclosed herein.

The following overview of the embodiments in conjunction with the drawings will make other aspects and advantages apparent to those of ordinary skill in the art. While the compounds and methods disclosed herein are susceptible to various forms of implementation, the following description includes specific implementations, with the understanding that the disclosure is illustrative and is not intended to limit the disclosure to the specific implementations described herein Way.

In some examples, disclosed herein are delivery vehicle compositions comprising hydroxyethyl-terminated cationic peptoids, including, for example, hydroxyethyl-terminated tertiary aminolipidated cationic peptoids. The delivery vehicle compositions of the present disclosure can form electrostatic interactions between the hydroxyethyl-terminated tertiary aminolipidated cationic peptoids of the delivery vehicle composition and polyanionic compounds, such as nucleic acids, to form the delivery vehicle Agent complex, wherein the polyanionic compound serves as the cargo of the complex. Delivery vehicle complexes can be used to deliver polyanionic compounds, such as nucleic acids (eg, mRNA) into cells. The delivery vehicle complexes of the disclosure comprising mRNA as a polyanionic cargo unexpectedly exhibit excellent in vitro and in vivo mRNA expression. When the mRNA of the delivery vehicle complex encodes, for example, a viral antigen, the delivery vehicle complex can elicit both humoral and cellular immune responses in vivo, thereby functioning as a vaccine. Additional advantages of the delivery vehicle complexes disclosed herein are that they are stable and exhibit good tolerability and low toxicity.

As used herein, "peptoid" refers to a peptidomimetic compound in which one or more nitrogen atoms of the peptide backbone are substituted with side chains. As used herein, "lipidated peptoid" or "lipidoid" refers to a peptide in which one or more side chains on the nitrogen atom comprise a lipid. As used herein, "polyanion" refers to a compound, such as a nucleic acid, that has at least two negative charges. delivery vehicle composition

Some exemplary delivery vehicle compositions of the present disclosure comprise one or more hydroxyethyl-terminated tertiary amino lipidated cationic peptoids. Such positively charged peptides can be associated with polyanionic compounds, such as nucleic acids, to form delivery vehicle complexes. In some implementations, the delivery vehicle composition further comprises one or more anionic or zwitterionic components, such as phospholipids; neutral lipids, such as sterols; and shielding lipids, such as pegylated lipids. In various implementations, the delivery vehicle composition further comprises anionic or zwitterionic components (eg, phospholipids), neutral lipids (eg, sterols), and shielding lipids (eg, pegylated lipids). In some cases, the delivery vehicle composition consists essentially of a hydroxyethyl-terminated tertiary amino lipidated cationic peptoid, anionic or zwitterionic components (e.g., phospholipids), neutral lipids (e.g., sterols) and shielding lipids (eg, pegylated lipids). Hydroxyethyl-terminated Tertiary Amino Lipidated Cationic Peptoid Fraction

，其中n為1、2、3、4、5或6；R ¹為H、C _1-3烷基或C _2-3羥烷基；且各R ²獨立地為C _8-24烷基或C _8-24烯基。如本文所用，「烷基」係指含有一至三十個碳原子，例如，一至四個碳原子（例如，1、2、3或4個）之直鏈及分支鏈飽和烴基。術語C _n意謂烷基具有「n」個碳原子。舉例而言，C ₃烷基係指具有3個碳原子之烷基。C _1-4烷基係指具有涵蓋整個範圍（亦即1至4個碳原子）以及所有子群（例如1-2、1-3、2-3、2-4、1、2、3及4個碳原子）之碳原子數的烷基。烷基之非限制性實例包括甲基、乙基、正丙基、異丙基、正丁基、二級丁基（2-甲基丙基）及三級丁基（1,1-二甲基乙基）。除非另外指示，否則烷基可為未經取代之烷基或經取代之烷基。如本文所用，「羥烷基」係指如本文所定義之經羥基取代之烷基。例如，「C ₂羥烷基」或「羥乙基」具有結構：

。如本文所用，「烯基」係指具有雙鍵且含有二至三十個碳原子，例如，二至四個碳原子（例如，1、2、3或4個）之直鏈及分支鏈烴基。術語C _n意謂烯基具有「n」個碳原子。例如，C ₃烯基係指具有3個碳原子之烯基。C ₂-C ₄烯基係指具有涵蓋整個範圍（亦即2至4個碳原子）以及所有子群（例如2-3、2-4、2、3及4個碳原子）之碳原子數的烯基。烯基之非限制性實例包括乙烯基、丙烯基及丁烯基。除非另外指示，否則烯基可為未經取代之烯基或經取代之烯基。 The delivery vehicle compositions of the present disclosure comprise hydroxyethyl-terminated tertiary aminolipidated cationic peptoids ("cationic components", sometimes referred to as "ionizable lipids"). In some implementations, the hydroxyethyl-terminated tertiary aminolipidated cationic peptoid comprises a compound of formula (I):

, wherein n is 1, 2, 3, 4, 5 or 6; R ¹ is H, C _1-3 alkyl or C _2-3 hydroxyalkyl; and each R ² is independently C _8-24 alkyl or C _8-24 alkenyl. As used herein, "alkyl" refers to straight and branched chain saturated hydrocarbon groups containing one to thirty carbon atoms, eg, one to four carbon atoms (eg, 1, 2, 3 or 4). The term _Cn means that the alkyl group has "n" carbon atoms. For example, _C3 alkyl refers to an alkyl group having 3 carbon atoms. C _1-4 alkyl refers to the group having the whole range (i.e. 1 to 4 carbon atoms) and all subgroups (such as 1-2, 1-3, 2-3, 2-4, 1, 2, 3 and 4 carbon atoms) of the alkyl group. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, secondary butyl (2-methylpropyl) and tertiary butyl (1,1-dimethyl ethyl ethyl). Unless otherwise indicated, an alkyl group can be unsubstituted or substituted. As used herein, "hydroxyalkyl" refers to a hydroxy-substituted alkyl group as defined herein. For example, "C ₂ hydroxyalkyl" or "hydroxyethyl" has the structure:

. As used herein, "alkenyl" refers to straight and branched chain hydrocarbon groups having a double bond and containing two to thirty carbon atoms, for example, two to four carbon atoms (for example, 1, 2, 3 or 4) . The term _Cn means that the alkenyl group has "n" carbon atoms. For example, _C3 alkenyl refers to an alkenyl group having 3 carbon atoms. C ₂ -C ₄ alkenyl means the number of carbon atoms having the entire range (i.e. 2 to 4 carbon atoms) and all subgroups (e.g. 2-3, 2-4, 2, 3 and 4 carbon atoms) the alkenyl. Non-limiting examples of alkenyl groups include ethenyl, propenyl, and butenyl. Unless otherwise indicated, an alkenyl group can be an unsubstituted alkenyl group or a substituted alkenyl group.

In some implementations, n is 2-5. In various implementations, n is 3-4. In some implementations, n is 1. In various implementations, n is two. In some cases, n is 3. n was 4 in each case. In some implementations, n is 5. In various implementations, n is 6.

（羥乙基）。在各種情況下，R ¹為乙基或羥乙基。 In some implementations, R ¹ is H. In various implementations, R ¹ is C _1-3 alkyl. In some instances, R ¹ is methyl or ethyl. In some implementations, R ¹ is ethyl. In various implementations, R ¹ is C _2-3 hydroxyalkyl. In some cases, ^R1 is

(hydroxyethyl). In each case, R ¹ is ethyl or hydroxyethyl.

。在一些情況下，各R ²獨立地選自由以下組成之群：

。在各種情況下，各R ²獨立地選自由以下組成之群：

。在一些實現方式中，各R ²獨立地為

。 In some implementations, each R ² is independently C _8-18 alkyl or C _8-18 alkenyl. In various implementations, each R ² is independently C _8-16 alkyl or C _10-18 alkenyl. In some instances, each R ² is independently C _10-12 alkyl or C _10-18 alkenyl. In some implementations, each R ² is independently: C _8-18 alkyl, or C _8-16 alkyl, or C _8-14 alkyl, or C _8-12 alkyl. In various implementations, each ^R is independently selected from the group consisting of:

. In some cases, each R ^is independently selected from the group consisting of:

. In each instance, ^each R is independently selected from the group consisting of:

. In some implementations, each ^R2 is independently

.

。 In some implementations, n is 4, ^R is H, and each ^R is

.

907.9 146

711.4 151

852.8 152

1128.0 160

935.8 161

1019.9 162

951.8 Contemplated compounds of formula (I) include, but are not limited to, those listed in Table 1. Table 1. Examples of hydroxyethyl-terminated tertiary amino lipidated cationic peptoids. compound structure Mass experimental value ( LC-MS ) 140

907.9 146

711.4 151

852.8 152

1128.0 160

935.8 161

1019.9 162

951.8

In some implementations, the compound of formula (I) is compound 140.

Compounds of the disclosure are defined herein by their chemical structures and/or chemical names. When a compound is referred to by its chemical structure and chemical name and the chemical structure and chemical name are contradictory, the chemical structure determines the identity of the compound.

Unless otherwise indicated, structures depicted herein are also intended to include all isomeric (eg, enantiomerically, diastereomerically, cis-trans, conformational, and rotational) forms of the structures. For example, each asymmetric center, ( Z ) and ( E ) double bond isomers and ( Z ) and ( E ) configurational isomers are included in this invention unless only one of the isomers is specifically indicated The R and S configurations of the body. Accordingly, single stereochemical isomers as well as enantiomerically, diastereomerically, cis/trans isomerically, conformationally and rotationally isomer mixtures of the compounds of the disclosure are within the scope of the disclosure. In some instances, compounds disclosed herein are stereoisomers. "Stereoisomer" refers to a compound that differs in the chirality of one or more stereogenic centers. Stereoisomers include mirror-image and diastereomers. The compounds disclosed herein may exist as single stereoisomers or as mixtures of stereoisomers. Unless otherwise stated, the stereochemistry of the compounds shown herein indicates relative stereochemistry, not absolute stereochemistry. As indicated herein, a single stereoisomer, diastereomer, or enantiomer refers to at least more than 50% of the indicated stereoisomer, diastereomer, or enantiomer and in some cases Compounds that are at least 90% or 95% of the indicated stereoisomers, diastereoisomers or enantiomers.

The compounds described herein may exist in free form or, where appropriate, in the form of a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" means, within the scope of sound medical judgment, suitable for use in contact with tissues of humans and lower animals without undue side effects (such as toxicity, irritation, allergic reactions, and the like) and satisfying Salts of compounds with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic acids and bases, as well as organic acids and bases. Such salts can be prepared in situ during the final isolation and purification of the compounds. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, trifluoroacetic acid, oxalic acid, cis- butenedioic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphoric acid Salt, Camphor Sulfonate, Citrate, Cyclopentane Propionate, Digluconate, Lauryl Sulfate, Ethane Sulfonate, Formate, Fumarate, Glucoheptin Sugarate, glycerophosphate, gluconate, glutamate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactic acid Salt, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, Oxalate, Palmitate, Pamoate, Pectate, Persulfate, 3-Phenylpropionate, Phosphate, Picrate, Pivalate, Propionate, Stearic Acid Salt, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and similar salts. Salts of compounds containing carboxylic acids or other acidic functional groups can be prepared by reaction with a suitable base. Such salts include, but are not limited to, alkali metal salts, alkaline earth metal salts, aluminum salts, ammonium salts, N ⁺ (C _1-4 alkyl) ₄ salts, and organic bases such as trimethylamine, triethylamine, morpholine, pyridine , piperidine, picoline, dicyclohexylamine, N,N'-benzhydrylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl base) amine, procaine (procaine), benzhydryl piperidine, dehydroabietylamine, N,N'-didehydroabietylamine, glucosamine, N-methylglucamine, trimethyl Pyridine, quinine, quinoline) and basic amino acids such as lysine and arginine. This disclosure also contemplates the quaternary ammonization of any basic nitrogen-containing groups of the compounds disclosed herein. Water-soluble or oil-soluble or dispersible products can be obtained by such quaternary ammonification. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Where appropriate, other pharmaceutically acceptable salts include the use of counter ions (such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates) Formed non-toxic ammonium, quaternary ammonium and amine cations.

In an implementation, the delivery vehicle composition comprises between about 25 mol % and about 70 mol % of hydroxyethyl-terminated tertiary amino esterification based on the total molar amount of components in the delivery vehicle composition. Cationic peptoids (eg, compounds of formula (I), such as compound 140). The unit "mol%" or "mole percent" refers to the number of moles of a particular component of a delivery vehicle composition divided by the total number of moles of all components in the delivery vehicle composition times 100%. The polyanionic cargo is not counted as part of the total moles of the delivery vehicle composition. In some cases, the delivery vehicle composition comprises, based on the total moles of components in the delivery vehicle composition, from about 30 mol% to about 60 mol%, or from about 35 mol% to about 55 mol%, or about 30 mol% to about 45 mol%, or about 35 mol% to about 40 mol%, or about 45 mol% to about 60 mol%, or about 50 mol% to about 55 mol%, or about 38 mol% to about 52 Between mol%, or about 38 mol%, or about 52 mol% of hydroxyethyl-terminated tertiary amino lipidated cationic peptoids (eg, compounds of formula (I), such as compound 140). In some implementations, the delivery vehicle composition comprises less than about 50 mol % of a hydroxyethyl-terminated tertiary aminolipidated cationic peptoid, based on the total moles of components in the delivery vehicle composition, Such as less than about 49 mol%, less than about 48 mol%, less than about 47 mol%, less than about 46 mol%, less than about 45 mol%, less than about 44 mol%, less than about 43 mol%, less than about 42 mol%, less than About 41 mol%, less than about 40 mol%, less than about 39 mol%, less than about 38 mol%, less than about 37 mol%, less than about 36 mol%, less than about 35 mol%, less than about 34 mol%, less than about 33 mol%, less than about 32 mol%, less than about 31 mol%, less than about 30 mol%; and greater than about 20 mol% of hydroxyethyl-terminated tertiary amino lipidated cationic peptoids, such as greater than about 21 mol% , greater than about 22 mol%, greater than about 23 mol%, greater than about 24 mol%, greater than about 25 mol%, greater than about 26 mol%, greater than about 27 mol%, greater than about 28 mol%, greater than about 29 mol%, greater than About 30 mol%, greater than about 31 mol%, greater than about 33 mol%, greater than about 34 mol%, greater than about 35 mol%, greater than about 36 mol%, greater than about 38 mol%, greater than about 39 mol%, greater than about 40 mol%, greater than about 41 mol%, greater than about 42 mol%, greater than about 43 mol%, or greater than about 44 mol% hydroxyethyl-terminated tertiary amino lipidated cationic peptoid. In some implementations, the delivery vehicle composition comprises less than about 50 mol % of a hydroxyethyl-terminated tertiary aminolipidated cationic peptoid, based on the total moles of components in the delivery vehicle composition, Such as less than about 49 mol%, less than about 48 mol%, less than about 47 mol%, less than about 46 mol%, less than about 45 mol%, less than about 44 mol%, less than about 43 mol%, less than about 42 mol%, less than About 41 mol%, less than about 40 mol%, less than about 39 mol%, less than about 38 mol%, less than about 37 mol%, less than about 36 mol%, less than about 35 mol%, less than about 34 mol%, less than about 33 mol%, less than about 32 mol%, less than about 31 mol%, less than about 30 mol%; and greater than about 20 mol% of hydroxyethyl-terminated tertiary aminolipidated cationic peptoids, such as greater than about 21 mol% , greater than about 22 mol%, greater than about 23 mol%, greater than about 24 mol%, greater than about 25 mol%, greater than about 26 mol%, greater than about 27 mol%, greater than about 28 mol%, greater than about 29 mol%, greater than About 30 mol%, greater than about 31 mol%, greater than about 33 mol%, greater than about 34 mol%, greater than about 35 mol%, greater than about 36 mol%, greater than about 38 mol%, greater than about 39 mol%, or greater than about 40 mol% of hydroxyethyl-terminated tertiary amino lipidated cationic peptoids. In some cases, the delivery vehicle composition comprises, based on the total moles of components in the delivery vehicle composition, from about 30 mol% to about 49.5 mol%, or from about 30 mol% to about 45 mol%, or about 30 mol% to about 35 mol%, or about 40 mol% to about 45 mol%, or about 35 mol% to about 49 mol%, or about 36 mol% to about 48 mol%, or about 38 mol% to about 45 mol %, or about 38 mol % to about 42 mol %, of a hydroxyethyl-terminated tertiary amino lipidated cationic peptoid (eg, a compound of formula (I), such as compound 140). In some cases, the delivery vehicle composition comprises, based on the total number of moles of components in the delivery vehicle composition, from about 30 mol% to about 49.5 mol%, or from about 35 mol% to about 49 mol%, or about 36 mol % to about 48 mol %, or about 38 mol % to about 45 mol %, or about 38 mol % to about 42 mol % of a hydroxyethyl-terminated tertiary aminolipidated cationic peptoid (e.g., the formula ( I) Compounds such as compound 140). In some instances, the delivery vehicle composition comprises from about 30 mol % to about 35 mol % of a hydroxyethyl-terminated tertiary aminoesterified cation, based on the total moles of components in the delivery vehicle composition Peptoids (eg, compounds of formula (I), such as compound 140). In some instances, the delivery vehicle composition comprises from about 40 mol % to about 45 mol % of a hydroxyethyl-terminated tertiary aminolipidated cation, based on the total number of moles of components in the delivery vehicle composition Peptoids (eg, compounds of formula (I), such as compound 140). In some instances, the delivery vehicle composition comprises from about 35 mol % to about 39 mol % of a hydroxyethyl-terminated tertiary aminolipidated cation, based on the total moles of components in the delivery vehicle composition Peptoids (eg, compounds of formula (I), such as compound 140). In each instance, the delivery vehicle composition comprises from about 39 mole % to about 52 mole % of the hydroxyethyl-terminated tertiary aminolipidated cation, based on the total moles of components in the delivery vehicle composition Peptoids (eg, compounds of formula (I), such as compound 140). In some implementations, the delivery vehicle composition comprises from about 42 mol% to about 49 mol% of a hydroxyethyl-terminated tertiary amino esterified compound, based on the total moles of components in the delivery vehicle composition. Cationic peptoids (eg, compounds of formula (I), such as compound 140). In various implementations, the delivery vehicle composition comprises from about 50 mol % to about 52 mol %, based on the total moles of components in the delivery vehicle composition, of a hydroxyethyl-terminated tertiary amino esterified Cationic peptoids (eg, compounds of formula (I), such as compound 140). In some cases, the delivery vehicle composition comprises about 30 mol %, about 31 mol %, about 32 mol %, about 33 mol %, about 34 mol %, based on the total number of moles of components in the delivery vehicle composition %, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol % or about 45 mol% of a hydroxyethyl-terminated tertiary amino lipidated cationic peptoid (eg, a compound of formula (I), such as compound 140). Anionic/Zwitterionic Components

In some implementations, the delivery vehicle composition further includes a component that is anionic or zwitterionic ("anionic/zwitterionic component"). The anionic/zwitterionic component can buffer the zeta potential of the particle or delivery vehicle complex formed from the delivery vehicle composition without affecting the ratio of cargo and/or facilitated by protonation in endosomes at low pH. Particle or delivery vehicle endosome escape. The zwitterionic component can provide another function of holding the particles together by interacting the hydroxyethyl-terminated tertiary amino lipidated cationic peptoid with the polyanionic cargo compound. The anionic component may also allow for the formation of a core-shell structure of the particle or delivery vehicle, where a net positive zeta potential particle is first created (e.g. by lipidation of a tertiary amine terminated with a hydroxyethyl group with a positive +/- charge ratio). cationic peptoids and cargo), which are then coated with anionic components. These negatively charged multicomponent system particles will avoid clearance by the reticuloendothelial system (RES) better than positively charged particles.

Examples of suitable anionic and zwitterionic components of delivery vehicle compositions are described in WO2020/069442 and WO2020/069445, each of which is incorporated herein by reference in its entirety. In some implementations, the zwitterionic component comprises one or more phospholipids. By virtue of their amphiphilic properties and ability to disrupt cell membranes, phospholipids can provide further stabilization of complexes in solution and facilitate endocytosis by cells.

In some implementations, the one or more phospholipids are selected from the group consisting of 1,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristyl -sn-glycero-phosphocholine (DMPC), 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- Phosphocholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Diundecyl-sn-glycero-phosphocholine (DUPC) , 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine ( 18:0 Diether PC), 1-oleyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine base (C 16 Lyso PC), 1,2-dilinoleyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-Di(docosyl)hexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- Dispalmityl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl -sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, 1 ,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diole Acyl-sn-glycerol-3-dioxaphosphoryl-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof. In some instances, the phospholipid is DSPC, DOPE, or a combination thereof. In various implementations, the phospholipid is DSPC. In each case, the phospholipid is DOPE.

In an implementation, the delivery vehicle composition comprises between about 1 mol % and about 40 mol % of a phospholipid (eg, DSPC or DOPE), based on the total moles of components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises, based on the total moles of components in the delivery vehicle composition, from about 3 mol% to about 30 mol%, or from about 5 mol% to about 15 mol%, or about 5 mol% to about 10 mol%, or about 10 mol% to about 15 mol%, or about 9 mol% to about 12 mol%, or about 7 mol% to about 11 mol%, or about 7 mol% to about 12 mol%, or about 10 mol% to about 14 mol%, or about 9 mol%, or about 12 mol% of phospholipids (such as DSPC or DOPE). In some cases, the delivery vehicle composition comprises between about 10 mol% and about 11 mol% of a phospholipid (eg, DSPC or DOPE), based on the total moles of components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises, based on the total moles of components in the delivery vehicle composition, about 10.0 mol%, about 10.1 mol%, about 10.2 mol%, about 10.3 mol%, about 10.4 mol% %, about 10.5 mol%, about 10.6 mol%, about 10.7 mol%, about 10.8 mol%, about 10.9 mol%, or about 11.0 mol% of phospholipids (eg, DSPC or DOPE). neutral lipid fraction

In some implementations, the delivery vehicle composition further includes a component that is a neutral lipid ("neutral lipid component"). The neutral lipid component can be designed to degrade or hydrolyze to facilitate in vivo clearance of the multicomponent delivery system. Contemplated neutral lipid components include, for example, naturally occurring lipids and lipidated peptoids comprising a lipid moiety at the N-position of the peptoid. Further examples of lipidated peptoids are described in WO2020/069442 and WO2020/069445, each of which is incorporated herein by reference in its entirety.

