2-aminopropane-1, 3-diol terminated cationic peptides for nucleic acid delivery

Abstract

The present disclosure provides delivery vehicle compositions comprising a hydroxyalkyl terminated cationic peptoid, such as a 2-aminopropane-1, 3-diol terminated cationic peptoid, and complexes of the delivery vehicle with a polyanionic compound, such as a nucleic acid. The 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 with the delivery vehicle complexes of the present disclosure.

Description

Background Art

[0001] Description of Related Technology

[0002] Therapeutic nucleic acids, such as mRNA, small interfering RNA (siRNA), small activating RNA (saRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids and immunostimulatory nucleic acids, have great prospects for preventing and treating diseases at the genetic level. However, nucleic acids are generally subject to rapid degradation, renal clearance, poor cell uptake and inefficient endosomal escape in the blood. Therefore, in order to make nucleic acids useful in treatment, a safe and effective system for delivering nucleic acids to the nucleus or cytosol is needed. Traditional methods for delivering polyanionic compounds such as oligonucleotides in cells and in vivo include viral vectors, cationic lipid nanoparticles (LNPs) and polycationic polymers. These delivery systems may be subject to problems such as poor stability, rapid clearance, poor toxicology, immune response problems and poor expression of their polyanionic cargo. Summary of the invention

[0003] Stable, safe and effective systems for delivering nucleic acids to cells are needed. Therefore, the present disclosure relates to delivery vehicle compositions comprising 2-aminopropane-1,3-diol-terminated cationic peptoids, and complexes of the delivery vehicle compositions with polyanionic compounds such as nucleic acids. The present disclosure further relates to methods of preparing and using the delivery vehicle compositions and complexes for intracellular delivery of polyanionic compounds such as mRNA, and methods of eliciting an immune response using the complexes of the present disclosure.

[0004] N-substituted glycine oligomers (referred to as peptoids) and their use as ionizable lipid components in delivery vehicle compositions are disclosed. By parameterizing peptoid structural changes (including modifications to both the charged and hydrophobic portions of the peptoids), experimental design (DOE) methods are used to investigate a wide range of chemical spaces to facilitate the discovery of trends between peptoid structural elements and particle properties. From initial compounds to final lead candidate compounds, the optimization process resulted in an increase in mRNA expression produced in vivo by >10,000 times, which enables them to be used in therapeutic regimens, including enzyme replacement and antibody drugs produced in vivo. The disclosed peptoids result in the expression of therapeutically relevant levels of proteins (e.g., anti-RSV antibodies) in mice, with minimal tolerability issues or induced immune responses. The disclosed compounds and compositions thereof provide an adjustable delivery platform that can be optimized for specific therapeutic regimens.

[0005] In one aspect, the present disclosure provides compounds having the structure of Formula (I):

[0006] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R1 is H or C optionally substituted by 1 to 3 OH 2-5 Alkyl; R 2 is C substituted with 1 to 3 additional OH groups 2-5 Alkylene -OH; and each R 3 Independently for C 6-24 Alkyl or C 6-24 In some cases, n is 3. In some cases, n is 4. In some cases, n is 6. In some cases, n is 8. In some cases, n is 9. In some cases, R 1 is H. In some cases, R 2 C is replaced by an additional OH 2-5 In some cases, C 2-5 The alkylene group is substituted with 2 or 3 additional OH groups. 2 is C substituted with 1 to 3 additional OH groups 3-4 In some cases, each R 3 Independently for C 6-18 Alkyl or C 6-18 In some cases, each R 3 Independently for C 8-18 Alkyl or C 8-18 In some cases, each R 3 Independently selected from the group consisting of:

[0007] In some cases, each R 3 Independently selected from the group consisting of: In some cases, at least one R 3 Select from the group consisting of: In some cases, at least one R 3 for In some cases, the compound of Formula (I) has a structure selected from the group consisting of:

[0008]

[0009] In some cases, the compound of formula (I) is selected from 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).

[0010] Another aspect of the present disclosure provides a delivery vehicle composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof. In some specific implementations, the composition further comprises one or more of a phospholipid, a sterol, and a pegylated lipid. In some specific 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 specific 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 specific 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 specific 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 cases, 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 specific 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 specific 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 specific implementations, the compound or salt of formula (I) is present in an amount of about 50 mol % to about 52 mol %.

[0011] In various specific implementations, the composition comprises phospholipids, sterols and PEGylated lipids. In some cases, the composition is substantially composed of compounds disclosed herein or salts thereof, phospholipids, sterols and PEGylated lipids. In some cases, the composition comprises about 30 mol % to about 60 mol % of the compound of formula (I); about 3 mol % to about 20 mol % of the phospholipids, about 25 mol % to about 60 mol % of the sterols and about 1 mol % to about 5 mol % of the PEGylated lipids. In various cases, 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 phospholipids, about 30 mol % to about 55 mol % of the sterols and about 1 mol % to about 3 mol % of the PEGylated lipids. In some embodiments, 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 phospholipids, about 35 mol% to about 50 mol% of the sterols, and about 1 mol% to about 2 mol% of the PEGylated lipids. In various embodiments, 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 phospholipids, about 30 mol% to about 55 mol% of the sterols, and about 1 mol% to about 3 mol% of the PEGylated lipids. In some cases, 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 phospholipids, about 35 mol% to about 50 mol% of the sterols, and about 1 mol% to about 2 mol% of the PEGylated lipids. In some cases, 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 phospholipids, about 40 mol % to about 55 mol % of the sterols, and about 1 mol % to about 3 mol % of the PEGylated lipids. In some cases, 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 phospholipids, about 50 mol % to about 55 mol % of the sterols, and about 2 mol % to about 3 mol % of the PEGylated lipids. In some cases, 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 phospholipids, about 40 mol % to about 45 mol % of the sterols, and about 1 mol % to about 2 mol % of the PEGylated lipids.In some cases, 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-di(undecanoyl)-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-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-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 (MEPE), 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, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some cases, 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 specific 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 cases, 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 implementations, 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 implementations, 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.

[0012] Further disclosed herein is a delivery vehicle complex comprising a delivery vehicle composition described herein and a polyanionic compound. In some cases, a compound of formula (I) or a salt thereof is compounded with a 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 specific 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 mass ratio of about 10:1. In various cases, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 12:1. The compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 13:1. In some specific implementations, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 15:1. In various specific implementations, the compound or salt of formula (I) and the polyanionic compound are present in a mass ratio of about 17:1. In some cases, the phospholipid and the polyanionic compound are present in a mass ratio of about 2:1 to about 10:1. In some cases, the phospholipid and the polyanionic compound are present in a mass ratio of about 2:1 to about 4:1. In various cases, the phospholipid and the polyanionic compound are present in a mass ratio of about 2:1 to about 3:1. In various cases, the phospholipid and the polyanionic compound are present in a mass ratio of about 4.0:1. In various cases, the phospholipid and the polyanionic compound are present in a mass ratio of about 2.7:1. In some specific implementations, sterols and polyanionic compounds are present in a mass ratio of about 5:1 to about 8:1. In some embodiments, the sterol and the polyanionic compound are present in a mass ratio of about 5:1 to about 6:1. In various embodiments, the sterol and the polyanionic compound are present in a mass ratio of about 5.4:1. In some cases, the sterol and the polyanionic compound are present in a mass ratio of about 8.1:1. In some cases, the sterol and the polyanionic compound are present in a mass ratio of about 6.7:1. In some cases, the PEGylated lipid and the 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 the polyanionic compound are present in a mass ratio of about 1:1 to about 2:1. In some cases, the phospholipid and the polyanionic compound are present in a mass ratio of about 2.1:1. In some cases, the phospholipid and the polyanionic compound are present in a mass ratio of about 1.4:1.In various cases, the delivery vehicle complex comprises a compound 1, 6, 21 or 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-PEG 2000 having a mass ratio of about 1.4: 1 to the polyanionic compound. In various cases, the delivery vehicle complex comprises a compound 1, 6, 21 or 30 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-PEG 2000 having 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 at a mass ratio of about 15:1 to the polyanionic compound, DSPC at a mass ratio of about 2.7:1 to the polyanionic compound, and cholesterol at a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG 2000 at 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 at a mass ratio of about 17:1 to the polyanionic compound, DSPC at a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol at a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG 2000 at a mass ratio of about 1.4:1 to the polyanionic compound. In various cases, the delivery vehicle complex comprises a 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-PEG 2000 having a mass ratio of about 1.4: 1 to the polyanionic compound. In various cases, the delivery vehicle complex comprises a 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-PEG 2000 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 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-PEG 2000 having a mass ratio of about 2.1: 1 to the polyanionic compound. In some cases, the compound of Formula (I) or a 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.

[0013] In some cases, the complex has a particle size of about 50nm to about 200nm and / or a polydispersity index (PDI) of less than 0.25. In various cases, the complex has a particle size of about 60nm to about 100nm. In some specific implementations, the complex has a particle size of about 60nm to about 90nm. In various specific implementations, the complex has a particle size of about 105nm to about 200nm. In various cases, the complex has a particle size of about 150nm to about 200nm. In some cases, the delivery vehicle complex has a particle size of about 105nm to about 200nm. In some cases, the delivery vehicle complex has a particle size of about 40nm to about 115nm, or about 55nm to about 95nm, or about 70nm to about 80nm, or about 75nm. In various cases, the delivery vehicle complex has a particle size of about 135nm to about 225nm, or about 155nm to about 195nm, or about 170nm to about 180nm, or about 175nm. In each case, at least 80% of the polyanionic compound is retained after storage at 4°C for 48 days, or the delivery vehicle complex retains at least 80% of its original size after storage at 4°C for 48 days, or both.

[0014] In some cases, the polyanionic compound includes at least one nucleic acid. In various cases, at least one nucleic acid includes RNA, DNA or a combination thereof. In various cases, at least one nucleic acid includes RNA. In some specific implementations, RNA is an mRNA encoding a peptide, a protein or a functional fragment of the aforementioned. In various specific implementations, mRNA encodes a viral peptide, a viral protein or a functional fragment of any of the aforementioned. In some cases, mRNA encodes a human papillomavirus (HPV) protein or a functional fragment thereof. In various cases, mRNA encodes HPV E6 protein and / or HPV E7 protein, a variant thereof or a functional fragment of any of the aforementioned. In some cases, HPV protein is from HPV subtypes HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68. In various cases, HPV protein is from HPV subtypes HPV 16 and / or HPV 18. In some cases, mRNA encodes a viral spike protein or a functional fragment thereof. In various cases, the mRNA encodes SARS-CoV spike (S) protein, its variants, or a functional fragment of any of the foregoing. In some cases, the RNA encodes SARS-related coronaviruses (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 OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1) or human coronavirus NL63 (HCoV-NL63)). In some cases, the mRNA encodes influenza hemagglutinin (HA), its variants, or a functional fragment of any of the foregoing. In some specific implementations, the influenza A virus has a HA of a 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 embodiments, the influenza subtype is HA strain H1, H2, H3 or H5. In various cases, one mRNA encodes the SARS-CoV spike (S) protein and one mRNA encodes influenza hemagglutinin (HA), a variant thereof, or a functional fragment of any of the foregoing.

[0015] Further disclosed herein are pharmaceutical compositions comprising the 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.

[0016] Also disclosed herein is a method of inducing an immune response in a subject in need, the method comprising administering to the subject an effective amount of a delivery vehicle complex as described herein or a pharmaceutical preparation comprising a delivery vehicle complex, thereby inducing an immune response in the subject. Also disclosed herein is a method of treating a viral infection in a subject in need, the method comprising administering to the subject an effective amount of a delivery vehicle complex as described herein or a pharmaceutical preparation comprising a delivery vehicle complex, thereby treating a viral infection in the subject. Also disclosed herein is a method of treating cancer in a subject in need, the method comprising administering to the subject an effective amount of a delivery vehicle complex as described herein or a pharmaceutical preparation comprising a delivery vehicle complex, thereby treating 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 carried out by intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

[0017] 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 as described herein or a pharmaceutical preparation comprising a 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, a protein, or a fragment of any of the foregoing, and the cell expresses the peptide, protein, or fragment after contacting the delivery vehicle complex.

[0018] Also disclosed herein are methods of forming a delivery vehicle complex disclosed herein, the method comprising contacting a compound or salt of formula (I) with a polyanionic compound. In some cases, the method comprises mixing a solution comprising a compound or salt of formula (I) with a solution comprising a polyanionic compound.

[0019] Also disclosed herein are vaccines comprising the delivery vehicle complex disclosed herein or the pharmaceutical composition disclosed herein. Also disclosed are vaccines comprising the delivery vehicle complex disclosed herein or the pharmaceutical composition disclosed herein for use in treating cancer. Also disclosed are methods of treating or preventing cancer in a patient, the methods comprising administering to the patient the delivery vehicle complex disclosed herein or the 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.

[0020] It should be understood that all combinations of the foregoing concepts and implementations, as well as the additional concepts and implementations discussed in more detail below, are considered part of the inventive subject matter disclosed herein and can be employed in any suitable combination to achieve the benefits described herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are considered part of the inventive subject matter disclosed herein.

[0021] By reading the following detailed description in conjunction with the accompanying drawings, other aspects and advantages will be apparent to those of ordinary skill in the art. Although the compounds and methods disclosed herein are susceptible to implementation in various forms, the following description includes specific embodiments, and it is understood that the disclosure is illustrative and is not intended to limit the disclosure to the specific embodiments described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown are the results of an in vivo assay of delivery vehicles comprising the peptoids disclosed herein and control peptoids assessed using fLuc expression in BALB / c mice 6 hours after IV administration.

[0023] Figure 2 The percent total expression of the peptoid preparations disclosed herein and a control peptoid in spleen, lung, and liver is shown.

[0024] Figure 3 Shown are the results of a firefly luciferase expression assay in BALB / c mice evaluating the delivery efficacy of the peptoid formulations disclosed herein.

[0025] Figure 4 Shown are contour plots evaluating the effects of carbon number and branching on delivery vehicle efficacy in a firefly luciferase expression assay.

[0026] Figure 5 Shown are the results of anti-RSV titers of delivery vehicles comprising the peptoids disclosed herein dosed intravenously at 0.75 mg / kg mRNA in BALB / c mice and measured by IgG ELISA 24 hours later.

[0027] Figure 6 Shown are the results of a dose escalation study of a delivery vehicle containing Compound 41 in a mouse model as measured by serum aRSV.

[0028] Figure 7 It was shown that aRSV was detectable in serum more than 5 days after dosing.

[0029] Figure 8 Shown are the results of a dose escalation study of a delivery vehicle containing Compound 41 in a mouse model as measured via a firefly luciferase expression assay.

[0030] Fig. 9 Shown is a cryoTEM image of particles of compound 41 ("41-D22") in the D22 formulation.

[0031] Fig.10Particles of 41-D22 were shown to remain stable when stored at 4°C or -80°C for more than 1 month, with no observed growth or encapsulation changes.

[0032] Fig.11 Comparison of firefly luciferase expression of delivery vehicles formulated with compounds 31 and 41 is shown.

[0033] Fig.12 Comparison of serum aRSV expression of delivery vehicles formulated with compounds 31 and 41 is shown. DETAILED DESCRIPTION

[0034] The successful delivery of mRNA remains a major challenge for its adoption as a mainstream treatment. The approval of mRNA vaccines against SARS-COV2 using lipid nanoparticle (LNP) technology shows significant progress in this field, however, the expansion of mRNA drugs to applications beyond vaccines, such as protein replacement or immuno-oncology therapy for generating circulating antibodies, requires new delivery strategies and platforms. In these lipid nanoparticles, mRNA molecules are encapsulated in a combination of cationic or ionizable lipids, auxiliary lipids (such as cholesterol and phospholipids) and shielding lipids containing polyethylene glycol (PEG). With the application of mRNA to a wider range of applications, including those that rely on IV delivery, the ability to regulate the properties of ionizable lipids in particular has become a key focus in this field. Many reports show that regulating the properties of ionizable lipids affects the expression and tissue selectivity of delivery. The generation of new ionizable lipids is usually limited by effective synthetic routes, and requires a lot of synthetic efforts and resources to expand the lipid library to include new safe and effective candidates. This article provides a new class of peptoid-based mRNA delivery vehicles, which utilize highly adjustable peptoids as ionizable lipid components to regulate the delivery vehicle properties. An experimental design approach was used to modulate peptoid structure to optimize delivery vehicle function and demonstrate optimization of delivery vehicles for systemic delivery of mRNA encoding, for example, aRSV.

[0035] In some examples, a delivery vehicle composition is disclosed herein, which includes a hydroxyalkyl-terminated cationic peptide, including, for example, a 2-aminopropane-1,3-diol-terminated cationic peptide. The delivery vehicle composition disclosed herein can form an electrostatic interaction between the 2-aminopropane-1,3-diol-terminated cationic peptide and a polyanionic compound (such as a nucleic acid) of the delivery vehicle composition to form a delivery vehicle complex, wherein the polyanionic compound serves as the cargo of the complex. The delivery vehicle complex can be used to deliver polyanionic compounds such as nucleic acids (e.g., mRNA) to cells. The delivery vehicle complex disclosed herein comprising mRNA as a polyanionic cargo unexpectedly has excellent in vitro and in vivo mRNA expression. When the mRNA of the delivery vehicle complex encodes, for example, a viral antigen, the delivery vehicle complex can trigger a humoral and cellular immune response in vivo, thereby playing the role of a vaccine. Other advantages of the delivery vehicle complex disclosed herein are that they are stable and show good tolerance and low toxicity.

[0036] As used herein, "peptoid" refers to a peptidomimetic compound in which one or more of the nitrogen atoms of the peptide backbone are replaced by a side chain. As used herein, "polyanion" refers to a compound having at least two negative charges, such as a nucleic acid.

[0037] Delivery vehicle composition

[0038] Some exemplary delivery vehicle compositions of the present disclosure include one or more hydroxyalkyl-terminated cationic peptoids, such as 2-aminopropane-1,3-diol-terminated cationic peptoids. These positively charged peptoids can be associated with polyanionic compounds such as nucleic acids to form delivery vehicle complexes. In some specific implementations, the delivery vehicle composition also includes one or more of anionic or zwitterionic components, such as phospholipids; neutral lipids, such as sterols; and shielded lipids, such as pegylated lipids. In various specific implementations, the delivery vehicle composition also includes anionic or zwitterionic components (e.g., phospholipids), neutral lipids (e.g., sterols) and shielded lipids (e.g., pegylated lipids). In some cases, the delivery vehicle composition is essentially composed of hydroxyalkyl-terminated cationic peptoids (such as 2-aminopropane-1,3-diol-terminated cationic peptoids), anionic or zwitterionic components (e.g., phospholipids), neutral lipids (e.g., sterols) and shielded lipids (e.g., pegylated lipids).

