Ionizable cationic compounds

Abstract

Provided is a novel ionizable lipid compound of formula I. The use of the compound in forming lipid nanoparticles is described. Lipid nanoparticles can encapsulate therapeutic agents such as nucleic acids, which can be used in the delivery of therapeutic agents and methods for treating certain conditions or in methods for inducing immune responses.

Description

[Technical Field]

[0001] The present disclosure relates to cationic and / or ionizable lipid compounds that can be combined with other lipid molecules to form lipid nanoparticles for delivering polynucleotides to a subject. [Background technology]

[0002] Nucleic acid-based therapies have shown substantial promise in a variety of therapeutic applications. However, delivery of polynucleotides such as messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotides, plasmids, and DNA presents several challenges. Free nucleic acids, such as RNA, are subject to rapid enzymatic degradation and generally do not persist systematically. Furthermore, due to their negative charge, nucleic acids may not be able to effectively cross cellular barriers to enter the necessary intracellular compartments, for example, for translation or to otherwise achieve their effects.

[0003] Therefore, lipid particles such as lipid nanoparticles (LNPs) have been used to formulate nucleic acids to protect them from degradation and improve cellular uptake and intracellular delivery. LNPs are generally formed from ionizable cationic lipids and other lipid components, such as neutral lipids, cholesterol, and sterols, such as PEGylated lipids. Ionizable cationic lipids are amphiphilic molecules with a lipophilic region containing one or more hydrocarbon groups and a hydrophilic region containing at least one positively charged or ionizable polar head group. Such cationic lipids can be ionized at an appropriate pH and then form a positively charged complex with nucleic acids, making it easier for the nucleic acids to cross the plasma membrane of cells and enter the cytoplasm.

[0004] Onpattro (patisiran), the first siRNA therapeutic to be approved, entered the market just a few years ago for the treatment of hereditary amyloidogenic transthyretin (TTR) amyloidosis. Patisiran's therapeutic efficacy relies on siRNA-mediated TTR gene silencing, preventing the production of mutant proteins and at least halting disease progression. Efficient delivery of siRNA relies on LNP technology. More recently, nucleic acid vaccines have emerged as a promising approach for the treatment and prevention of various diseases, including those against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the ongoing global pandemic of severe infectious coronavirus disease 2019 (COVID-19). mRNA vaccines rely on the delivery of mRNA into the cytoplasm of host cells, where it is transcribed into antigenic proteins and elicits the production of an immune response. The large size and negative charge of mRNA hinder cellular uptake, so LNP is again required for proper delivery.

[0005] It will be apparent to those skilled in the art that there is a continuing need for improved ionizable cationic lipid compounds that are suitable for forming lipid particles, such as lipid LNPs, for delivery of nucleic acids and polynucleotides, because different ionizable cationic lipids exhibit different physicochemical profiles, including their acid dissociation constant (pKa) values, which may affect their ability to form complexes with nucleic acids as well as exhibit different toxicity profiles in vivo. Summary of the Invention

[0006] The present invention provides novel ionizable cationic lipid compounds, and their pharmaceutically acceptable salts, prodrugs, and stereoisomers, which can form lipid particles (e.g., LNPs) in the presence of additional lipids, including one or more of neutral lipids, charged lipids, structured lipids, PEGylated lipids, and their analogs, and can be used to deliver polynucleotides.Compositions containing such lipid particles, methods for forming lipid particles, their use in delivering polynucleotides, and methods for using lipid particles in the treatment of various diseases, disorders, and conditions are provided.

[0007] Thus, in one embodiment, the present disclosure provides a compound of formula I: [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: R 1 is selected from the group consisting of formulas IA-II, [ka] Z, A, and B are each independently selected from the group consisting of -C(H)2-, -N(H)-, -O-, and -S-, and at least one of Z, A, and B is -N(H)-, -O-, or -S-; n and q are each independently an integer from 0 to 8; t, u, v, and w are each independently an integer from 0 to 10; D, E, F, and G are each independently a straight-chain or branched C(H)2-. 1-16 -alkyl, [ka] represents a single or double bond, and R 2 and R 3 are each independently C 1-8 -Alkyl and C 1-8 -alkanols.

[0008] In embodiments, lipid nanoparticles (LNPs) comprising a compound of formula I are provided.

[0009] In embodiments, the LNP further comprises a polynucleotide.

[0010] In embodiments, pharmaceutical compositions are provided comprising such LNPs and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0011] In another embodiment, a method of forming an LNP comprising a compound of formula I is provided.

[0012] In additional embodiments, methods are provided for delivering polynucleotides within the aforementioned LNPs to cells.

[0013] In a further embodiment, a method for producing a polypeptide of interest in a cell is provided.

[0014] In yet another embodiment, a method of treating a disease, disorder, or condition in a subject is provided by administering to a subject in need of such treatment one or more of the aforementioned LNPs comprising a polynucleotide, or pharmaceutical compositions comprising same.

[0015] An LNP comprising a polynucleotide, or a pharmaceutical composition comprising the same, can be delivered to a subject as a component of a vaccine. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure is based on the use of certain novel biodegradable lipid compounds as components of LNPs for polynucleotide delivery. The novel lipid compounds exhibit biodegradable groups that may help reduce toxicity or improve clearance in vivo.

[0017] In one embodiment, the LNPs may be components of a vaccine, although the therapeutic uses of the compounds described herein and the LNPs of which they form components are not so limited.

[0018] In some embodiments, the LNPs formed may be suitable for delivery of messenger RNA (mRNA).

[0019] In some embodiments, the LNPs formed may be suitable for delivery of mRNA as a component of an mRNA vaccine.

[0020] overview Throughout this specification, unless specifically stated otherwise or the context otherwise requires, reference to a single step, composition, group of steps, or group of compositions shall be interpreted as encompassing one and more (i.e., one or more) of that step, composition, group of steps, or group of compositions.

[0021] Those skilled in the art will understand that the present disclosure is susceptible to variations and modifications other than those specifically described. It should be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or indicated in this specification, as well as any and all combinations or any two or more of such steps or features.

[0022] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0023] Any embodiment of the present disclosure herein shall apply mutatis mutandis to any other embodiment of the present disclosure unless expressly stated otherwise.

[0024] Unless specifically defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., synthetic organic chemistry, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0025] Unless otherwise indicated, all recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0026] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or either meaning.

[0027] When the terms "from" and "to" are used to denote a range, it should be understood that the range includes the recited lower and upper limits. For example, "p is an integer from 0 to 6" should be understood to include the case where p is not present (p is 0), the case where p is 6, and each natural integer value therebetween, i.e., p is 1, 2, 3, 4, or 5.

[0028] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0029] As used herein, the term "derived from" shall be interpreted to indicate that a particular integer may be obtained from a particular source, but is not necessarily obtained directly from that source.

[0030] Selected Definitions As used herein, the terms "lipid particle," "lipid nanoparticle," or "LNP" are understood to refer to lipid-based particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and comprising a compound of any formula described herein. In embodiments, LNPs are formulated in compositions for delivery of polynucleotides to desired targets, such as cells, tissues, organs, tumors, etc. LNPs generally comprise an ionizable cationic compound of the present disclosure and one or more of a neutral lipid, a charged lipid, a sterol, and a PEGylated lipid. In embodiments, lipid particles or LNPs may be selected from liposomes or vesicles in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single; unilamellar, or multiple; multilamellar) micelle-like lipid nanoparticle having a non-aqueous core and solid lipid nanoparticles. In embodiments, lipid nanoparticles or LNPs may have a structure comprising a single monolayer or bilayer of lipids encapsulating a solid phase. In preferred embodiments, lipid nanoparticles or LNPs do not have an aqueous or other liquid phase within them.

[0031] The terms "cationic compound", "ionizable cationic compound", "cationic lipid compound", "ionizable cationic lipid compound" and the like refer to lipid compounds of any structural formula described herein, which can have a positive charge.The ionizable cationic lipids disclosed herein contain one or more nitrogen-containing groups that can have a positive charge.They are ionizable so that they can exist in a positively charged or neutral form depending on pH.The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions.

[0032] The term "neutral lipid" refers to any of several lipid species that exist in either uncharged or neutral zwitterionic form at selected pH.At physiological pH, such lipids include but are not limited to phosphotidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM).

[0033] The term "charged lipid" refers to any of several lipid species that exist in either positively or negatively charged form, independent of pH within a useful physiological range (e.g., from about pH 3 to about pH 9). Non-limiting examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylmositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (including DOTAP and DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoylsterol.

[0034] As used herein, the term "polynucleotide" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), self-amplifying messenger RNA (saRNA or samRNA), antisense RNA, miRNA, micRNA, polyvalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Polynucleotides include those containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring and have similar binding properties to the reference polynucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, the term encompasses polynucleotides containing known analogs of natural nucleotides that have similar binding properties to the reference polynucleotide.Unless otherwise indicated, a specific nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the explicitly indicated sequence.Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081(1991); Ohtsuka et al., J.Biol.Chem., 260:2605-2608(1985); Rossolini et al., Mol.Cell.Probes, 8:91-98(1994)).

[0035] An "effective amount" or "therapeutically effective amount" of a therapeutic polynucleotide is an amount sufficient to produce a desired effect, such as increased or inhibited expression of a target sequence, compared to normal expression levels detected in the absence of the polynucleotide. Suitable assays for measuring expression of a target gene or target sequence include examination of protein or RNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, and phenotypic assays.

[0036] As used herein, "prodrug" refers to a compound that can be converted into any one or more compounds of the formulas described herein under physiological conditions or by solvent decomposition. Thus, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of such a compound. A prodrug may be inactive when administered to a subject in need thereof, but is converted into an active form in vivo. This term may also include any covalently bonded carrier that releases an active compound in vivo when such a prodrug is administered to a mammalian subject. Prodrugs of compounds of Formula I or other formulas described herein can be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved to the parent compound either by routine manipulation or in vivo.

[0037] The term "pharmaceutically acceptable carrier, diluent, or excipient" refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving an active compound), and has substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example, anti-adhesives, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (pigments), softeners, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, glidants (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other species disclosed herein.

[0038] "Pharmaceutically acceptable salts" includes both acid and base addition salts. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977). Acid addition salts are not biologically or otherwise undesirable, and retain the biological effectiveness and properties of the free bases, and include those derived from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, Those formed with organic acids such as glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Base addition salts are those that retain the biological effectiveness and properties of the free acids which are not biologically or otherwise undesirable. These salts are prepared from the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like.Preferred inorganic salts are ammonium salt, sodium salt, potassium salt, calcium salt and magnesium salt.The salt derived from organic bases includes but is not limited to the salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dianol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzethine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc.

[0039] As used herein, "stereoisomers" refer to compounds that are composed of the same atoms connected by the same bonds, but have different three-dimensional structures and are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0040] As used herein, the term "biodegradable group" refers to a group that can promote faster metabolism of lipids in mammals. Esters are preferred biodegradable groups, and the compounds of the present disclosure exhibit two such groups for improved in vivo biodegradability.

[0041] As used herein, "encapsulation efficiency" refers to the amount of polynucleotide that becomes part of an LNP composition relative to the initial total amount of polynucleotide used to prepare the LNP composition. For example, if 92 mg of polynucleotide is encapsulated in the LNP composition out of a total of 100 mg of polynucleotide initially provided in the composition, the encapsulation efficiency may be expressed as 92%. As used herein, "encapsulation" may refer to complete, substantial, or partial entrapment, entrapment, surrounding, or envelopment.

[0042] As used herein, the term "subject" means any animal, such as a mammal, and is intended to include humans. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0043] As used herein, the term "mammal" includes both humans and domestic animals, such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wild animals.

[0044] As used herein, the term "alkyl" encompasses both straight chain (i.e., linear) and branched chain hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, pentyl, and hexyl groups. In one example, an alkyl group contains 1 to 16 carbon atoms (i.e., C 1-16 alkyl).

[0045] As used herein, the term "alkanol" encompasses both straight chain (i.e., linear) and branched chain hydrocarbon groups substituted with an -OH (alcohol) group. Examples of alkanol groups include methanol, ethanol, and propanol groups. In one example, an alkanol group contains 1 to 5 carbon atoms (i.e., C1-5 alkanol).

[0046] compound In embodiments, the compounds of the present disclosure may offer advantages over other selected prior art ionizable cationic lipid compounds, including one or more of improved complexation with polynucleotides, beneficial pKa properties, improved encapsulation efficiency as part of LNPs, reduced toxicity, improved biodegradability, improved in vivo clearance, desirable N:P ratios when complexed with polynucleotides, desirable polydispersity indexes of LNPs containing them, and improved LNP formation.

[0047] In one broad aspect, the disclosure provides a compound of formula I: [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: R 1 is selected from the group consisting of formulas IA-II, [ka] Z, A, and B are each independently selected from the group consisting of -C(H)2-, -N(H)-, -O-, and -S-, and at least one of Z, A, and B is -N(H)-, -O-, or -S-; n and q each independently represent an integer of 0 to 8; t, u, v, and w are each independently an integer of 0 to 10; D, E, F, and G are each independently a straight-chain or branched C 1-16 -alkyl, [ka] represents a single or double bond, R 2 and R 3 are each independently C 1-8 -Alkyl and C 1-8-alkanols.

[0048] In compounds of formula I, R 1 is selected from the group consisting of formulas IA-II. [ka]

[0049] Thus, in embodiments, in compounds of formula I, R 1 is a group of formula IA. [ka]

[0050] In another embodiment, in the compound of formula I, R 1 is a group of formula IB [ka]

[0051] In another embodiment, in the compound of formula I, R 1 is a group of formula IC. [ka]

[0052] In another embodiment, in the compound of formula I, R 1 is the basis of the formula ID. [ka]

[0053] In another embodiment, in the compound of formula I, R 1 is a group of formula IE. [ka]

[0054] In another embodiment, in the compound of formula I, R 1 is a group of formula IF. [ka]

[0055] In another embodiment, in the compound of formula I, R 1 is a group of formula IG. [ka]

[0056] In another embodiment, in the compound of formula I, R 1 is a group of formula IH. [ka]

[0057] In another embodiment, in the compound of formula I, R 1 is a group of formula II. [ka]

[0058] In compounds of Formula I, Z, A, and B are each independently selected from the group consisting of -C(H)2-, -N(H)-, -O-, and -S-.

[0059] In embodiments, Z is independently selected from the group consisting of -C(H)2-, -N(H)-, -O-, and -S-. In embodiments, Z is -C(H)2-. In embodiments, Z is -N(H)-. In embodiments, Z is -O-. In embodiments, Z is -S-. In one embodiment, Z is selected from the group consisting of -N(H)-, -O-, and -S-.

[0060] In embodiments, A is independently selected from the group consisting of -C(H)2-, -N(H)-, -O-, and -S-. In embodiments, A is selected from the group consisting of -N(H)-, -O-, and -S-. In embodiments, A is -C(H)2-. In embodiments, A is -N(H)-. In embodiments, A is -O-. In embodiments, A is -S-.

[0061] In embodiments, B is independently selected from the group consisting of -C(H)2-, -N(H)-, -O-, and -S-. In embodiments, B is selected from the group consisting of -N(H)-, -O-, and -S-. In embodiments, B is -C(H)2-. In embodiments, B is -N(H)-. In embodiments, B is -O-. In embodiments, B is -S-.

[0062] It will be understood that Z, A, and B are independently selected from one another. Thus, one or more of Z, A, and B can be the same (e.g., Z, A, and B are -S-, or Z is -S-, A is -S-, and B is -O-), or alternatively, each can be different (e.g., Z is -O-, A is -S-, and B is -N(H)-).

[0063] In the compound of formula I, at least one of Z, A, and B is -N(H)-, -O-, or -S-. That is, each of Z, A, and B cannot simultaneously be -C(H)2-. That is, the compound of formula I is [ka] It cannot be.

[0064] In compounds of Formula I, n and q are each independently an integer from 0 to 8. That is, in embodiments, n and q are each independently an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8. In embodiments, n is an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8. In embodiments, q is an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8. The integers determine the carbon length encompassed in the associated parentheses. In embodiments, n and q are each independently an integer from 0 to 4 (i.e., 0, 1, 2, 3, or 4). In embodiments, n is 2. In embodiments, q is 2. In embodiments, n is 2 and q is 2.

[0065] It will be understood that n and q are independently selected from one another, and thus n and q can be the same integer (e.g., n is 2 and q is 2), or alternatively, can each be a different integer (e.g., n is 2 and q is 3).

[0066] It will be understood that when n is 0, the carbon chain contained within the associated brackets is absent. Instead, adjacent carbon atoms are directly bonded to adjacent Z groups. Similarly, when q is 0, the carbon chain contained within the associated brackets is absent. Instead, adjacent Z groups are directly bonded to adjacent R groups. 1 is directly bonded to the group.

[0067] When n or q is an integer greater than 1, and thus there are at least two integer carbon chains (i.e., at least -C(H)2-C(H)2-), the carbon chain is linear in structure (i.e., linear C2-alkyl). For example, when n or q is 3, the carbon chain will have the structure -C(H)2-C(H)2-C(H)2- (i.e., linear C3-alkyl). Similarly, when n or q is 4, for example, the carbon chain will have the structure -C(H)2-C(H)2-C(H)2-C(H)2- (i.e., linear C4-alkyl). That is, when n or q is greater than 1, there is no branching of the carbon chain.

[0068] In compounds of Formula I, t, u, v, and w are each independently an integer between 0 and 10. That is, in embodiments, t, y, v, and w are each independently an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In embodiments, t is an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In embodiments, u is an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In embodiments, v is an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In embodiments, w is an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The integers determine the carbon length encompassed by the associated parentheses.

[0069] It will be understood that t, u, v, and w are independently selected from one another, and thus one or more of t, u, v, and w can be the same integer (e.g., t is 2, u is 2, v is 2, and w is 2), or alternatively, can each be a different integer (e.g., t is 2, u is 3, v is 4, and w is 5).

[0070] When t is 0, it will be understood that the carbon chain contained within the associated parentheses is absent. Instead, adjacent carbon atoms are directly bonded to adjacent A groups. Similarly, when u is 0, it will be understood that the carbon chain contained within the associated parentheses is absent. Instead, adjacent carbon atoms are directly bonded to B groups. When v is 0, it will be understood that the carbon chain contained within the associated parentheses is absent. Instead, adjacent carbon atoms are directly bonded to A groups. When w is 0, it will be understood that the carbon chain contained within the associated parentheses is absent. Instead, adjacent carbon atoms are directly bonded to B groups.

[0071] When t, u, v, or w is an integer greater than 1, and thus there are at least two integer carbon chains (i.e., at least -C(H)2-C(H)2-), the carbon chain is linear in structure (i.e., a linear C2-alkyl), as described above for n and q.

[0072] In the compound of formula I, D, E, F, and G are each independently a straight or branched C 1-16 In an embodiment, D is a straight or branched C 1-16 In an embodiment, E is a straight or branched C 1-16 In an embodiment, E is a straight or branched C 1-16 In embodiments, F is a straight or branched C 1-16 -alkyl, that is, D, E, F, and G can each independently be C1-alkyl, C2-alkyl, C3-alkyl, C4-alkyl, etc.

[0073] It will be understood that D, E, F, and G are independently selected from one another. Thus, one or more of D, E, F, and G may be selected from the same linear or branched C 1-16 - alkyl groups, or alternatively, each of which is a different linear or branched C 1-16 In embodiments, D, E, F, and G can each independently be a linear C1-16 In embodiments, D, E, F, and G are each independently a straight-chain C 1-8 In embodiments, at least one of D, E, F, and G is a branched C 1-16 In embodiments, at least one of D, E, F, and G is a branched C 1-8 - alkyl.

[0074] D, E, F, and G are all in branch C. 1-16 -alkyl group, it will be understood that at least one carbon chain continues within the structure to connect to the adjacent atom / group as shown in the structure of Formula I.

[0075] In the compounds of formula I, [ka] represents a single or double bond. [ka] are independent of each other and mean that compounds of formula I can contain 0, 1, or 2 double bonds. [ka] is a single bond. In an embodiment, [ka] is a double bond.

[0076] In compounds of formula I, and particularly of formula IA and formula IB, R 2 and R 3 are each independently C 1-8 -Alkyl and C 1-8 In an embodiment, R 2 is C 1-8 In embodiments, R 2 is C1-8 In embodiments, R 3 is C 1-8 In embodiments, R 3 is C 1-8 - alkyl.