、熊果酸、α-生育酚及其混合物。在一些情況下，固醇包含膽固醇。在實現方式中，遞送媒劑組合物包含以遞送媒劑組合物中組分之總莫耳數計，約10 mol%至約80 mol%之間的固醇（例如膽固醇）。在一些情況下，遞送媒劑組合物包含以遞送媒劑組合物中組分之總莫耳數計，約20 mol%至約70 mol%、或約25 mol%至約60 mol%、或約30 mol%至約55 mol%、或約35 mol%至約50 mol%、或約25 mol%至約45 mol%、或約40 mol%至約60mol%、或約30 mol%至約40 mol%、或約45 mol%至約55mol%之間，或約35mol%、或約50 mol%之固醇（例如，膽固醇）。在一些情況下，遞送媒劑組合物包含以遞送媒劑組合物中組分之總莫耳數計，約40 mol%至約55 mol%、或約40 mol%至約45 mol%、或約50 mol%至約55 mol%之間的固醇（例如，膽固醇）。在一些情況下，遞送媒劑組合物包含以遞送媒劑組合物中組分之總莫耳數計，約40 mol%、約41 mol%、約42 mol%、約43 mol%、約44 mol%、約45 mol%、約46 mol%、約47 mol%、約48 mol%、約49 mol%、約50 mol%、約51 mol%、約52 mol%、約53 mol%、約54 mol%或約55 mol%之固醇（例如，膽固醇）。 屏蔽組分 In some cases, the neutral lipid component of the delivery vehicle composition comprises one or more sterols. In some implementations, the one or more sterols are selected from the group consisting of cholesterol, coprosterol, phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomato

, ursolic acid, alpha-tocopherol and mixtures thereof. In some instances, the sterol comprises cholesterol. In an implementation, the delivery vehicle composition comprises between about 10 mol% and about 80 mol% of a sterol (eg, cholesterol), based on the total number of moles of components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises, based on the total moles of components in the delivery vehicle composition, from about 20 mol % to about 70 mol %, or from about 25 mol % to about 60 mol %, or about 30 mol% to about 55 mol%, or about 35 mol% to about 50 mol%, or about 25 mol% to about 45 mol%, or about 40 mol% to about 60 mol%, or about 30 mol% to about 40 mol %, or about 45 mol% to about 55 mol%, or about 35 mol%, or about 50 mol% of sterols (eg, cholesterol). In some cases, the delivery vehicle composition comprises, based on the total moles of components in the delivery vehicle composition, from about 40 mol% to about 55 mol%, or from about 40 mol% to about 45 mol%, or about Between 50 mol% and about 55 mol% sterols (eg, cholesterol). In some cases, the delivery vehicle composition comprises about 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %, based on the total number of moles of components in the delivery vehicle composition %, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol % or about 55 mol% of sterols (eg, cholesterol). shielding components

In some implementations, the delivery vehicle composition further comprises a barrier component. The shielding component can increase the stability of the particle or delivery vehicle in vivo by acting as a steric barrier, thereby increasing the circulation half-life. Examples of suitable shielding components are described in WO2020/069442 and WO2020/069445, each of which is incorporated herein by reference in its entirety.

In some implementations, the shielding component comprises one or more pegylated lipids. As used herein, "PEGylated lipid" includes any lipid or lipid-like compound covalently bonded to a polyethylene glycol moiety. Suitable lipid moieties for pegylated lipids may include, for example, branched or linear aliphatic moieties which may be unsubstituted or substituted, or unidentified lipid moieties derived from natural lipid compounds including fatty acids, sterols, and isoprenoids. substituted or substituted moieties.

In some implementations, lipid moieties can include branched or straight chain aliphatic moieties having about 6 to about 50 carbon atoms, or about 10 to about 50 carbon atoms. In some implementations, an aliphatic moiety can contain one or more heteroatoms and/or one or more double or triple bonds (ie, saturated or monounsaturated or polyunsaturated). In some cases, the lipid moiety can comprise an aliphatic, linear or branched moiety, each hydrophobic tail independently having from about 8 to about 30 carbon atoms or from about 6 to about 30 carbon atoms, wherein the aliphatic moiety can be Substituted or substituted. In various implementations, lipid moieties can include, for example, aliphatic carbon chains derived from fatty acids and fatty alcohols. In some implementations, each lipid moiety is independently a C ₈ -C ₂₄ alkyl or a C ₈ -C ₂₄ alkenyl, wherein the C ₈ -C ₂₄ alkenyl may in some cases be monounsaturated or polyunsaturated Saturated.

Natural lipid moieties used in the practice of the disclosure can be derived from, for example, phospholipids, glycerides (such as di- or triglycerides), glycoside glycerides, sphingolipids, ceramides, and saturated and unsaturated sterols, isoprenoids alkenes and other similar natural lipids.

Other suitable lipid moieties may include lipophilic aromatic groups, such as optionally substituted aryl or aralkyl moieties, including, for example, naphthyl or ethylbenzyl; or lipids containing ester functional groups which Groups include, for example, sterol esters and wax esters.

In some implementations, the one or more PEGylated lipids are selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified Modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and any combination thereof. In some implementations, the PEGylated lipids comprise PEG-modified sterols. In various implementations, the PEGylated lipids comprise PEG-modified cholesterol. In some implementations, the PEGylated lipid is PEG-modified ceramide. In some cases, the PEG-modified ceramide is selected from the group consisting of N-octyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]} and N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]} and any combination thereof.

In some implementations, the PEGylated lipid is a PEG-modified phospholipid, wherein the phospholipid is selected from the group consisting of: 1,2-Dilinoleyl-sn-glycero-3-phosphocholine ( DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- Dispalmityl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecyl yl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecene yl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1- Hexadecyl-sn-glycero-3-phosphocholine (C 16 Lyso PC), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl -sn-glycero-3-phosphocholine, 1,2-di(docosyl)hexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleyl-sn-glycerol- 3-Phosphoethanolamine (DOPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl yl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bis(docosyl)hexaenoyl-sn-glycerol -3-Phosphoethanolamine, 1,2-dioleyl-sn-glycerol-3-dioxaphosphoryl-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof. In various implementations, the phospholipid is DOPE.

In some implementations, the one or more PEGylated lipids comprise PEG-modified phosphatidylethanol. In some implementations, the PEGylated lipid is PEG-modified phosphatidyl alcohol selected from the group consisting of PEG-modified DMPE (DMPE-PEG), PEG-modified DSPE (DSPE- PEG), DPPE modified by PEG (DPPE-PEG) and DOPE modified by PEG (DOPE-PEG).

In various implementations, the pegylated lipid is selected from the group consisting of dimyrisylglycerol-polyethylene glycol (DMG-PEG), distearoylglycerol-polyethylene glycol (DSG- PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), and dioleoylglycerol-polyethylene glycol (DOG-PEG). In some implementations, the PEG lipid is DMG-PEG.

The molecular weight of the PEG chains in the aforementioned PEGylated lipids can be tuned as desired to optimize the properties of the delivery vehicle composition. In some implementations, the PEG chains have between 350 and 6,000 g/mol, between 1,000 and 5,000 g/mol, or between 2,000 and 5,000 g/mol, or between about 1,000 and 3,000 g/mol, or about 1,500 Molecular weight between 4,000 g/mol. In some cases, the PEG chain of the PEG lipid has about 350 g/mol, 500 g/mol, 600 g/mol, 750 g/mol, 1,000 g/mol, 2,000 g/mol, 3,000 g/mol, 5,000 g/mol mol or molecular weight of 10,000 g/mol. In some implementations, the PEG chains of the PEGylated lipids have a molecular weight of about 500 g/mol, 750 g/mol, 1,000 g/mol, 2,000 g/mol, or 5,000 g/mol. PEG chains can be branched or linear. In some instances, the PEGylated lipid is dimyristylglycerol-polyethylene glycol 2000 (DMG-PEG 2000).

In an implementation, the delivery vehicle composition comprises between about 1 mol % and about 5 mol % of a pegylated lipid (eg, DMG-PEG 2000 ) based on the total number of moles of components in the delivery vehicle composition. ). In some cases, the delivery vehicle composition comprises, based on the total moles of components in the delivery vehicle composition, from about 1 mol % to about 3 mol %, or from about 1 mol % to about 2 mol %, or about 2 mol% to about 5 mol%, or about 0.5 mol% to about 1.5 mol%, or about 1.5 mol% to about 2.5 mol%, or about 1.5 mol% to about 2.0 mol%, or about 2.0 mol% to about 2.5 Between mol%, or about 1 mol%, or about 1.5 mol%, or about 2 mol%, or about 2.5 mol%, or about 3 mol%, or about 3.5 mol%, or about 4 mol% or about 5 mol % of PEGylated lipids (eg, DMG-PEG 2000). In some cases, the delivery vehicle composition comprises, based on the total moles of components in the delivery vehicle composition, from about 1 mol % to about 3 mol %, or from about 1 mol % to about 2 mol %, or about 2 mol% to about 5 mol%, or about 0.5 mol% to about 1.5 mol%, or about 1.5 mol% to about 2.5 mol%, or about 1 mol%, or about 1.5 mol%, or about 2 mol% , or about 2.5 mol%, or about 3 mol%, or about 3.5 mol%, or about 4 mol%, or about 5 mol% of a PEGylated lipid (eg, DMG-PEG 2000). In some cases, the delivery vehicle composition comprises about 1.5 mol %, 1.6 mol %, 1.7 mol %, 1.8 mol %, 1.9 mol %, 2.0 mol %, based on the total number of moles of components in the delivery vehicle composition %, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol% or about 2.5 mol% of PEGylated lipids (eg, DMG-PEG 2000). representative example

Non-limiting delivery vehicle combinations are described below. As previously described, the unit "mol %" or "mole percent" refers to the number of moles of a particular component of the delivery vehicle composition divided by the total number of moles of all components in the delivery vehicle composition multiplied by 100 %.

In some implementations, the delivery vehicle composition comprises at least 99 mol% cationic component and less than about 1 mol% barrier component (eg, Formulation F1A in Table 2). In some cases, the delivery vehicle composition comprises less than about 20 mol% of a cationic component, less than about 5 mol% of a barrier component, and greater than about 75 mol% of an anionic/zwitterionic component and a neutral lipid component. Mixtures (eg, Formulation F2A and Formulation F4A in Table 2). In some cases, the delivery vehicle composition comprises about 30 to about 45 mol % of a cationic component, about 50 to about 70 mol % of a mixture of an anionic/zwitterionic component and a neutral lipid component, and about 1.5 to about 4.5 mol% of the shielding component (eg, Formulation F3A and Formulation F5A in Table 2). In each case, the delivery vehicle composition comprises about 15 to about 35 mol % of a cationic component, about 60 to about 80 mol % of a mixture of anionic/zwitterionic and neutral lipid components, and about 1.5 to about 3.0 mol% of the shielding component (eg, Formulation F2A and Formulation F3A in Table 2). In some implementations, the delivery vehicle composition comprises about 15 to about 35 mol % cationic component, about 10 to about 20 mol % anionic/zwitterionic component, about 50 to about 65 mol % neutral lipid Component and about 1.5 to about 3.0 mol% of the masking component (eg, Formulation F2A and Formulation F3A in Table 2). In various implementations, the delivery vehicle composition comprises about 10 to about 20 mol% of a cationic component, about 75 to about 89 mol% of a lipid component, and about 1 to about 5 mol% of a barrier component (such as Table Formulation F4A in 2). In some cases, the delivery vehicle composition comprises about 40 to about 50 mol % of a cationic component, about 50 to about 59 mol % of an anionic/zwitterionic component, and about 1 to about 5 mol % of a barrier component (e.g. Formulation F5A in Table 2). In each case, the delivery vehicle composition comprises from about 30 to about 50 mol% of a cationic component, from about 50 to about 70 mol% of a neutral lipid component, and from about 1 to about 5 mol% of a barrier component (e.g., Table Formulation F6A in 2). In each case, the delivery vehicle composition comprises about 40 to about 45 mol % of a cationic component, about 50 to about 60 mol % of a mixture of an anionic/zwitterionic component and a neutral lipid component, and about 1.5 to about 2.0 mol% of the shielding component (eg, Formulation F6.1 and Formulation F6.2 in Table 2). In some implementations, the delivery vehicle composition comprises about 40 to about 45 mol % cationic component, about 10 to about 15 mol % anionic/zwitterionic component, about 40 to about 45 mol % neutral lipid Component and about 1.5 to about 2.0 mol% of the masking component (eg, Formulation F6.1 and Formulation F6.2 in Table 2). In each case, the delivery vehicle composition comprises about 30 to about 35 mol % of a cationic component, about 60 to about 70 mol % of a mixture of an anionic/zwitterionic component and a neutral lipid component, and about 2.0 to about 3.0 mol% of shielding component (eg, formulation F6.3 in Table 2). In some implementations, the delivery vehicle composition comprises about 30 to about 35 mol % cationic component, about 10 to about 15 mol % anionic/zwitterionic component, about 50 to about 55 mol % neutral lipid Component and about 2.0 to about 3.0 mol% of the masking component (eg, formulation F6.3 in Table 2). The cationic component can be any cationic component described herein, such as any of the compounds of formula (I) (for example, the compounds listed in Table 1, such as compounds 140, 146, 151, 152, 160, 161 and 162 ). In some implementations, the cationic compound is Compound 140. The anionic/zwitterionic component can be any anionic/zwitterionic component described herein (eg, a phospholipid). In some implementations, the anionic/zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid (eg, sterol) described herein. In some implementations, the neutral lipid component is cholesterol. The barrier component can be any barrier component described herein (eg, pegylated lipids). In some implementations, the shielding component is DMG-PEG2000.

In some implementations, the delivery vehicle composition comprises about 30 mol% to about 60 mol% (e.g., about 35 mol% to about 39 mol%, or about 39 mol% to about 52 mol%, or about 42 mol% to about 49 mol%, or about 50 mol% to about 52 mol%) of cationic components; about 3 mol% to about 20 mol% of anionic/zwitterionic components, about 25 mol% to about 60 mol% Sexual lipid compound and about 1 mol% to about 5 mol% of the shielding component. In various implementations, the delivery vehicle composition comprises about 35 to about 55 mol% cationic component; about 5 mol% to about 15 mol% anionic/zwitterionic component, about 30 mol% to about 55 mol% A neutral lipid compound and about 1 mol% to about 3 mol% of a barrier component. In various implementations, the delivery vehicle composition comprises about 38 to about 52 mol% cationic component; about 9 to about 12 mol% anionic/zwitterionic component, about 35 mol% to about 50 mol% Sexual lipid compound and about 1 mol% to about 2 mol% of the shielding component. In some instances, the delivery vehicle composition comprises from about 30 mol% to about 49 mol% of a compound of formula (I); from about 5 mol% to about 15 mol% of phospholipids; from about 30 mol% to about 55 mol% of Alcohol and about 1 mol% to about 3 mol% of pegylated lipids. In some cases, the composition comprises about 35 mol% to about 49 mol% of a compound or salt of formula (I); about 7 mol% to about 12 mol% of phospholipids, about 35 mol% to about 50 mol% of solid Alcohol and about 1 mol% to about 2 mol% of pegylated lipids. The cationic component can be any cationic component described herein, such as any of the compounds of formula (I) (for example, the compounds listed in Table 1, such as compounds 140, 146, 151, 152, 160, 161 and 162 ). In some implementations, the cationic compound is Compound 140. The anionic/zwitterionic component can be any anionic/zwitterionic component described herein (eg, a phospholipid). In some implementations, the anionic/zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid (eg, sterol) described herein. In some implementations, the neutral lipid component is cholesterol. The barrier component can be any barrier component described herein (eg, pegylated lipids). In some implementations, the shielding component is DMG-PEG2000.

In some implementations, the delivery vehicle composition comprises about 30 mol% to about 45 mol% of a cationic component; about 5 mol% to about 15 mol% of an anionic/zwitterionic component; about 40 mol% to about 60 mol% mol % neutral lipid compound and about 1 mol % to about 5 mol % of a barrier component. In various implementations, the delivery vehicle composition comprises about 35 mol% to about 40 mol% of a cationic component; about 8 mol% to about 12 mol% of an anionic/zwitterionic component; about 45 mol% to about 50 mol% mol % neutral lipid compound and about 1 mol % to about 3 mol % of a barrier component. In various implementations, the delivery vehicle composition comprises about 38.2 mol% cationic component; about 11.8 mol% anionic/zwitterionic component, about 48.2 mol% neutral lipid compound and about 1.9 mol% shielding group ("Formulation F2"). The cationic component can be any cationic component described herein, such as any of the compounds of formula (I) (for example, the compounds listed in Table 1, such as compounds 140, 146, 151, 152, 160, 161 and 162 ). In some implementations, the cationic compound is Compound 140. The anionic/zwitterionic component can be any anionic/zwitterionic component described herein (eg, a phospholipid). In some implementations, the anionic/zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid (eg, sterol) described herein. In some implementations, the neutral lipid component is cholesterol. The barrier component can be any barrier component described herein (eg, pegylated lipids). In some implementations, the shielding component is DMG-PEG-2000. In some implementations, the delivery vehicle composition comprises Formulation F2, as shown in Table 2 below. In some implementations, the delivery vehicle composition comprises about 38.2 mol% of Compound 140, about 11.8 mol% of DSPC, about 48.2 mol% of cholesterol, and about 1.9 mol% of DMG-PEG-2000 (“DV-140- F2").

In some implementations, the delivery vehicle composition comprises about 45 to about 55 mol% cationic component; about 5 mol% to about 15 mol% anionic/zwitterionic component, about 35 mol% to about 55 mol% A neutral lipid compound and about 1 mol% to about 5 mol% of a barrier component. In various implementations, the delivery vehicle composition comprises about 48 mol% to about 52 mol% of a cationic component; about 5 mol% to about 12 mol% of an anionic/zwitterionic component; about 38 mol% to about 42 mol% mol % neutral lipid compound and about 1 mol % to about 3 mol % of a barrier component. In various implementations, the delivery vehicle composition comprises about 51.3 mol% cationic component; about 9.3 mol% anionic/zwitterionic component, about 38.0 mol% neutral lipid compound, and about 1.5 mol% shielding group ("Formulation F6/17"). The cationic component can be any cationic component described herein, such as any of the compounds of formula (I) (for example, the compounds listed in Table 1, such as compounds 140, 146, 151, 152, 160, 161 and 162 ). In some implementations, the cationic compound is Compound 140. The anionic/zwitterionic component can be any anionic/zwitterionic component described herein (eg, a phospholipid). In some implementations, the anionic/zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid (eg, sterol) described herein. In some implementations, the neutral lipid component is cholesterol. The barrier component can be any barrier component described herein (eg, pegylated lipids). In some implementations, the shielding component is DMG-PEG 2000. In some implementations, the delivery vehicle composition comprises Formulation F6/17, as shown in Table 2 below. In some implementations, the delivery vehicle composition comprises about 51.3 mol% of Compound 140, about 9.3 mol% of DSPC, about 38.0 mol% of cholesterol, and about 1.5 mol% of DMG-PEG 2000 (“DV-140-F6 /17").

In some implementations, the delivery vehicle composition comprises about 30 mol% to about 49 mol% of a cationic component; about 5 mol% to about 15 mol% of an anionic/zwitterionic component; about 30 mol% to about 55 mol% mol % neutral lipid compound and about 1 mol % to about 3 mol % of a barrier component. In various implementations, the delivery vehicle composition comprises about 48 mol% to about 52 mol% of a cationic component; about 5 mol% to about 12 mol% of an anionic/zwitterionic component; about 38 mol% to about 42 mol% mol % neutral lipid compound and about 1 mol % to about 3 mol % of a barrier component. In various implementations, the delivery vehicle composition comprises about 42.6 mol % of a cationic component; about 10.0 mol % of an anionic/zwitterionic component, about 44.7 mol % of a neutral lipid compound, and about 1.7 mol % of a shielding group point. The cationic component can be any cationic component described herein, such as any of the compounds of formula (I) (for example, the compounds listed in Table 1, such as compounds 140, 146, 151, 152, 160, 161 and 162 ). In some implementations, the cationic compound is Compound 140. The anionic/zwitterionic component can be any anionic/zwitterionic component described herein (eg, a phospholipid). In some implementations, the anionic/zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid (eg, sterol) described herein. In some implementations, the neutral lipid component is cholesterol. The barrier component can be any barrier component described herein (eg, pegylated lipids). In some implementations, the shielding component is DMG-PEG 2000. In some implementations, the delivery vehicle composition comprises Formulation F6/12 or Formulation F6/15, as shown in Table 2 below. In some implementations, the delivery vehicle composition comprises about 42.6 mol% of Compound 140, about 10.9 mol% of DSPC, about 44.7 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000 (“DV-140-F6 /12"). In some implementations, the delivery vehicle composition comprises about 48.1 mol% of Compound 140, about 9.9 mol% of DSPC, about 40.4 mol% of cholesterol, and about 1.6 mol% of DMG-PEG 2000 (“DV-140-F6 /15").

In some implementations, the delivery vehicle composition comprises about 40 mol% to about 49 mol% of a cationic component; about 5 mol% to about 15 mol% of an anionic/zwitterionic component; about 30 mol% to about 55 mol% mol % neutral lipid compound and about 1 mol % to about 3 mol % of a barrier component. In various implementations, the delivery vehicle composition comprises about 42 mol% to about 46 mol% of a cationic component; about 7 mol% to about 12 mol% of an anionic/zwitterionic component; about 41 mol% to about 45 mol% mol % neutral lipid compound and about 1 mol % to about 2 mol % of a barrier component. In various implementations, the delivery vehicle composition comprises about 44.4 mol% cationic component; about 10.6 mol% anionic/zwitterionic component, about 43.3 mol% neutral lipid compound, and about 1.7 mol% shielding group point. In various implementations, the delivery vehicle composition comprises about 44.4 mol% cationic component; about 10.6 mol% anionic/zwitterionic component, about 43.4 mol% neutral lipid compound and about 1.7 mol% shielding group point. The cationic component can be any cationic component described herein, such as any of the compounds of formula (I) (for example, the compounds listed in Table 1, such as compounds 140, 146, 151, 152, 160, 161 and 162 ). In some implementations, the cationic compound is Compound 140. The anionic/zwitterionic component can be any anionic/zwitterionic component described herein (eg, a phospholipid). In some implementations, the anionic/zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid (eg, sterol) described herein. In some implementations, the neutral lipid component is cholesterol. The barrier component can be any barrier component described herein (eg, pegylated lipids). In some implementations, the shielding component is DMG-PEG 2000. In some implementations, the delivery vehicle composition comprises F6.1 or F6.2, as shown in Table 2 below. In some implementations, the delivery vehicle composition comprises about 44.4 mol% of Compound 140, about 10.6 mol% of DSPC, about 43.3 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000 (“DV-140-F6 .1"). In some implementations, the delivery vehicle composition comprises about 44.4 mol% of Compound 140, about 10.6 mol% of DSPC, about 43.4 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000 (“DV-140-F6 .2").

In some implementations, the delivery vehicle composition comprises about 30 mol% to about 39 mol% of a cationic component; about 5 mol% to about 15 mol% of an anionic/zwitterionic component; about 30 mol% to about 55 mol% mol % neutral lipid compound and about 1 mol % to about 3 mol % of a barrier component. In various implementations, the delivery vehicle composition comprises about 30 mol% to about 35 mol% of a cationic component; about 7 mol% to about 12 mol% of an anionic/zwitterionic component; about 50 mol% to about 55 mol% mol% neutral lipid compound and about 2 mol% to about 3 mol% barrier component. In various implementations, the delivery vehicle composition comprises about 33.1 mol% cationic component; about 10.5 mol% anionic/zwitterionic component, about 53.8 mol% neutral lipid compound, and about 2.5 mol% shielding group point. The cationic component can be any cationic component described herein, such as any of the compounds of formula (I) (for example, the compounds listed in Table 1, such as compounds 140, 146, 151, 152, 160, 161 and 162 ). In some implementations, the cationic compound is Compound 140. The anionic/zwitterionic component can be any anionic/zwitterionic component described herein (eg, a phospholipid). In some implementations, the anionic/zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid (eg, sterol) described herein. In some implementations, the neutral lipid component is cholesterol. The barrier component can be any barrier component described herein (eg, pegylated lipids). In some implementations, the shielding component is DMG-PEG 2000. In some implementations, the delivery vehicle composition comprises F6.3, as shown in Table 2 below. In some implementations, the delivery vehicle composition comprises about 33.1 mol% of Compound 140, about 10.5 mol% of DSPC, about 53.8 mol% of cholesterol, and about 2.5 mol% of DMG-PEG 2000 (“DV-140-F6 .1").

