2-Aminopropane-1,3-diol-capped cationic peptoids for nucleic acid delivery

Abstract

The present disclosure provides delivery vehicle compositions comprising hydroxyalkyl-capped cationic peptoids (e.g., 2-aminopropane-1,3-diol-capped cationic peptoids), and complexes of the delivery vehicles with polyanionic compounds (e.g., nucleic acids). The present disclosure further provides methods of making and using the delivery vehicle compositions and complexes, such as for delivering polyanionic compounds (e.g., nucleic acids) to cells. The present disclosure also provides methods of eliciting an immune response using the delivery vehicle complexes of the present disclosure.

Description

[Technical Field]

[0001] Description of Related Art Therapeutic nucleic acids, such as mRNA, small interfering RNA (siRNA), small activating RNA (saRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, and immunostimulatory nucleic acids, hold great promise for the prevention and treatment of diseases at the genetic level. However, nucleic acids typically suffer from rapid degradation in the blood, renal clearance, poor cellular uptake, and inefficient endosomal escape. Therefore, for nucleic acids to be therapeutically useful, a safe and effective system for delivering nucleic acids to the cell nucleus or cytosol is required. Conventional methods for cellular and in vivo delivery of polyanionic compounds, such as oligonucleotides, include viral vectors, cationic lipid nanoparticles (LNPs), and polycationic polymers. These delivery systems can be plagued by limitations, such as poor stability, rapid clearance, poor toxicity, concerns about immune responses, and suboptimal expression of their polyanionic cargo. Summary of the Invention

[0002] There is a need for a stable, safe, and effective system for delivering nucleic acids into cells. Accordingly, the present disclosure relates to delivery vehicle compositions comprising 2-aminopropane-1,3-diol-capped cationic peptoids, and complexes of the delivery vehicle compositions with polyanionic compounds (e.g., nucleic acids). The present disclosure also relates to methods of making and using the delivery vehicle compositions and complexes for intracellular delivery of polyanionic compounds, e.g., mRNA, as well as methods of eliciting an immune response using the complexes of the present disclosure.

[0003] N-substituted glycine oligomers (termed peptoids) and their use as ionic lipid components in delivery vehicle compositions are disclosed. By parameterizing variations in peptoid structure, including modifications to both the charged and hydrophobic portions of the peptoid, a design of experiments (DOE) methodology was used to explore a broad chemical space, facilitating the discovery of trends between peptoid structural elements and particle properties. This optimization process resulted in a >10,000-fold increase in in vivo mRNA expression from initial compounds to final lead candidates, enabling their use in therapeutic programs involving enzyme replacement and in vivo-produced antibody drugs. The disclosed peptoids produce therapeutically relevant levels of protein expression (e.g., anti-RSV antibodies) in mice with minimal tolerability concerns or induced immune responses. The disclosed compounds and compositions thereof offer a tunable delivery platform that can be optimized for specific therapeutic programs.

[0004] In one aspect, the present disclosure provides a compound of formula (I):

[0005] [ka] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R 1 is C optionally substituted with H or 1 to 3 OH 2~5 alkyl, and R 2 is a C substituted with 1 to 3 additional OH groups 2~5 alkylene-OH, and each R 3 independently, C 6~24 Alkyl or C 6~24 Optionally, n is 3. Optionally, n is 4. Optionally, n is 6. Optionally, n is 8. Optionally, n is 9. Optionally, R 1 is H. In some cases, R 2 is a C substituted with one additional OH2~5 alkylene-OH. In some cases, C 2~5 The alkylene is substituted with 2 or 3 additional OH. 2 is a C substituted with 1 to 3 additional OH groups 3~4 In some cases, each R 3 independently, C 6~18 Alkyl or C 6~18 alkenyl. In some cases, each R 3 independently, C 8~18 Alkyl or C 8~18 alkenyl. In some cases, each R 3 is, independently,

[0006] [ka] Optionally, each R is selected from the group consisting of 3 is, independently,

[0007] [ka] In some cases, at least one R is selected from the group consisting of 3 teeth,

[0008] [ka] In some cases, at least one R is selected from the group consisting of 3 teeth,

[0009] [ka] Optionally, the compound of formula (I) is

[0010] [ka]

[0011] [ka]

[0012] [ka]

[0013] [ka] In some cases, the compound of Formula (I) has a structure selected from the group consisting of Compound 1, Compound 6, Compound 21, and Compound 30. In some cases, the compound of Formula (I) is Compound 1. In some cases, the compound of Formula (I) is Compound 6. In some cases, the compound of Formula (I) is Compound 21. In some cases, the compound of Formula (I) is Compound 30. In some cases, the compound of Formula (I) is Compound 12. In some cases, the compound of Formula (I) is Compound 34. In some cases, the compound of Formula (I) is Compound 35. In some cases, the compound of Formula (I) is Compound 41. Further disclosed herein are pharmaceutically acceptable salts of compounds of Formula (I).

[0014] Another aspect of the present 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 a phospholipid, a sterol, and a PEGylated lipid. In some implementations, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount of 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 of 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 of 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 of about 35 mol% to about 39 mol%. In some implementations, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount of 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 of 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 of about 40 mol% to about 45 mol%. In various cases, the compound or salt of Formula (I) is present in an amount of 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%.

[0015] In various implementations, the composition comprises a phospholipid, a sterol, and a PEGylated lipid. In some cases, the composition consists 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 a compound of Formula (I), about 3 mol% to about 20 mol% of a phospholipid, about 25 mol% to about 60 mol% of a sterol, and about 1 mol% to about 5 mol% of a PEGylated lipid. In various cases, the composition comprises about 35 mol% to about 55 mol% of a compound or salt of Formula (I), about 5 mol% to about 15 mol% of a phospholipid, about 30 mol% to about 55 mol% of a sterol, and about 1 mol% to about 3 mol% of a PEGylated lipid. In some embodiments, the composition comprises about 38 mol% to about 52 mol% of a compound or salt of Formula (I), about 9 mol% to about 12 mol% of a phospholipid, about 35 mol% to about 50 mol% of a sterol, and about 1 mol% to about 2 mol% of a PEGylated lipid. In various embodiments, the composition comprises about 30 mol% to about 49 mol% of a compound of Formula (I), about 5 mol% to about 15 mol% of a phospholipid, about 30 mol% to about 55 mol% of a sterol, and about 1 mol% to about 3 mol% of a PEGylated lipid. In some embodiments, 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 a phospholipid, about 35 mol% to about 50 mol% of a sterol, and about 1 mol% to about 2 mol% of a PEGylated lipid. 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 a phospholipid, about 40 mol% to about 55 mol% of a sterol, and about 1 mol% to about 3 mol% of a PEGylated lipid. 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 a phospholipid, about 50 mol% to about 55 mol% of a sterol, and about 2 mol% to about 3 mol% of a PEGylated lipid. 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 a phospholipid, about 40 mol% to about 45 mol% of a sterol, and about 1 mol% to about 2 mol% of a PEGylated lipid.In some cases, the phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-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-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), or 1,2-diisopropyl-sn-glycero-3-phosphocholine (DIPC). PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleyl The phospholipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof. In some cases, the phospholipid is DOPE, DSPC, or a combination thereof. In various cases, the phospholipid is DSPC.In some embodiments, the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the sterol is cholesterol. In some embodiments, the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified sterol, and PEG-modified phospholipid. In various implementations, the PEG-modified lipid is selected from the group consisting of PEG-modified cholesterol, N-octanoyl-sphingosine-1-{succinyl[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), dimyristoylglycerol-polyethylene glycol (DMG-PEG), distearoylglycerol-polyethylene glycol (DSG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), dioleoylglycerol-polyethylene glycol (DOG-PEG), and combinations thereof. In some embodiments, the PEG-modified lipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000). In various cases, the composition comprises about 38.2 mol% of Compound 1, 6, 21, or 30, about 11.8 mol% of DSPC, about 48.2 mol% of cholesterol, and about 1.9 mol% of DMG-PEG 2000. In some embodiments, the composition comprises about 42.6 mol% of Compound 1, 6, 21, or 30, about 10.9 mol% of DSPC, about 44.7 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.In some embodiments, the composition comprises about 48.2 mol% of Compound 1, 6, 21, or 30, about 9.9 mol% of DSPC, about 40.4 mol% of cholesterol, and about 1.6 mol% of DMG-PEG 2000. In various cases, the composition comprises about 51.3 mol% of Compound 1, 6, 21, or 30, about 9.3 mol% of DSPC, about 38 mol% of cholesterol, and about 1.5 mol% of DMG-PEG 2000. In various cases, the composition comprises about 44.4 mol% of Compound 1, 6, 21, or 30, about 10.6 mol% of DSPC, about 43.3 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000. In various cases, the composition comprises about 44.4 mol% of Compound 1, 6, 21, or 30, about 10.6 mol% of DSPC, about 43.4 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000. In various cases, the composition comprises about 33.1 mol% of Compound 1, 6, 21, or 30, about 10.6 mol% of DSPC, about 53.8 mol% of cholesterol, and about 2.5 mol% of DMG-PEG 2000.

[0016] Also disclosed herein is a delivery vehicle complex comprising a delivery vehicle composition described herein and a polyanionic compound. In some cases, the compound of Formula (I) or a salt thereof is complexed with the polyanionic compound. In various cases, 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. 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 various cases, 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. 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 weight ratio of about 10:1. In various cases, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 12:1. In various implementations, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 13:1. In some implementations, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 15:1. In various implementations, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 17:1. In some cases, the phospholipid and the polyanionic compound are present in a weight ratio of about 2:1 to about 10:1. In some cases, the phospholipid and the polyanionic compound are present in a weight ratio of about 2:1 to about 4:1. In various cases, the phospholipid and the polyanionic compound are present in a weight ratio of about 2:1 to about 3:1. In various cases, the phospholipid and polyanionic compound are present in a weight ratio of about 4.0:1. In various cases, the phospholipid and polyanionic compound are present in a weight ratio of about 2.7:1. In some implementations, the sterol and polyanionic compound are present in a weight ratio of about 5:1 to about 8:1. In some implementations, the sterol and polyanionic compound are present in a weight ratio of about 5:1 to about 6:1. In various implementations, the sterol and polyanionic compound are present in a weight ratio of about 5.4:1.In some cases, the sterol and polyanionic compound are present in a mass ratio of about 8.1:1. In some cases, the sterol and polyanionic compound are present in a mass ratio of about 6.7:1. In some cases, the PEGylated lipid and polyanionic compound are present in a mass ratio of about 0.5:1 to about 2.5:1. In various cases, 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 various cases, the delivery vehicle complex comprises compound 1, 6, 21, or 30 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 in a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 in a mass ratio of about 1.4:1 to the polyanionic compound. In various cases, the delivery vehicle complex comprises compound 1, 6, 21, or 30 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 in a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 in a mass ratio of about 1.4:1 to the polyanionic compound. In some cases, the delivery vehicle complex comprises compound 1, 6, 21, or 30 having a mass ratio of about 15:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound. In various cases, the delivery vehicle complex comprises compound 1, 6, 21, or 30 having a mass ratio of about 17:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.In various cases, the delivery vehicle complex comprises compound 1, 6, 21, or 30 having a mass ratio of about 13:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound. In various cases, the delivery vehicle complex comprises compound 1, 6, 21, or 30 having a mass ratio of about 19:1 to the polyanionic compound, DSPC having a mass ratio of about 4.0:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 2.1:1 to the polyanionic compound. In various cases, the delivery vehicle complex comprises Compound 1, 6, 21, or 30 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 in a mass ratio of about 6.7:1 to the polyanionic compound, and DMG-PEG2000 in a mass ratio of about 2.1:1 to the polyanionic compound. In some cases, the compound of Formula (I) or salt thereof, phospholipid, sterol, and PEGylated lipid are present in a mass ratio of about 20:1.8:7.2:1.8.

[0017] In some cases, the complexes exhibit a particle size of about 50 nm to about 200 nm and / or a polydispersity index (PDI) of less than 0.25. In various cases, the complexes exhibit a particle size of about 60 nm to about 100 nm. In some implementations, the complexes exhibit a particle size of about 60 nm to about 90 nm. In various implementations, the complexes exhibit a particle size of about 105 nm to about 200 nm. In various cases, the complexes exhibit a particle size of about 150 nm to about 200 nm. In some cases, the delivery vehicle complexes exhibit a particle size of about 105 nm to about 200 nm. In some cases, the delivery vehicle complexes exhibit a particle size of about 40 nm to about 115 nm, or about 55 nm to about 95 nm, or about 70 nm to about 80 nm, or about 75 nm. In various cases, 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 various cases, at least 80% of the polyanionic compound is retained after 48 days of storage at 4° C., or the delivery vehicle complex retains at least 80% of its original size after 48 days of storage at 4° C., or both.

[0018] In some cases, the polyanionic compound comprises at least one nucleic acid. In various cases, the at least one nucleic acid comprises RNA, DNA, or a combination thereof. In various cases, the at least one nucleic acid comprises RNA. In some implementations, the RNA is an mRNA encoding a peptide, a protein, or a functional fragment of the foregoing. 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 various cases, 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 cases, the HPV protein is derived from HPV subtypes HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68. In various cases, the HPV protein is from HPV subtypes HPV16 and / or HPV18. In some cases, the mRNA encodes a viral spike protein or a functional fragment thereof. In various cases, 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 cases, 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 an HA subtype 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 various cases, one mRNA encodes a SARS-CoV spike (S) protein and one mRNA encodes an influenza hemagglutinin (HA), a variant thereof, or a functional fragment of any of the foregoing.

[0019] Further disclosed herein is a pharmaceutical composition comprising a delivery vehicle complex of the present disclosure and a pharmaceutically acceptable excipient. In some cases, the pharmaceutical composition is an intratumoral (IT) or intramuscular (IM) composition.

[0020] Also disclosed herein is a method for inducing an immune response in a subject in need thereof, comprising administering to the subject an effective amount of a delivery vehicle conjugate described herein or a pharmaceutical formulation comprising the delivery vehicle conjugate, thereby inducing an immune response in the subject. Also disclosed herein is a method for treating a viral infection in a subject in need thereof, comprising administering to the subject an effective amount of a delivery vehicle conjugate described herein or a pharmaceutical formulation comprising the delivery vehicle conjugate, thereby treating the viral infection in the subject. Also disclosed herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a delivery vehicle conjugate described herein or a pharmaceutical formulation comprising the delivery vehicle conjugate, thereby treating the cancer in the subject. In some cases, the cancer is cervical cancer, head and neck cancer, B-cell lymphoma, T-cell lymphoma, prostate cancer, lung cancer, or a combination thereof. In various cases, administration is by intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

[0021] Also disclosed herein is a method of delivering a polyanionic compound to a cell, the method comprising contacting the cell with a delivery vehicle complex described herein or a pharmaceutical formulation comprising the delivery vehicle complex. In some cases, the cell is a muscle cell, a tumor cell, or a combination thereof. In some cases, the polyanionic compound is an mRNA encoding a peptide, protein, or a fragment of any of the foregoing, and the cell expresses the peptide, protein, or fragment after contact with the delivery vehicle complex.

[0022] 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. Optionally, the method comprises mixing a solution comprising the compound or salt of Formula (I) with a solution comprising the polyanionic compound.

[0023] Also disclosed herein is a vaccine comprising the delivery vehicle complex disclosed herein or the pharmaceutical composition disclosed herein. Also disclosed is a vaccine comprising the delivery vehicle complex disclosed herein or the pharmaceutical composition disclosed herein for use in treating cancer. Also disclosed is a method 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 cases, the cancer is cervical cancer, head and neck cancer, B-cell lymphoma, T-cell lymphoma, prostate cancer, lung cancer, or a combination thereof.

[0024] It should be appreciated that all combinations of the foregoing concepts and implementations, as well as additional concepts and implementations described in more detail below, are contemplated as being part of the inventive subject matter disclosed herein and may be used in any suitable combination to achieve the benefits described herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

[0025] Further aspects and advantages will become apparent to those skilled in the art from a consideration of the following detailed description in conjunction with the drawings. While the compounds and methods disclosed herein may be implemented in a variety of forms, the following description includes specific implementations, but it is done so with the understanding that the disclosure is illustrative and is not intended to limit the disclosure to the specific implementations described herein. [Brief explanation of the drawings]

[0026] [Figure 1] 1 shows the results of an in vivo assay of delivery vehicles comprising peptoids disclosed herein and control peptoids, assessed using fLuc expression in BALB / c mice 6 hours after IV administration. [Figure 2] Shown are the percentages of total expression in spleen, lung, and liver for formulations of peptoids disclosed herein and control peptoids. [Figure 3] 1 shows the results of a firefly luciferase expression assay in BALB / c mice assessing the delivery efficacy of peptoid formulations disclosed herein. [Figure 4] 1 shows a contour plot assessing the effect of carbon number and branching on the efficacy of a delivery vehicle in a firefly luciferase expression assay. [Figure 5] 1 shows anti-RSV titer results for delivery vehicles comprising peptoids disclosed herein administered intravenously to BALB / c mice at 0.75 mg / kg mRNA and measured by IgG ELISA 24 hours later. [Figure 6] 1 shows the results of a dose escalation study of a delivery vehicle containing Compound 41 in a mouse model as measured by serum aRSV. [Figure 7] Showing that aRSV is detectable in serum for more than 5 days after administration. [Figure 8] 1 shows the results of a dose escalation study of a delivery vehicle containing Compound 41 in a mouse model, as measured by a firefly luciferase expression assay. [Figure 9] 1 shows a cryo-TEM image of particles of compound 41 in the D22 formulation ("41-D22"). [Figure 10] These results show that particles of 41-D22 remained stable for more than one month when stored at 4°C or -80°C, and no changes in growth or encapsulation were observed. [Figure 11] 1 shows a comparison of firefly luciferase expression by delivery vehicles formulated with compounds 31 and 41. [Figure 12] 1 shows a comparison of serum aRSV expression with delivery vehicles formulated with compounds 31 and 41. DETAILED DESCRIPTION OF THE INVENTION

[0027] Successful delivery of mRNA remains a major challenge to its adoption as a mainstream therapeutic modality. The approval of an mRNA vaccine against SARS-CoV-2 using lipid nanoparticle (LNP) technology represents significant progress in this field, but extending mRNA drugs to applications beyond vaccines (e.g., protein replacement therapy to generate circulating antibodies or immuno-oncology therapy to generate circulating antibodies) requires new delivery strategies and platforms. In these lipid nanoparticles, mRNA molecules are encapsulated with a combination of cationic or ionic lipids, helper lipids (e.g., cholesterol and phospholipids), and poly(ethylene glycol) (PEG)-containing shielding lipids. As mRNA is applied to a wider range of applications, including those relying on intravenous delivery, the ability to tailor the properties of ionic lipids, in particular, has become a key focus in this field. Numerous reports have shown that tailoring the properties of ionic lipids affects the expression and tissue selectivity of delivery. The generation of novel ionic lipids is often limited by efficient synthetic routes and requires significant synthetic effort and resources to expand lipid libraries to include novel, safe, and effective candidates. Provided herein is a new class of peptoid-based mRNA delivery vehicles, which utilizes highly adjustable peptoids as ionic lipid components to adjust the properties of delivery vehicles.Experimental design is used to adjust the peptoid structure to optimize the function of delivery vehicles, for example, to demonstrate the optimization of delivery vehicles for the systemic delivery of mRNA encoding aRSV.

[0028] Disclosed herein, in some examples, is a delivery vehicle composition comprising a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid). The delivery vehicle composition of the present disclosure can form an electrostatic interaction between the 2-aminopropane-1,3-diol-capped cationic peptoid of the delivery vehicle composition and a polyanionic compound, such as a nucleic acid, to form a delivery vehicle complex, with the polyanionic compound serving as the cargo of the complex. The delivery vehicle complex is useful for delivering polyanionic compounds, such as nucleic acids (e.g., mRNA), to cells. The delivery vehicle complex of the present disclosure, which includes mRNA as the polyanionic cargo, unexpectedly exhibits excellent mRNA expression both in vitro and in vivo. When the mRNA of the delivery vehicle complex encodes, for example, a viral antigen, the delivery vehicle complex can elicit humoral and cellular immune responses in vivo, thereby functioning as a vaccine. The delivery vehicle complexes disclosed herein are further advantageous in that they are stable, well tolerated and exhibit low toxicity.

[0029] As used herein, "peptoid" refers to a peptidomimetic compound in which one or more nitrogen atoms of the peptide backbone are replaced by a side chain. As used herein, "polyanionic" refers to a compound (e.g., a nucleic acid) that has at least two negative charges.

[0030] Delivery Vehicle Composition Some exemplary delivery vehicle compositions of the present disclosure include one or more hydroxyalkyl-capped cationic peptoids (e.g., 2-aminopropane-1,3-diol-capped cationic peptoids). These positively charged peptoids can associate with polyanionic compounds (e.g., nucleic acids) to form delivery vehicle complexes. In some implementations, the delivery vehicle composition further includes one or more of an anionic or zwitterionic component (e.g., a phospholipid), a neutral lipid (e.g., a sterol), and a shielding lipid (e.g., a PEGylated lipid). In various implementations, the delivery vehicle composition further includes an anionic or zwitterionic component (e.g., a phospholipid), a neutral lipid (e.g., a sterol), and a shielding lipid (e.g., a PEGylated lipid). In some cases, the delivery vehicle composition consists essentially of a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid), an anionic or zwitterionic component (e.g., a phospholipid), a neutral lipid (e.g., a sterol), and a shielding lipid (e.g., a PEGylated lipid).

