Calixarene-based delivery system and method of use

Abstract

The present invention relates to a delivery system for delivering one or more cargoes to one or more cells, wherein the cargo delivery system comprises at least a calixarene, a phospholipid, and additional lipids such as sterols. The present invention further relates to a method for delivering cargoes to a subject using the delivery system and to a pharmaceutical composition comprising the delivery system. The present invention also relates to the use of calixarenes in immunogenic compositions, wherein the composition comprises an immunogenic component encapsulated in lipid nanoparticles (LNPs) comprising calixarenes, and the LNPs have an adjuvant effect in the immunogenic composition. The present invention also relates to a vaccine, wherein the vaccine comprises an immunogenic component encapsulated in lipid nanoparticles, the lipid nanoparticles comprising at least one calixarene molecule, and the lipid nanoparticles act as an adjuvant in the vaccine. The present invention further relates to a method for preparing an immunogenic composition and to a composition comprising a calixarene-containing lipid nanoparticle (LNP) adjuvant.

Description

Technical Field

[0001] The present invention relates to a delivery system, particularly a calixarene-based delivery system for delivering various cargos such as nucleic acids, proteins, chemical substances, polysaccharides, etc. to target cells. The present invention also encompasses a method for delivering a cargo to a subject using the described delivery system.

Background Art

[0002] Efficient and targeted delivery of therapeutic agents such as nucleic acids, proteins, and chemical substances to cells is an important aspect of modern medicine. To enhance the effectiveness and safety of these agents, various delivery systems have been developed. However, there remains a continuing need for improved delivery systems that can effectively deliver diverse types of cargos to target cells.

[0003] RNA therapeutics constitute a rapidly expanding drug category that revolutionizes standard treatments for many diseases and enables personalized medicine. These agents are cost-effective, relatively easy to manufacture, and can act on pathways that were previously difficult to target. However, using nucleic acids as therapeutic agents is difficult because they are susceptible to degradation by nucleases, contribute to immune activation, and have unfavorable physicochemical properties that impede easy transfection into cells. Therefore, safe and effective nucleic acid therapeutics require advanced delivery platform technologies.

[0004] Lipid nanoparticles (LNPs) are a major technology for non-viral nucleic acid delivery. Naked RNA is degraded by cellular ribonucleases (RNases) soon after administration. LNPs delay the degradation process, ensure the stability of RNA, while also promoting intracellular uptake via endocytosis and enabling the release of RNA into the cytoplasm for translation by intracellular machinery.

[0005] LNPs typically consist of ionizable cationic lipids and three neutral helper lipids: phospholipids, cholesterol, and lipid-anchored polyethylene glycol (PEGylated lipids). The ionizable cationic lipids form complexes with polyanionic RNA via ion pairing interactions, enabling encapsulation by neutral lipids and promoting cellular uptake and endosome escape.

[0006] LNPs were initially optimized for the formulation of siRNA (approximately 23 nt), and in recent years have evolved to encapsulate larger RNA agents, including mRNA (approximately 1000 nt). Recently, there has been increased activity with even larger RNA payloads, such as self-amplifying RNA (saRNA). SaRNA has been shown to induce immune responses at doses up to 1 / 100th that of mRNA, and to extend protein expression duration in vivo, making it a promising alternative to mRNA. However, saRNA (approximately 10,000 nt) is larger than mRNA (approximately 1000 nt) and has more secondary structures, making encapsulation and delivery more difficult. The inherent chemical and structural differences between mRNA and saRNA in terms of length, stability, and charge density suggest that LNP delivery formulations for saRNA may require significantly different conditions than those developed for mRNA delivery.

[0007] Ionizable lipids are considered the most important element for improving encapsulation efficiency because they are components that form complexes with RNA cargo. However, to obtain sufficient saRNA encapsulation efficiency, high concentrations of ionizable lipids are required (for example, using 20 times the amount of ionizable lipids compared to the amount of saRNA).

[0008] By optimizing the encapsulation efficiency of the cargo, it is possible to minimize waste of expensive materials (i.e., RNA) and produce drug formulations with higher concentrations.

[0009] In addition to influencing encapsulation efficiency, cellular uptake, and the promotion of nucleic acid cargo extrusion into endosomes, each component of LNPs is also important for promoting monodisperse nanoparticle formation and improving nanoparticle stability. The polydispersity index (PDI) is a normalized value that indicates the nanoparticle size range in a sample and is a useful indicator of sample quality. In highly dispersible samples, larger particles in the distribution tend to aggregate and settle, leading to a decrease in effective RNA concentration and dose heterogeneity. Typically, LNP formulations developed for biological applications should have a PDI of less than 0.2, indicating that the colloid is monodisperse within an acceptable range. Monodispersity of nanoparticle drugs is crucial for ensuring consistent behavior of the intended drug, as particle size affects its behavior in the body. However, obtaining a monodisperse population of LNPs, particularly those containing saRNA cargoes, requires the use of high concentrations of ionizable lipids.

[0010] The present invention aims to solve at least some of the above-mentioned problems and inconveniences. [Overview of the Initiative]

[0011] The present invention provides a delivery system for delivering one or more cargoes to one or more cells, as described in claim 1. The delivery system comprises additional lipids such as calixarenes, phospholipids, and sterols, and optionally PEGylated lipids. Optionally, the delivery system may include ionizable lipids and / or cationic lipids. The calixarenes may be ionizable calixarenes, cationic calixarenes, or any combination thereof.

[0012] The delivery system may include specific concentrations of additional lipids such as calixarenes, phospholipids, sterols, PEGylated lipids, ionizable lipids, and cationic lipids. Furthermore, as described in the claims, various types of phospholipids, sterols, PEGylated lipids, and cationic lipids may be incorporated into the delivery system. The delivery system may be modified to include ionizable or cationic calixarenes, particularly depending on the target cargo and target cells.

[0013] The present invention also provides a method for delivering one or more cargoes to a subject, comprising administering the above-described delivery system to the subject. The cargoes may be selected from the group consisting of nucleic acids, proteins, chemicals, polysaccharides, and combinations thereof. Preferably, the cargo is a nucleic acid, more preferably RNA or DNA.

[0014] The present invention further provides pharmaceutical compositions comprising the above-described delivery system used for the prevention and / or treatment of a disease or disorder in a subject.

[0015] The present invention further provides the use of calixarenes in immunogenic compositions. In further embodiments, the present invention also relates to vaccines, methods for preparing immunogenic formulations, compositions comprising lipid nanoparticle (LNP) adjuvants and antigens comprising calixarenes, and methods for enhancing immune responses.

[0016] This invention provides an improved delivery system that can effectively deliver diverse types of cargo to target cells, with potential applications in various therapeutic uses.

[0017] The description of the drawings of specific embodiments of the present invention is for illustrative purposes only and does not limit the teachings, applications, or uses of the present invention. Throughout the drawings, corresponding reference numerals indicate the same or corresponding parts and features. [Brief explanation of the drawing]

[0018] [Figure 1] The chemical structure of an ionizable calixarene used in a delivery system according to one embodiment of the present invention is shown. [Figure 2] This shows a visualization of an estimation model (mean value + 95% confidence interval) of in vivo luminescence as a function of the (lipid + CX) / RNA mass ratio, helper mass ratio, and PEG lipid mass ratio in a delivery system according to one embodiment of the present invention. [Figure 3]Using various delivery systems of the present invention, in vivo protein expression (geometric mean signal and 95% confidence interval) is shown at 3, 6, and 9 hours after intramuscular injection of 1 μg of Fluc mRNA into the left and right hind limbs of mice. [Figure 4] This invention demonstrates saRNA-FLuc encapsulation in nanoparticles having different CX / RNA mass ratios and in vitro protein expression after transfection of these nanoparticles, according to one embodiment of the present invention. [Figure 5] This paper compares the in vivo saRNA-flux delivery efficiency of a delivery system according to one embodiment of the present invention with a reference delivery system that does not contain calixarenes. [Figure 6] The following shows the results of measuring the viral neutralizing antibody titer (VNT) in mouse serum vaccinated using a delivery system equipped with an ionizable calixarene according to one embodiment of the present invention, and carrying mRNA encoding the rabies virus G protein, at 15, 35, and 64 / 65 days. A 21-day Prime Boost Resimen with a single dose of 0.6 μg or 2.5 μg of mRNA was used. [Figure 7] The image shows a cryoTEM image of mRNA encapsulated by the delivery system of the present invention. [Figure 8] The chemical structure of a cationic calixarene synthesized for use in a delivery system according to one embodiment of the present invention is shown. [Figure 9] This document presents an experimental design (DoE) for modeling the effect of the amount of cationic calixarene in an LNP according to one embodiment of the present invention (expressed as mg of cationic calixarene per mg of RNA dose) on the physicochemical properties of the resulting LNP. [Figure 10]Using a delivery system comprising a cationic calixarene (CX4 or CX12), a PEGylated lipid (DMG-PEG2000 or DSG-PEG2000), DOPE, and cholesterol, the results of measuring the virus neutralizing antibody titer (VNT) in the sera of vaccinated mice carrying mRNA encoding the rabies virus G protein are shown at 15 days and 35 days. A 21-day prime-boost regimen with a single dose of 0.6 μg or 2.5 μg of mRNA was used. [Figure 11] The intensity distributions when an LNP (DLin-DMA:DOPE:cholesterol:DMG-PEG2000:CX12 LNP in 20 mM TRIS buffer) comprising DLin-DMA and a cationic calixarene according to an embodiment of the present invention encapsulates saRNA encoding the SARS-CoV-2 spike protein (Figure 11A), and when a reference LNP without a cationic calixarene (SM-102:DSPC:cholesterol:DMG-PEG2000 (50:10:38.5:1.5 molar ratio)) encapsulates saRNA encoding an equivalently long unpublished gene (Figure 11B) are shown. [Figure 12] The log10 luminescence at the injection site 6 hours after intramuscular (IM) injection is shown in either the case where DOTAP is present in the LNP or the case where a cationic calixarene is present in the LNP according to an embodiment of the present invention. [Figure 13] IgG levels (dots are individual observations, lines are geometric mean and 95% confidence intervals) against the spike protein and the receptor binding domain (RBD) of the spike protein are shown. The DODAP / DOTAP of all animals was below the lower limit of quantification, whereas different results were obtained when a calixarene was present in the LNP according to an embodiment of the present invention. [Figure 14] The VNT levels in the sera measured on day 35 (after prime-boost) after an LNP of DODAP:CX12 according to an embodiment of the present invention encapsulated mRNA encoding the rabies virus G protein and was intramuscularly administered at 2.5 μg to BALB / c mice are shown. [Figure 15A] For each LNP containing the LNP according to an embodiment of the present invention, the VNT serum levels measured at 15 days (after prime), 35 days, and 64 / 65 days (after boost) are shown. [Figure 15B] Virus neutralization titers (VTN) in sera measured 15 days (after prime), 35 days, and 65 days (after boost) in mice vaccinated using SM-102 LNP (control), DLin-DMA LNP, and DLin-DMA / CX4 LNP, the latter being a delivery system according to an embodiment of the present invention, are shown. A 21-day prime-boost regimen using 2.5 μg of mRNA (RNA encoding the glycoprotein G of rabies virus) per administration was used. The results are shown as a comparison with the control delivery system (SM-102 LNP). UDL = upper limit of determination, LDL = lower limit of determination. All points beyond the limits indicate a significant difference compared to the control. [Figure 16] The chemical structure of a cationizable calixarene (CX5) that can be used in the LNP according to an embodiment of the present invention is shown. [Figure 17] For each nanoparticle (intramuscular administration to BALB / c mice at doses of A: 0.6 μg and B: 2.5 μg), the VNT serum levels measured at 15 days (after prime), 35 days, and 65 days (after boost) for the LNP using cationizable calixarene according to an embodiment of the present invention are shown.

Mode for Carrying Out the Invention

[0019] The following detailed description provides further information regarding the calixarene-based delivery system according to the present invention and methods of using the same. The present invention encompasses a delivery system for delivering various types of cargo to target cells and methods of administering the delivery system to a subject.

[0020] Unless otherwise defined, all terms used in the disclosure of this invention, including technical and scientific terms, have the meanings generally understood by those skilled in the art. For further guidance, definitions of terms are included to better understand the teachings of this invention.

[0021] The following terms used in this specification have the meanings set forth below.

[0022] In this specification, "A," "an," and "the" refer to both singular and plural nouns, unless the context clearly indicates otherwise. For example, "a compartment" refers to one or more compartments.

[0023] As used herein, "about" means that with respect to measurable values ​​such as parameters, quantities, and temporal durations, it includes a variation of ±20%, preferably ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from a specified value, and that such variation is within the range that is appropriately implemented in the disclosure of the present invention. However, it should be understood that the value to which "about" modifies is also specifically disclosed.

[0024] As used herein, “equipped,” “having equipped,” and “equipped with ~” are synonymous with “contains,” “contains,” or “contains,” and are comprehensive or open-ended terms that specify the presence of subsequent components, etc., and do not exclude or preclude the presence of additional, undescribed components, features, elements, components, processes, things known to those skilled in the art, or disclosed herein.

[0025] Furthermore, terms such as “first,” “second,” and “third” in the specification and claims are used to distinguish between similar elements and, unless otherwise specified, do not indicate order or chronology. Such terms are interchangeable under appropriate circumstances, and it should be understood that embodiments of the invention described herein may operate in an order other than that described or illustrated.

[0026] When a numerical range is specified using endpoints, it includes all numerical values ​​and fractions contained within that range, as well as the specified endpoints.

[0027] The expressions "mass %", "weight %", "%wt", "%(w / w)", or "wt%", unless otherwise defined, throughout the description, indicate the relative weight of each component based on the total weight of the formulation (e.g., the weight of the entire delivery system excluding cargo (e.g., RNA) and other excipients (e.g., sucrose, TRIS, etc.)). The components included in "formulation" and the components constituting "total weight of formulation" are context-dependent. In this invention, when the mass fraction of a calixarene component or lipid component is referred to in relation to the total weight of the formulation, the weight of the calixarene component or lipid component is relative to the total weight of calixarenes and lipids present in the formulation, more specifically in the delivery system, and even more specifically in the lipid nanoparticles. Therefore, "total weight of formulation" in such cases does not include cargo (e.g., RNA) or other excipients (e.g., sucrose, TRIS, etc.).

[0028] For example, the terms “one or more” or “at least one,” such as “one or more” or “at least one” in relation to one or more components of a group, are self-explanatory, but as a further example, the terms can also refer to any one of those components, or any two or more of those components, for example, any three or more, four or more, five or more, six or more, seven or more, etc., or even all of those components.

[0029] Unless otherwise defined, all terms used in the disclosure of this invention (including technical and scientific terms) have the meanings generally understood by those skilled in the art. For further guidance, definitions of terms used in the specification are provided for a better understanding of the teachings of this invention. Terms or definitions used herein are provided solely to aid in the understanding of this invention.

[0030] Throughout this specification, the phrase "one embodiment" or "a certain embodiment" means that any particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, where the expression "one embodiment" or "a certain embodiment" appears in various places in this specification, they may, though not necessarily, refer to the same embodiment. Furthermore, certain features, structures, or characteristics can be combined in any suitable way in one or more embodiments, as will be clearly understood by those skilled in the art from this disclosure. Furthermore, it will be understood by those skilled in the art that some embodiments described herein do not include features included in other embodiments, and that combinations of features included in other embodiments fall within the scope of the present invention and form different embodiments. For example, in the following claims, any claimed embodiment may be used in any combination.

[0031] As used herein, “as needed” or “as required” means that the events or situations described below may or may not occur, including both cases in which the events or situations occur and cases in which they do not occur. For example, “as needed substituted aryl” means that the aryl group may or may not be substituted, and includes both substituted and unsubstituted aryl groups.

[0032] "Salt" or "pharmaceutically acceptable salt" refers to salts derived from various organic and inorganic counterions well known to those skilled in the art. Pharmaceutically acceptable acid-added salts can be formed by reacting them with inorganic and organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Pharmaceutically acceptable base-added salts can be formed by reacting them with inorganic and organic bases. Examples of inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Examples of organic bases include primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins, particularly isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base-adding salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.

[0033] As used herein, the terms "extra-enteral administration" and "administered extra-enterally" refer to administration routes other than enteral and transdermal administration, usually by injection, including, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intracutaneous tissue, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

[0034] As used herein, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for use in a reasonable benefit-to-risk ratio without causing excessive toxicity, irritation, allergic reactions, or other problems or complications when in contact with human or animal tissue from a medical standpoint. “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” means herein a pharmaceutically acceptable material, composition, or medium (such as a liquid or solid filler, diluent, excipient, solvent, or encapsulant). Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; and (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. Examples of suitable substances used in pharmaceutical preparations include (10) propylene glycol and other glycols, (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters such as ethyl oleic acid and ethyl lauric acid, (13) agar, (14) buffering agents such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) atherless raw water, (17) isotonic saline solution, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer, and (21) other non-toxic and suitable substances used in pharmaceutical preparations.

[0035] In certain embodiments, the terms “prevention” or “preventive” may refer to a compound that, in relation to a disease or disorder, reduces the incidence of the disorder or condition in a treated sample compared to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0036] As used herein, “treatment,” “to treat,” or “procedure” may include reducing, alleviating, or improving the symptoms of a disease or condition; preventing the onset of additional symptoms; improving or preventing the underlying cause of symptoms; inhibiting a disease or condition, for example, stopping the progression of a disease or condition; alleviating a disease or condition; causing regression of a disease or condition; alleviating a condition caused by a disease or condition; or preventing and / or therapeutically cessating the symptoms of a disease or condition.

[0037] The term "lipids" refers to a group of organic compounds that are generally poorly soluble in water but soluble in many organic solvents, and which include, but are not limited to, esters of branched or straight-chain fatty acids. Lipids are usually divided into at least three categories: (1) "simple lipids" which include fats, oils, and waxes; (2) "complex lipids" which include phospholipids or glycolipids; and (3) "derived lipids" such as steroids.

[0038] In the context of this invention, the term "sterol" is also known as steroid alcohol and refers to a subgroup of steroids that are naturally present in plants, animals, and fungi, or that can be produced by certain bacteria.

[0039] The term "neutral lipids" refers to any lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, these lipids include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), steroids such as sphingomyelin (SM), ceramides, and sterols, and their derivatives. Neutral lipids can be either synthetic or naturally occurring.

[0040] In the context of this disclosure, the term “ionizable” in the context of a compound or lipid means that any uncharged group present in the compound or lipid becomes an ion (usually H + This means that it can associate with ions and can become positively charged itself (also called "cationicable"). Alternatively, any uncharged group in the compound or lipid can associate with ions (usually H + It can produce ions, and thus become negatively charged. In the context of this disclosure, any type of ionizable lipid can be used appropriately.

[0041] The term "lipid nanoparticles" refers to particles comprising multiple lipid molecules that have at least one dimension on the order of nanometers (e.g., 1 to 1000 nm) and are physically associated with each other by intermolecular forces. Activators or therapeutic agents, such as nucleic acids, are encapsulated within an aqueous space surrounded by the lipid portion of the lipid nanoparticles, or some or all of the lipid portion of the lipid nanoparticles, thereby protecting them from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as harmful immune responses.