Non-limiting exemplary delivery vehicle compositions (characterized by mol %) of the present disclosure based on Compound 140 as the cationic component can be found in Table 2 below. Table 2. Delivery Vehicle Compositions Molecular percentage (mol%) cationic component anionic or zwitterionic components non-cationic lipid components shielding components F1A 99.1 0 0 0.9 E1 17.9 16.4 62.9 2.8 F3A 32.9 13.4 51.7 2.0 F4A 17.1 0 80.2 2.7 F5A 42.3 53.3 0 4.4 F6A 38.0 0 59.7 2.3 F1 21.4 15.7 60.3 2.7 F2 38.2 11.8 48.2 1.9 F3 36.0 10.0 51.7 2.4 F4 30.2 29.6 39.5 0.7 F5 32.0 16.7 48.7 2.6 F6/12 42.6 10.9 44.7 1.7 F6/15 48.1 9.9 40.4 1.6 F6/17 51.3 9.3 38.0 1.5 F6.1 44.4 10.6 43.3 1.7 F6.2 44.4 10.6 43.4 1.7 F6.3 33.1 10.5 53.8 2.5

In some instances, the delivery vehicle composition is F6.1, F6.2, or F6.3. In some instances, the delivery vehicle composition is F1A, F2A, F3A, F4A, F5A, F6A, F1, F2, F3, F4, F5, F6/12, F6/15, or F6/17. Delivery Vehicle Complex (DV)

The delivery vehicle compositions disclosed herein can form complexes with one or more polyanionic compounds (eg, nucleic acids) via electrostatic interactions between the cationic components of the delivery vehicle composition and the polyanionic compounds. Thus, a delivery vehicle complex refers to a mixture comprising a delivery vehicle composition as disclosed herein and a polyanionic compound. In some cases, the complexes allowed encapsulation of large quantities of cargo, were stable, and exhibited excellent in vivo efficiency and tolerability. Accordingly, the delivery vehicle complex is useful as a delivery vehicle for transporting the polyanionic cargo encapsulated therein to target cells. Additionally or alternatively, the delivery vehicle complex may include a non-anionic cargo. Accordingly, another aspect of the disclosure relates to a delivery vehicle complex comprising: (1) a delivery vehicle composition as previously described herein, and (2) a polyanionic compound (or cargo). In some implementations, the delivery vehicle composition is complexed with a polyanionic compound (eg, an RNA). In various implementations, the delivery vehicle composition is complexed with two different polyanionic compounds (eg, two different RNAs, or RNA and DNA). In some implementations, the delivery vehicle composition is complexed with three or more different polyanionic compounds (eg, 3, 4, or 5 different RNAs). In some cases, the multi-component delivery vehicle system is complexed with one or more nucleic acids selected from DNA and RNA (eg, antigenic RNA and helper DNA, such as CpG).

The delivery vehicle complexes described herein can be characterized by the relative mass ratio of one of the components of the delivery vehicle composition relative to the cargo (eg, polyanionic compound) in the complex. The mass ratios of the components in the delivery vehicle complexes can be readily calculated based on the known concentrations and volumes of stock solutions of the components used in preparing the complexes. In addition, if the non-anionic cargo is present in the delivery vehicle complex, the mass ratio can provide a more accurate comparison of the relative amounts of the delivery vehicle components with respect to the overall cargo than the cation:anionic charge ratio (which does not take into account the non-anionic material) representation. Specifically, the mass ratio of a component refers to the ratio of the mass of this particular component in the system to the mass of the "goods" in the system. "Cargo" may refer to the total polyanionic compound present in the system. In one example, a polyanionic compound can refer to a nucleic acid. In one example, a polyanionic compound refers to mRNA encoding at least one protein.

In some implementations, the mass ratio of the cationic component of the delivery vehicle complex to the polyanionic compound is between about 0.5:1 and about 20:1, between about 0.5:1 and about 10:1, between about 0.5:1 to about 5:1, about 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, Between about 2:1 and about 20:1, between about 2:1 and about 10:1 or between about 2:1 and about 5:1. In some implementations, the mass ratio of the cationic component of the delivery vehicle complex to the polyanionic compound is between about 2:1 and about 5:1. In yet other implementations, the mass ratio of the cationic component of the delivery vehicle complex to the polyanionic compound is about 3:1. In other implementations, the mass ratio of the cationic component of the delivery vehicle complex to the polyanionic compound is about 19:1. In other implementations, the mass ratio of the cationic component of the delivery vehicle complex to the polyanionic compound is about 20:1. In other implementations, the mass ratio of the cationic component of the delivery vehicle complex to the polyanionic compound is about 13:1. In other implementations, the mass ratio of the cationic component of the delivery vehicle complex to the polyanionic compound is about 10:1. In some implementations, the cationic component can be a compound of formula (I), such as a compound listed in Table 1 (eg, compound 140).

In certain implementations wherein the delivery vehicle complex comprises nucleic acid as the polyanionic compound or cargo, the mass ratio of the cationic component to the nucleic acid is between about 0.5:1 and about 20:1, or between about 0.5:1 and between about 10:1, or between about 0.5:1 and about 5:1, or between about 1:1 and about 20:1, or between about 1:1 and about 10:1, or Between about 1:1 and about 5:1, or between about 2:1 and about 20:1, or between about 2:1 and about 10:1 or between about 2:1 and about 5:1 between 1. In certain implementations, the mass ratio of cationic component to nucleic acid is between about 2:1 and about 5:1. In still other implementations, the mass ratio of cationic component to nucleic acid is about 3:1. In other implementations, the mass ratio of cationic component to nucleic acid is about 19:1. In other implementations, the mass ratio of cationic component to nucleic acid is about 20:1. In other implementations, the mass ratio of cationic component to nucleic acid is about 13:1. In other implementations, the mass ratio of cationic component to nucleic acid is about 10:1. In some implementations, the cationic component can be a compound of formula (I), such as a compound listed in Table 1 (eg, compound 140).

In some implementations, the mass ratio of cationic component to nucleic acid is from about 5:1 to about 25:1, or from about 7:1 to about 20:1, or from about 10:1 to about 17:1, or about 9.5 :1 to about 10.5:1 or about 11:1 to about 17:1. In various implementations, the mass ratio of cationic component to nucleic acid is about 20:1. In various implementations, the mass ratio of cationic component to nucleic acid is about 19:1. In some implementations, the mass ratio of cationic component to nucleic acid is about 17:1. In various implementations, the mass ratio of cationic component to nucleic acid is about 15:1. In various implementations, the mass ratio of cationic component to nucleic acid is about 13:1. In various implementations, the mass ratio of cationic component to nucleic acid is about 12:1. In various implementations, the mass ratio of cationic component to nucleic acid is about 10:1. In some implementations, the cationic component can be a compound of formula (I) as previously described herein, such as the compounds listed in Table 1. In various implementations, the cationic component is compound 140. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

In some cases, the mass ratio of anionic/zwitterionic component to polyanionic compound is from about 2:1 to about 10:1, or from about 2:1 to about 3:1, or from about 2:1 to about 4:1 , or about 5:1 to about 10:1. In some cases, the mass ratio of anionic/zwitterionic component to polyanionic compound is from about 2:1 to about 10:1, or from about 2:1 to about 3:1, or from about 5:1 to about 10:1. In some implementations, the mass ratio of anionic/zwitterionic component to polyanionic compound is about 4:1. In some implementations, the mass ratio of anionic/zwitterionic component to polyanionic compound is about 2.7:1. In some implementations, the anionic/zwitterionic component can be a phospholipid as previously described herein. In various implementations, the anionic/zwitterionic component is DOPE, DSPC, or combinations thereof. In some cases, the anionic/zwitterionic component is DSPC. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

In some cases, the mass ratio of neutral lipid component to polyanionic compound is from about 5:1 to about 8:1, or from about 4:1 to about 7:1, or from about 5:1 to about 6:1, or About 1:1 to about 5:1. In some cases, the mass ratio of neutral lipid component to polyanionic compound is between about 4:1 to about 7:1, or about 5:1 to about 6:1, or about 1:1 to about 5:1 . In some implementations, the mass ratio of neutral lipid component to polyanionic compound is about 5.4:1. In some implementations, the mass ratio of neutral lipid component to polyanionic compound is about 8.1:1. In some implementations, the mass ratio of neutral lipid component to polyanionic compound is about 6.7:1. In some implementations, the neutral lipid component can be a sterol as previously described herein. In various implementations, the neutral lipid component is cholesterol. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

In some cases, the mass ratio of shielding component to polyanionic compound is between about 0.5:1 to about 2.5:1, or about 1:1 to about 2:1, or about 2:1 to about 3:1. In some implementations, the mass ratio of neutral lipid component to polyanionic compound is about 2.1:1. In some implementations, the mass ratio of neutral lipid component to polyanionic compound is about 1.4:1. In some implementations, the shielding component can be a pegylated lipid as previously described herein. In various implementations, the shielding component is DMG-PEG 2000. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

In some implementations, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 10:1; an anionic/zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1; comprising a neutral lipid component to a polyanionic cargo in a mass ratio of 5.4:1; and a shielding component to a polyanionic cargo in a mass ratio of approximately 1.4:1 ("Formulation F2"). In some implementations, the cationic component is a compound of formula (I), the anionic/zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a pegylated lipid. In some implementations, the polyanionic compound is a nucleic acid, such as RNA. In various implementations, the delivery vehicle complex comprises Compound 140 at a mass ratio to nucleic acid of about 10:1, DSPC at a mass ratio to nucleic acid of about 2.7:1, and DSPC at a mass ratio to nucleic acid of about 5.4 :1 cholesterol and DMG-PEG 2000 ("DV-140-F2") at a mass ratio to nucleic acid of about 1.4.

In some implementations, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 17:1; an anionic/zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1; comprising neutral lipid component and polyanionic cargo in a mass ratio of 5.4:1; and shielding component and polyanionic cargo in a mass ratio of approximately 1.4:1 ("Formulation F6/17"). In some implementations, the cationic component is a compound of formula (I), the anionic/zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a pegylated lipid. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA. In various implementations, the delivery vehicle complex comprises Compound 140 at a mass ratio to nucleic acid of about 17:1, DSPC at a mass ratio to nucleic acid of about 2.7:1, and DSPC at a mass ratio to nucleic acid of about 5.4 :1 cholesterol and DMG-PEG 2000 ("DV-140-F6/17") at a mass ratio to nucleic acid of about 1.4.

In some implementations, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 12:1; an anionic/zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1; comprising neutral lipid component and polyanionic cargo in a mass ratio of 5.4:1; and shielding component and polyanionic cargo in a mass ratio of approximately 1.4:1 ("Formulation F6/12"). In some implementations, the cationic component is a compound of formula (I), the anionic/zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a pegylated lipid. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA. In various implementations, the delivery vehicle complex comprises Compound 140 at a mass ratio to nucleic acid of about 12:1, DSPC at a mass ratio to nucleic acid of about 2.7:1, and DSPC at a mass ratio to nucleic acid of about 5.4 :1 cholesterol and DMG-PEG 2000 ("DV-140-F6/12") at a mass ratio to nucleic acid of about 1.4.

In some implementations, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 15:1; an anionic/zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1; comprising neutral lipid component and polyanionic cargo in a mass ratio of 5.4:1; and shielding component and polyanionic cargo in a mass ratio of approximately 1.4:1 ("Formulation F6/15"). In some implementations, the cationic component is a compound of formula (I), the anionic/zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a pegylated lipid. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA. In various implementations, the delivery vehicle complex comprises Compound 140 at a mass ratio to nucleic acid of about 15:1, DSPC at a mass ratio to nucleic acid of about 2.7:1, and DSPC at a mass ratio to nucleic acid of about 5.4 :1 cholesterol and DMG-PEG 2000 ("DV-140-F6/15") at a mass ratio to nucleic acid of about 1.4.

In some implementations, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 13:1; an anionic/zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1; comprising neutral lipid component and polyanionic cargo in a mass ratio of 5.4:1; and shielding component and polyanionic cargo in a mass ratio of approximately 1.4:1 ("F6.1"). In some implementations, the cationic component is a compound of formula (I), the anionic/zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a pegylated lipid. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA. In various implementations, the delivery vehicle complex comprises Compound 140 at a mass ratio to nucleic acid of about 13:1, DSPC at a mass ratio to nucleic acid of about 2.7:1, and DSPC at a mass ratio to nucleic acid of about 5.4 :1 cholesterol and DMG-PEG 2000 ("DV-140-F6.1") at a mass ratio to nucleic acid of about 1.4.

In some implementations, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 19:1; an anionic/zwitterionic component and a polyanionic cargo in a mass ratio of about 4.0:1; 8. comprising a neutral lipid component and a polyanion cargo in a mass ratio of 1:1; and comprising a shielding component and a polyanion cargo in a mass ratio of approximately 2.1:1 ("F6.2"). In some implementations, the cationic component is a compound of formula (I), the anionic/zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a pegylated lipid. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA. In various implementations, the delivery vehicle complex comprises Compound 140 at a mass ratio to nucleic acid of about 19:1, DSPC at a mass ratio to nucleic acid of about 4.0:1, and DSPC at a mass ratio to nucleic acid of about 8.1 Cholesterol at :1 and DMG-PEG 2000 ("DV-140-F6.2") at a mass ratio to nucleic acid of about 2.1.

In some implementations, the delivery vehicle complex comprises a cationic component to a polyanionic cargo in a mass ratio of about 9.7; an anionic/zwitterionic component to a polyanionic cargo in a mass ratio of about 2.7:1; and in a mass ratio of about 6.7: comprising a neutral lipid component and a polyanion cargo in a mass ratio of 1; and comprising a shielding component and a polyanion cargo in a mass ratio of about 2.1:1 ("F6.3"). In some implementations, the cationic component is a compound of formula (I), the anionic/zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a pegylated lipid. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA. In various implementations, the delivery vehicle complex comprises Compound 140 at a mass ratio to nucleic acid of about 9.7:1, DSPC at a mass ratio to nucleic acid of about 2.7:1, and DSPC at a mass ratio to nucleic acid of about 6.7 Cholesterol at :1 and DMG-PEG 2000 ("DV-140-F6.3") at a mass ratio to nucleic acid of about 2.1.

In still other implementations, the amount of polyanionic cargo present in the delivery vehicle complex can be determined by the delivery vehicle composition (e.g., the sum of hydroxyethyl-terminated lipidated cationic peptoids, phospholipids, cholesterol, and/or or shielding component) to the mass ratio of one or more polyanionic cargo compounds. In some implementations, the mass ratio of the delivery vehicle composition to the one or more polyanionic cargo compounds is between about 0.5:1 and about 20:1, between about 0.5:1 and about 10:1, between about 0.5:1 to about 5:1, about 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, Between about 2:1 and about 20:1, between about 2:1 and about 10:1 or between about 2:1 and about 5:1. In certain implementations, the mass ratio of the delivery vehicle composition to the one or more polyanionic cargo compounds is between about 5:1 and about 8:1 or between about 6:1 and about 7:1.

In some implementations, the delivery vehicle complexes described herein can be characterized by the ratio of the number of cationic groups on the cationic component of the delivery vehicle composition to the number of anionic phosphate groups on the nucleic acid cargo. In some implementations, the delivery vehicle complex comprises a cationic component and nucleic acid having a cation:anion charge ratio of between about 0.5:1 and about 20:1, between about 0.5:1 and about 10:1 between about 0.5:1 and about 5:1, between about 1:1 and about 20:1, between about 1:1 and about 10:1, between about 1:1 and about 5:1 1, between about 2:1 and about 20:1, between about 2:1 and about 10:1, or between about 2:1 and about 5:1, or about 3:1 and about 8:1, or between about 3:1 and about 7:1, or between about 3:1 and about 4:1, or between about 4:1 and about 5:1, Or between about 6:1 and about 7:1 or between about 7:1 and about 8:1. In some implementations, the delivery vehicle complex comprises a cationic component and a nucleic acid having a cation:anion charge ratio of between about 0.5:1 and about 20:1, between about 0.5:1 and about 10:1 between about 0.5:1 and about 5:1, between about 1:1 and about 20:1, between about 1:1 and about 10:1, between about 1:1 and about 5:1 1, between about 2:1 and about 20:1, between about 2:1 and about 10:1, or between about 2:1 and about 5:1, or about 3:1 and about 7:1, or between about 3:1 and about 4:1, or between about 6:1 and about 7:1. In certain implementations, the delivery vehicle complex comprises a cationic component and a nucleic acid having a cation:anion charge ratio of between about 2:1 and about 5:1. In still other implementations, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation:anion charge ratio of about 3:1. In some implementations, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation:anion charge ratio of about 3.7:1. In some implementations, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation:anion charge ratio of about 6.4:1. In some implementations, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation:anion charge ratio of about 4.8:1. In some implementations, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation:anion charge ratio of about 7.2:1. In some implementations, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation:anion charge ratio of about 3.6:1. In some implementations, the cationic component is a compound of formula (I), such as those listed in Table 1. For example, the compound of formula (I) can be compound 140.

Non-limiting exemplary delivery vehicle compositions characterized by mass and charge ratios can be found in Table 3 below. Table 3. Mass ratio and charge ratio of delivery vehicle system components to polyanion cargo mass ratio cationic component anionic or zwitterionic components neutral lipid fraction shielding components charge ratio F1A 5.0 0 0 0.1 3.0 F2A 5.0 3.0 6.0 1.8 3.0 F3A 10 2.7 5.4 1.4 6.0 F4A 5.0 0 8.0 1.8 3.0 F5A 8.5 7.0 0 2.0 5.0 F6A 10 0 5.4 1.4 6.0 F1 5.0 3.0 6.0 1.8 1.9 F2 10 2.7 5.4 1.4 3.8 F3 10 2.4 6.1 1.8 3.8 F4 10 8.0 5.6 0.69 3.8 F5 10 4.3 6.5 2.3 3.8 F6/12 12 2.7 5.4 1.4 4.5 F6/15 15 2.7 5.4 1.4 5.6 F6/17 17 2.7 5.4 1.4 6.36 F6.1 13 2.7 5.4 1.4 4.8 F6.2 19 4.0 8.1 2.1 7.2 F6.3 9.7 2.7 6.7 2.1 3.6 Delivery vehicle complex characterization

The delivery vehicle complexes disclosed herein can be characterized by various parameters, such as particle size, polydispersity index, and percent cargo encapsulation. In one implementation, the delivery vehicle complex is nanoparticle-like and includes at least one polyanionic compound (described further below) encapsulated by the delivery vehicle composition. In one implementation, such complexes are mRNA nanoparticles comprising a delivery vehicle composition that encapsulates at least one mRNA.

In some implementations, the average diameter of the delivery vehicle complexes disclosed herein can be less than 300 nm, or less than 275 nm, or less than 250 nm, or less than 225 nm, or less than 200 nm, or less than 175 nm, or Less than 150 nm, or less than 125 nm, or less than 100 nm, or less than 90 nm, or less than 80 nm, or less than 70 nm, or less than 60 nm, or less than 50 nm, or less than 40 nm. In some implementations, the diameter size of the delivery vehicle complexes disclosed herein can range from about 40 nm to about 200 nm, or from about 50 nm to about 175 nm, or from about 50 nm to about 200 nm , or about 60 nm to about 150 nm, or about 60 nm to about 100 nm, or about 60 nm to about 90 nm, or about 70 nm to about 125 nm, or about 80 nm to about 100 nm, or about 70 nm to about 90 nm, or about 75 nm to about 95 nm, or about 80 nm to about 110 nm, or about 90 nm to about 125 nm, or about 70 nm to about 90 nm. In another implementation, the complex may have a diameter size greater than about 100 nm—for example, between about 105 nm and about 250 nm, between about 110 nm and about 220 nm, between about 150 nm and about 200 nm between, between about 110 nm and about 200 nm. In one implementation, the complex can have a diameter size between about 105 nm and about 200 nm. In some cases, the delivery vehicle complex exhibits a particle size of about 40 nm to about 115 nm, or about 55 nm to about 95 nm, or about 70 to about 80 nm, or about 75 nm. In each instance, the delivery vehicle complex exhibits a particle size of about 135 nm to about 225 nm, or about 155 nm to about 195 nm, or about 170 to about 180 nm, or about 175 nm. In some cases, the particle size depends on the method used to prepare the complex (eg, via a microfluidic device or manually). Particle size/diameter can be determined by dynamic light scattering (DLS) and is described in Example 4.

In some implementations, the delivery vehicle complexes of the disclosure exhibit a polydispersity index (PDI) of less than about 0.3, 0.25, 0.2, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, or 0.10 .

In some implementations, at least about 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the nucleic acid (eg, RNA) cargo is completely encapsulated in the delivery vehicle complex. The percentage of mRNA encapsulated within the delivery vehicle complex can be determined using a modified RiboGreen assay as described in Example 4.

Table 4 below provides characterization data for exemplary compositions of the disclosure. The nomenclature of the delivery vehicle complex refers to the compound of formula (I) used as the cationic component (e.g. compound 140, 146, 151, 152, 160, 161 or 162) and the formulations from Tables 2 and 3 ("Form ") name (eg, formulation F2 or formulation F6 (eg, F6/12, F6/15, F6/17)). For example, DV-140-F6/17 refers to a delivery vehicle complex comprising Compound 140 at a mass ratio to nucleic acid of about 17:1, DSPC at a mass ratio to nucleic acid of about 2.7:1 , cholesterol at a mass ratio to nucleic acid of about 5.4:1 and DMG-PEG 2000 at a mass ratio to nucleic acid of about 1.4. As shown in Table 4, Figures 1A and 1B, the delivery vehicle complexes of the present disclosure advantageously allow the formation of small monodisperse particles that allow high encapsulation of nucleic acid cargo.