[0077] R 2 and R 3 It will be understood that R are independently selected from each other. 2 and R 3 Each is the same linear or branched C 1-8 -Alkyl group or linear or branched C 1-8 -alkanol groups, or alternatively, each of which may be a different linear or branched C 1-8 -Alkyl group or linear or branched C 1-8 -alkanol groups.

[0078] In embodiments, R 2 is C 1-8 In embodiments, R 2 is C 1-3 In one embodiment, R 2 is C-alkyl. In one embodiment, R 2 is C2-alkyl. In one embodiment, R 2 is a C3-alkyl.

[0079] R 1 is formula IA, and R 3 If there is R 3 is C 1-8 In embodiments, R 3 is C 1-3 In one embodiment, R 3 is C-alkyl. In one embodiment, R 3 is C2-alkyl. In one embodiment, R 3 is a C3-alkyl.

[0080] As noted above, in embodiments, R 1is selected from the group consisting of Formula IA and Formula IB.

[0081] In embodiments, formula IA is: [ka] is selected from the group consisting of:

[0082] In one embodiment, Formula IA is Formula II-A. In one embodiment, Formula IA is Formula II-B. In one embodiment, Formula IA is Formula II-C.

[0083] In embodiments, formula IB is: [ka] is selected from the group consisting of:

[0084] In one embodiment, Formula IB is Formula II-D. In one embodiment, Formula IB is Formula II-E. In one embodiment, Formula IB is Formula II-F.

[0085] In any embodiment of Formula I, A and B may be the same. For example, A and B may be selected from the group consisting of -C(H)2-, -O-, and -S-. Z may be selected from the foregoing group, independently of A and B.

[0086] In certain embodiments, when A and B are the same, Z may be selected from -C(H)2-, and -O-.

[0087] In an embodiment, the compound of formula I is a compound of formula III-A: [ka] In the formula, R 2 , R 3 , Z, A, B, D, E, F, and G are as defined herein.

[0088] In an embodiment, the compound of formula I is a compound of formula IV-A: [ka] In the formula, R 2 , R 3 , A, B, D, E, F, and G are as defined herein.

[0089] In embodiments, the compound of formula I is a compound of formula VA: [ka] In the formula, R 2 , R 3 , D, E, F, and G are as defined herein.

[0090] In embodiments, the compound of formula I is [ka] is selected from the group consisting of:

[0091] lipid nanoparticles The present disclosure provides LNPs for delivering polynucleotides, such as RNA, wherein the LNPs comprise a compound of the present disclosure.

[0092] In embodiments, the LNPs are about 30 nm to about 160 nm, about 40 nm to about 160 nm, about 50 nm to about 160 nm, about 60 nm to about 160 nm, about 70 nm to about 160 nm, about 50 nm~about 140nm, about 60nm~about 130nm, about 70nm~about 120nm, about 80nm~about 120nm, about 90nm~about 120nm, about 70~about 110nm, about 80nm~about The LNPs have an average diameter of 110 nm, or about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, or 160 nm. The diameter of the LNPs can be measured by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), or other methods as known in the art.

[0093] In some embodiments, LNPs can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of LNPs. A small polydispersity index, e.g., less than 0.3 or less than 0.2, generally indicates a narrow particle size distribution. LNP compositions described herein can have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of an LNP composition can be about 0 to about 0.20 or 0.05 to 0.20.

[0094] LNPs may, where appropriate, include two or more compounds of Formula I or IA-I-N or II-A-II-N. Including two or more such compounds can be used, for example, to achieve a desired pKa profile.

[0095] The LNPs can include a compound of Formula I and additional cationic and / or ionizable lipids, such as cationic and / or ionizable lipids that include cyclic or acyclic amines. Such additional cationic and / or ionizable lipids can include: 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), and It may be selected from the non-limiting group consisting of 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester.

[0096] In embodiments, the LNP further comprises one or more of a PEG lipid, a sterol structure lipid, and / or a neutral lipid.

[0097] PEGylated lipids In one embodiment, the present disclosure provides an LNP comprising a compound of the present disclosure and a PEGylated lipid.

[0098] It will be clear to those skilled in the art that the reference to PEGylated lipid refers to the lipid modified with polyethylene glycol.Exemplary PEGylated lipid includes, but is not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.For embodiments, PEG lipid includes PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipid, and combinations thereof.

[0099] neutral lipid In one embodiment, the present disclosure provides an LNP comprising a compound of the present disclosure and a neutral lipid.

[0100] For example, suitable neutral or zwitterionic lipids for use in the present disclosure will be apparent to those of skill in the art, and in embodiments include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-di Palmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero Lipids include 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. The lipids can be saturated or unsaturated.

[0101] structural lipids In one embodiment, the present disclosure provides an LNP comprising a compound of the present disclosure and a structured lipid.

[0102] Exemplary structural lipids include, but are not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol.

[0103] In one embodiment, the structured lipid is a sterol. In an embodiment, the structured lipid is cholesterol. In another embodiment, the structured lipid is campesterol.

[0104] In embodiments, the LNPs comprise an ionizable cationic lipid compound of the present disclosure, a neutral lipid, a sterol such as cholesterol, and a PEGylated lipid. The LNPs are formulated with a polynucleotide to be delivered to a subject.

[0105] Polynucleotides The compounds of the present disclosure can be complexed with a range of polynucleotides and formulated into LNPs, including, but not limited to, mRNA, siRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimia), messenger RNA interference complementary RNA (micRNA), DNA, polyvalent RNA, Dicer substrate RNA, complementary DNA (cDNA), etc. In this manner, LNPs and compositions, in some embodiments, can be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with LNPs comprising one or more novel compounds of the present disclosure, the LNPs encapsulating or associated with a polynucleotide, e.g., mRNA or a plasmid encoding the desired protein, that is expressed to produce the desired protein. In alternative embodiments, the LNPs and compositions may be used to reduce target gene and protein expression in vitro or in vivo by contacting cells with LNPs comprising one or more novel compounds of the present disclosure, where the LNPs encapsulate or are associated with a target gene expression-reducing polynucleotide, such as an antisense oligonucleotide or siRNA.

[0106] Therefore, in some embodiments, polynucleotide is mRNA that encodes the polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide.The polypeptide that is encoded by mRNA can be of any size and have any secondary structure or activity.In some embodiments, the polypeptide that is encoded by mRNA can have therapeutic effect when expressed in cells.

[0107] In other embodiments, the polynucleotide is an siRNA that can selectively knock down or downregulate the expression of a gene of interest.For example, the siRNA can be selected to silence a gene associated with a specific disease, disorder, or condition when the LNP composition containing the siRNA is administered to a subject in need thereof.The siRNA can comprise a sequence that is complementary to the mRNA sequence that codes for the gene or protein of interest.In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0108] In some embodiments, polynucleotide is shRNA or its encoding vector or plasmid.ShRNA can be produced in target cell when appropriate construct is delivered to nucleus.The construct and mechanism of shRNA are well known in the relevant technical field.

[0109] A polynucleotide useful for formulation with an LNP incorporating an ionizable cationic compound of the present disclosure may include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region (e.g., a 5'-UTR) located at the 5'-end of the first region, a second flanking region (e.g., a 3'-UTR) located at the 3'-end of the first region, at least one 5'-cap region, and a 3'-stabilizing region. In some embodiments, the polynucleotide further includes a polyA region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, the polynucleotide may contain one or more intron sequences that can be excised from the polynucleotide. In some embodiments, the polynucleotide (e.g., mRNA) may include a 5'-cap structure, a chain-terminating nucleotide, a stem-loop, a polyA sequence, and / or a polyadenylation signal. Any one of the regions of the polynucleotide may include one or more alternative components (e.g., alternative nucleosides). For example, the 3'-stabilizing region can contain alternative nucleosides, e.g., L-nucleosides, inverted thymidines, or 2'-O-methyl nucleosides, and / or the coding region, 5'-UTR, 3'-UTR, or cap region can include alternative nucleosides, e.g., 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudoisocytidine uridines (e.g., 1-methyl-pseudoisocytidine uridine or 1-ethyl-pseudoisocytidine uridine), and / or 5-substituted cytidines (e.g., 5-methyl-cytidine).

[0110] Exemplary polynucleotides useful for formulation with LNPs incorporating ionizable cationic compounds of the present disclosure include a first region of linked nucleosides encoding an antigenic polypeptide, a first flanking region (e.g., a 5'-UTR) located at the 5'-end of the first region, a second flanking region (e.g., a 3'-UTR) located at the 3'-end of the first region, at least one 5'-cap region, and a 3'-stabilizing region.

[0111] Polynucleotides suitable for use with the present LNPs may contain one or more naturally occurring components, including any of the standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3'-UTR, (d) the poly-A tail, and any combination (of a, b, c, or d above) contain the naturally occurring standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).

[0112] In some embodiments, polynucleotides may contain one or more alternative components as described herein, which confer useful properties, including improved stability of the cells into which the polynucleotide is introduced and / or the lack of substantial induction of natural immune responses.For example, alternative polynucleotides exhibit reduced degradation in the cells into which the polynucleotide is introduced, compared with the corresponding unmodified polynucleotide.These alternative species may enhance the efficiency of protein production, intracellular retention of the polynucleotide, and / or the viability of contacted cells, and may also have reduced immunogenicity.

[0113] Polynucleotides can be naturally occurring or non-naturally occurring. Polynucleotides can contain one or more modified (e.g., altered or substituted) nucleobases, nucleosides, nucleotides, or combinations thereof. Polynucleotides can contain any useful modifications or alterations, such as nucleobases, sugars, or internucleoside linkages (e.g., phosphate linkages / phosphodiester linkages / phosphodiester backbones). In some embodiments, one or more alterations are present in each of the nucleobases, sugars, and internucleoside linkages.

[0114] A polynucleotide may or may not be uniformly modified along the entire length of the molecule, for example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or any one or more or all of A, G, U, C) may or may not be uniformly modified in a polynucleotide or a given, predetermined sequence region thereof.

[0115] Different sugar modifications and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in a polynucleotide. Those skilled in the art will understand that nucleotide analogs or other modification(s) can be placed at any position(s) of a polynucleotide so as not to substantially reduce the function of the polynucleotide. Modifications can be 5'- or 3'-terminal modifications. In some embodiments, a polynucleotide comprises a modification at the 3'-terminus.

[0116] Nucleobase substitutes Alternative nucleosides and nucleotides can include alternative nucleic acid bases. The nucleic acid base of a polynucleotide is an organic base such as a purine or pyrimidine, or a derivative thereof. The nucleic acid base can be a standard base (for example, adenine, guanine, uracil, thymine, and cytosine). These nucleic acid bases can be modified or completely replaced to provide a polynucleotide molecule with enhanced properties, such as increased stability, for example, resistance to nucleases. Non-standard or modified bases can include one or more substitutions or modifications, including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more condensed or open rings; oxidation; and / or reduction.

[0117] Alternative nucleotide base pairing encompasses not only standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between alternative nucleotides, including nucleotides and / or non-standard or alternative bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard base and a standard base, or between two complementary non-standard base structures. One example of such non-standard base pairing is base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil.

[0118] In some embodiments, the nucleobase is a substituted uracil. Exemplary nucleobases and nucleosides having a substituted uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio-uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m3U). , 5-methoxy-uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm5s2U), 5-aminomethyl-2 -thio-uracil (nm5s2U), 5-methylaminomethyl-uracil (mnm5U), 5-methylaminomethyl-2-thio-uracil (mnm5s2U), 5-methylaminomethyl-2-seleno-uracil (mnm5se2U), 5-carbamoylmethyl-uracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uracil (cmnm5s2U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τ m5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (mψ), 1-ethyl-pseudouridine (Et1ψ), 5-methyl-2-thio-uracil (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm5s2U), 5,2'-O-di Methyl-uridine (m5Um), 2-thio-2'-O_methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)- Examples of modified uracils include 2'-O-methyl-uridine (inm5Um), 1-thio-uracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil, and 5-[3-(1-E-propenylamino)]uracil. In one example, the modified uracil is pseudouridine. In one example, the modified uracil is N1-methyl-pseudouridine.

[0119] In some embodiments, the nucleobase is a cytosine substitute. Exemplary nucleobases and nucleosides having a cytosine substitute include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza- Pseudoisocytidine, Zebularine, 5-Aza-Zebularine, 5-Methyl-Zebularine, 5-Aza-2-Thio-Zebularine, 2-Thio-Zebularine, 2-Methoxy-Cytosine, 2-Methoxy-5-Methyl-Cytosine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, Lysidine (k2C), 5,2'-O-Dimethyl-Cytidine (m5Cm), N4-Aza- These include cetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine. In one example, the modified cytosine is 5-methyl-cytosine.

[0120] In some embodiments, the nucleobase is an adenine substitute. Exemplary nucleobases and nucleosides having an adenine substitute include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methylpurine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8- Aza-2,6-diaminopurine, 1-methyl-adenine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarba moyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl- These include adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0121] In some embodiments, the nucleobase is a guanine substitute. Exemplary nucleobases and nucleosides having a guanine substitute include inosine (I), 1-methylinosine (mll), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OHyW), unmodified hydroxywyobutosine (OHyW*), 7-deaza-guanine, queuosine (Q ), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQ0), 7-aminomethyl-7-deaza-guanine (preQ1), archeosine (G+), 7-deaza-8-aza-guanine, 6-thioguanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl N-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl -6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (mlm), 1-thio-guanine, and O-6-methyl-guanine.

[0122] The alternative nucleobase of a nucleotide can independently be a purine, a pyrimidine, a purine, or a pyrimidine analog. For example, the nucleobase can be an alternative to adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase is selected from the group consisting of, for example, pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine, or 1,3,5-triazine.

[0123] Polynucleotides for formulation using LNPs containing compounds of the present disclosure can be prepared according to any available technique known in the art. For example, mRNA can be prepared by enzymatic synthesis, which provides a process for template-directed synthesis of RNA molecules from an engineered DNA template consisting of an upstream bacteriophage promoter sequence linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from several sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD, RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v.941 Conn GL (ed), New York, NY Humana Press, 2012).

[0124] RNA transcription occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using commercially available kits, including, but not limited to, the RiboMax Large-Scale RNA Production System (Promega) and the MegaScript Transcription Kit (Life Technologies), as well as commercially available reagents containing RNA polymerase and rNTPs. Methodologies for in vitro transcription of mRNA are well known in the art (e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v. 41 409-46; Kamaka, R. T. and Kraus, W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. and Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, J. L. and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology). v. 530, 101-114, all of which are incorporated herein by reference).

[0125] The desired in vitro transcribed mRNA is then purified from undesired components of the transcription or related reaction. Techniques for isolating mRNA transcripts are well known in the art and include either phenol / chloroform extraction or precipitation with alcohol in the presence of monovalent cations or lithium chloride.

[0126] Lipid nanoparticle formation The LNP comprising the compound of the present disclosure can be prepared by using the approach well known in the field of formulation.For example, suitable LNP can be formed by using a mixing process such as microfluidics, including herringbone micromixing, and T-junction mixing of two fluid streams, one of which typically contains polynucleotide in aqueous solution, and the other typically has various required lipid components in ethanol.

[0127] LNPs can then be prepared by combining, for example, a compound of Formula I, a phospholipid (such as DOPE or DSPC, which can be purchased from commercial sources including Avanti Polar Lipids (Alabaster, AL)), a PEGylated lipid (such as 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as PEG-DMG, which can be purchased from commercial sources including Avanti Polar Lipids (Alabaster, AL)), and a structural lipid / sterol (such as cholesterol, which can be purchased from commercial sources including Sigma-Aldrich) at a concentration of, for example, about 50 mM in ethanol. The solution should be refrigerated during storage, for example, at -20°C. Various lipids may be combined to obtain the desired molar ratio and diluted with water and ethanol to a final desired lipid concentration, for example, about 5.5 mM to about 25 mM.

[0128] Polynucleotide-containing LNP compositions are prepared (as described in the Examples) by combining the lipid solution described above with a polynucleotide-containing solution, for example, at a weight:weight ratio of lipid components to polynucleotide of about 5:1 to about 50:1. The lipid solution may be rapidly injected into the polynucleotide solution using a NanoAssemblr microfluidic system at a flow rate of about 3 ml / min to about 18 ml / min to produce a suspension having a water-to-ethanol ratio of about 1:1 to about 4:1, or about 2:1 to about 4:1.

[0129] For LNP compositions containing RNA, a solution of RNA at a concentration of 1.0 mg / ml in deionized water may be diluted in 50 mM sodium citrate buffer at pH 3-6 to form a stock solution.

[0130] The LNP composition may be further processed, as known in the art, by, for example, diluting it 10-fold into 50 mM citrate buffer at pH 6 and undergoing tangential flow filtration (TFF) using a 300k molecular weight cutoff membrane (mPES) until concentrated to the original volume. The citrate buffer may then be replaced with a buffer containing 20 mM Tris buffer at pH 7.5, 80 mM sodium chloride, and 3% sucrose using diafiltration with 10 times the volume of new buffer. The LNP solution may be concentrated to a volume of 5-10 mL, filtered using a 0.2 micron PES syringe filter, aliquoted into vials, and frozen at 1°C / min using a Corning® CoolCell® LX cell freezing container until the sample reaches -80°C. Samples may be stored at -80°C until needed.

[0131] The above method induces nanoprecipitation and particle formation. The same nanoprecipitation can be achieved using alternative processes, including, but not limited to, T-junction and direct injection.

[0132] In some embodiments, the lipid component of the LNP formulation comprises about 25 mol% to about 60 mol% of a compound of Formula I, about 2 mol% to about 25 mol% of a phospholipid (neutral lipid), about 18.5 mol% to about 60 mol% of a structural lipid (sterol), and about 0.2 mol% to about 10 mol% of a PEGylated lipid, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the LNP formulation comprises about 30 mol% to about 50 mol% of a compound of Formula I, about 5 mol% to about 20 mol% of a phospholipid, about 30 mol% to about 55 mol% of a structural lipid, and about 1 mol% to about 5 mol% of a PEGylated lipid. In certain embodiments, the lipid component comprises about 40 mol% of a compound of the present disclosure, about 10 mol% of a phospholipid, about 48 mol% of a structural lipid, and about 2.0 mol% of a PEGylated lipid. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and / or the structural lipid may be cholesterol.

[0133] The encapsulation efficiency of polynucleotides within LNPs comprising compounds of the present disclosure can be at least 50%, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0134] Lipid Nanoparticle Composition The LNPs comprising the compounds and polynucleotides of the present disclosure can be formulated for administration via any acceptable administration mode for lipid particles, including LNPs, liposomes, lipid vesicles, and similar lipid-based particles.The pharmaceutical compositions of the present disclosure can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols.Typical routes of administration of such pharmaceutical LNP compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques.The composition administered to a subject can be in the form of one or more dosage units, for example, a tablet or an injectable liquid volume can be a single dosage unit. Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

[0135] Accordingly, one embodiment of the present disclosure provides a composition (such as a pharmaceutical composition) comprising an LNP comprising a compound of the present disclosure in combination with a pharmaceutically acceptable carrier.

[0136] Generally, a "carrier" refers to a solid or liquid filler, binder, diluent, encapsulating material, emulsifier, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely administered to any subject, for example, a human. Depending on the particular route of administration, a variety of acceptable carriers known in the art can be used, as described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0137] LNPs are useful for parenteral, topical, oral, or local administration, intramuscular administration, aerosol administration, or transdermal administration for prophylactic or therapeutic treatment. In one embodiment, LNPs are administered parenterally, such as intramuscularly, subcutaneously, or intravenously. In some embodiments, LNPs are administered intramuscularly.

[0138] The formulation of the administered LNP will vary according to the selected route of administration and formulation (e.g., solution, emulsion, capsule). Suitable pharmaceutical compositions containing the administered LNP can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media in embodiments. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Those skilled in the art know a variety of suitable aqueous carriers, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles can include additives, preservatives, or fluid, nutrient, or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, and toxicity adjusting agents, in embodiments, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The LNPs can be stored in the liquid stage or lyophilized for storage and reconstituted in a suitable carrier prior to use according to lyophilization and reconstitution techniques known in the art.