[0031] Hydroxyalkyl-capped cationic peptoid moieties Peptoids are a unique class of N-substituted alpha-amino acids structurally related to peptides, but they incorporate their diverse side chain functionalities on the amide nitrogen rather than the alpha carbon, allowing for modular synthesis. In a non-limiting embodiment, peptoids can be synthesized by a submonomer approach, where repeated cycles of acylation using bromoacetic acid and nucleophilic addition of primary amines can be achieved on a solid support, producing peptoids in high yield and fidelity. Peptoids have been successfully used in many applications, including as antifouling and antibacterial agents, for drug delivery, and as antifreeze additives in tissue preservation, and even as complexing agents for nucleic acids, but they have not been explored as ionic components of lipid nanoparticles for mRNA delivery.

[0032] Given their favorable properties and modularity, peptoids are believed to be an excellent platform for discovering new ionic lipids and investigating their structure-activity relationships. Peptoids are easily synthesized using solid-phase synthesizers and have a much larger monomer pool than traditional peptides. Traditionally, the discovery of new lipids for nucleic acid delivery has been highly empirical; therefore, successful systems often incorporate a high degree of structural diversity and allow rapid exploration of a broad range of chemical space. Utilizing a solid-phase submonomer approach, the peptoid-based platform disclosed herein takes advantage of the tunable and programmable nature of peptoid structure to more easily cover a broad range of chemical space than can be achieved with traditional lipid synthesis. When formulated with mRNA, DSPC, cholesterol, and DMG-PEG, delivery vehicles are generated and characterized for their physical properties and biological activity. Through iterative synthesis and particle screening, the correlation between peptoid structure and delivery vehicle activity can be investigated.

[0033] The first stage of screening focused on optimizing the cationic group on the peptoid molecule. The identity of the basic group used in the ionic lipid significantly determines the particle's pKa, which correlates with the efficacy and specificity of nanoparticle expression. Generally, particles with the highest efficacy in selective liver expression have a pKa between 6 and 6.5. However, it is still unclear how the molecular pKa of the peptoid correlates with the observed pKa when formulated into the delivery vehicles disclosed herein; therefore, various headgroup amines with varying basicity were selected.

[0034] Without being bound by any particular theory, we believe that modifying the lipophilic portion of peptoids while maintaining the primary 1,3-diol (APD) head group may allow for further optimization of overall expression while maintaining or increasing liver selectivity. For this optimization, we selected six different aliphatic monomers ranging in length from C6 to C12, including unsaturated oleyl lipids and branched 2-ethylhexyls. Even with this relatively small monomer pool, there are 55,944 possible peptoid designs ranging in length from 3 to 8. Therefore, we employed a systematic approach to evaluate this large structural space using design of experiments (DOE) methodology. To allow chemical structure information to be accurately input into the DOE model, the properties of the lipid block can be parameterized using four key factors: 1) the total number of lipid-containing monomers in the structure, 2) the total number of carbon atoms contained on all lipid side chains, 3) the number of branched / unsaturated lipid monomers used, and 4) the identity of the branched / unsaturated monomers used. Each peptoid disclosed herein can be represented by a combination of these four factors, and their performance data can be fitted to a multivariate model to deconvolute the contribution of each factor to mRNA delivery performance. Because this method of parameterization does not attempt to capture the order of monomers within the sequence, peptoids were designed to have alternating or symmetrical motifs whenever possible.

[0035] The delivery vehicle compositions of the present disclosure include a hydroxyalkyl-capped cationic peptoid, such as a 2-aminopropane-1,3-diol-capped cationic peptoid (sometimes referred to as a "cationic component" or "ionic lipid"). In some implementations, the hydroxyalkyl-capped cationic peptoid is a compound of formula (I):

[0036] [ka] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R 1is C optionally substituted with H or 1 to 3 OH 2~5 alkyl, and R 2 is C substituted with H or 1 to 3 additional OH 2~5 alkylene-OH, and each R 3 independently, C 6~24 Alkyl or C 6~24 As used herein, "alkyl" refers to straight- and branched-chain saturated hydrocarbon groups containing 1 to 30 carbon atoms, e.g., 1 to 24 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms). n The term "alkyl" means that the alkyl group has "n" carbon atoms. For example, C3 alkyl refers to an alkyl group having 3 carbon atoms. C 1~24Alkyl refers to the entire range (i.e., 1-24 carbon atoms) as well as all subgroups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, , 2-24, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-21, 3-22, 3-23, 3-24, 4-5, 4-6, 4-7, 4-8, 4-9 , 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 4-21, 4-22, 4-23, 4-24, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16 6, 5-17, 5-18, 5-19, 5-20, 5-21, 5-22, 5-23, 5-24, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 6-21, 6-22, 6-23, 6-24, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20, 7-21, 7-22, 7-23, 7-24, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16 6, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 10-11, 1 0~12, 10~13, 10~14, 10~15, 10~16, 10~17, 10~18, 10~19, 10~20, 10~21, 10~22, 10~23, 10~24, 11~12, 11~13, 11~14, 11~15, 11~16, 11~17, 11~18, 11~19,11-20, 11-21, 11-22, 11-23, 11-24, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21 1, 13-22, 13-23, 13-24, 14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 16-17, 16-18, 16 ~19, 16~20, 16~21, 16~22, 16~23, 16~24, 17~18, 17~19, 17~20, 17~21, 17~22, 17~23, 17~24, 18~19, 18~20, 18~21, 18~22, 18~23, 18~24, 19~20, 19~21, 19~22, 19~23, 19~24, 20~21, " refers to an alkyl group having a number of carbon atoms inclusive of 20-22, 20-23, 20-24, 21-22, 21-23, 21-24, 22-23, 22-24, 23-24, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), and t-butyl (1,1-dimethylethyl). Unless otherwise specified, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group. As used herein, "alkenyl" refers to straight- and branched-chain hydrocarbon groups having a double bond and containing 2 to 30 carbon atoms, e.g., 2 to 24 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms). n The term "alkenyl" means that the alkenyl group has "n" carbon atoms. For example, C3 alkenyl refers to an alkenyl group having 3 carbon atoms. C2-C 24Alkenyl refers to the entire range (i.e., 2-24 carbon atoms) as well as all subgroups (e.g., 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-21, 3-22, 3-23, 3-24, 4 ~5, 4~6, 4~7, 4~8, 4~9, 4~10, 4~11, 4~12, 4~13, 4~14, 4~15, 4~16, 4~17, 4~18, 4~19, 4~20, 4~21, 4~22, 4~23, 4~24, 5~6, 5~7, 5~8, 5~9, 5~10, 5~11, 5~12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-21, 5-22, 5-23, 5-24, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20 0, 6-21, 6-22, 6-23, 6-24, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20, 7-21, 7-22, 7-23, 7-24, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 11-12, 11-13, 11-14, 11-15, 11-16, 1 1-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 13-14,13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 17-18, 17-19, 17-20, 17- 21, 17-22, 17-23, 17-24, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 19-20, 19-21, 19-22, 19-23, 19-24, 20-21, 20-22, 20-23, 20-24, 21-22, 21-23, 21-24, 22-23, 22-24, 23-24, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms). Non-limiting examples of alkenyl groups include ethenyl, propenyl, butenyl, geranyl, and oleyl. Unless otherwise specified, an alkenyl group can be an unsubstituted alkenyl group or a substituted alkenyl group.

[0037] In some implementations, n is between 2 and 5. In various implementations, n is between 3 and 4. In some implementations, n is 1. In various implementations, n is 2. In some cases, n is 3. In various cases, n is 4. In some implementations, n is 5. In various implementations, n is 6. In various cases, n is 7. In various cases, n is 8. In various cases, n is 9. In various cases, n is 10.

[0038] In some implementations, R 1 is H. In various implementations, R 1 is C optionally substituted with 1 to 3 OH 2~5 In some cases, R 1is methyl or ethyl. In some implementations, R 1 is ethyl. In various implementations, R 1 is a C substituted with 0 to 2 additional OH groups 2~5 alkylene-OH. In some cases, R 1 teeth,

[0039] [ka] (hydroxyethyl). In various cases, R 1 is ethyl or hydroxyethyl. 2~5 The alkyl is substituted with one OH. 2~3 The alkyl is substituted with two OH. 2~5 The alkyl is substituted with 3 OH groups.

[0040] R 2 is a C substituted with 1 to 3 additional OH groups 2~5 alkylene-OH. In some cases, R 2 is a C2 alkylene-OH substituted with 1 to 3 additional OH. 2 is a C3 alkylene-OH substituted with 1 to 3 additional OH. 2 is a C4 alkylene-OH substituted with 1 to 3 additional OH. 2 is a C alkylene-OH substituted with 1 to 3 additional OH. 2~5 The alkylene -OH is substituted with one additional OH. 2~5 The alkylene -OH is substituted with two additional OH. 2~5 The alkylene -OH is substituted with three additional OH. 2 is propyl-1,3-diol. In some cases, R 2 teeth,

[0041] [ka] is.

[0042] In some implementations, each R 3 independently, C 8~18 Alkyl or C 8~18 In various implementations, each R is an alkenyl. 3 independently, C 8~16 Alkyl or C 10~18 alkenyl. In some cases, each R 3 independently, C 6~18 Alkyl or C 6~18 alkenyl. In some cases, each R 3 independently, C 10~12 Alkyl or C 10~18 In some implementations, each R is an alkenyl. 3 independently, C 10~18 Alkyl or C 8~16 Alkyl or C 8~14 Alkyl or C 8~12 In various implementations, each R 3 is, independently,

[0043] [ka] Optionally, each R3 is independently selected from the group consisting of:

[0044] [ka] In various cases, each R is selected from the group consisting of 3 is, independently,

[0045] [ka] In some implementations, each R 3 is, independently,

[0046] [ka] is.

[0047] Contemplated compounds of formula (I) include, but are not limited to, those listed in Table 1.

[0048] [Table 1-1]

[0049] [Table 1-2]

[0050] [Table 1-3]

[0051] [Table 1-4]

[0052] [Table 1-5]

[0053] [Table 1-6]

[0054] [Table 1-7]

[0055] [Table 1-8]

[0056] [Table 1-9]

[0057]

Table 1-10

[0058]

Table 1-11

[0059]

Table 1-12

[0060]

Table 1-13

[0061]

Table 1-14

[0062]

Table 1-15

[0063]

Table 1-16

[0064]

Table 1-17

[0065]

Table 1-18

[0066]

Table 1-19

[0067] [Table 1-20]

[0068] [Table 1-21]

[0069] [Table 1-22]

[0070] [Table 1-23]

[0071] [Table 1-24]

[0072] The properties of the peptoids disclosed herein can be compared favorably with the properties of other peptoids, including the comparative peptoids disclosed in Table C.

[0073] [Table 2-1]

[0074] [Table 2-2]

[0075] [Table 2-3]

[0076] [Table 2-4]

[0077] [Table 2-5]

[0078] [Table 2-6]

[0079] [Table 2-7]

[0080] In some implementations, the compound of Formula (I) is Compound 1, 6, 21, or 30. In some cases, the compound of Formula (I) is Compound 1. In some cases, the compound of Formula (I) is Compound 6. In some cases, the compound of Formula (I) is Compound 21. In some cases, the compound of Formula (I) is Compound 30.

[0081] The compounds of the present disclosure are defined herein by their chemical structure and / or chemical name. If a compound is referred to by both its chemical structure and chemical name, and the chemical structure and chemical name conflict, the chemical structure shall be determinative of the identity of the compound.

[0082] Unless otherwise indicated, structures depicted herein are also intended to include all isomeric (e.g., enantiomers, diastereomers, cis-trans, conformational, and rotamer) forms of the structure. For example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in the disclosure unless only one of the isomers is specifically indicated. Thus, single stereochemical isomers of the present compounds as well as mixtures of enantiomers, diastereoisomers, cis / trans isomers, conformational, and rotamer forms are within the scope of the disclosure. In some cases, the compounds disclosed herein are stereoisomers. "Stereoisomer" refers to a compound that differs in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds disclosed herein can exist as single stereoisomers or as mixtures of stereoisomers. The stereochemistry of the compounds depicted herein denotes relative, not absolute, stereochemistry unless otherwise stated. As depicted herein, a single stereoisomer, single diastereomer, or single enantiomer refers to a compound that is at least 50% or more of the depicted stereoisomer, diastereomer, or enantiomer, and in some cases, at least 90% or 95% of the depicted stereoisomer, diastereomer, or enantiomer.

[0083] The compounds described herein can exist in free form or, where appropriate, as pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue adverse effects (e.g., toxicity, irritation, allergic response, etc.) and is commensurate 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 and organic acids and bases. These 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 salts of amino groups formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or by using other methods used in the art, such as, for example, ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutamate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. and the like. Examples of suitable salts include esters of acetone, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such salts include alkali metal salts, alkaline earth metal salts, aluminum salts, ammonium salts, N,N-dimethylformamide salts, and the like. + (C 1~4 Examples of suitable salts include, but are not limited to, salts of alkyl (alkyl) 4 salts and salts of organic bases, such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine. The present disclosure also contemplates the quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Water- or oil-soluble or dispersible products may be obtained by such quaternization. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0084] In implementations, the delivery vehicle composition comprises about 25 mole % to about 70 mole % of a hydroxyalkyl-capped cationic peptoid, e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid (e.g., a compound of Formula (I), such as Compounds 1, 6, 21, or 30), based on the total moles of the components in the delivery vehicle composition. The unit "mol %" or "mol percentage" 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%. Polyanionic cargo is not calculated as part of the total moles of the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 30 mol% to about 60 mol%, or 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 mol%, or about 38 mol%, or about 52 mol% of a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I), such as Compound 1, 6, 21, or 30), based on the total number of moles of components in the delivery vehicle composition.In some embodiments, the delivery vehicle composition comprises less than about 50 mol% hydroxyalkyl-capped cationic peptoid (e.g., 2-aminopropane-1,3-diol-capped cationic peptoid), e.g., 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 more than about 20 mol% hydroxyalkyl-capped cationic peptoid, based on the total number of moles of components in the delivery vehicle composition. hydroxyalkyl-capped cationic peptoids (e.g., 2-aminopropane-1,3-diol-capped cationic peptoids), for example, 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% hydroxyalkyl-capped cationic peptoids (e.g., 2-aminopropane-1,3-diol-capped cationic peptoids).In some embodiments, the delivery vehicle composition comprises less than about 50 mol% hydroxyalkyl-capped cationic peptoid (e.g., 2-aminopropane-1,3-diol-capped cationic peptoid), e.g., 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%, based on the total number of moles of components in the delivery vehicle composition. %, and greater than about 20 mol% hydroxyalkyl-capped cationic peptoids (e.g., 2-aminopropane-1,3-diol-capped cationic peptoids), e.g., 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% hydroxyalkyl-capped cationic peptoids (e.g., 2-aminopropane-1,3-diol-capped cationic peptoids). In some cases, the delivery vehicle composition comprises about 30 mol% to about 49.5 mol%, or 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 hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I), such as Compound 1, 6, 21, or 30), 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 number of moles of components in the delivery vehicle composition, about 30 mol% to about 49.5 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 hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I) such as Compound 1, 6, 21, or 30). In some cases, the delivery vehicle composition comprises, based on the total number of moles of components in the delivery vehicle composition, about 30 mol% to about 35 mol% of a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I) such as Compound 1, 6, 21, or 30). In some cases, the delivery vehicle composition comprises about 40 mole % to about 45 mole % of a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I) such as Compound 1, 6, 21, or 30), based on the total number of moles of the components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 35 mole % to about 39 mole % of a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I) such as Compound 1, 6, 21, or 30), based on the total number of moles of the components in the delivery vehicle composition. In various cases, the delivery vehicle composition comprises about 39 mole % to about 52 mole % of a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I), such as Compound 1, 6, 21, or 30), based on the total number of moles of components in the delivery vehicle composition.In some implementations, the delivery vehicle composition comprises about 42 mol % to about 49 mol % of a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I) such as Compound 1, 6, 21, or 30), based on the total number of moles of components in the delivery vehicle composition. In various implementations, the delivery vehicle composition comprises about 50 mol % to about 52 mol % of a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I) such as Compound 1, 6, 21, or 30), based on the total number of moles of components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, 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 hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) (e.g., a compound of Formula (I), such as Compound 1, 6, 21, or 30) based on the total number of moles of components in the delivery vehicle composition.

[0085] Anionic / Zwitterionic Components In some implementations, the delivery vehicle composition further comprises 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 cargo ratio and / or contributing to particle or delivery vehicle endosomal escape via protonation at low pH in the endosome. The zwitterionic component can serve the additional function of holding the particle together by interacting with both the hydroxyalkyl-capped cationic peptoid (e.g., 2-aminopropane-1,3-diol-capped cationic peptoid) and the polyanionic cargo compound. Anionic components can also enable the formation of core-shell structures of particles or delivery vehicles, where net positive zeta potential particles are first created (e.g., by mixing a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) with a cargo at a positive + / - charge ratio), which is then coated with anionic components. These negatively charged multicomponent particles avoid reticuloendothelial system (RES) clearance better than positively charged ones.

[0086] Examples of suitable anionic and zwitterionic components of the delivery vehicle composition are described in WO 2020 / 069442 and WO 2020 / 069445 (each of which is incorporated by reference in its entirety). In some implementations, the zwitterionic component comprises one or more phospholipids. Phospholipids can provide additional stabilization to the complex in solution and can facilitate cellular endocytosis due to their amphiphilic properties and ability to disrupt cell membranes.

[0087] In some implementations, the one or more phospholipids are selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-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-diundecanoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipentaerythritol ... choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-di Arachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-di The phospholipid is selected from the group consisting of linoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some cases, the phospholipid is DSPC, DOPE, or a combination thereof. In various implementations, the phospholipid is DSPC. In various cases, the phospholipid is DOPE.

[0088] In implementations, the delivery vehicle composition comprises about 1 mol% to about 40 mol% of a phospholipid (e.g., DSPC or DOPE) based on the total number of moles of components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 3 mol% to about 30 mol%, or 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 a phospholipid (e.g., DSPC or DOPE) based on the total number of moles of components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 10 mol% to about 11 mol% of a phospholipid (e.g., DSPC or DOPE) based on the total number of moles of components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises 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 a phospholipid (e.g., DSPC or DOPE) based on the total number of moles of components in the delivery vehicle composition.

[0089] Neutral lipid components In some implementations, the delivery vehicle composition further comprises 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 multi-component delivery system. Contemplated neutral lipid components include, for example, naturally occurring lipids and lipidated peptoids that include a lipid moiety at the N-position of the peptoid. Further examples of lipidated petoids are described in WO 2020 / 069442 and WO 2020 / 069445, each of which is incorporated herein by reference in its entirety.

[0090] In some embodiments, 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, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the sterol comprises cholesterol. In some implementations, the delivery vehicle composition comprises about 10 mol% to about 80 mol% of a sterol (e.g., cholesterol) based on the total number of moles of components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 20 mol% to about 70 mol%, or 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 a sterol (e.g., cholesterol) based on the total number of moles of the components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 40 mol% to about 55 mol%, or about 40 mol% to about 45 mol%, or about 50 mol% to about 55 mol% of a sterol (e.g., cholesterol) based on the total number of moles of the components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 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 a sterol (e.g., cholesterol) based on the total number of moles of components in the delivery vehicle composition.

[0091] shielding component In some implementations, the delivery vehicle composition further comprises a shielding component. The shielding component acts as a steric barrier, thereby increasing the stability of the particle or delivery vehicle in vivo and thereby improving its circulatory half-life. Examples of suitable shielding components are described in WO 2020 / 069442 and WO 2020 / 069445, each of which is incorporated herein by reference in its entirety.

[0092] 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 bound to a polyethylene glycol moiety. Suitable lipid moieties for PEGylated lipids can include, for example, branched or straight-chain aliphatic moieties, which can be unsubstituted or substituted, or moieties derived from natural lipid compounds, including fatty acids, sterols, and isoprenoids, which can be unsubstituted or substituted.

[0093] In some implementations, the lipid moiety can include a branched or straight-chain aliphatic moiety having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms. The aliphatic moiety, in some implementations, can include one or more heteroatoms and / or one or more double or triple bonds (i.e., saturated or mono- or polyunsaturated). In some implementations, the lipid moiety can include an aliphatic straight-chain or branched-chain moiety, where each hydrophobic tail independently has about 8 to about 30 carbon atoms or about 6 to about 30 carbon atoms, and the aliphatic moiety can be unsubstituted or substituted. In various implementations, the lipid moiety can include aliphatic carbon chains derived from, for example, fatty acids and fatty alcohols. In some implementations, each lipid moiety independently has a carbon number between C8 and C9. 24 Alkyl or C8-C 24 alkenyl, where C to C 24 Alkenyl can be optionally mono- or polyunsaturated.

[0094] Natural lipid moieties used in implementations of the present disclosure can be derived from, for example, phospholipids, glycerides (such as diglycerides or triglycerides), glycosylglycerides, sphingolipids, ceramides, as well as saturated and unsaturated sterols, isoprenoids, and other similar natural lipids.

[0095] Other suitable lipid moieties can include lipophilic aromatic groups such as optionally substituted aryl or arylalkyl moieties, including, for example, naphthalenyl or ethylbenzyl, or lipids containing ester functional groups, such as sterol esters and wax esters.

[0096] In some embodiments, 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 dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and any combination thereof. In some embodiments, the PEGylated lipid comprises a PEG-modified sterol. In various embodiments, the PEGylated lipid comprises a PEG-modified cholesterol. In some embodiments, the PEGylated lipid is a PEG-modified ceramide lipid. In some embodiments, the PEG-modified ceramide is selected from the group consisting of N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]} and N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]}, and any combination thereof.