MC3   

（市售） DV-112-F2   

DV-43-F2   

DV-137-F2

DV-138-F2

DV-139-F2

DV-141-F2

Table 5 below provides structural information for similar compositions wherein the cationic component is not a compound of formula (I). MC3 refers to the commercially available cationic lipid DLIN-MC3-DMA. Table 4. Delivery Vehicle Complex Characterization Complex diameter ( nm ) Polydispersity Index ( PDI ) DV-140-F2 155.9 0.189 DV-146-F2 83.2 0.114 DV-151-F2 124.2 0.167 DV-152-F2 139.4 0.259 DV-160-F2 102.7 0.161 DV-161-F2 168.3 0.176 DV-162-F2 110.6 0.130 DV-140-F6/17 123.1 0.178 DV-146-F6/17 80.3 0.106 DV-151-F6/17 109.8 0.209 DV-152-F6/17 168.5 0.211 DV-160-F6/17 104.3 0.161 DV-161-F6/17 127.4 0.184 DV-162-F6/17 100.2 0.132 DV-140-F6.1 72.8 0.262 DV-140-F6.2 69.5 0.141 DV-140-F6.3 67.1 0.133 Table 5. Comparison of delivery vehicle complexes Complex Structure of Cationic Components DV-140-F2 (compound of this disclosure)

MC3

(commercially available) DV-112-F2

DV-43-F2

DV-137-F2

DV-138-F2

DV-139-F2

DV-141-F2

The delivery vehicle complexes of the present disclosure exhibit good storage stability. For example, DV-140-F2 and DV-140-F6/17 each complexed with Fluc mRNA showed no change in particle size or encapsulation when stored at 4°C for 17 or 48 days. See Figure 2. As another example, DV-140-F2 exhibits superior stability over DV-112-F2 when stored at -20°C, 4°C, 25°C, and 37-40°C over an in vivo time point of 24 hours, and Shows better stability than DV-112-F2 when exposed to freeze/thaw cycles (3 times). See Figure 3A and Figure 3B. Thus, in some implementations, the delivery vehicle complexes of the present disclosure are stored at 4°C-10°C for at least 10 days - such as at least 20 days, 30 days, 40 days, 50 days, 60 days, 70 days or more After a number of days, at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% are retained % of polyanionic compounds. In one implementation, the complex retains the aforementioned amount of polyanionic compound at 4°C for 48 days. Furthermore, in some instances, the delivery vehicle complexes of the disclosure retain at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% of its original size. In one implementation, the delivery vehicle complex retains its aforementioned size after storage at 4°C for 48 days.

The delivery vehicle complexes disclosed herein have also been found to be well tolerated at high and low doses without systemic toxicity or adverse events. See Example 5. Other delivery vehicle complex components

The delivery vehicle complexes described herein may include other components to fine-tune the complex for a particular application. Examples of components may include components that facilitate endosome escape, including but not limited to buffer amines or polyamines; nitrogen-containing heterocyclic and/or nitrogen-containing heteroaryl groups such as imidazole, pyrrole, pyridine, pyrimidine; olefinic acid derivatives; or membrane lytic peptides.

The delivery vehicle complex may also optionally include moieties on the surface of the system. Targeting moieties can be peptides, antibody mimetics, nucleic acids (eg, aptamers), polypeptides (eg, antibodies), glycoproteins, small molecules, carbohydrates, or lipids. Non-limiting examples of targeting moieties include peptides such as somatostatin, octreotide, LHRH (an EGFR binding peptide), RGD-containing peptides; protein scaffolds such as fibrin domains, aptide or Bipodal peptides, single domain antibodies, stable scFv or bispecific T cell engagers, nucleic acids (eg aptamers), polypeptides (eg antibodies or fragments thereof), glycoproteins, small molecules, carbohydrates or lipids. Targeting moieties can be aptamers in the form of RNA or DNA or artificial nucleic acids; small molecules; carbohydrates such as mannose, galactose and arabinose; vitamins such as ascorbic acid, niacin, pantothenic acid, carnitine, inositol, pyridoxine Pyridoxal, lipoic acid, folic acid/folate, riboflavin, biotin, vitamin B12, vitamins A, E, and K; proteins that bind to cell surface receptors (such as those for thrombospondin) or Peptides; tumor necrosis factor (TNF); phospholipid-binding protein V; interferon; cytokines; transferrin; granulocyte-macrophage colony-stimulating factor (GM-CSF); VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).

The delivery vehicle complex may also optionally include small molecule drugs or other biologics incorporated into the delivery vehicle complex. Non-limiting examples include incorporation of drugs that disrupt the blood-brain barrier or enhance cellular uptake; drugs that affect intracellular migration or endosome escape; or that act as immunomodulators that affect antigen presentation when the delivery vehicle complex is used as a vaccine. drug. polyanionic compound

The delivery vehicle complexes of the present disclosure can comprise one or more polyanionic compounds (polyanionic cargoes) that can be delivered in vivo by the complex to a target, such as a cell. The polyanionic compound can complex with the cationic component of the delivery vehicle complex (eg, a compound of formula (I), such as compound 140) via electrostatic interactions.

In some implementations, the polyanionic compound comprises a nucleic acid. As used herein, nucleic acid includes naturally occurring nucleic acids such as DNA, RNA, and/or hybrids thereof, as well as non-naturally occurring nucleic acids. Non-limiting examples of unnatural amino acids are those comprising unnatural backbones, modified backbone backbones (such as phosphorothioate), unnatural or modified bases, and/or unnatural and modified termini amino acids. Exemplary nucleic acids include genomic DNA, complementary DNA (cDNA), messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), small activating RNA (saRNA), peptide nucleic acid (PNA), antisense oligonucleotides nucleotides, ribozymes, plastids, and immunostimulatory nucleic acids.

In some implementations, the polyanionic compound comprises RNA. The RNA may be selected from the group consisting of: chemically modified or unmodified RNA, single or double stranded RNA, coding or noncoding RNA, mRNA, oligoribonucleotides, viral RNA, retroviral RNA, self-replicating (replicon) RNA (srRNA), tRNA, rRNA, immunostimulatory RNA, microRNA, siRNA, small nuclear RNA (snRNA), small hairpin (sh) RNA riboswitch, RNA aptamer, RNA decoy, antisense RNA , a ribozyme, or any combination thereof. In some implementations, the nucleic acid cargo is RNA, which includes, but is not limited to, modified mRNA, self-amplifying RNA, and circular RNA. In some implementations, the RNA comprises coding RNA.

RNA is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, that is, a polymer composed of nucleotide monomers. These nucleotides are usually adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP) and cytidine monophosphate (CMP) monomers or their analogs, which are along the so-called Skeleton connection. The backbone is formed by a phosphodiester bond between the sugar (ie, ribose) of a first monomer and the phosphate moiety of a second adjacent monomer. The specific order of the monomers, that is, the order in which the bases are attached to the sugar/phosphate backbone, is called the RNA sequence. Typically, RNA can be obtained, for example, by transcribing a DNA sequence within a cell. In eukaryotic cells, transcription typically takes place within the nucleus or mitochondria. In vivo transcription of DNA usually produces so-called immature RNA (also called pre-mRNA, pre-mRNA or heterologous nuclear RNA), which has to be processed into so-called messenger RNA, often abbreviated to mRNA. For example, the processing of immature RNA in eukaryotic organisms involves a variety of different post-transcriptional modifications, such as splicing, 5'-capping, polyadenylation, export from the nucleus or mitochondria, and the like. The sum of these processes is also called the maturation of RNA. Mature messenger RNA generally provides a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Typically, a mature mRNA comprises a 5'-cap, optionally a 5'UTR, an open reading frame, an optional 3'UTR, and a poly(A) tail.

In addition to messenger RNA, there are several non-coding types of RNA that may be involved in regulation of transcription and/or translation, and immune stimulation. In this disclosure, the term "RNA" further encompasses any type of single-stranded (ssRNA) or double-stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, small Interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), ribozyme, aptamer, riboswitch, immunostimulatory/immunostimulatory RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), Small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA) and Piwi-interacting RNA (piRNA).

5'-cap structure: A 5'-cap is typically a modified nucleotide (cap analog), especially a guanine nucleotide, added to the 5' end of an mRNA molecule. In certain implementations, the 5'-cap is added using a 5'-5'-triphosphate linkage (also known as m7GpppN). Other examples of 5'-cap structures include glyceryl inverted deoxyabasic residues (parts), 4',5' methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides , 4'-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo- Pentofuranosyl Nucleotides, Acyclic 3',4'-Open Ring Nucleotides, Acyclic 3,4-Dihydroxybutyl Nucleotides, Acyclic 3,5 Dihydroxypentyl Nucleosides acid, 3'-3' reverse nucleotide part, 3'-3' reverse abasic part, 3'-2' reverse nucleotide part, 3'-2' reverse abasic part, 1,4-Butanediol Phosphate, 3'-Phosphate Aminoate, Hexyl Phosphate, Hexyl Amino Phosphate, 3'-Phosphate, 3' Phosphorothioate, Phosphorodithioate or bridged or non Bridging methyl phosphate moiety. These modified 5'-cap structures can be used in the context of the present disclosure to modify the RNA sequences of the present disclosure. Other modified 5'-cap structures that can be used in the context of the present disclosure are cap 1 (additional methylation of the ribose sugar of the adjacent nucleotide of m7GpppN), cap 2 (additional methylation of the ribose sugar of the 2nd nucleotide downstream of m7GpppN), additional methylation of ribose), cap 3 (extra methylation of ribose at the 3rd nucleotide downstream of m7GpppN), cap 4 (extra methylation of ribose at the 4th nucleotide downstream of m7GpppN), ARCA ( anti-reverse cap analogs), modified ARCA (e.g. phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine.

In the context of the present disclosure, the 5' cap structure can also be formed using cap analogs in chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping), or the cap structure can be formed using capping enzymes (e.g., commercially available capping enzymes). cap set) formed in vitro.

A cap analog refers to a non-polymerizable di-nucleotide that functions as a cap in that it facilitates translation or localization and/or prevents RNA molecule degradation when incorporated at the 5' end of the RNA molecule. Non-polymerizable means that the cap analog will only be incorporated at the 5' end, since it does not have a 5' triphosphate and thus cannot be extended in the 3' direction by a template-dependent RNA polymerase.

Cap analogs include, but are not limited to, chemical structures selected from the group consisting of: m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2, 7GpppG), trimethylated cap analogs (e.g. m2,2,7GpppG), dimethylated symmetric cap analogs (e.g. m7Gpppm7G) or anti-reverse cap analogs (e.g. ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives). The synthesis of ^N7- (4-chlorophenoxyethyl) substituted dinucleotide cap analogs has recently been described.

The poly(A) tail, also known as the "3'-poly(A) tail" or "poly(A) sequence", typically has up to about 400 adenosine nucleotides added to the 3' end of the mRNA, such as about 25 Long homopolyadenosine nucleotide sequences of to about 400, about 50 to about 400, about 50 to about 300, about 50 to about 250, or about 60 to about 250 adenosine nucleotides. In certain implementations of the disclosure, the poly(A) tail of an mRNA or srRNA is derived from a DNA template by in vitro transcription of the RNA. Alternatively, the poly(A) sequence can also be obtained in vitro by common chemical synthesis methods without transcribing from DNA protoparticles. In addition, poly(A) sequences or poly(A) tails can be generated by polyadenylation of RNases.

Stable nucleic acids typically exhibit modifications that increase resistance to degradation in vivo (e.g., by exonucleases or endonucleases) and/or ex vivo (e.g., by a manufacturing process prior to administration of the composition, e.g. during administration of the composition). Stabilization of RNA can be achieved, for example, by providing a 5'-cap structure, a poly(A) tail or any other UTR modification. Stabilization can also be achieved by backbone modification (for example using synthetic backbones such as phosphorothioates) or modifying the G/C content or C content of nucleic acids. Various other methods are known in the art and conceivable in the context of the present disclosure to stabilize or otherwise improve the function of nucleic acids. Accordingly, provided herein are polynucleotides that have been designed to improve one or more of: stability and/or clearance in tissues, receptor uptake and/or kinetics, cell entry, engagement with translational machinery, RNA Half-life, translation efficiency, immune evasion, immune induction (for vaccines), protein production capacity, secretion efficiency (where appropriate), circulatory accessibility, protein half-life and/or modulation of cell state, function and/or activity.

5'-UTR is typically understood as a specific part of RNA. It is located 5' of the open reading frame of the mRNA. In the case of srRNA, the open reading frame encodes viral nonstructural proteins, while the sequence of interest is encoded in a subgenomic segment of the viral RNA. Therefore, the 5'UTR is upstream of the nsP1 open reading frame. In addition, the subgenic somatic RNA of srRNA has a 5'UTR. Thus, a subgenomic RNA containing a sequence of interest encoding a protein of interest contains a 5' UTR. Typically, the 5'-UTR begins with the transcription start site and ends one nucleotide before the start codon of the open reading frame. The 5'-UTR may contain elements for controlling gene expression, also known as regulatory elements. Such regulatory elements may be, for example, ribosome binding sites or 5' oligopyrimidine tracts. The 5'-UTR can be modified post-transcriptionally, for example by adding a 5'-cap. In the context of the present disclosure, the 5'UTR corresponds to the sequence of a mature mRNA or srRNA located between the 5'-cap and the start codon. In one implementation, the 5'-UTR corresponds to a sequence extending from a nucleotide located 3' to a 5'-cap; and in certain implementations, extending from a nucleotide located immediately 3' to the 5'-cap, to the nucleotide located 5' to the start codon of the protein coding region; and in some cases, extending to the nucleotide located immediately 5' to the start codon of the protein coding region sub sequence. The nucleotides located immediately 3' to the 5'-cap of a mature mRNA or srRNA typically correspond to the transcription initiation site. The term "corresponding to" means that the 5'-UTR sequence may be an RNA sequence, such as in the mRNA sequence used to define the 5'-UTR sequence, or a DNA sequence corresponding to the RNA sequence. In the context of the present disclosure, the term "5'-UTR of a gene" such as "5'-UTR of the NYESO1 gene" corresponds to the mature mRNA derived from this gene (i.e. by transcribing the gene and maturing the immature mRNA The sequence of the 5'-UTR of the obtained mRNA). The term "5'-UTR of a gene" includes DNA sequence and RNA sequence of the 5'-UTR.

In general, the term "3'-UTR" refers to a portion of a nucleic acid molecule that is located 3' (ie, "downstream") of an open reading frame and that is not translated into protein. Typically, the 3'-UTR is the part of the RNA that is located between the protein-coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(A) sequence of the mRNA. In the context of the present disclosure, the term 3'-UTR may also encompass elements not encoded in the template from which the RNA is transcribed but which are added after transcription during maturation, such as poly(A) sequences. The 3'-UTR of RNA is not translated into amino acid sequence.

With regard to srRNA, the 3'-UTR sequence is generally encoded by the viral genomic RNA, which is transcribed into the corresponding mRNA during the process of gene expression. Genome sequences are first transcribed into immature mRNA. The immature mRNA is then further processed into mature mRNA during maturation. This maturation process involves 5' capping. In the context of the present disclosure, the 3'-UTR corresponds to the sequence of the mature mRNA or srRNA (and srRNA subgenomic RNA) located at the stop codon of the protein coding region, preferably immediately 3' to the protein from the sequence of interest. Between the stop codon of the coding region and the poly(A) sequence of the mRNA. The term "corresponding to" means that the 3'-UTR sequence may be an RNA sequence, such as in the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to the RNA sequence. In the context of the present disclosure, the term "3'-UTR of a gene" is the 3'-UTR corresponding to the mature mRNA derived from this gene (ie, the mRNA obtained by transcribing the gene and maturing the immature mRNA). Sequence of UTRs. The term "3'-UTR of a gene" encompasses both the DNA sequence and the RNA sequence (sense and antisense and mature and immature) of the 3'-UTR.

According to certain implementations of the present disclosure, the RNA used in the delivery vehicle complex herein comprises RNA comprising at least one region encoding a peptide (eg, polypeptide) or protein or a functional fragment of the foregoing. As used herein, "functional fragment" refers to a fragment of a peptide (eg, polypeptide) or protein that is still capable of inducing an immune response. In one implementation, the coding RNA is selected from the group consisting of mRNA, viral RNA, retroviral RNA, and self-replicating RNA. In some implementations, the RNA encodes a viral peptide (eg, a viral polypeptide), a viral protein, or a functional fragment of the foregoing. In each case, the RNA encodes a human papillomavirus (HPV) protein, a variant thereof, or a functional fragment of any of the foregoing. In some cases, the RNA encodes an HPV E6 protein (or a variant thereof), an HPV E7 protein (or a variant thereof), a combination thereof, or a functional fragment of any of the foregoing. In some instances, the HPV protein is from HPV subtypes HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and/or 68. In each case, the HPV protein is from HPV subtypes HPV 16 and/or 18. In some cases, the RNA encodes a viral spike protein or a functional fragment thereof. In some cases, the RNA encodes a SARS-associated coronavirus (e.g. severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV ), human coronavirus 229E (HCoV-229E), human coronavirus 0C43 (HCoV-0C43), human coronavirus HKU1 (HCoV-HKU1) and/or human coronavirus NL63 (HCoV-NL63)). In various implementations, the RNA encodes a SARS-CoV Spike (S) protein, a variant thereof, or a functional fragment of any of the foregoing. In some cases, the RNA encodes an influenza protein, a variant thereof, or a functional fragment of any of the foregoing. In various implementations, the RNA encodes influenza hemagglutinin (HA) or a functional fragment thereof. In some implementations, the influenza A virus has a subtype HA selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. In various implementations, the influenza subtype is HA strain H1, H2, H3 or H5. In some implementations, the RNA encodes a combination of the foregoing.

Contemplated viruses that can be encoded by the RNA of the delivery vehicle complex include, but are not limited to: influenza A and B, poliovirus, adenovirus, rabies virus, bovine parainfluenza 3, human respiratory contagious virus, bovine respiratory tract Fusion virus, canine parainfluenza virus, Newcastle disease virus, herpes simplex virus-1 and herpes simplex virus-2, human papillomavirus, hepatitis A, hepatitis B, hepatitis C and human immunodeficiency Viruses, cytomegalovirus, varicella-zoster virus, Epstein-Barr Virus, Kaposi's Sarcoma virus, human herpesvirus-6, human herpesvirus-7, Human herpesvirus-8, macaque alphaherpesvirus 1, canine herpesvirus, equine alphaherpesvirus 1, bovine alphaherpesvirus 1, human herpesvirus 2, herpes simplex virus (Virus del herpes simplex), gammaherpesviridae, avian alphaherpesvirus Herpes virus 1, Ebola virus, Marburg virus, alpha virus, flavivirus, yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile Viruses, Zika virus, Venezuelan Equine Encephalomyelitis virus, Chikungunya virus, Western Equine Encephalomyelitis Virus, Eastern Equine Encephalomyelitis Virus, Tick-Borne Encephalomyelitis Virus, Kyasanur Forest Disease virus, Alkhurma disease virus, Omsk hemorrhagic fever virus, Hendra virus, Nipah virus, Rubeola virus, Rubeola virus (Rubella virus), human parvovirus B19, pox, alphavirus, molluscum contagiosum, arenaviridae, bunyaviridae, filoviridae, flaviviridae, paramyxoviridae, Chlamydoviridae, flavivirus, Colorado tick fever virus/coltivirus, coxsackievirus, rotavirus, norovirus, astrovirus, adenovirus, adenovirus, Human metapneumoviruses, rhinoviruses or coronaviruses such as SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV NL63, HKU1, 229E and OC43 human papillomavirus, Ebola virus, Marburg virus, alpha Virus, Flavivirus, Yellow Fever, Dengue Fever, Japanese Encephalitis, West Nile Virus, Zika Virus, Venezuelan Equine Encephalomyelitis Virus, Chugong Virus, Western Equine Encephalomyelitis Virus, Oriental Equine Encephalomyelitis Virus, Tick-borne encephalitis, Kaisanu forest disease, Alkhurma disease, Omsk hemorrhagic fever, Hendra virus, Nipah virus, measles virus, rubella virus, human parvovirus B19, human herpesvirus type 6, varicella-zoster Herpes virus, cytomegalovirus, Epstein-Barr virus, Kaposi's sarcoma virus, human herpesvirus-7, human herpesvirus-8, macaque alphaherpesvirus 1, canine herpesvirus, equine alphaherpesvirus 1, bovine Alphaherpesvirus 1, human herpesvirus 2, herpes simplex virus, gammaherpesviridae, avian alphaherpesvirus 1, pox, alphavirus, molluscum contagiosum, hepatitis virus-A, hepatitis virus-B, hepatitis-C, Hepatitis-D, Hepatitis-E, Poliovirus, Arenaviridae, Bunyaviridae, Filoviridae, Flaviviruses, Paramyxoviridae or Togaviridae, Flaviviruses such as Zika virus, Colorado tick fever virus, Coxsackie virus, rotavirus, norovirus, astrovirus, adenovirus, adenovirus, influenza A virus, human metapneumovirus, rhinovirus coronavirus, varicellavirus, adenovirus Related viruses, Aichi virus, Australian bat lyssavirus, BK polyoma virus, Banna virus, Barmah forest virus, Bunyamvira virus (Bunyamwera virus), La Crosse Bunyavirus, Bunyavirus snowshoe hare, Rhesus herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus A , Subvirus, Coxsackie virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Du Wenhai Duvenhage virus, eastern equine encephalitis virus, Ebola virus, Echovirus, encephalomyocarditis virus, European bat rabies virus, type C GB virus/hepatitis G virus, Hantaan virus ), Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis D virus, Horsepox virus, Human adenovirus, Human Astrovirus, Human coronavirus, Human cytomegalovirus, human enterovirus 68, 70, human papillomavirus 1, human papillomavirus 2, human papillomavirus 16, 18, human parainfluenza, human parvovirus B19, human respiratory fusion virus, human rhinovirus, Human SARS coronavirus, human foamy virus, human T-lymphotropic virus, human torivirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyoma virus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus marburgvirus), Langat virus, Lassa virus, Lordsdale virus, jumping disease virus, lymphocytic choriomeningitis virus, Machupo virus ), Mayaro virus, MERS coronavirus, measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, molluscum contagiosum virus, Monkeypox virus, mumps virus, Murray valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus , Oropouche virus, Pichinde virus, Polio virus, Punta toro sandfly fever virus, Puumala virus, Rabies virus , East African Rift valley fever virus, Rosavirus A, Ross river virus, rotavirus A, rotavirus B, rotavirus C, rubella virus, Sagiyama virus, Salivirus A, Sandfly fever Sicilian virus, Sapporo virus, SARS coronavirus 2, Semliki forest virus, Seoul virus, Simian foamy virus, Simian virus-5, Sindbis virus, Southampton virus, St. louis encephalitis virus ), Tick-borne powassan virus, lenovirus, Toscana virus, Uukuniemi virus, vaccinia virus, varicella-zoster virus , pox virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, Western equine encephalitis virus, WU polyoma virus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, yellow fever virus, Zika virus, Bovine Herpesvirus, Pseudorabies Virus, Adenoviridae, Bovine Adenovirus BAdV-9 = Human Adenovirus C, Anoroviridae (proposed family), Letroviridae TTV, Bornaviridae, Borna Disease Virus BDV, Bunyaviridae, Aino virus, Cache valley virus CVV, Crimean Congo hemorrhagic fever CCHF, Hantaan virus HTNV, Jamestown Canyon virus JCV, LaCrosse virus LACV, Pomora virus, East African Raft Valley sheep fever virus RVFV , Caliciviridae, Norovirus, San Miguel Sea Lion Virus SMSV-5, Cycloviridae, Bovine Circovirus BCV = Evolved strain of Porcine Circovirus Type 2 PCV-2, Coronavirus family, bovine coronavirus BCoV-1, bovine toroidal virus BtoV, Flaviviridae, bovine viral diarrhea virus BVDV, Japanese encephalitis virus JEV, Kaisanu forest disease virus KFDV, sheep jumping disease virus, Mahler Valley encephalitis Viruses MVE, St. Louis encephalitis virus SLEV, tick-borne encephalitis virus TBEV, Wesselsbron virus, West Nile virus (including Kunjin), hepadnaviridae, hepatitis E virus HEV, herpes Viridae, Bovine Herpes Virus BHV-4, Equine Herpes Virus EHV-1, Infectious Bovine Rhinotracheitis Virus IBR=BHV-1, Pseudorabies Virus PRV, Orthomyxoviridae, Dhori virus, Influenza A Virus, Thogotovirus (Thogotovirus) THOV, papillomavirus, bovine papillomavirus BPV, paramyxoviridae, bovine parainfluenza virus BPIV3, bovine respiratory fusion virus BRSV, small ruminant virus PPRV, rinderpest virus RPV, Parvoviridae, bovine adeno-associated virus BAAV, bovine hokovirus BHoV, picornavirus, bovine enterovirus BEV-1, BEV-2, bovine spine virus BKV-1 U-1 strain, encephalomyocarditis virus EMC, foot-and-mouth disease virus FMDV, Seneca valley virus SVV, Polyomaviridae, Bovine Polyomavirus BPyV, Poxviridae, Aracatuba virus, Bovine papulo stomatitis virus BPSV, Cantagalou virus (Cantagalo virus), vaccinia virus, pseudo vaccinia virus PCPV, vaccinia virus, Reoviridae, Banner virus BAV, Bluetongue virus BTV, E epidemic hemorrhagic disease virus EHDV, Liaoning ( Liao Ning) virus LNV, reovirus, rotavirus, retroviridae, bovine foamy virus BFV, bovine leukemia virus BLV, rhabdoviridae, bovine transient fever virus BEFV, rabies virus, vesicular stomatitis virus VSV , Chlamyviridae, Eastern Equine Encephalitis Virus EEEV, Getah Virus, Ross River Virus RRV, Sindbis Virus, Venezuelan Equine Encephalomyelitis Virus (Venezuelan equine encephalomyelitis virus) VEE, Anthroviridae (proposed family), Leptospirosis TTV, Bunyaviridae, Crimean Congo hemorrhagic fever virus, CCHF, Hantavirus HTNV, Jamestown Canyon virus JCV, LaCrosse virus LCV, Cup Circoviridae, Norovirus, San Miguel Sea Lion Virus SMSV-5, Sapporo Virus, Circoviridae, Porcine Circoviruses PCV-1 and PCV-2, Coronaviridae, Bovine Coronavirus BCoV-1 , Severe Acute Respiratory Syndrome Virus SARS, Transmissible Gastroenteritis Virus TGEV, Filoviridae, Ebola Reston virus, Flaviviridae, Bovine Viral Diarrhea Virus BVDV, Dengue Virus, Illius Virus (Ilheus virus), Japanese encephalitis virus JEV, sheep jumping virus, Mahler Valley encephalitis virus MVE, Powassin virus, tick-borne encephalitis virus TBEV, Wessels Brown virus, West Nile virus WNV (including library Beijing), hepatitis virus family, hepatitis E virus HEV, herpes virus family, infectious bovine rhinotracheitis virus IBR= BHV-1, porcine cytomegalovirus PCMV (B. Potts personal communication), pseudorabies virus PRV, orthomyxovirus family, avian influenza virus (H5N1), swine influenza virus (H1N1, H1N2), Paramyxoviridae, bovine parainfluenza virus BPIV3, Menangle virus MENV, Nipah virus NiV, small ruminant virus PPRV, bovine parainfluenza virus Pestivirus RPV, Tioman virus (Tioman virus) TIOV, Parvoviridae, porcine hokovirus PHoV, porcine parvovirus PPV, picornaviridae, encephalomyocarditis virus EMC, foot-and-mouth disease virus FMDV, porcine enterovirus PEV-9 PEV- 10. Seneca valley virus (Seneca valley virus) SVV, porcine vesicular disease virus SVDV, reoviridae, Banner virus BAV, reovirus, rotavirus, retroviridae, porcine endogenous Retroviruses PERV, Rhabdoviridae, Rabies Virus, Vesicular Stomatitis Virus VSV, Chlamyviridae, Oriental Equine Encephalitis Virus EEEV, Getta Virus, Ross River Virus RRV, or Venezuelan Equine Encephalomyelitis VEE.