[0042] As used herein, the terms “oligonucleotide” or “polynucleotide” refer to polymers containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and which constitute DNA, RNA, and hybrids thereof. DNA may be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA may be in the form of self-amplified RNA (saRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA or viral RNA (vRNA), guide RNA (gRNA), and combinations thereof. Nucleic acids include known nucleotide analogs or nucleic acids containing modified backbone residues or linkages, which may be synthetic, natural, or unnatural, and which have similar binding properties to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methylribonucleotides, and peptide-nucleotides (PNAs). Unless otherwise specified, this term encompasses nucleotides, including known native nucleotide analogs having similar binding properties to a reference nucleotide. Unless otherwise indicated, a particular nucleotide sequence implicitly includes conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologues, single nucleotide polymorphisms, and complementary sequences, as well as explicitly indicated sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue. A “nucleotide” contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other via phosphate groups."Bases" include purines and pyrimidines, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications introducing novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides, which are natural compounds, and further include adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs.

[0043] Buffers used herein include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, pentanoic acid, calcium dihydrogen phosphate, phosphoric acid, calcium trihydrogen phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, potassium dihydrogen phosphate, potassium monohydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium dihydrogen phosphate, sodium monohydrogen phosphate, sodium phosphate mixture, tromethamine, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof.

[0044] As used herein, the term N / P ratio or N:P ratio refers to the molar ratio of nitrogen atoms in a complexed lipid (or other chemical structure having at least one amino group used for encapsulation) to the molar ratio of phosphate groups in RNA. This ratio represents the interaction between the cationic charge of the amino(N+) group in the aminolipid (or other chemical structure) and the anionic charge of the phosphate(PO4-) group in the nucleotide backbone, and is the basis for the complexation of RNA by aminolipids (or other chemical structures having at least one amino group). The N / P ratio of the lipid / nucleotide complex may also affect other properties such as its surface charge, size, and stability.

[0045] "Effective dose" or "therapeutic effective dose" refers to an amount of pharmaceutical composition sufficient to exert a therapeutic effect in a mammal, preferably a human, when administered to an animal, preferably a mammal, preferably a human. The amount constituting the "therapeutic effective dose" of the pharmaceutical composition of the present invention varies depending on the compound, its state and severity, the method of administration, and the age of the animal being treated, but can usually be determined by a person skilled in the art based on their knowledge and this disclosure.

[0046] As used herein, the terms “self-replicating” and “self-amplifying” are synonymous and relate to molecules such as RNA that possess specific signal or characteristic sequences within their sequence that enable self-replication or self-amplification.

[0047] In this specification, "encapsulation efficiency" refers to the ratio of encapsulated RNA to total RNA in a sample. Several methods exist for evaluating the encapsulation efficiency of LNP formulations to cargo, but the modified ribogreen assay is the most common. Ribogreen is an RNA quantification dye that fluoresces upon nucleic acid binding. To measure the encapsulation efficiency in LNPs, first, ribogreen is added to a sample containing LNPs as they are, and the concentration of unencapsulated RNA is measured. Next, a surfactant solution (e.g., Triton X-100) is added to break down the nanoparticles, releasing the encapsulated RNA, and the total amount of RNA in the sample is calculated from the ribogreen fluorescence. Encapsulated RNA is calculated by subtracting unencapsulated RNA from total RNA, and the encapsulation efficiency is determined as the ratio of encapsulated RNA to total RNA in the sample.

[0048] An "adjuvant" is typically defined as a compound that can enhance and / or modulate the inherent immunogenicity of an antigen. To reduce side effects, novel vaccines often have a more clearly defined composition and are less immunogenic compared to conventional whole-cell or virus-based vaccines. Therefore, adjuvants are necessary to help novel vaccines induce a strong and sustained immune response, and also offer the advantage of reducing the amount of antigen and the number of doses required.

[0049] "Adjuvant / Adjuvant component": Most broadly, an adjuvant or adjuvant component is an active substance or composition (e.g., pharmacological or immunological) that can modify, for example, enhance the effectiveness of other active substances, such as pharmaceuticals or vaccines. Conventionally, in the context of the present invention, it refers to a compound or composition that functions as a carrier or auxiliary substance for immunogens and / or other pharmacologically active compounds. In the context of the present invention, an adjuvant preferably enhances the specific immunogenic effect of the active ingredient of the present invention. Usually, "adjuvant" and "adjuvant component" are synonymous and can be used interchangeably. Adjuvants can be classified, for example, as immunoenhancing agents, antigen delivery systems, or combinations thereof. The term "adjuvant" is usually understood to comprise an active substance that confers immunity on its own. Adjuvants assist in nonspecifically enhancing antigen-specific immune responses, for example, by promoting the presentation of antigens to the immune system or inducing nonspecific innate immune responses. Furthermore, adjuvants can favorably regulate antigen-specific immune responses, for example, by inducing mucosal immune responses, increasing IgA titers, or shifting a dominant Th2-type antigen-specific response towards a Th1-type antigen-specific response, or vice versa. Therefore, adjuvants can favorably regulate cytokine expression / secretion, antigen presentation, and the type of immune response.

[0050] The benefits of adjuvants include enhancing the immunogenicity of antigens, modifying the nature of the immune response, reducing the amount of antigen required for immunity, decreasing the frequency of booster immunizations, and improving the immune response in the elderly and immunocompromised vaccinated individuals. These may be administered concurrently via any route, such as intramuscular, subcutaneous, intravenous, or intradermal injection. As used herein, the term “antigen” refers to a substance that can be recognized by the immune system and can induce an antigen-specific immune response as part of an adaptive immune response, such as antibody production or the formation of antigen-specific T cells.

[0051] As used herein, "epitope" (also called an antigenic determinant) refers to a T cell epitope, which is preferably a fragment having a length of about 6 to about 20 or more amino acids, for example, a fragment treated and presented by an MHC class I molecule, preferably about 8 to about 10 amino acids, for example 8, 9, 10 (or 11, 12 amino acids), or a fragment treated and presented by an MHC class II molecule, preferably comprising a fragment having about 13 or more amino acids, for example 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids, and these fragments can be selected from any site in the amino acid sequence. These fragments are usually recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule. B cell epitopes are usually fragments located on the outer surface of (native) protein or peptide antigens.

[0052] As used herein, "vaccine" refers to a preventive or therapeutic material that provides at least one antigen or antigenic function. The antigen or antigenic function may stimulate the body's adaptive immune system to produce an adaptive immune response.

[0053] "Immunogenicity" refers to the ability of an external substance, such as an antigen, to induce an immune response in the body of a human or other animal. The "immunogenic composition" used herein is capable of inducing an immune response in the body of a human or other animal.

[0054] (Compositions, pharmaceutical compositions, and formulations) The present invention relates to a (pharmaceutical) composition that functions as a delivery system for delivering one or more cargoes to one or more cells, the delivery system comprising at least additional lipids such as calixarenes, phospholipids, and sterols, and optionally PEGylated lipids.

[0055] In a preferred embodiment, the delivery system is a lipid nanoparticle (LNP) comprising at least a calixarene, a phospholipid, a sterol, and a PEGylated lipid.

[0056] Lipid nanoparticles are a leading technology for nonviral nucleic acid delivery. Naked RNA is rapidly degraded by cellular ribonucleases (RNases) after administration. LNPs delay the degradation process, ensuring RNA stability, and promote intracellular uptake via endocytosis, allowing RNA to be released into the cytoplasm for translation by intracellular mechanisms.

[0057] The compositions of conventionally known LNPs typically include an ionizable cationic lipid and three neutral helper lipids, namely phospholipids, cholesterol, and lipid-anchored polyethylene glycol (PEGylated lipids). The ionizable cationic lipid forms a complex with polyanionic RNA via ion pairing interactions, enabling encapsulation by the neutral lipids and promoting cellular uptake and endosomal escape.

[0058] Cationic or ionizable lipids (under acidic pH) facilitate ion-pairing interactions with the negatively charged backbone of nucleic acids. These interactions facilitate the encapsulation of nucleic acid cargo within the electron-dense LNP core. Permanent cationic lipids can cause undesirable toxicity and immune response problems, leading to increased adoption of ionizable lipids. Ionizable lipids are positively charged during LNP formation but are primarily neutral at physiological pH. Maintaining a neutral pH during circulation can prevent the adsorption of loaded biomolecules, hindering rapid clearance by immune cells and extending circulation time. Ionizable lipids also promote the release of nucleic acid cargo via electrostatic interactions with the anionic endosomal membrane by becoming protonated in the acidic microenvironment within endosomes under acidic pH.

[0059] LNPs were initially optimized for the formulation of siRNA (approximately 23 nucleotides), but in recent years they have evolved to encapsulate larger RNA agendas, such as mRNA (approximately 1000 nucleotides). More recently, activity towards even larger RNA payloads, such as self-amplifying mRNA (saRNA), has increased. SaRNA has been shown to induce immune responses at up to 1 / 100th the dose of mRNA and to extend the duration of protein expression in vivo, making it a promising alternative to mRNA. However, saRNA (approximately 10,000 nucleotides) is larger than mRNA (approximately 1,000 nucleotides) and has more secondary structures, making encapsulation and delivery more difficult. Given the essential chemical and structural differences between mRNA and saRNA in terms of length, stability, and charge density, it is suggested that LNP delivery formulations of saRNA may require conditions significantly different from those developed for mRNA delivery.

[0060] Ionizable lipids are considered the most important element for improving encapsulation efficiency, as they play a role in forming complexes with RNA cargo. However, a large amount of ionizable lipids is required to achieve sufficient saRNA encapsulation efficiency (for example, 20 times the amount of ionizable lipids is needed relative to the amount of saRNA).

[0061] In addition to influencing encapsulation efficiency, cellular uptake, and the promotion of nucleic acid cargo extrusion into endosomes, each component of LNPs is also important for promoting monodisperse nanoparticle formation and improving nanoparticle stability. The polydispersity index (PDI) is a normalized value that indicates the nanoparticle size range in a sample and is a useful indicator of sample quality. In highly dispersible samples, larger particles in the distribution are more likely to aggregate and settle, which reduces the effective RNA concentration and impairs the consistency of the dosage. LNP formulations developed for biological applications should typically have a PDI of less than 0.2, indicating that the colloid has acceptable monodispersity. Monodispersity of nanoparticle pharmaceuticals is critical to ensuring the consistent behavior of the intended drug, and particle size affects particle interactions in the body.

[0062] However, obtaining a monodisperse LNP population containing saRNA cargo requires a large amount of ionizable lipids.

[0063] The present invention centers on a delivery system (more specifically, an LNP) comprising at least a calixarene.

[0064] Calixarenes are macrocyclic molecules known for their ability to encapsulate and deliver various cargoes such as nucleic acids, proteins, and chemical substances. Calixarenes are macrocyclic molecules based on methylene-bonded phenols. In the present invention, the delivery system comprises calixarenes which may be ionizable calixarenes, cationic calixarenes, or any combination thereof.

[0065] This modification is expected to completely alter the behavior of the resulting delivery system, particularly its encapsulation and release capabilities.

[0066] In the present invention, the size of the calixarene can vary depending on several desired properties, but is preferably a calix[4]arene.

[0067] Calix[4]arenes exhibit several important properties suitable for nucleic acid delivery. Most importantly, their natural cone-shaped three-dimensional structure has been found to be crucial for lipid nanoparticles / ionizable lipids to achieve high endosomal escape ability and promote RNA release in the cytoplasm.

[0068] As described above, permanently charged cationic components can cause undesirable toxicity and immune response problems, and as a result, conventional LNPs known in the prior art incorporate ionizable lipids.

[0069] In one embodiment, the present invention provides a delivery system that replaces conventional ionizable lipids by using ionizable calixarenes in RNA lipid nanoparticles (RNA-LNPs). Therefore, the resulting delivery system comprises four components: ionizable calixarenes, helper lipids, sterols, and PEG lipids.

[0070] Calixarenes are a platform that facilitates the synthesis of ionized compounds with multiple amine heads, meaning they can easily increase charge density (amine count / molecule). This property not only reduces the amount of ionized component needed to efficiently encapsulate nucleic acids, but also facilitates the encapsulation of very long RNAs such as self-amplifying RNA (saRNA) by increasing the number of amines without changing the mass ratio of the ionized component to the RNA. However, this challenge remains difficult with current LNP technology formulations that do not contain calixarenes.

[0071] In another embodiment, the present invention provides a delivery system incorporating a cationic calixarene as a fifth component into lipid nanoparticles (LNPs) made from ionizable lipids, helper lipids, sterols, and PEG lipids. As described above, and similar to ionizable calixarenes, cationic calixarenes can easily increase the charge density (amine count / molecule) and can particularly promote the encapsulation of very long RNA nucleic acids, such as self-amplifying RNA (saRNA). Furthermore, by incorporating cationic calixarenes into conventional LNPs ("5-component delivery system"), the present invention can (i) minimize the inherent toxicity of these cationic components by combining them with non-toxic, biocompatible lipids, and (ii) reduce the nonspecific adsorption of proteins that usually limit their effectiveness.

[0072] The calixarene concentration in the delivery system may range from 0.1 to 60 mol% depending on the desired properties of the delivery system. For example, the calixarene concentration in the delivery system may be 0.1 to 50 mol%, 0.1 to 44 mol%, 0.1 to 40 mol%, 0.1 to 30 mol%, 0.1 to 20 mol%, 0.1 to 10 mol%, 0.1 to 5 mol%, or 1 to 60 mol%, 10 to 60 mol%, 20 to 60 mol%, 30 to 60 mol%, 35 to 60 mol%, and any of these ranges and subranges.

[0073] In the present invention, the size of the calixarene can vary depending on several desired features, such as the precise three-dimensional structure of the calixarene (e.g., cone-shaped or other three-dimensional structure), the selected cargo, and / or the target cell. Preferred calixarene structures, though not limited to those disclosed, include calixarenes comprising tetramers, hexamers, or octamers, i.e., calix[4]arenes, calix[6]arenes, and calix[8]arenes. In a preferred embodiment, the calixarene is calix[4]arene.

[0074] In a preferred embodiment, the delivery system according to the present invention comprises a calix[4]arene.

[0075] In one embodiment, the calix[4]arene used in the delivery system of the present invention may be a compound of formula (I), where "A" represents the hydrophobic part of the molecule and "B" represents the head group (ionizable or cationic) that forms the hydrophilic part of the molecule. [ka]

[0076] In one embodiment, cationic or ionizable head groups may be bonded to the macrocycle via various biodegradable or non-biodegradable groups.

[0077] In one embodiment, the calixarene used in the present invention, relating to formula (I), comprises four identical B groups. In another embodiment, the calixarene used in the present invention comprises three identical B groups and one different B group. In yet another embodiment, the calixarene used in the present invention comprises two identical B groups and two other identical B groups. The same applies to the A groups. As can be understood, many configurations are possible.

[0078] In the embodiment, when cationic calixarene is used, its concentration may be in the range of 0.1 to 10 mol% of the delivery system. For example, the concentration of cationic calixarene may be 0.1 to 10 mol%, 0.5 to 9 mol%, 1 to 8 mol%, 2 to 7 mol%, 3 to 6 mol%, 4 to 5 mol%, or 0.1 to 9 mol%, 0.1 to 8 mol%, 0.1 to 7 mol%, 0.1 to 6 mol%, 0.1 to 5 mol%, 0.1 to 4 mol%, 0.1 to 3 mol%, 0.1 to 2 mol%, 0.1 to 1 mol%, or 0.5 to 10 mol%, 1 to 10 mol%, 2 to 10 mol%, 3 to 10 mol%, 4 to 10 mol%, 5 to 10 mol%, 6 to 10 mol%, 7 to 10 mol%, 8 to 10 mol%, 9 to 10 mol%, and any of these ranges and subranges. In the embodiment, the number of cationic sites (head groups) of the calixarene is in the range of 1 to 8. For example, the calixarene may have 8, 7, 6, 5, 4, 3, 2, or 1 cationic site, preferably 1 to 4 cationic sites.

[0079] In this embodiment, the concentration of the ionizable calixarene in the delivery system is 10 to 40 mol%.

[0080] In this embodiment, the concentration of the ionizable calixarene in the delivery system is 10 to 60 mol%.

[0081] For example, the concentration of ionizable calixarenes may be 10-60 mol%, 10-50 mol%, 10-45 mol%, 10-40 mol%, 10-35 mol%, 10-30 mol%, 10-25 mol%, 10-20 mol%, 10-15 mol%, or 15-60 mol%, 20-60 mol%, 25-60 mol%, 30-60 mol%, 35-60 mol%, or 15-55 mol%, 15-45 mol%, 15-40 mol%, 20-35 mol%, 20-30 mol%, 20-25 mol%, 25-30 mol%, and any of these ranges and subranges.

[0082] Phospholipids are essential components of biological membranes and can be used in delivery systems to enhance stability and biocompatibility. Suitable phospholipids for delivery systems can be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, and sphingolipids. The concentration of phospholipids in the delivery system may range from 1 to 45 mol%. For example, the phospholipid concentration in the delivery system may be 1 to 40 mol%, 1 to 30 mol%, 1 to 20 mol%, 1 to 10 mol%, 1 to 5 mol%, or 5 to 45 mol%, 10 to 45 mol%, 20 to 45 mol%, 30 to 45 mol%, 35 to 45 mol%, or 5 to 40 mol%, 10 to 35 mol%, 15 to 30 mol%, 20 to 30 mol%, and any of these ranges and subranges.

[0083] In preferred embodiments, the additional lipid imparts rigidity to the delivery system. In embodiments, the additional lipid may be a sterol, fatty acid, glycerol monooleate, trioleate, or a saturated short-chain molecule selected for its rigidity properties. In embodiments, the saturated short-chain molecule has fewer than 17 carbon (C), i.e., a maximum of 16C, 15C, 14C, 13C, 12C, 11C, 10C, 9C, 8C, 7C, 6C, 5C, 4C, or 3C, and preferably a maximum of 10C to 3C.

[0084] In one embodiment, an additional lipid (e.g., a sterol compound) is present in the composition according to the present invention at a concentration of 20 to 70 mol%. In the embodiment, the concentration of the additional lipid may be 20 to 60 mol%, 20 to 50 mol%, 20 to 40 mol%, 20 to 30 mol%, 20 to 25 mol%, or 30 to 70 mol%, 40 to 70 mol%, 50 to 70 mol%, 60 to 70 mol%, 65 to 70 mol%, and any of these ranges and subranges, preferably 35 to 60 mol%, more preferably 40 to 60 mol%, and even more preferably 42 to 57 mol%.

[0085] In preferred embodiments, the additional lipid is a sterol. Sterols are known to regulate membrane fluidity and stability and are important components of the delivery system. Suitable sterols for the delivery system disclosed may be selected from the group consisting of cholesterol, sitosterol, sitosterol-amino acid conjugate, stigmamanol, campesterol, fucosterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, and hydroxycholesterol.

[0086] In the embodiment, the delivery system comprises a calixarene, which is an ionizable or cationic calixarene, a phospholipid, and an additional lipid, the additional lipid being selected from sterols, fatty acids, glycerol monooleates, trioleates, or saturated short-chain molecules, and is characterized in that, if the calixarene is cationic, the delivery system further comprises an ionizable lipid.

[0087] In the embodiment, the delivery system further comprises PEGylated lipids, PEG lipids, or PEG conjugates to extend the circulation time of the delivery system and reduce unwanted host responses.