[0097] In some implementations, the PEGylated lipid is a PEG-modified phospholipid, and the phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-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-diundecanoyl- sn-glycero-3-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-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine Phosphorus, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1 In various implementations, the phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0098] In some embodiments, the one or more PEGylated lipids comprise a PEG-modified phosphatidylethanol, hi some embodiments, the PEGylated lipid is a PEG-modified phosphatidylethanol selected from the group consisting of PEG-modified DMPE (DMPE-PEG), PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), and PEG-modified DOPE (DOPE-PEG).

[0099] In various implementations, the PEGylated lipid is selected from the group consisting of dimyristoylglycerol-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.

[0100] The molecular weight of the PEG chain in the PEGylated lipid can be adjusted as desired to optimize the properties of the delivery vehicle composition. In some implementations, the PEG chain has a molecular weight of 350-6000 g / mol, 1,000-5000 g / mol, 2,000-5000 g / mol, about 1,000-3,000 g / mol, or about 1,500-4,000 g / mol. In some cases, the PEG chain of the PEG lipid has a molecular weight of 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, or 10,000 g / mol. In some embodiments, the PEG chain of the PEGylated lipid has a molecular weight of about 500 g / mol, 750 g / mol, 1,000 g / mol, 2,000 g / mol, or 5,000 g / mol. The PEG chain can be branched or linear. In some cases, the PEGylated lipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000).

[0101] In implementations, the delivery vehicle composition comprises about 1 mol% to about 5 mol% of a PEGylated lipid (e.g., 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 about 1 mol% to about 3 mol%, or 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 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 (e.g., 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 about 1 mol% to about 3 mol%, or 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 (e.g., DMG-PEG2000) based on the total number of moles of components in the delivery vehicle composition. 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%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, or about 2.5 mol% of a PEGylated lipid (e.g., DMG-PEG2000) based on the total number of moles of components in the delivery vehicle composition.

[0102] Representative examples Non-limiting delivery vehicle combinations are described below: As previously noted, the unit "mol %" or "mol percentage" 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 multiplied by 100%.

[0103] In some implementations, the delivery vehicle composition comprises at least 99 mol% cationic component and less than about 1 mol% shielding component (e.g., Formula F1A in Table 2). In some cases, the delivery vehicle composition comprises less than about 20 mol% cationic component, less than about 5 mol% shielding component, and more than about 75 mol% mixture of anionic / zwitterionic component and neutral lipid component (e.g., Formula F2A and Formula F4A in Table 2). In some cases, the delivery vehicle composition comprises about 30 to about 45 mol% cationic component, about 50 to about 70 mol% mixture of anionic / zwitterionic component and neutral lipid component, and about 1.5 to about 4.5 mol% shielding component (e.g., Formula F3A and Formula F5A in Table 2). In various cases, 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 an anionic / zwitterionic component and a neutral lipid component, and about 1.5 to about 3.0 mol % of a shielding component (e.g., Formula F2A and Formula F3A in Table 2). In some implementations, the delivery vehicle composition comprises about 15 to about 35 mol % of a cationic component, about 10 to about 20 mol % of an anionic / zwitterionic component, about 50 to about 65 mol % of a neutral lipid component, and about 1.5 to about 3.0 mol % of a shielding component (e.g., Formula F2A and Formula 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 shielding component (e.g., Formula F4A in Table 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 shielding component (e.g., Formula F5A in Table 2). In various cases, the delivery vehicle composition comprises about 30 to about 50 mol % of a cationic component, about 50 to about 70 mol % of a neutral lipid component, and about 1 to about 5 mol % of a shielding component (e.g., Formula F6A in Table 2). In various cases, 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 anionic / zwitterionic and neutral lipid components, and about 1.5 to about 2.0 mol % of a shielding component (e.g., Formulas F6.1 and F6.2 in Table 2).In some implementations, the delivery vehicle composition comprises about 40 to about 45 mol % of a cationic component, about 10 to about 15 mol % of an anionic / zwitterionic component, about 40 to about 45 mol % of a neutral lipid component, and about 1.5 to about 2.0 mol % of a shielding component (e.g., Formulas F6.1 and F6.2 in Table 2). In various cases, 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 anionic / zwitterionic and neutral lipid components, and about 2.0 to about 3.0 mol % of a shielding component (e.g., Formula F6.3 in Table 2). In some implementations, the delivery vehicle composition comprises about 30 to about 35 mol % of a cationic component, about 10 to about 15 mol % of an anionic / zwitterionic component, about 50 to about 55 mol % of a neutral lipid component, and about 2.0 to about 3.0 mol % of a shielding component (e.g., Formula F6.3 in Table 2). The cationic component can be any cationic component described herein, such as any of the compounds of Formula (I) (e.g., compounds listed in Table 1, such as Compounds 1, 6, 21, or 30). In some implementations, the cationic compound is Compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some implementations, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some implementations, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some implementations, the shielding component is DMG-PEG2000.

[0104] In some embodiments, 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 a cationic component, about 3 mol% to about 20 mol% of an anionic / zwitterionic component, about 25 mol% to about 60 mol% of a neutral lipid compound, and about 1 mol% to about 5 mol% of a shielding component. In various embodiments, the delivery vehicle composition comprises about 35 mol% to about 55 mol% of a cationic component, about 5 mol% to about 15 mol% of an anionic / zwitterionic component, about 30 mol% to about 55 mol% of a neutral lipid compound, and about 1 mol% to about 3 mol% of a shielding component. In various embodiments, the delivery vehicle composition comprises about 38 to about 52 mol% of a cationic component, about 9 to about 12 mol% of an anionic / zwitterionic component, about 35 mol% to about 50 mol% of a neutral lipid compound, and about 1 mol% to about 2 mol% of a shielding component. In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 49 mol% of a compound of Formula (I), about 5 mol% to about 15 mol% of a phospholipid, about 30 mol% to about 55 mol% of a sterol, and about 1 mol% to about 3 mol% of a PEGylated lipid. In some embodiments, 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 a phospholipid, about 35 mol% to about 50 mol% of a sterol, and about 1 mol% to about 2 mol% of a PEGylated lipid. The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., compounds listed in Table 1, such as Compounds 1, 6, 21, or 30). In some implementations, the cationic compound is Compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some implementations, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some implementations, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some implementations, the shielding component is DMG-PEG2000.

[0105] In some embodiments, 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% of a neutral lipid compound, and about 1 mol% to about 5 mol% of a shielding component. In various embodiments, 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% of a neutral lipid compound, and about 1 mol% to about 3 mol% of a shielding component. In various embodiments, the delivery vehicle composition comprises about 38.2 mol% of a cationic component, about 11.8 mol% of an anionic / zwitterionic component, about 48.2 mol% of a neutral lipid compound, and about 1.9 mol% of a shielding component ("Formulation F2"). The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., compounds listed in Table 1, such as Compounds 1, 6, 21, or 30). In some implementations, the cationic compound is Compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some implementations, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some implementations, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some implementations, the shielding component is DMG-PEG2000. In some implementations, the delivery vehicle composition comprises Formulation F2 shown in Table 2 below. In some embodiments, the delivery vehicle composition comprises about 38.2 mol% of compound 1, 6, 21, or 30, about 11.8 mol% of DSPC, about 48.2 mol% of cholesterol, and about 1.9 mol% of DMG-PEG-2000.

[0106] In some embodiments, 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% neutral lipid compound, and about 1 mol% to about 5 mol% shielding component. In various embodiments, the delivery vehicle composition comprises about 48 mol% to about 52 mol% cationic component, about 5 mol% to about 12 mol% anionic / zwitterionic component, about 38 mol% to about 42 mol% neutral lipid compound, and about 1 mol% to about 3 mol% shielding component. In various embodiments, 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 component ("Formulation F6 / 17"). The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., compounds listed in Table 1, such as Compounds 1, 6, 21, or 30). In some implementations, the cationic compound is Compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some implementations, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some implementations, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some implementations, the shielding component is DMG-PEG2000. In some implementations, the delivery vehicle composition comprises Formulation F6 / 17, shown in Table 2 below. In some embodiments, the delivery vehicle composition comprises about 51.3 mol% of compound 1, 6, 21, or 30, about 9.3 mol% of DSPC, about 38.0 mol% of cholesterol, and about 1.5 mol% of DMG-PEG2000.

[0107] In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 49 mol% of the cationic component, about 5 mol% to about 15 mol% of the anionic / zwitterionic component, about 30 mol% to about 55 mol% of the neutral lipid compound, and about 1 mol% to about 3 mol% of the shielding component. In various embodiments, the delivery vehicle composition comprises about 48 mol% to about 52 mol% of the cationic component, about 5 mol% to about 12 mol% of the anionic / zwitterionic component, about 38 mol% to about 42 mol% of the neutral lipid compound, and about 1 mol% to about 3 mol% of the shielding component. In various embodiments, the delivery vehicle composition comprises about 42.6 mol% of the cationic component, about 10.0 mol% of the anionic / zwitterionic component, about 44.7 mol% of the neutral lipid compound, and about 1.7 mol% of the shielding component. The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., compounds listed in Table 1, such as Compounds 1, 6, 21, or 30). In some implementations, the cationic compound is Compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some implementations, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some implementations, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some implementations, the shielding component is DMG-PEG2000. In some implementations, the delivery vehicle composition comprises Formulation F6 / 12 or Formulation F6 / 15 shown in Table 2 below. In some embodiments, the composition comprises about 42.6 mol% of Compound 1, 6, 21, or 30, about 10.9 mol% of DSPC, about 44.7 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000. In some embodiments, the delivery vehicle composition comprises about 48.1 mol% of Compound 1, 6, 21, or 30, about 9.9 mol% of DSPC, about 40.4 mol% of cholesterol, and about 1.6 mol% of DMG-PEG 2000.

[0108] In some embodiments, the delivery vehicle composition comprises about 40 mol% to about 49 mol% of the cationic component, about 5 mol% to about 15 mol% of the anionic / zwitterionic component, about 30 mol% to about 55 mol% of the neutral lipid compound, and about 1 mol% to about 3 mol% of the shielding component. In various embodiments, the delivery vehicle composition comprises about 42 mol% to about 46 mol% of the cationic component, about 7 mol% to about 12 mol% of the anionic / zwitterionic component, about 41 mol% to about 45 mol% of the neutral lipid compound, and about 1 mol% to about 2 mol% of the shielding component. In various embodiments, the delivery vehicle composition comprises about 44.4 mol% of the cationic component, about 10.6 mol% of the anionic / zwitterionic component, about 43.3 mol% of the neutral lipid compound, and about 1.7 mol% of the shielding component. In various implementations, the delivery vehicle composition comprises about 44.4 mol% of a cationic component, about 10.6 mol% of an anionic / zwitterionic component, about 43.4 mol% of a neutral lipid compound, and about 1.7 mol% of a shielding component. The cationic component can be any cationic component described herein, such as any of the compounds of Formula (I) (e.g., compounds listed in Table 1, such as Compounds 1, 6, 21, or 30). In some implementations, the cationic compound is Compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some implementations, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some implementations, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some embodiments, the shielding component is DMG-PEG2000. In some embodiments, the delivery vehicle composition includes F6.1 or F6.2 as shown in Table 2 below. In some embodiments, the delivery vehicle composition includes about 44.4 mol% of Compound 1, 6, 21, or 30, about 10.6 mol% of DSPC, about 43.3 mol% of cholesterol, and about 1.7 mol% of DMG-PEG2000.In some embodiments, the delivery vehicle composition comprises about 44.4 mol% of compound 1, 6, 21, or 30, about 10.6 mol% of DSPC, about 43.4 mol% of cholesterol, and about 1.7 mol% of DMG-PEG2000.

[0109] In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 39 mol% of the cationic component, about 5 mol% to about 15 mol% of the anionic / zwitterionic component, about 30 mol% to about 55 mol% of the neutral lipid compound, and about 1 mol% to about 3 mol% of the shielding component. In various embodiments, the delivery vehicle composition comprises about 30 mol% to about 35 mol% of the cationic component, about 7 mol% to about 12 mol% of the anionic / zwitterionic component, about 50 mol% to about 55 mol% of the neutral lipid compound, and about 2 mol% to about 3 mol% of the shielding component. In various embodiments, the delivery vehicle composition comprises about 33.1 mol% of the cationic component, about 10.5 mol% of the anionic / zwitterionic component, about 53.8 mol% of the neutral lipid compound, and about 2.5 mol% of the shielding component. The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., compounds listed in Table 1, such as Compounds 1, 6, 21, or 30). In some implementations, the cationic compound is Compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some implementations, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some implementations, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some implementations, the shielding component is DMG-PEG2000. In some implementations, the delivery vehicle composition comprises F6.3 as shown in Table 2 below. In some embodiments, the delivery vehicle composition comprises about 33.1 mol% of compound 1, 6, 21, or 30, about 10.5 mol% of DSPC, about 53.8 mol% of cholesterol, and about 2.5 mol% of DMG-PEG2000.

[0110] Non-limiting examples of delivery vehicle compositions of the present disclosure based on a compound of formula (I) (e.g., Compounds 1, 6, 21, or 30) as the cationic component (characterized by mole %) can be found in Table 2 below.

[0111] [Table 3]

[0112] In some cases, the delivery vehicle composition is F6.1, F6.2, or F6.3. In some cases, the delivery vehicle composition is F1A, F2A, F3A, F4A, F5A, F6A, F1, F2, F3, F4, F5, F6 / 12, F6 / 15, or F6 / 17. In some cases, the delivery vehicle composition is D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30, D31, or D32.

[0113] Delivery Vehicle Complex (DV) The delivery vehicle compositions disclosed herein can form complexes with one or more polyanionic compounds (e.g., nucleic acids) through electrostatic interactions between the cationic component of the delivery vehicle composition and the polyanionic compound. Thus, a delivery vehicle complex refers to a mixture comprising a delivery vehicle composition disclosed herein and a polyanionic compound. The complexes can, in some cases, allow for high cargo loading, are stable, and exhibit excellent in vivo efficacy and tolerability. Thus, the delivery vehicle complexes are useful as delivery vehicles for transporting polyanionic cargoes encapsulated therein to target cells. Additionally or alternatively, the delivery vehicle complexes can include non-anionic cargoes. Thus, another aspect of the present disclosure relates to a delivery vehicle complex comprising (1) a delivery vehicle composition described hereinabove and (2) a polyanionic compound (or cargo). In some implementations, the delivery vehicle composition forms a complex with one polyanionic compound (e.g., one RNA). In various implementations, the delivery vehicle composition is complexed with two different polyanionic compounds (e.g., two different RNAs or RNA and DNA). In some implementations, the delivery vehicle composition is complexed with three or more different polyanionic compounds (e.g., three, four, or five different RNAs). In some cases, the multi-component delivery vehicle system is complexed with one or more nucleic acids selected from DNA and RNA (e.g., antigenic RNA and adjuvant DNA, e.g., CpG).

[0114] The delivery vehicle complexes described herein can be characterized by the relative mass ratio of one of the components of the delivery vehicle composition to the cargo (e.g., polyanionic compound) in the complex. The mass ratio of components in a delivery vehicle complex can be easily calculated based on the known concentrations and volumes of stock solutions of each component used in preparing the complex. Furthermore, when a non-anionic cargo is present in a delivery vehicle complex, the mass ratio can more accurately represent the relative amount of the delivery vehicle component to the total cargo than the cation-to-anion ratio (which does not take non-anionic materials into account). Specifically, the mass ratio of components refers to the ratio of the mass of this particular component in a system to the mass of the "cargo" in the system. "Cargo" can refer to the total polyanionic compound(s) present in a system. In one example, the polyanionic compound(s) can refer to nucleic acid(s). In one example, the polyanionic compound refers to mRNA encoding at least one protein.

[0115] In some implementations, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 0.5:1 to about 20:1, about 0.5:1 to about 10:1, 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, about 2:1 to about 20:1, about 2:1 to about 10:1, or about 2:1 to about 5:1. In some implementations, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 2:1 to about 5:1. In still other implementations, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 3:1. In other implementations, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 19:1. In other implementations, the cationic component and the polyanionic compound of the delivery vehicle complex have a mass ratio of about 20:1. In other implementations, the cationic component and the polyanionic compound of the delivery vehicle complex have a mass ratio of about 13:1. In other implementations, the cationic component and the polyanionic compound of the delivery vehicle complex have a mass ratio of about 10:1. In some implementations, the cationic component can be a compound of Formula (I), for example, a compound listed in Table 1 (e.g., Compounds 1, 6, 21, or 30).

[0116] In certain implementations where the delivery vehicle complex comprises a polyanionic compound or a nucleic acid as cargo, the mass ratio of cationic component to nucleic acid is about 0.5:1 to about 20:1, or about 0.5:1 to about 10:1, or about 0.5:1 to about 5:1, or about 1:1 to about 20:1, or about 1:1 to about 10:1, or about 1:1 to about 5:1, or about 2:1 to about 20:1, or about 2:1 to about 10:1, or about 2:1 to about 5:1. In certain implementations, the mass ratio of cationic component to nucleic acid is about 2:1 to 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 moiety to nucleic acid is about 13: 1. In other implementations, the mass ratio of cationic moiety to nucleic acid is about 10: 1. In some implementations, the cationic moiety can be a compound of Formula (I), e.g., a compound listed in Table 1 (e.g., compound 1, 6, 21, or 30).

[0117] In some implementations, the mass ratio of cationic component to nucleic acid is about 5:1 to about 25:1, or about 7:1 to about 20:1, or 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 moiety can be a compound of formula (I) described hereinabove, such as the compounds listed in Table 1. In various implementations, the cationic moiety is compound 1, 6, 21, or 30. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

[0118] In some embodiments, the mass ratio of the anionic / zwitterionic component to the polyanionic compound is about 2:1 to about 10:1, or about 2:1 to about 3:1, or about 2:1 to about 4:1, or about 5:1 to about 10:1. In some embodiments, the mass ratio of the anionic / zwitterionic component to the polyanionic compound is about 2:1 to about 10:1, or about 2:1 to about 3:1, or about 5:1 to about 10:1. In some implementations, the mass ratio of the anionic / zwitterionic component to the polyanionic compound is about 4:1. In some implementations, the mass ratio of the anionic / zwitterionic component to the polyanionic compound is about 2.7:1. In some implementations, the anionic / zwitterionic component can be a phospholipid, as described previously herein. In various implementations, the anionic / zwitterionic component is DOPE, DSPC, or a combination thereof. In some cases, the anionic / zwitterionic component is DSPC. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

[0119] In some embodiments, the weight ratio of the neutral lipid component to the polyanionic compound is about 5:1 to about 8:1, or 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 embodiments, the weight ratio of the neutral lipid component to the polyanionic compound is 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 weight ratio of the neutral lipid component to the polyanionic compound is about 5.4:1. In some implementations, the weight ratio of the neutral lipid component to the polyanionic compound is about 8.1:1. In some implementations, the weight ratio of the neutral lipid component to the polyanionic compound is about 6.7:1. In some implementations, the neutral lipid component can be a sterol as described herein. In various implementations, the neutral lipid component is cholesterol. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

[0120] In some cases, the weight ratio of the shielding component to the polyanionic compound is 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 weight ratio of the neutral lipid component to the polyanionic compound is about 2.1:1. In some implementations, the weight ratio of the neutral lipid component to the polyanionic compound is about 1.4:1. In some implementations, the shielding component can be a PEGylated lipid, as described previously herein. In various implementations, the shielding component is DMG-PEG2000. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

[0121] 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, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 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 1, 6, 21, or 30 at a mass ratio of about 10:1 to the nucleic acid, DSPC at a mass ratio of about 2.7:1 to the nucleic acid, cholesterol at a mass ratio of about 5.4:1 to the nucleic acid, and DMG-PEG2000 at a mass ratio of about 1.4 to the nucleic acid.

[0122] 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, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 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 1, 6, 21, or 30 in a mass ratio of about 17:1 to the nucleic acid, DSPC in a mass ratio of about 2.7:1 to the nucleic acid, cholesterol in a mass ratio of about 5.4:1 to the nucleic acid, and DMG-PEG2000 in a mass ratio of about 1.4 to the nucleic acid.

[0123] 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, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 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 1, 6, 21, or 30 in a mass ratio of about 12:1 to the nucleic acid, DSPC in a mass ratio of about 2.7:1 to the nucleic acid, cholesterol in a mass ratio of about 5.4:1 to the nucleic acid, and DMG-PEG2000 in a mass ratio of about 1.4 to the nucleic acid.

[0124] 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, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 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 1, 6, 21, or 30 in a mass ratio of about 15:1 to the nucleic acid, DSPC in a mass ratio of about 2.7:1 to the nucleic acid, cholesterol in a mass ratio of about 5.4:1 to the nucleic acid, and DMG-PEG2000 in a mass ratio of about 1.4 to the nucleic acid.

[0125] 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, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 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 1, 6, 21, or 30 in a mass ratio of about 13:1 to the nucleic acid, DSPC in a mass ratio of about 2.7:1 to the nucleic acid, cholesterol in a mass ratio of about 5.4:1 to the nucleic acid, and DMG-PEG2000 in a mass ratio of about 1.4 to the nucleic acid.

[0126] 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, a neutral lipid component and a polyanionic cargo in a mass ratio of about 8.1:1, and a shielding component and a polyanionic cargo in a mass ratio of about 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 1, 6, 21, or 30 in a mass ratio of about 19:1 to the nucleic acid, DSPC in a mass ratio of about 4.0:1 to the nucleic acid, cholesterol in a mass ratio of about 8.1:1 to the nucleic acid, and DMG-PEG2000 in a mass ratio of about 2.1 to the nucleic acid.