[0039] Hydroxyalkyl-terminated cationic peptide components

[0040] Peptoids are a unique class of N-substituted α-amino acids that are structurally related to peptides, but have their various side chain functionalities incorporated on the amide nitrogen rather than the α carbon, allowing for modular synthesis. In one non-limiting embodiment, peptoids can be synthesized via a submonomer approach, where repeated cycles of acylation with bromoacetic acid and nucleophilic addition of primary amines can be accomplished on a solid support to produce peptoids in high yield and fidelity. Peptoids have been successfully used in many applications, including antiscalants and antimicrobial agents, antifreeze additives for drug delivery and tissue storage, and even as complexing agents for nucleic acids, but they have never been explored as ionizable components of lipid nanoparticles for mRNA delivery.

[0041] In view of its favorable characteristics and modular nature, it is believed that peptoids provide an excellent platform for discovering new ionizable lipids and exploring structure-activity relationships. Peptoids are easy to synthesize using solid phase synthesizers and have a monomer library that is even larger than traditional peptides. Traditionally, the discovery of new lipids for nucleic acid delivery is highly empirical, so successful systems are generally those systems that can introduce high structural diversity and quickly explore a wide range of chemical spaces. Utilizing the solid phase sub-monomer method, the platform based on peptoids disclosed herein utilizes the adjustable and programmable properties of peptoid structures to more easily cover a wide range of chemical spaces than traditional lipid synthesis. When formulated with mRNA, DSPC, cholesterol and DMG-PEG, a delivery vehicle is generated and its physical properties and biological activity are characterized. By repeated synthesis and particle screening, the relationship between the peptoid structure and the activity of the delivery vehicle can be explored.

[0042] The first stage of screening focused on optimizing the cationic groups on the peptoid molecules. The properties of the basic groups used in the ionizable lipids significantly drive the particle pKa, which is related to the efficacy and specificity of nanoparticle expression. A particle pKa of 6-6.5 generally shows the highest efficacy in selective liver expression. However, it is not known how the molecular pKa of the peptoids is related to the observed pKa once formulated in the delivery vehicle disclosed herein, so a variety of head group amines with different basicities were selected.

[0043] Without being bound by any particular theory, it is believed that modification of the lipophilic portion of the peptoid, while maintaining a predominantly 1,3-diol (apd) head group, allows for further optimization of overall expression while maintaining or increasing liver selectivity. For this optimization, 6 different aliphatic monomers ranging in length from C6 to C12 were selected, including unsaturated oleyl lipids and branched 2-ethylhexyl. Even with this relatively small library of monomers, there are 55,944 possible peptoid designs of length 3-8, so a systematic approach using a design of experiments (DOE) approach was employed to evaluate this large structural space. In order to allow for accurate input of chemical structure information into the DOE model, the properties of the lipid block can be parameterized using 4 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. This parameterization approach does not attempt to capture the order of monomers within the sequence, so the peptoids are designed to have alternating and repeating or symmetric motifs wherever possible.

[0044] The delivery vehicle composition of the present disclosure comprises a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid ("cationic component", sometimes referred to as "ionizable lipid"). In some embodiments, the hydroxyalkyl-terminated cationic peptoid comprises a compound of formula (I): wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R 1 is H or C optionally substituted by 1 to 3 OH 2-5 Alkyl; R 2 H or C substituted with 1 to 3 additional OH 2-5 Alkylene -OH; and each R 3 Independently for C 6-24 Alkyl or C 6-24 As used herein, "alkyl" refers to straight and branched chain saturated hydrocarbon groups containing from one to thirty carbon atoms, such as from one to twenty-four 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). The term C n It means that the alkyl group has "n" carbon atoms. For example, C3 alkyl refers to an alkyl group with 3 carbon atoms. 1-24Alkyl refers to groups having carbon atoms that encompass the entire range (i.e., 1 to 24), as well as all subgroups (e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 1 to 11, 2 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 2 to 22, 2 to 23, 2 to 24, 2 to 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 11, 3 to 12, 3 to 13, 3 to 14, 3 to 15, 3 to 16, 3 to 17, 3 to 18, 3 to 19, 3 to 20, 3 to 21, 3 to 22, 3 to 23, 3 to 24, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 4 to 11, 4 to 12, 4 to 13, 4 to 14, 4 to 15, 4 to 16, 4 to 17, 4 to 18, 4 to 19, 4 to 20, 4 to 21, 4 to 22, 4 to 23, 4 to 24, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 11, 5 to 12, 5 to 13, 5 to 14, 5 to 15, 5 to 16, 5 to 17, 5 to 18 , 5 to 19, 5 to 20, 5 to 21, 5 to 22, 5 to 23, 5 to 24, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 6 to 11, 6 to 12, 6 to 13, 6 to 14, 6 to 15, 6 to 16, 6 to 17, 6 to 18, 6 to 19, 6 to 20, 6 to 21, 6 to 22, 6 to 23, 6 to 24, 7 to 8, 7 to 9, 7 to 10, 7 to 11, 7 to 12, 7 to 13, 7 to 14, 7 to 15, 7 to 16, 7 to 17, 7 to 18, 7 to 19, 7 to 20, 7 to 21, 7 to 22, 7 to 23, 7 to 24, 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18 , 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 10 to 11, 10 to 12, 10 to 13, 1 0 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21,11 to 22, 11 to 23, 11 to 24, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to The alkyl group may be an alkyl group having 1 to 24 carbon atoms (20 to 21, 20 to 22, 20 to 23, 20 to 24, 21 to 22, 21 to 23, 21 to 24, 22 to 23, 22 to 24, 23 to 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 tert-butyl (1,1-dimethylethyl). Unless otherwise specified, the alkyl group may be an unsubstituted alkyl group or a substituted alkyl group. As used herein, "alkenyl" refers to straight and branched chain hydrocarbon groups having double bonds and containing two to thirty carbon atoms, such as two to twenty-four 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 It means that the alkenyl group has "n" carbon atoms. For example, C3 alkenyl refers to an alkenyl group with 3 carbon atoms. 243-4, 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, 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, 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 -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, 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, 11-17, 11-18, 1 1-19, 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, 18, 19, 20, 21, 22, 23, 24, 25-24, 26-27, 27-28, 28-29, 30-31, 31-32, 32-33, 33-34, 34-35, 36-37, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 45-46, 47-48, 49-50, 50-51, 51-52, 52-53, 53-54, 54-55, 56-57, 57-58, 58-59, 60-61, 61-62, 61-63, 61-64, 62-64, 62-65 Non-limiting examples of alkenyl groups include ethenyl, propenyl, butenyl, geranyl, and oleyl. Unless otherwise specified, an alkenyl group may be an unsubstituted alkenyl group or a substituted alkenyl group.

[0045] In some implementations, n is 2 to 5. In various implementations, n is 3 to 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.

[0046] In some specific implementations, R 1 is H. In various specific implementations, R 1 is C optionally substituted by 1 to 3 OH groups 2-5 In some cases, R 1 is methyl or ethyl. In some embodiments, R 1 In various embodiments, R 1 C substituted with 0-2 additional OH groups 2-5 In some embodiments, R 1 for (hydroxyethyl). In each case, R 1 is ethyl or hydroxyethyl. In some cases, C2-5 The alkyl group is substituted with one OH group. 2-3 The alkyl group is substituted with 2 OH groups. 2-5 The alkyl group is substituted with 3 OH groups.

[0047] .R 2 is C substituted with 1 to 3 additional OH groups 2-5 In some cases, R 2 is C2 alkylene-OH substituted with 1 to 3 additional OH groups. 2 is C3 alkylene-OH substituted with 1 to 3 additional OH. 2 is C4 alkylene-OH substituted with 1 to 3 additional OH groups. 2 is C3 alkylene-OH substituted with 1 to 3 additional OH groups. 2-5 The alkylene-OH is substituted with an additional OH. 2-5 The alkylene-OH is substituted with 2 additional OH groups. 2-5 The alkylene-OH is substituted with 3 additional OH groups. 2 is propyl-1,3-diol. In some cases, R 2 for

[0048] In some implementations, each R 3 Independently for C 8-18 Alkyl or C 8-18 In various embodiments, each R 3 Independently for C 8-16 Alkyl or C 10-18 In some cases, each R 3 Independently for C 6-18 Alkyl or C 6-18 In some cases, each R 3 Independently for C 10-12 Alkyl or C 10-18 In some embodiments, each R 3 Independently: C 10-18 Alkyl, or C 8-16 Alkyl, or C 8-14 Alkyl, or C 8-12 In various embodiments, each R 3 Independently selected from the group consisting of:

[0049] In some cases, each R3 is independently selected from the group consisting of:

[0050] In each case, each R 3 Independently selected from the group consisting of: In some implementations, each R 3 Independently

[0051] Contemplated compounds of formula (I) include, but are not limited to, those listed in Table 1.

[0052] Table 1. Examples of hydroxyalkyl-terminated cationic peptoids .

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] The properties of the peptoids disclosed herein can be advantageously compared to the properties of other peptoids, including the comparative peptoids disclosed in Table C.

[0081] Table C. Comparative Peptoids

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] In some embodiments, 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.

[0090] The compounds of the present disclosure are defined herein by their chemical structure and / or chemical name. When a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name are inconsistent, the chemical structure determines the identity of the compound.

[0091] Unless otherwise indicated, the structures described herein are also intended to include all isomers (e.g., enantiomers, diastereomers, cis-trans, conformational and rotational) forms of the structure. For example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in the present disclosure unless only one of the isomers is specifically indicated. Therefore, single stereochemical isomers of the compounds of the present invention, as well as enantiomers, diastereomers, cis / trans, conformational and rotational mixtures are all within the scope of the present disclosure. In some cases, the compounds disclosed herein are stereoisomers. "Stereoisomers" refer to compounds with different chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds disclosed herein may exist in the form of a single stereoisomer or a mixture of stereoisomers. Unless otherwise indicated, the stereochemistry of the compounds shown herein indicates relative stereochemistry, rather than absolute stereochemistry. As used herein, a single stereoisomer, diastereomer or enantiomer refers to a compound that is at least greater than 50% of the indicated stereoisomer, diastereomer or enantiomer, and in some cases, at least 90% or 95% of the indicated stereoisomer, diastereomer or enantiomer.

[0092] The compounds described herein may exist in free form or, where appropriate, in the form of pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound that is suitable for contact with tissues of humans and lower animals without excessive side effects (such as toxicity, irritation, allergic reactions, etc.) and commensurate with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. 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 separation and purification of the compound. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed of an amino group with an inorganic acid (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with an organic acid (such as 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 ion exchange) formed salts. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutamate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, 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, valerate, and the like. Salts of compounds containing carboxylic acids or other acidic functional groups can be prepared by reaction with a suitable base. Such salts include, but are not limited to, alkali metal salts, alkaline earth metal salts, aluminum salts, ammonium salts, N + (C 1-4The present invention also contemplates quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Water-soluble or oil-soluble or dispersible products can be obtained by such quaternization. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates, where appropriate.

[0093] In a specific implementation, the delivery vehicle composition comprises about 25 mol % to about 70 mol % of a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of formula (I), such as compound 1, 6, 21 or 30), based on the total moles of components in the delivery vehicle composition. The unit "mol %" or "molar 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%. The 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-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of Formula (I), such as Compound 1, 6, 21, or 30), based on the total moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises less than about 50 mol% of a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid, such as less than about 49 mol%, less than about 48 mol%, less than about 47 mol%, less than about 46 mol%, less than about 45 mol%, less than about 44 mol%, less than about 43 mol%, less than about 42 mol%, less than about 41 mol%, less than about 40 mol%, less than about 39 mol%, less than about 38 mol%, less than about 37 mol%, less than about 36 mol%, less than about 35 mol%, less than about 34 mol%, less than about 33 mol%, less than about 32 mol%, less than about 31 mol%, less than about 30 mol% of a hydroxyalkyl-terminated cationic peptoid, based on the total moles of components in the delivery vehicle composition; and Greater than about 20 mol % of hydroxyalkyl terminated cationic peptoids, such as 2-aminopropane-1,3-diol terminated cationic peptoids, such as greater than about 21 mol %, greater than about 22 mol %, greater than about 23 mol %, greater than about 24 mol %, greater than about 25 mol %, greater than about 26 mol %, greater than about 27 mol %, greater than about 28 mol %, greater than about 29 mol %, greater than about 30 mol %, greater than about 31 mol %, greater than about 33 mol %, greater than about 34 mol %, greater than about 35 mol %, greater than about 36 mol %, greater than about 38 mol %, greater than about 39 mol %, greater than about 40 mol %, greater than about 41 mol %, greater than about 42 mol %, greater than about 43 mol % or greater than about 44 mol % of hydroxyalkyl terminated cationic peptoids, such as 2-aminopropane-1,3-diol terminated cationic peptoids.In some embodiments, the delivery vehicle composition comprises less than about 50 mol% of a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid, such as less than about 49 mol%, less than about 48 mol%, less than about 47 mol%, less than about 46 mol%, less than about 45 mol%, less than about 44 mol%, less than about 43 mol%, less than about 42 mol%, less than about 41 mol%, less than about 40 mol%, less than about 39 mol%, less than about 38 mol%, less than about 37 mol%, less than about 36 mol%, less than about 35 mol%, less than about 34 mol%, less than about 33 mol%, less than about 32 mol%, less than about 31 mol%, less than about 30 mol%, mol % of hydroxyalkyl terminated cationic peptides; and greater than about 20 mol % of hydroxyalkyl terminated cationic peptides, such as 2-aminopropane-1,3-diol terminated cationic peptides, such as greater than about 21 mol %, greater than about 22 mol %, greater than about 23 mol %, greater than about 24 mol %, greater than about 25 mol %, greater than about 26 mol %, greater than about 27 mol %, greater than about 28 mol %, greater than about 29 mol %, greater than about 30 mol %, greater than about 31 mol %, greater than about 33 mol %, greater than about 34 mol %, greater than about 35 mol %, greater than about 36 mol %, greater than about 38 mol %, greater than about 39 mol % or greater than about 40 mol % of hydroxyalkyl terminated cationic peptides, such as 2-aminopropane-1,3-diol terminated cationic peptides. 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-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of Formula (I), such as Compound 1, 6, 21, or 30), based on the total moles of components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises 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-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of Formula (I), such as Compound 1, 6, 21, or 30), based on the total moles of components in the delivery vehicle composition.In some cases, the delivery vehicle composition comprises about 30 mol% to about 35 mol% of a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of formula (I), such as compound 1, 6, 21, or 30), based on the total moles of the components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 40 mol% to about 45 mol% of a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of formula (I), such as compound 1, 6, 21, or 30), based on the total moles of the components in the delivery vehicle composition. In some cases, the delivery vehicle composition comprises about 35 mol% to about 39 mol% of a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of formula (I), such as compound 1, 6, 21, or 30), based on the total moles of the components in the delivery vehicle composition. In various cases, based on the total moles of components in the delivery vehicle composition, the delivery vehicle composition comprises about 39 mol% to about 52 mol% of a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of formula (I), such as compound 1, 6, 21, or 30). In some embodiments, based on the total moles of components in the delivery vehicle composition, the delivery vehicle composition comprises about 42 mol% to about 49 mol% of a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of formula (I), such as compound 1, 6, 21, or 30). In various embodiments, the delivery vehicle composition comprises about 50 mol % to about 52 mol % of a hydroxyalkyl-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of formula (I), such as compound 1, 6, 21 or 30), based on the total 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-terminated cationic peptoid, such as a 2-aminopropane-1,3-diol-terminated cationic peptoid (e.g., a compound of Formula (I), such as Compound 1, 6, 21, or 30), based on the total moles of components in the delivery vehicle composition.

[0094] Anionic / zwitterionic components

[0095] In some embodiments, the delivery vehicle composition further comprises an anionic or zwitterionic component ("anionic / zwitterionic component"). The anionic / zwitterionic component can buffer the zeta potential of the particles or delivery vehicle complexes formed by the delivery vehicle composition without affecting the ratio of the cargo and / or facilitating the particles or delivery vehicles to escape from the endosomal structure by protonation at low pH in the endosome. The zwitterionic component can further act to keep the particles together by interacting with both the hydroxyalkyl-terminated cationic peptoids (such as 2-aminopropane-1,3-diol-terminated cationic peptoids) and the polyanionic cargo compound. The anionic component can also allow the formation of a core-shell structure of the particle or delivery vehicle, wherein a net positive zeta potential particle is first prepared (e.g., by mixing the hydroxyalkyl-terminated cationic peptoids (such as 2-aminopropane-1,3-diol-terminated cationic peptoids) and the cargo in a positive + / - charge ratio), and then coated with the anionic component. These negatively charged multicomponent system particles will avoid reticuloendothelial system (RES) clearance better than positively charged multicomponent system particles.

[0096] Examples of suitable anionic and zwitterionic components of delivery vehicle compositions are described in WO2020 / 069442 and WO2020 / 069445, each of which is incorporated herein by reference in its entirety. In some embodiments, the zwitterionic component comprises one or more phospholipids. Due to the amphiphilic nature of phospholipids and their ability to disrupt cell membranes, phospholipids can further stabilize the complex in solution and promote endocytosis.

[0097] In some embodiments, 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-di(undecanoyl)-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-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-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-glyceryl-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glyceryl-3-phosphoethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phospho-racem-(1-glyceryl) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some cases, the phospholipid is DSPC, DOPE, or a combination thereof. In various specific implementations, the phospholipid is DSPC. In various cases, the phospholipid is DOPE.

[0098] In a specific implementation, based on the total molar number of components in the delivery vehicle composition, the delivery vehicle composition comprises about 1 mol % to about 40 mol % of phospholipids (e.g., DSPC or DOPE). In some cases, based on the total molar number of components in the delivery vehicle composition, 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 phospholipids (e.g., DSPC or DOPE). In some cases, based on the total molar number of components in the delivery vehicle composition, the delivery vehicle composition comprises about 10 mol % to about 11 mol % of phospholipids (e.g., DSPC or DOPE). 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 moles of components in the delivery vehicle composition.

[0099] Neutral lipid components

[0100] In some embodiments, the delivery vehicle composition also includes a component that is a neutral lipid ("neutral lipid component"). The neutral lipid component can be designed to degrade or hydrolyze to promote in vivo clearance of the multi-component delivery system. Contemplated neutral lipid components include, for example, naturally occurring lipids and lipidated peptoids comprising a lipid portion at the N-position of the peptoid. Other examples of lipidated peptoids are described in WO2020 / 069442 and WO2020 / 069445, each of which is incorporated herein by reference in its entirety.

[0101] In some cases, the neutral lipid component of the delivery vehicle composition includes one or more sterols. In some specific implementations, one or more sterols are selected from the group consisting of the following items: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some cases, sterol includes cholesterol. In specific implementations, based on the total molar number of components in the delivery vehicle composition, the delivery vehicle composition includes about 10 mole % to about 80 mole % of sterol (e.g., cholesterol). In some cases, based on the total moles of components in the delivery vehicle composition, 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 sterol (e.g., cholesterol). In some cases, based on the total moles of components in the delivery vehicle composition, 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 sterol (e.g., cholesterol). 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 moles of components in the delivery vehicle composition.