[0088] PEGylated lipids are lipids modified by the addition of polyethylene glycol (PEG) chains, which can improve the stability and circulation time of the delivery system. Suitable PEGylated lipids for the delivery system can be selected from the group consisting of PEG-diacylglycerol (PEG-DAG), PEG-dialkyloxypropyl (PEG-DAA), PEG-phospholipids, and PEG-ceramides. Preferred PEG-ceramides are those with a C16 alkyl chain and a PEG molecular weight of 500 to 2000, such as C16 PEG500, C16 PEG750, C16 PEG1000, C16 PEG1250, C16 PEG1500, C16 PEG1750, C16 PEG2000, and any of these ranges and subranges. The concentration of PEGylated lipids in the delivery system can be adjusted as needed.

[0089] PEG is a polymer with hydrophilic and flexible properties, making it frequently used in nanoparticle formulations for its stealth capabilities. Binding PEG to the delivery system reduces interaction between the delivery system and plasma proteins. This prevents plasma proteins from adsorbing to the liposome surface and prevents the delivery system from being taken up by the reticuloendothelial system (RES). PEG binding, or PEGylation, allows the delivery system to circulate in the body for longer periods, extending its circulating half-life and consequently increasing its accumulation within target tissues and tumor cells.

[0090] In one embodiment, the delivery system does not include PEG lipids, PEGylated lipids, or PEG conjugates. In another embodiment, the delivery system includes a polypeptide such as polysarcosine instead of PEG lipids or PEG conjugates. Polysarcosine (pSar) is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine). In certain embodiments, a combination of PEG lipids, PEGylated lipids, or PEG conjugates and polysarcosine is used.

[0091] In embodiments, ionizable lipids and / or cationic lipids may be included in the delivery system to enhance their interaction with negatively charged cargo molecules (e.g., nucleic acids) and facilitate their encapsulation and delivery.

[0092] Suitable cationic lipids may be selected from the group consisting of DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DC-cholesterol (3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol), DORI (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium bromide), DOSPA (2,3-dioleoyloxy-N-(2-(sperminecarboxamide)ethyl)-N,N-dimethyl-1-propanaminonium trifluoroacetate), ICE (imidazole cholesterol ester), DOTMA (1,2-di-O-octadecenyl-3-trimethylammoniumpropane), or any combination thereof.

[0093] However, in preferred embodiments, ionizable lipids may be included in the delivery system instead of cationic lipids.

[0094] In embodiments using ionizable lipids, the total concentration of ionizable lipids and cationic calixarenes in the delivery system is 10 to 60 mol%. In embodiments, the total concentration of ionizable lipids and cationic calixarenes is 10 to 60 mol%, 10 to 55 mol%, 10 to 50 mol%, 10 to 45 mol%, 10 to 40 mol%, 10 to 35 mol%, 10 to 30 mol%, 10 to 25 mol%, 10 to 20 mol%, 10 to 15 mol%, or 15 to 60 mol%, 20 to 60 mol%, 25 to 60 mol%, 30 to 60 mol%, 35 to 60 mol%, or 15 to 55 mol%, 15 to 45 mol%, 15 to 40 mol%, 20 to 35 mol%, 20 to 30 mol%, 20 to 25 mol%, 25 to 30 mol%, and any of these ranges and subranges.

[0095] Depending on the specific application and desired properties, the delivery system may be modified to include a specific composition of calixarenes, phospholipids, additional lipids (preferably sterols), PEGylated lipids, ionizable lipids, and cationic lipids.

[0096] In other embodiments, depending on the specific application and desired properties, the delivery system may be adjusted to include a specific composition of calixarenes, phospholipids, additional lipids (preferably sterols), ionizable lipids, and cationic lipids. The concentrations of each component may be as described above.

[0097] In another embodiment, the delivery system may be adjusted to include a specific composition of calixarenes, phospholipids, additional lipids (preferably sterols), and PEGylated lipids. The concentrations of each component may be as described above.

[0098] In another embodiment, the delivery system may be adjusted to include a specific composition of calixarenes, phospholipids, additional lipids (preferably sterols), cationic lipids, and optionally PEGylated lipids. The concentrations of each component may be as described above.

[0099] In another embodiment, the delivery system may be configured to include a specific composition of one or more calixarenes, phospholipids, additional lipids (preferably sterols), ionizable lipids, and optionally PEGylated lipids.

[0100] In one embodiment, the delivery system comprises at least an ionizable calixarene, a phospholipid, a sterol, and optionally a PEGylated lipid.

[0101] In one embodiment, the delivery system comprises an ionizable calixarene, DOPE, cholesterol, and PEGylated lipids.

[0102] In one embodiment, the delivery system comprises at least a cationic calixarene, a phospholipid, a sterol, an ionizable lipid, and optionally a PEGylated lipid.

[0103] In one embodiment, the delivery system comprises a cationic calixarene, DODAP, DOPE, cholesterol, and PEGylated lipids.

[0104] In a preferred embodiment, the delivery system of the present invention comprises a nucleic acid cargo such as mRNA or saRNA. In one embodiment, the mass of the calixarene is up to 25 times the mass of the nucleic acid cargo.

[0105] (4-component system) As described above, one embodiment of the present invention provides a delivery system that uses ionizable calixarene as a substitute for conventional ionizable lipids in RNA lipid nanoparticles (RNA-LNPs) (4 components: ionizable calixarene, helper lipid, sterol, PEG lipid).

[0106] It should be noted that PEGylated lipids are necessary to obtain stable, non-aggregating monodisperse nanoparticles by ensuring particle shielding and stabilizing the lipid-water interface. Therefore, the mass fraction of PEGylated lipids must be strictly controlled. Too little PEG induces particle aggregation, while too much PEGylated lipids limits transfection ability (see in vivo protein expression discussed in Example 2 below). While the presence of PEG is essential for improving the physicochemical properties of LNPs, its level must be kept to a minimum due to recorded cases of anaphylactic shock caused by PEG-induced hypersensitivity (HR). Indeed, PEG is considered one of the causes of anaphylaxis associated with COVID-19 vaccines, such as the mRNA vaccines from Pfizer BioNTech and Moderna.

[0107] To better understand the structure-activity relationship, a library of ionizable calixarenes was synthesized. Calixarenes with one (CX14, CX16, CX24) or four (CX1, CX2, CX3, CX5, CX6, CX29) ionizable head groups (i-heads) were synthesized (Figure 1). The ionizable head groups were selected from secondary amines (CX6, CX14) and tertiary amines (CX1, CX2, CX3, CX5, CX16, CX24, CX29), cyclic or substituted with methyl or hydroxyethyl groups. Two types of biodegradable groups (amide linkage of CX2 and ester linkage of CX3 / CX29) were also considered to link the ionizable head groups to a macrocyclic core, promoting metabolic degradation of the resulting compounds and avoiding in vivo accumulation. All of these calixarenes formed nanoparticles by self-assembly with helper lipids (phospholipids), sterols, and PEGylated lipids. Several helper lipids (DOPE, DSPC) and PEG lipids (DMG-PEG2000, DSG-PEG2000) were investigated. Examples of nanoparticles prepared with ionizable calixarenes and containing Fluc-mRNA (unless otherwise specified) or other RNA are shown in Table 1 of Example 2.

[0108] The ratios between these components were defined to generate stable, monodisperse nanoparticles. Interestingly, the N / P ratios and molar ratios conventionally used in this field proved unsuitable for this system. In particular, when transitioning from a single-head group to a four-head group calixarene, the mass ratio between components changes significantly due to the higher charge density (number of amines per molecule), and these parameters become extremely important for particle stability.

[0109] The inventors observed that calix[4]arenes having four ionizable head groups (e.g., indicated as the B group in formula I) exhibit particularly advantageous properties for nucleic acid incorporation compared to calix[4]arenes having only one ionizable head group (see examples below).

[0110] Therefore, in a preferred embodiment, the delivery system of the present invention comprises a calix[4]arene having four ionizable head groups.

[0111] The ionizable head group may comprise, for example, a secondary amine (see CX6 or CX14 in Figure 1) or a tertiary amine (see CX1, CX2, CX3, CX5, CX16, CX24, or CX29 in Figure 1). The inventors performed in vitro efficacy assays in cells using LNPs comprising various ionizable calixarenes and found that calixarene compounds having a tertiary amine exhibited higher efficacy than calixarene compounds having a secondary amine (see results in Table 2 of Example 2). Therefore, in a preferred embodiment, one or more of the ionizable head groups of the calixarene comprise one or more tertiary amines.

[0112] As described above, the ionizable head groups of the calixarene may be different or identical. In one embodiment, the calixarene comprises four identical ionizable head groups.

[0113] The inventors discovered that a minimum mass ratio of [calixarene + lipid] / RNA is required to obtain stable and monodisperse particles in LNPs and to optimize in vivo protein expression of RNA encapsulated within LNPs after administration (see Example 2). This minimum ratio is approximately 20 for single-headed calixarenes and can be reduced to approximately 10 for four-headed calixarenes. This interesting result indicates that less material is needed to achieve encapsulation efficiency and monodispersity comparable to conventionally used ionizable lipids (e.g., SM-102). This difference may be due to the increased charge density of four-headed ionizable calixarenes. An in vitro efficacy assay of LNPs containing ionizable calixarenes was also performed in Jurkat cells using Fluc mRNA as a reporter gene. The results (see Table 2 in Example 2) further supported the superiority of four-headed calixarenes over single-headed calixarenes.

[0114] Similarly, in vivo protein expression at 3, 6, and 9 hours after intramuscular injection of 1 μg of Fluc mRNA encapsulated in LNPs containing one-headed or four-headed calixarenes was also examined (see Example 2, Figure 3). Overall, this experiment demonstrated that the dynamics of protein expression differ between one-headed and four-headed calixarenes. The signal for CX16 (one-headed) decreased rapidly, while protein expression with four-headed calixarenes (CX1, CX2, CX3, CX5) remained stable over different time points (3–9 hours post-injection). This result again demonstrates that four-headed calixarenes are superior to one-headed calixarenes in achieving stable and high protein expression. This observation is further supported by the fact that less four-headed calixarene is needed to achieve comparable performance (approximately 5 μg of four-headed CX / μg RNA vs. approximately 10 μg of one-headed CX / μg RNA).

[0115] [Calyxarene + Lipid] refers to the total mass of the calixarene component and lipid component in the LNP. In one embodiment, this mass is equal to the total mass of the calixarene component, helper lipid component, sterol component, and PEGylated lipid component.

[0116] In one embodiment, when using a calixarene having one ionizable head group (such as CX14, CX16, CX24 shown in Figure 1), the mass ratio is less than 50, more preferably less than 40, even more preferably less than 30, and more preferably less than 29, 28, 27, 26, 25, 24, 23, 22, or 21, for example, 20. In this case, the total mass of the calixarene component and the lipid component can be reduced to as little as 20 times the mass of the RNA cargo.

[0117] In one embodiment, when using calixarenes having four ionizable head groups (such as CX1, CX2, CX3, CX5, CX6, CX29 shown in Figure 1), the mass ratio is less than 50, more preferably less than 40, even more preferably less than 30, even more preferably 20, and even more preferably less than 19, 18, 17, 16, 15, 14, 13, 12, or 11, for example, 10. In this case, the total mass of the calixarene component and the lipid component can be reduced to as little as 10 times the mass of the RNA cargo.

[0118] This intriguing result indicates that less material is required to achieve encapsulation efficiency and monodispersity comparable to conventionally used ionizable lipids (e.g., SM-102). This difference may be due to an increase in the charge density of the four-headed ionizable calixarene.

[0119] For helper lipids, sterols, and PEGylated lipids, their mass fraction is maintained even when they transition from a single-heading group to a four-heading group system. The mass fractions of these lipids (expressed as a percentage, with the total mass of the delivery system excluding cargo and other excipients being 100%) are preferably as follows. Ionizable calixarenes: 10-60% Helper lipids: 5-35% Sterols: 15-50% PEGylated lipids: 4-24%

[0120] Therefore, in one embodiment, when ionizable calixarene is used as a substitute for conventional ionizable lipids in RNA lipid nanoparticles (RNA-LNP) (4 components: ionizable calixarene, helper lipid, sterol, PEG lipid), the mass of the ionizable calixarene is 10-60% of the total mass of the delivery system.

[0121] As described above, when referring to the mass fraction of calixarene or lipid components relative to the total weight of the formulation, the weight of the calixarene or lipid component refers to the total weight of calixarene and lipids present in the delivery system (and therefore excluding cargo (e.g., RNA) and other excipients (e.g., sucrose, TRIS, etc.)). Thus, the mass fraction refers to the mass of lipids or calixarene relative to the total mass of "lipids + calixarene". Mass is a measure of the amount of substance contained in an object.

[0122] In one embodiment of the four-component system, the ionizable calixarene is present in the delivery system in a mass fraction of 10-60% (w / w), for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 3%. The ionizable calixarenes are present in the delivery system at a mass fraction of 1%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% (w / w) or any value in between. In one embodiment, the ionizable calixarenes are present in the delivery system at a mass fraction of 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, or 55-60% (w / w). In one embodiment, the ionizable calixarene is present in the delivery system at a mass fraction of 10-20%, 20-30%, 30-40%, 40-50%, or 50-60% (w / w).

[0123] In one embodiment, when RNA lipid nanoparticles (RNA-LNPs) (four components: ionizable calixarenes, helper lipids, sterols, and PEG lipids) are prepared using ionizable calixarenes as a substitute for conventional ionizable lipids, the mass of the phospholipids is 5-35%, more preferably 10-30%, of the total mass of the delivery system.

[0124] In one embodiment of the four-component system, the phospholipid is present in the delivery system in a mass fraction of 5-35% (w / w), more preferably 10-30% (w / w), for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% (w / w) or any value in between.

[0125] In one embodiment, when RNA lipid nanoparticles (RNA-LNPs) (four components: ionizable calixarene, helper lipid, sterol, and PEG lipid) are prepared using ionizable calixarene as a substitute for conventional ionizable lipids, the mass of the sterol is 15-50% of the total mass of the delivery system.

[0126] In one embodiment of the four-component system, the sterol is present in the delivery system in a mass fraction of 15-50% (w / w), for example, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% (w / w) or any value in between.

[0127] In one embodiment, when RNA lipid nanoparticles (RNA-LNPs) (four components: ionizable calixarene, helper lipid, sterol, and PEG lipid) are prepared using ionizable calixarene as a substitute for conventional ionizable lipids, the mass of the PEGylated lipid is 2-24%, more preferably 10-24%, of the total mass of the delivery system.

[0128] In one embodiment of the four-component system, the PEGylated lipid is present in the delivery system in a mass fraction of 2-24% (w / w), more preferably 10-24% (w / w), for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, or 24% (w / w), or any value in between.

[0129] The inventors investigated the optimal ratio of calixarene mass to cargo mass and conducted further experiments to optimize encapsulation efficiency (see Example 2).

[0130] In a preferred embodiment, the delivery system of the present invention comprises a nucleic acid cargo such as mRNA or saRNA.

[0131] The inventors found an optimized mass ratio (calixarene / saRNA) (see Example 2), which enabled them to obtain stable nanoparticles with high encapsulation efficiency (over 80%) (see Figure 4) and sufficient protein expression in vivo (see Figure 5).

[0132] In one embodiment, the delivery system comprises an ionizable calixarene, a phospholipid, a sterol, and a PEGylated lipid, and if the cargo comprises a nucleic acid, the mass of the ionizable calixarene is equal to or greater than the mass of the cargo.

[0133] In one embodiment, the delivery system comprises an ionizable calixarene, a phospholipid, a sterol, and a PEGylated lipid, and when the cargo comprises a nucleic acid, the mass of the ionizable calixarene is up to 15 times the mass of the nucleic acid cargo.

[0134] In one embodiment, a delivery system comprising a nucleic acid cargo comprising an ionizable calixarene, a phospholipid, a sterol, and a PEGylated lipid, wherein the mass of the ionizable calixarene is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times the mass of the nucleic acid cargo.

[0135] In one embodiment, a delivery system comprising a nucleic acid cargo comprising an ionizable calixarene, a phospholipid, a sterol, and a PEGylated lipid, wherein the mass of the ionizable calixarene is 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 times the mass of the nucleic acid cargo.

[0136] As described above, saRNA (approximately 10,000 base pairs) is larger than mRNA (approximately 1,000 base pairs) and has more secondary structures, making encapsulation and delivery more difficult. Essential chemical and structural differences in length, stability, and charge density between mRNA and saRNA suggest that LNP delivery formulations using saRNA may require significantly different conditions than those developed for mRNA delivery.

[0137] In a preferred embodiment, the delivery system comprises an ionizable calixarene, a phospholipid, a sterol, and a PEGylated lipid, and a saRNA cargo, wherein the mass of the ionizable calixarene is up to 15 times, more preferably up to 10 times, the mass of the saRNA cargo.

[0138] In one embodiment, the delivery system comprises an ionizable calixarene, a phospholipid, a sterol, and a PEGylated lipid, and further comprises a saRNA cargo, wherein the mass of the ionizable calixarene is 5 to 15 times the mass of the saRNA cargo.

[0139] In one embodiment, the delivery system comprises an ionizable calixarene, a phospholipid, a sterol, and a PEGylated lipid, and a saRNA cargo, wherein the mass of the ionizable calixarene is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times the mass of the saRNA cargo.

[0140] In one embodiment, the delivery system comprises an ionizable calixarene, a phospholipid, a sterol, and a PEGylated lipid, and a saRNA cargo, wherein the mass of the ionizable calixarene is 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 times the mass of the saRNA cargo.

[0141] It is noteworthy that this optimized mass ratio is smaller than the ratio (approximately 20 μg i-lipid / μgRNA) required to achieve similar properties (encapsulation efficiency and in vitro protein expression) with saRNA using ionizable lipids well known in the field (SM-102, ALC-0315).

[0142] The inventors further preclinically validated the delivery system of the present invention, comprising ionizable calixarene (CX5), phospholipids, sterols, and PEGylated lipids. Mice were vaccinated following a 21-day prime-boost regimen using either 0.6 μg or 2.5 μg of mRNA per single dose. As shown in Figure 6, the delivery system demonstrated strong VNT (viral neutralizing antibody titer) well above the protective correlation value (0.5 IU / mL) with both dosing regimens (0.6 or 2.5 μg). These results were obtained without any adverse events observed in the animals (weight loss, effects on spleen, liver, or kidney weight, inflammation, or excessive reactiongenic response).

[0143] (5-component system) In another embodiment, the present invention provides a delivery system in which a cationic calixarene is incorporated as a fifth component into lipid nanoparticles (LNPs) made from ionizable lipids, helper lipids, sterols, and PEG lipids (5 components).

[0144] In a preferred embodiment, the cationic calixarene is a calix[4]arene.

[0145] As described above, and also referring to formula (I), the calix[4]arene used in the present invention may comprise four identical B groups. In another embodiment, the calix[4]arene used in the present invention may comprise three identical B groups and one different B group. In yet another embodiment, the calix[4]arene used in the present invention may comprise two identical B groups and two other identical B groups. The same applies to the A groups.

[0146] In one embodiment of the five-component system, the calixarene is a calix[4]arene having four cationic head groups. In yet another embodiment, the calixarene comprises four identical cationic head groups.

[0147] In one embodiment, one or more of the cationic head groups comprise at least one quaternary amine group. In one embodiment, each of the cationic head groups comprises at least one quaternary amine group. In one embodiment, one or more of the cationic head groups comprise multiple quaternary amine groups.