[0127] In some implementations, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 9.7, an anionic / zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1, a neutral lipid component and a polyanionic cargo in a mass ratio of about 6.7:1, and a shielding component and a polyanionic 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 1, 6, 21, or 30 in a mass ratio of about 9.7:1 to the nucleic acid, DSPC in a mass ratio of about 2.7:1 to the nucleic acid, cholesterol in a mass ratio of about 6.7:1 to the nucleic acid, and DMG-PEG2000 in a mass ratio of about 2.1 to the nucleic acid.

[0128] In yet other implementations, the amount of polyanionic cargo present in a delivery vehicle complex can be characterized by the mass ratio of the delivery vehicle composition (e.g., the total combined hydroxyalkyl-capped cationic peptoid, such as a 2-aminopropane-1,3-diol-capped cationic peptoid, phospholipid, cholesterol, and / or shielding component) to one or more polyanionic cargo compounds. In some implementations, the mass ratio of the delivery vehicle composition to one or more polyanionic cargo compounds is about 0.5:1 to about 20:1, about 0.5:1 to about 10:1, 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, about 2:1 to about 20:1, about 2:1 to about 10:1, or about 2:1 to about 5:1. In certain implementations, the mass ratio of the delivery vehicle composition to the one or more polyanionic cargo compounds is from about 5:1 to about 8:1 or from about 6:1 to about 7:1.

[0129] In some implementations, the compound of Formula (I) or salt thereof, the phospholipid, the sterol, and the PEGylated lipid are present in a mass ratio of about 20:1.8:7.2:1.8 ("D22"). In some cases, the phospholipid is DSPC. In some cases, the sterol is cholesterol. In some cases, the PEGylated lipid is DMG-PEG2000. In some cases, the compound of Formula (I) can be Compound 12, 34, 35, or 41.

[0130] 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 embodiments, the delivery vehicle complex comprises a cationic component and a nucleic acid at a cation to anion charge ratio of about 0.5:1 to about 20:1, about 0.5:1 to about 10:1, 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, about 2:1 to about 20:1, about 2:1 to about 10:1, or about 2:1 to about 5:1, or about 3:1 to about 8:1, or about 3:1 to about 7:1, or about 3:1 to about 4:1, or about 4:1 to about 5:1, or about 6:1 to about 7:1, or about 7:1 to about 8:1. In some implementations, the delivery vehicle complex comprises a cationic component and a nucleic acid at a cation to anion charge ratio of about 0.5:1 to about 20:1, about 0.5:1 to about 10:1, 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, about 2:1 to about 20:1, about 2:1 to about 10:1, or about 2:1 to about 5:1, or about 3:1 to about 7:1, or about 3:1 to about 4:1, or about 6:1 to about 7:1. In certain implementations, the delivery vehicle complex comprises a cationic component and a nucleic acid at a cation to anion charge ratio of about 2:1 to about 5:1. In still other implementations, the delivery vehicle complex comprises a cationic component and a nucleic acid at a cation to anion charge ratio of about 3:1. In some embodiments, the delivery vehicle complex comprises a cationic compound and a nucleic acid at a cation to anion charge ratio of about 3.7:1. In some embodiments, the delivery vehicle complex comprises a cationic compound and a nucleic acid at a cation to anion charge ratio of about 6.4:1. In some embodiments, the delivery vehicle complex comprises a cationic compound and a nucleic acid at a cation to anion charge ratio of about 4.8:1. In some embodiments, the delivery vehicle complex comprises a cationic compound and a nucleic acid at a cation to anion charge ratio of about 7.2:1. In some embodiments, the delivery vehicle complex comprises a cationic compound and a nucleic acid at a cation to anion charge ratio of about 3.6:1. In some embodiments, the cationic component is a compound of Formula (I), such as a compound listed in Table 1.For example, the compound of formula (I) can be compound 1, 6, 21, or 30.

[0131] Non-limiting examples of delivery vehicle compositions, characterized by mass ratio and charge ratio, can be found in Table 3 below.

[0132] [Table 4]

[0133] Characterization of delivery vehicle complexes The delivery vehicle complexes disclosed herein can be characterized by a variety of different parameters (e.g., particle size, polydispersity index, and cargo encapsulation rate). In one implementation, the delivery vehicle complexes resemble nanoparticles and include at least one polyanionic compound (described further below) encapsulated by a delivery vehicle composition. In one implementation, such complexes are mRNA nanoparticles that include a delivery vehicle composition encapsulating at least one mRNA.

[0134] In some implementations, the delivery vehicle complexes disclosed herein may have an average diameter of 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 delivery vehicle complexes disclosed herein can range in size from about 40 nm to about 200 nm in diameter, or from about 50 nm to about 175 nm, or from about 50 nm to about 200 nm, or from about 60 nm to about 150 nm, or from about 60 nm to about 100 nm, or from about 60 nm to about 90 nm, or from about 70 nm to about 125 nm, or from about 80 nm to about 100 nm, or from about 70 nm to about 90 nm, or from about 75 nm to about 95 nm, or from about 80 nm to about 110 nm, or from about 90 nm to about 125 nm, or from about 70 nm to about 90 nm. In other implementations, the complexes can have a size greater than about 100 nm in diameter, e.g., from about 105 nm to about 250 nm, from about 110 nm to about 220 nm, from about 150 nm to about 200 nm, or from about 110 nm to about 200 nm. In one implementation, the complexes may have a size of about 105 nm to about 200 nm in diameter. In some cases, the delivery vehicle complexes exhibit 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 various cases, the delivery vehicle complexes exhibit 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 complexes (e.g., via a microfluidic device or by hand). Particle size / diameter can be determined by dynamic light scattering (DLS), as described in Example 3.

[0135] In some implementations, the delivery vehicle complexes of the present 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.

[0136] In some embodiments, 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 (e.g., RNA) cargo is fully 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 3.

[0137] In some embodiments, the delivery vehicle complexes of the present 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 the polyanionic compound f after storage for at least 10 days, e.g., at least 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, or more, at 4° C. to 10° C. In one embodiment, the complexes retain the aforementioned levels of polyanionic compound f for 48 days at 4° C. Further, in some cases, the delivery vehicle complexes of the present 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 their original size after storage for at least 10 days, e.g., at least 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, or more, at 4° C. In one implementation, the delivery vehicle complexes retain the aforementioned size after storage at 4° C. or for 48 days.

[0138] The delivery vehicle conjugates disclosed herein are well tolerated at high and low doses without systemic toxicity or adverse events.

[0139] Other Delivery Vehicle Complex Components The delivery vehicle complexes described herein may contain additional components to fine-tune the complex for a particular application. Examples of components may include those that promote endosomal escape, including, but are not limited to, buffering amines or polyamines, nitrogen-containing heterocyclic and / or nitrogen-containing heteroaryl groups such as imidazoles, pyrroles, pyridines, pyrimidines, maleic acid derivatives, or membrane-lytic peptides.

[0140] The delivery vehicle complex may also optionally include a moiety on the surface of the system. The targeting moiety may be a peptide, an antibody mimic, a nucleic acid (e.g., an aptamer), a polypeptide (e.g., an antibody), a glycoprotein, a small molecule, a carbohydrate, or a lipid. Non-limiting examples of targeting moieties include peptides such as somatostatin, octreotide, LHRH, EGFR-binding peptides, RGD-containing peptides, protein scaffolds such as fibronectin domains, aptides or bidentate peptides, single-domain antibodies, stable scFvs, or bispecific T-cell engagers, nucleic acids (e.g., aptamers), polypeptides (e.g., antibodies or fragments thereof), glycoproteins, small molecules, carbohydrates, or lipids. The targeting moiety can be an aptamer, which is either RNA or DNA or an artificial nucleic acid; a small molecule; a carbohydrate, such as mannose, galactose, arabinose, etc.; a vitamin, such as ascorbic acid, niacin, pantothenic acid, carnitine, inositol, pyridoxal, lipoic acid, folic acid (folate), riboflavin, biotin, vitamin B12, vitamin A, E, and K; a protein or peptide that binds to a cell surface receptor, such as a receptor for thrombospondin, tumor necrosis factor (TNF), annexin V, interferon, cytokines, transferrin, GM-CSF (granulocyte-macrophage colony-stimulating factor), or a receptor for vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (BGF), or a peptide that binds to a cell surface receptor. The receptors may be receptors for growth factors such as bFGF, epidermal growth factor (EGF), and the like.

[0141] The delivery vehicle complex may also optionally include a small molecule drug or other biologic incorporated into the delivery vehicle complex. Non-limiting examples include incorporating a drug that disrupts the blood-brain barrier or enhances cellular uptake, a drug that affects intracellular trafficking or endosomal escape, or, when the delivery vehicle complex is used as a vaccine, a drug that is an immunomodulator that affects antigen presentation.

[0142] Polyanionic Compounds The delivery vehicle complexes of the present disclosure can include one or more polyanionic compounds (polyanionic cargos) that can be delivered by the complex to an in vivo target, such as a cell. The polyanionic compounds can be complexed with the cationic component of the delivery vehicle complex (e.g., a compound of Formula (I), such as Compounds 1, 6, 21, or 30) via electrostatic interactions.

[0143] In some implementations, the polyanionic compound comprises a nucleic acid. As used herein, nucleic acid includes naturally occurring nucleic acids (e.g., DNA, RNA, and / or hybrids thereof) and non-naturally occurring nucleic acids. Non-limiting examples of non-natural amino acids include those containing non-natural backbones, modified backbone linkages such as phosphorothioates, non-natural or modified bases, and / or non-natural and modified termini. 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, ribozymes, plasmids, and immunostimulatory nucleic acids.

[0144] In some embodiments, the polyanionic compound comprises RNA. The RNA may be selected from the group consisting of chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA, mRNA, oligoribonucleotide, 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, ribozyme, or any combination thereof. In some embodiments, the nucleic acid cargo is RNA, including, but not limited to, modified mRNA, self-amplifying RNA, and circular RNA. In some embodiments, the RNA comprises coding RNA.

[0145] RNA is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., 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 linked together along a so-called backbone. The backbone is formed by a phosphodiester bond between the first sugar (i.e., ribose) and the phosphate moiety of the second adjacent monomer. The specific order of the monomers, i.e., the order of the bases linked to the sugar / phosphate backbone, is called the RNA sequence. RNA is usually obtained by transcription of a DNA sequence, for example, within a cell. In eukaryotic cells, transcription typically occurs in the nucleus or mitochondria. In vivo, transcription of DNA usually produces so-called immature RNA (also called pre-mRNA, precursor mRNA, or heteronuclear RNA), which must usually be processed into so-called messenger RNA, abbreviated as mRNA. For example, in eukaryotes, the processing of immature RNA involves a variety of different post-transcriptional modifications, such as splicing, 5'-capping, polyadenylation, and export from the nucleus or mitochondria. The sum of these processes is also called RNA maturation. Mature messenger RNA usually provides a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Typically, mature mRNA contains a 5'-cap, optionally a 5'-UTR, an open reading frame, optionally a 3'UTR, and a poly(A) tail.

[0146] In addition to messenger RNA, there are several non-coding types of RNA that may be involved in regulating transcription and / or translation, and immune stimulation. Within the present 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 (drcRNA), ribozymes, aptamers, riboswitches, immunostimulatory / immunostimulatory RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

[0147] 5'-CAP structure: 5'-CAP is typically a modified nucleotide (CAP analog), particularly a guanine nucleotide, added to the 5' end of an mRNA molecule. In certain implementations, 5'-CAP is added using a 5'-5'-triphosphate linkage (also referred to as m7GpppN). Further examples of 5'-CAP structures include glyceryl, inverted deoxyabasic residues (moieties), 4',5' methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, and the like. Modified 5'-CAP structures include nucleotides such as 3'-3'-inverted nucleotide moieties, 3'-3'-inverted abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, or bridged or non-bridged methylphosphonate moieties. These modified 5'-CAP structures can be used in the context of the present disclosure to modify the RNA sequences of the present disclosure. Further modified 5'-CAP structures that may be used in the context of the present disclosure are CAP1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), CAP2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), CAP3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), CAP4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse CAP analog), modified ARCA (e.g., phosphothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0148] In the context of the present disclosure, the 5' cap structure can also be formed during chemical RNA synthesis or in vitro transcription of RNA (co-transcriptional capping) using a cap analog, or the cap structure can be formed in vitro using a capping enzyme (e.g., a commercially available capping kit).

[0149] A cap analog refers to a non-polymerizable dinucleotide that possesses a cap functionality in that, when incorporated at the 5' end of an RNA molecule, it facilitates translation or localization and / or prevents degradation of the RNA molecule. Non-polymerizable means that the cap analog is incorporated only at the 5' end because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase.

[0150] 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). 7 The synthesis of -(4-chlorophenoxyethyl) substituted dinucleotide cap analogues has recently been described.

[0151] A poly(A) tail, also referred to as a "3'-poly(A) tail" or "poly(A) sequence," is typically a long homopolymeric sequence of adenosine nucleotides, e.g., about 25 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, added to the 3' end of an mRNA. In certain implementations of the present disclosure, the poly(A) tail of an mRNA or srRNA is derived from a DNA template by in vitro transcription of the RNA. Alternatively, a poly(A) sequence can also be obtained in vitro by common methods of chemical synthesis, without necessarily being transcribed from a DNA precursor. Furthermore, a poly(A) sequence or poly(A) tail can be generated by enzymatic polyadenylation of RNA.

[0152] Stabilized nucleic acids typically exhibit modifications that increase resistance to in vivo degradation (e.g., degradation by exonucleases or endonucleases) and / or ex vivo degradation (e.g., degradation due to manufacturing processes prior to administration of the composition, e.g., degradation during preparation of the composition to be administered). RNA stabilization 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 modifications (e.g., use of synthetic backbones such as phosphorothioates) or modifying the G / C or C content of the nucleic acid. A variety of other methods for stabilizing nucleic acids or otherwise improving the function of nucleic acids are known in the art and are contemplated in the context of the present disclosure. Thus, provided herein are polynucleotides designed to improve one or more of: tissue stability and / or clearance, receptor uptake and / or kinetics, cellular access, engagement with the translation machinery, RNA half-life, translation efficiency, immune evasion, immune induction (in the case of vaccines), protein production capacity, secretion efficiency (where applicable), accessibility to circulation, protein half-life, and / or modulation of cellular state, function and / or activity.

[0153] The 5'-UTR is typically understood to be a specific section of RNA. It is located 5' to the open reading frame of an mRNA. In the case of srRNA, the open reading frame encodes viral nonstructural proteins, but the sequence of interest is encoded in a subgenomic fragment of the viral RNA. Therefore, the 5'-UTR is upstream of the nsP1 open reading frame. Furthermore, subgenomic srRNA RNAs have a 5'-UTR. Therefore, subgenomic RNAs containing a sequence of interest encoding a protein of interest contain a 5'-UTR. Typically, the 5'-UTR begins at 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 called regulatory elements). Such regulatory elements may be, for example, a ribosome binding site or a 5'-terminal oligopyrimidine tract. The 5'-UTR may be post-transcriptionally modified, for example, by the addition of 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 the sequence extending from the nucleotide located 3' to the 5'-CAP, and in certain implementations, from the 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, to the nucleotide located immediately 5' to the start codon of the protein-coding region. The nucleotide located immediately 3' to the 5'-CAP of a mature mRNA or srRNA typically corresponds to the transcription start site. The term "corresponding to" means that the 5'-UTR sequence can be an RNA sequence, such as an mRNA sequence used to define the 5'-UTR sequence, or a DNA sequence corresponding to such an RNA sequence. In the context of the present disclosure, the term "5'-UTR of a gene," such as "5'-UTR of the NYESO1 gene," refers to a sequence corresponding to the 5'-UTR of a mature mRNA derived from this gene, i.e., an mRNA obtained by transcription of the gene and maturation of the premature mRNA. The term "5'-UTR of a gene" encompasses the DNA and RNA sequences of the 5'-UTR.

[0154] Generally, the term "3'-UTR" refers to a portion of a nucleic acid molecule that is located 3' (i.e., "downstream") of an open reading frame and is not translated into a protein. Typically, a 3'-UTR is a portion of an RNA that is located between the protein-coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(A) sequence of an mRNA. In the context of the present disclosure, the term 3'-UTR can also include elements that are not encoded in the template from which the RNA is transcribed, but are added post-transcriptionally during maturation, such as a poly(A) sequence. The 3'-UTR of an RNA is not translated into an amino acid sequence.

[0155] With respect to srRNA, the 3'-UTR sequence is generally encoded by the viral genomic RNA, which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into a premature mRNA. The premature mRNA is then further processed into a mature mRNA in a maturation process, which includes 5'-capping. In the context of the present disclosure, the 3'-UTR corresponds to the sequence of a mature mRNA or srRNA (and srRNA subgenomic RNA), which is located between the stop codon of the protein-coding region, preferably the stop codon of the protein-coding region of the sequence of interest, and the poly(A) sequence of the mRNA. The term "corresponding to" means that the 3'-UTR sequence can be an RNA sequence, such as the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to such an RNA sequence. In the context of the present disclosure, the term "3'-UTR of a gene" is the sequence corresponding to the 3'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the premature mRNA. The term "3'-UTR of a gene" encompasses the DNA and RNA sequences of the 3'-UTR (both sense and antisense strands, and both mature and premature).

[0156] According to certain implementations of the present disclosure, RNA for use in the delivery vehicle complexes herein includes RNA containing at least one region encoding a peptide (e.g., a polypeptide), or a protein, or a functional fragment of the foregoing. As used herein, a "functional fragment" refers to a fragment of a peptide (e.g., a polypeptide) or a protein that retains the ability to induce 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 (e.g., a viral polypeptide), a viral protein, or a functional fragment of the foregoing. In various cases, 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 cases, the HPV protein is from HPV subtype HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68. In various cases, the HPV protein is from HPV subtype HPV16 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 an influenza hemagglutinin (HA) or a functional fragment thereof. In some implementations, the influenza A virus has an HA subtype 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.