[0139] When the LNP composition is a vaccine composition, the carrier can be water, typically pyrogen-free water, isotonic saline, or a buffered (aqueous) solution, e.g., a phosphate, citrate, or other buffer solution. For injection of the LNP vaccine composition, water, or preferably a buffer, more preferably an aqueous buffer, containing sodium salts, preferably at least 50 mM sodium salts, calcium salts, preferably at least 0.01 mM calcium salts, and optionally potassium salts, e.g., at least 3 mM potassium salts, may be used. In one embodiment, the sodium, calcium, and optionally potassium salts may be present as their chlorides, iodides, or bromides, or in the form of their hydroxides, carbonates, bicarbonates, sulfates, or the like. Non-limiting examples of sodium salts include, for example, NaCl, NaI, NaBr, Na2CO3, NaHCO3, and Na2SO4; examples of optional potassium salts include, for example, KCl, KI, KBr, K2CO3, KHCO3, and K2SO4; examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO3, CaSO4, and Ca(OH)2. Additionally, organic anions of the aforementioned cations may be contained in the buffer. In certain embodiments, a buffer suitable for injection purposes may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2), and optionally potassium chloride (KCl), and additional anions may be present in addition to chloride. In embodiments, the salts in the injection buffer are present at concentrations of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl), and at least 0.01 mM calcium chloride (CaCl2). Injection buffers can be hypertonic, isotonic, or hypotonic with reference to a particular reference medium.

[0140] In some embodiments of vaccine, one or more suitable solid or liquid fillers or diluents or encapsulating compounds may be used that are suitable for human administration.Pharmaceutically acceptable carriers, fillers, and diluents have sufficiently high purity and sufficiently low toxicity to be suitable for administering to subjects.Some examples of compounds that can be used as pharmaceutically acceptable carriers, fillers, or their components include: sugars such as lactose, glucose, trehalose, and sucrose; starches such as corn starch or potato starch; dextrose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; glidants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and theobroma oil; polyols such as polypropylene glycol, glycol, sorbitol, mannitol, and polyethylene glycol; and alginic acid.

[0141] When the LNP composition is a vaccine composition, it may further comprise one or more pharmaceutically acceptable adjuvants to enhance the immunostimulatory properties of the composition. The adjuvant may be any compound suitable for supporting the administration and delivery of the LNP composition and capable of initiating or augmenting an immune response of the innate immune system, i.e., a nonspecific immune response.

[0142] Such adjuvants are known to those skilled in the art and may be selected from any adjuvant suitable for the particular properties of the vaccine, i.e., the induction of a suitable immune response in a mammal. In embodiments, the adjuvant is selected from MF59® (squalene-water emulsion), TDM, MDP, muramyl dipeptide, Pluronic, alum solution, aluminum hydroxide, ADJUMER™ (polyphosphazene); aluminum phosphate gel; algae-derived glucan; algammulin; aluminum hydroxide gel (alum); high protein absorption aluminum hydroxide gel; low viscosity aluminum hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate buffered saline, pH 7.4); AVRIDINE™ (propanediamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylamino-bD-glycopyranosyl)-N-octadecyl-dodecanoyl-amidohydroacetate); CALCITRIOL™ (l-alpha, 25-dihydroxy-vitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-AI-protein-AD-fragment fusion protein, subunit B of cholera toxin; CRL1005 (block copolymer P1205); cytokine-containing liposomes; DDA (dimethyldioctadecyl ammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyris dimyristoylphosphatidylcholine; DMPG (dimyristoylphosphatidylglycerol); DOC / alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma insulin; Gelb adjuvant (i) N-acetylglucosaminyl-(Pl-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), ii) dimethyldioctadecyl ammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF mixture; GMDP (N-acetylglucosaminyl-(bl-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine);Imiquimod (l-(2-methylpropyl)-lH-imidazo[4,5-c]quinolin-4-amine); ImmTher™ (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRV (immunoliposomes prepared from dehydrated-rehydrated vesicles); interferon-gamma; interleukin-l beta; interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP7.0.3™; liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E. coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MONTANIDE ISA51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA720™ (metabolizable oil adjuvant); MPL™ (3-Q-desacyl-4'-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMI TINE™ and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGIn-sn-glycerodipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISV (nonionic surfactant vesicles); PLEURAN™ (β-glucan); PLGA, PGA, and PLA (homo- and copolymers of lactic and glycolic acid; microspheres / nanospheres); PLURONIC L121™; PMMA (polymethyl methacrylate); PODDS™ (proteinoid microspheres); polyethylene carbamate derivatives; poly-rA:poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80);Protein cochleates (Avanti Polar Lipids, Inc., Alabaster, AL); STIMULON™ (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-lH-imidazo[4,5c]quinoline-l-ethanol); SAF-1™ ("Syntex adjuvant formulation"); Sendai proteoliposomes and Sendai containing lipid matrices; Span-85 (trioleate) sorbitan acetate; Specol (an emulsion of Marcol 52, Span 85, and Tween 85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosane and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyl tyrosine (octadecyl tyrosine hydrochloride); Theramid® (N-acetylglucosamine) Cosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide; threonyl-MDP (Termurtide™ or [thrl]-MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A absorbed on aluminum hydroxide), and lipopeptides containing Pam3Cys, especially aluminum salts, e.g., Adju-phos, Alhydrogel, Rehydragel; emulsions containing CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers containing Optivax (CRL1005), L121, poloxamer 4010, etc.; liposomes containing Stealth, cochelates containing BIORAL; QS21, Quil A, Iscomatrix, a plant-derived adjuvant containing ISCOM; an adjuvant suitable for costimulation containing tomatine, biopolymers including PLG, PMM, and inulin;The adjuvant may be selected from the group consisting of microbial adjuvants, including romurtide, DETOX, MPL, CWS, mannose, CpG polynucleotide sequences, CpG7909, ligands for human TLR1-10, ligands for mouse TLR1-13, ISS-1018, IC31, imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIV, VLPs, cholera toxin, heat-labile toxins, Pam3Cys, flagellin, GPI anchors, LNFPIII / Lewis X, antimicrobial peptides, UC-1V150, RSV fusion proteins, and cdiGMP; and adjuvants suitable as antagonists, including CGRP neuropeptides. In one preferred example, the adjuvant may be the oil-in-water emulsion adjuvant MF59®, particularly when the vaccine is an influenza vaccine.

[0143] Upon formulation, the compositions of the present disclosure will be administered in a manner compatible with the dosage formulation, and in a therapeutically / prophylactically effective amount. The dosage range for administration of LNPs will be large enough to produce the desired effect. For embodiments, the composition comprises an effective amount of encapsulated or associated RNA, such as mRNA or self-replicating RNA. In one embodiment, the composition comprises a therapeutically effective amount of RNA. In another embodiment, the composition comprises a prophylactically effective amount of RNA.

[0144] Dosage should not be so large as to cause harmful side effects.Generally, dosage varies according to age, condition, sex and degree of disease in patient, and can be determined by those skilled in the art.Dosage can be adjusted by individual physician in the event of any complication.

[0145] Methods for preparing the above compounds and compositions are further described herein and / or known in the art.

[0146] Methods of treatment and production of polypeptides of interest Diseases, disorders, and / or conditions that may be the result of or associated with abnormal proteins or polypeptides may be treated by the present LNPs comprising the compounds and polynucleotides of the present disclosure, and may include, but are not limited to, rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0147] The LNP composition can be formulated in a unit dosage form. The therapeutically or prophylactically effective dose for any particular patient will depend on a variety of factors, including the severity and identity of the disorder being treated; the particular composition used; the patient's age, weight, general health, sex, and diet; the administration time, route of administration, and excretion rate of the particular pharmaceutical composition used; the duration of treatment; drugs used in combination with or simultaneously with the particular pharmaceutical composition used; and similar factors well known in the medical arts.

[0148] The LNP compositions described herein can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents, which can be administered together in a single composition or separately in different compositions.

[0149] The present disclosure provides a method for producing a polypeptide of interest in mammalian cells. The polypeptide production method involves contacting cells with the LNP composition described herein, which comprises mRNA encoding the polypeptide of interest. When cells are contacted with the LNP composition, the mRNA can be taken up and translated within the cell to produce the polypeptide of interest.

[0150] The step of contacting a cell with an LNP composition containing mRNA may involve or cause transfection. The phospholipids contained in the lipid component of the LNP composition may facilitate transfection and / or increase transfection efficiency, for example, by interacting with and / or fusing with the cell membrane or intracellular membrane. Transfection may enable translation of the mRNA within the cell.

[0151] In some embodiments, the LNP compositions described herein can be used therapeutically. For example, the mRNA contained in the LNP composition can encode a therapeutic polypeptide (e.g., within a translatable region) and produce the therapeutic polypeptide upon contact with and / or entry into a cell (e.g., transfection). In other embodiments, the mRNA contained in the LNP composition can encode a polypeptide that can improve or increase the subject's immunity.

[0152] In embodiments, the mRNA contained in the LNP composition may encode a recombinant polypeptide that can replace one or more polypeptides that may be substantially absent in cells contacted with the LNP composition. The one or more substantially absent polypeptides may be absent due to a genetic mutation in the encoding gene or its regulatory pathway. Alternatively, the recombinant polypeptide produced by translation of the mRNA may antagonize the activity of an endogenous protein present within the cell, present on the surface of the cell, or secreted from the cell. Antagonistic recombinant polypeptides may be desirable to counteract deleterious effects caused by the activity of endogenous proteins, such as altered activity or localization caused by mutations. In another alternative, the recombinant polypeptide produced by translation of the mRNA may indirectly or directly antagonize the activity of a biological moiety present within the cell, present on the surface of the cell, or secreted from the cell. Antagonized biological moieties include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins. The recombinant polypeptide produced by translation of the mRNA can be engineered for localization within a specific compartment within the cell, for example, the nucleus, or can be engineered for secretion from the cell or translocation to the plasma membrane of the cell.

[0153] In some embodiments, contacting cells with an LNP composition containing mRNA may reduce the cellular innate immune response to an exogenous polynucleotide. Cells may be contacted with a first LNP composition containing a first amount of a first exogenous mRNA containing a translatable region, and the level of the cellular innate immune response to the first exogenous mRNA may be determined. Subsequently, the cells may be contacted with a second LNP composition containing a second amount of the first exogenous mRNA, the second amount being a lower amount of the first exogenous mRNA compared to the first amount. Alternatively, the second composition may contain a first amount of a second exogenous mRNA that is different from the first exogenous mRNA. The step of contacting cells with the first and second LNP compositions may be repeated one or more times. Additionally, the efficiency of polypeptide production (e.g., translation) in the cells may optionally be determined, and the cells may be repeatedly re-contacted with the first and / or second compositions until a target protein production efficiency is achieved.

[0154] In some embodiments, the present disclosure provides the use of LNPs comprising the disclosed compounds and polynucleotides in the manufacture of a medicament for the treatment of a disease, disorder, or condition. The disease, disorder, or condition may be as described in any one or more embodiments herein.

[0155] The medicament may be for the prevention or treatment of cancer, infectious disease, allergy, or autoimmune disease. In embodiments, the medicament is a vaccine. The vaccine may be a tumor vaccine, an influenza vaccine, or a SARS-CoV-2 vaccine.

[0156] Use of LNPs in vaccines In some embodiments, LNPs containing the disclosed compounds and polynucleotides can be components of vaccines. Vaccines include compounds and preparations that can provide immunity against one or more conditions associated with infectious diseases, and thus may include mRNA encoding antigens and / or epitopes derived from infectious diseases. Vaccines also include compounds and preparations that direct an immune response against cancer cells, and may include mRNA encoding antigens, epitopes, and / or neoepitopes derived from tumor cells. Compounds that induce immune responses include vaccines, corticosteroids (e.g., dexamethasone), and other species.

[0157] In embodiments, the vaccine may be an mRNA vaccine, such that the LNPs comprising the compounds of the present disclosure encapsulate or associate with an mRNA molecule comprising an mRNA sequence encoding an antigenic peptide or protein, or a fragment, variant, or derivative thereof.

[0158] The antigen peptide or protein can be a pathogenic antigen, a tumor antigen, an allergenic antigen, or an autoimmune autoantigen. Such a pathogenic antigen can be derived from a pathogenic organism, particularly a bacterial, viral, or protozoal (multicellular) pathogenic organism that induces an immunological response in a mammalian subject, such as a human. The pathogenic antigen can be a surface antigen, such as a protein or a fragment thereof, located on the surface of a virus, a bacterium, or a protozoal organism.

[0159] Pathogenic antigens of interest may include those derived from one or more of the following: Acinetobacter baumannii, Anaplasma spp., Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Area nobacteri urn haemolyticum, Ascaris lumbricoides, Aspergillus spp., Astroviridae, Babesia spp., Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocysts hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia spp., Brucella spp., Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, QD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcusneoformans, Cryptosporidium spp., cytomegalovirus (CMV), dengue viruses (DEN-1, DEN-2, DEN-3, and DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus spp., Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia spp., Entamoeba histolytica, Enterococcus spp., Enterovirus spp., enteroviruses, mainly Coxsackie A virus and enterovirus 71 (EV71), Epidermophyton spp., Epstein-Barr virus (EBV), Escherichia coli O157:H7, O11, and O104:H4, Fasciola hepatica and Fasciola gigantica, FFI prions, Filarioidea superfamily, flaviviruses, Francisella tularensis, Fusobacterium spp., Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipaviruses (Hendra virus, Nipah virus), Hepatitis A virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Koolupulion, Lassa virus, Legionellapneumophila, Leishmania spp., Leptospira spp., Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp., Marburg virus, measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp., Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (influenza), Paracoccidioides brasiliensis, Paragonimus spp., Paragonimus westermani, Parvovirus B19, Pasteurella spp., Plasmodium spp., Pneumocystis jirovecii, poliovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia spp., Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, rotavirus, rubella virus, Sabia virus, Salmonella spp., Sarcoptes scabiei, SARS coronavirus, Schistosoma spp., Shigella spp., Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus spp., Staphylococcus spp., Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia spp., Taenia solium, Tick-borne encephalitis (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp., Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella-zoster virus (VZV), Smallpox (high fatality rate) or Smallpox (low fatality rate), vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0160] In certain embodiments, the relevant antigen may be derived from a pathogen selected from: severe acute respiratory syndrome (SARS), severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-1 and SARS-CoV-2), influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, dengue virus, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus, and yellow fever virus.