[0148] The inventors demonstrated that these cationic calixarenes can be incorporated into lipid nanoparticles that encapsulate RNA (see Table 3). These are prepared from the following components. • Ionizable lipids: e.g., DODAP, DLin-DMA, DLin-MC3-DMA, ALC-0315, SM-102; • Helper lipids: e.g., DOPE, DOPC; ·Sterols: For example, cholesterol; • PEG lipids: For example, DMG-PEG2000, DSG-PEG2000.

[0149] Therefore, in one embodiment, when a cationic calixarene is incorporated as a fifth component into lipid nanoparticles (LNPs) made from ionizable lipids, helper lipids, sterols, and PEG lipids (5 components), the mass of the cationic calixarene is 0.1 to 60% (w / w), preferably 0.1 to 50% (w / w), of the total mass of the delivery system.

[0150] In one embodiment of the five-component system, the cationic calixarene is present in the delivery system at a mass fraction of 0.1-10% (w / w), 10-20% (w / w), 20-30% (w / w), 30-40% (w / w), 40-50% (w / w), or 50-60% (w / w).

[0151] In one embodiment of the five-component system, the cationic calixarene is present in the delivery system at a mass fraction of 0.1-50% (w / w), 0.1-40%, 0.1-30%, 0.1-20%, or 0.1-10% (w / w).

[0152] In one embodiment of the five-component system, the cationic calixarene is present in the delivery system at a mass fraction of 0.2 to 10% (w / w), for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% (w / w), or any value in between. In one embodiment, the cationic calixarene is present in the delivery system at a mass fraction of 0.1 to 10%, 10 to 20%, 20 to 30%, 30 to 40%, or 40 to 50% (w / w). In one embodiment, the cationic calixarene is present in the delivery system in mass fractions of 0.1-1%, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 10-11%, 11-12%, 12-13%, 13-14%, or 14-15% (w / w).

[0153] In one embodiment, when a cationic calixarene is incorporated as a fifth component into lipid nanoparticles (LNPs) made from ionizable lipids, helper lipids, sterols, and PEG lipids (5 components), the mass of the phospholipid is 5 to 35%, more preferably 10 to 30%, of the total mass of the delivery system.

[0154] In one embodiment of the five-component system, the phospholipid is present in the delivery system in a mass fraction of 5-35% (w / w), more preferably 10-30% (w / w), for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% (w / w) or any value in between.

[0155] In one embodiment, when a cationic calixarene is incorporated as a fifth component into lipid nanoparticles (LNPs) made from ionizable lipids, helper lipids, sterols, and PEG lipids (5 components), the mass of the sterol is 15-50% of the total mass of the delivery system.

[0156] In one embodiment of the five-component system, the sterol is present in the delivery system in a mass fraction of 15-50% (w / w), for example, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% (w / w) or any value in between.

[0157] In one embodiment, when a cationic calixarene is incorporated as a fifth component into lipid nanoparticles (LNPs) made from ionizable lipids, helper lipids, sterols, and PEG lipids (5 components), the mass of the PEGylated lipid is 2-24%, more preferably 10-24%, of the total mass of the delivery system.

[0158] In one embodiment of the five-component system, the PEGylated lipid is present in the delivery system in a mass fraction of 2-24% (w / w), more preferably 10-24% (w / w), for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, or 24% (w / w), or any value in between.

[0159] In one embodiment, when a cationic calixarene is incorporated as a fifth component into lipid nanoparticles (LNPs) made from ionizable lipids, helper lipids, sterols, and PEG lipids (5 components), the mass of the ionizable lipids is 15-50% of the total mass of the delivery system.

[0160] In one embodiment of the five-component system, the ionizable lipid is present in the delivery system in a mass fraction of 15-50% (w / w), for example, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% (w / w) or any value in between.

[0161] The inventors further demonstrated that LNPs containing 0.1 to 20.7 times more cationic calixarenes than mRNA (by mass ratio) were all monodisperse and encapsulated more than 95% of the RNA. It was found that the addition of cationic calixarenes increased both the size (77 to 129 nm) and the charge (5.8 to 11.0 mV) (see Table 4 and Figure 9, Example 3).

[0162] The inventors further demonstrated that potent LNPs can be obtained by combining cationic calixarenes with DLin-DMA as an ionizable lipid (Example 3). These results also suggest that the selection of PEGylated lipids affects the immune response. Specifically, simply increasing the number of carbon atoms in the two alkyl chains of the PEGylated lipid by four can significantly reduce the immune activity of LNPs under otherwise identical conditions (see Figure 10, Example 3).

[0163] The inventors further demonstrated that the five-component system can be used for efficient encapsulation of saRNA. The resulting LNPs were monodisperse, with a Z-mean of 120 nm (Figure 11A), and 76.6% of the saRNA was encapsulated. This result is particularly interesting because encapsulation of saRNA is difficult with common ionizable lipids. This was demonstrated using saRNA of similar length that encodes an unpublished target gene. The saRNA was prepared in the ratio SM-102:DSPC:cholesterol:DMG-PEG2000 (50:10:38.5:1.5 molar ratio). The resulting LNPs were polydisperse (Figure 11B).

[0164] According to the present invention, buffers and solvents can be further included in the delivery system, or their production can be assisted. For example, in the production of the delivery system, ethanol is preferred as the solvent for the lipid phase, and an acidic aqueous buffer is preferred as the solvent for the aqueous phase.

[0165] In the embodiment, preferred buffers are citrate buffers and acetate buffers with varying pH and concentration. In a specific embodiment, the pH of the buffer is 3 to 6 and the concentration is 1 to 100 mM.

[0166] In the embodiments, preferred buffers are citrate buffers and acetate buffers with varying pH and concentration. In certain embodiments, the pH of the buffer is 3 to 5.5 and the concentration is 10 to 50 mM. However, the buffers are not limited to those exemplified above, and various buffers known in the art for use in forming delivery systems may be used, as will be apparent to those skilled in the art. A preferred method of preparation, though not limited, is microfluidic mixing.

[0167] The (average) diameter of the delivery system can be quantified by any method known in the prior art, such as quasi-electric light scattering (QELS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and imaging methods (e.g., scanning electron microscopy (SEM), transmission electron microscopy (TEM), cryo-TEM). In one embodiment, DLS can measure the average particle size and polydispersity index (PDI, an index of particle size distribution), and NTA can measure the mean, mode, and span of the particle population.

[0168] In one embodiment, the delivery system comprises a monodisperse population. Therefore, in a preferred embodiment, the PDI of the delivery system is less than 0.25, more preferably less than 0.2.

[0169] Figure 7 shows cryo-TEM imaging of LNPs comprising the delivery system according to the present invention, more specifically, ionizable calixarenes, phospholipids, sterols, and PEGylated lipids (CX5:DOPE:cholesterol:DMG-PEG2000 nanoparticles encapsulating mRNA-FLuc). The imaged LNPs are monodisperse, with the minimum and maximum LNPs being approximately 40 nm and 80 nm, respectively, which is in good agreement with DLS measurements (Z mean approximately 50 nm).

[0170] In one embodiment, the average size and PDI of the delivery system of this disclosure can be estimated using advanced mathematical analysis (e.g., CUMULANT analysis). In addition to affecting the in vivo distribution, the diameter also affects the carrying capacity of the delivery system.

[0171] In one embodiment, the average diameter of the delivery system is in the range of 10 nm to 10,000 nm, more preferably 10 nm to 9,000 nm, even more preferably 10 nm to 8,000 nm, even more preferably 10 nm to 7,000 nm, even more preferably 10 nm to 6,000 nm, even more preferably 10 nm to 5,000 nm, even more preferably 10 nm to 4,000 nm, even more preferably 10 nm to 3,000 nm, even more preferably 10 nm to 2,000 nm, and even more preferably the average diameter of the delivery system is in the range of 10 nm to 1,000 nm, more preferably 50 nm to 200 nm. In one embodiment, the average diameter of the delivery system is at least 10 nm, preferably at least 20 nm, even more preferably at least 25 nm, even more preferably at least 30 nm, even more preferably at least 35 nm, even more preferably at least 40 nm, even more preferably at least 45 nm, and even more preferably at least 50 nm. In one embodiment, the average diameter of the delivery system is at most 900 nm, more preferably at most 800 nm, more preferably at most 700 nm, more preferably at most 600 nm, more preferably at most 500 nm, more preferably at most 400 nm, more preferably at most 300 nm, and more preferably at most 200 nm.

[0172] In one embodiment, the average diameter of the delivery system is 10nm~200nm, 10nm~190nm, 10nm~180nm, 10nm~170nm, 10nm~160nm, 10nm~150nm, 10nm~140nm, 10nm~130nm, 10nm~120nm, 10nm~110nm, 10nm~100nm, 10nm~90nm, 10nm~80nm, 10nm~70nm, 10nm~60nm, 10nm~50nm, 10nm~40nm, 10nm~30nm, 10nm~20nm, 20nm~200nm, 20nm~190nm, and 20nm~180nm. , 20nm~170nm, 20nm~160nm, 20nm~150nm, 20nm~140nm, 20nm~130nm, 20nm~1 20nm, 20nm~110nm, 20nm~100nm, 20nm~90nm, 20nm~80nm, 20nm~70nm, 20nm~6 0nm, 20nm~50nm, 20nm~40nm, 20nm~30nm, 30nm~200nm, 30nm~190nm, 30nm~1 80nm, 30nm~170nm, 30nm~160nm, 30nm~150nm, 30nm~140nm, 30nm~130nm, 30nm m~120nm, 30nm~110nm, 30nm~100nm, 30nm~90nm, 30nm~80nm, 30nm~70nm, 30 nm~60nm, 30nm~50nm, 30nm~40nm, 40nm~200nm, 40nm~190nm, 40nm~180nm, 4 0nm~170nm, 40nm~160nm, 40nm~150nm, 40nm~140nm, 40nm~130nm, 40nm~120 nm, 40nm~110nm, 40nm~100nm, 40nm~90nm, 40nm~80nm, 40nm~70nm, 40nm~60n m, 40nm~50nm, 50nm~200nm, 50nm~190nm, 50nm~180nm, 50nm~170nm, 50nm~1 60nm, 50nm~150nm, 50nm~140nm, 50nm~130nm, 50nm~120nm, 50nm~110nm, 50nm m~100nm, 50nm~90nm, 50nm~80nm, 50nm~70nm, 50nm~60nm, 60nm~200nm, 60n m~190nm, 60nm~180nm, 60nm~170nm, 60nm~160nm, 60nm~150nm, 60nm~140nm,60nm~130nm、60nm~120nm、60nm~110nm、60nm~100nm、60nm~90nm、60nm~80nm、60nm~70nm、70nm~200nm、70nm~190nm、70nm~180nm、70nm~170nm、70nm~160nm、70nm~150nm、70nm~140nm、70nm~130nm、70nm~120nm、70nm~110nm、70nm~100nm、70nm~90nm、70nm~80nm、80nm~200nm、80nm~190nm、80nm~180nm、80nm~170nm、80nm~160nm、80nm~150nm、80nm~140nm、80nm~130nm、80nm~120nm、80nm~110nm、80nm~100nm、80nm~90nm、90nm~200nm、90nm~190nm、90nm~180nm、90nm~170nm、90nm~160nm、90nm~150nm、90nm~140nm、90nm~130nm、90nm~120nm、90nm~110nm、90nm~100nm、100nm~200nm、100nm~190nm、100nm~180nm、100nm~170nm、100nm~160nm、100nm~150nm、100nm~140nm、100nm~130nm、100nm~120nm、100nm~110nm、110nm~200nm、110nm~190nm、110nm~180nm、110nm~170nm、110nm~160nm、110nm~150nm、110nm~140nm、110nm~130nm、110nm~120nm、120nm~200nm、120nm~190nm、120nm~180nm、120nm~170nm、120nm~160nm、120nm~150nm、120nm~140nm、120nm~130nm、130nm~200nm、130nm~190nm、130nm~180nm、130nm~170nm、130nm~160nm、130nm~150nm、130nm~140nm、140nm~200nm、140nm~190nm、140nm~180nm、140nm~170nm、140nm~160nm、140nm~150nm、150nm~200nm、150nm~190nm、150nm~180nm、150nm~170nm、The ranges are 150nm-160nm, 160nm-200nm, 160nm-190nm, 160nm-180nm, 160nm-170nm, 170nm-200nm, 170nm-190nm, 170nm-180nm, 170nm-180nm, 180nm-190nm, and 190nm-200nm.

[0173] In one embodiment, the average diameter of the delivery system is 200nm~1,000nm, 200nm~950nm, 200nm~900nm, 200nm~850nm, 200nm~800nm, 200nm~750nm, 200nm~700nm, 200nm~650nm, 200nm~600nm, 200nm~550nm, 200nm~500nm, 200nm~450nm, 200nm~400nm, 200nm~350nm, 200nm~300nm, 200nm~250nm, 250nm~1, 000nm, 250nm~950nm, 250nm~900nm, 250nm~850nm, 250nm~800nm, 250nm~750nm, 250nm~700nm, 250nm~650nm, 250nm~600nm, 250nm~5 50nm, 250nm~500nm, 250nm~450nm, 250nm~400nm, 250nm~350nm, 250nm~300nm, 300nm~1,000nm, 300nm~950nm, 300nm~900nm, 300nm~8 50nm, 300nm~800nm, 300nm~750nm, 300nm~700nm, 300nm~650nm, 300nm~600nm, 300nm~550nm, 300nm~500nm, 300nm~450nm, 300nm~40 0nm, 300nm~350nm, 350nm~1,000nm, 350nm~950nm, 350nm~900nm, 350nm~850nm, 350nm~800nm, 350nm~750nm, 350nm~700nm, 350nm~6 50nm, 350nm~600nm, 350nm~550nm, 350nm~500nm, 350nm~450nm, 350nm~400nm, 400nm~1,000nm, 400nm~950nm, 400nm~900nm, 400nm~ 850nm, 400nm~800nm, 400nm~750nm, 400nm~700nm, 400nm~650nm, 400nm~600nm, 400nm~550nm, 400nm~500nm, 400nm~450nm, 450nm~1,000nm, 450nm~950nm, 450nm~900nm, 450nm~850nm, 450nm~800nm, 450nm~750nm, 450nm~700nm, 450nm~650nm, 450nm~60 0nm, 450nm~550nm, 450nm~500nm, 500nm~1,000nm, 500nm~950nm, 500nm~900nm, 500nm~850nm, 500nm~800nm, 500nm~75 0nm, 500nm~700nm, 500nm~650nm, 500nm~600nm, 500nm~550nm, 550nm~1,000nm, 550nm~950nm, 550nm~900nm, 550nm~85 0nm, 550nm~800nm, 550nm~750nm, 550nm~700nm, 550nm~650nm, 550nm~600nm, 600nm~1,000nm, 600nm~950nm, 600nm~90 0nm, 600nm~850nm, 600nm~800nm, 600nm~750nm, 600nm~700nm, 600nm~650nm, 650nm~1,000nm, 650nm~950nm, 650nm~90 0nm, 650nm~850nm, 650nm~800nm, 650nm~750nm, 650nm~700nm, 700nm~1,000nm, 700nm~950nm, 700nm~900nm, 700nm~85 The range is 0nm, 700nm~800nm, 700nm~750nm, 750nm~1,000nm, 750nm~950nm, 750nm~900nm, 750nm~850nm, 750nm~800nm, 800nm~1,000nm, 800nm~950nm, 800nm~900nm, 800nm~850nm, 850nm~950nm, 850nm~900nm, 900nm~950nm, and 950nm~1,000nm.

[0174] Cargoes delivered using calixarene-based delivery systems may be selected from the group consisting of nucleic acids, proteins, chemicals, polysaccharides, and combinations thereof. Preferably, the cargo is a nucleic acid, more preferably RNA or DNA. Cargoes may include therapeutic agents such as gene therapies, siRNA, mRNA, CRISPR / Cas9 components, proteins, enzymes, antibodies, small molecules, or other chemicals, as well as imaging agents or contrast agents for diagnostic applications. Cargoes may be encapsulated within the delivery system or associated with the delivery system via electrostatic interactions, covalent bonds, or other binding means.

[0175] The cargo may be encapsulated within the delivery system or associated with the delivery system via ion pairing interactions, covalent bonds, or other binding means.

[0176] In some embodiments, the cargo is selected from long RNA, coding RNA, non-coding RNA, long non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), trans-amplified mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNA (gRNA), riboswitches, immunostimulatory RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, nuclear small RNA (snRNA), nucleolar small RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

[0177] In one embodiment, the cargo is microRNA, saRNA, circRNA, or mRNA.

[0178] In some embodiments, the cargo comprises a modified RNA molecule. In some embodiments, the modification of the RNA molecule comprises a chemical modification comprising a skeletal modification, a sugar modification, or a base modification. In this context, the modified RNA molecule as defined herein comprises a nucleotide analog / modification, e.g., a skeletal modification, a sugar modification, or a base modification. Skeletal modification as used in this disclosure is a modification in which the phosphate of the nucleotide backbone contained in the RNA molecule is chemically modified. Sugar modification as used in this disclosure is a chemical modification of the sugar of the nucleotide in the RNA molecule. Furthermore, base modification as used in this disclosure is a chemical modification of the base portion of the nucleotide in the RNA molecule. In this context, the nucleotide analog or modification is selected from nucleotide analogs applicable to transcription and / or translation. In further embodiments, the modified RNA may be 6-azacytidine, 2-thiocytidine, α-thiocytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methylpsoidouridine, 5,6-dihydrouridine, α-thiouridine, 4-thiouridine, 6-azauridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolocytidine, inosine, It features nucleoside modifications selected from α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, and 7-deaza-adenosine.

[0179] In one embodiment, the cargo is self-amplifying RNA (saRNA).

[0180] The delivery system has also been found to be suitable for use in combination with large-sized RNAs, such as self-amplifying RNA (saRNA). Because saRNA is larger than conventional mRNA, lipid nanoparticles described in conventional techniques often do not function properly, resulting in insufficient encapsulation and unoptimal in vivo delivery. The delivery system defined herein can solve this problem.

[0181] Preferably, the size of the (sa)RNA may be in the range of 500 to 50,000 nucleotides (Nt), more preferably 1,000 to 40,000 Nt, even more preferably 5,000 to 30,000 Nt, or 8,000 to 16,000 Nt.

[0182] The self-replicating nature of mRNA constructs is based on the genomic RNA of an RNA virus but lacks one or more genes encoding structural proteins. Self-replicating RNA molecules are translatable to produce non-structural proteins of RNA viruses and heterologous proteins encoded by the self-replicating RNA. Self-replicating RNA molecules are designed not to induce the production of infectious viral particles. One example of a suitable system for achieving self-replication is the use of alphavirus-based RNA replicons. These positive-strand replicons are translated after delivery to cells, yielding replicases (or replicase-transcriptases). The replicases are translated as polyproteins, which are automatically cleaved to provide a replication complex, which creates a genomic minus-strand copy of the positive-stranded RNA. These minus-strand transcripts can themselves transcribe further copies of the positive-strand parental RNA and also produce subgenomic transcripts encoding the desired gene product. Translation of these subgenomic transcripts then leads to in situ expression of the desired gene product by the cell. Suitable alphavirus replicacons may include replicases of cymbosvirus, semliki forest virus, eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Preferred self-replicating RNA molecules include (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule and (ii) encoding the target protein / peptide. The polymerase may be an alphavirus replicase comprising, for example, the alphavirus protein nsP4.