[0157] Contemplated viruses from which the RNA of the delivery vehicle complex may encode include, but are not limited to, influenza A and B, poliovirus, adenovirus, rabies virus, bovine parainfluenza 3, human respiratory syncytial virus, bovine respiratory syncytial virus, canine parainfluenza virus, Newcastle disease virus, herpes simplex virus 1 and herpes simplex virus 2, human papillomavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, cytomegalovirus, and varicella zoster. Herpes 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, gammaherpesvirinae, avian alphaherpesvirus 1, Ebola virus, Marburg virus, alphavirus, flavivirus, yellow fever virus, dengue virus, Japanese encephalitis virus , West Nile virus, Zika virus, Venezuelan equine encephalomyelitis virus, Chikungunya virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Tick-borne encephalitis virus, Kyasanur Forest disease virus, Alkouma disease virus, Omsk hemorrhagic fever virus, Hendra virus, Nipah virus, Measles virus, Rubella virus, Human parvovirus B19, Smallpox, Alphavirus, Molluscum contagiosum virus, Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, Paramyxoviridae, Togaviridae, Flavivirus, Colorado tick fever virus (coltivirus), coxsackievirus, rotavirus, norovirus, astrovirus, adenovirus, human metapneumovirus, rhinovirus, or coronavirus (e.g., SARS-CoV, SARS-CoV-2, MERS-CoV, HCoVNL63, HKU1, 229E, and OC43), human papillomavirus, Ebola virus, Marburg virus, alphavirus, flavivirus, yellow fever, dengue fever, Japanese encephalitis, West Nile virus, Zika virus, Venezuelan equine encephalomyelitis virus,Chikungunya virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Tick-borne encephalitis, Kyasanur Forest disease, Alkooma disease, Omsk hemorrhagic fever, Hendra virus, Nipah virus, Measles virus, Rubella virus, Human parvovirus B19, Human herpesvirus 6, Varicella-zoster 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 Herpesvirus 1, human herpesvirus 2, herpes simplex virus, gammaherpesviridae, avian alphaherpesvirus 1, smallpox, alphavirus, molluscum contagiosum virus, hepatitis A virus, hepatitis B virus, hepatitis C, hepatitis D, hepatitis E, poliovirus, Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, Paramyxoviridae, or Togaviridae, flavivirus (e.g., Zika virus), Colorado tick fever virus (coltivirus), coxsackievirus, rotavirus, norovirus s, astrovirus, adenovirus, influenza virus type A, human metapneumovirus, rhinovirus, coronavirus, varicella virus, adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah Forest virus, Bunyamwera virus, Lacrosse bunyavirus, snowshoe hare bunyavirus, long-tailed macaque herpesvirus, Chandipura virus, chikungunya virus, Cosavirus A, cowpox virus, coxsackievirus, Crimean Congo hemorrhagic fever virus, Dengue virus, Dori virus, Djugbe virus, Dubenhague virus, Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, European bat lyssavirus, GB virus C / G hepatitis virus, Hantaan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta 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 syncytial virus, human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos skomovirus Rivirus, Lake Victoria Marburg virus, Langat virus, Lassa virus, Rosedale virus, Louping ill 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, Pichin virus Devirus, Poliovirus, Punta Toro phlebovirus, Puumala virus, Rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Sarivirus A, Sicilian sandfly fever 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, Luctenovirus, Toscanavirus, Ukuniemivirus, Vaccinia virus, Varicella-zoster virus, Smallpox virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, 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, Anelloviridae (proposed family), Torque teno virus TTV, Bornaviridae,Borna disease virus BDV, Bunyaviridae, Aino virus, Cache Valley virus CW, Crimean-Congo hemorrhagic fever virus CCHF, Hantaan virus HTNV, Jamestown Canyon virus JCV, La Crosse virus LACV, Puumala virus, Rift Valley fever virus RVFV, Caliciviridae, Norovirus, San Miguel sea lion virus SMSV-5, Circoviridae, Bovine circovirus BCV = evolved strain of porcine circovirus type 2 PCV-2, Coronaviridae, Bovine coronavirus BCoV-1, Bovine torovirus B toV, Flaviviridae, bovine viral diarrhea virus BVDV, Japanese encephalitis virus JEV, Kyasanur Forest disease virus KFDV, louping ill virus, Murray Valley encephalitis virus MVE, St. Louis encephalitis virus SLEV, tick-borne encephalitis virus TBEV, Wesselsbron virus, West Nile virus (including Kunjin), Hepeviridae, hepatitis E virus HEV, Herpesviridae, bovine herpesvirus BHV-4, equine herpesvirus EHV-1, infectious bovine rhinotracheitis virus IBR=BHV-1, pseudorabies virus PRV, Orthomyxoviridae, Dorivirus, Influenza A virus, Thogotovirus THOV, Papillomaviridae, Bovine papillomavirus BPV, Paramyxoviridae, Bovine parainfluenza virus BPIV3, Bovine respiratory syncytial virus BRSV, Peste des petits ruminants virus PPRV, Rinderpest virus RPV, Parvoviridae, Bovine adeno-associated virus BAAV, Bovine hocovirus BHoV, Picornaviridae, Bovine enteroviruses BEV-1, BEV-2, Bovine kobuvirus BKV-1U-1 strain, Encephalomyocarditis virus EMC, Foot-and-mouth disease Viruses FMDV, Seneca Valley virus SW, Polyomaviridae, Bovine polyomavirus BPyV, Poxviridae, Araçatuba virus, Bovine papular stomatitis virus BPSV, Cantagalovirus, Cowpox virus, Pseudocowpox virus PCPV, Vaccinia virus, Reoviridae, Banna virus BAV, Bluetongue virus BTV, Epidemic hemorrhagic disease virus EHDV, Liaoning virus LNV, Reovirus, Rotavirus, Retroviridae, Bovine foamy virus BFV, Bovine leukemia virus BLV, Rhabdoviridae,Bovine ephemeral fever virus BEFV, rabies virus, vesicular stomatitis virus VSV, Togaviridae, Eastern equine encephalitis virus EEEV, Getah virus, Ross River virus RRV, Sindbis virus, Venezuelan equine encephalomyelitis virus VEE, Anelloviridae (proposed family), Torque teno virus TTV, Bunyaviridae, Crimean-Congo hemorrhagic fever virus CCHF, Hantaan virus HTNV, Jamestown Canyon virus JCV, La Crosse virus LCV, Caliciviridae, norovirus, San Miguel sea lion virus SMSV-5 , Sapovirus, Circoviridae, Porcine circovirus 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, Ileus virus, Japanese encephalitis virus JEV, Loop virus, Murray Valley encephalitis virus MVE, Powassan virus, Tick-borne encephalitis virus TBEV, Wesselsbron virus, West Nile virus W NV (including Kunjin), Hepeviridae, Hepatitis E virus HEV, Herpesviridae, Infectious bovine rhinotracheitis virus IBR = BHV-1, Porcine cytomegalovirus PCMV (B. Potts, personal communication), Pseudorabies virus PRV, Orthomyxoviridae, Avian influenza virus (H5N1), Swine influenza virus (H1N1, H1N2), Paramyxoviridae, Bovine parainfluenza virus BPIV3, Menangle virus MENV, Nipah virus NiV, Peste des petits ruminants virus PPRV, Rinderpest virus RPV, Tioma TIOV, Parvoviridae, Porcine Hocovirus PHoV, Porcine Parvovirus PPV, Picornaviridae, Encephalomyocarditis virus EMC, Foot-and-mouth disease virus FMDV, Porcine Enterovirus PEV-9PEV-10, Seneca Valley virus SW, Swine Vesicular Disease virus SVDV, Reoviridae, Bannavirus BAV, Reovirus, Rotavirus, Retroviridae, Porcine Endogenous Retrovirus PERV, Rhabdoviridae, Rabies virus, Vesicular Stomatitis virus VSV, Togaviridae, Eastern Equine Encephalitis virus EEEV, Getah virus,Ross River virus (RRV), or Venezuelan equine encephalomyelitis (VEE).

[0158] In some implementations, the RNA encodes an adenovirus, an alphavirus, a calicivirus (e.g., a calicivirus capsid antigen), a coronavirus polypeptide, a distemper virus, an Ebola virus polypeptide, an enterovirus, a flavivirus, a hepatitis virus (AE), a herpesvirus, an infectious peritonitis virus, a leukemia virus, a Marburg virus, an orthomyxovirus, a papillomavirus, a parainfluenza virus, a paramyxovirus, a parvovirus, a pestivirus, a picornavirus (e.g., a poliovirus), a poxvirus (e.g., a vaccinia virus), a rabies virus, a reovirus, a retrovirus, or a rotavirus. In certain implementations, the RNA encodes SARS-CoV-2, an HPV (e.g., E6 and / or E7 from HPV16 and / or HPV18), or an influenza (e.g., an influenza hemagglutinin (HA)).

[0159] In some implementations, delivery of two or more specific nucleic acids in combination can be particularly useful for therapeutic applications. For example, in some implementations, one or more polyanionic cargo compounds include a combination of sgRNA (single guide RNA) as a CRISPR sequence and mRNA encoding Cas9. In yet further implementations, the nucleic acid can also be complexed with a protein, such as a CRISPR / Cas9 ribonucleoprotein complex. In some cases, the multi-component delivery vehicle system is complexed with one or more nucleic acids selected from DNA and RNA (e.g., antigenic RNA and adjuvant DNA, e.g., CpG).

[0160] Polynucleotide Synthesis Methods for producing polynucleotides of predetermined sequences are well known. Solid-phase synthesis methods are known for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods for synthesizing DNA are also useful for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can be incorporated into polynucleotides as well.

[0161] Any method known in the art for producing RNA is contemplated herein for producing RNA. Exemplary methods for producing RNA include, but are not limited to, chemical synthesis and in vitro transcription.

[0162] In certain implementations, RNA for use in the methods herein is chemically synthesized. Chemical synthesis of relatively short fragments of oligonucleotides with defined chemical structures provides rapid and inexpensive access to 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 is most often performed in the opposite direction, i.e., 3' to 5'. In certain implementations, this process is implemented as solid-phase synthesis using the phosphoramidite method and phosphoramidite building blocks derived from protected nucleosides (A, C, G, and U) or chemically modified nucleosides.

[0163] In some embodiments, modifications are included in the modified nucleic acid or in one or more individual nucleosides or nucleotides. For example, modifications to a nucleoside may include one or more modifications to the nucleobase, sugar, and / or internucleoside linkage. In some embodiments, a polynucleotide having at least one modification includes a backbone portion containing the nucleobase, sugar, and internucleoside linkage of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-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-riboside-MP.

[0164] In other implementations having at least one modification, the polynucleotide comprises a backbone moiety containing nucleobases, sugars, and internucleoside linkages of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, or 5-bromo-cytidine-MP. Nucleoside and nucleotide modifications contemplated for use in this disclosure are known in the art.

[0165] To obtain the desired oligonucleotide, building blocks are sequentially coupled to a growing oligonucleotide chain on a solid phase in the order required by the product sequence in a fully automated process. Once chain assembly is complete, the product is released from the solid phase into solution, deprotected, and recovered. Because the number of errors increases with the length of the synthesized oligonucleotide, the occurrence of side reactions sets a practical limit to the length of synthetic oligonucleotides (up to approximately 200 nucleotide residues). The product is often isolated by HPLC, yielding the desired oligonucleotide in high purity.

[0166] In certain implementations, RNA is produced using in vitro transcription. The terms "RNA in vitro transcription" or "in vitro transcription" refer to a process in which RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as a template for the production of RNA transcripts. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which in certain implementations is a linearized plasmid DNA template. The promoter for controlling in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, and SP6 RNA polymerases. The DNA template for in vitro RNA transcription can be obtained by cloning a nucleic acid, particularly a cDNA corresponding to the respective RNA to be in vitro transcribed, and introducing it into an appropriate vector for in vitro transcription, such as a plasmid DNA. In one implementation of the present disclosure, the DNA template is linearized with an appropriate restriction enzyme before being transcribed in vitro. The cDNA can be obtained by reverse transcription of mRNA or chemical synthesis. Additionally, DNA templates for in vitro RNA synthesis can also be obtained by gene synthesis.

[0167] Methods for in vitro transcription are known in the art. Reagents used in the methods typically include: 1) a linearized DNA template bearing a promoter sequence with high binding affinity for its respective RNA polymerase, such as a bacteriophage-encoded RNA polymerase; 2) ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine, and uracil); 3) optionally, a cap analog as defined above (e.g., m7G(5')ppp(5')G(m7G)); 4) a DNA-dependent RNA polymerase (e.g., T7, T3, or SP6 RNA polymerase) capable of binding to the promoter sequence in the linearized DNA template; 5) optionally, an RNase inhibitor to inactivate any contaminating ribonucleases (RNases); 6) optionally, a pyrophosphatase to degrade pyrophosphate, which may inhibit transcription; and 7) Mg as a cofactor for the polymerase.2+ 8) Buffers to maintain an appropriate pH value, which may also contain antioxidants (e.g., DTT) and / or polyamines such as spermidine at optimal concentrations.

[0168] Methods for Making Delivery Vehicle Complexes The components of the delivery vehicle complex can be prepared by various physical and / or chemical methods to tailor their physical, chemical, and biological properties. These can include rapidly combining a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) in water or a water-miscible organic solvent with a desired polyanionic cargo compound (e.g., an oligonucleotide or nucleic acid) in water or an aqueous buffer. These methods can include simple mixing of the components by pipetting or microfluidic mixing processes, such as those involving a T-mixer, vortex mixer, or other chaotic mixing structure. In some implementations, the multi-component delivery system is prepared on a microfluidic platform.

[0169] It should be understood that the specific process conditions for preparing the delivery vehicle complexes described herein can be adjusted or selected as appropriate to provide the desired physical properties of the complex. For example, parameters for mixing the components of the delivery system complex that can affect the final composition can include, but are not limited to, the order of mixing, the temperature of mixing, the speed / rate of mixing, the flow rate, the physical dimensions of the mixing structure, the concentration of the starting solution, the molar ratio of the components, and the solvent used.

[0170] Formulation of the delivery vehicle complex 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 a uniform distribution of the components throughout the delivery particle.

[0171] In other cases, components can be added sequentially to generate a core-shell structure. For example, a cationic component can be added first to initiate particle condensation, followed by a lipid component to associate the particle surface with target cells, followed by a shielding component to prevent particle aggregation. For example, a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) can be premixed with a nucleic acid cargo to form the core structure. A lipid component (e.g., a lipid component containing phospholipids and cholesterol) can then be added to affect cell / endosomal membrane binding. Because the shielding component is primarily useful on the exterior of a multi-component delivery system, this component can be introduced last, so that it does not disrupt the internal structure of the system but rather provides a coating for the system after it has formed.

[0172] Additional components in the complexes and compositions (e.g., additional components of polymers, surfactants, targeting moieties, and / or excipients) may be mixed and combined with the remaining components before, during, or after the main component of the nucleic acid cargo, the cationic component, the lipid component, and the shielding component are combined.

[0173] 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 mixing a solution comprising the compound or salt of Formula (I) with a solution comprising the polyanionic compound.

[0174] Pharmaceutical Formulations and Modes of Administration Also provided herein is a pharmaceutical composition comprising a delivery vehicle complex of the present disclosure and an effective amount of one or more pharmaceutically acceptable excipients. "Effective amount" includes "therapeutically effective amount" and "prophylactically 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 to prevent and / or substantially reduce the likelihood of a disease or condition in a subject. As used herein, the terms "patient" and "subject" may be used interchangeably and refer to animals (e.g., dogs, cats, cows, horses, and sheep (i.e., non-human animals), as well as humans). A particular patient or subject is a mammal (e.g., a human). The terms "patient" and "subject" include male and female. As used herein, the term "excipient" means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than an active pharmaceutical ingredient (API), appropriately selected with regard to the intended form of administration and consistent with conventional pharmaceutical practice.

[0175] The conjugates of the present disclosure can be administered to a subject or patient in a therapeutically effective amount. The conjugates can be administered alone or as part of a pharmaceutically acceptable composition or formulation. Furthermore, the conjugates can be administered all at once, for example, by bolus injection, in multiple doses, or can be delivered substantially uniformly over a period of time. It should also be noted that the dose of the compound can vary over time.

[0176] The delivery vehicle conjugates and other pharmaceutically active compounds disclosed herein can be administered to a subject or patient by any suitable route, if desired, for example, orally, rectally, parenterally (e.g., intravenously, intramuscularly, or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, or as a buccal, inhalation, or nasal spray. Administration can be to provide a systemic effect (e.g., enteral or parenteral administration). All methods that can be used by those skilled in the art to administer pharmaceutically active agents are contemplated.

[0177] Compositions suitable for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous 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, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0178] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Microbial contamination can be prevented by adding various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[0179] The pharmaceutical compositions may be in the form of a sterile injectable solution, aqueous suspension, or oleaginous suspension. These suspensions can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents, as described above. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil, including synthetic mono- or diglycerides, may be used. Additionally, fatty acids, such as oleic acid, are used in the preparation of injectable solutions.

[0180] The composition for parenteral administration is administered in a sterile medium.Depending on the vehicle used and the drug concentration in the preparation, parenteral preparation can be either a suspension or a solution containing dissolved drug.For example, adjuvants such as local anesthetic, preservative and buffer can also be added to parenteral composition.

[0181] When the composition of the present disclosure is used as a vaccine, it may contain one or more immunoadjuvants. As used herein, the term "immunoadjuvant" refers to a compound or mixture of compounds that, when used in conjugation with an immunogen (e.g., a neoantigen), acts to accelerate, prolong, enhance, or modify the immune response. An adjuvant may be non-immunogenic when administered alone to a host, but when administered in conjunction with another antigen, enhances the host's immune response to that antigen. Specifically, the terms "adjuvant" and "immunological adjuvant" are used interchangeably in the present disclosure. Adjuvant-mediated enhancement and / or prolongation of the duration of the immune response can be assessed by any method known in the art, including, but not limited to, one or more of the following: (i) an increase in the number of antibodies produced in response to immunization with an adjuvant / antigen combination relative to the number of antibodies produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells that recognize the antigen or adjuvant; and (iii) an increase in the level of one or more cytokines. Adjuvants can 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 can depend on the antigen, vaccine, and route of administration.

[0182] In some implementations, adjuvants improve adaptive immune responses to vaccine antigens by modulating innate immunity or facilitating delivery and presentation. Adjuvants act directly or indirectly on antigen-presenting cells (APCs), including dendritic cells (DCs). Adjuvants may be ligands for Toll-like receptors (TLRs) and directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and scope of adaptive immunity. In other examples, adjuvants may signal through pro-inflammatory pathways to promote immune cell infiltration, antigen presentation, and effector cell maturation. This class of adjuvants includes inorganic salts, oil emulsions, nanoparticles, and polyelectrolytes, including colloids and molecular aggregates exhibiting complex heterogeneous structures. In one example, the composition further comprises pidotimod as an adjuvant. In another example, the composition further comprises CpG as an adjuvant.

[0183] The compounds of the present disclosure can be administered to a subject or patient at dosage levels ranging from about 0.1 to about 3,000 mg per day. For a normal adult weighing about 70 kg, a dosage ranging from about 0.01 to about 100 mg per kilogram of body weight is typically sufficient. The particular dosage and dosage range used can potentially depend on several factors, including the requirements of the subject or patient, the severity of the condition or disease being treated, and the pharmacological activity of the compound being administered. Determination of dosage ranges and optimal dosages for a particular subject or patient is within the skill of one of ordinary skill in the art.

[0184] How to use The delivery vehicle complexes disclosed herein can be used to deliver complex (or cargo) polyanionic compounds to cells. Accordingly, disclosed herein are methods for delivering polyanionic compounds, such as nucleic acids (e.g., RNA), to cells, comprising contacting the cells with a delivery vehicle complex or pharmaceutical composition disclosed herein. In some implementations, the cells can be contacted in vitro. In some implementations where the cells are contacted in vitro, the cells are HeLa cells. In other implementations where the cells are contacted in vivo, the multi-component delivery system of the present disclosure is administered to a mammalian subject. The mammalian subject may include, but is not limited to, a human or mouse subject. In yet other implementations where the cells are contacted ex vivo, the cells are obtained from a human or mouse subject. Optionally, the cells are tumor cells. Optionally, the cells are muscle cells.

[0185] In some implementations, one or more polyanionic cargo compounds can be delivered for therapeutic purposes. Non-limiting therapeutic applications include cancer, infectious diseases, autoimmune disorders, and neurological disorders. In certain implementations, a complex comprising a multicomponent delivery system and a polyanionic cargo compound is used as a vaccine. Genetic vaccination, or the administration of nucleic acid molecules (e.g., RNA) to a patient and subsequent transcription and / or translation of the encoded genetic information, is useful for the treatment and / or prevention of not only inherited genetic diseases, but also autoimmune diseases, infectious diseases, cancerous or tumor-related diseases, and inflammatory diseases. Genetic vaccination is useful for the treatment or prevention of coronaviruses. The vaccine target for most of these entities is the coronavirus spike (S) protein, a highly glycosylated trimeric class I fusion protein that coats the virus and is responsible for entry into host cells. The S protein of SARS-CoV-2 shares high structural homology with SARS-CoV-1 and contains several subunits essential for viral entry into host cells via the angiotensin-converting enzyme 2 (ACE2) receptor, including the S1 domain, S2 domain, and receptor binding domain (RBD). Therefore, the S protein and its subunits, as well as accessible peptide sequences within these domains, are attractive vaccine antigen targets. Furthermore, gene vaccination is particularly useful in cancer treatment because tumors are generally not easily recognized and eliminated by the host, as evidenced by the development of disease, and cancer cells express antigens.

[0186] Vaccines. The delivery vehicle complexes of the present disclosure are also useful as vaccines, where the polyanionic compound is RNA that can encode an immunogen, antigen, or neoantigen. This provides the host immune system with a means to rapidly and specifically mount a defensive response against pathogenic microorganisms and contribute to the rejection of malignant tumors. The immune response has generally been described as comprising a humoral response, in which antigen-specific antibodies are produced by differentiated B lymphocytes, and a cell-mediated response, in which various types of T lymphocytes eliminate antigens by various mechanisms. For example, CD4 (also called CD4+) helper T cells, which can recognize specific antigens, can respond by releasing soluble mediators, such as cytokines, to recruit additional cells of the immune system to participate in the immune response. CD8 (also called CD8+) cytotoxic T cells can also recognize 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 important for the elimination of tumor cells and cells infected with microorganisms (e.g., viruses, bacteria, or parasites). The delivery vehicle complexes of the present disclosure have been shown to induce immune responses when one or more of the polyanionic compounds of the complex encodes a viral peptide (e.g., a viral polypeptide), a viral protein, or a functional fragment of the foregoing.

[0187] Accordingly, the present disclosure includes a method for inducing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a delivery vehicle complex of the present disclosure (e.g., formulated as an antigenic composition). Also disclosed herein is a method for treating a viral infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a delivery vehicle complex of the present disclosure. In some implementations, administration is by intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

[0188] In various implementations, administering a delivery vehicle complex of the present disclosure (e.g., formulated as a composition, pharmaceutical preparation, or antigenic composition) to a subject can result in an increase in the amount of antibodies (e.g., neutralizing antibodies) produced against a viral antigen in the subject compared to the amount of antibodies (e.g., neutralizing antibodies) produced in a subject that was not administered the delivery vehicle complex. 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.

[0189] The immune response elicited by the methods of the present disclosure generally includes antibody responses, preferably neutralizing antibody responses, T cell and B cell maturation and memory, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (GDC), and T cell-mediated responses such as CD4+ and CD8+. The immune response generated by the delivery vehicle complexes comprising RNA encoding a viral antigen disclosed herein generates an immune response that recognizes and preferably ameliorate and / or neutralize viral infection, as described herein. Methods for assessing antibody responses after administration (immunization or vaccination) of an antigenic composition are known in the art and / or described herein. In some implementations, the immune response includes a T cell-mediated response (e.g., a peptide-specific response, such as a proliferative response or a cytokine response). In some implementations, the immune response includes both a B cell response and a T cell response. The antigenic composition can be administered in several suitable ways, including intramuscular injection, intratumoral injection, subcutaneous injection, intradermal administration, and mucosal administration, e.g., oral or intranasal. Additional modes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal, intravaginal, intrarectal, and oral administration. Combinations of different routes of administration in an immunized subject, e.g., simultaneous intramuscular and intranasal administration, are also contemplated by the present disclosure.

[0190] Cancer. Various cancers (e.g., cervical cancer) can be treated with polyanionic cargo compounds delivered by the delivery vehicle complexes of the present disclosure. As used herein, the term "cancer" refers to any of a variety of 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 pathological conditions characterized by such malignant neoplastic growth. Cancer can be a tumor or a hematological malignancy, including, but not limited to, all types of lymphoma / leukemia, carcinoma, and sarcoma, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, bone marrow, tailbone, testicles, thyroid, and uterus.