[0102] Shielding components

[0103] In some embodiments, the delivery vehicle composition further comprises a shielding component. The shielding component can increase the stability of the particle or delivery vehicle in vivo by acting as a spatial barrier, thereby improving the circulation half-life. Examples of suitable shielding components are described in WO2020 / 069442 and WO2020 / 069445, each of which is incorporated herein by reference in its entirety.

[0104] In some embodiments, 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 may include, for example, branched or linear aliphatic moieties that may be unsubstituted or substituted, or moieties derived from natural lipid compounds, including unsubstituted or substituted fatty acids, sterols, and isoprenoids.

[0105] In some embodiments, the lipid portion may include a branched or straight chain aliphatic portion having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms. In some embodiments, the aliphatic portion may contain one or more heteroatoms and / or one or more double bonds or triple bonds (i.e., saturated or monounsaturated or polyunsaturated). In some cases, the lipid portion may include an aliphatic, straight chain or branched chain portion, each hydrophobic tail independently having about 8 to about 30 carbon atoms or about 6 to about 30 carbon atoms, wherein the aliphatic portion may be unsubstituted or substituted. In various embodiments, the lipid portion may include, for example, an aliphatic carbon chain derived from a fatty acid and a fatty alcohol. In some embodiments, each lipid portion is independently C8-C 24 -alkyl or C8-C 24 -alkenyl, wherein C8-C 24 An -alkenyl group may in some cases be monounsaturated or polyunsaturated.

[0106] The natural lipid moieties used in the practice of the present disclosure can be derived from, for example, phospholipids, glycerides (such as diglycerides or triglycerides), glycoglycerides, sphingolipids, ceramides, as well as saturated and unsaturated sterols, isoprenoids, and other similar natural lipids.

[0107] Other suitable lipid moieties may include lipophilic aromatic groups, such as optionally substituted aryl or arylalkyl moieties, including, for example, naphthyl or ethylbenzyl, or lipids comprising ester functionalities, including, for example, sterol esters and wax esters.

[0108] In some specific implementations, 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 specific implementations, the PEGylated lipid comprises a PEG-modified sterol. In various specific implementations, the PEGylated lipid comprises a PEG-modified cholesterol. In some specific implementations, the PEGylated lipid is a PEG-modified ceramide. In some cases, 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.

[0109] In some embodiments, the PEGylated lipid is a PEG-modified phospholipid, wherein 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-di(undecanoyl)-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-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-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 (ME 16.0PE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glyceryl-3-phosphoethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phospho-racem-(1-glyceryl) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In various specific implementations, the phospholipid is DOPE.

[0110] In some embodiments, the one or more PEGylated lipids include PEG-modified phosphatidylethanol. In 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).

[0111] In various specific 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 specific implementations, the PEG lipid is DMG-PEG.

[0112] The molecular weight of the PEG chain in the aforementioned PEGylated lipid can be adjusted as needed to optimize the characteristics of the delivery vehicle composition. In some specific implementations, the PEG chain has 350g / mol to 6,000g / mol, 1,000g / mol to 5,000g / mol or 2,000g / mol to 5,000g / mol or about 1,000g / mol to 3,000g / mol or about 1,500g / mol to 4,000g / mol molecular weight. In some cases, the PEG chain of PEG lipid has about 350g / mol, 500g / mol, 600g / mol, 750g / mol, 1,000g / mol, 2,000g / mol, 3,000g / mol, 5,000g / mol or 10,000g / mol molecular weight. 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-PEG 2000).

[0113] In embodiments, 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 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 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-PEG 2000), based on the total 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-PEG 2000) based on the total moles of components in the delivery vehicle composition.

[0114] Representative Examples

[0115] Non-limiting delivery vehicle compositions are described below. As previously described, the unit "mol %" or "mole percent" refers to the number of moles of a particular component of the delivery vehicle composition divided by the total number of moles of all components in the delivery vehicle composition multiplied by 100%.

[0116] In some embodiments, the delivery vehicle composition comprises at least 99 mol% of the cationic component and less than about 1 mol% of the shielding component (e.g., Formulation F1A in Table 2). In some cases, the delivery vehicle composition comprises less than about 20 mol% of the cationic component, less than about 5 mol% of the shielding component, and greater than about 75 mol% of a mixture of anionic / zwitterionic components and neutral lipid components (e.g., Formulation F2A and Formulation F4A in Table 2). In some cases, the delivery vehicle composition comprises about 30 mol% to about 45 mol% of the cationic component, about 50 mol% to about 70 mol% of the mixture of anionic / zwitterionic components and neutral lipid components, and about 1.5 mol% to about 4.5 mol% of the shielding component (e.g., Formulation F3A and Formulation F5A in Table 2). In various cases, the delivery vehicle composition comprises about 15 mol% to about 35 mol% of a cationic component, about 60 mol% to about 80 mol% of a mixture of an anionic / zwitterionic component and a neutral lipid component, and about 1.5 mol% to about 3.0 mol% of a shielding component (e.g., Formulations F2A and F3A in Table 2). In some implementations, the delivery vehicle composition comprises about 15 mol% to about 35 mol% of a cationic component, about 10 mol% to about 20 mol% of an anionic / zwitterionic component, about 50 mol% to about 65 mol% of a neutral lipid component, and about 1.5 mol% to about 3.0 mol% of a shielding component (e.g., Formulations F2A and F3A in Table 2). In various implementations, the delivery vehicle composition comprises about 10 mol% to about 20 mol% of a cationic component, about 75 mol% to about 89 mol% of a lipid component, and about 1 mol% to about 5 mol% of a shielding component (e.g., Formulation F4A in Table 2). In some cases, the delivery vehicle composition comprises about 40 mol% to about 50 mol% of the cationic component, about 50 mol% to about 59 mol% of the anionic / zwitterionic component, and about 1 mol% to about 5 mol% of the shielding component (e.g., Formulation F5A in Table 2). In various cases, the delivery vehicle composition comprises about 30 mol% to about 50 mol% of the cationic component, about 50 mol% to about 70 mol% of the neutral lipid component, and about 1 mol% to about 5 mol% of the shielding component (e.g., Formulation F6A in Table 2). In various cases, the delivery vehicle composition comprises about 40 mol% to about 45 mol% of the cationic component, about 50 mol% to about 60 mol% of a mixture of anionic / zwitterionic components and neutral lipid components, and about 1.5 mol% to about 2.0 mol% of the shielding component (e.g., Formulation F6.1 and Formulation F6.2 in Table 2).In some embodiments, the delivery vehicle composition comprises about 40 mol% to about 45 mol% of the cationic component, about 10 mol% to about 15 mol% of the anionic / zwitterionic component, about 40 mol% to about 45 mol% of the neutral lipid component, and about 1.5 mol% to about 2.0 mol% of the shielding component (e.g., Formulations F6.1 and F6.2 in Table 2). In various cases, the delivery vehicle composition comprises about 30 mol% to about 35 mol% of the cationic component, about 60 mol% to about 70 mol% of the mixture of the anionic / zwitterionic component and the neutral lipid component, and about 2.0 mol% to about 3.0 mol% of the shielding component (e.g., Formulation F6.3 in Table 2). In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 35 mol% of a cationic component, about 10 mol% to about 15 mol% of an anionic / zwitterionic component, about 50 mol% to about 55 mol% of a neutral lipid component, and about 2.0 mol% to about 3.0 mol% of a shielding component (e.g., Formulation F6.3 in Table 2). The cationic component can be any cationic component described herein, such as any compound of formula (I) (e.g., compounds listed in Table 1, such as compounds 1, 6, 21, or 30). In some embodiments, the cationic compound is compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., phospholipids). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., PEGylated lipids). In some implementations, the shielding component is DMG-PEG2000.

[0117] 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 mol% to about 52 mol% of a cationic component; about 9 mol% 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 cases, 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 the phospholipids, about 30 mol% to about 55 mol% of the sterols, and about 1 mol% to about 3 mol% of the PEGylated lipids. In some cases, the composition comprises about 35 mol% to about 49 mol% of a compound or salt of formula (I); about 7 mol% to about 12 mol% of the phospholipids, about 35 mol% to about 50 mol% of the sterols, and about 1 mol% to about 2 mol% of the PEGylated lipids. The cationic component can be any cationic component described herein, such as any compound of formula (I) (e.g., compounds listed in Table 1, such as compounds 1, 6, 21 or 30). In some specific 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., phospholipids). In some specific implementations, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., sterol). In some specific implementations, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., pegylated lipids). In some specific implementations, the shielding component is DMG-PEG2000.

[0118] In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 45 mol% of the cationic component; about 5 mol% to about 15 mol% of the anionic / zwitterionic component, about 40 mol% to about 60 mol% of the neutral lipid compound, and about 1 mol% to about 5 mol% of the shielding component. In various embodiments, the delivery vehicle composition comprises about 35 mol% to about 40 mol% of the cationic component; about 8 mol% to about 12 mol% of the anionic / zwitterionic component, about 45 mol% to about 50 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 38.2 mol% of the cationic component; about 11.8 mol% of the anionic / zwitterionic component, about 48.2 mol% of the neutral lipid compound, and about 1.9 mol% of the shielding component ("Form F2"). The cationic component can be any cationic component described herein, such as any compound of formula (I) (e.g., compounds listed in Table 1, such as compounds 1, 6, 21, or 30). In some specific 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., phospholipids). In some specific implementations, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., sterol). In some specific implementations, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., PEGylated lipids). In some specific implementations, the shielding component is DMG-PEG-2000. In some specific implementations, the delivery vehicle composition comprises Form F2 as shown in Table 2 below. In some implementations, 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.

[0119] In some embodiments, the delivery vehicle composition comprises about 45 mol% to about 55 mol% of the cationic component; about 5 mol% to about 15 mol% of the anionic / zwitterionic component, about 35 mol% to about 55 mol% of the neutral lipid compound and about 1 mol% to about 5 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 51.3 mol% of the cationic component; about 9.3 mol% of the anionic / zwitterionic component, about 38.0 mol% of the neutral lipid compound and about 1.5 mol% of the shielding component ("Form F6 / 17"). The cationic component can be any cationic component described herein, such as any compound of formula (I) (e.g., compounds listed in Table 1, such as compounds 1, 6, 21, or 30). In some embodiments, the cationic compound is compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., phospholipids). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., PEGylated lipids). In some embodiments, the shielding component is DMG-PEG 2000. In some embodiments, the delivery vehicle composition comprises form F6 / 17 as shown in Table 2 below. In some implementations, 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-PEG 2000.

[0120] In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 49 mol% of cationic components; about 5 mol% to about 15 mol% of anionic / zwitterionic components, about 30 mol% to about 55 mol% of neutral lipid compounds, and about 1 mol% to about 3 mol% of shielding components. In various embodiments, the delivery vehicle composition comprises about 48 mol% to about 52 mol% of cationic components; about 5 mol% to about 12 mol% of anionic / zwitterionic components, about 38 mol% to about 42 mol% of neutral lipid compounds, and about 1 mol% to about 3 mol% of shielding components. In various embodiments, the delivery vehicle composition comprises about 42.6 mol% of cationic components; about 10.0 mol% of anionic / zwitterionic components, about 44.7 mol% of neutral lipid compounds, and about 1.7 mol% of shielding components. The cationic component can be any cationic component described herein, such as any compound of formula (I) (e.g., compounds listed in Table 1, such as compounds 1, 6, 21, or 30). In some embodiments, the cationic compound is compound 1, 6, 21 or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., phospholipids). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., PEGylated lipids). In some embodiments, the shielding component is DMG-PEG 2000. In some embodiments, the delivery vehicle composition comprises form F6 / 12 or form F6 / 15 as shown in Table 2 below. In some embodiments, the delivery vehicle 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 implementations, 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.

[0121] 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 embodiments, 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 compound of formula (I) (e.g., compounds listed in Table 1, such as compounds 1, 6, 21, or 30). In some embodiments, the cationic compound is compound 1, 6, 21, or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., phospholipids). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., pegylated lipids). In some embodiments, the shielding component is DMG-PEG 2000. In some implementations, the delivery vehicle composition comprises F6.1 or F6.2 as shown in Table 2 below. In some implementations, the delivery vehicle composition comprises about 44.4 mol% of Compound 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 some implementations, 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-PEG 2000.

[0122] In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 39 mol% of cationic components; about 5 mol% to about 15 mol% of anionic / zwitterionic components, about 30 mol% to about 55 mol% of neutral lipid compounds and about 1 mol% to about 3 mol% of shielding components. In various embodiments, the delivery vehicle composition comprises about 30 mol% to about 35 mol% of cationic components; about 7 mol% to about 12 mol% of anionic / zwitterionic components, about 50 mol% to about 55 mol% of neutral lipid compounds and about 2 mol% to about 3 mol% of shielding components. In various embodiments, the delivery vehicle composition comprises about 33.1 mol% of cationic components; about 10.5 mol% of anionic / zwitterionic components, about 53.8 mol% of neutral lipid compounds and about 2.5 mol% of shielding components. The cationic component can be any cationic component described herein, such as any compound of formula (I) (e.g., compounds listed in Table 1, such as compounds 1, 6, 21 or 30). In some embodiments, the cationic compound is compound 1, 6, 21 or 30. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., phospholipids). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., PEGylated lipids). In some embodiments, the shielding component is DMG-PEG 2000. In some embodiments, 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-PEG 2000.

[0123] Non-limiting examples of delivery vehicle compositions of the present disclosure based on a compound of Formula (I) (eg, Compound 1, 6, 21, or 30) as the cationic component (characterized in mole %) can be found in Table 2 below.

[0124] Table 2. Delivery vehicle composition

[0125]

[0126] 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.

[0127] Delivery Vehicle Complex (DV)

[0128] The delivery vehicle compositions disclosed herein can form a complex with one or more polyanionic compounds (e.g., nucleic acids) through electrostatic interactions between the cationic components of the delivery vehicle compositions and the polyanionic compounds. Therefore, a delivery vehicle complex refers to a mixture comprising a delivery vehicle composition as disclosed herein and a polyanionic compound. In some cases, the complex allows a large amount of cargo to be encapsulated, is stable, and exhibits excellent efficiency and tolerance in vivo. Therefore, the delivery vehicle complex can be used as a delivery vehicle for transporting polyanionic cargo encapsulated therein to target cells. Additionally or alternatively, the delivery vehicle complex can include non-anionic cargo. Therefore, another aspect of the present disclosure relates to a delivery vehicle complex, which comprises: (1) a delivery vehicle composition as previously described herein, and (2) a polyanionic compound (or cargo). In some specific implementations, the delivery vehicle composition is complexed with a polyanionic compound (e.g., an RNA). In various specific 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., 3, 4 or 5 different RNAs). In some cases, the multi-component delivery vehicle system is complexed with one or more of nucleic acids selected from DNA and RNA (e.g., antigenic RNA and auxiliary DNA, such as CpG).

[0129] The delivery vehicle complex described herein may 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 each component in the delivery vehicle complex can be easily calculated based on the known concentration and volume of the stock solution of each component used when preparing the complex. In addition, if a non-anionic cargo is present in the delivery vehicle complex, the mass ratio can provide a more accurate representation of the relative amount of the delivery vehicle component to the total cargo compared to the cation: anion charge ratio without considering the non-anionic material. Specifically, the mass ratio of the component refers to the ratio of the mass of the specific component in the system to the mass of the "cargo" in the system. "Cargo" can refer to the total polyanionic compound present in the system. In one example, the polyanionic compound can refer to a nucleic acid. In one example, the polyanionic compound refers to an mRNA encoding at least one protein.

[0130] In some implementations, the cationic component and the 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 the polyanionic compound of the delivery vehicle complex have a mass ratio of about 2:1 to about 5:1. In other implementations, the cationic component and the polyanionic compound of the delivery vehicle complex have a mass ratio of about 3:1. In other implementations, the cationic component and the 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 embodiments, the cationic component of the delivery vehicle complex and the polyanionic compound have a mass ratio of about 13: 1. In other embodiments, the cationic component of the delivery vehicle complex and the polyanionic compound have a mass ratio of about 10: 1. In some embodiments, the cationic component can be a compound of formula (I), such as a compound listed in Table 1 (e.g., Compound 1, 6, 21 or 30).

[0131] In certain embodiments in which the delivery vehicle complex comprises a nucleic acid as a polyanionic compound or cargo, the mass ratio of the cationic component to the 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 embodiments, the mass ratio of the cationic component to the nucleic acid is about 2:1 to about 5:1. In other embodiments, the mass ratio of the cationic component to the nucleic acid is about 3:1. In other embodiments, the mass ratio of the cationic component to the nucleic acid is about 19:1. In other embodiments, the mass ratio of the cationic component to the nucleic acid is about 20:1. In other embodiments, the mass ratio of the cationic component to the nucleic acid is about 13:1. In other embodiments, the mass ratio of the cationic component to the nucleic acid is about 10:1. In some implementations, the cationic component can be a compound of formula (I), such as a compound listed in Table 1 (eg, Compound 1, 6, 21, or 30).

[0132] In some implementations, the mass ratio of the cationic component to the 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 the cationic component to the nucleic acid is about 20: 1. In various implementations, the mass ratio of the cationic component to the nucleic acid is about 19: 1. In some implementations, the mass ratio of the cationic component to the nucleic acid is about 17: 1. In various implementations, the mass ratio of the cationic component to the nucleic acid is about 15: 1. In various implementations, the mass ratio of the cationic component to the nucleic acid is about 13: 1. In various implementations, the mass ratio of the cationic component to the nucleic acid is about 12: 1. In various implementations, the mass ratio of the cationic component to the nucleic acid is about 10: 1. In some implementations, the cationic component can be a compound of formula (I) as previously described herein, such as the compounds listed in Table 1. In various implementations, the cationic component is Compound 1, 6, 21, or 30. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

[0133] In some cases, 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 cases, 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 previously described 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.

[0134] In some cases, the mass ratio of the neutral lipid component and 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 cases, the mass ratio of the neutral lipid component and 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 specific implementations, the mass ratio of the neutral lipid component and the polyanionic compound is about 5.4: 1. In some specific implementations, the mass ratio of the neutral lipid component and the polyanionic compound is about 8.1: 1. In some specific implementations, the mass ratio of the neutral lipid component and the polyanionic compound is about 6.7: 1. In some specific implementations, the neutral lipid component can be a sterol as previously described herein. In various specific implementations, the neutral lipid component is cholesterol. In some specific implementations, the polyanionic cargo is a nucleic acid, such as RNA.