[0183] In embodiments, a pharmaceutical composition comprising a delivery system may be stabilized in a lyophilized or dried form. “Lyophilization” as used herein refers to the lyophilization of a liquid or pre-lyophilized formulation. Lyophilization is carried out by freezing the formulation and then sublimating the ice from the frozen contents at a suitable primary drying temperature. Under these conditions, the product temperature is below the disintegration temperature of the formulation. A secondary drying step may then be performed to produce a suitable lyophilized cake. Lyophilization is commonly used in the manufacture of pharmaceutical compounds because it enhances the stability of the active ingredient (API) by removing the solvent. Lyophilization has many advantages, including enabling the processing and development of pharmaceutical compounds that are unstable in solution and thus improving shelf life. This technique can accelerate the development, use, distribution, and commercialization of new drugs.

[0184] (Instructions for use and treatment) The present invention also provides a method for delivering one or more cargoes to a subject using a calixarene-based delivery system. The method comprises the following steps: Preparation of the delivery system: Combining delivery system components, including calixarenes, phospholipids, additional lipids such as sterols, optionally PEGylated lipids, and optionally ionizable lipids, to formulate a delivery system. The cargoes to be delivered are incorporated into the delivery system by encapsulation, association by ion pairing interactions, or other binding means. The cargoes may be incorporated during or after the combination of the delivery system components. In the following, "delivery system" refers to a delivery system containing cargoes.

[0185] Administration of Delivery Systems: The prepared delivery systems are administered to subjects, such as patients, who require therapeutic effects or diagnostic information on the cargo. Administration may be carried out intravenously, intramuscularly, subcutaneously, orally, by inhalation, or by other appropriate routes, depending on the target tissue, cargo, and desired therapeutic or diagnostic outcome. The systems detailed herein may be formulations suitable for any available route of administration, including oral, mucosal (e.g., nasal cavity, sublingual, vaginal, buccal mucosa, rectal), parenteral (e.g., intra-articular, intravenous, intraperitoneal, intramuscular, intradermal, or subcutaneous injection), topical, or transdermal delivery forms. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injections or suspensions, which may contain antioxidants, buffers, preservatives, and solutes to be isotonic with the recipient's blood. Aqueous and non-aqueous sterile suspensions may also contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In carrying out the present invention, the composition is preferably administered, for example, by intravenous drip, oral, topical, intraperitoneal, intravesical, or intrathecal infusion. While not limited to, a TRIS buffer containing sucrose is preferred as the buffer for injection.

[0186] The delivery system can be prepared in unit dose form. In some embodiments, the unit dose may have a volume of about 0.1 to 1.0 ml, for example, about 0.5 ml. The pharmaceutical composition or vaccine may be provided in sealed containers of unit dose or multiple doses, such as ampoules or vials. The injectable solution and suspension may be prepared from sterile powder, granules, or tablets. Cells transduced with the pharmaceutical composition of the present invention may also be administered intravenously or parenterally.

[0187] The delivery system can be administered as a single dose or multiple doses. In the case of multiple doses, two or more doses are administered within a specified period. This period can be any of the following: 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or up to 1 year.

[0188] Cargo delivery to target cells: When a delivery system is administered, it may facilitate the delivery of cargo to target cells. Calixarene-based delivery systems may facilitate intracellular uptake, endosomal extrusion, and release of cargo into the cytoplasm or other intracellular compartments, depending on the cargo and target cells.

[0189] Evaluation of therapeutic or diagnostic effects: After the cargo is delivered to target cells, therapeutic or diagnostic effects can be evaluated. This may include monitoring changes in gene expression, protein levels, cellular function, disease progression, and other related outcomes. For diagnostic applications, imaging and other techniques can be used to visualize the distribution and effects of the cargo.

[0190] Furthermore, the present invention also relates to a (pharmaceutical) composition comprising a delivery system having the cargo described above. Furthermore, the present invention also relates to a method for treating a disease or disorder using the (pharmaceutical) composition. In some embodiments, the composition is used as a human or veterinary drug.

[0191] In one embodiment, the pharmaceutical composition is used in gene therapy. Gene therapy may include protein replacement strategies (e.g., by DNA or mRNA delivery) or gene silencing (e.g., by siRNA or miRNA delivery). In a preferred embodiment, the pharmaceutical composition may be a vaccine or used for immunizing or vaccinating a subject.

[0192] In one embodiment, the disclosure provides an RNA (e.g., mRNA) vaccine comprising at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one antigenic polypeptide or its immunogenic fragment (e.g., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide). While not bound by theory, RNA (e.g., mRNA) vaccines, e.g., mRNA polynucleotides, are designed to produce appropriate protein conformations during translation, and RNA (e.g., mRNA) vaccines utilize the natural mechanisms of cells. Conventional vaccines are manufactured ex vivo and can evoke undesirable cellular responses, whereas RNA (e.g., mRNA) vaccines are presented to cell lines in a more natural manner.

[0193] The pharmaceutical composition comprises an immunologically effective amount of polynucleotides and, optionally, other components. “Immunologically effective amount” means that the amount is effective for treatment or prevention when administered to a subject as a single dose or as part of a series of doses. This amount may vary depending on the subject's health status and physical condition, age, taxonomy (e.g., non-human primates, primates, etc.), the subject's immune system’s antibody production capacity, the desired level of protection, the vaccine formulation, the physician’s medical judgment, and other relevant factors. The amount is expected to fall within a relatively wide range that can be determined by standard testing. The RNA content of the pharmaceutical compositions described herein is generally expressed as the amount of RNA per dose. In one embodiment, the dose contains approximately 100 μg or less of RNA (e.g., 10-100 μg, for example, about 10 μg, 25 μg, 50 μg, 75 μg, or 100 μg), but expression may also be observed at much lower levels, for example, 1 μg / dose or less, 100 ng / dose or less, 10 ng / dose or less, or 1 ng / dose or less.

[0194] In preferred embodiments, the polynucleotide in the pharmaceutical composition encodes an antigen, preferably an antigen associated with an infectious disease or infectious agent.

[0195] In certain embodiments, the antigen is a target-specific antigen and may be a tumor antigen, bacterial antigen, viral antigen, or fungal antigen. The target-specific antigen may originate from whole mRNA isolated from target cells, one or more specific target mRNA molecules, a protein resaturation from target cells, a specific protein from target cells, or a target-specific peptide or synthetic mRNA or DNA encoding a protein and a target-specific antigen or its derivative peptide. To avoid misunderstanding, the pharmaceutical compositions disclosed herein may comprise a single mRNA molecule or a combination of one or more mRNA molecules encoding an immunomodulatory protein and / or one or more mRNA molecules encoding an antigen-specific or disease-specific protein.

[0196] In one embodiment, the polynucleotide encodes an immunogen. The immunogen can induce an immune response to bacteria, viruses, fungi, parasites, allergens, or tumor antigens. The immune response may comprise an antibody response (usually including IgG) and / or a cellular immune response. The immunogen may be, for example, a surface polypeptide (e.g., adhesin, hemagglutinin, envelope glycoprotein, spike glycoprotein, etc.), an internal protein (e.g., a nucleoprotein), or a combination of either.

[0197] In some cases, immunogens induce an immune response against one of the following bacteria: • Neisseria meningitidis (Meningococcus): Useful immunogens include, but are not limited to, adhesion molecules, autotransporters, toxins, and membrane proteins such as iron-acquiring proteins and factor H-binding proteins. • Streptococcus pneumoniae: Useful immunogens include, but are not limited to, the RrgB pyrus subunit, β-N-acetylhexosaminidase precursor (spr0057), spr0096, general stress protein GSP-781 (spr2021, SP2216), serine / threonine kinase StkP (SP1732), and pneumococcal surface adhesion factor PsaA. Streptococcus pyogenes (Streptococcus pyogenes). Moraxella catarrhalis. • Bordetella pertussis (Pertussis bacterium): Useful pertussis immunogens include, but are not limited to, pertussis toxin or toxoid (PT), filamentous hemagglutinin (FHA), partactin, and agglutinogens 2 and 3. • Staphylococcus aureus: Useful immunogens include, but are not limited to, hemolysin, esxA, esxB, ferrichrome-binding protein (sta006) and / or sta011 lipoprotein. • Clostridium tetani (Tetanus bacterium): Its typical immunogen is tetanus toxoid. • Corynebacterium diphtheria (diphtheria bacterium): The typical immunogen is diphtheria toxoid. Haemophilus influenzae (Haemophilus influenzae). ·Pseudomonas aeruginosa. ·Streptococcus agalactiae (group B hemolytic streptococcus). Chlamydia trachomatis: Useful immunogens include, but are not limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7 / L12, OmcA, AtoS, CT547, Eno, HtrA, and MurG. Chlamydia pneumoniae. • Helicobacter pylori: Useful immunogens include, but are not limited to, CagA, VacA, NAP, and / or urease. • Escherichia coli: Useful immunogens include, but are not limited to, immunogens derived from enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EaggEC), diffuse adherent Escherichia coli (DAEC), enteropathogenic Escherichia coli (EPEC), extraenteropathogenic Escherichia coli (ExPEC), and / or enterohemorrhagic Escherichia coli (EHEC). ExPEC strains include urinary tract pathogenic Escherichia coli (UPEC) and meningitis / sepsis-associated Escherichia coli (MNEC). AcfD is an immunogen useful for multiple Escherichia coli types. ·Bacillus anthracis • Yersinia pestis (plague bacterium) • Staphylococcus epidermis (Staphylococcus epidermis) Clostridium perfringens or Clostridium botulinum Legionella pneumophila • Coxiella burwetii • Brucella genus: B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis, B. pinnipediae, etc. • Francisella genus: F. novicida, F. philomiragia, F. tularensis, etc. Neisseria gonorrhoeae (gonorrhea) • Treponema pallidum (Syphilis Treponema) ·Haemophilus ducreyi (chancroid) Enterococcus faecalis or Enterococcus faecium (Enterococcus) • Staphylococcus saprophyticus • Yersinia enterocolitica ·Mycobacterium tuberculosis • Rickettsia • Listeria monocytogenes Vibrio cholera (cholera bacterium) Salmonella typhi (Typhi bacterium) Borrelia burgdorferi Porphyromonas gingivalis • Klebsiella (genus)

[0198] In some cases, immunogens induce an immune response to one of the following viruses: • Orthomyxoviruses: Useful immunogens can be derived from influenza A, B, or C viruses, such as hemagglutinin, neuraminidase, or matrix M2 protein. If the immunogen is hemagglutinin from influenza A virus, it can be any subtype, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. Paramyxoviridae viruses: Viral immunogens include, but are not limited to, those derived from pneumonia viruses (e.g., respiratory syncytial virus, RSV), rubra viruses (e.g., mumps virus), paramyxoviruses (e.g., parainfluenza virus), metapneumoviruses, and morbilliviruses (e.g., measles virus). • Poxviridae: Viral immunogens include those derived from orthopoxviruses (e.g., smallpox virus, Variola vera), including, but not limited to, Variola major and Variola minor. • Picornaviruses: Viral immunogens include, but are not limited to, those derived from the Picornaviridae family, such as enteroviruses, rhinoviruses, heparnaviruses, cardioviruses, and aftviruses. In one embodiment, the enterovirus is a poliovirus (e.g., type 1, type 2, and / or type 3 poliovirus). In another embodiment, the enterovirus is an EV71 enterovirus. In yet another embodiment, the enterovirus is a coxsackie A or B virus. • Bunyaviruses: Viral immunogens include, but are not limited to, those derived from orthobunyaviruses (e.g., California encephalitis virus), phleboviruses (e.g., Rift Valley fever virus), and orthonailoviruses (e.g., Crimean-Congo hemorrhagic fever virus). • Heparnavirus: Viral immunogens include, but are not limited to, those derived from heparnavirus (e.g., hepatitis A virus (HAV)). • Filoviruses: Viral immunogens include, but are not limited to, those derived from filoviruses (e.g., Ebola virus (including Zaire, Ivory Coast, Reston, or Sudan Ebola virus) or Marburg virus). • Togaviruses: Viral immunogens include, but are not limited to, those derived from togaviruses (e.g., rubivirus, alphavirus, or arterivirus). This includes rubella virus. Flaviviruses: Viral immunogens include, but are not limited to, those derived from flaviviruses (e.g., tick-borne encephalitis (TBE) virus, dengue virus (types 1, 2, 3, or 4), yellow fever virus, Japanese encephalitis virus, Kaisanur forest virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Poissant encephalitis virus). • Pestiviruses: Viral immunogens include, but are not limited to, those derived from pestiviruses (e.g., bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV)). • Hepadnavirus: Viral immunogens include, but are not limited to, those derived from hepadnavirus (e.g., hepatitis B virus). The composition may contain hepatitis B virus surface antigen (HbsAg). Other hepatitis viruses: The composition may contain immunogens derived from hepatitis C virus, hepatitis delta virus, hepatitis E virus, or hepatitis G virus. • Rhabdoviruses: Viral immunogens include, but are not limited to, those derived from rhabdoviruses (e.g., lyssavirus (e.g., rabies virus), vesiculovirus (VSV)). • Caliciviridae: Viral immunogens include, but are not limited to, those derived from Caliciviridae (e.g., Norwalk virus (norovirus)) and Norwalk-like viruses (e.g., Hawaii virus, Snow Mountain virus). Coronaviruses: Viral immunogens include, but are not limited to, those derived from SARS coronavirus, avian bronchitis virus (IBV), mouse hepatitis virus (MHV), and porcine gastroenteritis virus (TGEV). Coronavirus immunogens may be spike polypeptides. • Retroviruses: Viral immunogens include, but are not limited to, those derived from oncoviruses, lentiviruses (e.g., HIV-1 or HIV-2), or spumaviruses. • Reoviruses: Viral immunogens include, but are not limited to, orthoreoviruses, rotaviruses, orbiviruses, or cortiviruses. • Parvovirus: Viral immunogens include, but are not limited to, those derived from parvovirus B19. Herpesviruses: Viral immunogens include, but are not limited to, those derived from human herpesviruses (e.g., herpes simplex virus (HSV) (e.g., HSV-1 and 2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), human herpesvirus 8 (HHV8)). • Papovaviruses: Viral immunogens include, but are not limited to, those derived from papillomaviruses and polyomaviruses. (Human) papillomaviruses may be serotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63, or 65, for example, one or more serotypes 6, 11, 16, and / or 18. • Adenovirus: The viral immunogens include those derived from adenovirus type 36 (Ad-36).

[0199] The fungal immunogens are derived from dermatophytes, including Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, and Trichophyton megnini. Trichophyton megnini), Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, Trichophyton verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum and / or Trichophyton fabiforme faviforme); or Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger (AspergillusCandida niger), Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glablata, Candida krusei, Candida parapsilosis, Candida stellatoidea (Candida Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum Capsulatum), Klebsiella pneumonia, Microsporidia, Encephalitozoon spp., Septata intestinalisRarely found species include Brachiola spp., Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp., Paracoccidioides brasiliensis, Pneumocystis carinii, Pythium insidiosum, Pityrosporum ovale, and Sacharomyces. Saccharomyces boulardii, fission yeast (Saccharomyces pombe), Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp, Fonsecaea spp, Wangiella spp, Sporothrix spp, Basidiobolus spp, Conidiobolus genus spp), Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghanlella spp, Saksenaea spp, Alternaria spp, CurvulariaThis includes genera such as Helminthosporium, Fusarium, Aspergillus, Penicillium, Monolinia, Rhizoctonia, Paecilomyces, Pithomyces, and Cladosporium.

[0200] In some cases, immunogens induce an immune response against malaria parasites (Plasmodium) such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale. Therefore, this disclosure can also be used for immunization against malaria. In some cases, immunogens induce an immune response against parasites of the family Caligidae, particularly against sea lice of the genus Lepeophtheirus and Caligus, such as Lepeophtheirus salmonis or Caligus rogercresseyi.

[0201] In some cases, immunogens induce immune responses to pollen allergens (tree, herb, weed, and grass pollen allergens); insect or spider allergens (inhalant, salivary, and venomous allergens, e.g., dust mite allergens, cockroach and midge allergens, hymenoptera venomous allergens); animal hair and dander allergens (e.g., derived from dogs, cats, horses, rats, mice, etc.); and food allergens (e.g., gliadin). Important pollen allergens from trees, grasses, and herbs originate from the Fagales, Oleales, Pinales, and Platanaceae families, including, but not limited to, birch (Betula), alder (Alnus), hazel (Corylus), birch (Carpinus), olive (Olea), cedar (Cryptomeria and Juniperus), and plane tree (Platanus). The Poales order includes rye, oak grass, long-leaved grass, timothy grass, white grass, reed grass, rye, and sorghum, while the Asterales and Urticales order include ragweed, mugwort, and herbs of the genus Lamiaceae. Other important inhaled allergens include house dust mites of the genera Dermatophagoides and Euroglyphus, storage mites (e.g., dust mites, oribatid mites, flour mites), cockroaches, midges and fleas (e.g., German cockroaches, cockroaches, midges, dog fleas), mammals (cats, dogs, horses, etc.), and venomous allergens from biting insects (Hymenoptera, e.g., honeybees (Apidae), wasps (Vespidea), ants (Formicoidae)).

[0202] In some cases, immunogens include (a) cancer-testis antigens, e.g., NY-ESO-1, SSX2, SCP1, RAGE, BAGE, GAGE, and MAGE family polypeptides, e.g., GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, MAGE-12 (these can be used for melanoma, lung, head and neck, non-small cell lung cancer, breast cancer, gastrointestinal cancer, bladder tumors, etc.); (b) mutant antigens, e.g., p53 (associated with various solid tumors, e.g., colorectal cancer, lung cancer, head and neck cancer), p21 / Ras (e.g., associated with melanoma, pancreatic cancer, colorectal cancer), CDK4 (e.g., associated with melanoma), MUM1 (e.g., associated with melanoma), caspase-8 (e.g., associated with head and neck cancer), CIA 0205 (e.g., associated with bladder cancer), HLA-A2-R1701, β-catenin (e.g., associated with melanoma), TCR (e.g., associated with T-cell non-Hodgkin lymphoma), BCR-abl (e.g., associated with chronic myeloid leukemia), triose phosphate isomerase, KIA 0205, CDC-27, LDLR-FUT; (c) Overexpressed antigens, e.g., Galectin 4 (e.g., associated with colorectal cancer), Galectin 9 (e.g., associated with Hodgkin's disease), proteinase 3 (e.g., associated with chronic myeloid leukemia), WT1 (e.g., associated with various types of leukemia), carbonic anhydrase (e.g., associated with renal cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., associated with breast cancer, colorectal cancer, lung cancer, ovarian cancer), mammoglobulin, alpha-fetoprotein (e.g., associated with hepatocellular carcinoma), K SA (e.g., associated with colorectal cancer), gastrin (e.g., associated with pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (e.g., associated with breast and ovarian cancer), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast and colorectal cancer), carcinoembryonic antigen (e.g., associated with breast, lung, and gastrointestinal cancers (colorectal cancer, etc.)); (d) Co-antigens, e.g., melanoma-melanocyte differentiation antigens (MART-1 / Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1 and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma));(e) prostate-related antigens, e.g., PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2 (e.g., associated with prostate cancer); (f) tumor antigens selected from immunoglobulin idiotypes (e.g., associated with multiple myeloma and B-cell lymphoma). In certain embodiments, the tumor immunogens include p15, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigen (including E6 and E7), hepatitis B and C virus antigens, human T-cell leukemia virus antigen, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, β-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA This includes, but is not limited to, 27.29 (BCAA), CA 195, CA 242, CA-50, CAM43, CD68 (KP1), CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophyllin C-related protein), TAAL6, TAG72, TLP, TPS, etc.