[0161] In some embodiments, the relevant virulence antigens may be selected from the following: outer membrane protein A OmpA, biofilm-associated protein Bap, transport protein MucK (Acinetobacter baumannii, Acinetobacter infection); variable surface glycoprotein VSG, microtubule-associated protein MAPP15, trans-sialidase TSA (Trypanosoma brucei, sleeping sickness (African trypanosomiasis)); HIV p24 antigen, HIV envelope proteins (Gp120, Gp41, Gp160), polyprotein GAG, negative factor protein Nef, transactivator of transcription Tat (HIV (human immunodeficiency virus), AIDS (acquired immune deficiency syndrome)); galactose-inhibitory adherence protein GIAP, 29 kDa antigen Eh29, Gal / GalNAc lectin, protein CRT, 125 kDa immunodominant antigen, protein M17, adhesin ADH112, protein STIRP (Entamoeba histolytica, amebiasis); major surface proteins 1-5 (MSPla, MSPlb, MSP2, MSP3, MSP4, MSP5), type IV secretion system proteins (VirB2, VirB7, VirBll, VirD4) (Anaplasma genus, Anaplasmosis); protective antigen PA, edema factor EF, lethal factor LF, S-layer homology protein SLH (Bacillus anthracis, anthrax); acranolisin, phospholipase D, collagen-binding protein CbpA (Area nobacteri urn haemolyticum, Area nobacteri urn haemolyticum infection); nucleocapsid protein NP, glycoprotein precursor GPC, glycoprotein GP1, glycoprotein GP2 (Junín virus, Argentine hemorrhagic fever); chitin-protein layer protein, 14 kDa surface antigen A14, major sperm protein MSP, MSP polymerization assembly protein MPOP, MSP fiber protein 2 MFP2, MSP polymerization activating kinase MPAK, ABA-l-like protein ALB, protein ABA-1, cuticulin CUT-1 (Ascaris lumbricoides, ascariasis);41 kDa allergen Asp vl3, allergen Asp f3, major conidial surface protein rodlet A, protease Peplp, GPI-anchored protein Gellp, GPI-anchored protein Crflp (Aspergillus genus, aspergillosis); family VP26 protein, VP29 protein (Astroviridae, astrovirus infection); rhoptry-associated protein 1 RAP-1, merozoite surface antigens MSA-1, MSA-2 (al, a2, b, c), 12D3, 11C5, 21B4, P29, variant erythrocyte surface antigen VESA1, apical membrane antigen 1 AMA-1 (Babesia genus, babesiosis); hemolysin, enterotoxin C, PXO1-51, glycolate oxidase, ABC transporter, penicillin-binding protein, zinc transporter family protein, pseudouridine synthase Rsu, plasmid replication protein RepX, oligoendopeptidase F, prophage membrane protein, protein HemK, flagellar antigen H, 28.5-kDa cell surface antigen (Bacillus cereus, Bacillus cereus infection); large T antigen LT, small T antigen, capsid protein VP1, capsid protein VP2 (BK virus, BK virus infection); 29-kDa protein, caspase-3-like antigen, glycoprotein (Blastocysts hominis, Blastocystis hominis infection); yeast surface adhesin WI-1 (Blastomyces dermatitidis, blastomycosis); nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP (Machupo virus, Bolivian hemorrhagic fever); surface protein A OspA, surface protein OspB, surface protein OspC, decorin-binding protein A DbpA, decorin-binding protein B DbpB, flagellar filament 41 kDa core protein Fla, base membrane protein A precursor BmpA (immunodominant antigen P39), surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE (Borrelia genus, Borrelia infections);Botulinum neurotoxins BoNT / Al, BoNT / A2, BoNT / A3, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, recombinant botulinum toxin F He domain FHc (Clostridium botulinum, botulism (and infant botulism)); nucleocapsid, glycoprotein precursor (Sabia virus, Brazilian hemorrhagic fever); copper / zinc superoxide dismutase SodC, bacterioferritin Bfr, ​​50S ribosomal protein RpIL, OmpA-like transmembrane domain-containing protein Omp31, immunogenic 39-kDa protein M5 P39, zinc ABC transporter periplasmic zinc-binding protein znuA, periplasmic immunogenic protein Bp26, 30S ribosomal protein S12 RpsL, glyceraldehyde-3-phosphate dehydrogenase Gap, 25 kDa outer membrane immunogenic protein precursor Omp25, invasion protein B lalB, trigger factor Tig, molecular chaperone DnaK, putative peptidyl-prolyl cis-trans isomerase SurA, lipoprotein Ompl9, outer membrane protein MotY Ompl6, conserved outer membrane protein D15, malate dehydrogenase Mdh, component of type IV secretion system (T4SS) VirJ, lipoprotein of unknown function BAB1_0187 (Brucella genus, Brucellosis); members of the ABC transporter family (LolC, OppA, and PotF), putative lipoprotein release system transmembrane protein LolC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial elongation factor Tu EF-Tu, 17 kDa OmpA-like protein, boaA-encoded protein, boaB-encoded protein (Burkholderia cepacia and other Burkholderia species, Burkholderia infections); mycolyltransferase Ag85A, heat shock protein Hsp65, protein TB10.4, 19 kDa antigen, protein PstS3, heat shock protein Hsp70 (Mycobacterium ulcerans, Buruli ulcer);Norovirus major and minor viral capsid proteins VP1 and VP2, genome polyprotein, Sapovirus capsid protein VP1, protein Vp3, genome polyprotein (Caliciviridae family, Calicivirus infections (Norovirus and Sapovirus)); major outer membrane protein PorA, flagellin FlaA, surface antigen CjaA, fibronectin-binding protein CadF, aspartate / glutamate-binding ABC transporter protein PeblA, protein FspAl, protein FspA2 (Campylobacter genus, campylobacteriosis); glycolytic enzyme enolase, secreted aspartyl proteinase SAPl-10, glycosylphosphatidylinositol (GPI)-linked cell wall protein, protein Hyrl, complement receptor 3-related protein CR3-RP, adhesin Als3p, heat shock protein 90 kDa hsp90, cell surface hydrophobic protein CSH (typically associated with Candida albicans and other Candida species, candidiasis); 17-kDa antigen, protein P26, trimeric autotransporter adhesin TAA, Bartonella adhesin A BadA, variably expressed outer membrane protein Vomp, protein Pap3, protein HbpA, envelope-associated protease HtrA, protein OMP89, protein GroEL, protein LalB, protein OMP43, dihydrolipoamide succinyltransferase SucB (Bartonella henselae, cat scratch disease); amastigote surface protein-2, amastigote-specific surface protein SSP4, cruzipain, trans-sialidase TS, trypomastigote surface glycoprotein TSA-1, complement regulatory protein CRP-10, protein G4, protein G2, paraxonemal rod protein PAR2, paraflagellar rod) component Pari, mucin-associated surface protein MPSP (Trypanosoma cruzi, Chagas disease (American trypanosomiasis)); envelope glycoproteins (gB, gC, gE, gH, gl, gK, gL), (varicella-zoster virus (VZV), chickenpox);Major outer membrane protein MOMP, likely outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock protein Hsp60 HSP10, protein IncA, protein from type III secretion apparatus, ribonucleotide reductase short chain protein NrdB, plasmid protein Pgp3, chlamydial outer membrane protein N CopN, antigen CT521, antigen CT425, antigen CT043, antigen TC0052, antigen TC0189, antigen TC0582, antigen TC0660, antigen TC0726, antigen TC0816, antigen TC0828 (Chlamydia trachomatis); low calcium response protein E LCrE, chlamydial outer membrane protein N CopN, serine / threonine-protein kinase PknD, acyl-carrier-protein S-malonyltransferase FabD, single-stranded DNA binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, polymorphic membrane protein family (Pmpl, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, PmplO, Pmpll, Pmpl2, Pmpl3, Pmpl4, Pmpl5, Pmpl6, Pmpl7, Pmpl8, Pmpl9, Pmp20, Pmp21), (Chlamydophila pneumoniae, Chlamydophila pneumoniae infection); cholera toxin B CTB, toxin co-expressing pilin A TcpA, toxin co-expressing pilin TcpF, toxin co-expressing pilus biogenesis protein F TcpF, cholera enterotoxin subunit A, cholera enterotoxin subunit B, heat-stable enterotoxin ST, mannose-sensitive hemagglutinin MSHA, outer membrane protein U porin ompU, Poring B protein, polymorphic membrane protein D (Vibrio cholerae, cholera); propionyl-CoA carboxylase PCC, 14-3-3 protein, prohibitin, cysteine ​​protease, glutathione transferase, gelsolin, cathepsin L proteinase CatL, coat protein 20.8 kDa TP20.8, coat protein 31.8 kDa TP31.8, lysophosphatidic acid phosphatase LPAP, (Clonorchis sinensis, liver fluke disease);Surface layer protein SLP, glutamate dehydrogenase antigen GDH, toxin A, toxin B, cysteine ​​protease Cwp84, cysteine ​​protease Cwpl3, cysteine ​​protease Cwpl9, cell wall protein CwpV, flagellar protein FliC, flagellar protein FliD (Clostridium difficile, Clostridium difficile infection); rhinovirus: capsid proteins VP1, VP2, VP3, VP4; coronavirus: spike protein S, envelope protein E, membrane protein M, nucleocapsid protein N (usually rhinoviruses and coronaviruses, common cold (acute viral nasopharyngitis; acute rhinitis)); prion protein Prp (CJ; D prion, Creutzfeldt-Jakob disease (CJD); envelope protein Gc, envelope protein Gn, nucleocapsid protein (Crimean-Congo hemorrhagic fever virus, Crimean-Congo hemorrhagic fever (CCHF)); pathogenesis-associated DEAD-box RNA helicase VAD1, galactoxylomannan-protein GalXM, glucuronoxylomannan GXM, mannoprotein MP (Cryptococcus neoformans, cryptococcosis); acidic ribosomal protein P2 CpP2, mucin antigens Mucl, Muc2, Muc3 Muc4, Muc5, Muc6, Muc7, surface adherence protein CP20, surface adherence protein CP23, surface protein CP12, surface protein CP21, surface protein CP40, surface protein CP60, surface protein CP15, surface-associated glycopeptide gp40, surface-associated glycopeptide gpl5, oocyst wall protein AB, profilin PRF, apyrase (Cryptosporidium spp., cryptosporidiosis); fatty acid and retinol binding protein-1 FAR-1, tissue inhibitor of metalloproteinase TIMP (TMP), cysteine ​​proteinase ACEY-1, cysteine ​​proteinase ACCP-1, surface antigen Ac-16, secreted protein 2 ASP-2, metalloproteinase 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, adult-specific secretory factor Xa serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1 (usually Ancylostoma braziliense; several other parasites, cutaneous larva migrans (CLM)); cathepsin L-like protease, 53 / 25-kDa antigen, 8-kDa family member, cysticercal protein TsAg5 with little trypsin-like activity, oncocysticercosis protein TSOL18, oncocysticercosis protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia solium, cysticercosis);pp65 antigen, membrane protein ppl5, capsid-proximal tegument protein ppl50, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glycoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99 (cytomegalovirus (CMV), cytomegalovirus infection); capsid protein C, pre-membrane protein prM, membrane protein M, envelope protein E (domain I, domain II, domain II), protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A, protein 2K, protein NS4B, protein NS5 (dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4) - flavivirus, dengue fever); 39 kDa protein (Dientamoeba fragilis, diphtheria toxin precursor Tox, diphtheria toxin DT, pilin-specific sortase SrtA, shaft pilin protein SpaA, tip pilin protein SpaC, minor pilin protein SpaB, surface-associated protein DIP1281 (Corynebacterium diphtheriae, diphtheriae); glycoprotein GP, ​​nucleoprotein NP, minor matrix protein VP24, major matrix protein VP40, transcription activator VP30, polymerase cofactor VP35, RNA polymerase L (Ebola virus (EBOV), Ebola hemorrhagic fever); prion protein (vQD prion, variant Creutzfeldt-Jakob disease (vCJD, nvCJD)); UvrABC system protein B, protein Flpl, protein Flp2, protein Flp3, protein TadA, hemoglobin receptor HgbA, outer membrane protein TdhA, protein CpsRA, regulator CpxR, protein SapA, 18kDa antigen, outer membrane protein NcaA, protein LspA, protein LspAl, protein LspA2, protein LspB, outer membrane component DsrA, lectin DltA, lipoprotein Hip, major outer membrane protein OMP, outer membrane protein OmpA2 (Haemophilus ducreyi, soft chancre);Aspartyl protease 1 Pepl, phospholipase B PLB, alpha-mannosidase 1 AMN1, glucanosyltransferase GEL1, urease URE, peroxisomal matrix protein Pmpl, proline-rich antigen Pra, human T cell reactive protein TcrP (Coccidioides immitis and Coccidioides posadasii, coccidioidomycosis); allergen Tri r2, heat shock protein 60 Hsp60, fungal actin Act, antigen Tri r2, antigen Tri r4, antigen Tri tl, protein IV, glycerol-3-phosphate dehydrogenase Gpdl, osmosensor HwSholA, osmosensor HwSholB, histidine kinase HwHhk7B, allergen Mala s1, allergen Mala s11, thioredoxin Trx Mala s13, allergen Mala f, allergen Mala s (usually Trichophyton spp., Epidermophyton spp., Malassezia spp., Hortaea werneckii, dermatophytosis); protein EG95, protein EG10, protein EG18, protein EgA31, protein EM18, antigen EPC1, antigen B, antigen 5, protein P29, protein 14-3-3, 8-kDa protein, myophilin, heat shock protein 20 HSP20, glycoprotein GP-89, fatty acid binding protein FAPB (Echinococcus spp., echinococcosis); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigenic protein MAPI, major antigenic protein MAP1-2, major antigenic protein MAP1B, major antigenic protein MAP1-3, Erum2510-encoded protein, protein GroEL, protein GroES, 30-kDA major outer membrane protein, GE100-kDa protein, GE130-kDa protein, GE160-kDa protein (Ehrlichia genus, ehrlichiosis);Secreted antigen SagA, sagA-like proteins SalA and SalB, collagen adhesin Scm, surface protein Fmsl (EbpA(fm)), Fms5 (EbpB(fm)), Fms9 (EpbC(fm) and FmslO), protein EbpC(fm), 96 kDa immune defense glycoprotein Gl (Enterococcus genus, Enterococcus infections); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein Protein VP4, protease 2A, protease 3C (Enterovirus genus, Enterovirus infections); outer membrane protein OM, 60-kDa outer membrane protein, cell surface antigen OmpA, cell surface antigen OmpB (sca5), 134-kDa outer membrane protein, 31-kDa outer membrane protein, 29.5-kDa outer membrane protein, cell surface protein SCA4, cell surface protein Adrl (RP827), cell surface protein Adr2 (RP828), cell surface protein SCA1, invasion protein invA, cell division protein fts, secreted protein secO family, virulence proteins virB, tlyA, tlyC, parvulin-like protein Pip, preprotein translocase SecA, 120-kDa surface protein antigen SPA, 138-kD complex antigen, major 100-kD protein (protein I), cytoplasmic protein D, defensive surface protein antigen SPA (Rickettsia prowazekii, typhus; Epstein-Barr nuclear antigens (EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP)), latent membrane proteins (LMP-1, LMP-2A, LMP-2B), early antigen EBV-EA, membrane antigen EBV-MA, viral capsid antigen EBV-VCA, alkaline nuclease EBV-AN, glycoprotein H, glycoprotein gp350, glycoprotein gpllO, glycoprotein gp42, glycoprotein gHgL, glycoprotein gB (Epstein-Barr virus (EBV), Epstein-Barr virus infectious mononucleosis); capsid protein VP2, capsid protein VP1, major protein NS1 (parvovirus B19, erythema infectiosum (fifth disease));pp65 antigen, glycoprotein 105, major capsid protein, envelope glycoprotein H, protein U51 (human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), exanthem subitum); thioredoxin-glutathione reductase TGR, cathepsins LI and L2, Kunitz-type protein KTM, leucine aminopeptidase LAP, cysteine ​​proteinase Fas2, saposin-like protein-2 SAP-2, thioredoxin peroxidase TPx, Prx-1, Prx-2, cathepsin I cysteine ​​proteinase CL3, protease cathepsin L CLl, phosphoglycerate kinase PGK, 27-kDa secretory protein, 60-kDa protein HSP35 alpha, glutathione transferase GST, 28.5-kDa tegument antigen 28.5 kDa TA, cathepsin B3 protease CatB3, type I cystatin stefin-1, cathepsin L5, cathepsin Llg, and cathepsin B, fatty acid-binding protein FABP, leucine aminopeptidase LAP (Fasciola hepatica and Fasciola gigantica, fascioliasis); prion protein (FFI prion, fatal familial insomnia (FFI)); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, vespid allergen homolog VAH, thioredoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N, Nl, N2, and N3), activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, vespid allergen homolog VAH, 175kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, epidermal collagen Col-4, secretory larval acidic protein SLAP, chitinase CHI-1, maltose-binding protein MBP, glycolytic enzyme fructose-l,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, onchocystatin CPI-2, Cox-2 (Filarioidea superfamily, filariasis);Phospholipase C PLC, heat-labile enterotoxin B, iota toxin component lb, protein CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, perfringolysin 0 Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, fructose-bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha toxoid ATd, epsilon-toxoid ETd, protein HP, large cytotoxin TpeL, endo-beta-N-acetylglucosaminidase Naglu, phosphoglycerin mutase Pgm (Clostridium perfringens, Clostridium perfringens ingestion); toxin); leukotoxin IktA, adhesin FadA, outer membrane protein RadD, high molecular weight arginine-binding protein (Fusobacterium spp., Fusobacterium infections); phospholipase C PLC, heat-labile enterotoxin B, iota toxin component lb, protein CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, perfringolysin 0 Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, fructose-bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha toxoid ATd, epsilon toxoid ETd, protein HP, large cytotoxin TpeL, endo-beta-N-acetylglucosaminidase Naglu, phosphoglycerin mutase Pgm (typically Clostridium perfringens; other Clostridium species, Gas gangrene (Clostridial myonecrosis); lipase A, lipase B, peroxidase Decl (Geotrichum candidum, geotrichum disease); prion protein (GSS prion, Gerstmann-Straussler-Scheinker syndrome (GSS)); cyst wall proteins CWP1, CWP2, CWP3, variant surface proteins VSP, VSP1, VSP2, VSP3, VSP4, VSP5, VSP6, 56 kDa antigen, pyruvate ferredoxin oxidoreductase PFOR, alcohol dehydrogenase E ADHE, alpha-giardin, alpha 8-giardin, alpha l-giardin (guiardin), beta-giardin, cysteine ​​protease, glutathione S-transferase GST, arginine deiminase ADI, fructose-l,6-bisphosphate aldolase FBA, Giardia Trophozoite antigen GTA (GTA1, GTA2), ornithine carboxyltransferase OCT, striated fiber-asseblin-like protein SALP, uridine phosphoryl-like protein UPL, alpha-tubulin, beta-tubulin (Giardia intestinalis, giardiasis);Members of the ABC transporter family (LolC, OppA, and PotF), putative lipoprotein release system transmembrane protein LolC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial elongation factor-Tu EF-Tu, a 17-kDa OmpA-like protein, and a boaA-encoded protein (Burkholderia mallei, Glanders); cyclophilin CyP, a 24-kDa third-stage larval protein GS24, and excretory-secretory products ESP (40, 80, 120, and 208 kDa) (Gnathostoma spinigerum and Gnathostoma hispidum, gnathostomiasis); pilin proteins, the minor pilin-related subunit pilC, the major pilin subunit and variant pilE, pilS, the phase variant protein porA, and porin B. PorB, protein TraD, Neisserial outer membrane antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin-binding protein TbpA, transferrin-binding protein TbpB, PBP2, mtrR-encoded protein, ponA-encoded protein, membrane permease FbpBC, FbpABC protein system, LbpAB protein, outer membrane protein Opa, outer membrane transporter FetA, iron-repressible regulator MpeR (Neisseria gonorrhoeae, gonorrhea); outer membrane protein A OmpA, outer membrane protein C OmpC, outer membrane protein K17 OmpK17 (Klebsiella granulomatis, granuloma inguinale (donovanosis)); fibronectin-binding protein Sfb, fibronectin / fibrinogen-binding protein FBP54, fibronectin-binding protein FbaA, M Protein type 1 Emml, M protein type 6 Emm6, immunoglobulin-binding protein 35 Sib35, surface protein R28 Spr28, superoxide dismutase SOD, C5a peptidase ScpA, antigen I / II Agl / II, adhesin AspA, G-related alpha2-macroglobulin-binding protein GRAB, surface filamentous protein M5 (Streptococcus pyogenes, group A streptococcal infections);C protein beta antigen, arginine deiminase protein, adhesin BibA, 105 kDa protein BPS, surface antigen c, surface antigen R, surface antigen X, trypsin-resistant protein Rl, trypsin-resistant protein R3, trypsin-resistant protein R4, surface immunogenic protein Sip, surface protein Rib, leucine-rich repeat protein LrrG, serine-rich repeat protein Srr-2, C protein alpha-antigen Bca, beta antigen Bag, surface antigen epsilon, alpha-like protein ALP1, alpha-like protein ALP5, surface antigen delta, alpha-like protein ALP2, alpha-like protein ALP3, alpha-like protein ALP4, C beta protein Bac (Streptococcus agalactiae, group B streptococcus infections); transferrin-binding protein 2 Tbp2, phosphatase P4, outer membrane protein P6, peptidoglycan-associated lipoprotein Pal, protein D, protein E, adherence and permeability protein Hap, outer membrane protein 26 Omp26, outer membrane protein P5 (fimbrin), outer membrane protein D15, outer membrane protein OmpP2, 5'-nucleotidase NucA, outer membrane protein PI, outer membrane protein P2, outer membrane lipoprotein Pep, lipoprotein E, outer membrane protein P4, fuculokinase FucK, [Cu,Zn]-superoxide dismutase SodC, protease HtrA, protein O145, alpha-galactosylceramide (Haemophilus influenzae, Haemophilus influenzae infection); polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (enteroviruses, mainly Coxsackievirus A and enterovirus 71 (EV71), hand, foot, and mouth disease (HFMD)); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein G1, nucleoprotein NP, protein N, polyprotein M (Sin Nombre virus, hantavirus, hantavirus pulmonary syndrome (HPS));Heat shock protein HspA, heat shock protein HspB, citrate synthase GltA, protein UreB, heat shock protein Hsp60, neutrophil-activating protein NAP, catalase KatA, cell vacuolating toxin VacA, urease alpha UreA, urease beta Ureb, protein CpnlO, protein groES, heat shock protein HsplO, protein MopB, cytotoxicity-associated 10 kDa protein CAG, 36 kDa antigen, beta-lactamase HcpA, beta-lactamase HcpB (Helicobacter pylori infection); integral membrane protein, aggregation-prone protein, O-antigen, toxin-antigen Stx2B, toxin-antigen StxlB, adhesin-antigen fragment Int28, protein EspA, protein EspB, intimin, protein Tir, protein IntC300, protein Eae (Escherichia coli O157:H7, O111 and O104:H4, hemolytic uremic syndrome (HUS); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein Gl, nucleoprotein NP, protein N, polyprotein M (Bunyaviridae family, hemorrhagic fever with renal syndrome (HFRS)); glycoprotein G, matrix protein M, nucleoprotein N, fusion protein F, polymerase L, protein W, protein C, phosphoprotein p, nonstructural protein V (Henipavirus (Hendra virus Nipah virus), Henipavirus infection); polytan Protein, glycoprotein Gp2, hepatitis A surface antigen HBAg, protein 2A, viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4, protein P1B, protein P2A, protein P3AB, protein P3D (hepatitis A virus, hepatitis A); hepatitis B surface antigen HBsAg, hepatitis B core antigen HbcAg, polymerase, protein Hbx, preS2 intermediate surface protein, surface protein L, large S protein, viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4 (hepatitis B virus (HBV), hepatitis B);Envelope glycoprotein El gp32 gp35, envelope glycoprotein E2 NSl gp68 gp70, capsid protein C, core protein core, polyprotein, viral protein VPl, viral protein VP2, viral protein VP3, viral protein VP4, antigen G, protein NS3, protein NS5A (hepatitis C virus, hepatitis C); viral protein VPl, viral protein VP2, viral protein VP3, viral protein VP4, large hepatitis delta antigen, small hepatitis delta antigen (hepatitis D virus, hepatitis D); viral protein VPl, viral protein VP2, viral protein VP3, viral protein VP4, capsid protein E2 (hepatitis E virus, hepatitis E);Glycoprotein L UL1, uracil-DNA glycosylase UL2, protein UL3, protein UL4, DNA replication protein UL5, portal protein UL6, virion maturation protein UL7, DNA helicase UL8, replication origin binding protein UL9, glycoprotein M UL10, protein UL11, alkaline endonuclease UL12, serine-threonine protein kinase UL13, tegument protein UL14, terminase UL15, tegument protein UL16, protein UL17, capsid protein VP23, UL18, major capsid protein VP5 UL19, membrane protein UL20, tegument protein UL21, glycoprotein H (UL22), thymidine kinase UL23, protein UL24, protein UL25, capsid protein P40 (UL26, VP24, VP22A), glycoprotein B (UL27), ICP18.5 protein (UL28), major DNA-binding protein ICP8 (UL29), DNA polymerase UL30, nuclear matrix protein UL31, envelope glycoprotein UL32, protein UL33, inner nuclear membrane protein UL34, capsid protein VP26 (UL35), large tegument protein UL36, capsid assembly protein UL37, V P19C protein (UL38), ribonucleotide reductase (large subunit) UL39, ribonucleotide reductase (small subunit) UL40, tegument protein / virion host shutoff VHS protein (UL41), DNA polymerase processivity factor UL42, membrane protein UL43, glycoprotein C (UL44), membrane protein UL45, tegument protein VP11 / 12 (UL46), tegument protein VP13 / 14 (UL47), virion maturation protein VP16 (UL48, alpha-TIF), envelope protein UL49, dUTP diphosphatase UL50, tegument protein; Protein UL51, DNA helicase / primase complex protein UL52, glycoprotein K (UL53), transcriptional regulatory protein IE63 (ICP27, UL54), protein UL55, protein UL56, viral replication protein ICP22 (IE68, US1), protein US2, serine / threonine-protein kinase US3, glycoprotein G (US4), glycoprotein J (US5), glycoprotein D (US6), glycoprotein I (US7), glycoprotein E (US8), tegument protein US9, capsid / tegument protein US10, Vmw21 protein (US11), ICP47 protein (IE12, US12), major transcriptional activator ICP4 (IE175, RSI), E3 ubiquitin ligase ICPO (IE110), latency-associated protein 1 (LRP1), and latency-associated protein 2 (LRP2). LRP2, neurovirulence factor RL1 (ICP34.5), latency-associated transcript LAT (herpes simplex virus 1 and 2 (HSV-1 and HSV-2), herpes simplex); heat shock protein Hsp60, cell surface protein H1C, dipeptidyl peptidase type IV DppIV, M antigen, 70 kDa protein, 17 kDa histone-like protein (Histoplasma capsulatum, histoplasmosis); fatty acid and retinol binding protein-1 FAR-1, tissue inhibitor of metalloproteinases TIMP (TMP), cysteine ​​proteinase ACEY-1, cysteine ​​proteinase ACCP-1, surface antigen Ac-16, secreted protein 2 ASP-2, metalloproteinase 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, surface-associated antigen SAA-2, adult-specific factor Xa, serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1, glutathione S-transferase GST, aspartic protease APR-1, acetylcholinesterase AChE (Ancylostoma duodenale and Necator americanus, hookworm infections); protein NSl, protein NP1, protein VP1, protein VP2, protein VP3 (human bocavirus (HBoV), human bocavirus infection);Major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigenic protein MAPI, major antigenic protein MAP1-2, major antigenic protein MAP1B, major antigenic protein MAP1-3, Erum2510-encoded protein, protein GroEL, protein GroES, 30-kDA major outer membrane protein, GE100-kDa protein, GE130-kDa protein, GE160-kDa protein (Ehrlichia ewingii, human ehrlichiosis); major surface proteins 1-5 (MSPla, MSPlb, MSP2, MSP3, MSP4, MSP5), type IV secretion system proteins VirB2, VirB7, VirBll, and VirD4 (Anaplasma phagocytophilum, human granulocytic anaplasmosis (HGA); protein NSl, small hydrophobic protein NS2, SH protein, fusion protein F, glycoprotein