[0135] In some cases, the mass 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 mass ratio of the neutral lipid component to the polyanionic compound is about 2.1:1. In some implementations, the mass 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 previously described herein. In various implementations, the shielding component is DMG-PEG 2000. In some implementations, the polyanionic cargo is a nucleic acid, such as RNA.

[0136] In some embodiments, 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 ("Form F2"). In some embodiments, 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 embodiments, the polyanionic compound is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises a mass ratio of about 10:1 of compound 1, 6, 21 or 30 to the nucleic acid, a mass ratio of about 2.7:1 of DSPC to the nucleic acid, a mass ratio of about 5.4:1 of cholesterol to the nucleic acid, and a mass ratio of about 1.4 of DMG-PEG 2000 to the nucleic acid.

[0137] In some embodiments, 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 ("Form F6 / 17"). In some embodiments, 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 embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises a mass ratio of about 17:1 of compound 1, 6, 21 or 30 to the nucleic acid, a mass ratio of about 2.7:1 of DSPC to the nucleic acid, a mass ratio of about 5.4:1 of cholesterol to the nucleic acid, and a mass ratio of about 1.4 of DMG-PEG 2000 to the nucleic acid.

[0138] In some embodiments, 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 ("Form F6 / 12"). In some embodiments, 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 embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises a mass ratio of about 12:1 of compound 1, 6, 21 or 30 to the nucleic acid, a mass ratio of about 2.7:1 of DSPC to the nucleic acid, a mass ratio of about 5.4:1 of cholesterol to the nucleic acid, and a mass ratio of about 1.4 of DMG-PEG 2000 to the nucleic acid.

[0139] In some embodiments, 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 ("Form F6 / 15"). In some embodiments, 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 embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises a mass ratio of about 15:1 of compound 1, 6, 21 or 30 to the nucleic acid, a mass ratio of about 2.7:1 of DSPC to the nucleic acid, a mass ratio of about 5.4:1 of cholesterol to the nucleic acid, and a mass ratio of about 1.4 of DMG-PEG 2000 to the nucleic acid.

[0140] In some embodiments, 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 embodiments, 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 embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises a mass ratio of about 13:1 of compound 1, 6, 21 or 30 to the nucleic acid, a mass ratio of about 2.7:1 of DSPC to the nucleic acid, a mass ratio of about 5.4:1 of cholesterol to the nucleic acid, and a mass ratio of about 1.4 of DMG-PEG 2000 to the nucleic acid.

[0141] In some embodiments, 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 embodiments, 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 embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises a mass ratio of about 19:1 of compound 1, 6, 21 or 30 to the nucleic acid, a mass ratio of about 4.0:1 of DSPC to the nucleic acid, a mass ratio of about 8.1:1 of cholesterol to the nucleic acid, and a mass ratio of about 2.1 of DMG-PEG 2000 to the nucleic acid.

[0142] In some embodiments, 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 embodiments, 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 embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises a mass ratio of about 9.7:1 of compound 1, 6, 21 or 30 to the nucleic acid, a mass ratio of about 2.7:1 of DSPC to the nucleic acid, a mass ratio of about 6.7:1 of cholesterol to the nucleic acid, and a mass ratio of about 2.1 of DMG-PEG 2000 to the nucleic acid.

[0143] In other embodiments, the amount of polyanionic cargo present in the delivery vehicle complex can be characterized by the mass ratio of the delivery vehicle composition (e.g., the total amount of hydroxyalkyl-terminated cationic peptides such as 2-aminopropane-1,3-diol-terminated cationic peptides, phospholipids, cholesterol and / or shielding components) to one or more polyanionic cargo compounds. In some embodiments, the mass ratio of the delivery vehicle composition to the 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 some embodiments, the mass ratio of the delivery vehicle composition to the one or more polyanionic cargo compounds is about 5:1 to about 8:1 or about 6:1 to about 7:1.

[0144] In some embodiments, the compound of formula (I) or a salt thereof, phospholipid, sterol and 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.

[0145] In some embodiments, the delivery vehicle complex 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 having a cation: 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 having a cation: 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 having a cation: anion charge ratio of about 2: 1 to about 5: 1. In other implementations, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation: anion charge ratio of about 3: 1. In some implementations, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation: anion charge ratio of about 3.7: 1. In some embodiments, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation: anion charge ratio of about 6.4: 1. In some embodiments, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation: anion charge ratio of about 4.8: 1. In some embodiments, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation: anion charge ratio of about 7.2: 1. In some embodiments, the delivery vehicle complex comprises a cationic compound and a nucleic acid having a cation: anion charge ratio of about 3.6: 1. In some 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.

[0146] Non-limiting examples of delivery vehicle compositions characterized by mass ratios and charge ratios can be found in Table 3 below.

[0147] Table 3. Mass and charge ratios of delivery vehicle system components and polyanionic cargo

[0148]

[0149]

[0150] Delivery vehicle complex characterization

[0151] The delivery vehicle complex disclosed herein can be characterized by various parameters such as particle size, polydispersity index, and encapsulation percentage of cargo. In one embodiment, the delivery vehicle complex is similar to a nanoparticle, including at least one polyanionic compound (described further below) encapsulated by a delivery vehicle composition. In one embodiment, such a complex is an mRNA nanoparticle, including a delivery vehicle composition encapsulating at least one mRNA.

[0152] In some embodiments, the delivery vehicle complex 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 diameter of the delivery vehicle complex disclosed herein can range in size from about 40 nm to about 200 nm, or from about 50 nm to about 175 nm, or from about 50 nm to about 200 nm, or 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 another implementation, the complex can have a diameter greater than about 100 nm, for example, from about 105 nm to about 250 nm, from about 110 nm to about 220 nm, from about 150 nm to about 200 nm, from about 110 nm to about 200 nm. In one implementation, the complex can have a diameter of about 105 nm to about 200 nm. In some cases, the delivery vehicle complex has a particle size of about 40nm to about 115nm, or about 55nm to about 95nm, or about 70nm to about 80nm, or about 75nm. In various cases, the delivery vehicle complex has a particle size of about 135nm to about 225nm, or about 155nm to about 195nm, or about 170nm to about 180nm, or about 175nm. In some cases, the particle size depends on the method used to prepare the complex (e.g., via a microfluidic device or manually). The particle size / diameter can be determined by dynamic light scattering (DLS) and is described in Example 3.

[0153] In some implementations, the delivery vehicle complexes of the present disclosure have 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.

[0154] 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 completely encapsulated in the delivery vehicle complex. As described in Example 3, the percentage of mRNA encapsulated in the delivery vehicle complex can be determined using a modified RiboGreen assay.

[0155] In some embodiments, the delivery vehicle complex of the present disclosure retains 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 after storage at 4°C-10°C for at least 10 days, such as at least 20 days, 30 days, 40 days, 50 days, 60 days, 70 days or more. In one embodiment, the complex retains the above-mentioned level of polyanionic compound for 48 days at 4°C. In addition, in some cases, after being stored at 4° C. for at least 10 days, such as at least 20 days, 30 days, 40 days, 50 days, 60 days, 70 days or more, the delivery vehicle complex of the present disclosure retains at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% of its original size. In a specific implementation, the delivery vehicle complex retains the above size after being stored at 4° C. for 48 days.

[0156] The delivery vehicle complexes disclosed herein are well tolerated at both high and low doses without systemic toxicity or adverse events.

[0157] Other Delivery Vehicle Complex Components

[0158] The delivery vehicle complexes described herein may include other components for fine-tuning the complexes for specific applications. Examples of components may include those that promote endosomal escape, including but not limited to buffering amines or polyamines; nitrogen-containing heterocyclic groups and / or nitrogen-containing heteroaryl groups, such as imidazole, pyrrole, pyridine, pyrimidine; maleic acid derivatives; or membrane lytic peptides.

[0159] 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 mimetic, 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 bipedal 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 RNA or DNA or an artificial nucleic acid; a small molecule; a carbohydrate such as mannose, galactose, arabinose; a vitamin such as ascorbic acid, niacin, pantothenic acid, carnitine, inositol, pyridoxal, lipoic acid, folic acid, riboflavin, biotin, vitamin B12, vitamin A, E, K; a protein or peptide that binds to a cell surface receptor such as thrombospondin, tumor necrosis factor (TNF), annexin V, interferon, cytokine, transferrin, GM-CSF (granulocyte-macrophage colony stimulating factor), or a growth factor such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).

[0160] The delivery vehicle complex may also optionally include small molecule drugs or other biologics incorporated into the delivery vehicle complex. Non-limiting examples include incorporation of drugs that disrupt the blood-brain barrier or enhance cellular uptake; drugs that affect intracellular trafficking or endosomal escape; or drugs that are immunomodulators that affect antigen presentation when the delivery vehicle complex is used as a vaccine.

[0161] Polyanionic compounds

[0162] The delivery vehicle complex of the present disclosure may include one or more polyanionic compounds (polyanionic cargoes), which can be delivered to a target in vivo, such as a cell, via the complex. The polyanionic compound can be complexed with the cationic component (e.g., a compound of formula (I), such as compound 1, 6, 21 or 30) of the delivery vehicle complex via electrostatic interactions.

[0163] In some specific implementations, the polyanionic compound includes nucleic acid. As used herein, nucleic acid includes naturally occurring nucleic acid (e.g., DNA, RNA and / or its hybrid) and non-naturally occurring nucleic acid. Non-limiting examples of non-natural amino acids are those amino acids comprising non-natural backbones, modified backbone connections (such as thiophosphates), non-natural or modified bases and / or non-natural and modified ends. 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.

[0164] In some implementations, the polyanionic compound comprises RNA. RNA can be selected from the group consisting of chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA, mRNA, oligoribonucleotides, viral RNA, retroviral RNA, self-replicating (replicon) RNA (srRNA), tRNA, rRNA, immunostimulatory RNA, microRNA, siRNA, small nuclear RNA (snRNA), small hairpin (sh) RNA riboswitch, RNA aptamer, RNA bait, antisense RNA, ribozyme or any combination thereof. In some implementations, the nucleic acid cargo is RNA, including but not limited to modified mRNA, self-amplification RNA and circular RNA. In some implementations, RNA comprises coding RNA.

[0165] RNA is a 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 connected to each other along the so-called main chain. The main chain is formed by a phosphodiester bond between the sugar (i.e., ribose) of the first monomer and the phosphate portion of the second adjacent monomer. The specific order of monomers, i.e., the order of bases connected to the sugar / phosphate main chain, is called the RNA sequence. Generally, RNA can be obtained by transcription of a DNA sequence, for example, in a cell. In eukaryotic cells, transcription is usually carried out in the nucleus or mitochondria. In vivo, transcription of DNA usually results in so-called immature RNA (also referred to as pre-mRNA, pre-mRNA or heterologous nuclear RNA), which must be processed into so-called messenger RNA, usually abbreviated as mRNA. The processing of immature RNA, for example in eukaryotic organisms, includes a variety of different post-transcriptional modifications, such as splicing, 5'-capping, polyadenylation, export from the nucleus or mitochondria, etc. The sum of these processes is also referred to as the maturation of RNA. The mature messenger RNA generally provides a nucleotide sequence that can be translated into an amino acid sequence of a specific peptide or protein. Typically, the mature mRNA comprises a 5'-cap, an optional 5'UTR, an open reading frame, an optional 3'UTR and a poly(A) tail.

[0166] In addition to messenger RNA, there are several non-coding types of RNA that may be involved in the regulation of transcription and / or translation and immune stimulation. In the present disclosure, the term "RNA" further encompasses any type of single-stranded (ssRNA) or double-stranded RNA (dsRNA) molecules known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), 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).

[0167] 5'-cap structure: The 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, the 5'-cap is added using a 5'-5'-triphosphate bond (also known as m7GpppN). Other examples of 5'-cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4',5' methylene nucleotide, 1-(β-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, α-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-sequenced nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3' phosphorothioate, phosphorodithioate, or a bridged or non-bridged methylphosphonate moiety. These modified 5'-cap structures can be used in the context of the present disclosure to modify the RNA sequences of the present disclosure. Further modified 5'-cap structures that can be used in the context of the present disclosure are CAP1 (additional methylation of the ribose of the adjacent nucleotide of 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., phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0168] In the context of the present disclosure, a 5' cap structure can also be formed using a cap analog in chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping), or a capping enzyme (eg, commercially available capping kits) can be used to form the cap structure in vitro.

[0169] A cap analog refers to a non-polymerizable dinucleotide that has a cap function because, when incorporated into the 5' end of an RNA molecule, it promotes translation or localization of the RNA molecule, and / or prevents degradation of the RNA molecule. Non-polymerizable means that the cap analog will only be incorporated 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.

[0170] 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). N has recently been described. 7 Synthesis of -(4-chlorophenoxyethyl)-substituted dinucleotide cap analogs.

[0171] The poly(A) tail, also referred to as the "3'-poly(A) tail" or "poly(A) sequence" is typically a long homopoly(adenosine) nucleotide sequence of up to about 400 adenosine nucleotides, such as 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 the mRNA. In certain embodiments of the present disclosure, the poly(A) tail of the mRNA or srRNA is derived from a DNA template by in vitro transcription of RNA. Alternatively, the poly(A) sequence can also be obtained in vitro by conventional chemical synthesis methods without having to be transcribed from a DNA progenitor cell. In addition, the poly(A) sequence or poly(A) tail can be produced by enzymatic polyadenylation of RNA.

[0172] Stable nucleic acids generally have modifications that increase resistance to degradation in vivo (e.g., degradation by exonucleases or endonucleases) and / or in vitro degradation (e.g., by a manufacturing process before the composition is administered, e.g., in the process of preparing a composition to be administered). The stabilization of RNA can be achieved, for example, by providing a 5'-cap structure, a poly (A) tail, or any other UTR modification. Stabilization can also be achieved by backbone modification (e.g., using a synthetic backbone such as phosphorothioate) or by modifying the G / C content or C content of the nucleic acid. Various other methods for stabilizing or otherwise improving the function of nucleic acids are known in the art and are conceivable in the context of the present disclosure. Therefore, provided herein are polynucleotides designed to improve stability and / or clearance in tissues, receptor uptake and / or kinetics, cell entry, binding to translation mechanisms, RNA half-life, translation efficiency, immune escape, immune induction (for vaccines), protein production capacity, secretion efficiency (when applicable), accessibility of circulation, protein half-life, and / or cell state, function, and / or regulation of activity.

[0173] 5'-UTR is generally understood as a specific part of RNA. It is located at 5' of the open reading frame of mRNA. In the case of srRNA, the open reading frame encodes viral nonstructural proteins, and the sequence of interest is encoded in the subgenomic fragment of the viral RNA. Therefore, 5'UTR is located upstream of the nsP1 open reading frame. In addition, the subgenomic RNA of srRNA has a 5'UTR. Therefore, the subgenomic RNA containing the sequence of interest encoding the protein of interest contains a 5'UTR. Typically, 5'-UTR starts with a transcription start site and ends at one nucleotide before the start codon of the open reading frame. 5'-UTR may include elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements may be, for example, ribosome binding sites or 5'-terminal oligopyrimidine regions. 5'-UTR may be modified post-transcriptionally, for example, by adding a 5'-cap. In the context of the present disclosure, 5'UTR corresponds to the sequence of a mature mRNA or srRNA located between a 5'-cap and a start codon. In one embodiment, the 5'-UTR corresponds to a sequence extending from the nucleotides located 3' to the 5'-cap, and in certain embodiments from the nucleotides located immediately adjacent to the 3' to the 5'-cap to the nucleotides located 5' to the start codon of the protein coding region, and in some cases to the nucleotides located immediately adjacent to the start codon of the protein coding region. The nucleotides located immediately adjacent to the 3' to the 5'-cap of the mature mRNA or srRNA generally correspond to the transcription start site. The term "corresponding to" means that the 5'-UTR sequence can be an RNA sequence, such as in an mRNA sequence used to define a 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", is a sequence corresponding to the 5'-UTR of a mature mRNA derived from the gene (i.e., an mRNA obtained by transcription of the gene and maturation of the immature mRNA). The term "5'-UTR of a gene" includes DNA sequences and RNA sequences of the 5'-UTR.

[0174] Typically, 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 a protein coding region (open reading frame (ORF) or coding sequence (CDS)) and a poly(A) sequence of an mRNA. In the context of the present disclosure, the term 3'-UTR may also include elements that are not encoded in the template for transcribed RNA 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.

[0175] In the case of srRNA, the 3'-UTR sequence is usually encoded by the viral genomic RNA, which is transcribed into the corresponding mRNA during gene expression. The genomic sequence is first transcribed into immature mRNA. Then in the maturation process, the immature mRNA is further processed into mature mRNA. The maturation process includes 5' capping. In the context of the present disclosure, the 3'-UTR corresponds to the sequence of mature mRNA or srRNA (and srRNA subgenomic RNA), which is located in the termination codon of the protein coding region, preferably between the 3' to the termination codon of the protein coding region and the poly (A) sequence of the mRNA next to the sequence of interest. The term "corresponding to" means that the 3'-UTR sequence can be an RNA sequence, such as in the mRNA sequence for defining 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 a sequence corresponding to the 3'-UTR of the mature mRNA derived from the gene (i.e., the mRNA obtained by transcription of the gene and maturation of the immature mRNA). The term "3'-UTR of a gene" includes the DNA sequence and the RNA sequence (sense and antisense strands and mature and immature) of the 3'-UTR.

[0176] According to certain specific implementations of the present disclosure, the RNA used for the delivery vehicle complex herein comprises RNA containing at least one region encoding a peptide (e.g., 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., polypeptide) or a protein that retains the ability to induce an immune response. In one specific implementation, the coding RNA is selected from the group consisting of mRNA, viral RNA, retroviral RNA, and self-replicating RNA. In some specific implementations, the RNA encodes a viral peptide (e.g., 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 subtypes HPV 16, 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 HPV 16 and / or 18. In some cases, the RNA encodes a viral spike protein or a functional fragment thereof. In some cases, the RNA encodes SARS-related coronaviruses (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 OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1) and / or human coronavirus NL63 (HCoV-NL63)). In various specific 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 specific implementations, the RNA encodes an influenza hemagglutinin (HA) or a functional fragment thereof. In some implementations, the influenza A virus has an HA of a 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.