[0191] By way of non-limiting example, carcinomas that may be treated include acute granulocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal carcinoma, adrenocortical carcinoma, anal carcinoma, anaplastic astrocytoma, angiosarcoma, appendix carcinoma, astrocytoma, basal cell carcinoma, B-cell lymphoma), bile duct carcinoma, bladder carcinoma, bone cancer, intestinal cancer, brain tumor, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct carcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, uterine Endometrial cancer, ependymoma, epithelial sarcoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid cancer, 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 cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, invasive / infiltrating breast cancer, pancreatic islet cell cancer, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, submeningeal metastasis, leukemia, lip cancer, lip cancer Liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous cell neck cancer, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, hordeolum, eye cancer, ocular melanoma, oligodendroglioma, oral cavity cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma, bone Sarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian sex cord-stromal tumor, Paget's disease, pancreatic cancer, papillary cancer, sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, microcytic astrocytoma, pineal tumor, pineoblastoma, pituitary cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, osteosarcoma, sarcoma, soft tissue cancer, uterine sarcoma, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal column cancer, spinal cord cancer, spinal cord tumor,These include squamous cell carcinoma, gastric cancer, synovial sarcoma, T-cell lymphoma), testicular cancer, pharyngeal cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsillar cancer, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, ureteral cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, endometrial cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0192] In some implementations, the delivery vehicle complexes of the present disclosure are 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, hi some implementations, the delivery vehicle complexes can be used to treat cervical cancer.

[0193] Infectious diseases. In some implementations, the delivery vehicle complexes of the present disclosure are used to treat infectious diseases, such as microbial infections, e.g., viral, bacterial, fungal, or parasitic infections. Non-limiting examples of infectious diseases include hepatitis (such as HBV or HCV infection), RSV, influenza, adenovirus, rhinovirus, or other viral infections.

[0194] Autoimmune Diseases. A variety of autoimmune and autoimmune-related diseases can be treated with the delivery vehicle complexes of the present disclosure. As used herein, the term "autoimmune disease" refers to a disease in which the body produces antibodies that attack its own tissues. As non-limiting examples, autoimmune diseases include acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agamaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, and antiphospholipid syndrome. syndrome, APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AlED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura. purpura, ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuropathic neuropathy, Baro's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome ** , chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed eosinophilic globulinemia, demyelinating neuritis, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans' syndrome, fibromyalgia **, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA) (formerly called Wegener's granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura purpura, ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosis, lichen conjunctivitis, linear IgA disease (LAD), lupus (SLE), chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease Mullen's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatoid arthritis, PANDAS (Streptococcal childhood autoimmune neuropsychiatric disorders), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuriahemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, peripheral squamous cell inflammation, pemphigus, peripheral neuropathy, periencephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyarthritis type I, II, III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, septicemia psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis The following conditions may be present: bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmopathy, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular dermatosis, vitiligo, and Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)).

[0195] Neurological Diseases. A variety of neurological diseases may be treated with the delivery vehicle system of the present disclosure. By way of non-limiting example, neurological diseases include septum pellucidum defect, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupil, Addie syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome, AIDS-neurological complications, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (AMLS), and the like. Sclerosis, ALS), anencephaly, aneurysm, Angelman syndrome, hemangiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger's syndrome, ataxia, ataxia-telangiectasia, ataxia and cerebellar or spinocerebellar degeneration, atrial fibrillation and stroke, attention deficit hyperactivity disorder, autism spectrum disorder, autonomic dysfunction, lower back pain, Barth syndrome, Batten disease, Becker's syndrome Toni, Behçet's disease, Bell's palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhard-Ross syndrome, Binswanger's disease, blepharospasm, Block-Sulzberger syndrome, brachial plexus birth injury, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal cord tumors, cerebral aneurysms, brain injury, Brown-Séquard syndrome, spinal-bulbar muscular atrophy, autosomal dominant cerebral arteriopathy with subcortical infarction and leukoencephalopathy Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, causalgia, cavernous hemangioma, cavernous malformation, central cervical spinal cord syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelinolysis, head disease, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous malformation, cerebral gigantism, cerebral hypoxia, cerebral palsy, cerebro-oculo-facio-skeletal syndrome (COFS),Charcot-Marie-Tooth disease, Chiari malformation, cholesterol ester storage disease, chorea, choreoacanthocytosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, colpocephaly, coma, complex regional pain syndrome, congenital facial paralysis, congenital myasthenia, congenital myopathy, congenital vascular cavernous malformation, corticobasal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob disease, cumulative trauma disease, Cushing's syndrome, cytomegalovirus inclusion body disease, dancing eyes-dancing feet syndrome, Dan De Walker syndrome, Dawson's disease, De Morcier syndrome, Degerine-Krampke palsy, dementia, dementia-multiple infarct, dementia-semantic, subcortical dementia, dementia with Lewy bodies, dentatocerebellar ataxia, dentatorubral atrophy, dermatomyositis, developmental dyspraxia, Devic's syndrome, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, autonomic dysfunction, dysgraphia, dyslexia, dysphagia, dyspraxia, myoclonic cerebellar dyscoordination, progressive cerebellar dyscoordination, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, brain inflammation, encephalitis lethargica, encephalocele, encephalopathy, encephalopathy (familial infantile), cerebral trigeminal angiomatosis, epilepsy, epileptic hemiplegia, Erb's palsy, Erb-Duchenne palsy and Degerines-Krampke palsy, essential tremor, extrapontine myelolysis, Fabry disease, Farr's syndrome, syncope, familial dysautonomia, familial hemangiomas, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber's disease, febrile seizures, fibromuscular dysplasia, Fisher's syndrome, floppy infant syndrome, foot drop, Friedreich's ataxia dementia, frontotemporal dementia, Gaucher disease, systemic gangliosidosis, Gerstmann syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, spherical cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head trauma, headache, persistent migraine, hemifacial spasm, alternating hemiplegia, hereditary neuropathy, hereditary spastic paraplegia, polyneuritis-like hereditary ataxia, shingles, herpes zoster oticus,Hirayama syndrome, Holmes-Addie syndrome, holoprosencephaly, HTLV-1-associated myelopathy, Hughes syndrome, Huntington's disease, hydranencephaly, hydrocephalus, normal pressure hydrocephalus, hydromyelopathy, hypercortisolism, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, infantile phytanic acid storage disease, infantile Refsum's disease, infantile convulsions, inflammatory myopathy, encephalopathy, intestinal lipodystrophy, intracranial cyst, intracranial hypertension, Isaacs syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbourne syndrome, Klein-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome Syndrome (KTS), Klüver-Bucy syndrome, Korsakoff amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, Kuru disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, Lateral femoral cutaneous nerve entrapment, Lateral bulbar syndrome, Learning disability, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, Leukodystrophy, Levin-Critchley syndrome, Dementia with Lewy bodies, Lipid storage disease, Lipoid proteinosis, Lissencephaly, Locked-ln syndrome, Lou Gehrig's disease, Lupus - neurological sequelae, Lyme disease - neurological complications, Machado-Joseph disease, Megaencephaly, Megalencephaly, Melkerson-Rosenthal syndrome, Meningitis, Meningitis and encephalitis, Menkes disease, Dysesthesias mergia, Metachromatic leukodystrophy, Microcephaly , migraine, Miller-Fisher syndrome, mini-stroke, mitochondrial myopathy, Moebius syndrome, monolimb muscular atrophy, motor neuron 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, spinal cord destructive diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, congenital myopathy, myopathy-thyrotoxicosis, myotonia, myotonia congenita, narcolepsy, neuroacanthosis, neurodegeneration due to cerebral iron accumulation, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological consequences of cytomegalovirus infection, neurological signs of Pompe disease,Neurological sequelae of lupus, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, hereditary neuropathy, neurosarcoidosis, neurosyphilis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, Ohtahara syndrome, olivopontocerebellar atrophy, blepharoclonic myoclonus, orthostatic hypotension, overuse syndrome, chronic pain, pantothenate kinase-associated neurodegeneration, paraneoplastic syndrome, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal migraine, Parry-Romberg disease, Pelizaeus-Merzbach disease, Pena-Chocaire II syndrome, perineural cyst, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytanic acid storage disease, Pick's disease, nerve compression, piriformis syndrome, pituitary tumor, polymyositis, Pompe disease, porencephaly, post-polio syndrome, postherpetic neuralgia, post-infectious encephalomyelitis , orthostatic hypotension, orthostatic dysregulation, orthostatic tachycardia syndrome, primary dentate atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacensis, progressive ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, pseudotorch syndrome, pseudotoxoplasmosis syndrome, pseudotumor cerebri, psychogenic movement disorder, Ramsay-Hunt syndrome I, Ramsay-Hunt syndrome II, Rasmussen's encephalitis, reflex Sympathetic dystrophy syndrome, Refsum's disease, Refsum's disease - childhood, repetitive motion disorder, repetitive stress disorder, restless legs syndrome, retroviral-associated myelopathy, Rett's syndrome, Reye's syndrome, rheumatoid encephalitis, Riley-Day syndrome, sacral nerve root cyst, St. Vitus's dance, salivary gland disease, Sandhoff's disease, Schilder's disease, schizencephaly, Seitelberger's disease, seizure disorders, semantic dementia, septo-optic dysplasia, severe myoclonic infantile epilepsy Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjögren's Syndrome, Sleep Apnea Syndrome, Sleeping Sickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumor, Spinal Muscular Atrophy, Spinocerebellar Atrophy, Spinocerebellar Degeneration, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis,Subcortical arteriosclerotic encephalopathy, Shortlasting, Unilateral, Neuralgiform (SUNCT), Headache, Dysphagia, Chorea minor, Syncope, Syphilitic Spinal Sclerosis, Hydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes dorsalis, Tardive Dyskinesia, Tarlov Cyst, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen's Myotonia, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic-Dourou, Todd's Palsy, Tourette's Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathy, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Troyer's Syndrome, Tuberous Sclerosis Complex, Vascular Erectile Tumors, Vasculitic Syndromes of the Central and Peripheral Nervous System, Von Economo Disease, Von Hippel-Lindau Disease Disease (VHL), von Recklinghausen's disease, Wallenberg syndrome, Werdnig-Hoffmann disease, Wernicke-Korsakoff syndrome, West syndrome, whiplash syndrome, Whipple's disease, Williams syndrome, Wilson's disease, Wolman's disease, and X-linked spinal-bulbar muscular atrophy.

[0196] In jurisdictions that prohibit the patenting of methods performed on the human body, the meaning of "administering" a composition to a human subject or patient shall be limited to prescribing a controlled substance to the human subject or patient to self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with the statute or regulation defining patentable subject matter is intended. In jurisdictions that do not prohibit the patenting of methods performed on the human body, "administering" a composition includes both the method and the aforementioned activities performed on the human body. [Example]

[0197] The following examples are provided for illustrative purposes and are not intended to limit the scope of the present disclosure.

[0198] Example 1 - General Synthesis of Hydroxyalkyl Cationic Peptoids To apply the delivery vehicles disclosed herein to the systemic expression of antibody-based drugs, the peptoid structure was optimized to maximize desired characteristics, including, but not limited to, liver selectivity and expression in mice. When producing secreted proteins, liver-selective expression is preferred for high-level production in circulating serum. A two-step strategy can be applied to optimize peptoid-based delivery systems. First, the optimal ionizable group is evaluated using the common lipophilic portion of the molecule, and then the lipid portion is separately optimized with its ionizable group. This approach takes advantage of the modularity of the peptoid structure. This allows for easy modification of the ionizable N-terminal domain while the peptoid core remains constant, and because interactions between the lipid and cationic portions are typically minimal, the entire molecule can be efficiently optimized without overlooking important lead candidates.

[0199] General protocols for synthesizing the cationic peptoids disclosed herein can be found in WO 2020 / 069442 and WO 2020 / 069445, each of which is incorporated by reference in its entirety. The following examples illustrate general protocols for the synthesis of cationic peptoids.

[0200] All polymers were synthesized via a submonomer approach using bromoacetic acid and primary amines. Fmoc-Rink amide resin was used as the solid support. The Fmoc group on the resin was deprotected with 20% (v / v) piperidine-dimethylformamide (DMF). The amino resin was then amidated with bromoacetic acid. Following amidation, amination of the α-carbon was achieved by nucleophilic displacement of the bromide with a primary amine. The two steps were repeated sequentially to generate the desired cationic peptide sequences.

[0201] All reactions and washes were performed at room temperature unless otherwise noted. Resin washing refers to adding the wash solvent (usually DMF or dimethyl sulfoxide (DMSO)) to the resin, stirring the resin to obtain a homogeneous slurry, and then allowing the solvent to completely drain from the resin. Solvent was removed by vacuum filtration through the fritted bottom of the reaction vessel until the resin appeared dry. In all syntheses, the resin slurry was stirred by bubbling argon through the bottom of the fritted vessel.

[0202] Initial resin deprotection. A fritted reaction vessel was loaded with Fmoc-Rink amide resin. DMF was added to the resin, and the solution was agitated to swell the resin. The DMF was then drained. The Fmoc group was removed by adding 20% ​​piperidine in DMF to the resin, agitating the resin, and draining the resin. 20% piperidine in DMF was added to the resin, agitated for 15 minutes, and then drained. The resin was then washed six times with DMF.

[0203] Acylation / amidation. The deblocked amine was then acylated by adding bromoacetic acid in DMF to the resin, followed by N,N'-diisopropylcarbodiimide (DIG) in DMF. The solution was stirred at room temperature for 30 minutes and then drained. This step was repeated twice. The resin was then washed twice with DMF and once with DMSO. This was one completed reaction cycle.

[0204] Nucleophilic Substitution / Amination. The acylated resin was treated with the desired primary or secondary amine to effect nucleophilic substitution at the bromine leaving group on the α-carbon. This acylation / substitution cycle was repeated until the desired peptide sequence was obtained.

[0205] Peptide cleavage from resin. The dried resin was placed in a glass scintillation vial containing a Teflon-coated microstir bar, and 95% trifluoroacetic acid (TEA) 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% TEA. The combined filtrate was then lyophilized three times from 1:1 acetonitrile:water. The lyophilized peptide was redissolved in 5% acetonitrile in water to a concentration of 5 mM.

[0206] Purification and characterization. The redissolved crude peptide was purified by preparative HPLC. The purified peptide was characterized by LC-MS analysis. In one specific case, aminolipidated peptoids were synthesized by the submonomer method using bromoacetic acid and N,N'-diisopropylcarbodiimide (DIG). Polystyrene-supported MBHA Fmoc-protected Rink amide resin (200 mg representative scale, 0.64 mmol / g loading) was used as the 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 (DIG) for 15 min. Amine substitution was performed using a 1 M solution of the amine in DMF for 45 min. After synthesis, the crude peptoid was cleaved from the resin using 5 mL of a 95:5 trifluoroacetic acid (TEA):water mixture at room temperature for 40 min. The resin was removed by filtration, and the filtrate was diluted with water to form a milky suspension, followed by addition of polystyrene resin for (heterogeneous) solid-phase extraction of the peptoid. The solution was mixed thoroughly and incubated at room temperature for 20 minutes. The solid-phase extracted peptoids were then released by the addition of ethanol and concentrated using vacuum centrifugation. The crude peptoids were further purified by reverse-phase flash chromatography using a C4 column and a gradient of 60-95% ACN / HO containing 0.1% TEA. Purity and identity were assayed using an LC-MS system consisting of a TOE mass spectrometer (LC / TOF). Chromatographic separation was performed at 40°C using a C8 column (2.1 x 100 mm). A gradient system was used at a flow rate of 0.2 ml / min. Initially, the mobile phase consisted of acetonitrile-water-formic acid (60:40:0.1) containing 10 mM ammonium formate. It was then linearly programmed over 11 minutes to isopropanol-acetonitrile-formic acid (80:20:0.1) containing 10 mM ammonium formate.

[0207] For this screen, a lipid block containing four dodecyl lipid (N-dodecylglycine) monomers was selected, and 12 peptoids containing these groups were synthesized on a solid-phase synthesizer using a submonomer approach. The resulting materials were purified, and their identities were confirmed by LC-MS. Varying both the number of lipid monomers and the total lipid carbon content was observed to have a significant effect on the hydrophobicity of the peptoid molecules, as measured by HPLC retention time. When fitted to a multiparameter model, both the calculated logP and empirical reverse-phase HPLC retention time increased as the number of lipid carbons increased, as expected for molecules with increasing lipid content. Furthermore, as the number of lipid monomers increased (while maintaining a similar total number of lipid carbons), the predicted logP and retention time decreased. This is likely due to the increased contribution of the hydrophilic amide backbone to the overall molecular polarity (i.e., peptoids with 6 × 6 carbon lipid monomers are much more hydrophilic than peptoids with 3 × 12 carbon monomers). Collectively, these parameters yielded a library of peptoids with a range of physical properties that were used for optimization.

[0208] Characterization data for the peptoids disclosed herein is provided in Table 4.

[0209] [Table 5]

[0210] Example 2 - Synthesis of Delivery Vehicle Conjugates Synthesis. Hydroxyalkyl-capped cationic peptoids (e.g., 2-aminopropane-1,3-diol-capped cationic peptoids) can be evaluated in vitro or in vivo for therapeutic and / or prophylactic purposes. Without being bound by any particular theory, the cationic moiety(s) of the amino-lipidated peptoid bind to the negatively charged phosphodiester backbone of a polyanionic cargo (e.g., a nucleic acid cargo) primarily through electrostatic interactions, forming a mixed coacervate complex. Hydrophobic interactions between lipid chains on the hydroxyalkyl-capped cationic peptoid (e.g., 2-aminopropane-1,3-diol-capped cationic peptoid) may act to stabilize microparticle formation and aid in membrane association.

[0211] Delivery vehicle complexes can be prepared by any physical and / or chemical method known in the art to tailor their physical, chemical, and biological properties. These methods typically involve rapidly combining a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) in water or a water-miscible organic solvent with an oligonucleotide in water or an aqueous buffer. These methods can include simple mixing of the components by pipetting, or microfluidic mixing processes such as those involving a T-mixer, vortex mixer, or other chaotic mixing structure.

[0212] In a standard formulation, a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) and additional lipid are dissolved in absolute ethanol at a concentration of 10 mg / mL, resulting in a solution stable 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 to a final concentration of 1-2 mg / mL. These solutions can be stored long-term at -20°C or -78°C.

[0213] To prepare the delivery vehicle compositions disclosed herein, a hydroxyalkyl-capped cationic peptoid (e.g., a 2-aminopropane-1,3-diol-capped cationic peptoid) and additional lipid components were first premixed in an ethanol phase at the required mass ratio. The nucleic acid cargo(s) were diluted with ethanol and an acidic buffer (10 mM phosphate / citrate, pH 5.0). The ethanol and aqueous phases were mixed in a 3:1 volume ratio and then immediately diluted with PBS to a 1:1 volume ratio to obtain a final mRNA concentration of 0.1 μg / uL. Non-limiting exemplary delivery vehicle compositions prepared by the aforementioned method include those listed in Table 2 above (e.g., compositions F2, F6 / 17, F6 / 12, and F6 / 15).

[0214] The delivery vehicle compositions were combined with a polyanionic compound, e.g., firefly luciferase (Fluc), RNA encoding the COVID-19 spike protein, functional fragments thereof, and / or variants thereof, or E6 / E7 oncogene (e.g., from HPV16, HPV18, functional fragments thereof, and / or variants thereof) in the ratios shown in Table 3 to form delivery vehicle complexes that were evaluated in vitro or in vivo for therapeutic and / or prophylactic purposes. In Table 3, w / w refers to the mass ratio of the indicated component to mRNA.

[0215] In one particular case, the peptoids disclosed herein were formulated into delivery vehicles by first dissolving them in ethanol with DSPC, cholesterol, and DMG-PEG2000 in a weight ratio of 20:1.8:7.2:1.8 (formulation D22 in Table 3 above), then mixing them with firefly luciferase (fLuc) mRNA dissolved in aqueous buffer at a volume ratio of 1:3 via microfluidic nanoprecipitation on a turbulent mixing device to obtain small, poorly dispersed particles. Physical particle properties were characterized. Delivery vehicles with initial sizes greater than 120 nm were not advanced to in vivo screening because the liver window primarily accommodates LNPs smaller than 100 nm in size. The in vivo expression of delivery vehicles containing peptoids with various head groups was assessed using fLuc expression in BALB / c mice 6 hours after IV administration (Figure 1). This panel of headgroup-modified peptoids showed significant differences in both total expression levels and organ selectivity, highlighting the importance of screening a wide chemical diversity in the ionic moiety of peptoids. Both the aminoethyl and aminopropyl headgroups exhibited preferential expression in the lung (Figure 2). This is likely due to the high basicity of the pendant primary amine, as groups with higher pKas are known to accumulate in that organ. Interestingly, the piperidinylpropyl and methoxypropyl headgroups shifted selectivity toward the spleen. These results provide evidence that simply tailoring the peptoid headgroup can result in a dramatic shift in selective expression. While spleen and lung selectivity are worthy of further optimization for future therapeutic applications, liver-selective targeting was used here for further studies to prepare antibodies for systemic delivery and maximize their serum expression with anti-RSV cargo. The 1,3-diol (apd) headgroup exhibited the highest liver expression and selectivity of all peptoids tested and was selected for further lipid block optimization.

[0216] The HiScribe® T7 High Yield RNA Synthesis Kit was used to transcribe mRNA from a DNA template encoding firefly luciferase. The reaction contained N instead of UTP. 1-methylpseudo-UTP (mΨTP) was used, and CleanCap® was used for co-transcriptional capping during IVT. The NTP concentrations used were calculated according to the template sequence, and a 4:1 Cap to GTP ratio was used in the IVT. The reaction was carried out at 37°C for 4 hours.