G, matrix protein M, matrix protein M2-1, matrix protein M2-2, phosphoprotein P, nucleoprotein N, polymerase L (human metapneumovirus (hMPV), human metapneumovirus infection); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigenic protein MAPI, major antigenic protein MAP1-2, major antigenic protein MAP1B, major antigenic protein MAP1-3, Erum2510-encoded protein, protein GroEL, protein GroES, 30-kDA major outer membrane protein, GE100-kDa protein, GE130-kDa protein, GE160-kDa protein (Ehrlichia chaffeensis, human monocytic ehrlichiosis);Replication protein E1, regulatory protein E2, protein E3, protein E4, protein E5, protein E6, protein E7, protein E8, major capsid protein LI, minor capsid protein L2 (human papillomavirus (HPV), human papillomavirus (HPV) infection); fusion protein F, hemagglutinin-neuraminidase HN, glycoprotein G, matrix protein M, phosphoprotein P, nucleoprotein N, polymerase L (human parainfluenza virus (HPIV), human parainfluenza virus infection); hemagglutinin (HA), neuraminidase (NA), nucleoprotein Protein (NP), Ml protein, M2 protein, NSl protein, NS2 protein (NEP protein: nuclear transport protein), PA protein, PB1 protein (polymerase basal 1 protein), PB1-F2 protein and PB2 protein (Orthomyxoviridae family, influenza virus (influenza)); genome polyprotein, protein E, protein M, capsid protein C (Japanese encephalitis virus, JE); RTX toxin, type IV pilus, major pilus subunit PilA, regulatory transcription factors PilS and PilR, protein sigma 54, outer membrane protein (Kingella kingae, Kingella kingae infection); prion protein (kuru-purin, kuru); nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP (Lassa virus, Lassa fever); peptidoglycan-associated lipoprotein PAL, 60 kDa chaperonin Cpn60 (groEL, HspB), type IV pilin PilE, outer membrane protein MIP, major outer membrane protein Momps, zinc metalloproteinase MSP (Legionella pneumophila, Legionellosis (Legionnaires' disease, Pontiac fever));P4 nuclease, protein WD, ribonucleotide reductase M2, surface membrane glycoprotein Pg46, cysteine ​​proteinase CP, glucose-regulated protein 78 GRP-78, stage-specific S antigen-like protein A2, ATPase Fl, beta-tubulin, heat shock protein 70 Hsp70, KMP-11, glycoprotein GP63, protein BT1, nucleoside hydrolase NH, cell surface protein Bl, ribosomal protein Pl-like protein PI, sterol 24-c-methyltransferase SMT, LACK protein, histone HI, SPB1 protein, thiol-specific antioxidant TSA, protein antigen STI1, signal peptidase SP, histone H2B, surface antigen PSA-2, cysteine ​​proteinase b Cpb (Leishmania genus, leishmaniasis); major membrane protein I, serine-rich antigen-45 kDa, 10 kDa chaperonin GroES, HSP kDa antigen, amino-oxononanoic acid synthase AONS, protein recombinase A RecA, acetyl- / propionyl-coenzyme A carboxylase alpha, alanine racemase, 60 kDa chaperonin 2, ESAT-6-like protein EcxB (L-ESAT-6), protein Lsr2, protein ML0276, heparin-binding hemagglutinin HBHA, heat shock protein 65 Hsp65, mycPl or ML0041-encoded protein, htrA2 or ML0176-encoded protein, htrA4 or ML2659-encoded protein, gcp or ML0379-encoded protein, clpC or ML0235-encoded protein (Mycobacterium leprae and Mycobacterium lepromatosis, leprosy);Outer membrane protein LipL32, membrane protein LIC10258, membrane protein LP30, membrane protein LIC12238, Ompa-like protein Lsa66, surface protein LigA, surface protein LigB, major outer membrane protein OmpLl, outer membrane protein LipL41, protein LigAni, surface protein LcpA, adhesin protein LipL53, outer membrane protein UpL32, surface protein Lsa63, flagellin FlaBl, membrane lipoprotein LipL21, membrane tag Protein pL40, Leptospiral surface adhesin Lsa27, outer membrane protein OmpL36, outer membrane protein OmpL37, outer membrane protein OmpL47, outer membrane protein OmpL54, acyltransferase LpxA (Leptospira genus, leptospirosis); listeriolysin O precursor Hly (LLO), invasion-related protein lap (P60), listeriolysin regulatory protein PrfA, zinc metalloproteinase Mpl, phosphatidylinositol-specific phospholipase C PLC (PlcA, PlcB), O-acetyltransferase Oat, ABC-transporter permease Im.G_1771, adhesin protein LAP, LAP receptor Hsp60, adhesin LapB, hemolysin listeriolysin OLLO, protein ActA, internalin A InIA, protein InIB (Listeria monocytogenes, listeriosis); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin-binding protein A DbpA, decorin-binding protein B DbpB, flagellar filament 41 kDa core protein Fla, base membrane protein A BmpA (immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE (typically Borrelia burgdorferi and other Borrelia species, Lyme disease (Lyme borreliosis));Venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, vespid allergen homolog VAH, thioredoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N, Nl, N2, and N3), activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, vespid allergen homolog VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, epidermal collagen Col-4, secretory larval acidic protein SLAP, chitinase CHI-1, maltose-binding protein MBP, glycolytic enzyme fructose-l,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, onchocystatin CPI-2, protein Cox-2 (Wuchereria bancrofti and Brugia malayi, lymphatic filariasis (elephantiasis); glycoprotein GP, ​​matrix protein Z, polymerase L, nucleoprotein N (lymphocytic choriomeningitis virus (LCMV), lymphocytic choriomeningitis); thrombospondin-related unspecified protein TRAP, SSP2 sporozoite surface protein 2, apical membrane antigen 1 AMA1, rhoptry membrane antigen RMA1, acidic-basic repeat antigen ABRA, cell passage protein PF, protein Pvs25, merozoite surface protein 1 MSP-1, merozoite surface protein 2 MSP-2, ring-infected erythrocyte surface antigen RESA Liver stage antigen 3 LSA-3, protein Eba-17; 5, serine repeat antigen 5 SERA-5, circumsporozoite protein CS, merozoite surface protein 3 MSP3, merozoite surface protein 8 MSP8, enolase PF10, hepatocyte erythrocyte protein 17kDa HEP17, erythrocyte membrane protein 1 EMP1, protein K beta merozoite surface protein 4 / 5 MSP4 / 5, heat shock protein Hsp90, glutamine-rich protein GLURP, merozoite surface protein 4 MSP-4, protein STARP, circumsporozoite protein-related antigen precursor CRA (Plasmodium spp., Malaria); nucleoprotein N, membrane-associated protein VP24, minor nucleoprotein VP30, polymerase cofactor VP35, polymerase L, matrix protein VP40, envelope glycoprotein GP (Marburg virus, Marburg hemorrhagic fever (MHF)); protein C, matrix protein M, phosphoprotein P, nonstructural protein V, hemagglutinin glycoprotein H, polymerase L, nucleoprotein N, fusion protein F (measles virus, Measles); members of the ABC transporter family (LolC, OppA, and PotF), putative lipoprotein release system transmembrane protein LolC / E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA-encoded protein, boaB-encoded protein (Burkholderia pseudomallei, melioidosis (Whitmore's disease);Pilin protein, minor pilin-related subunit pilC, major pilin subunit and variant pilE, pilS, phase variant protein porA, porin B PorB, protein TraD, Neisserial outer membrane antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin-binding protein TbpA, transferrin-binding protein TbpB, PBP2, mtrR-encoded protein, ponA-encoded protein, membrane permease FbpBC, FbpABC protein system, LbpAB protein, outer membrane protein Opa, outer membrane transporter FetA, iron repression regulator MpeR, factor H-binding protein fHbp, adhesin NadA, protein NhbA, repressor FarR (Neisseria meningitidis, meningococcal infection); 66 kDa protein, 22 kDa protein (usually Metagonimus yokagawai, Yokogawa fluke disease); polar tube proteins (34, 75, and 170 kDa in Glugea, 35, 55, and 150 kDa in Encephalitozoon), kinesin-related proteins, RNA polymerase II largest subunit, similar integral membrane protein YIPA, anti-silencing protein 1, heat shock transcription factor HSF, protein kinase, thymidine kinase, NOP-2-like nucleolar protein (Microsporidia phylum, microsporidia disease); CASP8 and FADD-like apoptosis regulators, glutathione peroxidase GPX1, RNA helicase NPH-II NPH2, poly(A) polymerase catalytic subunit PAPL, major envelope protein P43K, early transcription factor 70 kDa subunit VETFS, early transcription factor 82 kDa subunit VETFL, metalloendopeptidase G1 type, nucleoside triphosphatase I NPH1, replication protein A28-like MC134L, RNA polymerase 7-kDa subunit RP07 (molluscum contagiosum virus (MCV), molluscum contagiosum (MC));Matrix protein M, phosphoprotein P / V, small hydrophobic protein SH, nucleoprotein N, protein V, fusion glycoprotein F, hemagglutinin-neuraminidase HN, RNA polymerase L (mumps virus, epidemic parotitis); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, cytoplasmic protein D, crystalline surface layer protein SLP, protective surface protein antigen SPA (Rickettsia typhi, murine typhus (scrub typhus)); adhesin PI, adhesin P30, protein pll6, protein P40, cytoskeletal protein HMW1, cytoskeletal protein HMW2, cytoskeletal protein HMW3, MPN152-encoded protein, MPN426-encoded protein, MPN456-encoded protein, MPN-500-encoded protein (Mycoplasma pneumoniae, Mycoplasma pneumoniae); NocA, iron-dependent regulatory protein; VapA, VapD, VapF, VapG, caseinolytic proteases, filament tip-associated 43-kDa protein, protein P24, protein P61, 15-kDa protein, 56-kDa protein (usually Nocardia asteroides and other Nocardia species, nocardiosis);Venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, vespid allergen homolog VAH, thioredoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N, Nl, N2, and N3), activation-associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferase GST, myosin, vespid allergen homolog VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, epidermal collagen Col-4, secretory larval acidic protein SLAP, chitinase CHI-1, maltose-binding protein MBP, glycolytic enzyme fructose-l,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode-specific gene product OvB20, onchocystatin CPI-2, Cox-2 (Onchocerca volvulus, onchocerciasis (river blindness); 43-kDa secreted glycoprotein, glycoprotein gpO, glycoprotein gp75, antigen Pb27, antigen Pb40, heat shock protein Hsp65, heat shock protein Hsp70, heat shock protein Hsp90, protein P10, triosephosphate isomerase TPI, N-acetyl-glucosamine-binding lectin paracoccin, 28-kDa protein Pb28 (Paracoccidioides brasiliensis, paracoccidioidomycosis (South American blastomycosis)); 28-kDa cruzipain-like cysteine ​​protease Pw28CCP (typically Paragonimus westermani and other Paragonimus species, paragonimiasis);Outer membrane protein OmpH, outer membrane protein Omp28, protein PM1539, protein PM0355, protein PM1417, repair protein MutL, protein BcbC, protein PM0305, formate dehydrogenase-N, protein PM0698, protein PM1422, DNA gyrase, lipoprotein PlpE, adhesion protein Cp39, heme acquisition system receptor HasR, 39-kDa capsule protein, iron-regulated OMP IROMP, outer membrane protein OmpA87, fimbrial protein Ptf, fimbrial subunit protein PtfA, transferrin-binding protein Tbpl, esterase enzyme MesA, Pasteurella multocida toxin PMT, adhesion protein Cp39 (Pasteurella genus, pasteurellosis); filamentous hemagglutinin FhaB, adenylate cyclase CyaA, pertussis toxin subunit 4 precursor PtxD, pertactin precursor Prn, toxin subunit 1 PtxA, protein Cpn60, protein brkA, pertussis toxin subunit 2 precursor PtxB, pertussis toxin subunit 3 precursor PtxC, pertussis toxin subunit 5 precursor PtxE, pertactin Prn, protein Fim2, protein Fim3; (Bordetella pertussis, whooping cough);"Fl capsule antigen, virulence-associated V antigen, secreted effector protein LcrV, V antigen, outer membrane protease Pla, secreted effector protein YopD, putative secreted protein-tyrosine phosphatase Yoph, needle complex major subunit YscF, protein kinase YopO, putative autotransporter protein YapF, inner membrane ABC-transporter YbtQ (Irp7), putative carbohydrate-binding protein YPO0612, heat shock protein 90 HtpG, putative sulfatase protein YdeN, outer membrane lipoprotein carrier protein LolA, secretion chaperone YerA, putative lipoprotein YPO0420, hemolysin activator protein HpmB, pesticin / yersiniabactin outer membrane receptor Psn, secreted effector protein YopE, secreted effector protein YopF, secreted effector protein YopK, outer membrane protein YopN, outer membrane protein YopM, coagulase / fibrinolysin precursor Pla;" (Yersinia pestis, plague); protein PhpA, surface adhesin PsaA, pneumolysin Ply, ATP-dependent protease CIp, lipoic acid-protein ligase LplA, cell wall surface-anchored protein psrP, sortase SrtA, glutamyl-tRNA synthetase GltX, choline-binding protein A CbpA, pneumococcal surface protein A PspA, pneumococcal surface protein C PspC, 6-phosphogluconate dehydrogenase Gnd, iron-binding protein PiaA, murein hydrolase LytB, proteon LytC, protease Al (Streptococcus pneumoniae, pneumococcal infection); major surface protein B, kexin-like protease KEX1, protein A12, 55 kDa antigen P55, major surface glycoprotein Msg (Pneumocystis jirovecii, Pneumocystis pneumoniae (PCP); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (poliovirus, polio);Protein Nfal, exendin-3, secretory lipase, cathepsin B-like protease, cysteine ​​protease, cathepsin, peroxiredoxin, protein CrylAc (usually Naegleria fowleri, primary amebic meningoencephalitis (PAM)); agnoprotein, large T antigen, small T antigen, major capsid protein VP1, minor capsid protein Vp2 (JC virus, progressive multifocal leukoencephalopathy); low calcium response protein E LCrE, chlamydial outer membrane protein N CopN, serine / threonine-protein kinase PknD, acyl-carrier-protein S-malonyltransferase FabD, single-stranded DNA-binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, polymorphic membrane protein family (Pmpl, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, PmplO, Pmpll, Pmpl2, Pmpl3, Pmpl4, Pmpl5, Pmpl6, Pmpl7, Pmpl8, Pmpl9, Pmp20, Pmp21) (Chlamydophila psittaci, psittacosis); outer membrane protein PI, heat shock protein B HspB, peptide ABC transporter, GTP-binding protein, protein IcmB, ribonuclease R, phosphata SixA, protein DsbD, outer membrane protein TolC, DNA-binding protein PhoB, ATPase D; otB, heat shock protein B HspB, membrane protein ComI, 28 kDa protein, DNA-3-methyladeniglycosidase I, outer membrane protein OmpH, outer membrane protein AdaA, glycine cleavage system T-protein (Coxiella burnetii, Q fever); nucleoprotein N, large structural protein L, phosphoprotein P, matrix protein M, glycoprotein G (rabies virus, rabies); fusion protein F, nucleoprotein N, matrix protein M, matrix protein M2-1, matrix protein M2-2, phophoprotein P, small hydrophobic protein SH, major surface glycoprotein G, polymerase L, nonstructural protein 1 NS1, nonstructural protein 2 NS2 (respiratory syncytial virus (RSV), respiratory syncytial virus infection); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (rhinovirus, rhinovirus infection); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, protein PS120, cytoplasmic protein D, defensive surface protein antigen SPA (Rickettsia genus, Rickettsial infection); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, cytoplasmic protein D (Rickettsia akari, Rickettsialpox); envelope glycoprotein GP, ​​polymerase L, nucleoprotein N, nonstructural protein NSS (Rift Valley fever virus, Rift Valley fever (RVF)); outer membrane protein OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, cytoplasmic protein D (Rickettsia rickettsii, Rocky Mountain spotted fever (RMSF));Nonstructural protein 6 NS6, nonstructural protein 2 NS2, intermediate capsid protein VP6, inner membrane capsid protein VP2, nonstructural protein 3 NS3, RNA-directed RNA polymerase L, protein VP3, nonstructural protein 1 NS1, nonstructural protein 5 NS5, outer membrane capsid glycoprotein VP7, nonstructural glycoprotein 4 NS4, outer membrane capsid protein VP4; (rotavirus, rotavirus infection); polyprotein P200, glycoprotein El, glycoprotein E2, protein NS2, capsid protein C (rubella virus, rubella); chaperonin GroEL (MopA), inositol phosphate phosphatase SopB, heat shock protein HsIU, chaperone protein DnaJ, protein TviB, protein IroN, flagellin FliC, invasion protein SipC, glycoprotein gp43, outer membrane protein LamB, outer membrane protein PagC, outer membrane protein TolC, outer membrane protein NmpC, outer membrane protein FadL, transport protein SadA, transferrin enzyme WgaP, effector proteins SifA, SteC, SseL, SseJ, and SseF (Salmonella genus, salmonellosis); protein 14, nonstructural protein NS7b, nonstructural protein NS8a, protein 9b, protein 3a, nucleoprotein N, nonstructural protein NS3b, nonstructural protein NS6, protein 7a, nonstructural protein NS8b, membrane protein M, envelope small membrane protein EsM, replicase polyprotein la, spike glycoprotein S, replicase polyprotein lab; SARS coronavirus, SARS (severe acute respiratory syndrome); serine protease, atypical Sarcoptes antigen 1 ASA1, glutathione S-transferase GST, cysteine ​​protease, serine protease, apolipoprotein (Sarcoptes scabiei, scabies);Glutathione S-transferase GST, paramyosin, hemoglobinase SM32, major egg antigen, 14 kDa fatty acid-binding protein Sml4, major larval surface antigen P37, 22.6 kDa tegument antigen, calpain CANP, triphosphate isomerase Tim, surface protein 9B, outer membrane capsid protein VP2, 23 kDa integral membrane protein Sm23, Cu / Zn-superoxide dismutase, glycoprotein Gp, myosin (Schistosoma spp., Schistosomiasis (Bilharziosis)); 60 kDa chaperonin, 56 kDa type-specific antigen, pyruvate phosphate dikinase, 4-hydroxybenzoate octaprenyltransferase (Orientia tsutsugamushi disease; dehydrogenase GuaB, invasion protein Spa32, invasin IpaA, invasin IpaB, invasin IpaC, invasin IpaD, invasin IpaH, invasin IpaJ (Shigella spp., Shigellosis (Bacillary dysentery); protein P53, virion protein US10 homolog, transcription factor IE63, transcription transactivator IE62, protease P33, alpha transinducer 74 kDa protein, deoxyuridine 5'-triphosphate nucleotidyl hydrolase, transcription transactivator IE4, membrane protein UL43 homolog, nuclear phosphoprotein UL3 homolog, nuclear protein UL4 homolog, replication origin binding protein, membrane protein 2, phosphoprotein 32, protein 57, DNA polymerase processivity factor, portal protein 54, DNA primase, tegument protein UL14 homolog, tegument protein UL21 homolog, tegument protein UL55 homolog, tripartite terminase subunit UL33 homolog, tripartite partite) terminase subunit UL15 homolog, capsid-associated protein 44, virion packaging protein 43 (Varicella zoster virus (VZV), Shingles (Herpes zoster));Cleaved 3-beta-hydroxy-5-enesteroid dehydrogenase homolog, virion mature membrane protein A13, protein A19, protein A31, cleaved protein A35 homolog, protein A37.5 homolog, protein A47, protein A49, protein A51, semaphorin-like protein A43, serine proteinase inhibitor 1, serine proteinase inhibitor 2, serine proteinase inhibitor 3, protein A6, protein B15, protein CI, protein C5, protein C6, protein F7, protein F8, protein F9, protein F11, protein F14, protein F15, protein F16 (Variola (major) or Variola (minor)), Variola); adhesin / glycoprotein gp70, protease (Sporothrix schenckii, sporotrichosis); heme-iron-binding protein IsdB, collagen adhesin Cna, coagulation factor A ClfA, protein MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntCl, enterotoxin type C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, toxic shock syndrome toxin-1 TSST-1, staphylokinase, penicillin-binding protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus genus, staphylococcal food poisoning); heme-iron-binding protein IsdB, collagen adhesin Cna, coagulation factor A ClfA, protein MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntCl, enterotoxin type C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, toxic shock syndrome toxin-1 TSST-1, staphylokinase, penicillin-binding protein 2a PBP2a (MecA), secretory antigen SssA (Staphylococcus genus, e.g., aureus, Staphylococcal infections);Antigen Ss-IR, antigen NIE, strongylastacin, Na+-K+ATPase Sseat-6, tropomycin SsTmy-1, protein LEC-5, 41-kDa antigen P5, 41-kDa larval protein, 31-kDa larval protein, 28-kDa larval protein (Strongyloides stercoralis, Strongyloidiasis); glycerophosphodiester phosphodiesterase GlpQ (Gpd), outer membrane protein TmpB, protein Tp92, antigen TpFl, repeat protein Tpr, repeat protein F TprF, repeat protein G TprG, repeat protein I Tprl, repeat protein J TprJ, repeat protein K TprK, treponemal membrane protein A TmpA, lipoprotein, 15 kDa Tppl5, 47-kDa membrane antigen, miniferritin TpFl, adhesin Tp0751, lipoprotein TP0136, protein TpN17, protein TpN47, outer membrane protein TP0136, outer membrane protein TP0155, outer membrane protein TP0326, outer membrane protein TP0483, outer membrane protein TP0956 (Treponema pallidum, syphilis); cathepsin L-like protease, 53 / 25-kDa antigen, 8-kDa family member, cysticercus protein TsAg5 with little trypsin-like activity, oncocyst protein TSOL18, oncocyst protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB (Taenia spp., cestode disease); tetanus toxin TetX, tetanus toxin C TTC, 140 kDa S-layer protein, flavoprotein beta-subunit CT3, phospholipase (lecithinase), phosphocarrier protein HPr (Clostridium tetani, Tetanus (Lockjaw)); genomic polyprotein, protein E, protein M, capsid protein C (tick-borne encephalitis virus (TBEV), tick-borne encephalitis);58-kDa antigen, 68-kDa antigen, Toxocara larvae excretory-secretory antigen TES, 32-kDa glycoprotein, glycoprotein TES-70, glycoprotein GP31, excretory-secretory antigen TcES-57, periintestinal fluid antigen Pe, soluble extract antigen Ex, excretory / secretory larval antigen ES, antigen TES-120, polyprotein allergen TBA-1, cathepsin L-like cysteine ​​protease c-cpl-1, 26-kDa protein (Toxocara canis or Toxocara cati, toxocariasis (ocular larva migrans (OLM) and visceral larva migrans (VLM)); microneme proteins (MICl, MIC2, MIC3, MIC4, MIC5, MIC6, MIC7, MIC8), rhoptry protein Rop2, rhoptry proteins (Ropl, Rop2, Rop3, Rop4, Rop5, Rop6, Rop7, Ropl6, Rjopl7), protein SRl, surface antigen P22, major antigen p24, major surface antigen p30, dense granule proteins (GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA9, GRA10), 28 kDa antigen, surface antigen SAG1, SAG2-related antigen, nucleoside-triphosphatase 1, nucleoside-triphosphatase 2, protein Stt3, HesB-like domain-containing protein; Protein, rhomboid-like protease 5, toxomepsin 1 (Toxoplasma gondii, toxoplasmosis); 43-kDa secreted glycoprotein, 53-kDa secreted glycoprotein, paramyosin, antigen Ts21, antigen Ts87, antigen p46000, TSL-1 antigen, caveolin-1 (CAV-1), 49-kDa neonatal larval antigen, prosaposin homolog, serine protease, serine proteinase inhibitor, 45-kDa glycoprotein Gp45 (Trichinella spiralis, trichinosis); Myb-like transcription factors (Mybl, Myb2, Myb3), adhesion protein AP23, adhesion protein AP33, adhesion protein AP33-3, adhesion AP51, adhesion AP65, adhesion protein AP65-1, alpha-actinin, kinesin-related protein, teneurin, 62-kDa proteinase, subtilisin-like serine protease SUB1, cysteine ​​proteinase gene 3 CP3, alpha-enolase Enol, cysteine ​​proteinase CP30, heat shock proteins (Hsp70, Hsp60), immunogenic protein P270, (Trichomonas vaginalis, trichomoniasis); beta-tubulin, 47-kDa protein, secretory leukocyte-like proteinase-1 SLP-1, 50-kDa protein TT50, 17-kDa antigen, 43 / 47-kDa protein (Trichuris trichiura, Trichuriasis (Whipworm infection));Protein ESAT-6 (EsxA), 10 kDa filtrate antigen EsxB, secreted antigen 85-B FBPB, fibronectin-binding protein A FbpA (Ag85A), serine protease PepA, PPE family protein PPE18, fibronectin-binding protein D FbpD, immunogenic protein MPT64, secreted protein MPT51, catalase-peroxidase-peroxynitritase T KATG, periplasmic phosphate-binding lipoprotein PSTS3 (PBP-3, Phos-1), iron-regulated heparin-binding hemagglutinin Hbha, PPE family protein PPE14, PPE family protein PPE68, protein Mtb72F, protein Apa, immunogenic protein MPT63, periplasmic phosphate-binding lipoprotein PSTS1 (PBP-1), molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permease protein pstA, 14 kDa antigen, fibronectin-binding protein C FbpCl, alanine dehydrogenase TB43, glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like protein, protein MTB32, protein MTB39, crystallin, heat shock protein HSP65, protein PST-S (usually found in Mycobacterium tuberculosis, tuberculosis); outer membrane protein FobA, outer membrane protein FobB, intracellular multiplication site IglCl, intracellular multiplication site IglC2, aminotransferase Wbtl, chaperonin GroEL, 17 kDa major membrane protein TUL4, lipoprotein LpnA, chitinase family 18 protein, isocitrate dehydrogenase, Nif3 family protein, type IV pyrimyl glycosylation protein, outer membrane protein tolC, FAD-binding family protein, type IV pilin multimeric outer membrane protein, two-component sensor protein KdpD, chaperone protein DnaK, protein TolQ (Francisella tularensis, tularemia);"MB antigen, urease, protein GyrA, protein GyrB, protein ParC, protein ParE, lipid-associated membrane protein LAMP, thymidine kinase TK, phospholipase PL-Al, phospholipase PL-A2, phospholipase PL-C, surface-expressed 96-kDa antigen; (Ureaplasma urealyticum, Ureaplasma urealyticum infection); nonstructural polyprotein, structural polyprotein, capsid protein CP, protein El, protein E2, protein E3, protease PI, protease P2, protease P3 (Venezuelan equine encephalitis virus, Venezuelan equine encephalitis); glycoprotein GP, ​​matrix protein Z, polymerase L, nucleoprotein N (Guanarito virus, Venezuelan hemorrhagic fever); polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein N S2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (West Nile virus, West Nile fever); capsid protein CP, protein El, protein E2, protein E3, protease P2 (Western equine encephalitis virus, Western equine encephalitis); genomic polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (yellow fever virus, Yellow fever);Putative Yop targeting protein YobB, effector protein YopD, effector protein YopE, protein Yoph, effector protein YopJ, protein translocation protein YopK, effector protein YopT, protein YpkA, flagellar biosynthesis protein FlhA, peptidase M48, potassium efflux system KefA, transcription factor RovA, adhesin Ifp, translocator protein LcrV, protein PcrV, invasin Inv, outer membrane protein OmpF-like porin, adhesin YadA, protein kinase C, phospholipase CI, protein PsaA, mannosyltransferase-like protein WbyK, protein YscU, antigen YPMa (Yersinia pseudotuberculosis, Yersinia pseudotuberculosis infection); effector protein YopB, 60 kDa chaperonin, protein WbcP, tyrosine-protein phosphatase Yoph, protein YopQ, enterotoxin, galactoside permease, reductase NrdE, protein YasN, invasin Inv, adhesin YadA, outer membrane porin F OmpF, protein UspAl, protein EibA, protein Hia, cell surface protein Ail, chaperones SycD, protein LcrD, protein LcrG, protein LcrV, protein SycE, protein YopE, regulator protein TyeA, protein YopM, protein YopN, protein YopO, protein YopT, protein YopD, protease ClpP, protein MyfA, protein FilA, and protein PsaA (Yersinia enterocolitica, yersiniosis);