In some implementations, the RNA encodes an adenovirus, an alphavirus, a calicivirus (e.g., a calicivirus capsid antigen), a coronavirus polypeptide, a canine distemper virus, an Ebola virus polypeptide, an enterovirus, a flavivirus, a hepatitis Virus (AE), herpes virus, infectious peritonitis virus, leukemia virus, Marburg virus, orthomyxovirus, papillomavirus, parainfluenza virus, paramyxovirus, parvovirus, pestivirus, picornavirus (such as poliovirus), poxvirus (such as poxvirus), rabies virus, reovirus, retrovirus, and rotavirus. In certain implementations, the RNA encodes SARS-CoV-2, HPV (eg, E6 and/or E7 from HPV16 and/or HPV18), or influenza (eg, influenza hemagglutinin (HA)).

In some implementations, delivery of two or more specific nucleic acids together in combination may be particularly useful for therapeutic applications. For example, in some implementations, the one or more polyanionic cargo compounds include a single guide RNA (sgRNA) as a CRISPR sequence in combination with mRNA encoding Cas9. In yet other implementations, nucleic acids can also be complexed with proteins, such as CRISPR/Cas9 ribonucleoprotein complexes. In some cases, the multi-component delivery vehicle system is complexed with one or more nucleic acids selected from DNA and RNA (eg, antigenic RNA and helper DNA, such as CpG). polynucleotide synthesis

Methods for preparing polynucleotides of predetermined sequence are well known. Methods for solid phase synthesis of polyribonucleotides and polydeoxyribonucleotides are known (well known methods for the synthesis of DNA are also applicable to the synthesis of RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases may also be incorporated into polynucleotides.

Any method known in the art for preparing RNA is contemplated herein for use in preparing RNA. Illustrative methods for preparing RNA include, but are not limited to, chemical synthesis and in vitro transcription.

In certain implementations, the RNA used in the methods herein is chemically synthesized. The chemical synthesis of relatively short fragments of oligonucleotides with defined chemical structures provides a fast and inexpensive method of obtaining custom oligonucleotides of any desired sequence. While enzymes synthesize DNA and RNA only in the 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, but it most often proceeds in the opposite (ie, 3' to 5') direction. In certain implementations, the process uses the phosphoramidate approach and phosphoramidate building blocks derived from protected nucleosides (A, C, G, and U) or chemically modified nucleosides to Implemented in the form of solid-phase synthesis.

In some implementations, modifications are included in the modified nucleic acid or one or more individual nucleosides or nucleotides. For example, modifications to nucleosides may include one or more modifications to nucleobases, sugars, and/or internucleoside linkages. In some implementations having at least one modification, the polynucleotide comprises a backbone moiety comprising the following nucleobases, sugars, and internucleoside linkages: pseudouridine-α-thio-MP, 1-methyl-pseudouridine Glycoside-α-thio-MP, 1-ethyl-pseudouridine-MP, 1-propyl-pseudouridine-MP, 1-(2,2,2-trifluoroethyl)-pseudouridine- MP, 2-amino-adenine-MP, xanthosine-MP, 5-bromo-cytidine-MP, 5-aminoallyl-cytidine-MP or 2-aminopurine-nucleoside-MP.

In other implementations having at least one modification, the polynucleotide comprises a backbone moiety comprising the following nucleobases, sugars, and internucleoside linkages: pseudouridine-α-thio-MP, 1-methyl-pseudouridine Glycoside-α-thio-MP or 5-bromo-cytidine-MP. Nucleoside and nucleotide modifications contemplated for use in the present disclosure are known in the art.

To obtain the desired oligonucleotides, the building blocks are coupled sequentially in a fully automated process to the growing oligonucleotide chain on the solid phase in the order required for the sequence of the product. After chain assembly is complete, the product is released from the solid phase into solution, deprotected, and collected. The presence of side reactions sets a practical limit on the length of synthetic oligonucleotides (up to about 200 nucleotide residues), since the number of errors increases with the length of the synthesized oligonucleotides. The product is usually isolated by HPLC to obtain the desired oligonucleotide in high purity.

In certain implementations, in vitro transcription is used to prepare RNA. The term "RNA in vitro transcription" or "in vitro transcription" relates to a process in which RNA is synthesized in an episomal system (in vitro). DNA, especially plastid DNA, is used as a template for the production of RNA transcripts. RNA can be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which in some implementations is a linearized plastid DNA template. The promoter used to control transcription in vitro can be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3 and SP6 RNA polymerases. DNA templates for in vitro transcription of RNA can be obtained by cloning the nucleic acid to be transcribed in vitro, in particular cDNA corresponding to the corresponding RNA, and introducing it into a suitable vector for in vitro transcription, e.g. plastid DNA. In one implementation of the disclosure, DNA templates are linearized with suitable restriction enzymes and subsequently transcribed in vitro. cDNA can be obtained by reverse transcription of mRNA or chemical synthesis. In addition, DNA templates for in vitro RNA synthesis can also be obtained by gene synthesis.

Methods for in vitro transcription are known in the art. Reagents used in these methods typically include: 1) a linearized DNA template with a promoter sequence that has high binding affinity for its corresponding RNA polymerase, such as that of a bacteriophage; ribonucleoside triphosphate (NTP) of a base (adenine, cytosine, guanine, and uracil); 3) in some cases, a cap analog as defined above (e.g., m7G(5')ppp (5')G (m7G)); 4) a DNA-dependent RNA polymerase (such as T7, T3 or SP6 RNA polymerase) capable of binding to a promoter sequence within a linearized DNA template; 5) optional use RNase inhibitors to inactivate any contaminating ribonucleases (RNases); 6) Optional pyrophosphatase to degrade pyrophosphate, which inhibits transcription; 7) MgCl ₂ , which supplies Mg ²⁺ ions as cofactors for the polymerase; 8) buffers to maintain a suitable pH, which may also contain optimal concentrations of antioxidants (such as DTT) and/or polyamines (such as spermidine). Methods of making delivery vehicle complexes

The components of the delivery vehicle complex can be prepared by various physical and/or chemical methods to adjust their physical, chemical and biological properties. These may involve hydroxyethyl-terminated tertiary aminolipidated cationic peptoids in water or a water-miscible organic solvent with the desired polyanionic cargo compound (e.g., oligonucleotide or nucleic acid) in water. Or quick combination in buffered aqueous solution. Such methods may include simple mixing of components by pipetting or microfluidic mixing processes, such as those involving T-blenders, vortex mixers, or other hybrid mixing structures. In some implementations, the multi-component delivery system is prepared on a microfluidic platform.

It is understood that the particular processing conditions used to prepare the delivery vehicle complexes described herein can be adjusted or selected accordingly to provide the desired physical properties of the complexes. For example, parameters for mixing components of a delivery system complex that can affect the final composition can include, but are not limited to, order of mixing, temperature of mixing, speed/rate of mixing, flow rate, physical dimensions of the mixing structure, starting solution The concentration, the molar ratio of the components and the solvent used.

Formulation of delivery vehicle complexes can be accomplished in a number of ways. In some cases, all components can be premixed prior to addition of the nucleic acid cargo, which can result in an even distribution of the components throughout the delivery particle.

In other cases, the components may be added sequentially to produce a core-shell structure. For example, a cationic component can be added first to initiate particle aggregation, followed by a lipid component to associate the particle surface with target cells, followed by a shielding component to prevent particle aggregation. For example, hydroxyethyl-terminated tertiary aminolipidated cationic peptoids can be premixed with nucleic acid cargo to form the core structure. Next, lipid components, such as those comprising phospholipids and cholesterol, can be added to affect cell/endosomal membrane association. Since the barrier component is primarily suitable for use on the outside of the multi-component delivery system, this component can be introduced last so that it does not disrupt the internal structure of the system, but provides a coating for it after the system is formed.

Other components in complexes and compositions, such as additional components of polymers, surfactants, targeting moieties, and/or excipients may be present in the main components of the nucleic acid cargo, cationic components, lipid components, and shielding components. The components are blended before, during or after they are combined and combined with the rest of the components.

Accordingly, also provided herein is a method of forming a delivery vehicle complex disclosed herein comprising contacting a compound or salt of formula (I) with a polyanionic compound. In some implementations, the method comprises admixing a solution comprising a compound or salt of formula (I) with a solution comprising a polyanionic compound. Pharmaceutical formulations and modes of administration

Also provided herein are pharmaceutical compositions comprising a delivery vehicle complex of the disclosure and an effective amount of one or more pharmaceutically acceptable excipients. "Effective amount" includes "therapeutic effective amount" and "prophylactic effective amount". The term "therapeutically effective amount" refers to an amount effective to treat and/or ameliorate a disease or condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and/or substantially reducing the chance of a disease or condition in a subject. As used herein, the term "patient" and "individual" are used interchangeably and mean animals such as dogs, cats, cows, horses, and sheep (ie, non-human animals), as well as humans. A particular patient or individual is a mammal (eg, a human). The terms "patient" and "subject" include males (men) and females (women). As used herein, the term "excipient" means any pharmaceutically acceptable additive, carrier, diluent, adjuvant or other ingredient other than an active pharmaceutical ingredient (API), which is appropriately selected according to the intended form of administration And in line with conventional medical practice.

The complexes of the disclosure can be administered to an individual or patient in a therapeutically effective amount. The complexes can be administered alone or as part of a pharmaceutically acceptable composition or formulation. Additionally, the complex can be administered once, such as, for example, by bolus injection; administered multiple times; or delivered substantially uniformly over a period of time. It should also be noted that the dosage of the compound may vary over time.

Delivery vehicle complexes and other pharmaceutically active compounds disclosed herein may be by any suitable route, such as oral, rectal, parenteral (e.g., intravenous, intramuscular, or subcutaneous), intracisternal, Administration to an individual or patient intravaginally, intraperitoneally, intravesically, or as buccal, inhalation, or nasal spray. Administration can provide a systemic effect (eg, enteral or parenteral). All methods of administering pharmaceutically active agents available to those skilled in the art are contemplated.

Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol and similar alcohols), suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Microbial contamination can be prevented by the addition of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents which delay absorption, for example, aluminum monostearate and gelatin.

Pharmaceutical compositions may be in the form of sterile injectable solutions, aqueous or oleaginous suspensions. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Compositions for parenteral administration are administered in sterile media. Depending on the vehicle used and the concentration (the concentration of drug in the formulation), parenteral formulations can be either suspensions or solutions containing dissolved drug. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to parenteral compositions.

When the composition of the disclosure is used as a vaccine, it may contain one or more immunological adjuvants. As used herein, the term "immune adjuvant" refers to a compound or mixture of compounds used to accelerate, prolong, enhance or modulate an immune response when used in combination with an immunogen, such as a neoantigen. An adjuvant may be non-immunogenic when administered alone to a host, but when administered in combination with another antigen enhances the host's immune response to that other antigen. In particular, the terms "adjuvant" and "immune adjuvant" are used interchangeably in this disclosure. Adjuvant-mediated enhancement and/or duration of immune response can be assessed by any method known in the art, including (but not limited to) one or more of the following: (i) in response to the use of an adjuvant/ An increase in the number of antibodies produced by immunization with antigen combinations compared to the number of antibodies produced in response to immunization with antigens alone; (ii) an increase in the number of T cells that recognize the antigen or adjuvant; and (iii) the presence of one or more cytokines The level increases. Adjuvants may be aluminum-based adjuvants including, but not limited to, aluminum hydroxide and aluminum phosphate; saponins, such as steroidal saponins and triterpenoid saponins; bacterial flagellin, and some cytokines, such as GM-CSF. The choice of adjuvant depends on the antigen, vaccine and route of administration.

In some implementations, adjuvants improve the innate immune response to vaccine antigens by modulating innate immunity or facilitating transport and presentation. Adjuvants act directly or indirectly on antigen presenting cells (APCs) including dendritic cells (DCs). Adjuvants can be ligands for Toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, bias, breadth and scope of acquired immunity. In other instances, adjuvants can signal via pro-inflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation. This class of adjuvants includes mineral salts, oil emulsions, nanoparticles, and polyelectrolytes, and includes colloids and molecular assemblies that exhibit complex heterostructures. In one example, the composition further comprises pidotimod as an adjuvant. In another example, the composition further comprises CpG as an adjuvant.

Compounds of the disclosure can be administered to an individual or patient at dosage levels ranging from about 0.1 to about 3,000 mg/day. For the average adult human having a body weight of about 70 kg, a dosage in the range of about 0.01 to about 100 mg per kilogram of body weight is usually adequate. The particular dosage and dosage range to be used may depend on a variety of factors, including individual or patient requirements, the severity of the condition or disease being treated, and the pharmacological activity of the compound being administered. The determination of dosage ranges and optimal dosages for a particular individual or patient is within the ordinary skill of the art. Instructions

The delivery vehicle complexes disclosed herein can be used to deliver the polyanionic compound (or cargo) of the complex to cells. Accordingly, disclosed herein are methods of delivering a polyanionic compound, such as a nucleic acid (eg, RNA), to a cell comprising contacting the cell with a delivery vehicle complex or pharmaceutical composition disclosed herein. In some implementations, the cells can be contacted ex vivo. In some implementations in which the cells are contacted ex vivo, the cells are HeLa cells. In other implementations in which cells are contacted in vivo, the multi-component delivery system of the disclosure is administered to a mammalian individual. Mammalian subjects may include, but are not limited to, human or mouse subjects. In yet other implementations in which cells are contacted ex vivo, the cell line is obtained from a human or mouse individual. In some instances, the cells are tumor cells. In some instances, the cells are muscle cells.

In some implementations, one or more polyanionic cargo compounds can be delivered for therapeutic use. Non-limiting therapeutic uses include cancer, infectious diseases, autoimmune disorders, and neurological disorders. In certain implementations, a complex comprising a multi-component delivery system and a polyanionic cargo compound is used as a vaccine. Genetic vaccination or administration of nucleic acid molecules (such as RNA) and subsequent transcription and/or translation of encoded genetic information to patients is suitable for the treatment and/or prevention of inherited genetic diseases and autoimmune diseases, infectious diseases, cancerous or Tumor-related diseases and inflammatory diseases. Genetic vaccination is suitable for treating or preventing coronaviruses. Vaccines for most of these entities target the spike (S) protein of coronaviruses, a heavily glycosylated, trimeric class I fusion protein that coats the outside of the virus and is responsible for host cell entry. The S protein of SARS-CoV-2 shares high structural homology with SARS-CoV-1 and contains receptors (including S1 domain, S2 domain and receptor binding domain (RBD) ) into several subunits that are critical for entry into host cells. Therefore, the S protein and its subunits, as well as the accessible peptide sequences within these domains, are attractive vaccine antigen targets. Furthermore, genetic vaccination is particularly useful in the treatment of cancer, because cancer cells express antigens, so tumors are generally not easily recognized and eliminated by the host, as evidenced by disease progression.

Vaccines . The delivery vehicle complexes of the disclosure are also suitable for use as vaccines, wherein the polyanionic compound is RNA encoding an immunogen, antigen or neoantigen. The host's immune system provides the means for rapidly and specifically mounting a protective response against pathogenic microorganisms and promoting rejection of malignancies. Immune responses are generally described as including humoral responses, in which antibodies specific to antigens are produced by differentiating B lymphocytes, and cell-mediated responses, in which various types of T lymphocytes eliminate antigens through a variety of mechanisms. For example, CD4 (also known as CD4+) helper T cells capable of recognizing specific antigens can respond by releasing soluble mediators such as interleukins to recruit additional cells of the immune system to participate in the immune response. CD8 (also known as CD8+) cytotoxic T cells are also capable of recognizing specific antigens and can bind to and destroy or damage antigen-bearing cells or particles. In particular, cell-mediated immune responses, including cytotoxic T lymphocyte (CTL) responses, can be critical for eliminating tumor cells and cells infected by microorganisms such as viruses, bacteria or parasites. Delivery vehicle complexes of the disclosure have been found to induce an immune response when one or more of the polyanionic compounds of the complexes encodes a viral peptide (eg, a viral polypeptide), a viral protein, or a functional fragment of the foregoing. For example, a delivery vehicle complex comprising an oncogene encoding HPV E6/E7 (e.g., from HPV 16 and/or HPV 18), a SARS-CoV spike (S) protein construct, and/or influenza hemagglutinin DV-140-F2 or DV-140-F6/17 compounded with the mRNA of HA, triggers a strong humoral and cellular immune response. See , eg, Examples 8-9 and Figures 8-12.

Accordingly, the disclosure includes methods for inducing an immune response in an individual in need thereof comprising administering to the individual an effective amount of a delivery vehicle complex of the disclosure (eg, formulated as an antigenic composition). Also disclosed herein is a method of treating a viral infection in an individual in need thereof comprising administering to the individual an effective amount of a delivery vehicle complex of the disclosure. In some implementations, the administration is by intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

In various implementations, administration of a delivery vehicle complex of the present disclosure (e.g., formulated as a composition, pharmaceutical formulation, or antigenic composition) to an individual can result in an increase in The amount of antibodies produced increases the amount of antibodies (eg, neutralizing antibodies) produced in an individual against viral antigens. In some implementations, the increase is a 2-fold increase, a 5-fold increase, a 10-fold increase, a 50-fold increase, a 100-fold increase, a 200-fold increase, a 500-fold increase, a 700-fold increase, or a 1000-fold increase.

The immune response elicited by the methods of this disclosure generally includes antibody responses (preferably neutralizing antibody responses), maturation and memory of T and B cells, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody cell-mediated Phagocytosis (ADCP), complement dependent cytotoxicity (CDC) and T cell mediated responses such as CD4+, CD8+. The immune response generated by the delivery vehicle complex comprising RNA encoding a viral antigen as disclosed herein generates an immune response that recognizes and preferably ameliorates and/or neutralizes a viral infection as described herein. Methods for assessing antibody responses following administration of an antigenic composition (immunization or vaccination) are known in the art and/or described herein. In some implementations, the immune response comprises a T cell-mediated response (eg, a peptide-specific response, such as a proliferative response or a cytokine response). In some implementations, the immune response includes B cell and T cell responses. The antigen composition can be administered in various suitable ways, such as intramuscular injection, intratumoral injection, subcutaneous injection, intradermal administration, and mucosal administration, such as oral or intranasal administration. Other modes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal, intravaginal, intrarectal, and oral administration. This disclosure also contemplates combinations of different routes of administration in immunized individuals, such as simultaneous intramuscular and intranasal administration.