[0217] The DNA template was removed by TURBO™ DNase digestion at 37°C for 30 minutes in a 0.15 U / µL reaction volume. The transcription product was then purified using RNACIean™ XP beads. The RNA-bound beads were washed three times with freshly prepared 70% ethanol, air-dried for 15 minutes, and then eluted in 200 µL of nuclease-free water. The purified mRNA was quantified spectrophotometrically and analyzed using a Labchip® GX Touch™ nucleic acid analyzer.

[0218] Delivery vehicles were formulated by rapid microfluidic mixing using a high-throughput microfluidic system. Briefly, an ethanol solution containing peptoid, DSPC, cholesterol, and DMG-PEG2000 in a weight ratio of 20:1.79:7.16:1.84 (formulation D22 from Table 3 above) was mixed with synthetic mRNA in a 1:3 volume ratio in 10 mM sodium citrate buffer (pH 5.0). The peptoid-based LNPs were dialyzed overnight at 4°C against 1-2 liters of Tris-sucrose buffer (TS7, pH 7.4).

[0219] Apd lipid-block mutant peptoids were formulated into delivery vehicles, and their physical properties were characterized and analyzed. Overall, it was observed that monomer length and total carbon number significantly affected particle physical properties, and that the longest monomer and maximum total carbon number did not necessarily demonstrate the best performance, suggesting that the range of monomer lengths tested was sufficient. Particle size was found to be difficult to correlate with any single factor, except that particles were significantly larger when oleyl (Ole) monomers were used compared to 2-ethylhexyl (Ehx) monomers. The percentage of entrapped mRNA was highly dependent on both the total number of lipid carbons on the peptoid and the type of branched / unsaturated monomer used, with maximal encapsulation observed at approximately 55 total lipid carbons and with 2-ethylhexyl (Ehx) monomers. The pKa of the particles varied between pH 5 and 7, with a lower pKa observed with more monomers and a higher total carbon number. Additionally, we observed that increasing the number of branched monomers slightly decreased the pKa of the delivery vehicle, even when the pKa of the headgroup was similar. While conventional LNP materials achieve an optimal particle pKa of 6–6.5 by utilizing tertiary or secondary amines with predicted pKas of 9.5–10.5, Apd peptoids have a predicted pKa of approximately 5.5–5.7, which is very similar to their measured particle pKa of 5–7, indicating that the pKa does not shift upon formulation into lipid nanoparticle complexes. This may be due to several factors, including the hydrophilicity of the peptoid backbone, which provides a more favorable environment for amine protonation, or the packing dynamics of the peptoids within the particles, and will be the focus of further research. Fusogenicity, or the ability of LNPs to fuse with endosomal membranes and avoid degradation, has also been shown to vary depending on the lipid structure of the LNP. RBC hemolysis at neutral and acidic pH was used as a measure of fusogenicity. At low pH, which is most predictive of membrane interactions in endosomes, increasing branching / unsaturation leads to greater hemolysis at pH 5 for peptoids with fewer total monomers overall. Interestingly, the introduction of oleyl lipids was observed to frequently increase hemolysis at both pH 5 and 7.This suggests that oleyl-based peptoids may not be well tolerated due to background levels of hemolysis at neutral pH.

[0220] Example 3 - Particle characterization The resulting delivery vehicle complexes were evaluated by dynamic light scattering (DLS) to determine the volume average particle size / diameter (nm) and size polydispersity index (PDI) within the delivery vehicle complexes.

[0221] Particle size. Particle size and size distribution were measured using a Wyatt DynaPro® Plate Reader III. Typically, formulated samples were diluted to 2 ng / uL with 100 μL of PBS. Data are reported as the hydrodynamic diameter (in nm) of the cumulant fit of the correlation function and the polydispersity of the measurements.

[0222] Encapsulation rate. The percentage of mRNA encapsulated within delivery vehicle complexes containing Fluc mRNA was determined using a modified RiboGreen® assay. Typically, 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. 840 nm / Em. 520 nm, and the encapsulated mRNA was calculated by taking the ratio of fluorescence of undissolved particles to dissolved particles. Complexes containing increasing amounts of cationic components resulted in higher encapsulation.

[0223] Particle Stability: The delivery vehicle complexes were stored at 4° C., and the particle size and encapsulation efficiency of the resulting particles were determined after 17 and 48 days.

[0224] In certain cases, the size and polydispersity (PDI) of the delivery vehicle were measured by dynamic light scattering in PBS using a DynaPro® plate reader III. The zeta potential of the delivery vehicle was determined using a Nano-ZS™ particle analyzer. Zeta samples were diluted to 30 μM total lipid in a weak buffer solution of 10 mM HEPES, 10 mM MES, and 10 mM ammonium acetate (pH 7). The potential was manually set to 120 mV, and measurements were collected in duplicate at least 30 times.

[0225] The mRNA concentration and percentage of entrapped mRNA were determined using a fluorescence-based Quant-iT™ RiboGreen® RNA reagent kit using literature procedures. To determine the mRNA concentration, the delivery vehicle was incubated with 2% Triton™ X-100 for 10 minutes to release the captured mRNA, followed by the addition of RiboGreen® RNA reagent. The fluorescence intensity (excitation at 485 nm and emission at 530 nm) of the mRNA-RiboGreen® reagent complex was detected using a microplate reader. The ratio of fluorescence with and without Triton™ X-100 was used to calculate the percentage of entrapped mRNA.

[0226] The retention time and purity of the synthetic peptoids were determined using HPLC-UV, using UV absorbance at 214 nm and an injection volume of 3 μL. RP-HPLC was performed using a 1.7 μm 4-protein column (pore size: 300 Å, 2.1 (ID) × 100 mm) at 55 °C with a flow rate of 0.4 mL / min, using a mixture of solvent A and solvent B (solvent A: water / isopropanol (98 / 2, containing 0.1% v / v trifluoroacetic acid); solvent B: isopropanol / acetonitrile (80 / 20, containing 0.1% v / v trifluoroacetic acid)), running a gradient from 35% to 90% solvent B in 8 min, followed by a 4-min wash with 100% solvent B and a recovery to 35% solvent B.

[0227] The apparent particle pKa was assessed by TNS fluorescence assay. Briefly, particles were diluted to 130 μM total lipid in TNS buffers ranging from pH 3 to 11 with 10 mM TNS probe for 10 min. Fluorescence was measured at room temperature on a plate reader (excitation 320 / emission 445). Fluorescence intensity was normalized to the minimum and maximum values, and the pKa was determined by a three-parameter fit to determine the half-maximum of the curve.

[0228] RBC hemolysis was used as a measure of LNP fusogenicity at low pH. Sheep RBCs were washed with 2x PBS by pelleting at 800xg and resuspended at 5% v / v in either 20mM citrate, 140mM NaCl (pH 5.5) or PBS. Particles were diluted to 150mM total lipid with 5% RBC solution in wells and incubated at 37°C for 45 minutes. Supernatants were collected and absorbance was read at 425nm on a microplate reader. Fusion was normalized to fully lysed samples, including 2% Triton™ and buffer-only controls. Hemolysis (%) = 100x [(ABS T-background) - (ABS sample-background) / (ABS T-background)].

[0229] Characterization data for delivery vehicles containing peptoids disclosed herein are shown in Table 5. Characterization data for delivery vehicles containing comparative compounds C1-C13 are shown in Table 6.

[0230] [Table 6]

[0231] [Table 7]

[0232] Example 4 - Efficacy of Delivery Vehicle Complexes - Firefly Luciferase Expression in Vitro The efficacy of complexes comprising the delivery vehicle composition disclosed herein and mRNA encoding firefly luciferase (Fluc mRNA) is evaluated in vitro based on their ability to deliver the firefly luciferase (Fluc) reporter gene to cultured cells. 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 (total volume: 150 μL). The resulting luciferase expression was measured by a luminescence plate reader 6 and 18 hours after treatment. The delivery vehicle of the present disclosure showed high luciferase expression.

[0233] Example 5 - Luciferase Expression In Vivo General Methods. All animal studies were performed by Lumigenics LLC (Hercules, CA) under accreditation by the California State Department of Public Health and oversight by IACUC and Veterinarian Supervision. Prior to all studies, animals (e.g., mice and / or rats) were acclimated for a minimum of 3 days before use. Animals were maintained in a temperature- and humidity-controlled room on a 12-hour light cycle. Health checks and food and water checks were performed daily.

[0234] Injections were performed subcutaneously (50–200 μL), intraperitoneally (up to 1000 μL), intravenously (50–200 μL), intramuscularly (≥50 μL), or intratumorally (≥50 μL) using a 26–30 gauge needle depending on the injection site. Animals were under isoflurane anesthesia for all injections / implantations.

[0235] For imaging, animals were anesthetized, injected intraperitoneally with D-luciferin (15 mg / mL) at a dose of 10 μL per gram of body weight, placed in a camera chamber, and imaged for up to 30 minutes. Imaging was performed 15 minutes after substrate injection.

[0236] Intravenous administration. The delivery vehicle complexes disclosed herein are effective for administering Fluc mRNA to BALB / c mice in vivo via multiple routes of administration. Generally, the delivery system complexes were administered via tail vein injection at a dose of 0.5 mg / kg, and the resulting bioluminescence was quantified 6 hours later. Treated animals were sacrificed, the organs of interest were dissected, and the resulting bioluminescence was separately quantified to measure organ-specific bioluminescence levels.

[0237] 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 measured 6 hours later.

[0238] In one particular case, to investigate how the peptoid lipid block affected in vivo expression, delivery vehicles larger than 120 nm in diameter were excluded, and the remaining candidates were administered IV to BALB / c mice. Luminescence was measured 6 hours later, and the resulting expression was quantified in the liver, lung, and spleen. Even without changing the cationic headgroup, luciferase expression spanned four orders of magnitude as a result of altering the lipid block in this series. Compounds 12, 41, 34, and 35 emerged as top expressors from this screen (Figure 3). Interestingly, these peptoids are all six monomers long and contain an ethylhexyl (Ehx) branched monomer. The in vivo expression of all candidates was then fitted to a multiparameter model containing four input parameters to understand the individual contribution of each factor to overall performance. This model suggests that expression has a very strong quadratic dependence on both the number of total lipid carbons and the number of unsaturated or branched monomers, a strong dependence on whether the peptoid uses oleyl or Ehx as the unsaturated / branched monomer, and a weak dependence on the total number of lipid monomers. Contour plots of the effects of these factors on expression (all using Ehx as the branched / unsaturated monomer) are shown in Figure 4.

[0239] Based on this model, maximum predicted expression was achieved by using peptoids containing 5–6 total lipid monomers, 55 total lipid carbons, and 2–3 Ehx monomers as non-linear lipids. Notably, three of the four best-performing peptoids (compounds 41, 34, and 35) had attributes highly consistent with this prediction. The fourth top candidate (compound 12) had three of the four optimal attributes (number of lipid monomers, non-linear monomers, and Ehx as branched / unsaturated monomers), with the total number of lipid carbons slightly outside this range (Figure 4). This was promising and suggested that the utilized parameterization scheme fairly predicts the activity of peptoid-based ionic lipids. Trusting the lipid block optimization process, these top four candidates were advanced to further experiments to evaluate the in vivo production of secreted proteins.

[0240] For imaging studies, female BALB / c mice (6-8 weeks old) were obtained and allowed to acclimate for a minimum of 3 days before experimentation. Animals were maintained in a temperature- and humidity-controlled room with a 12-hour light cycle. Health checks and food and water checks were performed daily. For dosing, mice were intravenously injected with 2.5 μg of fLuc mRNA-containing delivery vehicle in a total injection volume of 100 μL. Six hours after injection, mice were anesthetized with isoflurane and intraperitoneally injected with 30 mg / mL D-luciferin at a dose of 10 μL / gram body weight. Imaging was performed 10 minutes after D-luciferin injection using an IVIS® Spectrum Imaging System. Immediately after in vivo imaging, mice were terminally bled by cardiac puncture and euthanized. Organs of interest were harvested, placed in black 24-well plates, and imaged. Living Image® software was used to quantify total photon flux in the region of interest. For secreted protein studies, mice were intravenously injected with aRSV mRNA-containing delivery vehicle in a total injection volume of 100 μL. Mice were euthanized 24 hours later, and serum was collected at 6 and 24 hours post-injection. Serum was used to quantify secreted aRSV levels using ELISA according to the manufacturer's recommendations for IgG, using a standard curve generated from aRSV proteins.

[0241] Cryo-TEM was also performed. 3 μL of approximately 1E14 particles / mL was applied to a clean grid (perforated carbon film on 400-mesh copper), blotted with filter paper, and then immediately vitrified in liquid ethane. Micrographs were acquired using a cryo-transmission electron microscope (cryo-TEM) operated at 200 kV, equipped with a direct electron detector and software for automated data collection. High-magnification images were acquired at nominal magnifications of 73,000× (0.200 nm / pixel) and 28,000× (0.524 nm / pixel). Images were acquired with a nominal underfocus of -5.5 μm to -3.5 μm and an electron dose of approximately 10 to 25 e / Å.

[0242] Example 6 - Efficacy and Toxicity Studies The compound of formula (I) was tested in mouse efficacy and rat toxicity studies. Briefly, Sprague-Dawley rats were treated with, for example, 0.03 or 0.3 mg / kg F2 or control mRNA formulated in PBS vehicle. Injections were administered intramuscularly into the hind leg four times over a 13-day period. Whole blood was collected for hematology 6 hours after the first and final doses, and 2 weeks after the final dose, and serum was collected for clinical chemistry and cytokine analysis. In addition, two animals were sacrificed 6 hours after the final dose and 2 weeks after the final dose, and gross necropsies were performed. Tissue samples were retained, and histopathological examinations were performed on selected organs.

[0243] Example 7 - Protein Expression Studies To understand how the delivery vehicles disclosed herein (e.g., delivery vehicles containing a compound of Formula (I)) function as systemic delivery vehicles, the top four candidates for firefly luciferase expression from the optimized DOE model (compounds 12, 41, 34, and 35) were evaluated for their ability to generate anti-RSV antibodies in BALB / c mice. Anti-RSV was selected as the model target because therapeutic benchmark information was available and the sequence was also available. Delivery vehicles containing aRSV were formulated from two mRNA molecules encoding heavy and light chains at a 2:1 mass ratio. All delivery vehicles were less than 120 nm in size and had encapsulation rates of greater than 85% (Table 7).

[0244] [Table 8]

[0245] Delivery vehicles were administered intravenously to BALB / c mice at a total dose of 0.75 mg / kg mRNA, and 24 hours later, serum aRSV levels were compared by IgG ELISA. Compounds 34 and 41 had the highest levels of secreted protein expression (Figure 5). Interestingly, compound 12, which had the highest overall fLuc expression, had the lowest aRSV expression among the top four candidates. This indicates that there is not a perfect correlation between the expression of intracellular proteins such as fLuc and the expression of secreted proteins such as aRSV. Further PK and dose characterization was performed on compound 41 formulated in formulation D22, which showed the highest overall aRSV titer (Figure 6). Using this candidate, aRSV titers were observed in serum after 5 days, and serum titers showed a generally linear dependence on the injected dose at 0.3, 0.75, and 1.5 mg / kg (Figure 7). The overall tolerability of the compound 41-D22 formulation was further investigated by dose escalation from 0.3 mg / kg to 7.5 mg / kg using fLuc cargo mRNA, and no immediate tolerability concerns were observed across this wide dose range (Figure 8).

[0246] The particle structure of the compound 41-D22 formulation was characterized by cryo-TEM, and its stability was monitored over time. The majority of compound 41-D22 particles exhibit a dense core morphology with no observable bleb characteristics (Figure 9). Stability was monitored after storage at 4°C or -80°C for over a month, and no changes in growth or encapsulation were observed (Figure 10). Taken together, the spherical, dense particle morphology, along with stability at 4°C and in frozen conditions, supports the practical use of the delivery vehicle disclosed herein for therapeutic applications.

[0247] We also investigated the effect of monomer lipid order on delivery vehicle performance. To achieve this, we synthesized compound 31, in which the order of the C12 and Ehx monomers in the lipid block was slightly altered compared to the monomers used in compound 41. Physical characteristics such as size, encapsulation, and hemolysis were all nearly identical between these two isomers (Table 5). In vivo, both luciferase expression and secreted protein expression were found to be within error for the two sequence variations. This suggests that monomer composition, rather than sequence, is responsible for the differences between peptoids (Figures 11 and 12). This further validates the screening funnel used and allows future peptoid mutations to consider sequence variations only as a secondary factor.

[0248] The foregoing disclosure demonstrates that peptoid-based lipid nanoparticles provide a highly tunable platform for optimizing mRNA delivery for both intracellular and secreted protein expression. Through extensive headgroup screening, tailoring the headgroup was found to alter delivery vehicle expression among major organs, including the lung, spleen, and liver. From this initial screening, the 1,3-diol headgroup with the highest liver selectivity was selected for lipid block optimization. The previously described parameterization strategy for lipid blocks identified the ideal combination of the number of lipid monomers, total lipid carbon count, number of branched or unsaturated monomers, and whether 2-ethylhexyl or oleyl monomers were used. The optimized delivery vehicle demonstrated greater than 90% liver selectivity and was used to generate sustained titers of aRSV in mouse serum, validating the platform for systemic delivery without observed tolerability concerns. Although further studies in higher species are underway to investigate tolerability, these data support delivery vehicles comprising the compounds disclosed herein (e.g., compounds of Formula (I)) as a demonstrable platform for mRNA therapeutic delivery that can be optimized for a given therapeutic target.

[0249] It should be appreciated that all combinations of the foregoing concepts and implementations, as well as additional concepts and implementations described in more detail below, are contemplated as part of the inventive subject matter disclosed herein and may be used in any suitable combination to achieve the benefits described herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein. The foregoing description has been given for clarity of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the present disclosure may be apparent to those skilled in the art.

[0250] As used throughout this specification, the terms "substantially" and "about" are used to describe and explain small variations, for example, they can refer to ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0251] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not to the exclusion of any other integer or step or group of integers or steps.

[0252] Throughout this specification, when a composition is described as comprising components or materials, it is contemplated that the composition can consist essentially of, or consist of, any combination of the listed components or materials, unless otherwise stated. Similarly, when a method is described as including particular steps, it is contemplated that the method can also consist essentially of, or consist of, any combination of the listed steps, unless otherwise stated. The disclosure illustratively disclosed herein may suitably be practiced in the absence of any element or step not specifically disclosed herein.

[0253] The methods disclosed herein and their individual steps can be performed manually and / or with the aid of, or using automation provided by, electronic devices. While described with reference to specific implementations, those skilled in the art will readily appreciate that other ways of performing the acts associated with the methods can be used. For example, the order of various steps can be changed without departing from the scope or spirit of the methods. In addition, some of the individual steps can be combined, omitted, or further subdivided into additional steps.

[0254] All patents, publications, and references cited herein are hereby incorporated by reference in their entirety. In the event of a conflict between the present disclosure and the incorporated patents, publications, and references, the present disclosure shall control.

Claims

1. A compound having the structure of formula (I): 【Chemical 1】 During the ceremony, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R １ is H or C optionally substituted with 1 to 3 OH ２～５ is alkyl, R ２ is a C substituted with 1 to 3 additional OH ２～５ alkylene-OH, Each R ３ are independently ６～２４ Alkyl or C ６～２４ A compound which is alkenyl.

2. 2. The compound of claim 1, wherein n is 3.

3. 2. The compound of claim 1, wherein n is 4.

4. 2. The compound of claim 1, wherein n is 6.

5. 2. The compound of claim 1, wherein n is 8.

6. 2. The compound of claim 1, wherein n is 9.

7. R １ The compound of any one of claims 1 to 6, wherein is H.

8. R ２ is replaced by one additional OH ２～５ The compound of any one of claims 1 to 7, which is alkylene-OH.

9. C ２～５ The compound of any one of claims 1 to 7, wherein the alkylene is substituted with 2 or 3 additional OH.

10. R ２ is substituted with 1 to 3 additional OH ３～４ 10. The compound of claim 8 or 9, which is alkylene.

11. Each R ３ But independently, C ６～１８ Alkyl or C ６～１８ The compound of any one of claims 1 to 10, which is alkenyl.

12. Each R ３ But independently, C ８～１８ Alkyl or C ８～１８ The compound of any one of claims 1 to 11, which is alkenyl.

13. Each R ３ But independently, 【Chemistry 2】 The compound according to any one of claims 1 to 12, selected from the group consisting of:

14. Each R ３ But independently, 【Chemistry 3】 The compound according to any one of claims 1 to 13, selected from the group consisting of:

15. At least one R ３ but, 【Chemistry 4】 The compound according to any one of claims 1 to 14, selected from the group consisting of:

16. At least one R ３ but, 【Chemistry 5】 The compound according to any one of claims 1 to 15,

17. 10. The compound of claim 1 having a structure listed in Table 1.

18. 18. The compound of claim 17 having the structure of Compound 1.

19. 18. The compound of claim 17 having the structure of compound 6.

20. 18. The compound of claim 17 having the structure of compound 21.

21. 18. The compound of claim 17 having the structure of compound 30.

22. 18. The compound of claim 17 having the structure of compound 12.

23. 18. The compound of claim 17 having the structure of compound 34.

24. 18. The compound of claim 17 having the structure of compound 35.

25. 18. The compound of claim 17 having the structure of compound 41.

26. A pharmaceutically acceptable salt of the compound of any one of claims 1 to 25.

27. A delivery vehicle composition comprising a compound according to any one of claims 1 to 25 or a salt according to claim 26.

28. 28. The delivery vehicle composition of claim 27, wherein the composition further comprises one or more of a phospholipid, a sterol, and a PEGylated lipid.