[0177] Contemplated viruses that the RNA of the delivery vehicle complex may encode include, but are not limited to, influenza A and B, polio virus, adenovirus, rabies virus, bovine parainfluenza virus 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 and human immunodeficiency virus, cytomegalovirus, varicella-zoster virus, Epstein-Barr virus (Epstein-Barr virus), and herpes simplex virus-2. 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, chicken alphaherpesvirus-1, Ebola virus, Marburg virus, alphavirus, flavivirus, yellow fever virus, dengue virus, Japanese Enchephalitis virus, West Nile Viruses, Zika virus, Venezuelan Equine Encephalomyelitis virus, Chikungunya virus, Western Equine Encephalomyelitis virus, Eastern Equine Encephalomyelitis virus, Tick-borne Encephalitis virus, Kyasanur Forest Disease virus virus), Alkhurma 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 virusvirus) (Colorado tick-borne fever virus), coxsackievirus, rotavirus, norovirus, astrovirus, adenovirus, adenovirus, human metapneumovirus, rhinovirus or coronaviruses such as SARS-CoV, SARS-CoV-2, MERS-CoV, HCoVNL63, HKU1, 229E and OC43, human papillomavirus, Ebolavirus, Marburgvirus, alphavirus, flavivirus, yellow fever, dengue, Japanese encephalitis, West Nile virus, Zika virus, Venezuelan equine encephalitis virus, chikungunya virus, western equine encephalitis virus, eastern equine encephalitis virus, tick-borne encephalitis virus, Casanore Forest disease, Alkhurma 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 1, human herpesvirus 2, herpes simplex virus, gammaherpesvirus subfamily, chicken alphaherpesvirus 1, smallpox, alphavirus, molluscum contagiosum virus, hepatitis A virus, hepatitis B virus, hepatitis C Inflammation, hepatitis D, hepatitis E, poliovirus, Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, Paramyxoviridae or Togaviridae, Flaviviruses such as Zika virus, Colorado tick fever virus (Colorado tick fever virus), Coxsackievirus, Rotavirus, Norovirus, Astrovirus, Adenovirus, Adenovirus, Influenza A virus, Human metapneumovirus, Rhinovirus, Coronavirus, Varicella virus, Adeno-associated virus, Aichi virus (Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, macaque herpesvirus, Chandipura virus, Chikungunya virus, Coxsackie virus A, Vaccinia virus, Coxsackie virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Duvenhage virusvirus), Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, European bat rabies virus, GB virus C / hepatitis G virus, Hantaan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis D virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68, 70, Human papillomavirus 1, Human papillomavirus 2, Human papillomavirus 16, 18, Human parainfluenza, Human parvovirus B19, Human respiratory syncytial virus, Human rhinovirus, Human SARS coronavirus, Human foamy retrovirus, Human T-lymphotropic virus, Human torovirus, Influenza A virus, Influenza B virus, Influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus arenavirus), KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria marburgvirus, Langat virus, Lassa virus, Lordsdale virus, jumping disease virus, lymphocytic choriomeningitis virus, Machupovirus, Mayaro virus, coronavirus, measles virus, Mengoencephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, molluscum contagiosum virus, Murray valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus, Oropouche virus virus), Pichindevirus, 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, Salivirus A, Sandfly fever Sicilian virusSicilian virus), Sapporovirus, SARS coronavirus 2, Semliki forest virus, Seoul virus, simian foamy virus, simian virus type 5, Sindbis virus, Southampton virus, St.louis encephalitis virus, Tick-borne powassan virus, Parvovirus, Toscana virus, Uukuniemi virus, vaccinia virus, varicella-zoster virus, smallpox virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yabamonkey tumor virus, Yaba-like disease virus virus), yellow fever virus, Zika virus, bovine herpes virus, pseudorabies virus, Adenoviridae, bovine adenovirus BAdV-9 = human adenovirus type C, Anelloviridae (proposed family), TTV, Bornaviridae, Borna disease virus BDV, Bunyaviridae, Ainovirus, Cache valley virus CW, Crimean-Congo hemorrhagic fever virus CCHF, Hantavirus HTNV, Jamestown Canyon virus JCV, LaCrosse virus LACV, Puumala virus, Rift Valley fever virus RVFV, Caliciviridae, Norovirus, San Miguel sea lion virus virus) SMSV-5, Circoviridae, Bovine circovirus BCV = Evolution of porcine circovirus type 2 PCV-2, Coronaviridae, Bovine coronavirus BCoV-1, Bovine circovirus BtoV, Flaviviridae, Bovine viral diarrhea virus BVDV, Japanese encephalitis virus JEV, Casanol Forest disease virus KFDV, Jumping disease virus, Murray Valley encephalitis virus MVE, Saint Louis encephalitis virus (SLEV), Tick-borne encephalitis virus TBEV, Wesselsbron virus (Wesselsbronvirus), West Nile virus (including Kunjin), Hepaciviridae, Hepatitis E virus HEV, Herpesviridae, Bovine herpesvirus BHV-4, Equine herpesvirus EHV-1, Bovine infectious rhinotracheitis virus IBR=BHV-1, Pseudorabies virus PRV, Orthomyxoviridae, Dory virus, 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 enterovirus BEV-1, BEV-2, Bovine Kupchak virus (Bovine kobuvirus) BKV-1U-1 strain, encephalomyocarditis virus EMC, foot-and-mouth disease virus FMDV, Seneca valley virus SW, Polyomaviridae, bovine polyomavirus BPyV, Poxviridae, Aracatuba virus, bovine papular stomatitis virus BPSV, Cantagalo virus, vaccinia virus, pseudocowpox virus PCPV, vaccinia virus, Reoviridae, Banna virus BAV, bluetongue virus BTV, epidemic hemorrhagic disease virus EHDV, Liaoning virus (LiaoNing virus) LNV, reovirus, rotavirus, Retroviridae, bovine foamy virus BFV, bovine leukemia virus BLV, Rhabdoviridae, bovine epidemic fever virus BEFV, rabies virus, vesicular stomatitis virus VSV, Togaviridae, Eastern equine encephalitis virus EEEV, Getah virus, Ross River virus (Ross River virus) Rivervirus RRV, Sindbis virus, Venezuelan equine encephalitis virus VEE, Anelloviridae (proposed family), Tenovirus TTV, Bunyaviridae, Crimean Congo hemorrhagic fever virus CCHF, Hantavirus HTNV, James City 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, Lysis virusvirus), Japanese encephalitis virus JEV, jumping disease virus, Murray Valley encephalitis virus MVE, Powassan virus, tick-borne encephalitis virus TBEV, Wessels Brown virus, West Nile virus WNV (including Kunjin), Hepaciviridae, Hepatitis E virus HEV, Herpesviridae, bovine infectious 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, Tioman virus ( virus)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, Banna virus BAV, reovirus, rotavirus, Retroviridae, porcine endogenous retrovirus PERV, Rhabdoviridae, rabies virus, vesicular stomatitis virus VSV, Togaviridae, eastern equine encephalitis virus EEEV, Geta virus, Ross River virus RRV, or Venezuelan equine encephalitis VEE.

[0178] In some embodiments, RNA encodes adenovirus, alphavirus, calicivirus (e.g., calicivirus capsid antigen), coronavirus polypeptide, canine distemper virus, Ebola virus polypeptide, enterovirus, flavivirus, hepatitis virus (AE), herpes virus, infectious peritonitis virus, leukemia virus, Marburg virus, orthomyxovirus, papillomavirus, parainfluenza virus, paramyxovirus, parvovirus, pestivirus, picornavirus (e.g., poliovirus), poxvirus (e.g., vaccinia virus), rabies virus, reovirus, retrovirus and rotavirus. In some embodiments, RNA encodes SARS-CoV-2, HPV (e.g., E6 and / or E7 from HPV16 and / or HPV18) or influenza (e.g., influenza hemagglutinin (HA)).

[0179] In some implementations, two or more specific nucleic acids are delivered in combination together, and are 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 an mRNA encoding Cas9. In other implementations, nucleic acids can also be compounded with proteins, such as with CRISPR / Cas9 ribonucleoprotein complexes. In some cases, a multi-component delivery vehicle system is compounded with one or more of a nucleic acid selected from DNA and RNA (e.g., antigen RNA and auxiliary DNA, such as CpG).

[0180] Polynucleotide synthesis

[0181] Methods for preparing polynucleotides of predetermined sequence are well known. Solid phase synthesis methods for polyribonucleotides and polydeoxyribonucleotides are known (well-known methods for synthesizing DNA can also be used to synthesize RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can also be incorporated into polynucleotides.

[0182] Any method known in the art for preparing RNA is contemplated herein for use in preparing RNA. Exemplary methods for preparing RNA include, but are not limited to, chemical synthesis and in vitro transcription.

[0183] In some specific implementations, the RNA used in the methods herein is chemically synthesized. Chemical synthesis of relatively short oligonucleotide fragments with a definite chemical structure provides a fast and inexpensive approach to obtain the custom oligonucleotides of any desired sequence. Enzymes synthesize DNA and RNA only on the 5' to 3' direction, and chemical oligonucleotide synthesis does not have this limitation, but it is most usually carried out in the opposite direction, i.e. 3' to 5' direction. In some specific implementations, the method is implemented as solid phase synthesis using phosphoramidite methods and phosphoramidite structural units derived from protected nucleosides (A, C, G and U) or chemically modified nucleosides.

[0184] In some embodiments, the modification is included in the modified nucleic acid or in one or more individual nucleosides or nucleotides. For example, the modification of the nucleoside may include one or more modifications to the nucleobase, sugar and / or internucleoside bond. In some embodiments with at least one modification, the polynucleotide includes a backbone portion containing the following nucleobases, sugars and internucleoside bonds: 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-nucleoside-MP.

[0185] In other embodiments having at least one modification, the polynucleotide includes a backbone portion comprising the following nucleobases, sugars, and internucleoside linkages: pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, or 5-bromo-cytidine-MP. Nucleoside and nucleotide modifications contemplated for use in the present disclosure are known in the art.

[0186] In order to obtain the desired oligonucleotide, in a fully automated process, the building blocks are coupled successively to the oligonucleotide chains grown on the solid phase in the order required for the product sequence. After the chain assembly is complete, the product is released from the solid phase into the solution, deprotected and collected. The occurrence of side reactions sets practical restrictions on the length of the synthetic oligonucleotide (up to about 200 nucleotide residues) because the number of errors increases with the length of the synthesized oligonucleotide. The product is usually separated by HPLC to obtain highly purified desired oligonucleotide.

[0187] In some embodiments, RNA is prepared using in vitro transcription. The term "RNA in vitro transcription" or "in vitro transcription" relates 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 generating RNA transcripts. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which in some embodiments is a linearized plasmid DNA template. The promoter used to control in vitro transcription can be any promoter of 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 nucleic acids, particularly cDNA corresponding to the corresponding RNA to be transcribed in vitro, and introducing it into a suitable vector for in vitro transcription, such as plasmid DNA. In a specific embodiment of the present disclosure, before the in vitro transcription of the DNA template, it is linearized with a suitable restriction enzyme. cDNA can be obtained by reverse transcription or chemical synthesis of mRNA. In addition, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis.

[0188] Methods for in vitro transcription are known in the art. The reagents used in the method generally include: 1) a linearized DNA template having a promoter sequence that has a high binding affinity for its corresponding RNA polymerase, such as a bacteriophage-encoded RNA polymerase; 2) ribonucleoside triphosphates (NTPs) of the four bases (adenine, cytosine, guanine, and uracil); 3) in some cases, a cap analog as defined above (e.g., m7G(5')ppp(5')G(m7G)); 4) a DNA-dependent RNA polymerase (e.g., T7, T3, or SP6 RNA polymerase) that is capable of binding to the promoter sequence within the linearized DNA template; 5) an optional ribonuclease (RNase) inhibitor that inactivates any contaminating RNase; 6) an optional pyrophosphatase that degrades pyrophosphates, which can inhibit transcription; 7) MgCl2, which provides Mg 2+ ions as cofactors for the polymerase; 8) a buffer to maintain an appropriate pH value, which may also contain an optimal concentration of an antioxidant (eg DTT) and / or a polyamine, such as spermidine.

[0189] Methods for preparing delivery vehicle complexes

[0190] The components of the delivery vehicle complex can be prepared by a variety of physical and / or chemical methods to adjust their physical, chemical and biological properties. These can include hydroxyalkyl-terminated cationic peptides (such as 2-aminopropane-1,3-diol-terminated cationic peptides) in water or water-miscible organic solvents and the rapid combination of the required polyanionic cargo compounds (for example, oligonucleotides or nucleic acids) in water or aqueous buffer solutions. These methods may include simply mixing components by pipetting or microfluidic mixing methods (such as those involving T-mixers, vortex mixers or other chaotic mixing structures). In some specific implementations, a multi-component delivery system is prepared on a microfluidic platform.

[0191] It should be understood that the specific process conditions for preparing the delivery vehicle complex described herein can be adjusted or selected accordingly to provide the desired physical properties of the complex. For example, the parameters for mixing the components of the delivery system complex that can affect the final composition can include, but are not limited to, the mixing order, mixing temperature, mixing speed / rate, flow rate, physical dimensions of the mixed structure, concentration of the starting solution, molar ratio of the components, and the solvent used.

[0192] The formulation of the delivery vehicle complex can be accomplished in a number of ways. In some cases, all components can be premixed prior to adding the nucleic acid cargo, which can result in a uniform distribution of the components throughout the delivery particle.

[0193] In other cases, components can be added sequentially to produce a core-shell structure. For example, a cationic component can be added first to start particle coagulation, followed by a lipid component to allow the particle surface to bind to the target cell, followed by a shielding component to prevent particle aggregation. For example, a hydroxyalkyl-terminated cationic peptoid (such as a 2-aminopropane-1,3-diol-terminated cationic peptoid) can be premixed with a nucleic acid cargo to form a core structure. Then, a lipid component (such as a lipid component comprising phospholipids and cholesterol) can be added to affect intracellular / endosome membrane binding. Because the shielding component can be mainly used on the outside of a multi-component delivery system, the component can be introduced last so that it does not destroy the internal structure of the system, but provides a coating of the system after it is formed.

[0194] Additional components in complexes and compositions, such as polymers, surfactants, targeting moieties and / or excipients, can be mixed and combined with the remaining components before, during, or after the main components of the nucleic acid cargo, cationic component, lipid component, and shielding component have been combined.

[0195] Thus, also provided herein is a method of forming a delivery vehicle complex disclosed herein, the method comprising contacting a compound or salt of formula (I) with a polyanionic compound. In some specific implementations, the method comprises mixing a solution comprising a compound or salt of formula (I) with a solution comprising a polyanionic compound.

[0196] Drug formulation and administration

[0197] 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. An "effective amount" includes a "therapeutically effective amount" and a "prophylactic effective amount". The term "therapeutically effective amount" refers to an amount that effectively treats and / or improves a disease or condition of a subject. The term "prophylactic effective amount" refers to an amount that effectively prevents a disease or condition of a subject and / or substantially reduces its chance. As used herein, the terms "patient" and "subject" are used interchangeably and refer to animals, such as dogs, cats, cattle, horses and sheep (i.e., non-human animals) and humans. A specific patient or subject is a mammal (e.g., a human). The terms "patient" and "subject" include males and females. As used herein, the term "excipient" means any pharmaceutically acceptable additive, carrier, diluent, adjuvant or other ingredient other than an active pharmaceutical ingredient (API), which is appropriately selected according to the intended form of administration and is in accordance with conventional pharmaceutical practice.

[0198] The compounds of the present disclosure can be administered to a subject or patient in a therapeutically effective amount. The compounds can be administered alone or as part of a pharmaceutically acceptable composition or formulation. In addition, the compounds can be administered all at once, for example, by bolus injection, multiple administrations, or delivered substantially uniformly over a period of time. It should also be noted that the dosage of the compound can vary over time.

[0199] If desired, the delivery vehicle complexes disclosed herein and other pharmaceutically active compounds can be administered to a subject or patient by any suitable route, such as oral, rectal, parenteral (e.g., intravenous, intramuscular or subcutaneous), intracisternal, intravaginal, intraperitoneal, intravesical, or in the form of an oral, inhaled or nasal spray. Administration can provide a systemic effect (e.g., enteral or parenteral). All methods that can be used by those skilled in the art to administer pharmaceutically active agents are contemplated.

[0200] 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 (such as olive oil) and injectable organic esters such as ethyl oleate. For example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant, appropriate fluidity can be maintained.

[0201] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Microbial contamination may be prevented by adding various antibacterial and antifungal agents, such as 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 may be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0202] The pharmaceutical composition can be in the form of a sterile injection, an aqueous suspension or an oily suspension. The suspension can be prepared according to known techniques using those suitable dispersants or wetting agents and suspending agents mentioned above. Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, for example as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections.

[0203] Compositions for parenteral administration are applied in sterile media. Depending on the vehicle used and the concentration of the drug in the preparation, parenteral preparations can be suspensions or solutions containing dissolved drugs. Auxiliary agents such as local anesthetics, preservatives and buffers can also be added to parenteral compositions.

[0204] When the composition of the present disclosure is used as a vaccine, it may include one or more immunoadjuvants. As used herein, the term "immunoadjuvant" refers to a compound or mixture of compounds that accelerates, prolongs, enhances or changes the immune response when used to conjugate with an immunogen (e.g., a new antigen). When administered alone to a host, the adjuvant may be non-immunogenic, but when administered in combination with another antigen, the adjuvant enhances the host's immune response to the antigen. Specifically, the terms "adjuvant" and "immunoadjuvant" are used interchangeably in the present disclosure. The enhancement and / or extension of the duration of an adjuvant-mediated immune response can be assessed by any method known in the art, including but not limited to one or more of the following: (i) the number of antibodies produced in response to immunization with an adjuvant / antigen combination compared to the number of antibodies produced in response to immunization with an antigen alone Increase; (ii) an increase in the number of T cells that recognize antigens or adjuvants; and (iii) an increase in one or more cytokine levels. The adjuvant may be an aluminum-based adjuvant, 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 may depend on the antigen, vaccine and route of administration.

[0205] In some specific implementations, adjuvants improve the adaptive immune response to vaccine antigens by regulating innate immunity or promoting transport and presentation. The adjuvant acts directly or indirectly on antigen presenting cells (APCs) including dendritic cells (DCs). The adjuvant can be a ligand of toll-like receptors (TLRs), and can directly affect DC to change the intensity, efficacy, speed, duration, bias, breadth and scope of adaptive immunity. In other cases, the adjuvant can send signals via proinflammatory pathways and promote immune cell infiltration, antigen presentation and effector cell maturation. Such adjuvants include mineral salts, oil emulsions, nanoparticles and polyelectrolytes, and include colloids and molecular assemblies with complex, heterogeneous structures. In one example, the composition also includes pidotimod as an adjuvant. In another example, the composition also includes CpG as an adjuvant.

[0206] The compounds of the present disclosure can be administered to a subject or patient at a dosage level in the range of about 0.1 mg to about 3,000 mg per day. For a normal adult weighing about 70 kg, a dosage in the range of about 0.01 mg to about 100 mg per kilogram of body weight is generally sufficient. The specific dosage and dosage range to be used may potentially depend on a variety of factors, including the needs of the subject or patient, the severity of the condition or disease being treated, and the pharmacological activity of the compound being administered. It is within the scope of ordinary technicians in the art to determine the dosage range and optimal dosage for a specific subject or patient.