[0203] In one embodiment, the delivery system can be used to efficiently deliver the cancer vaccine to a target site in the body.

[0204] In one embodiment, the pharmaceutical composition comprises one polynucleotide. In another embodiment, the pharmaceutical composition comprises multiple polynucleotides.

[0205] This disclosure also provides delivery devices (e.g., syringes, nebulizers, sprayers, inhalers, transdermal patches, etc.) containing the pharmaceutical compositions disclosed herein. These devices can be used to administer the pharmaceutical compositions to subjects.

[0206] Furthermore, methods of treatment are disclosed herein. More specifically, methods for using the pharmaceutical compositions or delivery systems described herein as human or veterinary pharmaceuticals are disclosed herein. More specifically, the pharmaceutical compositions described herein may be used in methods of treating or prophylactic treatment of a disorder in a human or non-human animal, which include administering a therapeutically effective amount of the pharmaceutical composition to a human or non-human animal.

[0207] The animal may be a terrestrial animal, an aquatic animal, a bird, or an amphibian. The animal may be a mammal or a non-mammalian. In a preferred embodiment, the animal is a human. In another embodiment, the animal is a non-human animal. The non-human animal may be an animal raised for food or a pet. Examples of animals to which the compositions of this disclosure may be administered include ruminant species (e.g., sheep, goats, cattle, deer, bison, buffalo, elk, alpaca, camel, llama), ungulates (e.g., horses, donkeys, pigs), birds (e.g., chickens (including laying hens and broilers), turkeys, geese, ducks, Cornish game hen, quail, partridges, pheasants, guinea fowl, ostriches, emus, swans, pigeons), aquatic animals (e.g., farmed species), fish (e.g., salmon, trout, tilapia, sea bream, carp, cod, halibut, snapper, herring, catfish, flounder, hake, smelt, anchovies, rinkott, moi, perch, orange roughy, bass, tuna, mahi-mahi, mackerel, eel, barracuda, marlin, Atlantic ocean perch, nike Examples include luperch, Arctic char, haddock, hoki, Alaska pollock, turbot, freshwater drum, walleye, skate, sturgeon, Dover sole, common sole, wolffish, sablefish, American shad, John Dory, grouper, anglerfish, pompano, lake whitefish, tilefish, wahoo, casque, bowfin, kingclip, opah, mako shark, swordfish, cobia, croaker, or their hybrids), crustaceans (e.g., lobster, shrimp, prawn, crab, krill, crayfish, barnacles, copepods, etc.), and mollusks (e.g., squid, octopus, abalone, conch, rock whelk, turban shell, clam, oyster, mussel, cockle, etc.). Furthermore, or alternatively, the animal may be a companion animal (e.g., dog, cat, rabbit, rodent (rat, mouse, hamster, gerbil, guinea pig, chinchilla, etc.), bird (parrot, canary, parakeet, finch, macaw, lovebird, etc.), reptile (snake, lizard, tortoise, turtle, etc.), fish, crustacean, or amphibian (frog, toad, newt, etc.)).

[0208] Accordingly, the present invention also provides a method for the prevention and / or treatment of a disorder, the method comprising administering the pharmaceutical composition of the present invention to an individual (subject) in need of the present invention. In some embodiments, the compound is administered orally or parenterally. In some embodiments, the compound is administered topically.

[0209] The compounds and compositions may also be used in in vitro methods, such as in vitro methods for administering the compounds or compositions to cells for screening purposes and / or quality control assays.

[0210] The compounds and compositions can also be used in ex vivo therapies (e.g., chimeric antigen receptor (CAR) T-cell therapy).

[0211] In certain embodiments, the pharmaceutical compositions of the present invention may be administered alone or in combination with other types of therapeutic agents. As used herein, the term “combined administration” includes any form of administration in which two or more different therapeutic compounds are administered while a previously administered therapeutic compound is still effective in the body (e.g., the two compounds are effective simultaneously in the patient, which may include a synergistic effect between the two compounds). For example, different therapeutic compounds may be administered simultaneously or sequentially in the same formulation or in separate formulations. In certain embodiments, different therapeutic compounds may be administered to each other within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 1 week. Thus, an individual receiving such treatment may benefit from the combined effects of the different therapeutic compounds.

[0212] In some embodiments, the individual is an animal, preferably a mammal. In some embodiments, the individual is a primate, cattle, sheep, pig, horse, dog, cat, or rodent. In some embodiments, the individual is a human. In some embodiments, the individual has one of the diseases or disorders disclosed herein. In some embodiments, the individual is at risk of developing one of the diseases or disorders disclosed herein. In some embodiments, the individual is a human. In some embodiments, the human is about 21, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 85 years of age or older or about that age. In some embodiments, the human is a child. In some embodiments, the human is about 20, 18, 15, 12, 10, 8, 6, 5, 4, 3, 2 or less than 1 year of age or about that age.

[0213] (Manufactured goods and kits) This disclosure further provides manufactured articles containing the compounds or salts thereof described herein, the (pharmaceutical) compositions described herein, or one or more unit doses described herein in appropriate packaging. In certain embodiments, the manufactured articles are used in any of the methods described herein. Suitable packaging is well known to those skilled in the art and includes, for example, vials, containers, ampoules, bottles, jars, flexible packaging, etc. The manufactured articles may be further sterilized and / or sealed.

[0214] This disclosure further provides kits for carrying out the methods of the present invention, each comprising one or more compounds or (pharmaceutical) compositions described herein. The kit may use any of the compounds disclosed herein. In one embodiment, the kit may use a compound or a salt thereof described herein. The kit may be used for one or more of the uses described herein. The kit generally comprises appropriate packaging. The kit may comprise one or more containers comprising any of the compounds described herein. Each component (if there are multiple components) may be packaged in a separate container, and some components may be combined in a single container if cross-reactivity and shelf life are acceptable. The kit may be in unit dose form, bulk packaging (e.g., multi-dose packaging), or sub-unit dose form. For example, a kit may be provided that contains a sufficient dose of the compounds disclosed herein and / or additional pharmacologically active compounds useful for diseases described in detail herein, and that can provide an effective treatment to an individual over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months or longer. The kit may also include multiple unit doses of the compound and instructions for use, and may be packaged in quantities sufficient for storage and use in a pharmacy (e.g., a hospital pharmacy or a compounding pharmacy).

[0215] The kit may include a set of instructions for using the components of the method of the present invention, which are generally written instructions, but instructions on an electronic storage medium (e.g., magnetic diskette or optical disc) are also acceptable, as needed. The instructions included in the kit generally include information on the components and their administration to an individual.

[0216] (Advantages and applications) A major challenge in the field of mRNA therapeutics is the development of efficient delivery vectors that can (i) aggregate cargo such as mRNA, (ii) protect it from degradation, and (iii) ensure intracellular delivery. Of all non-viral vectors, lipid nanoparticles (LNPs) are the most studied and have the most clinical development.

[0217] These lipid nanoparticles consist of four components: ionizable lipids, helper lipids, sterols, and PEGylated lipids. Over the past 20 years, ionizable lipids have been intensively studied as the main component of this system. While structure-activity relationships are still in their early stages, several parameters have been identified as significantly influencing the encapsulation and release of multiple nucleic acids (e.g., pKa, end length, shape, etc.). Efforts have also been made to improve tolerability and biodegradability, resulting in several lead compounds, including the well-known DLin-MC3-DMA, SM-102, and ALC-0315 lipids. However, these systems still have room for improvement, particularly in terms of in vivo distribution (off-target), (thermal) stability, and reactogenicity, and therefore, next-generation ionizable components for RNA delivery are currently under development. This invention offers an unexpected solution to this challenge by providing a novel hybrid calixarene-lipid delivery system that overcomes the shortcomings of conventional LNPs.

[0218] Calixarenes offer a unique opportunity to design highly variable ionizable components for cargo delivery because each phenolic unit of a calixarene can be individually derivatized (particularly in the case of calix[4]arenes), allowing for the design of molecules with a variable number of ionizable head groups and hydrophobic ends. By combining multiple chemically distinct head groups or ends, encapsulation / delivery properties can be precisely controlled, or the immunogenicity of the resulting delivery particles can be improved by introducing adjuvant patterns.

[0219] Furthermore, the inventors unexpectedly discovered that calixarenes themselves have an adjuvant effect when used in LNPs, and that calixarenes can be used in immunogenic compositions, which comprise immunogenic components encapsulated within lipid nanoparticles containing the calixarenes, and that the LNPs have an adjuvant effect in the immunogenic compositions. The adjuvant effect is described in Examples 4-8.

[0220] In one embodiment, the lipid nanoparticles further comprise PEGylated lipids. In a further embodiment, the lipid nanoparticles further comprise sterols and / or phospholipids. In one embodiment, the lipid nanoparticles comprise ionizable calixarenes and further comprise PEGylated lipids, sterols and phospholipids. In one embodiment, the lipid nanoparticles comprise cationic calixarenes and further comprise PEGylated lipids, sterols, ionizable lipids and phospholipids.

[0221] In a preferred embodiment, the lipid nanoparticle comprises at least one cationic calixarene having at least one positively charged moiety and / or at least one ionizable calixarene having a moiety capable of associating with at least one ion and becoming positively charged. In a further embodiment, the positively charged moiety is an amine-containing group comprising a secondary, tertiary, and / or quaternary amine. In one embodiment, the calixarene is an ionizable calix[4]arene having four head groups, with at least one head group comprising at least one secondary or tertiary amine (for example, CX5 shown in Figure 16, where four identical head groups each comprise one tertiary amine group). In a further embodiment, the calixarene is a cationic calix[4]arene having four head groups, with at least one of the head groups comprising at least one quaternary amine. In another further embodiment, the calixarene is a cationic calix[4]arene having four head groups, where at least one of the head groups comprises at least one quaternary amine (for example, CX4 and CX12, shown in Figure 8, where four identical head groups each have one quaternary amine group).

[0222] When used in an immunogenic composition, the calixarene is preferably present in the lipid nanoparticles at a mass fraction of 0.1 to 60% (w / w). In one embodiment, if the calixarene is ionizable, it is present in the lipid nanoparticles at a mass fraction of 10 to 60% (w / w). In one embodiment, when used in an immunogenic composition, the ionizable calixarene is present in the lipid nanoparticles at a mass fraction of 10 to 60% (w / w), for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 2 9%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% (w / w) or any value in between. In one embodiment, when used in an immunogenic composition, the ionizable calixarenes are present in the lipid nanoparticles in a mass fraction of 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, or 55-60% (w / w). In one embodiment, when used in an immunogenic composition, the ionizable calixarene is present in the lipid nanoparticles in a mass fraction of 10-20%, 20-30%, 30-40%, 40-50%, or 50-60% (w / w).

[0223] In one embodiment, when the calixarene is cationic, it is present in the lipid nanoparticles at a mass fraction of 0.1 to 50% (w / w). In one embodiment, when used in an immunogenic composition, the cationic calixarene is present in the lipid nanoparticles at a mass fraction of 0.1 to 50% (w / w), preferably 0.2 to 10% (w / w), for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% (w / w), or any value in between. In one embodiment, when used in an immunogenic composition, the cationic calixarene is present in the lipid nanoparticles in a mass fraction of 0.1-10%, 10-20%, 20-30%, 30-40%, or 40-50% (w / w). In one embodiment, when used in an immunogenic composition, the cationic calixarene is present in the lipid nanoparticles in a mass fraction of 0.1-1%, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 10-11%, 11-12%, 12-13%, 13-14%, or 14-15% (w / w).

[0224] When used in the immunogenic composition, the mass of calixarenes contained in the lipid nanoparticles is preferably less than 10 times, more preferably less than 5 times, and even more preferably less than that, the mass of nucleic acids encapsulated in the lipid nanoparticles.

[0225] Thus, the calixarenes provided in the delivery system of the present invention not only offer advantages related to efficient delivery as described above (efficient encapsulation of nucleic acids, promotion of encapsulation of very long RNAs and / or minimization of the intrinsic toxicity of cationic components and / or reduction of nonspecific adsorption of proteins that normally limit their effectiveness), but also function at the level of immunogenic response in immunogenic compositions comprising immunogenic components encapsulated in lipid nanoparticles containing the calixarenes.

[0226] Similar to the delivery system described above, the lipid nanoparticles may further comprise ionizable lipids. In one embodiment, the ionizable lipids are present in the immunogenic composition at a mass fraction of up to 60% (w / w).

[0227] When calixarenes are used in an immunogenic composition, the immunogenic component preferably comprises at least one nucleic acid molecule encoding at least one epitope of at least one antigen. In a preferred embodiment, the encoding nucleic acid molecule is an mRNA or saRNA molecule.

[0228] Preferably, the immunogenic composition is administered intramuscularly to the subject.

[0229] In a further embodiment, the Disclosure relates to a vaccine comprising an immunogenic component encapsulated in lipid nanoparticles, the lipid nanoparticles comprising at least one calixarene molecule, the LNP having an adjuvant effect in the vaccine.

[0230] In one embodiment, the vaccine is a DNA vaccine, and the immunogenic component comprises DNA. In one embodiment, the vaccine is an RNA vaccine, and the immunogenic component comprises RNA. In one embodiment, the vaccine is a cancer vaccine. In a preferred embodiment, the calixarene enhances the immunogenicity of the immunogenic component, as measured by an increase in viral neutralizing antibody titer (VNT) after in vivo administration to a subject, compared to the same immunogenic composition without the calixarene.

[0231] In a further embodiment, the present invention relates to a method for preparing an immunogenic preparation such as a vaccine, the method comprising the step of encapsulating an immunogenic component in lipid nanoparticles, the lipid nanoparticles comprising at least one calixarene molecule, the LNP having an adjuvant effect in the lipid nanoparticles.

[0232] As described above, the calixarene is preferably an ionizable or cationic calixarene, and the immunogenic component is preferably a nucleotide such as an mRNA or saRNA molecule.

[0233] In comparison, conventional lipid or lipoid platforms exhibit far less flexibility. Conventional ionizable lipids typically have only one ionizable head group, and introducing additional hydrophobic ends to emphasize the cone-shaped structure requires multi-step synthesis, a property that is essential for calix[4]arenes. With lipoids, their derivatization usually results in symmetric compounds, and the properties of the ionizable head group and hydrophobic ends cannot be modulated relative to each other.

[0234] As recently demonstrated with several lipoid structures, increasing the number of ionizable head groups typically improves encapsulation efficiency and RNA release by increasing the N / P ratio (charge density) without changing the mass ratio of RNA to ionizable component. In calix[4]arenes, the number of these ionizable head groups can easily reach four, making it possible to consider the delivery of very long RNA molecules such as saRNA, which is extremely difficult with current LNP technologies based on ionizable lipids. At the same time, for conventional non-replicating mRNA, increasing the number of ionizable functions can reduce the amount of ionizable component required for good encapsulation and release, thereby lowering the overall cost of the delivery system.

[0235] Furthermore, the present invention provides another delivery system utilizing cationic calixarenes and ionizable lipids. Calixarenes are macrocyclic or cyclic oligomers based on methylene-crosslinked phenols and are represented by the following general structural formula. [ka]

[0236] These compounds exhibit several key characteristics that make them suitable for nucleic acid delivery when used in delivery systems similar to five-component systems. This allows for controllable size and shape. Compounds with a fixed number of phenol units (4, 6, 8…) can be obtained. Furthermore, calix[4]arenes readily adopt a conical three-dimensional structure. This structure is a key characteristic of conventional ionizable lipids, promoting RNA release via membrane destabilization and endosomal escape.

[0237] The encapsulation of negatively charged nucleotides primarily depends on interactions with positively charged compounds. The selection of such positively charged compounds is crucial, as they contribute to the capture of RNA molecules and the promotion of endosomal escape.

[0238] As described above, the calixarene-based delivery system of this disclosure has several advantages over conventional delivery systems. The delivery system can be designed for specific applications by adjusting the composition of calixarenes, phospholipids, sterols, PEGylated lipids, and ionizable lipids.

[0239] Calixarene compounds can be controlled in size and shape. Compounds with a fixed number of phenolic units (4, 6, or 8) can be obtained. Furthermore, calixarene compounds readily adopt a conical three-dimensional structure. This structure is an important property that promotes nucleotide (RNA) release via membrane destabilization and endosomal escape.

[0240] Furthermore, calixarene compounds can be readily derivatized (at the lower and upper edges), thereby yielding amphiphilic compounds that self-assemble micelles in water. These compounds may also contain cargo within the cavity or by forming ion pairs between functional groups and charged cargo (e.g., nucleotides).

[0241] In addition, the number of cationic or ionizable head groups on a single molecule of calixarene compounds can be adjusted. By increasing the number of cationic head groups, it is possible to improve encapsulation efficiency and nucleotide (e.g., mRNA) release by increasing the N / P ratio without changing the mass ratio of mRNA to cationic components.

[0242] Furthermore, calixarenes offer a unique opportunity to design highly variable ionizable components for RNA delivery. In fact, each phenolic unit of a calixarene can be individually derivatized (particularly evident in calix[4]arenes), allowing for the design of molecules with a variable number of ionizable head groups and hydrophobic ends. It is also possible to combine multiple chemically different head groups or ends, allowing for precise control of encapsulation / delivery properties or improving the immunogenicity of delivery particles obtained by introducing adjuvant patterns. In comparison, the flexibility of conventional lipid or lipoid platforms is far less. Conventional ionizable lipids typically have only one ionizable head group, and multi-step synthesis is required to introduce additional hydrophobic ends to enhance the cone-shaped conformation, a property inherent in calixarenes. With lipoids, derivatization usually results in symmetric compounds, and the properties of the ionizable head groups and hydrophobic ends cannot be modulated with each other.

[0243] As demonstrated in several lipoid structures in recent years, increasing the number of ionizable head groups typically improves encapsulation efficiency and RNA release by increasing the N / P ratio (charge density) without altering the mass ratio of RNA to ionizable components. With calixarenes, the number of ionizable head groups can be easily increased to eight, making it possible to explore the delivery of very long RNA molecules such as saRNA, which is extremely difficult with current ionizable lipid-based LNP technologies. Simultaneously, for conventional non-replicating mRNA, increasing the number of ionizable functional groups can reduce the amount of ionizable component required for good encapsulation and release, thereby lowering the overall cost of the delivery system.

[0244] Furthermore, calixarene compounds can be used to modify the properties of their hydrophobic ends or to combine different hydrophobic motifs on the same molecule. These motifs can, for example, confer adjuvant activity to molecules or assist in endosomal escape.

[0245] The compounds of the present invention can easily incorporate biodegradable functional groups (such as esters, amides, and disulfide bridges) to bond the hydrophobic end and cationic head group to the macrocyclic core.

[0246] This invention is widely applicable to various therapeutic and diagnostic settings, including gene therapy, RNA interference, genome editing, protein replacement therapy, drug delivery, and imaging. Improved cargo delivery systems can enhance the efficacy and safety of these interventions, ultimately benefiting patients and contributing to advancements in the medical field.

[0247] Although the present invention has been described in detail with reference to specific embodiments, it should be understood that various modifications and substitutions are possible without departing from the spirit and scope of the invention. Accordingly, the scope of the invention should not be limited to the specific embodiments described herein, but rather encompasses all embodiments within the scope described in the appended claims.