[0162] In embodiments in which the infectious disease is influenza, the mRNA molecule may have a coding region encoding at least one antigenic peptide or protein derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2) of an influenza virus, or a fragment or variant thereof.

[0163] In certain embodiments, the coding region encodes at least one antigenic peptide or protein derived from the hemagglutinin (HA) and / or neuraminidase (NA) of an influenza virus, or a fragment or variant thereof, wherein the HA and / or NA may independently be derived from an influenza A virus or an influenza B virus, or a fragment of either.

[0164] In embodiments where the infectious disease is influenza, the mRNA molecule may have a coding region that encodes at least one antigenic peptide or protein derived from the spike (S) protein. [Example]

[0165] Example 1: Synthetic approach to compounds SL26-SL33 The following synthetic procedures for compounds SL26-SL33 illustrate approaches by which the compounds of the present disclosure can be made. These synthetic approaches will be known to those skilled in the art. Simple modification of the conditions or the nature of any particular substrate can be altered to achieve any compound within the scope of Formula I.

[0166] Synthesis of compound 2 [ka] To a solution of compound 1 (99.5 g, 1.27 mol, 88.8 mL, 1.00 equiv.) and imidazole (104 g, 1.53 mol, 1.20 equiv.) in DCM (800 mL), TBSCl (211 g, 1.40 mol, 171 mL, 1.10 equiv.) was added portionwise at 25° C. After the addition, the resulting suspension was stirred at 25° C. for 10 h. TLC (petroleum ether / ethyl acetate=5 / 1, R of compound 1) showed a 5:1 ratio. f = 0.74, R of compound 2 f =0.43) indicated the reaction was complete. The reaction solution was poured into water (300 mL) and extracted with EtOAc (100 mL). The combined organic layers were washed with brine (200 mL), dried over sodium sulfate, and concentrated to give the product. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to give compound 2 (100 g, 519 mmol, 40.8% yield) as a colorless oil. 1 H NMR:ET61249-5-P1A(400MHz, CDCl3) δ 3.73(t,J=6.4Hz,2H), 2.60-2.65(m,2H), 1.53(t,J=8.4Hz,2H), 0.87(s,9H), 0.05(s,6H).

[0167] Synthesis of compound 3 [ka] To a solution of compound 2 (35.0 g, 181 mmol, 1.00 equiv.) in DCM (70.0 mL) was added K2CO3 (1.26 g, 9.10 mmol, 0.05 equiv.). Compound 2a (17.2 g, 200 mmol, 18.0 mL, 1.10 equiv.) was added dropwise to the solution. The solution was stirred at 25 °C for 12 h. TLC (petroleum ether / ethyl acetate = 3 / 1, R of compound 2) f = 0.7, R of compound 3 f=0.38) indicated that the reaction solution was complete. The reaction was poured into water (300 mL). The solution was extracted with EtOAc (100 mL × 2), and the organic layers were combined and concentrated in vacuo to obtain a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to obtain compound 3 (48.0 g, crude) as a colorless oil. 1 H NMR:ET61249-6-P1A(400MHz,CDCl3)δ 5.29(s,1H), 3.68-3.78(m,2H), 3.68(s,3H), 2.67-2.84(m,2H), 2.59-2.65(m,4H), 0.87(s,9H), 0.05(s,6H).

[0168] Synthesis of compound 4 [ka] To a solution of compound 3 (35.0 g, 125 mmol, 1.00 equiv.) in THF (175 mL) under N was added LiBH (3.40 g, 156 mmol, 1.24 equiv.) at 0 °C. The solution was stirred at 15 °C for 3 h. TLC (petroleum ether / ethyl acetate = 5 / 1, R of compound 3) was performed. f = 0.73, R of compound 4 f =0.42) indicated that the reaction was complete. The solution was poured into NH4Cl (saturated aqueous solution 200 mL). The solution was extracted with EtOAc (50.0 mL × 2). The organic layers were combined and concentrated in vacuo to give compound 4 (28.0 g, 112 mmol, 88.9% yield) as a colorless oil. 1 H NMR:ET61249-12-P1A(400MHz,CDCl3)δ 3.74-3.79(m,4H), 2.64-2.70(m,4H), 1.82-1.88(m,2H), 0.90(s,9H), 0.08(s,6H).

[0169] Synthesis of compound 5 [ka] To a solution of compound 4 (40.0 g, 160 mmol, 1.00 equiv.) in DCM (400 mL) was added CBr (106 g, 319 mmol, 2.00 equiv.), pyridine (126 g, 1.60 mol, 129 mL, 10.0 equiv.), and PPh (83.8 g, 319 mmol, 2.00 equiv.). The mixture was stirred at 20 °C for 5 h. TLC (petroleum ether / ethyl acetate = 10 / 1) showed the R of compound 4. f = 0.23, R of compound 5 f =0.65) indicated that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0). Compound 5 (25.0 g, 79.8 mmol, 50.0% yield) was obtained as a colorless oil. 1 H NMR:ET60350-30-P1A1(400MHz,CDCl3)δ 0.04-0.10(m,6H), 0.86-0.92(m,9H), 2.06-2.15(m,2H), 2.61-2.73(m,4H), 3.47-3.53(m,2H), 3.73-3.79(m,2H).

[0170] Synthesis of compound 6 [ka] To a suspension of Mg (10.3 g, 426 mmol, 6.67 equiv.) and I2 (1.62 g, 6.38 mmol, 1.29 mL, 0.10 equiv.) in THF (40.0 mL), compound 5 (20.0 g, 63.8 mmol, 1.00 equiv.) was added dropwise over 30 min at 50 °C. After the addition, the solution was stirred at 50 °C for 30 min. Compound 5a (2.56 g, 25.5 mmol, 2.30 mL, 0.4 equiv.) was then added dropwise at 50 °C. The resulting solution was stirred at 25 °C for 2 h. TLC (petroleum ether / ethyl acetate = 1 / 1, R of compound 5) showed no significant difference. f = 0.76, R of compound 6 f =0.32) indicated that the reaction solution was complete. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 50 / 1 to 0 / 1) to give compound 6 (14.0 g, 24.6 mmol, 77.1% yield) as a colorless oil.1 H NMR:ET61249-20-P1A(400MHz,CDCl3)δ 3.76(t,J=6.8Hz,4H), 3.67(s,2H), 2.64(t,J=7.2Hz,4H), 2.55-2.26(m,4H), 1.52-1.57(m,10H), 0.90(s,18H), 0.08(s,12H).

[0171] Synthesis of compound 7 [ka] To a solution of compound 6 (3.50 g, 6.15 mmol, 1.00 equiv.) and TEA (1.24 g, 12.3 mmol, 1.71 mL, 2.00 equiv.) in DCM (15.0 mL), MsCl (1.00 g, 8.73 mmol, 676 uL, 1.42 equiv.) was added dropwise at 0° C. After the addition, the solution was stirred at 0° C. for 6 hours. TLC (petroleum ether / ethyl acetate=1 / 1, R of compound 6) f = 0.24, R of compound 7 f = 0.37) indicated that the reaction mixture was complete. The solution was poured into NH4Cl (saturated aqueous solution 50.0 mL). The solution was extracted with EtOAc (20.0 mL × 2). The organic layers were combined and concentrated in vacuo to give compound 7 (3.90 g, 6.03 mmol, 97.9% yield) as a yellow oil.

[0172] Synthesis of compound 8 [ka] To a solution of compound 3a (2.19 g, 21.6 mmol, 2.98 mL, 4.00 equiv.) in DCM (8.00 mL) was added compound 7 (3.50 g, 5.41 mmol, 1.00 equiv.). The solution was stirred at 50° C. for 12 hours. TLC (DCM / MeOH=8 / 1, R of compound 7) f = 0.65, R of compound 8 f=0.40), which indicated the reaction was complete. The organic layers were combined and concentrated in vacuo to give the crude product. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to give compound 8 (3.00 g, 4.60 mmol, 85.1% yield) as a yellow oil.

[0173] Synthesis of compound 9 [ka] To a solution of compound 8 (3.00 g, 4.60 mmol, 1.00 equiv.) in THF (10.5 mL) was added TBAF (1.00 M, 13.8 mL, 3.00 equiv.). The solution was stirred at 25° C. for 3 hours. TLC (DCM / MeOH=8 / 1, R of compound 8) f = 0.52, R of compound 9 f =0.26) indicated that the reaction was complete. The reaction solution was concentrated in vacuo to give compound 9 (6.00 g, crude) as a yellow oil.

[0174] Synthesis of SL26 [ka] To a solution of compound 9 (2.00 g, 4.72 mmol, 1.00 equiv) in Py (10.0 mL) was added oleic acid (4.00 g, 14.2 mmol, 4.00 mL, 3.00 equiv) and EDCI (3.62 g, 18.9 mmol, 4.00 equiv). The mixture was stirred at 45 °C for 10 h. LCMS showed that compound 9 was completely consumed. Several new peaks were detected by LCMS, indicating the desired compound. The reaction mixture was partitioned between EtOAc (200 mL) and water (100 mL). The organic layer was dried over NaSO and concentrated under reduced pressure to give the crude product. The crude product was purified by preparative HPLC (column: Welch Xtimate C1 100 x 30 mm x 5 um; mobile phase: [Water (0.225% FA)-(ACN-THF 2 / 1)]; B%: 40%-85%, 20 min) to give SL26 (1.00 g, 1.05 mmol, 22.3% yield) as a yellow oil. LCMS: ET60350-32-P1A (RT = 2.916 min, M+H + =952.9). 1 H NMR:ET60350-32-P1A(400MHz,CDCl3)δ 5.31-5.39(m,4H), 4.19-4.25(m,4H), 2.72-2.78(m,4H), 2.54-2.61(m,4H), 2.28-2.47(m,10H), 1 .98-2.05(m,8H), 1.61-1.68(m,10H), 1.43-1.55(m,12H), 1.26-1.34(m,40H), 0.85-0.91(m,12H).