[0207] How to use

[0208] The delivery vehicle complex disclosed herein can be used to deliver the polyanionic compound of the complex (or cargo) to a cell. Therefore, disclosed herein is a method for delivering a polyanionic compound such as a nucleic acid (e.g., RNA) to a cell, the method comprising contacting the cell with a delivery vehicle complex or a pharmaceutical composition disclosed herein. In some embodiments, the cell can be contacted in vitro. In some embodiments in which the cell is contacted in vitro, the cell is a HeLa cell. In other embodiments in which the cell is contacted in vivo, the multi-component delivery system of the present disclosure is applied to a mammalian subject. Mammalian subjects may include, but are not limited to, human or mouse subjects. In other embodiments in which the cell is contacted ex vivo, the cell is obtained from a human or mouse subject. In some cases, the cell is a tumor cell. In some cases, the cell is a muscle cell.

[0209] In some embodiments, one or more polyanionic cargo compounds can be delivered for therapeutic use. Non-limiting therapeutic uses include cancer, infectious diseases, autoimmune disorders, and neurological disorders. In some embodiments, a complex comprising a multi-component delivery system and a polyanionic cargo compound is used as a vaccine. Genetic vaccination, or administration of nucleic acid molecules (e.g., RNA) to a patient and subsequent transcription and / or translation of the encoded genetic information, can be used to treat and / or prevent hereditary genetic diseases, as well as autoimmune diseases, infectious diseases, cancer or tumor-related diseases, and inflammatory diseases. Genetic vaccination can be used to treat or prevent coronaviruses. The vaccine target for most of these entities is the spike (S) protein of the coronavirus, a highly glycosylated trimeric class I fusion protein that is coated on the outside of the virus and is responsible for host cell entry. The S protein of SARS-CoV-2 has a high degree of structural homology with SARS-CoV-1 and contains several subunits that are critical for the virus to enter host cells through the angiotensin-converting enzyme 2 (ACE2) receptor, including the S1 domain, the S2 domain, and the 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. In addition, genetic vaccination is particularly useful for treating cancer, because cancer cells express antigens and tumors are often not easily recognized and eliminated by the host, as evidenced by the development of the disease.

[0210] Vaccines. The delivery vehicle complex of the present disclosure can also be used as a vaccine, wherein the polyanionic compound is an RNA that can encode an immunogen, an antigen, or a neoantigen. The host's immune system provides a means to quickly and specifically produce a protective response to pathogenic microorganisms and promote the rejection of malignant tumors. The immune response is generally described as including a humoral response and a cell-mediated response, in which antibodies specific to the antigen are produced by differentiated B lymphocytes, and in which various types of T lymphocytes eliminate the antigen through a variety of mechanisms. For example, CD4 (also known as CD4+) helper T cells that 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 known as CD8+) cytotoxic T cells can also recognize specific antigens and can bind to and destroy or damage cells or particles carrying antigens. In particular, cell-mediated immune responses including cytotoxic T lymphocyte (CTL) responses may be important for eliminating tumor cells and cells infected by microorganisms (such as viruses, bacteria, or parasites). It has been found that the delivery vehicle complexes of the present disclosure induce an immune response when one or more of the polyanionic compounds of the complex encodes a viral peptide (eg, a viral polypeptide), a viral protein, or a functional fragment of the foregoing.

[0211] Therefore, the present disclosure includes methods 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 antigen 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 specific implementations, administration is carried out by intramuscular, intratumoral, intravenous, intraperitoneal or subcutaneous delivery.

[0212] In various specific implementations, relative to the amount of antibodies produced in a subject to which the delivery vehicle complex is not administered, administering the delivery vehicle complex of the present disclosure (e.g., formulated as a composition, a pharmaceutical preparation, or an antigen composition) to a subject may result in an increase in the amount of antibodies (e.g., neutralizing antibodies) produced in the subject to a viral antigen. In some specific 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.

[0213] The immune response caused by the method disclosed herein generally includes an antibody response, preferably a neutralizing antibody response, maturation and memory of T cells and B cells, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (GDC) and T cell-mediated responses, such as CD4+, CD8+. The immune response generated by the delivery vehicle complex comprising RNA encoding viral antigens disclosed herein produces an immune response that recognizes and preferably improves and / or neutralizes the viral infection described herein. The method for assessing the antibody response after administering the antigen composition (immunization or vaccination) is known in the art and / or described herein. In some specific implementations, the immune response includes a T cell-mediated response (e.g., a peptide-specific response, such as a proliferation response or a cytokine response). In some specific implementations, the immune response includes both B cells and T cell responses. The antigen composition can be administered in a variety of suitable ways, such as intramuscular injection, intratumor injection, subcutaneous injection, intradermal administration and mucosal administration, such as oral or intranasal administration. Additional modes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal, intravaginal, rectal, and oral administration. The present disclosure also contemplates the combination of different routes of administration in an immunized subject, such as simultaneous intramuscular and intranasal administration.

[0214] 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 malignancies characterized by the proliferation of anaplastic cells that tend to invade surrounding tissues and metastasize to new body sites, and also refers to pathological conditions characterized by the growth of such malignancies. Cancer can be a tumor or a hematologic malignancy, and includes, but is not limited to, all types of lymphomas / leukemias, carcinomas, and sarcomas, such as those found in the anus, bladder, bile duct, bone, brain, breast, uterus, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), oral cavity, ovary, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, coccyx, testicle, and uterus.

[0215] As non-limiting examples, the cancer that can be treated can be acute myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal cancer, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B-cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, cancer), fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, general germ cell tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin lymphoma, Hodgkin's disease, Hodgkin's lymphoma, hypopharyngeal cancer, infiltrating ductal carcinoma, infiltrating lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, infiltrating breast cancer, islet cell carcinoma, jaw cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer cancer), leiomyosarcoma, leptomeningeal metastasis, leukemia, 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, mesenchymous, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, nasal cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell cancer, ocular cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cancer cavitycancer), oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian sex cord-stromal tumor, Paget's diseasedisease), pancreatic cancer, papillary cancer, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pinealoblastoma, pituitary gland cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, osteosarcoma, soft tissue sarcoma (Sarcomasoft tissue), uterine sarcoma (Sarcoma uterine), venous sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma (Soft tissue sarcoma), spinal cancer, spinal cancer, spinal cord cancer, spinal tumors, squamous cell carcinoma, stomach cancer (Stomach cancer), synovial sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer (Throat cancer) cancer), thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, tubule cancer, ureteral cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma (Uterinesarcoma), vaginal cancer, and vulvar cancer.

[0216] In some implementations, the delivery vehicle complex of the present disclosure is used to treat a cancer selected from the group consisting of cervical cancer, head and neck cancer, B-cell lymphoma, T-cell lymphoma, prostate cancer, and lung cancer. In some implementations, the delivery vehicle complex can be used to treat cervical cancer.

[0217] Infectious diseases. In some embodiments, the delivery vehicle complex of the present disclosure is used to treat infectious diseases, such as microbial infections, e.g., viral infections, bacterial infections, fungal infections, or parasitic infections. Non-limiting examples of infectious diseases include hepatitis (e.g., HBV infection or HCV infection), RSV, influenza, adenovirus, rhinovirus, or other viral infections.

[0218] Autoimmune diseases. Various autoimmune diseases 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, the autoimmune disease can be acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (ED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, disease), celiac disease, Chagas disease, chronic fatigue syndrome**, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (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 ryoglobulinemia, demyelinating neuropathy, 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 Wegener's granulomatosis)Granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosis, immunomodulatory 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 sclerosus, woody conjunctivitis, linear IgA disease (LAD), systemic lupus erythematosus (SLE), Lyme disease disease), chronic, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, Palindromic rheumatism, PANDAS (childhood autoimmune neuropsychiatric disorders caused by Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonnage-Turner syndrome, Pars planus planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndromes types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndromesyndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu'sarteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)).

[0219] Nervous System Diseases. Various neurological diseases can be treated with the delivery vehicle systems disclosed herein. As non-limiting examples, the neurological diseases can be Septum Periwinkle, Acid Lipase Deficiency, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit Hyperactivity Disorder (ADHD), Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS-Neurological Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Disease), ALS, Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Hypoxia, Antiphospholipid Syndrome, Aphasia, Dyspraxia, Arachnoid Cyst, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxia and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit Hyperactivity Disorder, Autism Spectrum Disorder, Dysautonomia, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, Behcet's Disease, Bell's Palsy Palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, blepharospasm, Bloch-Sulzberger Syndrome, brachial plexus birth injury, brachial plexus injury, Bradbury-Eggleston Syndrome, brain and spinal tumors, cerebral aneurysm, brain injury, lateral spinal cord compression-segmental cord syndrome (Brown-Sequard syndrome),Syndrome), spinal bulbar muscular atrophy, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, causalgia, cavernoma, cavernous hemangioma, cavernous malformation, central cervical cord syndrome, central cord syndrome, central pain syndrome, mid-pontine myelinolysis, brain disorder, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, brain atrophy, cerebral beriberi, cerebral cavernous malformation, cerebral gigantism, cerebral hypoxia, cerebral palsy, cerebro-oculo-facio-skeletal syndrome (Cerebro-Oculo-Facio-Skeletal Syndrome) (COFS), Charcot-Marie-Tooth Disease, Chiari Malformation, cholesterol ester storage disease, chorea, choreacanthocytosis, 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 cavernous malformation, corticobasal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, cumulative trauma, Cushing's Syndrome, cytomegalovirus infection, dancing eyes-dancing feet syndrome Syndrome, Dandy-Walker Syndrome, Dawson Disease, DeMorsier's Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia-Multi-Infarct, Dementia-Semantic, Dementia Subcortical, Dementia with Lewy Bodies, Dentate-Cerebellar Ataxia, Dentatorrubral Atrophy, Dermatomyositis, Developmental Dyskinesia, Devic'sSyndrome), diabetic neuropathy, diffuse sclerosis, Dravet Syndrome, familial dysautonomia, dysgraphia, dysphagia, dyspraxia, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, dystonia, early pediatric epileptic encephalopathy, empty sella syndrome, encephalitis, encephalitis lethargica, encephalocele, encephalopathy, encephalopathy (familial infantile), encephalotrigeminal angiomatosis, epilepsy, epileptic hemiplegia, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, essential tremor, extrapontine myelinolysis, Fabry disease Disease), Fahr's Syndrome, Syncope, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralysis, Familial Spastic Paralysis, Farber's Disease, Febrile Seizures, Fiber Muscular Dysplasia, Fisher Syndrome, Infantile Hypotonia Syndrome, Foot Drop, Friedreich's Ataxia, Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidosis, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease Disease), Giant Axonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen Storage Disease, Guillain-Barré Syndrome, Hallervorden-Spatz Disease, Head Injury, Headache, Migraine Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathy, Hereditary Spastic Paraplegia, Hereditary Ataxia, Polyneuritis Type, Herpes Zoster, Herpes Zoster Otic, Hirayama Syndrome, Holmes-Adiesyndrome, Holoprosencephaly, HTLV-1-Associated Myelopathy, Hughes SyndromeSyndrome), Huntington's Disease, Anencephaly with Hydrops, Hydrocephalus, Hydrocephalus-Normal Pressure, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinence Pigmenti, Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathy, Occipital Schistencephaly, Intestinal Lipodystrophy, Intracranial Cyst, Intracranial Hypertension, Isaacs' Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne Syndrome, Kleine-Levin Syndrome, Klippel-Feil Syndrome Syndrome, Klippel-Trenaunay Syndrome (KTS), Klüver-Bucy Syndrome, Korsakoff's Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome Syndrome, Dementia with Lewy Bodies, Lipid Storage Disease, Lipoprotein Deposition Disease, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus - Neurological Sequelae, Lyme Disease - Neurological Complications, Machado-Joseph Disease, Megalecephaly, Megalecephaly, Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis, Menkes DiseaseDisease), meralgia paresthesia, metachromatic leukodystrophy, microcephaly, migraine, Miller Fisher syndrome, mini-stroke, mitochondrial myopathy, Moebius syndrome, monomelic muscular dystrophy, motor neuron disease, Moyamoya disease Disease), Mucolipidoses, Mucopolysaccharidoses, Multi-infarct dementia, Multifocal motor neuropathy, Multiple sclerosis, Multiple system atrophy, Multiple system atrophy with postural hypotension, Muscular dystrophy, Myasthenia - congenital, Diffuse sclerosis with myelin sheath destruction, Myoclonic encephalopathy of infancy, Myoclonus, Myopathy, Myopathy - congenital, Myopathy - thyrotoxic, Myotonia, Myotonia congenita, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with brain iron accumulation, Neurofibromatosis, Neuropsychiatric malignant syndrome, Neurological complications of AIDS, Neurological consequences of cytomegalovirus infection, Neurological manifestations of Pompe disease, Neurological sequelae of lupus, Neuromyelitis optica, Neuromyotonia, Neuronal ceroid lipofuscinosis, Neuronal migration disorders, Neuropathy - hereditary, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Cavernous hemangioma, Niemann-Pick disease Disease), O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus-Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain - Chronic, Pantothenate Kinase-Related Neurodegeneration, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis, Paroxysmal Migraine, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome 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, Hole-in-the-Brain, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Dentin Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia, Prion Disease, Progressive Facial Hemitrophy, Progressive Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Spinal Dystrophy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudo-Torch Syndromesyndrome), Pseudo-toxoplasmosis Syndrome, Pseudotumor Cerebri, Psychogenic Movements, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen's Encephalitis, Reflex Sympathetic Dystrophy, Refsum's Disease, Refsum's Disease-Infant, Repetitive Movement Disorders, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome, Sacral Root Cyst, Saint Vitus Dance, Salivary Gland Diseases, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seitelberger Disease Disease), seizure disorders, semantic dementia, septo-optic dysplasia, severe myoclonic epilepsy of infancy (SMEI), shaken baby syndrome, herpes zoster, Shy-Drager Syndrome, Sjogren's syndrome Syndrome), sleep apnea, sleep sickness, Soto's 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, short-duration, unilateral, neuromorphic (SUNCT) headache, dysphagia, Sydenham's chorea, syncope, syphilitic myelosclerosis, syringomyelia, syringomyelia, systemic lupus erythematosus, tabes dorsalis, tardive dyskinesia, Tarlov cysts, Tay-Sachs disease, Disease), temporal arteritis, tethered cord syndrome, Thomsen's Myotonia, thoracic outlet syndrome, thyrotoxic myopathy, trigeminal neuralgia, Todd's Paralysis, Tourette Syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, Troyer Syndrome, tuberous sclerosis, angioerectile tumors, vasculitis syndromes of the central and peripheral nervous systems, von Economo's Disease, von Hippel-Lindau Disease (VHL), von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease (VHL), Disease), Wernicke-Korsakoff Syndrome, West Syndrome, Whipple's Disease, Williams Syndrome, Wilson Disease, Wolman's Disease, X-linked spinal bulbar muscular atrophy.

[0220] In jurisdictions that prohibit patenting of methods performed on humans, the meaning of "administering" a composition to a human subject or patient shall be limited to providing that the human subject or patient will self-administer the controlled substance by any technique (e.g., oral, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with the law or regulation defining patentable subject matter is intended. In jurisdictions that do not prohibit patenting of methods performed on humans, "administering" a composition includes both methods performed on humans and the aforementioned activities.

[0221] Example

[0222] The following examples are provided for illustration and are not intended to limit the scope of the present disclosure.

[0223] Example 1 - General Synthesis of Hydroxyalkyl Cationic Peptides

[0224] In order to apply the delivery vehicles disclosed herein to systemic expression of antibody-based drugs, the peptoid structure is optimized to maximize the desired characteristics, including but not limited to liver selectivity and expression in mice. For secretory protein production, selective liver expression is conducive to producing high levels in circulating serum. A two-stage strategy can be applied to optimize the peptoid-based delivery system. First, the best ionizable group is evaluated using the common lipophilic part of the molecule, and then the lipid part is optimized separately with the ionizable group. This method takes advantage of the modularity of the peptoid structure, which allows the ionizable N-terminal domain to be easily modified when the peptoid core is unchanged, thereby allowing the effective optimization of the entire molecule without losing important lead candidates, because the interaction between the lipid and the cationic part is usually minimal.

[0225] General protocols for the synthesis of cationic peptoids disclosed herein can be found in WO2020 / 069442 and WO2020 / 069445, each of which is incorporated herein by reference in its entirety. The following examples describe general protocols for the synthesis of cationic peptoids.

[0226] All polymers were synthesized by the submonomer method using bromoacetic acid and primary amines. Fmoc-Rink amide resin was used as a solid support. The Fmoc group on the resin was deprotected using 20% ​​(V / V) piperidine-dimethylformamide (DMF). The amino resin was then amidated with bromoacetic acid. After amidation, the α-carbon was aminated by nucleophilic displacement of the bromide with a primary amine. Both steps were repeated continuously to produce the desired cationic peptide sequence.

[0227] Unless otherwise stated, all reactions and washes were performed at room temperature. Washing of the resin refers to adding a wash solvent (usually DMF or dimethyl sulfoxide (DMSO)) to the resin, stirring the resin to obtain a uniform slurry, and then exhausting the solvent from the resin. The solvent was removed by vacuum filtration through the sintered bottom of the reaction vessel until the resin appeared dry. In all syntheses, the resin slurry was stirred by bubbling argon gas upward through the bottom of the sintered vessel.

[0228] Initial Resin Deprotection. Charge the Fmoc-Rink amide resin to a sintered reaction vessel. Add DMF to the resin and stir the solution to swell the resin. Then drain the DMF. Remove the Fmoc group by adding a 20% piperidine solution in DMF to the resin, stirring the resin, and draining the resin. Add a 20% piperidine solution in DMF to the resin and stir for 15 minutes, then drain. Then wash the resin six times with DMF.

[0229] Acylation / amidation. The unblocked amine is then acylated by adding bromoacetic acid in DMF to the resin followed by N,N-isopropylcarbodiimide (DIG) in DMF. The solution is stirred at room temperature for 30 minutes and then drained. This step is repeated once more. The resin is then washed twice with DMF and once with DMSO. This is a complete reaction cycle.

[0230] Nucleophilic displacement / amination. The acylated resin is treated with the desired primary or secondary amine to effect nucleophilic displacement at the bromine leaving group on the α-carbon. This acylation / displacement cycle is repeated until the desired peptide sequence is obtained.

[0231] Cleavage of peptide from resin. The dried resin was placed in a glass scintillation vial containing a polytetrafluoroethylene coated micro stir 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 equipped 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 with 1:1 acetonitrile:water. The lyophilized peptide was redissolved in 5% acetonitrile in water to a concentration of 5 mM.

[0232] Purification and characterization. The re-dissolved crude peptide was purified by preparative HPLC. The purified peptide was characterized by LC-MS analysis.