Cancer . Various cancers, such as cervical cancer, can be treated with polyanionic cargo compounds delivered by the delivery vehicle complexes of the disclosure. As shown in Example 8, DV-140-F2 complexed with mRNA encoding HPV E6/E7 (from HPV 16 and/or HPV 18) elicited strong cellular and humoral immune responses, illustrating the delivery vehicle complexes of the present disclosure Can treat cancer. As used herein, the term "cancer" refers to any of various malignant neoplasms characterized by the proliferation of undifferentiated cells that tend to invade surrounding tissues and metastasize to new body sites, and also refers to cancers characterized by such malignant neoplasms. Pathological condition of natural growth. Cancers may be tumors or hematological malignancies and include but are not limited to all types of lymphomas/leukaemias, carcinomas and sarcomas such as those found in: anus, bladder, bile duct, bone, brain, breast, Cervix, large intestine/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (thoracic cavity), mouth, ovary, pancreas, penis, prostate, skin, small intestine, stomach, Spinal cord, coccyx, testes, thyroid and uterus.

As a non-limiting example, treatable cancers may be acute granular leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal carcinoma, polymorphic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma), cholangiocarcinoma, bladder cancer, bone cancer, bowel cancer, brain cancer, brainstem glioma, brain tumor , breast cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse Sexual astrocytoma, ductal carcinoma in situ, endometrial carcinoma, ependymoma, epithelioid sarcoma, esophageal carcinoma, Ewing sarcoma, extrahepatic bile duct carcinoma, eye cancer, fallopian tube carcinoma, fibrosarcoma , Gallbladder Cancer, Gastric Cancer, Gastrointestinal Cancer, Gastrointestinal Carcinoid, Gastrointestinal Stromal Tumor, General, Germ Cell Tumor, Glioblastoma Multiforme, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Hemangioendothelioma, Hodgkin's lymphoma, Hodgkin's disease, Hodgkin's lymphoma, hypopharyngeal carcinoma, invasive breast ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, bowel cancer, intrahepatic cholangiocarcinoma, Invasive/invasive breast cancer, pancreatic islet cell carcinoma, carcinoma of the palate, Kaposi sarcoma, renal carcinoma, laryngeal carcinoma, leiomyosarcoma, leptomeningeal metastases, leukemia, lip carcinoma, liposarcoma, liver carcinoma, lobular carcinoma Carcinoma, low-grade astrocytoma, lung cancer, lymph node carcinoma, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal cell carcinoma Mesenchymous, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, nasal cavity cancer , nasopharyngeal carcinoma, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-small cell lung cancer, oat cell carcinoma, eye cancer , Ocular Melanoma, Oligodendroglioma, Mouth Cancer, Oral Cancer, Oropharyngeal Cancer, Osteogenic Sarcoma, Osteosarcoma, Ovarian Cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Primary Peritoneal Cancer tumor, ovarian sex cord stromal tumor, Paget's disease, pancreatic cancer, mastoid cancer, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, Pheochromocytoma, pilioastrocytoma, pineal region tumor, pinealoblastoma, pituitary gland carcinoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis carcinoma , rhabdomyosarcoma, salivary gland cancer, sarcoma, sarcoma, bone sarcoma, soft tissue sarcoma, uterine tumor, sinus cancer, skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, spinal cord cancer, spinal cancer, spinal cord cancer (Spinal cancer) cord cancer), spinal cord tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, T-cell lymphoma), testicular cancer, throat cancer, thymoma/thymus carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma Cell carcinoma, transitional cell carcinoma, triple negative breast cancer, fallopian tube carcinoma, tubular carcinoma, ureteral carcinoma, ureteral carcinoma, urethral carcinoma, uterine adenocarcinoma, uterine carcinoma, uterine sarcoma, vaginal carcinoma and vulvar carcinoma.

In some implementations, a delivery vehicle complex of the disclosure is used to treat a cancer selected from the group consisting of cervical cancer, head and neck cancer, B-cell lymphoma, T-cell lymphoma, prostate cancer, and lung cancer. In some implementations, the delivery vehicle complexes can be used to treat cervical cancer.

Infectious Diseases . In some implementations, the delivery vehicle complexes of the disclosure are used to treat infectious diseases, such as microbial infections, eg, viral, bacterial, fungal, or parasitic infections. Non-limiting examples of infectious diseases include hepatitis (such as HBV infection or HCV infection), RSV, influenza, adenovirus, rhinovirus or other viral infections.

Autoimmune Diseases . A variety of autoimmune and autoimmune-related diseases can be treated with the delivery vehicle complexes of the disclosure. As used herein, the term "autoimmune disease" refers to a disease in which the body produces antibodies that attack its own tissues. As a non-limiting example, the autoimmune disease can be acute diffuse encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, Amylosis, Adhesive Spondylitis, Anti-GBM/Anti-TBM Nephritis, Antiphospholipid Syndrome (APS), Autoimmune Angioedema, Autoimmune Aplastic Anemia, Autoimmune Autonomic Disorder, Autoimmune Autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immune deficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreas Inflammation, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy, Balo disease ), Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome**, chronic inflammatory demyelinating Polyneuropathy (CIDP), Chronic Relapsing Multifocal Osteomyelitis (CRMO), Churg-Strauss Syndrome, Cicatricial Pemphigoid/Benign Mucosal Pemphigoid, Crohn's disease, Kogan Cogans syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, primary mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis , Devick's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, nodules Erythema, experimental allergic encephalomyelitis, Ivan's syndrome, fibromyalgia**, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Cuban-Steel syndrome , granulomatosis with polyangiitis (GPA) (formerly known as Wegener's Granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosis Diseases, immunomodulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome Eaton syndrome), leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, chronic Meniere's disease ( chronic, Meniere's disease), microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann's disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy optic neuritis, neuromyelitis optica (Devic's), neutropenia, ocular scarring pemphigoid, optic neuritis, paroxysmal rheumatism, PANDAS (streptococcus-associated pediatric autoimmune Neuropsychiatric disorders), with neoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, cycloplanitis (peripheral uveal inflammation), pemphigus, peripheral neuropathy, venous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, type II and type III autoimmune polyglandular syndrome, polymyalgia rheumatica, Polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis , pyoderma gangrenosum, pure red cell aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs Syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autogenesis Body Immunity, Stiff Man Syndrome, Subacute Bacterial Endocarditis (SBE), Susac's Syndrome, Sympathetic Ophthalmia, Takayasu's Arteritis, Temporal Arteritis/Giant Cell Artery Inflammation, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular skin disease, leukoplakia and Wegener's granulomatosis (now called granulomatous polyangiitis (GPA)).

Neurological Disorders . A variety of neurological diseases can be treated with the delivery vehicle systems of the disclosure. As a non-limiting example, the neurological disorder may be loss of septum pellucidum, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute diffuse encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Gutiers ( Aicardi-Goutieres Syndrome, AIDS-Neurologic Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Muscular Dystrophy Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Hypoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cyst, Arachnoid Arnold-Chiari malformation, arteriovenous malformation, Asperger syndrome, ataxia, telangiectatic disorder, ataxia with cerebellar or spinocerebellar degeneration, atrial fibrillation and Stroke, ADHD, ASD, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, White Behcet's Disease, Bell's Palsy, benign spontaneous blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhardt-Roth Syndrome, Bell's Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injury, Brachial Plexus Injury, Bradbury-Eggleston Syndrome, Brain and Spinal Cord Tumors, Brain Aneurysm, Brain Injury, Brown-Squay Brown-Sequard Syndrome, Bulbar Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarction and Leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Causalgia , Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Syndrome, Central Spinal Cord Syndrome, Central Pain Syndrome, Central Pontine Myelin Disorders, Head Disorders, Neurons Aminase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral spongiform deformity, cerebral gigantism, cerebral hypoxia, cerebral palsy, brain-eye-facial -Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Chiari Malformation, Cholesterol Storage Disease, Chorea, Choroidal Spike Cell Syndrome, Chronic Inflammation Chronic demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, enlarged occipital horn of lateral ventricle Colpocephaly, coma, complex regional pain syndrome, congenital bilateral facial paralysis, congenital myasthenia, congenital myopathy, congenital vascular cavernous malformation, corticobasal degeneration, cranial arteritis, craniosynostosis Cree encephalitis, Creutzfeldt-Jakob Disease, cumulative trauma disorder, Cushing's Syndrome, giant cell inclusion disease, cytomegalovirus infection, dancing eyes and dancing feet syndrome , Dandy-Walker syndrome, Dawson disease, De Morsier syndrome, Dejerine-Klumpke palsy, dementia, multi-infarct dementia, semantic dementia, subcortical dementia, Lewy body dementia (Dementia With Lewy Bodies), as a non-limiting example, neurological disorders can be loss of lucid septum, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute diffuse encephalomyelitis, attention deficit hyperactivity ADHD, Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Hypoplastic Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi- Aicardi-Goutieres Syndrome, AIDS-Neurologic Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease ), Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Hypoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Spider Membranous cysts, arachnoiditis, Arnold-Chiari Malformation, arteriovenous malformations, Asperger Syndrome, ataxia, telangiectasia, ataxia, and cerebellar or spinocerebellar degeneration , atrial fibrillation and stroke, attention-deficit-hyperactivity disorder, autism spectrum disorder, autonomic dysfunction, back pain, Barth Syndrome, Batten Disease, Baker's myotonia ( Becker's Myotonia), Behcet's Disease, Bell's Palsy, benign spontaneous blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhardt- Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injury, Brachial Plexus Injury, Bradbury-Eggleston Syndrome, Bradbury-Eggleston Syndrome, Brain and Spinal Cord Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome, Bulbar Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarction and Leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome , causalgia, cavernomas, cavernous angioma, cavernous malformation, central cervical cord syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelinopathy, head Head disease, neuroamidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, spongiform deformity, gigantism, cerebral hypoxia, cerebral palsy, Cerebral-ocular-facial-skeletal syndrome (COFS), Charcot-Marie-Tooth Disease, Chiari Malformation, cholesteryl ester storage disease, chorea, choreic spike cells Polyneuropathy, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, coma, complex regional pain syndrome, congenital bilateral facial paralysis, congenital myasthenia, congenital myopathy, congenital vascular cavernous malformation, corticobasal degeneration, cranial arteritis , craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, cumulative trauma disorder, Cushing's Syndrome, giant cell inclusion body disease, cytomegalovirus infection, Dancing eye and dancing foot syndrome, Dandy-Walker syndrome, Dawson disease, De Morsier syndrome, Dejerine-Klumpke palsy, dementia, multi-infarct dementia, semantic dementia, subcortical dementia, Lewy bodies Dementia With Lewy Bodies, dentate-cerebellar ataxia, dentate atrophy, dermatomyositis, developmental coordination disorder, Devic's Syndrome, diabetic neuropathy, diffuse Sexual sclerosis, Dravet syndrome, autonomic disturbances, dysgraphia, dyslexia, dysphagia, coordination disorders, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome , encephalitis, lethargic encephalitis, encephalocele, encephalopathy, encephalopathy (familial infant), cerebral trigeminal angiomatosis, epilepsy, epileptic hemiplegia, Erb's palsy, Erb-Duchenne and Dejerine-Klumpke palsy, idiopathic Familial tremor, extrapontine myelination, Fabry's disease, Fahr's syndrome, syncope, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spasticity Sexual paralysis, Farber's disease, febrile convulsions, fibromuscular dysplasia, Fisher Syndrome, infantile flaccid syndrome, foot drop, Friedreich's Ataxia, frontotemporal Dementia, Gaucher Disease, systemic gangliosides, Gerstmann's Syndrome, Gerstmann-Straussler-Schenk disease Scheinker Disease), giant axon neuropathy, giant cell arteritis, giant cell inclusion disease, leukosphere cell dystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré Syndrome ), Hallervorden-Spatz Disease, head injury, headache, persistent hemifacial headache, hemifacial spasm, crossed hemiplegia, hereditary neuropathy, hereditary spastic paraplegia, hereditary polyneuritis Disorders, herpes zoster, otic shingles, Hirayama Syndrome, Holmes-Adie syndrome, holoprosencephaly, HTLV-1-related myelopathy, Hughes Syndrome, Huntington's Huntington's Disease, hydrocephalus, hydrocephalus, normal pressure hydrocephalus, hydrospinal hydrocephalus, hypercortisolism, lethargy, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, containment Body myositis, Incontinence pigmentosa, Infantile hypotonia, Infantile axonal degeneration, Infantile phytanic acid storage disease, Infantile Refsum disease, Infantile spasms, Inflammatory myopathy, Schizocephaly, Intestinal lipid dystrophy Syndrome, intracranial cyst, intracranial hypertension, Isaacs' Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kingsburn Syndrome ( Kinsbourne syndrome), Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Klüver-Bucy syndrome, Korsakoff's Amnesic Syndrome, Krabbe Disease, Kugelberg - Welander disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner syndrome, lateral vastus cutaneous nerve entrapment, lateral bulbar syndrome, learning disabilities, Leigh's disease Disease), Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukoencephalopathy, Levine-Critchley Syndrome, Lewy Body Dementia, Lipid Storage Disease, Lipoproteinosis, Planencephaly, Locked-in Syndrome, Gray Lou Gehrig's Disease, Lupus - Neurological Sequelae, Lyme Disease - Neurological Complication, Machado-Joseph Disease, Megalencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis Inflammation, Menkes Disease, Allodynia, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini-Stroke, Mitochondrial Myopathy, Moebius Syndrome, Single Limb Muscle Atrophy, motor neuron disease, hairy cerebrovascular disease (Moyamoya Disease), mucolipidosis, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, multiple system atrophy with Orthostatic hypotension, muscular dystrophy, congenital myasthenia, myasthenia gravis, demyelinating diffuse sclerosis, infantile myospasm encephalopathy, myoclonus, myopathy, congenital myopathy, thyrotoxic myopathy, myotonia , congenital myotonia, narcolepsy, neurogenic spike erythrocytosis, cerebral siderosis neurodegeneration, neurofibroma, antipsychotic malignant syndrome, AIDS neurological complications, Lyme disease neurological complications, giant Neurological consequences of cellular viral infection, neurological manifestations of Pompe disease, neurological sequelae of lupus, neuromyelitis optica, neuromuscular rigidity, neurotic celipoidosis, neuronal migration disorders, hereditary neuropathies, neurosarcoidosis , neurosyphilis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, Ohtahara syndrome, olivopontocerebellar atrophy, ocular myoclonus, orthostatic hypotension , Overuse Syndrome, Chronic Pain, Pantothenate Kinase-Associated Neurodegeneration, Paraneoplastic Syndrome, Paresthesias, Parkinson's Disease, Paroxysmal Chorea, Paroxysmal Migraine, Parry-Romberg Disease , Pelizaeus-Merzbacher Disease, Pena Shokeir Syndrome II, Perineural Cyst, Periodic Paralysis, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorder, Phytanic Acid Storage Disease, Pick's Disease ), nerve crush, piriformis syndrome, pituitary gland tumors, polymyositis, Pompe disease, perforating malformation, postpolio syndrome, postherpetic neuralgia, postinfectious encephalomyelitis, postural hypotension , orthostatic orthostatic tachycardia syndrome, orthostatic tachycardia syndrome, primary dentate nucleus atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacial atrophy , progressive motor ataxia, progressive multisector leukoencephalopathy, progressive sclerosing gray matter atrophy, progressive supranuclear palsy, prosopagnosia, pseudo-torch syndrome (Pseudo-Torch syndrome), pseudotoxoplasmosis syndrome (Pseudotoxoplasmosis syndrome), pseudotumor of the brain, psychogenic movement, Ramsay Hunt syndrome I, Ramsay Hunt syndrome II, Rasmussen's encephalitis, reflex sympathetic dystrophy syndrome, Refsum disease, infant Refsum disease, repetitive motion injury, Repetitive Strain Injury, Restless Leg Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome, Sacral Root Cyst, Saint Vitus Chorea, salivary gland disease, Sandhoff's disease, Schilder's disease, split-brain syndrome, Seitelberger's disease, epilepsy, semantic dementia, ventricular septal defect with optic nerve hypoplasia, infant Severe Myoclonic Epilepsy (SMEI), Shaken Baby Syndrome, Herpes Zoster, Shy-Drager Syndrome, Sjögren's Syndrome, Sleep Apnea, Sleeping Sickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Infarction, Spinal Cord Injury, Spinal cord tumors, spinal muscular atrophy, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, stiff man syndrome, striatonigral degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis , subcortical arteriosclerotic encephalopathy, transient unilateral neuralgia-like (SUNCT) headache, Swallowing Disorder, Sydenham Chorea, syncope, syphilitic myelosclerosis, Syringohydromyelia, spinal cord Cavitation syndrome, systemic lupus erythematosus, tabes dorsalis, tardive akinesia, Tarlov Cysts, Tay-Sachs Disease, temporal arteritis, tethered cord syndrome, Thomsen's myotonia , thoracic outlet syndrome, thyrotoxic myopathy, trigeminal neuralgia, Todd's palsy, Tourette Syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraplegia, Troyer Syndrome, Tuberous Sclerosis, Vascular Erectile Neoplasms, Vasculitis Syndrome of Central and Peripheral Nervous System, Von Economo's Disease, Von Hippel-Lindau's Disease (VHL), Von Recklinghausen's Disease , Wallenberg's syndrome, Werdnig-Hoffman's disease, Wernicke-Korsakoff's syndrome, Western syndrome, whiplash injury (Whiplash), Whipple's disease, Williams' syndrome, Wilson's disease, Wolman's disease, X-linked spinobulbar muscular atrophy.

In the purview of prohibiting the patenting of methods practiced on humans, the meaning of "administering" a composition to a human subject or patient should be limited to prescribing (medicating) the human subject or patient to be administered by any technique (e.g., oral, Inhalation, topical application, injection, insertion, etc.) self-administration of controlled substances. The broadest reasonable interpretation consistent with a law or regulation defining patentable subject matter is expected. To the extent that the patenting of methods of practice on humans is not prohibited, "administering" a composition includes both the method of practice on humans and the aforementioned activities. example

The following examples are provided for illustration and are not intended to limit the scope of the disclosure. Example 1 - General Synthesis of Tertiary Amino Lipidated Cationic Peptoids

General protocols for the synthesis of the tertiary aminolipidated cationic peptoids disclosed herein can be found in WO2020/069442 and WO2020/069445, each of which is incorporated herein by reference in its entirety. The following example describes a general scheme for the synthesis of tertiary aminolipidated cationic peptoids

All polymers were synthesized using bromoacetic acid and primary amines. Fmoc-Rink amide resin was used as a solid support. The Fmoc group on the resin was deprotected with 20% (v/v) piperidine-dimethylformamide (DMF). The amino resin is then aminated with bromoacetate. Amidation is followed by amination of the α-carbon by nucleophilic displacement of the bromide with a primary amine. The two steps are repeated consecutively to generate the desired cationic peptide sequence.

All reactions and washes were performed at room temperature unless otherwise stated. Resin washing refers to the addition of a washing solvent (usually DMF or dimethylsulfoxide (DMSO)) to the resin, stirring the resin so that a homogeneous slurry is obtained, followed by complete drainage of the solvent from the resin. Solvent was removed by vacuum filtration through the sintered bottom of the reaction vessel until the resin appeared dry. In all syntheses, the resin slurry was agitated by bubbling argon through the bottom of the sintering vessel.

Initial Resin Removal of Protecting Groups . Fmoc-Rink Amide Resin was charged to the sintered reaction vessel. DMF was added to the resin and the solution was stirred to swell the resin. DMF is then drained. The Fmoc group was removed by adding 20% piperidine in DMF to the resin, stirring the resin and draining the resin. 20% piperidine in DMF was added to the resin and stirred for 15 minutes and then drained. The resin was then washed six times with DMF.

Acylation / Amidation . The dehindered amide was then acylated by adding bromoacetic acid in DMF to the resin followed by N,N-diisopropylcarbodiimide (DIC) in DMF. This solution was stirred at room temperature for 30 minutes and then drained. Repeat this step a second time. The resin was then washed twice with DMF and once with DMSO. This is a completed reaction cycle.

Nucleophilic Displacement / Amination . The acylated resin is treated with the desired primary or secondary amine to effect nucleophilic displacement at the bromine leaving group on the α-carbon. This cycle of acylation/displacement is repeated until the desired peptide sequence is obtained.

Peptide cleavage from resin . Dried resin was placed in a glass scintillation vial containing a teflon-coated micro stir bar and 95% trifluoroacetic acid (TFA) in water was added. The solution was stirred for 20 minutes and then filtered through a solid phase extraction (SPE) column fitted with a polyethylene frit into a polypropylene conical centrifuge tube. The resin was washed with 1 mL of 95% TFA. The combined filtrates were then lyophilized three times from 1:1 acetonitrile:water. Lyophilized peptides were redissolved to a concentration of 5 mM in 5% acetonitrile/water.

Purification and Characterization . The redissolved crude peptide was purified by preparative HPLC. The purified peptides were characterized by LC-MS analysis. Synthesis of Example 2-Hydroxyethyl-terminated Tertiary Amino Lipidated Cationic Peptoids

Hydroxyethyl-terminated lipidated peptoids were synthesized by the submonomer method described in Example 1 with bromoacetic acid and N,N'-diisopropylcarbodiimide (DIC). Polystyrene-supported MBHA Fmoc-protected Rink amide (200 mg representative scale, 0.64 mmol/g loading, Protein Technologies) resin was used as a solid support. For bromoacetylation, the resin was combined with a 1:1 mixture of 0.8 M bromoacetic acid and 0.8 M N,N'-diisopropylcarbodiimide (DIC) for 15 minutes. Amine displacement was performed using 1 M amine in DMF for 45 minutes. After synthesis, the crude peptoid was cleaved from the resin using 5 mL of a 95:2.5:2.5 mixture of trifluoroacetic acid (TFA):water:triisopropylsilane for 40 minutes at room temperature. The resin was removed by filtration and the filtrate was concentrated using a vacuum centrifuge. Crude peptoids were further purified by reverse phase flash chromatography (Biotage Selekt) using a C4 column and a gradient of 60-95% ACN/ _H2O +0.1% TFA. Purity and identity were analyzed on a Waters Acquity UPLC Peptide BEH C4 column within a 5-95% gradient using a Waters Acquity UPLC system with an Acquity diode array UV detector and a Waters SQD2 mass spectrometer. Selected peptoids were further purified on a Waters XBridge BEH300 Prep C4 column using a gradient of 40-85% acetonitrile and 0.1% TFA in water by a preparative Waters Prep150LC system with a Waters 2489 UV/Visible detector over 30 minutes. Example 3 - Synthesis of Delivery Vehicle Complex

Synthesis . Hydroxyethyl-terminated tertiary aminolipidoids can be combined with polyanionic compounds, such as nucleic acids, to form delivery vehicle complexes that can be assessed in vitro or in vivo for therapeutic and/or prophylactic purposes. Without being bound by any particular theory, the cationic portion of the amine-lipidated peptoid binds to the negatively charged phosphodiester backbone of the polyanionic cargo (eg, nucleic acid cargo) via predominantly electrostatic interactions, forming a mixed coacervate complex. Hydrophobic interactions between lipid chains on hydroxyethyl-terminated tertiary amino lipidated peptoids can serve to stabilize particle formation and assist membrane association.

The delivery vehicle complex can be prepared by any physical and/or chemical method known in the art to adjust its physical, chemical and biological properties. These methods typically involve the rapid combination of a hydroxyethyl-terminated tertiary aminolipidopeptoid in water or a water-miscible organic solvent with the oligonucleotide in water or a buffered aqueous solution. Such methods may include simple mixing of components by pipetting or microfluidic mixing processes, such as those involving T-blenders, vortex mixers, or other hybrid mixing structures.