[0248] The present invention is further illustrated by the following non-limiting examples. These are for further illustration of the present invention and are not intended to limit the scope of the present invention, nor should they be construed as such.

Example

[0249] Example 1: An example of a delivery system according to an embodiment of the present invention: Use of cationic calixarenes in a delivery system.

[0250] Molar ratios of components used in the formation of the delivery system: · Ionizable + cationic component = 25 - 40% · Part of the ionizable component = 80 - 95% · Calixarene = 1.25 - 8% · Helper lipid (phospholipid) = 25 - 35% · Additional lipid (sterol) = 25 - 50%

[0251] The delivery system is prepared using cationic calixarenes according to the above molar ratios. Specifically, DODAP is used as the ionizable component, calixarene as the cationic component, DOPE as the phospholipid, and cholesterol as the additional lipid.

[0252] The main physicochemical properties of the prepared delivery system were evaluated, and the results are shown below. · Particle size = 75 - 150 nm (PDI (polydispersity index): less than 0.3) · Encapsulation efficiency %: more than 85% · Protein expression increases by combining calixarene with DODAP

[0253] Example 2: Development and characterization of lipid nanoparticles according to an embodiment of the present invention comprising ionizable calix[4]arene

[0254] (Introduction) This example focuses on the design of ionizable calix[4]arene and its utilization in RNA lipid nanoparticles (RNA-LNPs), aiming to replace conventional ionizable lipids. It is expected that this modification will completely change the behavior of the resulting delivery system, particularly its encapsulation and release capabilities. Calix[4]arene has several important characteristics suitable for nucleic acid delivery. The most important is its natural cone-shaped three-dimensional structure, which has been found to be crucial for lipid nanoparticles / ionizable lipids to achieve high endosomal escape ability and promote the release of RNA in the cytoplasm. Furthermore, calix[4]arene is a platform that facilitates the synthesis of ionizable calix[4]arene with one, two, three, four or more amine head groups, i.e., the charge density (number of amines / molecule) can be easily increased. This property helps to encapsulate very long RNAs such as self-amplifying RNA (saRNA) by increasing the number of amines without changing the mass ratio of the ionizable component to RNA, which is a difficult problem with current LNP technology without calixarene.

[0255] Synthesis of ionizable calix[4]arene according to an embodiment of the present invention and self-assembly into monodisperse nanoparticles encapsulating mRNA-FLuc

[0256] To better understand the structure-activity relationship, a library of ionizable calixarenes was synthesized. Calixarenes with one (CX14, CX16, CX24) or four (CX1, CX2, CX3, CX5, CX6, CX29) ionizable head groups (i-heads) were synthesized (see Figure 1). The ionizable head groups were selected from secondary amines (CX6, CX14) and tertiary amines (CX1, CX2, CX3, CX5, CX16, CX24, CX29), those substituted with cyclic or methyl or hydroxyethyl groups. Furthermore, two types of biodegradable groups (amide bond (CX2) and ester bond (CX3 / CX29)) were also investigated to bind the ionizable head group to the macrocyclic core and promote the metabolic degradation of the resulting compound to avoid in vivo accumulation.

[0257] All of these calixarenes were self-assembled into nanoparticles together with helper lipids (phospholipids), sterols, and PEGylated lipids according to one embodiment of the present invention. Multiple helper lipids (DOPE, DSPC) and PEG lipids (DMG-PEG2000, DSG-PEG2000) were investigated. The ratios of these components were defined to produce stable monodisperse nanoparticles.

[0258] To obtain stable and monodisperse particles, a minimum mass ratio of [calixarene + lipid] / RNA is required. This minimum ratio is approximately 20 for single-headed calixarenes and can be reduced to 10 for four-headed calixarenes. This intriguing result suggests that less material is needed to achieve encapsulation efficiency and monodispersity comparable to commonly used ionizable lipids (e.g., SM-102). This difference is obtained due to the increased charge density in four-headed ionizable calixarenes.

[0259] For helper lipids, sterols, and PEGylated lipids, their mass fraction is maintained even when they are transferred from a single-headed group to a four-headed group. The mass fractions of these lipids (expressed as a percentage, with the mass of the delivery system excluding cargo being 100%) are preferably as follows. Ionizable calixarenes: 10-60% Helper lipids: 5-35% Sterols: 15-50% PEGylated lipids: 2-24%

[0260] It should be noted that PEGylated lipids are essential for obtaining stable, non-aggregating monodisperse nanoparticles because they play a role in shielding particles and stabilizing the lipid-water interface. Therefore, the mass fraction of PEGylated lipids must be strictly controlled; too little PEG induces particle aggregation, while too much PEGylated lipids limit transfection ability (see in vivo protein expression results below). An example of nanoparticles using ionizable calixarenes according to one embodiment of the present invention is shown in Table 1 below.

[0261] [Table 1]

[0262] In vitro transfection of mRNA-FLuc using a delivery system according to one embodiment of the present invention In vitro activity assays were performed in Jurkat cells using Fluc mRNA as a reporter gene. Average luminescence was measured 24 hours after transfecting Jurkat cells with 100 ng of mRNA. Cells were seeded on day 1 (10,000 cells per well in a 96-well plate), transfected on day 2, and luminescence was measured after luciferin addition on day 3. The initial results (Table 2) revealed that CX compounds with tertiary amines showed higher activity. Simultaneously, these assays provided evidence of the superiority of tetraheaded calixarenes over tetraheaded calixarenes. For certain groups of tetraheaded calixarenes, it was also shown that increasing the number of hydroxyethyl groups improved activity.

[0263] [Table 2]

[0264] mRNA-FLuc in vivo protein expression is regulated by mass ratio. Within the range of optimal mass ratios for obtaining stable monodisperse particles as defined above, experiments were designed to further optimize these ratios and maximize protein expression in vivo. The one that showed the highest activity in vitro (CX5) was selected, and nanoparticles were tested with varying (lipid + CX) / RNA total mass ratios ranging from 15 to 45, helper content from 10 to 30%, and PEGylated lipid content from 10 to 24%.

[0265] 1 μg of Fluc mRNA was administered intramuscularly, and the effects of the above factors were estimated using luminescence at the injection site. The estimated model (mean + 95% confidence interval) is visualized in Figure 2, clearly showing the favorable mass ratios of helper and PEG lipids for increasing in vivo protein expression. As mentioned above, the minimum amount of PEG is preferable, but a lower limit is necessary to achieve colloidal stability. Figure 2 shows the visualization of the estimated model (mean + 95% confidence interval) of in vivo luminescence as a function of (lipid + CX) / RNA mass ratio, helper mass ratio, and PEG lipid mass ratio.

[0266] After optimizing the molar ratio in vivo using CX5, different calixarenes selected from those shown in Figure 1 were compared. CX1, CX2, CX3, and CX5, each with four head groups, were prepared in the optimal ratio defined for CX5. CX16, with one head group, was also included in this study in its own optimal ratio defined in vitro. Protein expression results (geometric mean signal and 95% confidence interval) for each calixarene delivery system at 3, 6, and 9 hours after intramuscular injection of 1 μg of FlucmRNA are shown in Figure 3. 1 μg of RNA was administered to both the left and right hind limbs of mice. Sample randomization was performed using DoE to avoid interactions between delivery systems, and luminescence was estimated by integrating radiance at the injection site. These data indicate that CX5, a calixarene with four head groups containing the most hydroxyethyl groups, exhibits the best performance. Overall, this experiment suggests that the dynamics of protein expression differ between calixarenes with one head group and those with four head groups. While the signal for CX16 (one head group) decreased rapidly, protein expression induced by calixarenes with four head groups (CX1, CX2, CX3, and CX5) remained stable over different time points (3–9 hours post-injection). This result again demonstrates that calixarenes with four head groups are superior to those with one head group in achieving stable and high protein expression. This observation is supported by the fact that less calixarene with four head groups is required to achieve comparable performance (approximately 5 μg of CX / μgRNA with four head groups versus approximately 10 μg of CX / μgRNA with one head group).

[0267] Encapsulation of saRNA-FLuc in nanoparticles according to one embodiment of the present invention While maintaining an optimized mass ratio between the four components (CX, helper, cholesterol, and PEG lipid), saRNA encoding FLuc was encapsulated using CX5 by varying the CX / saRNA mass ratio. Stable nanoparticles with high encapsulation efficiency (over 80%) were obtained across the entire screening region (CX / saRNA mass ratio 2.3 to 9.2) (see Figure 4). Figure 4 shows the encapsulation efficiency and in vitro protein expression for various delivery systems according to one embodiment of the present invention, for different CX5 / saRNA mass ratios. Two independent samples were prepared for each condition. Interestingly, as shown in Figure 4, when the CX / saRNA mass ratio was increased from 2.3 to 5.8, the encapsulation efficiency improved, but then remained stable at a mass ratio of 9.2, indicating a saturation effect on the amount of CX required to reach maximum encapsulation efficiency. However, it was possible to increase protein expression by further increasing the amount of CX5. It is worth noting that this optimized mass ratio (9.2) is smaller than the mass ratio (approximately 20 μg i-lipid / μgRNA) required for ionizable lipids (SM-102, ALC-0315) commonly used in this field to achieve similar properties (encapsulation efficiency and in vitro protein expression) with saRNA.

[0268] Comparison of the in vivo delivery efficiency of saRNA-Fluc by a delivery system according to one embodiment of the present invention with a reference delivery system that does not contain calixarenes. The superior performance demonstrated in vitro was also confirmed in vivo. Intramuscular injection of 0.2 μg of FLuc-encoding saRNA induced strong luciferase production, which increased for at least one week (Figure 5). Similar results were obtained with a reference system (SM-102-based LNP), but with approximately twice the amount of ionized component (20.9 vs. 9.2). This again demonstrates the superiority of using calixarenes with four head groups that have a high charge density. Figure 5 shows the geometric mean signal (positive signal for each mouse) over time for each delivery system (blue for delivery systems with CX5, red for the reference delivery system). 0.2 μg of saRNA was administered to the right hind limb of each mouse. Luminescence was estimated by integrating the radiance at the injection site.

[0269] Preclinical evaluation of a delivery system comprising mRNA encoding a rabies virus G protein, according to one embodiment of the present invention. The delivery system containing CX5 was evaluated in an immunogenicity study using rabies virus glycoprotein G as a model antigen. Mice were vaccinated with a 21-day prime-boost regimen using either 0.6 μg or 2.5 μg of mRNA per dose, followed by vaccination. As shown in Figure 6, CX5, using the two dosing regimens (0.6 or 2.5 μg), showed strong VNT (viral neutralizing antibody titer) well above the protective correlation value (0.5 IU / mL) and did not induce any adverse events in the animals (weight loss, effects on spleen, liver, or kidney weight, inflammation, or excessive reactivity). Figure 6 shows serum VNT values ​​measured on day 15 (after initial dose), day 35, and day 65 (after additional dose) (left: 0.6 μg dose, right: 2.5 μg dose).

[0270] Cryo-TEM observation of nanoparticles prepared with ionizable calix[4]arenes according to one embodiment of the present invention CX5:DOPE:cholesterol:mRNA-FLuc was encapsulated in DMG-PEG2000 nanoparticles and observed by cryo-TEM (Error! Reference source not found.). A Lacey Formvar / Si monoxide grid (300 mesh Cu, Ted Pella Inc, 01887-F) was glow-discharged with an ELMO glow discharge device at 4.5 mA for 20 seconds. 3.5 μl of the sample was dropped onto the grid and rapidly frozen in liquid ethane in a chamber at 95% humidity and 20 °C, and double-sided blotting for 4 seconds was performed using Gatan Cp-3. The cryo-grid was observed with a JEOL 1400 microscope (60,000x magnification) equipped with TVIPS F416 and a Gatan 626 side-entry holder (60k magnification). The 60k magnification image was 4k × 4k, and the pixel size was 0.194 nm / px. The sample was applied without dilution. The LNP was monodisperse, and the minimum and maximum LNP sizes were approximately 40 nm and 80 nm, respectively, which were in good agreement with the DLS measurement value (Z-average of approximately 50 nm).

[0271] Example 3: Development and Characterization of Lipid Nanoparticles According to an Embodiment of the Present Invention Comprising Cationic Calix[4]arenes

[0272] (Introduction) This example focuses on cationic calixarenes and their incorporation into lipid nanoparticles (LNP) made with ionizable lipids, helper lipids, sterols, and PEG lipids as the fifth component.

[0273] The addition of cationic calixarenes significantly alters the behavior of the resulting delivery system, particularly its encapsulation and release capabilities. As described above, calix[4]arenes possess several important characteristics that make them suitable for nucleic acid delivery. Most importantly, they have a natural cone-shaped structure, which is thought to be crucial for lipid nanoparticles / ionizable lipids to achieve high endosomal escape ability and promote RNA release in the cytoplasm. Furthermore, as a platform, calix[4]arenes facilitate access to cationic compounds having one, two, three, or four or more quaternary amine head groups, thus easily increasing the charge density (amine count / molecule). This property suggests the possibility of achieving encapsulation of very long RNAs, such as self-amplified RNA (saRNA), which is difficult with current LNP technology, by adding small amounts of cationic calixarenes to LNPs. Furthermore, incorporating cationic calixarenes into LNPs allows for (i) minimizing the intrinsic toxicity of these cationic components by combining them with non-toxic, biocompatible lipids, and (ii) reducing nonspecific protein adsorption, which typically limits their effectiveness.

[0274] Synthesis of cationic calix[4]arenes according to one embodiment of the present invention and addition to monodisperse lipid nanoparticles encapsulating mRNA-FLuc Two cationic calixarenes (CX4 and CX12) were synthesized and tested (see Figure 8). Experiments showed that these cationic calixarenes could be prepared with the following compositions and incorporated into lipid nanoparticles encapsulating RNA (Fluc-mRNA or other RNA unless otherwise specified) (Table 3). - Ionizable lipids: DODAP, DLin-DMA, DLin-MC3-DMA, ALC-0315, SM-102 - Helper lipids: DOPE, DOPC - Sterols: Cholesterol -PEG lipids: DMG-PEG2000, DSG-PEG2000

[0275] [Table 3]

[0276] LNPs remain stable even when they contain high levels of cationic calixarenes. Using Design of Experiments (DoE), we modeled the effect of CX12 mass per LNP (expressed as mg of CX12 per mg of RNA dose) on its physicochemical properties. The masses of other lipids (DLin-DMA, DOPE, cholesterol, DMG-PEG) and mRNA (firefly luciferase) were kept constant. The experimental and modeling results are shown in Table 4 and Figure 9. LNPs produced with CX12 in amounts ranging from 0 to 20.7 times greater than mRNA were found to be monodisperse and encapsulate over 95% of the RNA. The addition of CX12 was found to increase both the size (77–129 nm) and the charge (5.8–11.0 mV).

[0277] [Table 4]

[0278] Preclinical evaluation of candidate LNPs comprising cationic calixarene and DLin-DMA according to one embodiment of the present invention. Four types of LNPs were prepared using combinations of CX4 and CX12, and DMG-PEG2000 and DSG-PEG2000. In all cases, DLin-DMA was used as the ionizable lipid. mRNA encoding the G protein of the rabies virus was used. The physicochemical properties of the prepared LNPs are shown in Table 5.

[0279] [Table 5]

[0280] BALB / c mice were vaccinated with DLin-DMA:CX4 or DLin-DMA:CX12 LNPs along with two types of PEGylated lipids, after a priming-boosting regimen at 21-day intervals (initial dose on day 0, replenishment dose on day 21). Serum levels of VNTs were measured at 15 or 35 days for RNA doses of 0.6 or 2.5 μg. LNPs were frozen at -80°C for delivery and storage. The same batch was used for the initial and replenishment doses of the mice. Geometric mean antibody levels are shown in Figure 10. Strong VNTs were obtained at 35 days for all LNPs. Raw data showed a dose-response effect, with the 2.5 μg dose group showing higher average values ​​than the 0.6 μg dose group at both 15 and 35 days. VNTs significantly exceeded the protective correlation value (shown as a dashed black line of 0.5 au) for all LNPs and doses. These results indicate that the combination of cationic calixarenes and the ionizable lipid DLin-DMA yields potent LNPs. Furthermore, the selection of PEGylated lipids suggests that it influences the immune response; even a difference of just four carbon atoms in the two alkyl chains of a PEGylated lipid can significantly reduce immune activity, even in LNPs that are otherwise identical.

[0281] Encapsulation of saRNA by LNP comprising DLin-DMA and CX12 according to one embodiment of the present invention LNPs of DLin-DMA:DOPE:cholesterol:DMG-PEG2000:CX12 were used to encapsulate saRNA encoding the full-length spike protein of SARS-CoV-2. The resulting LNPs were monodisperse, with a Z-mean of 120 nm (see Figure 11A), and the saRNA encapsulation rate was 76.6%. This result indicates that this composition can be used for larger RNAs such as saRNA. Particularly interesting is the difficulty in encapsulating saRNA with common ionizable lipids. This was demonstrated using saRNA encoding an undisclosed target gene of comparable length. The saRNA was prepared using SM-102:DSPC:cholesterol:DMG-PEG2000 (50:10:38.5:1.5 molar ratio). The resulting LNPs were polydisperse (see Figure 11B).

[0282] Example 4: LNPs comprising cationic calix[4]arenes act as adjuvants using SARS-CoV-2 prefusion stabilization spike proteins.

[0283] Experiments were conducted to prepare LNPs using ionizable lipids (i-lipids) together with helper lipids, cholesterol, PEGylated lipids, and mRNA. The inventors confirmed that LNPs prepared with DODAP (1,2-dioleoyl-3-dimethylaminopropane) had low cytoplasmic delivery and encapsulation efficiency. However, they found that this problem could be improved by adding cationic lipids to the LNPs and carefully selecting the helper lipids (phospholipids such as DOPE, DOPC, and DSPC), the molar ratio between lipids, and the amine-to-phosphate (N:P) ratio. The cationic lipids tested included DOTAP, DDAB, and DC-cholesterol. Furthermore, cationic calix[4]arenes were also tested as a novel molecular class for incorporation into LNPs.

[0284] After optimizing the manufacturing process and molar composition, two types of LNPs were used to encapsulate mRNA encoding firefly luciferase. LNP I005-034-08 contained DOTAP as the cationic component, and LNP I005-203-401 contained cationic calixarene CX12 as the cationic component (Figure 8B), both using DODAP as the i-lipid. The LNPs were prepared in TRIS 20 mM, sucrose 8% (w / v), pH 7.4 buffer. Both LNPs encapsulated over 95% of the mRNA and exhibited monodisperse (see Table 6 for physicochemical properties).

[0285] [Table 6]

[0286] 1 μg of RNA encapsulated in each LNP type was administered intramuscularly (IM) to BALB / c mice. Luciferin was administered at 6, 12, and 24 hours after LNP administration, and luminescence was measured using an in vivo imaging system (IVIS). Luciferase expression levels were estimated by integrating the radiance across the entire region of interest (ROI). The results at 6 hours are shown in Figure 12. Both LNPs showed similar levels of protein expression.