[0233] In a specific case, amino lipidated peptoids were synthesized by a submonomer method with bromoacetic acid and N, N'-diisopropylcarbodiimide (DIG). Rink amide (200 mg representative scale, 0.64 mmol / g loading) resins protected by MBHA Fmoc- were used as solid supports. For bromoacetylation, the resin was mixed with a 1:1 mixture of 0.8 M bromoacetic acid and 0.8 M N, N'-diisopropylcarbodiimide (DIG) for 15 minutes. Amine displacement was performed using a 1 M solution of amine in DMF for 45 minutes. After synthesis, a mixture of 5 mL 95:5 trifluoroacetic acid (TEA): water was used to cleave the crude peptoids from the resin at room temperature for 40 minutes. The resin was removed by filtration, and the filtrate was diluted in water to form a milky white suspension, followed by addition of polystyrene resin for (heterogeneous) solid phase extraction of peptoids. The solution was fully mixed and incubated at room temperature for 20 min. Then, the peptoids of solid phase extraction were released by adding ethanol and concentrated using a vacuum centrifuge. The crude peptoid was further purified by reverse phase flash chromatography using a C4 column and a gradient of 60%-95% ACN / H2O containing 0.1% TEA. Purity and identity were determined using an LC-MS system consisting of a TOE mass spectrometer (LC / TOF). Chromatographic separation was performed using a C8 column (2.1×100 mm) at 40° C. 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 programmed to be isopropanol-acetonitrile-formic acid (80:20:0.1) containing 10 mM ammonium formate in a linear manner within 11 min.

[0234] For this screening, a lipid block containing 4 dodecyl lipid (N-dodecylglycine) monomers was selected, and 12 peptoids containing these groups were synthesized on a solid phase synthesizer using a submonomer method. The resulting material was purified and its identity confirmed by LC-MS. It was observed that changing the number of lipid monomers and total lipid carbons had a significant effect on the hydrophobicity of the peptoid molecules, as measured by HPLC retention time. When fitted to a multi-parameter model, both the calculated logP and the empirical reversed-phase HPLC retention time increased as the number of lipid carbons increased, as expected for molecules with increased lipid content. Additionally, as the number of lipid monomers increased (the number of total lipid carbons was similar), the predicted logP and retention time decreased, which may be 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). These parameters together produce a library of peptoids with different physical properties for optimization.

[0235] Characterization data for the peptoids disclosed herein are provided in Table 4.

[0236] Table 4: Peptoid characterization data

[0237]

[0238]

[0239]

[0240] Example 2 - Synthesis of delivery vehicle complexes

[0241] Synthesis. Hydroxyalkyl-terminated cationic peptoids, such as 2-aminopropane-1,3-diol-terminated cationic peptoids, can be evaluated in vitro or in vivo for therapeutic and / or preventive purposes. Without being bound by any particular theory, the cationic portion of the aminolipidated peptoid binds to the negatively charged phosphodiester backbone of a polyanionic cargo (e.g., a nucleic acid cargo) primarily through electrostatic interactions to form a mixed coacervate complex. Hydrophobic interactions between lipid chains on hydroxyalkyl-terminated cationic peptoids (such as 2-aminopropane-1,3-diol-terminated cationic peptoids) can serve to stabilize particle formation and aid in membrane binding.

[0242] Delivery vehicle complex can be prepared by any physical and / or chemical method known in the art, to adjust their physical, chemical and biological properties. These methods generally relate to the rapid combination of hydroxyalkyl-terminated cationic peptides (such as 2-aminopropane-1,3-diol-terminated cationic peptides) in water or water-miscible organic solvents with oligonucleotides in water or aqueous buffer solutions. These methods may include simply mixing components by pipetting or microfluidic mixing methods (such as those involving T-mixers, vortex mixers or other chaotic mixing structures).

[0243] In a standard formulation, a hydroxyalkyl-terminated cationic peptoid (such as a 2-aminopropane-1,3-diol-terminated cationic peptoid) and an additional lipid are dissolved in anhydrous ethanol at a concentration of 10 mg / mL to obtain a solution that is stable at room temperature. In some specific implementations, the solution is stored at -20°C. The nucleic acid cargo is dissolved in water that does not contain DNase or RNase at a final concentration of 1 mg / mL-2 mg / mL. These solutions can be stored for a long time at -20°C or -78°C.

[0244] In order to prepare the delivery vehicle composition disclosed herein, the hydroxyalkyl-terminated cationic peptide (such as 2-aminopropane-1,3-diol-terminated cationic peptide) and the additional lipid component are first premixed in the ethanol phase in the desired mass ratio. The nucleic acid cargo is diluted in ethanol and an acidic buffer (10mM phosphate / citrate, pH 5.0). Ethanol and the aqueous phase are mixed in a 3: 1 volume ratio and then immediately diluted with 1: 1 volume ratio of PBS to obtain a final mRNA concentration of 0.1 μg / uL. Non-limiting exemplary delivery vehicle compositions prepared by the above method include the compositions listed in Table 2 above (e.g., compositions F2, F6 / 17, F6 / 12, and F6 / 15).

[0245] The delivery vehicle composition is combined with a polyanionic compound (such as RNA encoding, for example, firefly luciferase (Fluc), COVID-19 spike protein, functional fragments thereof and / or variants thereof, or E6 / E7 oncogenes (e.g., from HPV16, HPV18, functional fragments thereof and / or variants thereof)) at the ratios shown in Table 3 to form a delivery vehicle complex to be evaluated for therapeutic and / or preventive purposes in vitro or in vivo. The w / w in Table 3 is the mass ratio of the components shown to the mRNA.

[0246] In one specific case, the peptoids disclosed herein were formulated into delivery vehicles by first dissolving the peptoids disclosed herein with DSPC, cholesterol, and DMG-PEG2000 in ethanol at a weight ratio of 20:1.8:7.2:1.8 (Formulation D22 in Table 3 above), and then mixing it with firefly luciferase (fLuc) mRNA dissolved in an aqueous buffer at a volume ratio of 1:3 via microfluidic nanoprecipitation on a turbulent mixing device to obtain small, low-dispersibility particles. Physical particle properties were characterized. Delivery vehicles with an initial size greater than 120 nm were not entered into the in vivo screening because the liver window mainly accommodated LNPs with a size less than 100 nm. At 6 hours after IV administration, fLuc expression was used in BALB / c mice to evaluate the in vivo expression of delivery vehicles containing peptoids with different head groups ( Figure 1 ). This panel of headgroup-modified peptoids showed significant differences in both overall expression levels as well as organ selectivity, highlighting the importance of screening a broad range of chemical diversity in the ionizable portion of the peptoid. Both the aminoethyl and aminopropyl headgroups showed preferential expression in the lung ( Figure 2), which may be due to the high basicity of the side chain primary amine, as groups with higher pKa are known to accumulate in this organ. Interestingly, the piperazinylpropyl and methoxypropyl head groups shifted selectivity toward the spleen. These results provide evidence that simply modulating the peptoid head group leads to significant changes in selective expression. While spleen and lung selectivity warrant further optimization for future therapeutic applications, the liver-selective target was used in this article for further investigation to prepare the antibody target for systemic delivery and maximize its serum expression with the anti-RSV cargo. Of all the peptoids tested, the 1,3 diol (apd) head group showed the highest liver expression and selectivity and was selected for further lipid-block optimization.

[0247] use The T7 High Yield RNA Synthesis Kit transcribes mRNA from a DNA template encoding firefly luciferase. 1 -Methyl pseudo-UTP (m1ΨTP) is used to replace UTP for the reaction and is used during IVT For co-transcriptional capping. The NTP concentration used was calculated based on the template sequence, and a 4:1 cap to GTP ratio was used for IVT. The reaction was carried out at 37°C for 4 hours.

[0248] By TURBO TM DNA enzyme was digested at 0.15U / μL reaction volume at 37℃ for 30min to remove DNA template. Then RNACIean TM XP beads were used to purify transcripts. RNA-bound beads were washed three times with freshly prepared 70% ethanol, air-dried for 15 minutes, and subsequently eluted in 200 μL of nuclease-free water. Purified mRNA was quantified by spectrophotometry and analyzed using GX Touch TM Nucleic acid analyzer for analysis.

[0249] The delivery vehicle was prepared by rapid microfluid mixing using a high-throughput microfluidic system. In short, the ethanol solution (preparation D22 of the upper table 3) containing the peptoids, DSPC, cholesterol and DMG-PEG2000 with a weight ratio of 20:1.79:7.16:1.84 was mixed with 10mM sodium citrate buffer (pH 5.0) containing synthetic mRNA at a volume ratio of 1:3. The LNP based on the peptoids was then dialyzed overnight at 4°C with 1 liter-2 liters of tris-sucrose buffer (TS7, pH 7.4).

[0250] Apd lipid block variant peptoids are formulated into delivery vehicles, and physical properties are characterized and analyzed. In general, it is observed that monomer length and total carbon number have a significant impact on the physical properties of particles, and the longest monomer and the maximum total carbon number are not the best performing, which shows that the tested monomer length range is sufficient. It is found that particle size is difficult to be associated with any single factor, except that when oleyl (Ole) monomers are used relative to 2-ethylhexyl (Ehx), particles are significantly larger. The percentage of the mRNA encapsulated depends significantly on the total number of lipid carbons on the peptoid and the type of the branched / unsaturated monomers used, wherein the maximum encapsulation occurs at about 55 total lipid carbons, and 2-ethylhexyl (ehx) monomers are used. It is observed that particle pKa changes between pH 5-7, and higher monomers and higher total carbon numbers lead to lower particle pKa. Additionally, it is observed that even with similar head group pKa, increasing the number of branched monomers also results in a slightly lower pKa in the delivery vehicle. 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, the pKa of the Apd peptoids has been predicted to be about 5.5-5.7, which is very similar to the particle pKa of 5-7 measured by them, indicating that the pKa does not change when formulated into lipid nanoparticle complexes. This may be due to several factors, including the hydrophilicity of the peptoid backbone that provides an environment more conducive to amine protonation, or the packing dynamics of the peptoids within the particles, and will be the focus of further research. It has also been shown that fusogenicity, or the ability of LNPs to fuse with endosomal membranes and escape degradation, varies with the lipid structure of the LNPs. Hemolysis of RBCs at neutral and acidic pHs is used as a measure of fusogenicity. At low pHs that best predict membrane interactions in endosomes, for peptoids with overall lower total monomers, increasing the branched / unsaturated groups results in higher hemolysis at pH 5. Interestingly, it has been observed that the introduction of oleyl lipids at pH 5 and 7 often increases hemolysis. This suggests that oleyl peptoids may not be well tolerated due to background levels of hemolysis at neutral pH.

[0251] Example 3 - Particle Characterization

[0252] 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.

[0253] Granularity. Using Wyatt Plate Reader III measures particle size and size distribution. Typically, formulated samples were diluted to 2 ng / μL in 100 μL PBS. Data are reported as the hydrodynamic diameter (in nm) of the cumulant fit of the correlation function and the polydispersity of the measurement.

[0254] Encapsulation percentage. Use improved The assay determines the percentage of mRNA encapsulated within the delivery vehicle complex containing Fluc mRNA. Generally, the formulated mRNA samples are mixed with or without Triton-X. TM Dilute to 500 ng / mL in Tris-EDTA buffer. Add 200-fold dilution (Invitrogen), and the plate was incubated for 5 minutes. Fluorescence was measured at Ex.840nm / Em.520nm, and the encapsulated mRNA was calculated by calculating the fluorescence ratio of uncleaved particles to cleaved particles. Complexes with increased amounts of cationic components resulted in higher encapsulation efficiency.

[0255] Particle stability. The delivery vehicle complexes were stored at 4°C and the resulting particle size and percent encapsulation were determined after 17 and 48 days.

[0256] In one specific case, using Plate Reader III was used to measure the size and polydispersity (PDI) of the delivery vehicle by dynamic light scattering in PBS. TM The particle analyzer determines the zeta potential of the delivery vehicle. Zeta samples were diluted to 30 μM total lipid in a weak buffer solution of 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate pH 7. Measurements were collected in duplicate for more than 30 times with the voltage manually set to 120 mV.

[0257] Using fluorescence-based Quant-iT TM RNA kit, and determine the mRNA concentration and the percentage of encapsulated mRNA using the literature procedure. To determine the mRNA concentration, the delivery vehicle was mixed with 2% Triton TM X-100 was incubated for 10 min to release the embedded mRNA, and then RNA reagents. Detection using a microplate reader Fluorescence intensity of the reagent complex (excitation at 485 nm and emission at 530 nm). TM The percentage of encapsulated mRNA was calculated by comparing the fluorescence ratio of X-100.

[0258] The retention time and purity of the synthesized peptoids were monitored using HPLC-UV with UV absorption at 214 nm and injection volume (3 μL). A 1.7 μm 4 protein column (pore size: RP-HPLC was performed at 55°C with 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% trifluoroacetic acid)) in 8 min with a running gradient from 35% to 90% solvent B, followed by a 4 min wash with 100% solvent B and a return to 35% solvent B at a flow rate of 0.4 ml / min.

[0259] Apparent particle pKa was assessed by TNS fluorescence assay. Briefly, particles were diluted to 130 μM total lipid with 10 mM TNS probe in TNS buffer in the pH range of 3-11 for 10 minutes. Fluorescence was measured in a plate reader ex 320 / em 445 at room temperature. Fluorescence intensity was normalized to minimum and maximum values ​​and pKa was determined by 3-parameter fitting to determine the half-maximum of the curve.

[0260] RBC hemolysis was used as a measure of LNP fusibility at low pH. Sheep RBCs were pelleted at 800×g, washed with 2×PBS, and resuspended to 5% v / v in 20mM citrate 140mM NaCl pH 5.5 or PBS. The particles were diluted to 150mM total lipids with 5% RBC solution in the wells and incubated at 37°C for 45min. The supernatant was collected and the absorbance was read at 425nm on a microplate reader. The supernatant was washed with 2% Triton TM Fusion was normalized to fully lysed samples with a buffer only control. % Hemolysis = 100*[(ABS T-Background)-(ABS Sample-Background) / (ABS T-Background)].

[0261] The characterization data for delivery vehicles comprising the peptoids disclosed herein are shown in Table 5. The characterization data for delivery vehicles comprising comparative compounds C1-C13 are shown in Table 6.

[0262] Table 5: Delivery vehicle characterization data

[0263]

[0264]

[0265] Table 6: Comparative Peptoid Delivery Vehicle Characterization Data .

[0266]

[0267] Example 4 - Delivery Vehicle Complex Efficacy - In vitro Firefly Luciferase Expression

[0268] The ability of firefly luciferase (Fluc) reporter gene delivery to cultured cells based on the compound comprising the delivery vehicle composition disclosed herein and the mRNA (FlucmRNA) encoding firefly luciferase is evaluated in vitro for the efficacy of the compound. In a representative experiment, the delivery vehicle composition is combined with Fluc mRNA, and the resulting particles are added to the cultured HEK-293 cells with a dosage of 50ng / well (in 150 μL total volume). After processing for 6 hours and 18 hours, the resulting luciferase expression is measured by a luminescent plate reader. The delivery vehicle disclosed herein shows high luciferase expression.

[0269] Example 5 - In vivo luciferase expression

[0270] General Methods. All animal experiments were performed by Lumigenics LLC (Hercules, CA) under the supervision of the California Department of Public Health, IACUC and veterinary supervision. Prior to all studies, animals (e.g., mice and / or rats) were allowed to acclimate for a minimum of 3 days prior to use. The animals were maintained in a temperature and humidity controlled room with a 12-hour light cycle. Daily health checks and food and water checks were performed.

[0271] Depending on the injection site, injections were given subcutaneously (50 μL-200 μL), intraperitoneally (up to 1000 μL), intravenously (50 μL-200 μL), intramuscularly (50-μL), or intratumorally (50-μL) using a 26-30 gauge needle. Animals were anesthetized with isoflurane for all injections / implants.

[0272] For imaging, animals were placed under anesthesia, injected intraperitoneally with D-luciferin (15 mg / mL) at a dose of 10 μL per gram of body weight, and placed in a camera chamber and imaged for up to 30 minutes. Imaging was performed 15 minutes after substrate injection.

[0273] Intravenous administration. The delivery vehicle complex disclosed herein is effective for in vivo administration of Fluc mRNA to BALB / c mice by a variety of administration routes. Generally, the delivery system complex is administered via tail vein injection at a dose of 0.5 mg / kg, and the resulting bioluminescence is quantified after 6 hours. Organ-specific bioluminescence is quantified by sacrificing the treated animals, dissecting the organs of interest, and quantifying the resulting bioluminescence separately.

[0274] Local administration. In addition to IV administration, the delivery vehicle complexes described herein can also be effectively used to locally administer mRNA via intratumoral (IT), subcutaneous (SC), or intramuscular (IM) administration routes. For these examples, mRNA was administered intratumorally at a dose of 0.1 mpk, or subcutaneously and intramuscularly at a dose of 0.01 mpk, and the resulting bioluminescence was quantified after 6 hours.

[0275] In one specific case, to investigate how the peptoid lipid block affects in vivo expression, delivery vehicles with diameters greater than 120 nm were excluded and the remaining candidates were administered IV to BALB / c mice. Luminescence was measured after 6 hours and the resulting expression was quantified in the liver, lungs, and spleen. Even without changing the cationic head group, luciferase expression spanned 4 orders of magnitude due to changing the lipid block in the series. Compounds 12, 41, 34, and 35 emerged as the best expressors in this screen ( Figure 3 ). Interestingly, these peptoids were all 6 monomers in length and contained ethylhexyl (Ehx) branching monomers. The in vivo expression of all candidates was then fit to a multi-parameter model with 4 input parameters to understand the individual contribution of each factor to the overall performance. The model showed that expression had a very strong second-order dependence on the number of total lipid carbons and the number of unsaturated or branching monomers, a strong dependence on whether the peptoid used oleyl or Ehx as the unsaturated / branching monomer, and a moderate dependence on the total number of lipid monomers. Contour plots of the effects of these factors on expression (all using Ehx as the branching / unsaturated monomer) are shown in Figure 4 middle.

[0276] Based on this model, the maximum predicted expression was achieved using peptoids containing 5-6 total lipid monomers, 55 total lipid carbons, and 2-3 ehx monomers as nonlinear lipids. Notably, 3 of the 4 best performing peptoids (compounds 41, 34, and 35) had properties very close to this prediction. The fourth best candidate (compound 12) had 3 of the 4 best properties (number of lipid monomers, nonlinear monomers, and Ehx as a branched / unsaturated monomer) and the total number of lipid carbons was just outside this range ( Figure 4 ). This is encouraging and suggests that the parameterization scheme utilized can predict the activity of peptoid-based ionizable lipids well. With confidence in the lipid block optimization process, these top 4 candidates were advanced to additional experiments to evaluate their in vivo production of secreted proteins.