In the standard formulation, hydroxyethyl-terminated tertiary aminolipidopeptoids and additional lipids were dissolved in absolute ethanol at a concentration of 10 mg/mL to yield a stable solution at room temperature. In some implementations, the solution is stored at -20°C. The nucleic acid cargo is dissolved in DNase or RNase free water at a final concentration of 1-2 mg/mL. These solutions can be stored at -20°C or -78°C for longer periods of time.

To prepare the delivery vehicle compositions disclosed herein, the hydroxyethyl-terminated tertiary amino lipidated peptoid and additional lipid components are first premixed in the ethanol phase in the desired mass ratio. The nucleic acid cargo is diluted in ethanol and acidic buffer (10 mM phosphate/citrate, pH 5.0). The ethanol and aqueous phases were mixed at a 3:1 volume ratio and then immediately diluted with a 1:1 volume ratio of PBS to give a final mRNA concentration of 0.1 µg/uL. Non-limiting exemplary delivery vehicle compositions prepared by the foregoing methods include the compositions listed in Table 2 above (eg, compositions F2, F6/17, F6/12, and F6/15).

Delivery vehicle compositions with polyanionic compounds, such as those encoding, for example, firefly luciferase (Fluc), COVID-19 spike proteins, functional fragments thereof, and variants thereof, and E6/E7 oncogenes (e.g., from HPV16, RNAs of HPV18, functional fragments thereof and/or variants thereof) were combined in the ratios indicated in Table 3 to form delivery vehicle complexes to be evaluated in vitro or in vivo for therapeutic and/or prophylactic purposes. The w/w in Table 3 is the mass ratio of the indicated components to mRNA. Example 4 - Particle Characterization

The resulting delivery vehicle complexes were evaluated by dynamic light scattering (DLS) to determine volume average particle size/diameter (nm) and size polydispersity index (PDI) within the delivery vehicle complexes.

Particle Size . Particle size and size distribution were measured using a Wyatt DynaPro Disk Reader III. Typically, reconstituted samples are diluted to 2 ng/uL in 100 µL PBS. Data are reported as the cumulative fit of the correlation function to the hydrodynamic diameter in nm and the polydispersity of this measurement. Complexes with increased amounts of cationic components result in smaller particle sizes. See Figure 1A.

Percent Encapsulation . The percentage of mRNA encapsulated within delivery vehicle complexes containing Fluc mRNA was determined using a modified RiboGreen assay. In general, formulated mRNA samples were diluted to 500 ng/mL in Tris-EDTA buffer with or without Triton-X. RiboGreen (Invitrogen) was added at a 200-fold dilution and the plate was incubated for 5 minutes. Fluorescence was measured at Ex. 840nm/Em. 520nm, and encapsulated mRNA was calculated by taking the ratio of fluorescence of non-lysed particles to lysed particles. Complexes with increased amounts of cationic components resulted in higher encapsulation. See Figure 1B.

Particle stability . The delivery vehicle complexes were stored at 4°C and the resulting particle size and percent encapsulation were determined after 17 and 48 days. The complexes did not exhibit significant changes in particle size or encapsulation over the storage periods tested. See Figure 2.

DV-140-F2 and DV-112-F2 each complexed with Fluc mRNA were stored at -20°C, 4°C, 25°C, and 37-40°C, and after a 24-hour in vivo time point and freeze/thaw cycles The particle size (Fig. 3A) and polydispersity (Fig. 3B) of the composites were evaluated within (3 times, 4°C/25°C). DV-140-F2 exhibited better stability than DV-112-F2 in all experiments in this example.

In another example, complexes DV-140-F2, DV-140-F6/17, and DV-112-F2 were subjected to a storage temperature of 5°C and monitored for particle size and encapsulation over time according to the conditions in Table 6 below. . Table 6. Stability Study Parameters Complex number of days point in time Storage temperature Encapsulation ( % ) Z mean ( nm ) PDI DV-140-F2* 60 3 5°C 89.9 ± 1.6 76.8±2.2 0.12±0.03 DV-140-F6/17 ⁺ 35 4 5°C 90.4 ± 2.7 91.3 ± 1.7 0.20±0.02 DV-112-F2 ^# 175 19 5°C 96.5±0.6 55.7 ± 3.6 0.12±0.03 *Monthly monitoring, 0.22 µm filtered samples + weekly monitoring, unfiltered samples #0.22 µm filtered samples Example 5 - Toxicology Study

The complex DV-140-F2 with mRNA as polyanionic component was administered to rats by intramuscular injection at doses of 0.1 mg/kg and 0.3 mg/kg according to the parameters shown in Table 7 below. Table 7. Toxicology Study Parameters N dose mRNA (µg/rat) mRNA volume (µL/rat) Formulation Concentration (mg/ml) Rat body weight (g) mRNA dose level (µg/kg) DV dose level (µg/kg) Dose volume (mL/kg) 5 0.1 mg/kg 30 167 0.2 ~350 ~100 ~1000 ~0.5 8 0.3 mg/kg 100 167 0.6 ~350 ~300 ~3000 ~0.5

Histopathology, macroscopic organ assessment, necropsy, hematology, clinical chemistry, cytokine analysis, body weight, clinical observations in the Spergdoori rat model were assessed using methods well known in the art for DV-140-F2 and food intake effects (data not shown). The results are shown in Table 8 below. Table 8. Toxicological Evaluation Evaluate Therapeutic dose in mice Scaled therapeutic dose for rats high dose in rats plan 3 doses, 7 days apart, approximately 1-2 µg RNA 4 doses, 30 µg RNA over 2 weeks 4 doses, 30 µg RNA over 2 weeks vote IM or IT IM IM weight no change from control no change from control Reduced weight gain in some animals Injection site reactions or other clinical observations not observed not observed not observed Gross Organ Pathology not assessed not observed not observed Hematological parameters not assessed no change from control Some transient changes in serum chemistries compared to controls, most of which remain within normal ranges clinical chemistry not evaluated Serum chemistries did not change meaningfully compared to controls; most remained within normal range Serum chemistries did not change meaningfully compared to controls; most remained within normal range serum cytokines not evaluated no change from control Levels of a subset of cytokines increased but returned to baseline by day 30

DV-140-F2 complexed with Fluc mRNA was well tolerated at both high and low doses without systemic toxicity or adverse events. Example 6 - Delivery Vehicle Complex Efficacy - In Vitro Firefly Luciferase Performance

The efficacy of complexes comprising the delivery vehicle compositions disclosed herein and mRNA encoding firefly luciferase (Fluc mRNA) is based on their ability to deliver the firefly luciferase (Fluc) reporter gene to cultured cells in vivo external assessment. In a representative experiment, the delivery vehicle composition was combined with Fluc mRNA, and the resulting particles were added to cultured HEK-293 cells at a dose of 50 ng/well (150 µL total volume). The resulting luciferase expression was measured by luminescent disc reader after 6 and 18 hours of treatment. Delivery vehicles of the present disclosure (eg, DV-140-F2) exhibit high luciferase expression. Example 7 - In vivo luciferase expression

General Methods . Prior to all studies, animals (eg mice and/or rats) were acclimatized for a minimum of 3 days prior to use. Animals were maintained in a temperature and humidity controlled room on a 12 hour light cycle. Conduct daily health checks and food and water checks.

Subcutaneous (50-200 µL), intraperitoneal (up to 1000 µL), intravenous (50-200 µL), intramuscular (50-µL), or intratumoral (50-µL) using a 26-30 gauge needle depending on the injection site -µL) injection. Animals were anesthetized with isoflurane for all injections/implantations.

For imaging, animals were mounted under anesthesia, injected intraperitoneally with D-luciferin (15 mg/mL) at a dose of 10 µL per gram of body weight, and placed in a camera chamber and imaged for up to 30 minutes. Imaging was performed 15 min after substrate injection.

Intravenous Administration . The delivery vehicle complexes disclosed herein were effective for in vivo administration of Fluc mRNA to Balb/c mice via a variety of routes of administration. In general, the delivery system complexes were administered via tail vein injection at a dose of 0.5 mg/kg, and the resulting bioluminescence was quantified after 6 hours. Organ-specific bioluminescence was quantified by sacrificing the treated animals, dissecting the relevant organs and quantifying the resulting bioluminescence individually.

Local Administration . In addition to IV administration, the delivery vehicle complexes described herein are also effective for local administration of mRNA via intratumoral (IT), subcutaneous (SC) or intramuscular (IM) routes of administration. For these examples, mRNA was administered at a dose of 0.1 mpk for intratumoral administration, or 0.01 mpk for subcutaneous and intramuscular administration, and the resulting bioluminescence was quantified 6 hours later.

The delivery vehicle complexes of the disclosure showed high luciferase expression when administered via intramuscular injection (see Figures 4, 5, and 6) and via intratumoral injection (see Figure 7). Example 8 - RNA-based vaccines for cervical cancer

The delivery vehicle complexes of the present disclosure were evaluated for their efficacy as RNA-based vaccines for cancer.

Cellular responses . The vaccines described herein were assessed by formulating mRNA encoding ovalbumin (OVA) or the HPV E6/E7 oncogene (from HPV16 and/or HPV18) with representative peptoids and administering this vaccine to C57B1/6 mice Efficacy of the delivery vehicle complexes in disease models. Vaccine candidates are administered twice, with a prime dose on Day 0 and a booster dose on Day 7 at the end of the study. Resulting immune responses to epitopes of defined characteristics were determined on day 14 by measuring antigen-specific CD8+ T cell levels in peripheral blood and spleen with fluorescent MHC-I tetramer conjugates (MBL International) . The DV-140-F2 complex elicited a stronger cellular response than the other complexes tested. See Figure 8A.

Humoral responses . Humoral responses to candidate vaccines were assessed by E7-IgG ELISA. Briefly, MaxiSorp ELISA plates (Thermo Scientific) were coated with 1 ug/mL E7-His protein (Abcam) overnight at 4C. Plates were then washed and blocked with 10% FBS. Plasma samples were plated at a 1:5 dilution with five 10-fold dilutions in blocking buffer (10% FCS). Samples were added to plates and incubated overnight at 4C. Detection was with donkey anti-mouse IgG-HRP (Jackson Immunology) at 1:1000 in blocking buffer for 1 hour, followed by detection with HRP substrate and reading at 450 nm. The DV-140-F2 complex elicited a stronger IgGr response than the other complexes tested. See Figure 8B. Example 9 - RNA-based vaccines for COVID-19

DV-140-F2 was complexed with RNA encoding the COVID-19 spike protein (SARS-CoV Spike (S) protein), and the immune response was assessed. DV-140-F2, a construct comprising the full-length Spike protein, exhibited stable humoral and cellular responses to the Spike protein. Furthermore, spike protein-specific antibodies were detected in serum and lung, and spike protein-specific CD4 and CD8 T cells produced IFNγ, TNFα and IL2. The range of humoral and cellular responses induced by the DV-140-F2 complex was similar to that shown in mice treated with mRNA-1273. Furthermore, the combination of spike protein and influenza hemagglutinin (HA) RNA induces responses against influenza HA and spike protein

Induction of spike protein-specific neutralizing antibodies . Balb/c mice (n=8) were immunized intramuscularly with 1 µg of different full-length spike protein constructs on day 0 and day 21—construct No. 1, COVID RNA of Construct No. 2 and Construct No. 3 (which included some mutations from South African and British variants), reference full-length Spike protein or scrambled complexed DV-140-F2. Recombinant spike protein (10 µg) in oil-in-water squalene emulsion (Addavax, Invivogen) was also included as a control. Serum and bronchoalveolar lavage fluid were collected on day 50. Spike protein-specific antibodies in serum ( FIG. 9A ) and lung ( FIG. 9B ) were assessed by ELISA using the spike protein receptor binding domain (RBD) region and secondary anti-mouse IgG for coating. Absorbance was measured at OD450. The endpoint titers of sera were calculated using background mean + 5x std of background wells as cutoff. Neutralizing antibody levels in sera were assessed at a 1:100 dilution using the SASRS-CoV2 Surrogate Virus Neutralization Test from GenScript according to the instructions (Fig. 9C). Complexes containing DV-140-F2 and COVID RNA induce spike protein-specific neutralizing antibodies. Antibodies specific to the spike protein were detectable in serum and lung.

Induction of spike protein-specific cellular immune responses . Balb/c mice (n=8) were immunized intramuscularly with 1 µg of different full-length spike protein constructs on day 0 and day 21—construct No. 1, COVID RNA of Construct No. 2 and Construct No. 3 (which included some mutations from South African and British variants), reference full-length Spike protein, or scrambled complexed DV-140-F2. Recombinant spike protein (10 µg) in oil-in-water squalene emulsion (Addavax, Invivogen) was also included as a control. Spleens were harvested on day 50 and processed into single cell suspensions. Spike-specific immune responses in splenocytes were assessed by IFNg ELISpot ( FIG. 10A ) and intracellular interleukin staining for IFNγ, TNFα, and IL2 ( FIG. 10B ) using overlapping peptide libraries of the full-length Spike protein. See also Fig. 10C. Complexes comprising DV-140-F2 and COVID RNA induce spike protein-specific T cell responses.

Comparison of spike protein-specific titers induced by mRNA-1273 and vaccines of the present disclosure . Balb/c mice (n=32) were immunized intramuscularly with 1 µg of mRNA-1273 and full-length spike on days 0 and 21 Protein Constructs - RNA of Construct No. 1, Construct No. 2, and Construct No. 3 (which included some mutations from South African and British variants), or DV-140-F2 complexed with scrambled RNA control. Serum was collected on day 50. Spike protein-specific antibodies in serum were assessed by ELISA using the spike protein receptor binding domain (RBD) region and secondary anti-mouse IgG for coating. The endpoint titers of sera were calculated using background mean + 5x std of background wells as cutoff. Shows mean +/- geometric standard deviation. See Figure 11A. Complexes comprising DV-140-F2 and COVID RNA induced spike protein-specific antibody levels in mice that were within the range of responses reported in mice using mRNA-1273 with a similar protocol. See Figure 1 IB, which is a diagram from Corbett et al., Nature 586 567-571 (2020), where spike protein-specific antibodies were induced in Balb/c mice after 1 or 2 doses of mRNA-1273.

Influenza combination vaccine . Balb/c mice (n=8) were immunized intramuscularly on days 0 and 21 with 1 µg of DV complexed with full-length spike protein RNA, influenza hemagglutinin (HA) or scrambled -140-F2. Serum ( FIG. 12A ) and spleen ( FIG. 12B ) were collected on day 50. Humoral immune response: Spike protein-specific (blue) and influenza HA-specific (green) antibodies in serum were assessed by ELISA. The endpoint titers of sera were calculated using background mean + 5x std of background wells as cutoff. See Figure 12A. Cellular immune responses: Immune responses in splenocytes were assessed by intracellular interleukin staining using overlapping peptide libraries of full-length Spike protein (blue), influenza HA protein (green), or medium-only (unstimulated, yellow). See Figure 12B. Vaccines targeting influenza HA of the disclosure induce influenza HA-specific humoral and cellular immune responses. Combination vaccines targeting both the Spike protein and influenza HA induced similar Spike protein and influenza HA-specific immune responses as vaccines targeting either the Spike protein or influenza HA alone. Example 10 - Comparison of Delivery Vehicles in RNA-Based Vaccines for COVID-19

A variety of delivery vehicles (MC3, commercial vehicles SM-102 and ALC-0315, DV-140-F6.1, DV-140-F6.3 and PBS control) and encoding COVID-19 spike protein (SARS-CoV spike Spike (S) protein) was complexed with RNA and the immune response was assessed. DV-140-F6.1 and DV-140-F6.3, constructs comprising the full-length Spike protein, exhibited stable humoral and cellular responses to the Spike protein. The range of humoral and cellular responses induced by the DV-140-F6.3 complex was similar to that shown in mice treated with commercial Moderna and Pfizer vehicles.

Induction of spike protein-specific neutralizing antibodies . Balb/c mice (n=8) were immunized intramuscularly with 1 µg of different full-length spike protein constructs on day 0 and day 21—construct No. 1, Construct No. 2 and Construct No. 3 COVID RNA (which includes some mutations from South African and UK variants), reference full-length Spike protein or delivery vehicle for scrambling complex (MC3, commercially available from Moderna and Pfizer agents, DV-140-F6.1 and DV-140-F6.3). Recombinant Spike protein (10 µg) in phosphate buffered saline (PBS) was also included as a control. Serum and bronchoalveolar lavage fluid were collected on day 35. Spike protein-specific antibodies in sera (Figure 13A) were assessed by ELISA using the spike protein receptor binding domain (RBD) region and secondary anti-mouse IgG for coating. Absorbance was measured at OD450. The endpoint titers of sera were calculated using background mean + 5x std of background wells as cutoff. Neutralizing antibody levels in serum were assessed at a 1:100 dilution using the SARS-CoV2 Surrogate Virus Neutralization Test from GenScript according to the instructions. Complexes comprising DV-140-F6.1 and COVID RNA and DV-140-F6.3 and COVID RNA induce spike protein-specific neutralizing antibodies. Antibodies specific to the spike protein were detectable in serum and lung.

Induction of spike protein-specific cellular immune responses . Balb/c mice (n=8) were immunized intramuscularly with 1 µg of different full-length spike protein constructs on day 0 and day 21—construct No. 1, Construct No. 2 and Construct No. 3 COVID RNA (which includes some mutations from South African and UK variants), reference full-length Spike protein or delivery vehicle for scrambling complex (MC3, commercially available from Moderna and Pfizer agents, DV-140-F6.1 and DV-140-F6.3). Recombinant Spike protein (10 µg) in phosphate buffered saline (PBS) was also included as a control. Spleens were harvested on day 50 and processed into single cell suspensions. Spike-specific immune responses in splenocytes were assessed by IFNg ELISpot using an overlapping peptide library of the full-length Spike protein ( FIG. 13B ). Complexes containing DV-140-F6.1 and COVID RNA and DV-140-F6.3 and COVID RNA induce spike protein-specific T cell responses, and complexes containing DV-140-F6.3 and COVID RNA The induced response was higher than MC3 and comparable to commercial Moderna and Pfizer vehicles. Example 10 - Efficacy and Toxicology Studies

Compound 140 was found to be well tolerated in mouse efficacy and rat toxicology studies. Briefly, Sprague-Dawley rats (n=8) were treated with control mRNA formulated in DV-140-F2 (0.03 or 0.3 mg/kg) or PBS vehicle. Injections were performed 4 times over a 13-day period and were administered intramuscularly into the hindlimb. Whole blood was drawn for hematology and serum for clinical chemistry and cytokine analysis at 6 hours after the first and final dose and 2 weeks after the final dose. In addition, 2 animals were sacrificed 6 hours after the final dose and 2 weeks after the final dose, and gross necropsy was performed. Tissue samples were retained and histopathological procedures were performed on selected organs. Table 9: Efficacy and Toxicology Studies Evaluate Therapeutic dose in mice Scaled therapeutic dose for rats high dose in rats plan 3 doses, 7 days apart; approximately 1-2 µg RNA, IT or IM 4 doses over 2 weeks; 30 µg RNA, IM 4 doses over 2 weeks; 100 µg RNA, IM weight no change from control no change from control Reduced weight gain in some animals Injection site reactions or other clinical observations not observed not observed not observed Gross Organ Pathology not assessed not observed not observed Hematological parameters not assessed no change from control Some transient changes in hematology; return to baseline by D30 clinical chemistry not assessed Serum chemistries did not change meaningfully compared to controls; most remained within normal range Serum chemistries did not change meaningfully compared to controls; most remained within normal range serum cytokines not evaluated no change from control Levels of a subset of cytokines increased; returned to baseline by D30

It is to be appreciated that all combinations of the foregoing concepts and implementations, as well as additional concepts and implementations discussed in greater detail below, are contemplated as part of the subject matter of the disclosure disclosed herein and may be employed in any suitable combination to achieve a goal as described herein. benefit. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are intended to be a part of the subject matter of this disclosure disclosed herein. The foregoing description has been given for clarity of understanding only, and no unnecessary limitations should be read therefrom, as modifications within the scope of the present disclosure may become apparent to those of ordinary skill in the art.

The terms "substantially" and "about" are used throughout this specification for purposes of description and to allow for minor fluctuations. For example, such minor fluctuations may mean less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, Such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

Throughout this specification and subsequent claims, unless otherwise specified herein, the word "comprise" and variations (such as "comprises/comprising") should be read to imply inclusion of one of the integer or step or set of integers or steps, but does not exclude any other integer or step or any other set of integers or steps.

Throughout this specification, unless otherwise stated, when a composition is described as comprising components or materials, it is contemplated that the composition may also consist essentially of or consist of any combination of the listed components or materials . Likewise, when methods are described as comprising specific steps, it is contemplated that such methods may also consist essentially of or consist of any combination of the recited steps, unless otherwise described. The present disclosure, illustratively disclosed herein, may be suitably practiced in the absence of any element or step not specifically disclosed herein.

The methods disclosed herein and practice of the individual steps thereof can be performed manually and/or with the aid of or automation provided by electronic equipment. Although the processes have been described with reference to particular implementations, those of ordinary skill in the art will readily appreciate that actions can be performed in other ways in relation to the methods. For example, unless otherwise described, the order of various steps may be changed without departing from the scope or spirit of the method. Additionally, some of the individual steps may be combined, omitted, or further subdivided into other steps.

All patents, publications and references cited herein are hereby incorporated by reference in their entirety. In the event of conflict between the present disclosure and incorporated patents, publications, and references, the present disclosure shall control.

Fig. 1A shows that at 10:1 (formulation F2, see Table 3), 12:1 (F6/12, see Table 3), 15:1 (F6/15, see Table 3), 17:1 (formulation F6 /17, see Table 3) and a mass ratio of 17:1 (all scaled) wt:wt comprising Compound 140 as the delivery vehicle for the cationic component complexed with RNA encoding firefly luciferase ("Fluc mRNA") The particle size of the compound. Delivery vehicle complexes with increased amounts of cationic components result in smaller particle sizes. For the "all scaled" condition, all components of the delivery vehicle composition (eg, DSPC, cholesterol, DMG-PEG 2000) were increased to match the 17:1 ratio of the DV-140-F6/17 complex.

Figure 1B shows the ratio of 10:1 (F2, see Table 3), 12:1 (F6/12, see Table 3), 15:1 (F6/15, see Table 3), 17:1 (F6/17, see Table 3) Table 3), 20:1 and 25:1 wt:wt mass ratios comprising compound 140 as the cationic component and the encapsulation percentage of the delivery vehicle complex complexed with RNA encoding firefly luciferase (Fluc). Complexes with increased amounts of cationic components resulted in higher encapsulation.

Figure 2 shows the particle size of DV-140-F2 and F6 formulations (DV-140-F6/17) after storage at 4°C for 17 and 48 days.

Figure 3 shows that the complexes of DV-140-F2 and DV-112-F2 (each complexed with Fluc mRNA) were subjected to in vivo time points of 24 hours and freeze/thaw cycles (3 times, 4°C/25°C). Resulting particle size (Figure 3A) and polydispersity index (Figure 3B) after temperatures of -20°C, 4°C, 25°C and 37°C. DV-140-F2 exhibited better stability than DV-112-F2 in all experiments.