[0175] Synthesis of compound 2-2 [ka] To a solution of compound 2-1 (50.0 g, 657 mmol, 47.6 mL, 1.00 equiv.) in DCM (750 mL) was added imidazole (35.7 g, 525 mmol, 0.80 equiv.) at 15° C. Then, TBSCl (59.4 g, 394 mmol, 48.3 mL, 0.60 equiv.) was added portionwise to the mixture at 0° C., maintaining the internal temperature below 10° C. The resulting solution was stirred at 15° C. for 2 h. TLC (petroleum ether / ethyl acetate=1 / 1, R of compound 2-1) was performed. f = 0.10, and R of compound 2-2 f =0.50) showed that none of the TBSCl remained, and two new major spots with lower polarity were detected. The reaction solution was poured into a saturated aqueous solution of NH4Cl (500 ml), and the organic phase was washed with a half-saturated aqueous solution of NaCl (200 ml). The organic phase was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / MTBE = 100 / 1 to 50 / 1) to give compound 2-2 (51.0 g, 267 mmol, 40.7% yield) as a colorless oil. 1 HNMR:ET60053-22-P1A3(400MHz, CDCl3)δ 3.77-3.83(m,4H), 1.74-1.79(m,2H), 0.89(s,9H), 0.07(s,6H).

[0176] Synthesis of Compound 2-2A [ka] To a solution of compound 2A (230 g, 1.51 mol, 215 mL, 1 equiv.) and TEA (229 g, 2.27 mol, 316 mL, 1.5 equiv.) in THF (1.5 L) was added TsCl (317 g, 1.66 mol, 1.1 equiv.) in portions at 0° C., maintaining the internal temperature below 20° C. After the addition, the reaction was stirred at 20° C. for 12 h. TLC (petroleum ether / ethyl acetate=5 / 1, R of compound 2A) was performed. f = 0.4, R of compound 2-2A f= 1.0) indicated complete consumption of the starting material. The reaction mixture was poured into water (1.0 L). The resulting solution was extracted with MTBE (500 mL × 3). The combined organic phases were washed with brine (1.5 L), dried over anhydrous NaSO, and concentrated under vacuum to give the crude product as a brown solid. The crude product was triturated with a solution of petroleum ether / MTBE (2 / 1, 600 mL) at 20 °C for 1 hour. The resulting suspension was filtered, and the filter cake was rinsed with MTBE (100 mL). The filter cake was dried under vacuum to give compound 2-2A (440 g, 1.44 mol, 95.0% yield) as an off-white solid. 1 H NMR:ET61271-17-P1A,(400MHz,CDCl3)δ 7.78(d,J=8.4Hz,2H), 7.22-7.35(m,7H), 4.47(s,2H), 4.18(t,J=4.8Hz,2H), 3.65(t,J=4.8Hz,2H), 2.42(s,3H).

[0177] Synthesis of Compound 2-3 [ka] To a solution of compound 2-2 (29.8 g, 156 mmol, 1.20 equiv.) in THF (280 mL), t-BuOK (17.5 g, 156 mmol, 1.20 equiv.) was added at 0° C. under a N atmosphere, maintaining the internal temperature below 20° C. After the addition, the reaction solution was stirred at 20° C. for 0.5 h. Compound 2-2A (40.0 g, 130 mmol, 1.0 equiv.) was then added dropwise to the reaction solution at 20° C. The resulting solution was stirred at 20° C. for 2 h. TLC (petroleum ether / ethyl acetate=5 / 1, R of compound 2-2A) f = 0.30, and R of compound 2-3 f= 0.60) indicated that approximately 20% of compound 2-2A remained, and two new major spots with lower polarity were detected. The reaction was washed with a saturated aqueous solution of NH4Cl (500 mL), and the organic phase was washed with a half-saturated aqueous solution of NaCl (500 mL). The organic phase was then concentrated under reduced pressure to give compound 2-3 (45.0 g, crude) as a colorless oil, which was used in the next step without further purification.

[0178] Synthesis of Compounds 2-4 [ka] To a solution of compound 2-3 (180 g, 554 mmol, 1.00 equiv.) in THF (300 mL), TBAF (1 M, 721 mL, 1.30 equiv.) was added at 20° C. The reaction solution was stirred at 20° C. for 2 hours. TLC (petroleum ether / ethyl acetate=5:1, R of compound 2-3) was performed. f = 0.9, and R of compounds 2-4 f =0.5) showed that none of compound 2-3 remained, and one new major spot with lower polarity was detected. The reaction solution was poured into a saturated aqueous solution of NH4Cl (600 ml), and the organic phase was washed with a half-saturated aqueous solution of NaCl (300 ml). The final organics were concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 400 / 1 to 300 / 1) to give compound 2-4 (45 g, 214 mmol, 38.5% yield) as a brown oil. 1 HNMR:ET60053-40-P1A1(400MHz,CDCl3)δ 7.34-7.38(m,5H), 4.57(s,2H), 3.77-3.80(m,2H), 3.63-3.70(m,6H), 1.85-1.88(m,2H).

[0179] Synthesis of Compounds 2-5 [ka] To a solution of compound 2-4 (45.0 g, 214 mmol, 1.00 equiv.) in THF (225 mL) was added CBr (141 g, 428 mmol, 2.0 equiv.). Then, a solution of PPh (112 g, 428 mmol, 2.00 equiv.) in THF (500 mL) was added to the reaction solution at 0 °C under a N atmosphere, maintaining the internal temperature below 15 °C. The resulting solution was stirred at 20 °C for 2 h. TLC (petroleum ether / ethyl acetate = 10:1, R of compound 2-4) showed no significant difference. f = 0.10, and R for compounds 2-5 f =0.50) showed that none of compound 2-4 remained, and one new major spot with lower polarity was detected. The reaction solution was added to a saturated aqueous solution of NH4Cl (1000 ml), and the organic phase was washed with a half-saturated aqueous solution of NaCl (500 ml). The final organic phase was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1-20 / 1) to give compound 2-5 (50.0 g, 183 mmol, 85.5% yield) as a brown oil. 1 HNMR:ET60053-42-P1A4(400MHz,CDCl3)δ 7.29-7.36(m,5H), 4.58(s,2H), 3.60-3.65(m,6H), 3.51-3.54(t,J=6.8Hz,2H), 2.01-2.16(m,2H).

[0180] Synthesis of Compounds 2-6 [ka] To a suspension of Mg (5.34 g, 220 mmol, 5 equiv) in THF (50 mL) was added I2 (112 mg, 439 umol, 0.01 equiv) in one portion at 25°C. The resulting suspension was stirred at 25°C for 1 hour. The suspension was then heated to 50°C, and compound 2-5 (12 g, 43.9 mmol, 1 equiv) was added dropwise to the reaction at 50°C, maintaining the internal temperature below 60°C. After the addition, the resulting suspension was stirred at 25°C for 1 hour. Compound 5a (1.76 g, 17.6 mmol, 0.4 equiv) was then added portionwise to the reaction at 25°C. The reaction was stirred at 25°C for 2 hours. TLC (petroleum ether / ethyl acetate = 10 / 1, R of compound 2-5) f =0.6, R of Compound 2-6 f = 0.0) indicated complete consumption of the starting material. The reaction mixture was poured into an aqueous solution of NH4Cl (saturated, 30 mL). The resulting suspension was extracted with MTBE (20 mL × 3). The combined organic phases were washed with brine (30 mL) and concentrated in vacuo to give the crude product as a brown oil. The crude product was purified by column chromatography on silica gel (petroleum ether:ethyl acetate = 10:1-3:1) to give compound 2-6 (4.5 g, 9.21 mmol, 21.0% yield) as a pale yellow oil. 1 H NMR:ET61271-26-P1A,(400MHz,CDCl3)δ 7.27-7.37(m,10H), 4.57(s,4H), 3.59-3.62(m,10H), 3.49(t,J=6.0Hz,4H), 1.38-1.68(m,14H).

[0181] Synthesis of compounds 2-7 [ka] To a solution of compound 2-6 (6.00 g, 12.3 mmol, 1.00 equiv.) in DCM (30 mL), TEA (3.73 g, 36.8 mmol, 5.13 mL, 3.00 equiv.) and MsCl (2.67 g, 23.3 mmol, 1.80 mL, 1.90 equiv.) were added at 0° C. The solution was stirred at 0° C. for 1 h. TLC (petroleum ether / ethyl acetate=0 / 1, R of compound 2-6) f = 0.47, R of Compound 2-7f =0.65) indicated that the reaction was complete. The reaction solution was poured into water (50 mL). The aqueous layer was extracted with DCM (30 mL × 2). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. Compound 2-7 (7.00 g, crude) was obtained as a yellow oil, which was used directly in the next step without further purification. 1 H NMR:ET60190-19-P1A(400MHz,CDCl3)δ 7.28-7.35(m,10H), 4.56(s,4H), 4.18-4.24(m,2H), 3.62(s,8H), 3.43-3.50(m,4H), 2.98(s,3H), 1.35-1.83(m,14H).

[0182] Synthesis of Compounds 2-8 [ka] A solution of compound 2-7 (6.00 g, 10.6 mmol, 1.00 equiv.) and compound 3a (5.36 g, 52.9 mmol, 7.30 mL, 5.00 equiv.) was stirred at 50° C. for 16 hours. TLC (petroleum ether / ethyl acetate=0 / 1) showed the R f =0.69, R of compound 2-8 f =0.03) indicated that the reaction was complete. The reaction solution was concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO, DCM / MeOH = 500 / 1 to 5 / 1) to give compound 2-8 (4.00 g, 7.00 mmol, 66.1% yield) as a yellow oil. 1 H NMR:ET60190-20-P1A2(400MHz,CDCl3)δ 7.28-7.35(m,10H), 4.58(s,4H), 3.62(s,8H), 3.44-3.50(m,4H), 2.36-2.45(m,6H) ), 1.60-1.66(m,6H), 1.44-1.53(m,10H), 1.26-1.28(m,2H), 0.88(t,J=7.2Hz,6H).

[0183] Synthesis of Compounds 2-9 [ka] A suspension of compound 2-8 (4.00 g, 7.00 mmol, 1.00 equiv) and Pd / C (1 g, 10% purity) in MeOH (50 mL) was degassed under vacuum and degassed three times with H (50 psi). The suspension was stirred under H (50 psi) at 50 °C for 24 h. LCMS showed the reaction was complete. The reaction suspension was filtered, and the filtrate was concentrated under vacuum to give compound 2-9 (2.50 g, 6.38 mmol, 91.3% yield) as a yellow oil, which was used directly in the next step without further purification. LCMS: ET60190-21-P1A2 (RT = 0.381 min, M+H + =392.2). 1 H NMR:ET60190-21-P1A2(400MHz,CDCl3)δ 3.72(t,J=4.4Hz,4H), 3.52-3.56(m,4H), 3.46-3.51(m,4H), 2.34-2.39(m,6H), 1.60-1.6 4(m,4H), 1.51-1.55(m,2H), 1.42-1.48(m,8H), 1.24-1.27(m,4H), 0.86(t,J=7.2Hz,6H).

[0184] Synthesis of SL27 [ka] To a solution of compound 2-9 (2.00 g, 5.11 mmol, 1.00 equiv) and oleic acid (4.33 g, 15.3 mmol, 4.33 mL, 3.00 equiv) in Py (10 mL) was added EDCI (2.94 g, 15.3 mmol, 3.00 equiv). The solution was stirred at 40 °C for 16 h. LCMS showed the reaction was complete. The reaction solution was concentrated in vacuo to give a residue. The residue was poured into a saturated aqueous solution of NH4Cl (100 mL), and the aqueous layer was extracted with EtOAc (50 mL × 3). The combined organic layers were concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to give SL27 (1.00 g, 1.09 mmol, 21.3% yield) as a yellow oil. LCMS:ET60190-23-P1A2(RT=2.831min, M+H+ =921.0). 1 H NMR:ET60190-23-P1A06(400MHz,CDCl3)δ 5.33-5.37(m,4H), 4.22(t,J=4.8Hz,4H), 3.64(t,J=5.2Hz,4H), 3.48(t,J=6.0Hz,4H), 2.80-2.86(m,6H), 2.34(t,J= 7.6Hz,4H), 1.60-1.64(m,14H), 1.48-1.52(m,8H), 1.25-1.32(m,40H), 0.99(t,J=6.8Hz,6H), 0.89(t,J=6.8Hz,6H).

[0185] Synthesis of compound 3-2 [ka] To a solution of compound 3-1 (90.0 g, 497 mmol, 65.2 mL, 1.00 equiv.) and imidazole (84.5 g, 1.24 mol, 2.50 equiv.) in DMF (200 mL) was added a solution of TBSCl (89.9 g, 596 mmol, 73.1 mL, 1.20 equiv.) in DMF (250 mL) dropwise at 0° C. The solution was stirred under nitrogen protection at 0° C. for an additional 1 h. TLC (petroleum ether / ethyl acetate=10:1, R of compound 3-1) was performed. f =0.48, R of compound 3-2 f =0.72), indicating that the reactant was completely consumed, and one new spot with lower polarity was detected. The reaction solution was poured into a saturated aqueous solution of NH4Cl (550 mL). The solution was extracted three times with MTBE (500 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 50 / 1) to give compound 3-2 (143 g, 487 mmol, 98.0% yield) as a colorless oil. 1H NMR:ET60052-3-P1A(400MHz,CDCl3)δ 3.61(t,J=6.4Hz,2H), 3.41(t,J=7.2Hz,2H), 1.91-1.84(m,2H), 1.56-1.5 1(m,2H), 1.49-1.42(m,2H), 1.40-1.34(m,2H), 0.90(s,9H), 0.05(s,6H).

[0186] Synthesis of Compound 3-4 [ka] To a suspension of Mg (32.9 g, 1.35 mol, 5.00 equiv) in THF (80 mL) was added I2 (688 mg, 2.71 mmol, 546 uL, 0.01 equiv) in one portion under N2 at 20 °C. The suspension was stirred at 40 °C for 1 h. Then, a solution of compound 3-2 (80.0 g, 271 mmol, 1.00 equiv) in THF (80 mL) was added dropwise to the above suspension at 40 °C. The suspension was stirred at 40 °C for 3 h. Then, compound 3-3 (12.6 g, 108 mmol, 318 uL, 0.40 equiv) was added dropwise to the suspension at 0 °C. The suspension was stirred at 40 °C for 16 h. TLC (petroleum ether / ethyl acetate = 10 / 1, R of compound 3-2) f = 0.61, R of compound 3-4 f =0.03) indicated the reaction was complete. The reaction solution was poured into a saturated aqueous solution of NH4Cl (300 mL). The aqueous layer was extracted with EtOAc (200 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 800 / 1 to 10 / 1) to give compound 3-4 (50.0 g, 91.1 mmol, 67.2% yield) as a colorless oil. 1 H NMR:ET60190-8-P1A(400MHz,CDCl3)δ 3.75(s,2H), 3.68-3.73(m,2H), 3.57-3.65(m,6H), 2.16(s,2H), 1.77(t,J=6. 0Hz,2H), 1.44-1.54(m,8H), 1.26-1.33(m,12H), 0.93(s,18H), 0.05(s,12H).

[0187] Synthesis of compounds 3-5 [ka] To a solution of compound 3-4 (25.0 g, 45.5 mmol, 1.00 equiv.) in DCM (100 mL), TEA (13.8 g, 137 mmol, 19.0 mL, 3.00 equiv.) and MsCl (6.39 g, 55.8 mmol, 4.32 mL, 1.22 equiv.) were added at 0° C. The solution was stirred at 20° C. for 1 h. TLC (petroleum ether / ethyl acetate = 2 / 1, R of compound 3-4) was performed. f =0.38, R of compounds 3-5 f =0.48) indicated the reaction was complete. The reaction solution was poured into NH4Cl (saturated aqueous solution 200 mL). The aqueous layer was extracted with DCM (100 mL × 2). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give compound 3-5 (26.0 g, crude) as a pale yellow oil, which was used directly in the next step. 1 H NMR:ET60190-13-P1A(400MHz,CDCl3)δ 4.35-4.38(m,2H), 3.70-3.74(m,4H), 3.61(t,J=6.8Hz,4H), 3.06(s,3H), 1.78(t,J=6.0Hz, 2H), 1.48-1.54(m,4H), 1.42-1.45(m,4H), 1.26-1.33(m,12H), 0.90(s,18H), 0.06(s,12H).

[0188] Synthesis of compounds 3-6 [ka] A solution of compound 3-5 (10.0 g, 15.9 mmol, 1.00 equiv.) and compound 1a (2 M, 31.8 mL, 4.00 equiv.) was stirred in a 100 mL sealed tube at 40° C. for 12 hours. TLC (petroleum ether / ethyl acetate=1 / 1, R of compound 3-5) f =0.87, R of compounds 3-6 f=0.02) indicated that the starting material was completely consumed. The reaction was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 400 / 1 to 0 / 1) to give compound 3-6 (7.70 g, 13.3 mmol, 83.8% yield) as a pale yellow oil. 1 H NMR:ET60044-12-P1A(400MHz,CDCl3)δ 3.66-6.52(m,8H), 2.49(t,J=5.6Hz,2H), 2.25(s,6H), 1.72(t,J=5.6Hz, 2H), 1.57-1.53(m,8H), 1.30-1.26(m,12H), 0.89(s,18H), 0.04(s,12H).

[0189] Synthesis of compounds 3-7 [ka] To a solution of compound 3-6 (7.0 g, 12.1 mmol, 1.00 equiv.) in THF (14 mL), TBAF (1 M, 36.4 mL, 3.00 equiv.) was added, and the solution was stirred at 20° C. for 2 h. TLC (DCM / MeOH=10 / 1, R of compound 3-6) f = 0.34, R of compounds 3-7 f =0.34) indicated that the starting material was completely consumed. The reaction solution was concentrated under reduced pressure to give compound 3-7 (4.50 g, crude) as a brown oil.

[0190] Synthesis of SL28 [ka] To a solution of compound 3-7 (4.50 g, 12.9 mmol, 1.00 equiv) in Py (23 mL) was added oleic acid (20.1 g, 71.2 mmol, 20.1 mL, 5.50 equiv). EDCI (12.4 g, 64.7 mmol, 5.00 equiv) was added to the solution. The resulting solution was stirred at 45 °C for 12 hours. LCMS showed that the reaction was complete. The reaction solution was poured into water (150 mL), and the aqueous phase was extracted with EtOAc (50 mL × 2). The combined organic phase was washed with brine (100 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 - 0 / 1) to give the crude product, which was purified by preparative HPLC (column: Welch Xtimate C1 100 x 30 mm x 5 um; mobile phase: [water (FA)-ACN]; B%: 50%-85%, 20 min) to give SL28 (1.0 g, 1.14 mmol, 8.81% yield) as a yellow oil. LCMS: ET60044-18-P1A (RT = 2.942 min, M+Na + =898.80). 1 H NMR:ET60044-18-P1A(400MHz,CDCl3)δ 5.36-5.33(m,4H), 4.05(t,J=6.4Hz,4H), 3.66-3.62(m,4H), 3.12(br.s,1H), 2.68-2.66(m,2H), 2.40(s,6H), 2.29(t,J=7.2Hz,4H) , 2.01-1.98(m,7.2Hz,8H), 1.74(t,J=5.6Hz,2H), 1.62-1.60(m,8H), 1.55-1.45(m,4H), 1.30-1.27(m,52H), 0.88(t,J=8.0Hz,6H).

[0191] Synthesis of compounds 3-8 [ka] A solution of compound 3-5 (10.0 g, 16.0 mmol, 1.00 equiv.) and compound 2a (5.83 g, 79.7 mmol, 8.21 mL, 5.00 equiv.) in DCM (10 mL) was stirred at 50° C. for 16 hours. TLC (petroleum ether / ethyl acetate=2 / 1, R of compound 3-5) f = 0.56, R of compounds 3-8 f =0.03) indicated that the reaction was complete. The reaction solution was concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO, DCM / MeOH = 1 / 0 to 5 / 1) to give compound 3-8 (7.20 g, 11.9 mmol, 74.7% yield) as a pale yellow oil. 1 H NMR:ET60190-15-P1A(400MHz,CDCl3)δ 3.54-3.65(m,8H), 2.58-3.67(m,6H), 1.73(s,2H), 1.25-1.52(m,20H), 1.03-1.06(m,6H), 0.90(s,18H), 0.05(s,12H).