[0277] For imaging studies, female BALB / c mice (6-8 weeks old) were obtained and acclimated for a minimum of 3 days before the experiment. The animals were maintained on a 12-hour light cycle in a temperature and humidity controlled room. Daily health checks and food and water checks were performed. For dosing, mice were injected intravenously with 2.5 μg of the delivery vehicle containing fLuc mRNA in a total injection volume of 100 μL. Six hours after injection, mice were anesthetized with isoflurane and injected intraperitoneally with 30 mg / mL D-luciferin at a dose of 10 μL per gram of body weight. The D-luciferin was injected 10 minutes after injection. Imaging was performed using the Living Spectrum Imaging System. Immediately after in vivo imaging, mice were terminally bled and euthanized by cardiac puncture. Organs of interest were harvested, placed in black 24-well plates, and imaged. Software was used to quantify the total photon flux in the region of interest. For secretory protein studies, mice were injected intravenously with a delivery vehicle containing aRSV mRNA in a total injection volume of 100 μL. Mice were euthanized 24 hours later and serum was collected at 6 hours and 24 hours after injection. Serum was used to quantify secreted aRSV levels using ELISA using a standard curve generated by aRSV protein according to the manufacturer's recommendations for IgG.

[0278] CryoTEM was also performed. 3 μL of approximately 1E14 particles / mL were applied to a clean grid (holey carbon film on 400 mesh copper), blotted with filter paper, and then repeated once more before immediate vitrification in liquid ethane. Micrographs were obtained using a cryogenic transmission electron microscope (Cryo-TEM) operated at 200 kV and equipped with a direct electron detector and software for automatic data collection. High magnification images were collected at nominal magnifications of 73,000× (0.200 nm / pixel) and 28,000× (0.524 nm / pixel). The images were taken at nominal underfocus of -5.5 μm to -3.5 μm and approximately The images were collected at an electron dose of .

[0279] Example 6 - Efficacy and Toxicology Studies

[0280] Compounds of formula (I) were tested in mouse efficacy and rat toxicology studies. In short, control mRNA or PBS vehicle prepared at 0.03mg / kg or 0.3mg / kg in, for example, F2 was used to treat Sprague-Dawley rats. Four injections were performed over the course of 13 days and administered intramuscularly to the hind limbs. Whole blood was collected for hematology 6 hours after the first and last administrations and 2 weeks after the last administration, and serum was collected for clinical chemistry and cytokine analysis. Additionally, two animals were killed 6 hours after the last administration and 2 weeks after the last administration, and macroscopic autopsy was performed. Tissue samples were retained, and histopathological examinations were performed on the selected organs.

[0281] Example 7 - Protein expression studies

[0282] In order to understand how the delivery vehicles disclosed herein (e.g., delivery vehicles comprising compounds of formula (I)) work as systemic delivery vehicles, the ability of the first four candidates (compounds 12, 41, 34 and 35) expressed by firefly luciferase from the optimized DOE model to produce anti-RSV antibodies in BALB / c mice was evaluated. Due to the availability of treatment benchmark information and sequence availability, anti-RSV was selected as a model target. A delivery vehicle comprising aRSV was prepared in a mass ratio of 2: 1 by two mRNA molecules encoding heavy and light chains. The size of all delivery vehicles was less than 120nm, and the encapsulation efficiency was greater than 85% (Table 7).

[0283] Table 7: Characterization data of delivery vehicles containing compounds 12, 41, 34 and 35 in formulation D22 .

[0284] Delivery vehicle diameter PDI Encapsulation efficiency Encapsulation efficiency standard deviation 34-D22 71.8 0.087 88.5 0.3 35-D22 71.7 0.09 92.3 0.6 12-D22 67.6 0.065 95.8 0.9 41-D22 80.2 0.078 96.1 0.2

[0285] The delivery vehicle was administered intravenously in BALB / c mice at a total dose of 0.75 mg / kg mRNA and serum levels of aRSV were compared by IgG ELISA 24 hours later. 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 4 candidates, suggesting that there is no perfect correlation between the expression of intracellular proteins (such as fLuc) and secreted proteins (such as aRSV). Additional 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 over the past 5 days, and serum titers showed a generally linear dependence on the injected dose at 0.3 mg / kg, 0.75 mg / kg, and 1.5 mg / kg ( Figure 7The overall tolerability of the compound 41-D22 formulation was further probed using fLuc cargo mRNA by gradually increasing the dose from 0.3 mg / kg to 7.5 mg / kg, and no immediate tolerability issues were observed over this wide dose range ( Figure 8 ).

[0286] The particle structure of compound 41-D22 formulations was characterized by cryo-TEM and their stability was monitored over time. Most of the compound 41-D22 particles showed a dense core morphology, and no bubble characteristics were observed ( Fig. 9 Stability was monitored over 1 month of storage at 4°C or -80°C and no growth or encapsulation changes were observed ( Fig.10 ). In summary, in addition to the 4°C and frozen stability, the spherical dense particle morphology supports the practical use of the delivery vehicles disclosed herein for therapeutic applications.

[0287] The effect of monomer lipid order on the performance of the delivery vehicle was also explored. To achieve this, compound 31 was synthesized, which subtly changed the order of C12 and Ehx monomers in the lipid block compared to the monomers used in compound 41. The physical properties of the two isomers, such as size, encapsulation efficiency, and hemolysis, were almost identical (Table 5). In vivo, luciferase expression and secreted protein expression were found to be within the error of the two order changes, indicating that monomer composition rather than order drives the differences between the peptoids ( Fig.11 and Fig.12 ). This further validates the screening funnel employed and that future peptoid variants can consider sequence variation only as a secondary factor.

[0288] The foregoing disclosures demonstrate that peptoid-based lipid nanoparticles provide a highly tunable platform for optimizing mRNA delivery for intracellular and secretory protein expression. Through extensive head group screening, it was found that head group regulation altered the expression of delivery vehicles in major organs, including the lungs, spleen, and liver. From this initial screening, 1,3 diol head groups with the highest liver selectivity were selected for lipid block optimization. The parameterization strategy for the above lipid blocks determined the ideal combination of the number of lipid monomers, the total number of lipid carbons, the number of branched or unsaturated monomers, and whether 2-ethylhexyl or oleyl monomers were used. The optimized delivery vehicle showed liver selectivity greater than 90% and was used to express sustained titers of aRSV in mouse serum, thereby validating the platform for systemic delivery, with no observed tolerance issues. Although further studies in higher species are underway to explore tolerance, these data support delivery vehicles comprising compounds disclosed herein (e.g., compounds of formula (I)) as a demonstration platform for therapeutic delivery of mRNA, which can be optimized for a given therapeutic target.

[0289] It should be understood that all combinations of the foregoing concepts and implementations, as well as the additional concepts and implementations discussed in more detail below, are considered part of the inventive subject matter disclosed herein and may be employed in any suitable combination to achieve the benefits described herein. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are considered part of the inventive subject matter disclosed herein. The foregoing description is given only for clarity of understanding, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the present disclosure are obvious to those of ordinary skill in the art.

[0290] The terms "substantially" and "approximately" used throughout the specification are used to describe and illustrate small fluctuations. For example, these terms may refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0291] Throughout the specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprising" and "including" and "having", will be understood to imply the inclusion of a stated integer or steps or groups of integers or steps but not the exclusion of any other integer or steps or groups of integers or steps.

[0292] Throughout the specification, where compositions are described as including components or materials, unless otherwise described, it is contemplated that compositions may also consist essentially of or consist of any combination of said components or materials. Similarly, where methods are described as including specific steps, it is contemplated that these methods may also consist essentially of or consist of any combination of said steps, unless otherwise described. The disclosures illustratively disclosed herein may be suitably implemented in the absence of any element or step not specifically disclosed herein.

[0293] The implementation of the methods disclosed herein and their various steps can be performed manually and / or with the aid of an electronic device or automation provided by an electronic device. Although the processes have been described with reference to specific embodiments, it will be readily appreciated by those of ordinary skill in the art that other ways of performing the actions associated with these methods may be used. For example, unless otherwise described, the order of the various steps may be changed without departing from the scope or spirit of the present method. In addition, some of the various steps may be combined, omitted, or further subdivided into additional steps.

[0294] All patents, publications, and references cited herein are hereby fully incorporated by reference. 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 a structure of formula (I): in n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R 1 is H or C optionally substituted by 1 to 3 OH 2-5 alkyl; R 2 is C substituted with 1 to 3 additional OH groups 2-5 Alkylene-OH; and Each R 3 Independently for C 6-24 Alkyl or C 6-24 Alkenyl.

2. The compound according to claim 1, wherein n is 3.

3. The compound according to claim 1, wherein n is 4. The compound according to claim 1 , wherein n is 6. The compound according to claim 1 , wherein n is 8. The compound according to claim 1 , wherein n is 9.

7. A compound according to any one of claims 1 to 6, wherein R 1 For H.

8. A compound according to any one of claims 1 to 7, wherein R 2 C is replaced by an additional OH 2-5 Alkylene-OH.

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

10. The compound according to claim 8 or 9, wherein R 2 is C substituted with 1 to 3 additional OH groups 3-4 Alkylene.

11. A compound according to any one of claims 1 to 10, wherein each R 3 Independently for C 6-18 Alkyl or C 6-18 Alkenyl.

12. A compound according to any one of claims 1 to 11, wherein each R 3 Independently for C 8-18 Alkyl or C 8-18 Alkenyl.

13. A compound according to any one of claims 1 to 12, wherein each R 3 Independently selected from the group consisting of:

14. A compound according to any one of claims 1 to 13, wherein each R 3 Independently selected from the group consisting of:

15. A compound according to any one of claims 1 to 14, wherein at least one R 3 Select from the group consisting of:

16. A compound according to any one of claims 1 to 15, wherein at least one R 3 for 17. The compound of claim 1, having the structure as described in Table 1. The compound according to claim 17 , which has the structure of Compound 1. The compound according to claim 17 , which has the structure of Compound 6.

20. The compound according to claim 17, which has the structure of Compound 21.

21. The compound according to claim 17, which has the structure of Compound 30.

22. The compound according to claim 17, which has the structure of Compound 12.

23. The compound according to claim 17, which has the structure of Compound 34.

24. The compound according to claim 17, which has the structure of Compound 35.

25. The compound according to claim 17, which has the structure of Compound 41.

26. A pharmaceutically acceptable salt of a compound according to 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. 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. The delivery vehicle composition of claim 28, wherein the composition comprises a phospholipid, a sterol, and a PEGylated lipid.

30. The delivery vehicle composition of claim 28, wherein the composition consists essentially of phospholipids, sterols, pegylated lipids, and a compound according to any one of claims 1 to 25 or a salt according to claim 26.

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

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

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

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

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

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

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

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

40. 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. A delivery vehicle composition according to 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. A delivery vehicle composition according to 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. 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. A delivery vehicle composition according to 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. A delivery vehicle composition according to 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. ​​A delivery vehicle composition according to 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. A delivery vehicle composition according to 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. according to the delivery vehicle composition described in any one of claims 28 to 47, wherein the phospholipid is selected from the group consisting of: 1,2-dilinoleoyl-sn-glyceryl-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glyceryl-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-di(undecanoyl)-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-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-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-glyceryl-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glyceryl-3-phosphoethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phospho-racemic-(1-glyceryl) sodium salt (DOPG), sphingomyelin, and combinations thereof.

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

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

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

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

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

56. 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-PEG 2000.

57. 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-PEG 2000.

58. 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-PEG 2000.

59. 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-PEG 2000.

60. 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-PEG 2000.

61. 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-PEG 2000.

62. 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-PEG 2000.

63. The delivery vehicle composition of any one of claims 27 to 30, comprising about 38.2 mol% of Compound 6, about 11.8 mol% of DSPC, about 48.2 mol% of cholesterol, and about 1.9 mol% of DMG-PEG 2000.

64. The delivery vehicle composition of any one of claims 27 to 30, comprising about 42.6 mol% of Compound 6, about 10.9 mol% of DSPC, about 44.7 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.

65. The delivery vehicle composition of any one of claims 27 to 30, comprising about 48.2 mol% of Compound 6, about 9.9 mol% of DSPC, about 40.4 mol% of cholesterol, and about 1.6 mol% of DMG-PEG 2000.

66. The delivery vehicle composition of any one of claims 27 to 30, comprising about 51.3 mol% of Compound 6, about 9.3 mol% of DSPC, about 38 mol% of cholesterol, and about 1.5 mol% of DMG-PEG 2000.

67. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% of Compound 6, about 10.6 mol% of DSPC, about 43.3 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.

68. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% of Compound 6, about 10.6 mol% of DSPC, about 43.4 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.

69. The delivery vehicle composition of any one of claims 27 to 30, comprising about 33.1 mol% of Compound 6, about 10.6 mol% of DSPC, about 53.8 mol% of cholesterol, and about 2.5 mol% of DMG-PEG 2000.

70. The delivery vehicle composition of any one of claims 27 to 30, comprising about 38.2 mol% of Compound 21, about 11.8 mol% of DSPC, about 48.2 mol% of cholesterol, and about 1.9 mol% of DMG-PEG 2000.

71. The delivery vehicle composition of any one of claims 27 to 30, comprising about 42.6 mol% of Compound 21, about 10.9 mol% of DSPC, about 44.7 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.

72. The delivery vehicle composition of any one of claims 27 to 30, comprising about 48.2 mol% of Compound 21, about 9.9 mol% of DSPC, about 40.4 mol% of cholesterol, and about 1.6 mol% of DMG-PEG 2000.

73. The delivery vehicle composition of any one of claims 27 to 30, comprising about 51.3 mol% of Compound 21, about 9.3 mol% of DSPC, about 38 mol% of cholesterol, and about 1.5 mol% of DMG-PEG 2000.

74. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% of Compound 21, about 10.6 mol% of DSPC, about 43.3 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.

75. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% of Compound 21, about 10.6 mol% of DSPC, about 43.4 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.

76. The delivery vehicle composition of any one of claims 27 to 30, comprising about 33.1 mol% of Compound 21, about 10.6 mol% of DSPC, about 53.8 mol% of cholesterol, and about 2.5 mol% of DMG-PEG 2000.

77. The delivery vehicle composition of any one of claims 27 to 30, comprising about 38.2 mol% of Compound 30, about 11.8 mol% of DSPC, about 48.2 mol% of cholesterol, and about 1.9 mol% of DMG-PEG 2000.

78. The delivery vehicle composition of any one of claims 27 to 30, comprising about 42.6 mol% of Compound 30, about 10.9 mol% of DSPC, about 44.7 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.

79. The delivery vehicle composition of any one of claims 27 to 30, comprising about 48.2 mol% of Compound 30, about 9.9 mol% of DSPC, about 40.4 mol% of cholesterol, and about 1.6 mol% of DMG-PEG 2000.

80. The delivery vehicle composition of any one of claims 27 to 30, comprising about 51.3 mol% of Compound 30, about 9.3 mol% of DSPC, about 38 mol% of cholesterol, and about 1.5 mol% of DMG-PEG 2000.

81. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% of Compound 30, about 10.6 mol% of DSPC, about 43.3 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.

82. The delivery vehicle composition of any one of claims 27 to 30, comprising about 44.4 mol% of Compound 30, about 10.6 mol% of DSPC, about 43.4 mol% of cholesterol, and about 1.7 mol% of DMG-PEG 2000.

83. The delivery vehicle composition of any one of claims 27 to 30, comprising about 33.1 mol% of Compound 30, about 10.6 mol% of DSPC, about 53.8 mol% of cholesterol, and about 2.5 mol% of DMG-PEG 2000.

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

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

1.

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

8.

111. The delivery vehicle complex of any one of claims 86 to 88, 91, 96, 98, 100 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

112. The delivery vehicle complex of any one of claims 86 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

113. The delivery vehicle complex of any one of claims 86 to 88, 94, 96 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

114. The delivery vehicle complex of any one of claims 86 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

115. The delivery vehicle complex of any one of claims 86 to 88, 93, 96 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

116. The delivery vehicle complex of any one of claims 86, 87, 89, 96, 97, 99, 101, 103, and 106 to 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-PEG 2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

117. A delivery vehicle complex according to any one of claims 86, 87, 91, 96 to 98, 100, 101 and 105 to 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-PEG 2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

118. A delivery vehicle complex according to any one of claims 86 to 88, 91, 96, 98, 100 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

119. A delivery vehicle complex according to any one of claims 86 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

120. The delivery vehicle complex of any one of claims 86 to 88, 94, 96 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

121. A delivery vehicle complex according to any one of claims 86 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

122. A delivery vehicle complex according to any one of claims 86 to 88, 93, 96 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

123. A delivery vehicle complex according to any one of claims 86, 87, 89, 96, 97, 99, 101, 103 and 106 to 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-PEG 2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

124. A delivery vehicle complex according to any one of claims 86, 87, 91, 96 to 98, 100, 101 and 105 to 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-PEG 2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

125. A delivery vehicle complex according to any one of claims 86 to 88, 91, 96, 98, 100 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

126. A delivery vehicle complex according to any one of claims 86 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

127. A delivery vehicle complex according to any one of claims 86 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

128. A delivery vehicle complex according to any one of claims 86 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

129. A delivery vehicle complex according to any one of claims 86 to 88, 93, 96 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

130. The delivery vehicle complex of any one of claims 86, 87, 89, 96, 97, 99, 101, 103, and 106 to 108, comprising compound 21 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-PEG 2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

131. A delivery vehicle complex according to any one of claims 86, 87, 91, 96 to 98, 100, 101 and 105 to 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-PEG 2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

132. A delivery vehicle complex according to any one of claims 86 to 88, 91, 96, 98, 100 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

133. A delivery vehicle complex according to any one of claims 86 to 88, 92, 96, 98, 103, 106, 107 and 109, comprising compound 30 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

134. A delivery vehicle complex according to any one of claims 86 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

135. A delivery vehicle complex according to any one of claims 86 to 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

136. A delivery vehicle complex according to any one of claims 86 to 88, 93, 96 to 98, 103, 106, 107 and 109, comprising compound 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-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.

137. A delivery vehicle complex according to any one of claims 86, 87, 89, 96, 97, 99, 101, 103 and 106 to 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-PEG 2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

138. A delivery vehicle complex according to any one of claims 86, 87, 91, 96 to 98, 100, 101 and 105 to 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-PEG 2000 having a mass ratio of about 2.1:1 to the polyanionic compound.

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

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

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

146. The delivery vehicle complex of any one of claims 84 to 145, wherein the complex has a particle size of about 50 nm to about 200 nm and / or a polydispersity index (PDI) of less than about 0.

25.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

164. The pharmaceutical composition of claim 163, which is in the form of an intratumoral (IT) or intramuscular (IM) composition.

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

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

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

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

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

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

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

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

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

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

176. The vaccine of claim 175, for use in treating cancer.

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

178. The vaccine for use according to claim 176 or the method according to 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.

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