Figure 4 shows the complexation of Fluc mRNA with compounds 140, 146, 151, 152, 160, 161 or 162 as cationic components in formulation F2 or F6 formulations (F6/17, see Tables 2, 3 and 4) In vivo expression of firefly luciferase (Fluc) in C57BI/6 mice treated (via intramuscular injection ("IM")) with delivery vehicle complexes. The data indicate that the delivery vehicle complexes of the present disclosure exhibit strong Fluc expression in vivo.

Figure 5 shows the formulation at 10:1 (formulation F2), 12:1 (formulation F6/12), 15:1 (formulation F6/15), 17:1 (formulation F6/17) and 17:1 (formulation F6/17) In vivo expression of firefly luciferase (Fluc) in C57BI/6 mice treated (IM) with delivery vehicle complexes containing Compound 140 complexed with Fluc mRNA. The delivery vehicle complexes of the disclosure exhibit high luciferase expression.

Figure 6 shows treatment (IM) with DV-140-F2 complexed with Fluc mRNA compared to treatment with a delivery vehicle complex comprising a cationic peptoid without hydroxyethyl cap (see Table 5 for structure) In vivo expression of firefly luciferase (Fluc) in C57BI/6 mice. The data indicate that the delivery vehicle complexes of the present disclosure outperform similar delivery vehicle complexes that do not include cationic peptoids with hydroxyethyl capping.

Figure 7 shows in vivo expression of Fluc mRNA in C57BI/6 mice treated intratumorally ("IT") with DV-140-F2 complexed with Fluc mRNA and other delivery vehicle complexes as indicated (see Table 5) . The data indicate that the delivery vehicle complexes of the present disclosure induce strong Fluc expression via intratumoral injection.

Figure 8A is a diagram depicting C57BI treated (IM) with DV-140-F2 complexed with mRNA encoding HPV E6 and/or HPV E7 from E16 and/or E18 ("HPV E6/E7 RNA") /6 mice elicited cellular immune responses. DV-140-F2/HPV E6/E7 complexes elicited stronger cellular responses compared to delivery vehicle complexes comprising cationic components without hydroxyethyl capping (see Table 5).

Figure 8B is a graph depicting the humoral immune response elicited in C57BI/6 mice treated (via IM) with DV-140-F2 containing HPV E6/E7 RNA. DV-140-F2 complexes elicited stronger IgGr responses compared to delivery vehicle complexes comprising cationic components without hydroxyethyl capping (see Table 5).

Figure 9 shows the immune response elicited in Balb/c mice treated (via IM) with DV-140-F2 complexed with COVID RNA constructs. The DV-140-F2/COVID complex elicited the production of spike protein-specific antibodies in serum (Fig. 9A) and lung (Fig. 9B), and also induced neutralizing antibodies (Fig. 9C).

Figure 10 shows the immune response elicited in Balb/c mice treated (via IM) with DV-140-F2 complexed with COVID RNA constructs. Site-specific immune responses in splenocytes were assessed ( FIG. 10A ) and intracellular interleukin staining for IFNγ, TNFα, and IL2 ( FIGS. 10B-1 and 10B-2 ). The DV-140-F2/COVID complex triggers the production of spike protein-specific T cell-reactive antibodies in serum (CD8: Figure 10C-1, Figure 10C-2, Figure 10C-3; CD4: 10D-1, Figure 10D- 2. Figure 10D-3).

Figure 11 shows that the immune response elicited in Balb/c mice treated (IM) with DV-140-F2 complexed with the COVID RNA construct (Figure 11A) is similar to that reported in mice with mRNA-1273 ( Figure 11B).

Figure 12 shows that DV-140-F2 complexed with RNA encoding influenza hemagglutinin (HA) (Flu Ha RNA) induces influenza HA-specific humoral (Figure 12A-1 and Figure 12A-2) and cells (Figure 12B-1 and Fig. 12B-2), and the combination vaccine targeting spike protein and influenza HA induced similar spike protein and influenza HA-specific immune responses (Fig. 12A-1, Fig. 12A-1, Fig. 12B-1, Fig. 12B-2).

13A is a graph showing a comparison of Spike protein-specific immunoglobulin responses in mice immunized with SARS-CoV-2 with vaccine formulations comprising various delivery vehicles. Delivery Vehicle 140-F6.3 performed comparable to commercial formulation SM-102 and ALC-0315 at day 35.

Figure 13B is a graph showing a comparison of Spike protein-specific T cell responses in mice immunized with SARS-CoV-2 with vaccine formulations comprising various delivery vehicles. Delivery Vehicle 140-F6.3 performed comparable to commercial formulations SM-102 and ALC-0315.

Claims (115)

A compound having a structure of formula (I):

, wherein n is 1, 2, 3, 4, 5 or 6; R ¹ is H, C _1-3 alkyl or hydroxyethyl; and each R ² is independently C _8-24 alkyl or C _8-24 Alkenyl. The compound as claimed in item 1, wherein n is 3. The compound as claimed in item 1, wherein n is 4. The compound according to any one of claims 1 to 3, wherein R ¹ is H. The compound according to any one of claims 1 to 3, wherein R ¹ is ethyl or hydroxyethyl. The compound according to any one of claims 1 to 5, wherein each R ² is independently C _8-18 alkyl or C _8-18 alkenyl. The compound as claimed in any one of items 1 to 6, wherein each ^R is independently selected from the group consisting of:

. The compound as claimed in any one of items 1 to 7, wherein each ^R is independently selected from the group consisting of:

. The compound as claimed in any one of items 1 to 8, wherein each ^R is independently selected from the group consisting of:

. The compound as claimed in any one of items 1 to 9, wherein each R ² is independently

. The compound as claimed in item 1, which has a structure selected from the group consisting of:

. As the compound of claim 11, it has the structure

. A pharmaceutically acceptable salt of the compound according to any one of claims 1 to 12. A delivery vehicle composition comprising the compound according to any one of claims 1 to 12 or the salt according to claim 13. The delivery vehicle composition according to claim 14, wherein the composition further comprises one or more of phospholipids, sterols and pegylated lipids. The delivery vehicle composition according to claim 14, wherein the composition comprises phospholipids, sterols and pegylated lipids. The delivery vehicle composition according to claim 14, wherein the composition consists essentially of the compound according to any one of claims 1 to 12 or the salt according to claim 13, phospholipids, sterols and pegylated lipids. The delivery vehicle composition according to any one of claims 14 to 17, wherein the compound or salt of formula (I) is present in an amount of about 30 mol% to about 60 mol%. The delivery vehicle composition according to claim 18, wherein the compound or salt of formula (I) is present in an amount of about 35 mol% to about 55 mol%. The delivery vehicle composition according to claim 18, wherein the compound or salt of formula (I) is present in an amount of about 30 mol% to about 45 mol%. The delivery vehicle composition according to claim 18 or 19, wherein the compound or salt of formula (I) is present in an amount of about 35 mol% to about 39 mol%. The delivery vehicle composition according to claim 18 or 19, wherein the compound or salt of formula (I) is present in an amount of about 39 mol% to about 52 mol%. The delivery vehicle composition according to claim 20, wherein the compound or salt of formula (I) is present in an amount of about 30 mol% to about 35 mol%. The delivery vehicle composition according to claim 22, wherein the compound or salt of formula (I) is present in an amount of about 40 mol% to about 45 mol%. The delivery vehicle composition according to claim 22, wherein the compound or salt of formula (I) is present in an amount of about 42 mol% to about 49 mol%. The delivery vehicle composition according to claim 22, wherein the compound or salt of formula (I) is present in an amount of about 50 mol% to about 52 mol%. The delivery vehicle composition according to any one of claims 15 to 17, wherein the composition comprises about 30 mol% to about 60 mol% of the compound of formula (I); about 3 mol% to about 20 mol% of the phospholipid, about 25 mol% to about 60 mol% of the sterol and about 1 mol% to about 5 mol% of the pegylated lipid. The delivery vehicle composition as claimed in item 27, wherein the composition comprises about 35 mol% to about 55 mol% of the compound or salt of formula (I); about 5 mol% to about 15 mol% of the phospholipid, about 30 mol% to about 55 mol% of the sterol and about 1 mol% to about 3 mol% of the pegylated lipid. The delivery vehicle composition as claimed in item 28, wherein the composition comprises about 38 mol% to about 52 mol% of the compound or salt of formula (I); about 9 mol% to about 12 mol% of the phospholipid, about 35 mol% to about 50 mol% of the sterol and about 1 mol% to about 2 mol% of the pegylated lipid. The delivery vehicle composition according to any one of claims 15 to 17, wherein the composition comprises about 30 mol% to about 49 mol% of the compound of formula (I); about 5 mol% to about 15 mol% of the phospholipid, about 30 mol% to about 55 mol% of the sterol and about 1 mol% to about 3 mol% of the pegylated lipid. The delivery vehicle composition as claimed in item 30, wherein the composition comprises about 35 mol% to about 49 mol% of the compound or salt of formula (I); about 7 mol% to about 12 mol% of the phospholipid, about 35 mol% to about 50 mol% of the sterol and about 1 mol% to about 2 mol% of the pegylated lipid. The delivery vehicle composition as claimed in item 30, wherein the composition comprises about 30 mol% to about 45 mol% of the compound or salt of formula (I); about 7 mol% to about 12 mol% of the phospholipid, about 40 mol% to about 55 mol% of the sterol and about 1 mol% to about 3 mol% of the pegylated lipid. The delivery vehicle composition as claimed in item 30, wherein the composition comprises about 30 mol% to about 35 mol% of the compound or salt of formula (I); about 7 mol% to about 12 mol% of the phospholipid, about 50 mol% to about 55 mol% of the sterol and about 2 mol% to about 3 mol% of the pegylated lipid. The delivery vehicle composition as claimed in item 30, wherein the composition comprises about 40 mol% to about 45 mol% of the compound or salt of formula (I); about 7 mol% to about 12 mol% of the phospholipid, about 40 mol% to about 45 mol% of the sterol and about 1 mol% to about 2 mol% of the pegylated lipid. The delivery vehicle composition according to any one of claims 15 to 34, wherein the phospholipid is selected from the group consisting of: 1,2-dilinoleyl-sn-glycero-3-phosphocholine (DLPC) , 1,2-Dimyristyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl Fattyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecyl- sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl- sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl 1,2-dilinoleyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn -Glycero-3-phosphocholine, 1,2-bis(docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleyl-sn-glycero-3- Phosphoethanolamine (DOPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE ), 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl- sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bis(docosyl)hexaenoyl-sn-glycerol-3 - Phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-dioxaphosphoryl-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and combinations thereof. The delivery vehicle composition according to claim 35, wherein the phospholipid is DOPE, DSPC or a combination thereof. The delivery vehicle composition according to claim 36, wherein the phospholipid is DSPC. The delivery vehicle composition according to any one of claims 15 to 37, wherein the sterol is selected from the group consisting of cholesterol, coprosterol, phytosterol, ergosterol, campesterol, stigmasterol , Brassinosterol, Tomato

(tomatidine), ursolic acid, alpha-tocopherol and mixtures thereof. The delivery vehicle composition according to claim 38, wherein the sterol is cholesterol. The delivery vehicle composition according to any one of claims 15 to 39, wherein the pegylated lipid is selected from the group consisting of phosphatidylethanolamine modified with PEG, phosphatidic acid modified with PEG, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified sterol, and PEG-modified Phospholipids. The delivery vehicle composition of claim 40, wherein the PEG-modified lipid is selected from the group consisting of PEG-modified cholesterol, N-octyl-sphingosine-1-{butanediyl [Methoxy (polyethylene glycol)]}, N-palmitoyl-sphingosine-1-{succinyl[methoxy (polyethylene glycol)]}, PEG-modified DMPE (DMPE-PEG), PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), PEG-modified DOPE (DOPE-PEG), dimyristyl glycerol- Polyethylene Glycol (DMG-PEG), Distearoylglycerol-Polyethylene Glycol (DSG-PEG), Dipalmitoylglycerol-Polyethylene Glycol (DPG-PEG), Dioleoylglycerol- Polyethylene glycol (DOG-PEG) and combinations thereof. The delivery vehicle composition according to claim 41, wherein the PEG-modified lipid is dimyristyl glycerol-polyethylene glycol 2000 (DMG-PEG 2000). The delivery vehicle composition of any one of claims 14 to 20, comprising about 38.2 mol% of

, about 11.8 mol% of DSPC, about 48.2 mol% of cholesterol and about 1.9 mol% of DMG-PEG 2000. The delivery vehicle composition of any one of claims 14 to 20, comprising about 42.6 mol% of

, about 10.9 mol% of DSPC, about 44.7 mol% of cholesterol and about 1.7 mol% of DMG-PEG 2000. The delivery vehicle composition of any one of claims 14 to 20, comprising about 48.2 mol% of

, about 9.9 mol% of DSPC, about 40.4 mol% of cholesterol and about 1.6 mol% of DMG-PEG 2000. The delivery vehicle composition of any one of claims 14 to 20, comprising about 51.3 mol% of

, about 9.3 mol% of DSPC, about 38 mol% of cholesterol and about 1.5 mol% of DMG-PEG 2000. The delivery vehicle composition of any one of claims 14 to 20, comprising about 44.4 mol% of

, about 10.6 mol% of DSPC, about 43.3 mol% of cholesterol and about 1.7 mol% of DMG-PEG 2000. The delivery vehicle composition of any one of claims 14 to 20, comprising about 44.4 mol% of

, about 10.6 mol% of DSPC, about 43.4 mol% of cholesterol and about 1.7 mol% of DMG-PEG 2000. The delivery vehicle composition of any one of claims 14 to 20, comprising about 33.1 mol% of

, about 10.6 mol% of DSPC, about 53.8 mol% of cholesterol and about 2.5 mol% of DMG-PEG 2000. A delivery vehicle complex comprising the delivery vehicle composition according to any one of claims 14 to 49 and a polyanionic compound. The delivery vehicle complex according to claim 50, wherein the compound of formula (I) or its salt is complexed with the polyanionic compound. The delivery vehicle complex according to claim 51, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 5:1 to about 25:1. The delivery vehicle complex according to claim 52, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 7:1 to about 20:1. The delivery vehicle complex according to claim 53, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 10:1 to about 17:1. The delivery vehicle complex according to claim 53, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 19:1. The delivery vehicle complex according to claim 53, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 20:1. The delivery vehicle complex according to claim 54, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 10:1. The delivery vehicle complex according to claim 54, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 12:1. The delivery vehicle complex according to claim 54, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 13:1. The delivery vehicle complex according to claim 54, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 15:1. The delivery vehicle complex according to claim 54, wherein the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 17:1. The delivery vehicle complex according to any one of claims 50 to 61, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 2:1 to about 10:1. The delivery vehicle complex according to claim 62, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 2:1 to about 4:1. The delivery vehicle complex according to claim 62, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 2:1 to about 3:1. The delivery vehicle complex according to claim 63, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 4:1. The delivery vehicle complex according to claim 64, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 2.7:1. The delivery vehicle complex of any one of claims 50 to 66, wherein the sterol and the polyanionic compound are present in a mass ratio of about 5:1 to about 8:1. The delivery vehicle complex of any one of claims 50 to 67, wherein the sterol and the polyanionic compound are present in a mass ratio of about 5:1 to about 6:1. The delivery vehicle complex of claim 68, wherein the sterol and the polyanionic compound are present in a mass ratio of about 5.4:1. The delivery vehicle complex according to claim 67, wherein the sterol and the polyanionic compound are present in a mass ratio of about 8.1:1. The delivery vehicle complex according to claim 67, wherein the sterol and the polyanionic compound are present in a mass ratio of about 6.7:1. The delivery vehicle complex according to any one of claims 50 to 71, wherein the pegylated lipid and the polyanionic compound are present in a mass ratio of about 0.5:1 to about 2.5:1. The delivery vehicle complex of claim 72, wherein the pegylated lipid and the polyanionic compound are present in a mass ratio of about 1:1 to about 2:1. The delivery vehicle complex according to claim 72, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 2.1:1. The delivery vehicle complex according to claim 73, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 1.4:1. The delivery vehicle complex according to any one of claims 50 to 54, 57, 63, 64, 66 to 69, 71 to 73 and 75, comprising: the polyanionic compound in a mass ratio of about 10:1

, DSPC with a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol with a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG with a mass ratio to the polyanionic compound of about 1.4:1 2000. The delivery vehicle complex according to any one of claims 50 to 54, 58, 62 to 64, 66 to 69, 72, 73 and 75, comprising: the polyanionic compound in a mass ratio of about 12:1

, DSPC with a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol with a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG with a mass ratio to the polyanionic compound of about 1.4:1 2000. The delivery vehicle complex according to any one of claims 50 to 54, 60, 62 to 64, 66 to 69, 72, 73 and 75, comprising: the mass ratio of the polyanionic compound is about 15:1

, DSPC with a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol with a mass ratio of about 5.4:1 to the polyanionic compound, and DMG with a mass ratio to the polyanionic compound of about 1.4:1- PEG 2000. The delivery vehicle complex according to any one of claims 50 to 54, 61 to 64, 66 to 69, 72, 73 and 75, comprising: the polyanionic compound in a mass ratio of about 17:1

, DSPC with a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol with a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG with a mass ratio to the polyanionic compound of about 1.4:1 2000. The delivery vehicle complex according to any one of claims 50 to 54, 59, 62 to 64, 66 to 69, 72, 73 and 75, comprising: the polyanionic compound in a mass ratio of about 13:1

, DSPC with a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol with a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG with a mass ratio to the polyanionic compound of about 1.4:1 2000. The delivery vehicle complex according to any one of claims 50 to 55, 62, 63, 65, 67 to 69 and 72 to 74, comprising: the mass ratio of the polyanionic compound is about 19:1

, DSPC with a mass ratio of about 4:1 to the polyanionic compound, cholesterol with a mass ratio of about 8.1:1 to the polyanionic compound, and DMG-PEG with a mass ratio to the polyanionic compound of about 2.1:1 2000. The delivery vehicle complex according to any one of claims 50 to 53, 57, 62 to 64, 66 to 68, and 71 to 74, comprising: the mass ratio of the polyanionic compound is about 9.7:1

, DSPC with a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol with a mass ratio of about 6.7:1 to the polyanionic compound, and DMG-PEG with a mass ratio to the polyanionic compound of about 2.1:1 2000. The delivery vehicle complex of any one of claims 50 to 82, wherein the complex exhibits a particle size of about 50 nm to about 200 nm and/or a polydispersity index (PDI) of less than about 0.25. The delivery vehicle complex of claim 83, wherein the complex exhibits a particle size of about 60 nm to about 100 nm. The delivery vehicle complex of claim 84, wherein the complex exhibits a particle size between about 60 nm and about 90 nm. The delivery vehicle complex of claim 83, wherein the complex exhibits a particle size of about 105 nm to about 200 nm. The delivery vehicle complex of claim 86, wherein the delivery vehicle complex exhibits a particle size of about 155 nm to about 195 nm. The delivery vehicle composite of any one of claims 50 to 87, wherein at least 80% of the polyanionic compound remains after storage at 4°C for 48 days, or the delivery vehicle composite is stored at 4°C for 48 days Thereafter retain at least 80% of its original size, or both. The delivery vehicle complex of any one of claims 50 to 88, wherein the polyanionic compound comprises at least one nucleic acid. The delivery vehicle complex according to claim 89, wherein the at least one nucleic acid comprises RNA, DNA or a combination thereof. The delivery vehicle complex of claim 90, wherein the at least one nucleic acid comprises RNA. The delivery vehicle complex according to claim 91, wherein the RNA is mRNA encoding a peptide, protein or a functional fragment thereof. The delivery vehicle complex according to claim 92, wherein the mRNA encodes a viral peptide, a viral protein or a functional fragment of any of the foregoing. The delivery vehicle complex according to claim 93, wherein the mRNA encodes a human papillomavirus (HPV) protein or a functional fragment thereof. The delivery vehicle complex according to claim 94, wherein the mRNA encodes HPV E6 protein and/or HPV E7 protein, or the aforementioned functional fragments. The delivery vehicle complex according to claim 95, wherein the mRNA encodes a viral spike protein or a functional fragment thereof. The delivery vehicle complex as claimed in claim 96, wherein the mRNA encodes a SARS-CoV spike (S) protein or a functional fragment thereof. The delivery vehicle complex according to claim 97, wherein the mRNA encodes influenza hemagglutinin (HA) or a functional fragment thereof. The delivery vehicle complex according to claim 98, which comprises mRNA encoding SARS-CoV spike (S) protein and mRNA encoding influenza hemagglutinin (HA) or the aforementioned functional fragments. A pharmaceutical composition comprising the delivery vehicle complex according to any one of claims 50 to 99 and a pharmaceutically acceptable excipient. The pharmaceutical composition according to claim 100, which is in the form of an intratumoral (IT) or intramuscular (IM) composition. A method of inducing an immune response in an individual in need thereof, comprising administering to the individual an effective amount of a delivery vehicle complex according to any one of claims 70 to 79 or a pharmaceutical formulation according to claim 100 or 101, An immune response is thus induced in the individual. A method of treating a virus infection in an individual in need thereof, comprising administering to the individual an effective amount of the delivery vehicle complex according to any one of claims 92 to 101 or any one of claims 100 or 101 A pharmaceutical formulation whereby the viral infection in the individual is treated. A method of treating cancer in an individual in need thereof, comprising administering to the individual an effective amount of the delivery vehicle complex according to any one of claims 92 to 101 or the medicine according to any one of claims 100 or 101 Formulations, thereby treating the cancer in the individual. The method of claim 104, wherein the cancer is cervical cancer, head and neck cancer, B-cell lymphoma, T-cell lymphoma, prostate cancer, lung cancer, or a combination thereof. The method of any one of claims 102 to 105, wherein the administering is by intramuscular, intratumoral, intravenous, intraperitoneal or subcutaneous delivery. A method of delivering a polyanionic compound to a cell, comprising contacting the cell with a delivery vehicle complex according to any one of claims 50-99 or a pharmaceutical composition according to claim 100 or 101. The method according to claim 107, wherein the cells are muscle cells, tumor cells or a combination thereof. The method of claim 107 or 108, wherein the polyanionic compound is an mRNA encoding a peptide, a protein, or a fragment of any of the foregoing, and the cell expresses the peptide, the protein, or the protein after being contacted with the delivery vehicle complex fragment. A method of forming a delivery vehicle complex according to any one of claims 50 to 99, comprising contacting the compound or salt of formula (I) with the polyanionic compound. The method according to claim 110, comprising mixing a solution comprising the compound or salt of formula (I) and a solution comprising the polyanionic compound. A vaccine comprising the delivery vehicle complex according to any one of claims 92 to 101 or the pharmaceutical formulation according to any one of claims 100 or 101. The vaccine according to claim 112, which is used for treating cancer. A method of treating or preventing cancer in a patient, comprising administering the vaccine according to claim 112 to the patient. The vaccine according to claim 113 or the method according to claim 114, wherein the cancer is cervical cancer, head and neck cancer, B-cell lymphoma, T-cell lymphoma, prostate cancer, lung cancer or a combination thereof.

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