[0287] Using a SARS-CoV-2 model, we discovered that LNPs containing cationic calixarenes (CX12 in this example) exhibit an unexpected adjuvant effect. Nanoparticles combining the known low-efficiency ionizable lipid DODAP with CX12 were demonstrated to induce a potent immune response. After a 21-day prime-boost regimen using 1 μg of RNA per dose, measured antibody titers (anti-spike and anti-RBD) were slightly lower compared to the Spikevax vaccine (Moderna, spike-coding RNA encapsulated in SM-102LNP), but the difference was not statistically significant (see Figures 13A and 13B).

[0288] Previous studies have shown that protein expression using the DODAP / CX12 system is three orders of magnitude lower than that of the SM-102LNP control, demonstrating the potent adjuvant effect of CX12. This effect is clearly attributable to calixarene; other DODAP-based systems with added cationic agents (DOTAP) did not show any immunogenicity, and instead showed slightly higher protein expression in mice. This result is particularly useful because it allows this performance to be achieved with an extremely small amount of CX12 required (approximately 0.35 μg per 1 μg of RNA).

[0289] Example 5: Expansion to other antigens - High titer LNP for rabies vaccine using cationic calixarenes according to one embodiment of the present invention

[0290] To further demonstrate the adjuvant effect of LNPs containing the cationic calixarene CX12, different preclinical models were used. The same LNPs were used to encapsulate mRNA encoding the rabies virus G protein. 2.5 μg of mRNA was administered to the hind limbs of mice, and a 21-day prime-boost regimen was performed. Viral neutralizing antibody titers (VNTs) were measured in serum 35 days post-priming. The results are shown in Figure 14, where the neutralizing antibody titer (VNT) was significantly above the protective correlation value (0.5 IU / mL). These results indicate that LNPs containing cationic calixarenes such as CX12 can act as adjuvants against different antigens and induce immune responses.

[0291] These results are particularly noteworthy because they require an extremely small amount of cationic calixarene to achieve the desired performance (approximately 0.35 μg / μg of RNA). For comparison, the literature reports that when cationic calixarene is used as a DNA transfection agent, the amount of calixarene required is at least 25 times greater (10 μg / μg of DNA). This indicates a synergistic effect between cationic calixarene and the LNP architecture.

[0292] Example 6: Extension to other LNPs and other cationic calix[4]arenes - Use according to one embodiment of the present invention

[0293] The use of cationic calix[4]arenes can be extended to other LNPs. LNPs were prepared with and without cationic calix[4]arenes using N,N-dimethyl-2,3-bis[(9Z,12Z)-9,12-octadecadiene-1-yloxy]-1-propanamine (DLin-DMA), a helper, cholesterol, and PEGylated lipids. Two different calix[4]arenes, namely CX12 and CX4 (see Figure 8), were tested. All LNPs exhibited monodispersity and encapsulated mRNA (see Table 7). All LNPs encapsulated mRNA encoding the rabies virus G protein. 0.6 or 2.5 μg of mRNA was administered to the hind limbs of mice, followed by a 21-day prime-boost regimen. Viral neutralizing antibody titers (VNT) were measured in serum at 15, 35, and 64 / 65 days. The results are shown in Figures 15A and 15B.

[0294] [Table 7]

[0295] As shown in Figure 15A, VNT obtained using DLin-DMA LNP boosted with CX4 or CX12 significantly exceeds the protective correlation value (0.5 IU / mL).

[0296] Figure 15B shows the viral neutralizing antibody titers (VNT) measured at 15 days (post-priming), 35 days, and 65 days (post-boost) in the serum of mice vaccinated with SM-102 LNP (control), DLin-DMA LNP, and DLin-DMA / CX4 LNP. A 21-day prime-boost regimen using 2.5 μg of mRNA (RNA encoding rabies virus glycoprotein G) per dose was used. Results are shown in comparison to the control delivery system (SM-102 LNP). UDL = upper limit, LDL = lower limit. A significant difference compared to the control is indicated if all points exceed these limits.

[0297] These results again demonstrate the potent adjuvant and synergistic effects of adding cationic calixarenes to lipid nanoparticles. Furthermore, the strong response obtained with CX4 indicates that the adjuvant effect is not limited to specific cationic calix[4]arenes.

[0298] Example 7: Stable LNPs can be obtained with a wide range of calixarene fractions.

[0299] Using Design of Experiments (DoE), the effect of the mass of cationic calixarene CX12 per LNP (expressed as mg of CX12 per mg of RNA) on its physicochemical properties was modeled. The masses of other lipids (DLin-DMA, DOPE, cholesterol, DMG-PEG) and mRNA (firefly luciferase) were kept constant. The experimental and modeling results are shown in Table 8 and Figure 9. LNPs prepared with CX12 in amounts ranging from 0.1 to 20.7 times more than mRNA all exhibited monodispersity and encapsulated more than 95% of the RNA. It was confirmed that the addition of CX12 increased both the size (77–129 nm) and the charge (5.8–11.0 mV).

[0300] [Table 8]

[0301] Example 8: Cationizable calix[4]arene for use in immunogenic compositions

[0302] The findings of this invention are not limited to cationic calix[4]arenes, and similar adjuvant effects were observed for cationizable calix[4]arenes, i.e., calix[4]arenes having a secondary or tertiary amine (preferably a tertiary amine). CX5 was synthesized (Figure 16) and self-assembled with helper lipids (phospholipids), sterols, and PEGylated lipids to form hybrid nanoparticles. Unlike cationic calixarenes, which self-assembled with i-lipids, cationizable calixarenes were used in place of i-lipids in these nanoparticles. The molar ratio was again optimized to produce monodisperse nanoparticles that encapsulate mRNA. In the best nanoparticles generated with CX5 and tested as part of the optimization study, protein expression was limited to 5% of that of LNPs using SM-102 or ALC-0315 as i-lipids (1 μg Fluc mRNA, intramuscular administration to BALB / c mice, estimated radiance 6 hours after administration - data not shown).

[0303] CX5 was evaluated in an immunogenicity study using mRNA encoding rabies-derived glycoprotein G as a model antigen. BALB / c mice were vaccinated following a 21-day prime-boost regimen with either 0.6 μg or 2.5 μg of RNA per dose. As shown in Figure 17, CX5 showed potent viral neutralizing antibody titers (VNT) significantly above the protective correlation value (0.5 IU / mL) in both dosing regimens (0.6 or 2.5 μg). After 15 days (post-priming), the response with CX5 was comparable to that obtained with the two control groups (SM-102 LNP and ALC-0315 LNP), regardless of the dosing regimen (0.6 or 2.5 μg). At 0.6 μg, this response was comparable to that of SM-102 and ALC-0315 LNP even after boosting (35 days) and at the end of the study (65 days). In contrast, at 2.5 μg, the two control groups were shown to slightly outperform CX5 after boosting (35 and 65 days). The observation that CX5 achieved equivalent or slightly lower VNT despite having 1 / 20th the protein expression compared to other LNPs is another demonstration that calix[4]arenes, i.e., cationizable calix[4]arenes in this example, can be used in potent immunogenic compositions with adjuvant effects in nucleic acid-based vaccines.

[0304] Nanoparticles produced with ionizable calixarenes were also confirmed not to induce side effects (weight loss, effects on spleen, liver, and kidney weight, inflammation, or excessive reactionogenic responses) in animals used in in vivo studies.

[0305] The present invention is not limited in any way to the embodiments described and / or illustrated in the examples. Rather, the methods according to the present invention can be carried out in a variety of forms without departing from the scope of the invention.

Claims

1. A delivery system for delivering one or more cargoes to one or more cells, wherein the delivery system comprises a calixarene, a phospholipid, and an additional lipid, the additional lipid being selected from sterols, fatty acids, glycerol monooleate, trioleic acid, or short saturated molecules.

2. The delivery system according to claim 1, wherein the calixarene is a calix[4]arene.

3. The delivery system according to claim 1 or 2, wherein the calixarene is an ionizable calixarene, a cationic calixarene, or any combination thereof.

4. The delivery system according to any one of claims 1 to 3, further comprising an ionizable lipid.

5. The delivery system according to any one of claims 1 to 4, further comprising PEGylated lipids and / or polysarcosine.

6. The delivery system according to any one of claims 1 to 5, wherein the system comprises a calixarene, a phospholipid, a sterol, and a PEG-modified lipid, and the calixarene is an ionizable calixarene.

7. The delivery system according to claim 6, wherein the calixarene is a calix[4]arene having one or more ionizable head groups.

8. The delivery system according to claim 7, wherein one or more of the one or more ionizable head groups comprises a tertiary amine group.

9. The delivery system according to claim 7 or 8, wherein the calixarene comprises four identical ionizable head groups.

10. The delivery system according to any one of claims 6 to 9, wherein the ionizable calixarene is present in the delivery system at a mass fraction of 10 to 60% (w / w).

11. The delivery system according to any one of claims 6 to 10, wherein the phospholipid is present in the delivery system at a mass fraction of 5 to 35% (w / w), more preferably 10 to 30% (w / w).

12. The delivery system according to any one of claims 6 to 11, wherein the sterol is present in the delivery system at a mass fraction of 15 to 50% (w / w).

13. The delivery system according to any one of claims 6 to 12, wherein the PEGylated lipid is present in the delivery system at a mass fraction of 2 to 24% (w / w), more preferably 10 to 24% (w / w).

14. The delivery system according to any one of claims 1 to 5, wherein the system comprises a calixarene, and the calixarene is a cationic calixarene, a phospholipid, a sterol, a PEG-modified lipid, and an ionizable lipid.

15. The delivery system according to claim 14, wherein the calixarene is a calix[4]arene having four cationic head groups.

16. The delivery system according to claim 15, wherein the one or more cationic head groups comprise at least one quaternary amine group.

17. The delivery system according to claim 15 or 16, wherein the calixarene comprises four identical cationic head groups.

18. The delivery system according to any one of claims 14 to 17, wherein the cationic calixarene is present in the delivery system at a mass fraction of 0.1 to 50% (w / w).

19. The delivery system according to any one of claims 14 to 18, wherein the phospholipid is present in the delivery system at a mass fraction of 5 to 35% (w / w), more preferably 10 to 30% (w / w).

20. The delivery system according to any one of claims 14 to 19, wherein the sterol is present in the delivery system at a mass fraction of 15 to 50% (w / w).

21. The delivery system according to any one of claims 14 to 20, wherein the ionizable lipid is present in the delivery system at a mass fraction of 15 to 50% (w / w).

22. The delivery system according to any one of claims 1 to 21, wherein the calixarene is present in the delivery system at a concentration of 0.1 to 60 mol%.

23. The delivery system according to claim 14, wherein the cationic calixarene is present in the delivery system at a concentration of 0.1 to 10 mol%.

24. The delivery system according to claim 6, wherein the ionizable calixarene is present in the delivery system at a concentration of 10 to 60 mol%.

25. The delivery system according to any one of claims 1 to 24, wherein the delivery system comprises a sterol, and the sterol is present in the delivery system at a concentration of 20 to 70 mol%.

26. The delivery system according to any one of claims 1 to 25, wherein the phospholipid is present in the delivery system at a concentration of 1 to 45 mol%.

27. The delivery system according to any one of claims 1 to 26, wherein the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, and sphingolipids.

28. The delivery system according to any one of claims 1 to 27, wherein the delivery system comprises an ionizable lipid and a cationic calixarene, and the total concentration of the ionizable lipid and the cationic calixarene is 10 to 60 mol% of the delivery system.

29. The delivery system according to any one of claims 1 to 28, wherein the system comprises a cargo, the cargo being selected from the group consisting of nucleic acids, proteins, chemical substances, polysaccharides and combinations thereof, preferably the cargo being a nucleic acid, and more preferably the cargo being RNA or DNA.

30. The delivery system according to claim 29, wherein the cargo is self-amplifying RNA (saRNA).

31. The delivery system according to claim 30, comprising an ionizable calixarene, a phospholipid, a sterol, and a PEG-modified lipid, wherein the mass of the ionizable calixarene is 5 to 15 times the mass of the saRNA cargo.

32. A method for delivering one or more cargoes to a subject, comprising administering to the subject a delivery system according to any one of claims 1 to 31.

33. A pharmaceutical composition comprising a delivery system according to any one of claims 1 to 31, used for the treatment and / or prevention of a disease or disorder in a subject.

34. The composition comprises an immunogenic component encapsulated in lipid nanoparticles (LNPs) comprising the calixarene, wherein the LNPs comprising the calixarene have an adjuvant effect in the immunogenic composition, and the use of calixarene in the immunogenic composition.

35. The use according to claim 34, wherein the lipid nanoparticles comprise at least one calixarene which is a cationic calixarene having at least one positively charged portion, or at least one calixarene which is an ionizable calixarene having a portion which can associate with at least one ion and become positively charged.

36. The use according to claim 34 or 35, wherein the positively charged portion is an amine-containing group comprising a secondary, tertiary, and / or quaternary amine.

37. The use according to any one of claims 34 to 36, wherein the calixarene is present in the lipid nanoparticles at a mass fraction of 0.1 to 60% (w / w).

38. The use according to any one of claims 35 to 37, wherein the ionizable calixarene is present in the lipid nanoparticles at a mass fraction of 10 to 60% (w / w).

39. The use according to any one of claims 35 to 37, wherein the cationic calixarene is present in the lipid nanoparticles at a mass fraction of 0.1 to 50% (w / w).

40. The use according to any one of claims 34 to 39, wherein the lipid nanoparticles further comprise ionizable lipids.

41. The use according to claim 40, wherein the ionizable lipid is present in the lipid nanoparticles at a mass fraction of up to 60% (w / w).

42. The use according to any one of claims 35 to 41, wherein the calixarene is an ionizable calix[4]arene having four head groups, and at least one head group comprises at least one secondary or tertiary amine, preferably at least one tertiary amine.

43. The ionizable calix[4]arene has the following structure 【Chemistry 1】 The use according to claim 42, comprising a CX5 having

44. The use according to any one of claims 35 to 41, wherein the calixarene is a cationic calix[4]arene having four head groups, and at least one head group comprises at least one quaternary amine.

45. The cationic calix[4]arene has the following structure 【Chemistry 2】 The use according to claim 44, comprising a CX12 having the following characteristics.

46. The cationic calix[4]arene has the following structure 【Transformation 3】 The use according to claim 44, comprising a CX4 having

47. The use according to any one of claims 34 to 46, wherein the immunogenic component comprises at least one nucleic acid molecule encoding at least one epitope of at least one antigen.

48. The use according to claim 47, wherein the nucleic acid molecule is an mRNA or saRNA molecule.

49. The use according to claim 47 or 48, wherein the mass of the calixarenes contained in the lipid nanoparticles is less than 10 times the mass of the nucleic acids encapsulated in the lipid nanoparticles.

50. The use according to claim 49, wherein the mass of the calixarenes contained in the lipid nanoparticles is less than five times the mass of the nucleic acids encapsulated in the lipid nanoparticles.

51. The use according to claim 50, wherein the mass of calixarenes contained in the lipid nanoparticles is less than the mass of nucleic acids encapsulated in the lipid nanoparticles.

52. The use according to any one of claims 34 to 51, wherein the lipid nanoparticles are lipid nanoparticles further comprising PEG-modified lipids.

53. The use according to claim 52, wherein the lipid nanoparticles further comprise sterols and / or phospholipids.

54. The use according to any one of claims 34 to 53, wherein the lipid nanoparticles comprise an ionizable calixarene and further comprise a PEGylated lipid, a sterol, and a phospholipid.

55. The use according to any one of claims 34 to 53, wherein the lipid nanoparticles comprise a cationic calixarene and further comprise a PEG-modified lipid, a sterol, an ionizable lipid, and a phospholipid.

56. The use according to any one of claims 34 to 55, wherein the immunogenic composition is administered intramuscularly to a subject.

57. A vaccine comprising an immunogenic component encapsulated in lipid nanoparticles, wherein the lipid nanoparticles comprise at least one calixarene molecule, and the lipid nanoparticles act as an adjuvant in the vaccine.

58. The vaccine according to claim 57, wherein the vaccine is a DNA vaccine.

59. The vaccine according to claim 57, wherein the vaccine is an RNA vaccine.

60. The vaccine according to any one of claims 57 to 59, wherein the vaccine is a cancer vaccine.

61. The vaccine according to any one of claims 57 to 60, wherein the lipid nanoparticles enhance the immunogenicity of the immunogenic component, as measured by an increase in viral neutralizing antibody titer (VNT), after in vivo administration to a subject, compared to when a subject is administered a vaccine comprising the same immunogenic component encapsulated in lipid nanoparticles that do not contain calixarenes.

62. A method for preparing an immunogenic composition, comprising the step of encapsulating an immunogenic component in lipid nanoparticles, wherein the lipid nanoparticles comprise at least one calixarene molecule, and the lipid nanoparticles comprising the calixarene have an adjuvant effect.

63. The method according to claim 62, wherein the calixarene is an ionizable calixarene or a cationic calixarene.

64. The method according to claim 62 or 63, wherein the immunogenic component is a nucleotide, such as an mRNA or saRNA molecule.

65. (a) A lipid nanoparticle (LNP) adjuvant containing calixarenes, (b) comprising an antigen, The composition induces an enhanced antibody response to the antigen after administration of the composition, compared to an antibody response to the antigen when the antigen is encapsulated in an LNP that does not have calixarenes.

66. The aforementioned LNP adjuvant is Ionizable lipids, Cationic calix[4]arene and Phospholipids and The composition according to claim 65, comprising a PEGylated lipid.

67. The composition according to claim 66, wherein the ionizable lipid is selected from DODAP, DLin-DMA, Dlin-MC3-DMA, ALC-0315, or SM-102 and any combination thereof.

68. The composition according to any one of claims 65 to 67, further comprising sterols.

69. The cationic calix[4]arene has the following structure 【Chemistry 4】 The composition according to any one of claims 66 to 68, comprising CX12 having

70. The cationic calix[4]arene has the following structure 【Transformation 5】 The composition according to any one of claims 66 to 68, comprising CX4 having

71. The aforementioned LNP adjuvant is Ionizable calix[4]arenes and Phospholipids and PEG-converted lipids and The composition according to claim 65, comprising:

72. The composition according to claim 71, further comprising sterols.

73. The ionizable calix[4]arene has the following structure 【Transformation 6】 The composition according to claim 71 or 72, comprising CX5 having the following.

74. The composition according to claim 73, wherein the concentration of CX5 is 12 mol% in the LNP, the phospholipid comprises DOPE at a concentration of 25 mol% in the LNP, the sterol comprises cholesterol at a concentration of 60 mol% in the LNP, and the PEGylated lipid comprises DMG-PEG2000 at a concentration of 3 mol% in the LNP.

75. The composition according to claim 74, wherein the antigen is an RNA molecule, and the RNA molecule is encapsulated in the LNP with an N:P ratio of 4 to 8, for example, 6.

76. The composition according to any one of claims 65 to 75, wherein the composition does not induce a toxic response in a subject when administered to a subject.

77. The composition according to any one of claims 65 to 70, wherein the cationic calixarene in the LNP adjuvant has a mass of about 20% to 500% of the mass of the antigen.

78. The composition according to claim 77, wherein the mass of the cationic calixarene in the LNP adjuvant is about 35% of the antigen mass.

79. The composition according to claim 76, wherein the toxic response comprises weight loss, effects on organ weight, inflammation, or an excessive reactiongenic response.

80. A method for enhancing the immune response in a subject, comprising administering a therapeutically effective dose of the composition according to any one of claims 65 to 79, the delivery system according to any one of claims 1 to 31, the pharmaceutical composition according to claim 33, or the vaccine according to any one of claims 57 to 61.

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