29. 30. The delivery vehicle composition of claim 28, wherein the composition comprises a phospholipid, a sterol, and a PEGylated lipid.

30. 29. The delivery vehicle composition of claim 28, wherein the composition consists essentially of a compound of any one of claims 1 to 25 or a salt of claim 26, a phospholipid, a sterol, and a PEGylated lipid.

31. 31. The delivery vehicle composition of any one of claims 27 to 30, wherein the compound or salt of formula (I) is present in an amount of about 30 mol % to about 60 mol %.

32. 32. The delivery vehicle composition of claim 31, wherein the compound or salt of formula (I) is present in an amount of about 35 mole % to about 55 mole %.

33. 32. The delivery vehicle composition of claim 31, wherein the compound or salt of formula (I) is present in an amount of about 30 mole % to about 45 mole %.

34. 32. The delivery vehicle composition of claim 31, wherein the compound or salt of formula (I) is present in an amount of about 35 mole % to about 39 mole %.

35. 32. The delivery vehicle composition of claim 31, wherein the compound or salt of formula (I) is present in an amount of about 39 mole % to about 52 mole %.

36. 32. The delivery vehicle composition of claim 31, wherein the compound or salt of formula (I) is present in an amount of about 30 mole % to about 35 mole %.

37. 32. The delivery vehicle composition of claim 31, wherein the compound or salt of formula (I) is present in an amount of about 40 mole % to about 45 mole %.

38. 32. The delivery vehicle composition of claim 31, wherein the compound or salt of formula (I) is present in an amount of about 42 mole % to about 49 mole %.

39. 32. The delivery vehicle composition of claim 31, wherein the compound or salt of formula (I) is present in an amount of about 50 mole % to about 52 mole %.

40. 32. The delivery vehicle composition of claim 31 , 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.

41. 41. The delivery vehicle composition of claim 40, 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.

42. 41. The delivery vehicle composition of claim 40, 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.

43. 32. The delivery vehicle composition of claim 31 , 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.

44. 44. The delivery vehicle composition of claim 43, 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.

45. 44. The delivery vehicle composition of claim 43, 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.

46. 44. The delivery vehicle composition of claim 43, 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.

47. 44. The delivery vehicle composition of claim 43, 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.

48. The phospholipid may be 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-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-diundecanoyl-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-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine , 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine 48. The delivery vehicle composition of any one of claims 28-47, wherein the glycerol-containing compound is selected from the group consisting of 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof.

49. 49. The delivery vehicle composition of claim 48, wherein the phospholipid is DOPE, DSPC, or a combination thereof.

50. 50. The delivery vehicle composition of claim 49, wherein the phospholipid is DSPC.

51. 51. The delivery vehicle composition of any one of claims 28 to 50, wherein the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

52. 52. The delivery vehicle composition of claim 51, wherein the sterol is cholesterol.

53. 53. The delivery vehicle composition of any one of claims 28 to 52, wherein the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified sterol, and PEG-modified phospholipid.

54. 54. The delivery vehicle composition of claim 53, wherein the PEG-modified lipid is selected from the group consisting of PEG-modified cholesterol, N-octanoyl-sphingosine-1-{succinyl[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), dimyristoylglycerol-polyethylene glycol (DMG-PEG), distearoylglycerol-polyethylene glycol (DSG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), dioleoylglycerol-polyethylene glycol (DOG-PEG), and combinations thereof.

55. 55. The delivery vehicle composition of claim 54, wherein the PEG-modified lipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000).

56. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 38.2 mol% Compound 1, about 11.8 mol% DSPC, about 48.2 mol% cholesterol, and about 1.9 mol% DMG-PEG2000.

57. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 42.6 mol% Compound 1, about 10.9 mol% DSPC, about 44.7 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

58. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 48.2 mol% Compound 1, about 9.9 mol% DSPC, about 40.4 mol% cholesterol, and about 1.6 mol% DMG-PEG2000.

59. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 51.3 mol% Compound 1, about 9.3 mol% DSPC, about 38 mol% cholesterol, and about 1.5 mol% DMG-PEG2000.

60. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% Compound 1, about 10.6 mol% DSPC, about 43.3 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

61. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% Compound 1, about 10.6 mol% DSPC, about 43.4 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

62. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 33.1 mol% Compound 1, about 10.6 mol% DSPC, about 53.8 mol% cholesterol, and about 2.5 mol% DMG-PEG2000.

63. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 38.2 mol% Compound 6, about 11.8 mol% DSPC, about 48.2 mol% cholesterol, and about 1.9 mol% DMG-PEG2000.

64. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 42.6 mol% Compound 6, about 10.9 mol% DSPC, about 44.7 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

65. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 48.2 mol% Compound 6, about 9.9 mol% DSPC, about 40.4 mol% cholesterol, and about 1.6 mol% DMG-PEG2000.

66. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 51.3 mol% Compound 6, about 9.3 mol% DSPC, about 38 mol% cholesterol, and about 1.5 mol% DMG-PEG2000.

67. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% Compound 6, about 10.6 mol% DSPC, about 43.3 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

68. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% Compound 6, about 10.6 mol% DSPC, about 43.4 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

69. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 33.1 mol% Compound 6, about 10.6 mol% DSPC, about 53.8 mol% cholesterol, and about 2.5 mol% DMG-PEG2000.

70. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 38.2 mol% Compound 21, about 11.8 mol% DSPC, about 48.2 mol% cholesterol, and about 1.9 mol% DMG-PEG2000.

71. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 42.6 mol% Compound 21, about 10.9 mol% DSPC, about 44.7 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

72. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 48.2 mol% Compound 21, about 9.9 mol% DSPC, about 40.4 mol% cholesterol, and about 1.6 mol% DMG-PEG2000.

73. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 51.3 mol% Compound 21, about 9.3 mol% DSPC, about 38 mol% cholesterol, and about 1.5 mol% DMG-PEG2000.

74. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% Compound 21, about 10.6 mol% DSPC, about 43.3 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

75. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% Compound 21, about 10.6 mol% DSPC, about 43.4 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

76. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 33.1 mol% Compound 21, about 10.6 mol% DSPC, about 53.8 mol% cholesterol, and about 2.5 mol% DMG-PEG2000.

77. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 38.2 mol% Compound 30, about 11.8 mol% DSPC, about 48.2 mol% cholesterol, and about 1.9 mol% DMG-PEG2000.

78. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 42.6 mol% Compound 30, about 10.9 mol% DSPC, about 44.7 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

79. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 48.2 mol% Compound 30, about 9.9 mol% DSPC, about 40.4 mol% cholesterol, and about 1.6 mol% DMG-PEG2000.

80. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 51.3 mol% Compound 30, about 9.3 mol% DSPC, about 38 mol% cholesterol, and about 1.5 mol% DMG-PEG2000.

81. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% Compound 30, about 10.6 mol% DSPC, about 43.3 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

82. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% Compound 30, about 10.6 mol% DSPC, about 43.4 mol% cholesterol, and about 1.7 mol% DMG-PEG2000.

83. 31. The delivery vehicle composition of any one of claims 27 to 30, comprising about 33.1 mol% Compound 30, about 10.6 mol% DSPC, about 53.8 mol% cholesterol, and about 2.5 mol% DMG-PEG2000.

84. A delivery vehicle complex comprising the delivery vehicle composition of any one of claims 20 to 83 and a polyanionic compound.

85. 85. The delivery vehicle complex of claim 84, wherein the compound of formula (I) or a salt thereof is complexed with the polyanionic compound.

86. 86. The delivery vehicle complex of claim 85, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 5:1 to about 25:

1.

87. 87. The delivery vehicle complex of claim 86, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 7:1 to about 20:

1.

88. 88. The delivery vehicle complex of claim 87, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 10:1 to about 17:

1.

89. 88. The delivery vehicle complex of claim 87, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 19:

1.

90. 88. The delivery vehicle complex of claim 87, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 20:

1.

91. 89. The delivery vehicle complex of claim 88, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 10:

1.

92. 89. The delivery vehicle complex of claim 88, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 12:

1.

93. 89. The delivery vehicle complex of claim 88, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 13:

1.

94. 89. The delivery vehicle complex of claim 88, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 15:

1.

95. 89. The delivery vehicle complex of claim 88, wherein the compound or salt of formula (I) and the polyanionic compound are present in a weight ratio of about 17:

1.

96. 96. The delivery vehicle complex of any one of claims 86 to 95, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 2:1 to about 10:

1.

97. 97. The delivery vehicle complex of claim 96, wherein the phospholipid and the polyanionic compound are present in a weight ratio of about 2:1 to about 4:

1.

98. 98. The delivery vehicle complex of claim 97, wherein the phospholipid and the polyanionic compound are present in a weight ratio of about 2:1 to about 3:

1.

99. 97. The delivery vehicle complex of claim 96, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 4:

1.

100. 98. The delivery vehicle complex of claim 97, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 2.7:

1.

101. 101. The delivery vehicle complex of any one of claims 86 to 100, wherein the sterol and the polyanionic compound are present in a mass ratio of about 5:1 to about 8:

1.

102. 102. The delivery vehicle complex of any one of claims 86 to 101, wherein the sterol and the polyanionic compound are present in a mass ratio of about 5:1 to about 6:

1.

103. 103. The delivery vehicle complex of claim 102, wherein the sterol and the polyanionic compound are present in a mass ratio of about 5.4:

1.

104. 102. The delivery vehicle complex of claim 101, wherein the sterol and the polyanionic compound are present in a mass ratio of about 8.1:

1.

105. 102. The delivery vehicle complex of claim 101, wherein the sterol and the polyanionic compound are present in a mass ratio of about 6.7:

1.

106. 106. The delivery vehicle complex of any one of claims 86 to 105, wherein the PEGylated lipid and the polyanionic compound are present in a mass ratio of about 0.5:1 to about 2.5:

1.

107. 107. The delivery vehicle complex of claim 106, wherein the PEGylated lipid and the polyanionic compound are present in a mass ratio of about 1:1 to about 2:

1.

108. 108. The delivery vehicle complex of claim 107, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 2.1:

1.

109. 108. The delivery vehicle complex of claim 107, wherein the phospholipid and the polyanionic compound are present in a mass ratio of about 1.4:

1.

110. 86. The delivery vehicle complex of claim 84 or 85, wherein the compound of formula (I) or salt thereof, phospholipid, sterol, and PEGylated lipid are present in a mass ratio of about 20:1.8:7.2:1.

8.

111. 110. The delivery vehicle complex of any one of claims 86-88, 91, 96, 98, 100-103, 106, 107, and 109, comprising Compound 1 having a mass ratio of about 10:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

112. 110. The delivery vehicle complex of any one of claims 86-88, 92, 96, 98, 103, 106, 107, and 109, comprising Compound 1 having a mass ratio of about 12:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

113. 110. The delivery vehicle complex of any one of claims 86-88, 94, 96-98, 103, 106, 107, and 109, comprising Compound 1 having a mass ratio of about 15:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

114. 110. The delivery vehicle complex of any one of claims 86-88, 95, 98, 103, 106, 107, and 109, comprising Compound 1 having a mass ratio of about 17:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

115. 110. The delivery vehicle complex of any one of claims 86-88, 93, 96-98, 103, 106, 107, and 109, comprising Compound 1 having a mass ratio of about 13:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

116. 109. The delivery vehicle complex of any one of claims 86, 87, 89, 96, 97, 99, 101, 103, and 106-108, comprising Compound 1 having a mass ratio of about 19:1 to the polyanionic compound, DSPC having a mass ratio of about 4:1 to the polyanionic compound, cholesterol having a mass ratio of about 8.1:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

117. 109. The delivery vehicle complex of any one of claims 86, 87, 91, 96-98, 100, 101, and 105-108, comprising Compound 1 having a mass ratio of about 9.7:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 6.7:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

118. 110. The delivery vehicle complex of any one of claims 86-88, 91, 96, 98, 100-103, 106, 107, and 109, comprising compound 6 having a mass ratio of about 10:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

119. 110. The delivery vehicle complex of any one of claims 86-88, 92, 96, 98, 103, 106, 107, and 109, comprising compound 6 having a mass ratio of about 12:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

120. 110. The delivery vehicle complex of any one of claims 86-88, 94, 96-98, 103, 106, 107, and 109, comprising compound 6 having a mass ratio of about 15:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

121. 110. The delivery vehicle complex of any one of claims 86-88, 95, 98, 103, 106, 107, and 109, comprising compound 6 having a mass ratio of about 17:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

122. 110. The delivery vehicle complex of any one of claims 86-88, 93, 96-98, 103, 106, 107, and 109, comprising compound 6 having a mass ratio of about 13:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

123. 109. The delivery vehicle complex of any one of claims 86, 87, 89, 96, 97, 99, 101, 103, and 106-108, comprising compound 6 having a mass ratio of about 19:1 to the polyanionic compound, DSPC having a mass ratio of about 4:1 to the polyanionic compound, cholesterol having a mass ratio of about 8.1:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

124. 109. The delivery vehicle complex of any one of claims 86, 87, 91, 96-98, 100, 101, and 105-108, comprising compound 6 having a mass ratio of about 9.7:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 6.7:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

125. 110. The delivery vehicle complex of any one of claims 86-88, 91, 96, 98, 100-103, 106, 107, and 109, comprising compound 21 having a mass ratio of about 10:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

126. 110. The delivery vehicle complex of any one of claims 86-88, 92, 96, 98, 103, 106, 107, and 109, comprising compound 21 having a mass ratio of about 12:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

127. 110. The delivery vehicle complex of any one of claims 86-88, 94, 96, 98, 103, 106, 107, and 109, comprising compound 21 having a mass ratio of about 15:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

128. 110. The delivery vehicle complex of any one of claims 86-88, 95, 98, 103, 106, 107, and 109, comprising compound 21 having a mass ratio of about 17:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

129. 110. The delivery vehicle complex of any one of claims 86-88, 93, 96-98, 103, 106, 107, and 109, comprising compound 21 having a mass ratio of about 13:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

130. 109. The delivery vehicle complex of any one of claims 86, 87, 89, 96, 97, 99, 101, 103, and 106-108, comprising compound 21 having a mass ratio of about 19:1 relative to the polyanionic compound, DSPC having a mass ratio of about 4:1 relative to the polyanionic compound, cholesterol having a mass ratio of about 8.1:1 relative to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 2.1:1 relative to the polyanionic compound.

131. 109. The delivery vehicle complex of any one of claims 86, 87, 91, 96-98, 100, 101, and 105-108, comprising compound 21 having a mass ratio of about 9.7:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 6.7:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

132. 110. The delivery vehicle complex of any one of claims 86-88, 91, 96, 98, 100-103, 106, 107, and 109, comprising compound 30 having a mass ratio of about 10:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

133. 110. The delivery vehicle complex of any one of claims 86-88, 92, 96, 98, 103, 106, 107, and 109, comprising compound 30 having a mass ratio of about 12:1 relative to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 relative to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 relative to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 relative to the polyanionic compound.

134. 110. The delivery vehicle complex of any one of claims 86-88, 94, 96, 98, 103, 106, 107, and 109, comprising compound 30 having a mass ratio of about 15:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

135. 110. The delivery vehicle complex of any one of claims 86-88, 95, 98, 103, 106, 107, and 109, comprising compound 30 having a mass ratio of about 17:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

136. 110. The delivery vehicle complex of any one of claims 86-88, 93, 96-98, 103, 106, 107, and 109, comprising compound 30 having a mass ratio of about 13:1 relative to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 relative to the polyanionic compound, cholesterol having a mass ratio of about 5.4:1 relative to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 1.4:1 relative to the polyanionic compound.

137. 109. The delivery vehicle complex of any one of claims 86, 87, 89, 96, 97, 99, 101, 103, and 106-108, comprising compound 30 having a mass ratio of about 19:1 to the polyanionic compound, DSPC having a mass ratio of about 4:1 to the polyanionic compound, cholesterol having a mass ratio of about 8.1:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

138. 109. The delivery vehicle complex of any one of claims 86, 87, 91, 96-98, 100, 101, and 105-108, comprising compound 30 having a mass ratio of about 9.7:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol having a mass ratio of about 6.7:1 to the polyanionic compound, and DMG-PEG2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

139. 111. The delivery vehicle complex of claim 110, comprising compound 12.

140. The delivery vehicle complex of claim 110, comprising compound 34.

141. The delivery vehicle complex of claim 110, comprising compound 35.

142. The delivery vehicle complex of claim 110, comprising compound 41.

143. 143. The delivery vehicle complex of any one of claims 139 to 142, wherein the phospholipid is DSPC.

144. 144. The delivery vehicle complex of any one of claims 139 to 143, wherein the sterol is cholesterol.

145. 145. The delivery vehicle complex of any one of claims 139 to 144, wherein the PEGylated lipid is DMG-PEG2000.

146. 146. The delivery vehicle complex of any one of claims 84 to 145, 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.

147. 147. The delivery vehicle complex of claim 146, wherein the complex exhibits a particle size of about 60 nm to about 100 nm.

148. The delivery vehicle complex of claim 147, wherein the complex exhibits a particle size of about 60 nm to about 90 nm.

149. 147. The delivery vehicle complex of claim 146, wherein the complex exhibits a particle size of about 105 nm to about 200 nm.

150. The delivery vehicle complex of claim 146, wherein the delivery vehicle complex exhibits a particle size of about 155 nm to about 195 nm.

151. 151. The delivery vehicle complex of any one of claims 84-150, wherein at least 80% of the polyanionic compound is retained after 48 days of storage at 4°C, or the delivery vehicle complex retains at least 80% of its original size after 48 days of storage at 4°C, or both.

152. 152. The delivery vehicle complex of any one of claims 84-151, wherein the polyanionic compound comprises at least one nucleic acid.

153. 153. The delivery vehicle complex of claim 152, wherein the at least one nucleic acid comprises RNA, DNA, or a combination thereof.

154. 154. The delivery vehicle complex of claim 153, wherein the at least one nucleic acid comprises RNA.

155. 155. The delivery vehicle complex of claim 154, wherein the RNA is an mRNA encoding a peptide, protein, or a functional fragment of the foregoing.

156. 156. The delivery vehicle complex of claim 155, wherein the mRNA encodes a viral peptide, a viral protein, or a functional fragment of any of the foregoing.

157. 157. The delivery vehicle complex of claim 156, wherein the mRNA encodes a human papillomavirus (HPV) protein or a functional fragment thereof.

158. 158. The delivery vehicle complex of claim 157, wherein the mRNA encodes HPV E6 and / or HPV E7 proteins, or functional fragments thereof.

159. The delivery vehicle complex of claim 156, wherein the mRNA encodes a viral spike protein or a functional fragment thereof.

160. 160. The delivery vehicle complex of claim 159, wherein the mRNA encodes the SARS-CoV spike (S) protein or a functional fragment thereof.

161. 157. The delivery vehicle complex of claim 156, wherein the mRNA encodes influenza hemagglutinin (HA) or a functional fragment thereof.

162. 157. The delivery vehicle complex of claim 156, comprising mRNA encoding the SARS-CoV spike (S) protein and mRNA encoding influenza hemagglutinin (HA), or functional fragments of the foregoing.

163. 163. A pharmaceutical composition comprising the delivery vehicle complex of any one of claims 84 to 162 and a pharmaceutically acceptable excipient.

164. 164. The pharmaceutical composition of claim 163 as an intratumoral (IT) or intramuscular (IM) composition.

165. 164. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject an effective amount of a delivery vehicle complex of any one of claims 84 to 162, or the pharmaceutical formulation of claim 163 or 164, thereby inducing an immune response in the subject.

166. 164. A method of treating a viral infection in a subject in need thereof, comprising administering to the subject an effective amount of a delivery vehicle complex of any one of claims 84 to 162, or the pharmaceutical formulation of any of claims 163 or 164, thereby treating the viral infection in the subject.

167. 164. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a delivery vehicle complex of any one of claims 84 to 162, or the pharmaceutical formulation of any of claims 163 or 164, thereby treating said cancer in said subject.

168. 168. The method of claim 167, wherein the cancer is cervical cancer, head and neck cancer, B-cell lymphoma, T-cell lymphoma, prostate cancer, lung cancer, or a combination thereof.

169. 169. The method of any one of claims 165-168, wherein the administering is by intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

170. 164. A method of delivering a polyanionic compound to a cell, comprising contacting said cell with a delivery vehicle complex of any one of claims 84 to 162, or a pharmaceutical composition of claim 163 or 164.

171. 171. The method of claim 170, wherein the cells are muscle cells, tumor cells, or a combination thereof.

172. 172. The method of claim 170 or 171, wherein the polyanionic compound is an mRNA encoding a peptide, protein, or a fragment of any of the foregoing, and the cell expresses the peptide, protein, or fragment after contact with the delivery vehicle complex.

173. 163. A method of forming the delivery vehicle complex of any one of claims 84 to 162, comprising contacting the compound or salt of formula (I) with the polyanionic compound.

174. 184. The method of claim 183, comprising mixing a solution containing the compound or salt of formula (I) with a solution containing the polyanionic compound.

175. 168. A vaccine comprising a delivery vehicle complex according to any one of claims 84 to 147 or a pharmaceutical formulation according to any one of claims 163 or 164.

176. 176. The vaccine of claim 175 for use in the treatment of cancer.

177. 176. A method of treating or preventing cancer in a patient, comprising administering to said patient the vaccine of claim 175.

178. 178. The vaccine for use of claim 176 or the method of claim 177, wherein the cancer is cervical cancer, head and neck cancer, B cell lymphoma, T cell lymphoma, prostate cancer, lung cancer, or a combination thereof.

Images