[0192] Synthesis of compounds 3-9 [ka] To a solution of compound 3-8 (6.50 g, 10.7 mmol, 1.00 equiv.) in THF (18.0 mL) was added TBAF (1 M, 32.2 mL, 3.00 equiv.) at 20° C. The solution was stirred under a N atmosphere at 20° C. for 2 hours. TLC (petroleum ether / ethyl acetate=10 / 1, R of compound 3-8) f = 0.52, R of compound 3-9 f =0.20) indicated that the reactant was completely consumed, and one new spot with higher polarity was detected. The reaction solution was poured into water (100 mL). The solution was extracted with EtOAc (150 mL × 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product compound 3-9 (4 g, crude) as a colorless oil, which was used in the next step without further purification.

[0193] Synthesis of SL29 [ka] To a solution of compound 3-9 (4.00 g, 10.6 mmol, 1.00 equiv.) and oleic acid (17.0 g, 60.2 mmol, 5.66 equiv.) in Py (20.0 mL) was added a solution of EDCI (12.1 g, 63.5 mmol, 5.97 equiv.) dropwise at 40 °C. The solution was stirred under nitrogen protection at 40 °C for an additional 16 h. LCMS showed that none of compound 3-9 remained. Several new peaks were observed by LCMS, and 60% of the desired compound SL29 was detected. The reaction solution was poured into water (150 mL). The solution was extracted with EtOAc (300 mL × 3). The crude product was purified by column chromatography (SiO2, DCM / MeOH = 1 / 0-10 / 1) to give compound SL29 (1.00 g, 1.11 mmol, 10.4% yield) as a colorless oil. LCMS:ET60052-23-P1A3(RT=3.854min, M+H + =904.8). 1 H NMR:ET60052-23-P1A(400MHz,CDCl3)δ 5.36-5.33(m,4H), 4.05(d,J=6.0Hz,4H), 3.65(d,J=6.0Hz,2H), 3.60(s,2H), 2.71(s,2H), 2.65(s,4H), 2.29(d,J=8.0Hz,4H), 2.03-1.99(m,8H), 1.72(d,J=6.0Hz,2H), 1.63-1.61(m,8H), 1.47-1.27(m,57H), 1.07(d,J=8.0Hz,6H), 0.88(d,J=6.0Hz,6H).

[0194] Synthesis of compounds 3-10 [ka] A solution of compound 3-5 (4.40 g, 7.02 mmol, 1.00 equiv) and compound 3a (4.26 g, 42.1 mmol, 5.80 mL, 6.00 equiv) in DCM (12.0 mL) was stirred at 50 °C for 16 h under a N atmosphere. LCMS showed 20% of compound 3-5 remained. Several new peaks were observed by LCMS, and 50% of the desired compound was detected. The reaction solution was poured into water (150 mL). The solution was extracted with EtOAc (300 mL × 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (SiO, DCM / MeOH = 1 / 0-10 / 1) to give compound 3-10 (3.02 g, 4.78 mmol, 68.1% yield) as a colorless oil. LCMS:ET60052-12-P1A1(RT=1.030min, M+H + =632.54). 1 H NMR:ET60052-12-P1A(400MHz,CDCl3)δ 3.66-3.58(m,6H), 3.51(s,2H), 2.64(s,2H), 2.43(m,4H), 1.72(t,J=5.8Hz,2H), 1. 53-1.47(m,12H), 1.30(s,12H), 0.89(s,18H), 0.87(t,J=9.0Hz,6H), 0.05(s,12H).

[0195] Synthesis of Compounds 3-11 [ka] To a solution of compound 3-10 (2.80 g, 4.43 mmol, 1.00 equiv.) in THF (9.00 mL) was added TBAF (1 M, 13.3 mL, 3.00 equiv.) dropwise at 0° C. The solution was stirred under a N atmosphere at 0° C. for 2 hours. TLC (petroleum ether / ethyl acetate=10:1, R of compound 3-11) was performed. f = 0.52, R of compound 3-11 f=0.2) indicated that the reactant was completely consumed, and one new spot with higher polarity was detected. The reaction solution was poured into water (100 mL). The solution was extracted with EtOAc (150 mL × 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography (SiO2, DCM / MeOH = 1 / 0-10 / 1) to give compound 3-11 (1.30 g, 3.22 mmol, 72.7% yield) as a colorless oil. 1 H NMR:ET60052-13-P1A1(400MHz,CDCl3)δ 3.65-3.62(m,6H), 3.51(s,2H), 2.64(s,2H), 2.42(s,4H), 1.72(t,J=6. 0Hz,2H), 1.61-1.54(m,5H), 1.50-1.27(m,21H), 0.85(t,J=7.4Hz,6H).

[0196] Synthesis of SL30 [ka] To a solution of compound 3-11 (1.00 g, 2.48 mmol, 1.00 equiv.) and oleic acid (2.25 g, 7.97 mmol, 2.25 mL, 3.22 equiv.) in Py (3.00 mL) was added EDCI (2.37 g, 12.4 mmol, 5.00 equiv.) at 40 °C. The solution was stirred under nitrogen protection at 40 °C for an additional 4 h. LCMS showed that none of compound 3-11 remained. Several new peaks were observed by LCMS, and 80% of the desired SL30 was detected. The reaction solution was poured into water (150 mL). The solution was extracted with (300 mL × 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography (SiO, DCM / MeOH = 1 / 0-10 / 1) to give crude SL30 (1.80 g) as a colorless oil. The crude product was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 100 / 1-5 / 1) to give SL30 (1.00 g, 1.07 mmol, 43.3% yield) as a colorless oil. LCMS: ET60052-18-P1A3 (RT = 3.985 min, M+H + =932.86). 1 H NMR:ET60052-19-P1A3(400MHz,CDCl3)δ 5.36-5.33(m,4H), 4.05(d,J=6.8Hz,4H), 3.64(d,J=5.6Hz,2H), 3.52(s,2H), 2.66(s,2H), 2.65(s,4H), 2.29(d,J =7.4Hz,4H), 2.02-1.99(m,8H), 1.72(d,J=5.8Hz,2H), 1.62-1.55(m,8H), 1.47-1.27(m,62H), 0.89-0.86(m,12H).

[0197] Synthesis of compound 4-3 [ka] To a suspension of Mg (22.3 g, 915 mmol, 5.00 equiv) in THF (50 mL) was added I2 (465 mg, 1.83 mmol, 369 uL, 0.01 equiv) in one portion under N2 at 20 °C. The suspension was stirred at 40 °C for 1 h. Compound 2-5 (50.0 g, 183 mmol, 1.00 equiv) was then added dropwise to the above suspension at 40 °C. The suspension was stirred at 40 °C for 3 h. Compound 3-3 (8.50 g, 73.2 mmol, 0.40 equiv) was then added dropwise to the suspension at 0 °C. The suspension was stirred at 40 °C for 16 h. TLC (petroleum ether / ethyl acetate = 10 / 1, R of compound 2-5) f =0.56, R of compound 4-3 f =0.04) indicated the reaction was complete. The reaction solution was poured into a saturated aqueous solution of NH4Cl (200 mL). The aqueous layer was extracted with EtOAc (100 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 800 / 1-0 / 1) to give compound 4-3 (32.0 g, 63.4 mmol, 69.3% yield) as a yellow oil. 1 H NMR:ET60190-17-P1A(400MHz,CDCl3)δ 7.27-7.35(m,10H), 4.57(s,4H), 3.61-3.69(m,12H), 3.47-3.53(m,5H), 1.76-1.79(m,2H), 1.57-1.65(m,8H).

[0198] Synthesis of compound 4-4 [ka] To a solution of compound 4-3 (23.0 g, 45.6 mmol, 1.00 equiv.) and TEA (13.8 g, 136 mmol, 19.0 mL, 3.00 equiv.) in DCM (120 mL), MsCl (11.8 g, 103 mmol, 7.97 mL, 2.26 equiv.) was added dropwise at 0° C. After the addition, the solution was stirred at this temperature for 6 h. TLC (petroleum ether / ethyl acetate=1 / 1, R of compound 4-3) f =0.26, R of compound 4-4 f=0.37) indicated the reaction was complete. The solution was poured into NH4Cl (saturated aqueous solution 200 mL). The solution was extracted with DCM (50.0 mL × 2). The organic layers were combined and concentrated under vacuum to give the crude product. The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 0 / 1) to give compound 4-4 (26.0 g, 44.6 mmol, yield 97.9%) as a yellow oil.

[0199] Synthesis of compounds 4-5 [ka] To a solution of compound 4-4 (14.1 g, 24.2 mmol, 1.00 equiv.) was added compound 1a (2.0 M, 48.4 mL, 4.0 equiv.) in a sealed tube at 20° C. The reaction solution was stirred at 60° C. for 14 hours. TLC (ethyl acetate / MeOH=8 / 1, R of compound 4-4) f =0.92, R of compound 4-5 f = 0.52) showed that none of compound 4-4 remained, and one new major spot with higher polarity was detected. The reaction solution was poured into a saturated aqueous solution of NH4Cl (20.0 mL), and the organic phase was washed with a half-saturated aqueous solution of NaCl (10 mL). The final organic phase was concentrated under reduced pressure to give compound 4-5 (6.70 g, 12.6 mmol, 51.9% yield) as a brown oil. 1 HNMR:ET60053-52-P1A2(400MHz,CDCl3)δ 7.27-7.35(m,10H), 4.55(s,4H), 3.62-3.63(m,4H), 3.61(s,2H), 3.53-3.56(t,J=1.6Hz,6H), 3 .45-3.48(m,4H), 2.53-2.55(t,J=5.6Hz,2H), 2.30(s,6H)1.60-1.63(m,2H), 1.52-1.55(m,8H).

[0200] Synthesis of compounds 4-6 [ka] To a solution of compound 4-5 (6.00 g, 11.2 mmol, 1.00 equiv.) in THF (40.0 mL) was added Pd / C (10.0% purity, 3.0 g) under N2 atmosphere. The suspension was degassed and purged with H2 (50 psi) three times. The mixture was stirred under H2 (50.0 psi) at 40 °C for 16 h. TLC (petroleum ether / ethyl acetate = 1 / 1, R of compound 4-5) f =0.92, R of compounds 4-6 f =0.41) showed that none of compound 2-8 remained, and one new major spot with higher polarity was detected. The reaction solution was filtered, and the filtrate was poured into a saturated aqueous solution of NH4Cl (20 ml). The organic phase was washed with a half-saturated aqueous solution of NaCl (20.0 ml) and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1-1 / 1) to obtain compound 4-6 (3.70 g, 10.5 mmol, 93.2% yield) as a brown oil. 1 HNMR:ET60053-54-P1A1(400MHz,CDCl3)δ 3.70-3.73(m,4H), 3.66(s,2H), 3.52-3.55(m,8H), 3.47-3.49(m,4H), 2.48- 2.51(t,J=5.6Hz), 2.26(s,6H), 1.74-1.77(t,J=6Hz,2H), 1.56-1.63(m,8H).

[0201] Synthesis of SL31 [ka] To a solution of compound 4-6 (3.70 g, 10.5 mmol, 1.00 equiv.) in Py (18.0 mL) was added EDCI (12.1 g, 63.1 mmol, 6.0 equiv.) at 20° C., and then oleic acid (17.8 g, 63.1 mmol, 17.4 mL, 6.0 equiv.) was added dropwise to the reaction solution at 20° C. The resulting solution was stirred at 40° C. for 16 hours. TLC (R of compound 4-6) f =0.1, and R of SL31 f=0.5) showed that none of compounds 4-6 remained, and one new major spot with higher polarity was detected. The reaction solution was concentrated to remove insoluble materials. The organic phase was poured into a saturated aqueous solution of NH4Cl (20.0 ml), and the organic phase was washed with a half-saturated aqueous solution of NaCl (20 ml). The final organic phase was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 10 / 1). Compound SL31 (1.00 g, 1.14 mmol, 10.8% yield) was obtained as a yellow oil. 1 HNMR:ET60053-55-P1A29(400MHz,CDCl3)δ 0.86-0.92(m,6H), 1.23-1.36(m,42H), 1.50-1.56(m,4H), 1.58-1.67(m,8H), 1.74-1.78(m,3H), 1.99-2.05(m,8H) , 2.50-2.58(m,2H), 3.43-3.50(m,4H), 3.54-3.59(m,2H), 3.60-3.68(m,6H), 4.19-4.23(m,4H), 5.27-5.40(m,4H).

[0202] Synthesis of compounds 4-7 [ka] To a solution of compound 2a (4.52 g, 61.8 mmol, 6.36 mL, 4.00 equiv.) in DCM (9.00 mL) was added compound 4-4 (9.00 g, 15.4 mmol, 1.00 equiv.). The resulting solution was stirred at 50° C. for 12 hours. TLC (dichloromethane / methanol=10 / 1, R of compound 4-4) f =0.52, R of compounds 4-7 f =0.40) indicated that the reaction was complete. The reaction solutions were combined and concentrated in vacuo to give the crude product. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to give compound 4-7 (7.00 g, 12.5 mmol, 80.9% yield) as a yellow oil. 1H NMR:ET61249-31-P1A(400MHz,CDCl3)δ 7.29-7.35(m,10H), 4.57(s,4H), 3.36-3.66(m,10H), 3.46-3.52(m,6H), 2.54-2.63(m,6H), 1.53-1.75(m,11H), 1.02(t,J=7.2Hz,6H).

[0203] Synthesis of compounds 4-8 [ka] To a solution of compound 4-7 (7.00 g, 12.5 mmol, 1.00 equiv) in MeOH (35.0 mL) and THF (10.0 mL) was added Pd / C (3.50 g, 10% purity) under N2 atmosphere. The suspension was degassed and purged with H2 three times. The solution was stirred under H2 (50 Psi) at 40 °C for 12 h. LCMS showed the reaction was complete. The suspension was then filtered through a pad of silica gel, and the filter cake was washed with MeOH (100 mL x 3). The combined filtrate was concentrated to dryness to give compound 4-8 (4.50 g, 11.9 mmol, 94.8% yield) as a yellow oil. LCMS: ET61249-32-P1A (RT = 0.784 min, M+H + =381.3). 1 H NMR:ET61249-32-P1A(400MHz,CDCl3)δ 3.55-3.72(m,4H), 3.52-3.54(m,2H), 3.48-3.49(m,12H), 2.63-2.63(m,6H), 1.55-1.75(m,12H), 1.02(t,J=7.2Hz,6H).

[0204] Synthesis of SL32 [ka] To a solution of compound 4-8 (3.50 g, 9.22 mmol, 1.00 equiv.) and (Z)-octadec-9-enoic acid (7.81 g, 27.6 mmol, 7.81 mL, 3.00 equiv.) in Py (20.0 mL) was added EDCI (7.07 g, 36.9 mmol, 4.00 equiv.). The resulting solution was stirred at 45 °C for 12 h. LCMS showed the reaction was complete. The reaction solution was poured into NH4Cl (saturated, 50.0 mL, aqueous solution), and the solution was extracted with EtOAc (20.0 mL × 2). The organic layers were combined and concentrated in vacuo to give the crude product. The crude product was purified by reverse-phase HPLC (column: Welch Xtimate C1 100 x 30 mm x 5 um; mobile phase: [water (FA)-ACN]; B%: 40%-80%, 15 min) to give compound 4-7 (1.00 g, 1.10 mmol, 11.9% yield) as a pale yellow oil. LCMS: ET61249-35-P1A (RT = 2.347 min, M+H + =908.8). 1 H NMR:ET61249-32-P1A(400MHz,CDCl3)δ 5.33-5.37(m,4H), 4.21(t,J=7.2Hz,4H), 3.61-3.67(m,6H), 3.45-3.53(m,6H), 2.35-2.60(m,5H), 2.33(t,J=7.6 Hz,4H), 1.99-2.20(m,8H), 1.63-1.75(m,16H), 1.27-1.30(m,40H), 1.04(t,J=7.2Hz,6H), 0.88(t,J=6.8Hz,6H).

[0205] Synthesis of compounds 4-9 [ka] To a solution of compound 4-4 (9.00 g, 15.4 mmol, 1.00 equiv.) in DCM (18.0 mL) was added compound 3a (6.25 g, 61.8 mmol, 8.52 mL, 4.00 equiv.). The mixture was stirred at 50 °C for 10 h. LCMS showed that compound 4-4 was completely consumed. Several new peaks were detected by LCMS, indicating the desired compound. The reaction mixture was partitioned between EtOAc (200 mL) and water (100 mL). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1). Compound 4-9 (5.00 g, 8.51 mmol, 55.1% yield) was obtained as a colorless oil. LCMS:ET60350-33-P1A(RT=0.401min, M+H + =588.4). 1 H NMR:ET60350-33-P1A(400MHz,CDCl3)δ 0.87(t,J=7.39Hz,6H), 1.42-1.70(m,12H), 1.72-1.79(m,2H), 2.45(s,4H), 2.66 (s,2H), 3.42-3.55(m,6H), 3.55-3.69(m,10H), 4.58(s,4H), 7.34-7.36(m,10H).

[0206] Synthesis of Compounds 4-10 [ka] To a solution of compound 4-9 (5.00 g, 8.51 mmol, 1.00 equiv.) in MeOH (35.0 mL) was added Pd(OH) (597 mg, 851 μmol, 20% purity, 0.10 equiv.) under N atmosphere. The suspension was degassed and purged with H three times. The solution was stirred under H (50.0 Psi) at 40 °C for 24 h. LCMS showed that compound 4-9 was completely consumed. Several new peaks were observed by LCMS, indicating the desired compound. The suspension was then filtered through a pad of Celite, and the filter cake was washed with MeOH (10.0 mL × 3). The combined filtrate was concentrated in vacuo to give the crude product. The crude product was used directly in the next step without purification. Compound 4-10 (3.00 g, 7.36 mmol, 86.5% yield) was obtained as a colorless oil. LCMS:ET60350-34-P1A(RT=1.281min, M+H + =406.4). 1 H NMR:ET60350-34-P1A(400MHz,CDCl3)δ 0.81-0.91(m,6H), 1.41-1.48(m,4H), 1.52-1.68(m,9H), 1.72-1.81(m,3H), 2.34-2.46(m,5H), 2.57-2.68(m,2H), 3.46-3.51(m,6H), 3.53-3.56(m,4H), 3.63-3.67(m,2H), 3.70-3.74(m,4H).

[0207] Synthesis of SL33 [ka] To a solution of compound 4-10 (3.00 g, 7.36 mmol, 1.00 equiv) in Py (15.0 mL) was added oleic acid (6.24 g, 22.1 mmol, 6.24 mL, 3.00 equiv) and EDCI (5.64 g, 29.4 mmol, 4.00 equiv). The mixture was stirred at 45 °C for 10 h. LCMS showed that compound 4-9 was completely consumed. Several new peaks were detected by LCMS, indicating the desired compound. The reaction mixture was partitioned between EtOAc (200 mL) and water (100 mL). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by reverse-phase HPLC (column: Welch Xtimate C1 100 × 30 mm × 5 um; mobile phase: [water (FA)-ACN]; B%: 30%-65%, 10 min). Compound SL33 (1.00 g, 1.07 mmol, 14.5% yield) was obtained as a yellow oil. LCMS: ET60350-35-P1A (RT = 2.824 min, M+H + =937.0). 1 H NMR:ET60350-35-P1A8(400MHz,CDCl3)δ 0.75-1.04(m,12H), 1.24-1.35(m,40H), 1.45-1.68(m,16H), 1.73-1.78(m,2H), 1.99-2.03(m,8H), 2.31-2.37(m,4H) ), 2.38-2.51(m,3H), 2.56-2.78(m,2H), 3.42-3.58(m,6H), 3.59-3.68(m,6H), 4.16-4.25(m,4H), 5.28-5.40(m,4H).

[0208] Example 2: Formulation of ionizable cationic lipids into LNPs with mRNA This is a general description of how each ionizable cationic lipid was used to formulate samRNA LNPs. Details of each lipid are listed in Table 1. Ionizable cationic lipids (see Table 1), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG) were combined in a molar ratio of 40:10:48:2 in ethanol at a concentration of 3.2 mM (for 0.5 mg samRNA scale) or 6.4 mM (for 1.5 mg samRNA scale). A solution of samRNA expressing the antigen of interest in 50 mM citrate buffer at pH 6 with 10 mM tris(2-carboxyethyl)phosphine (TCEP) was prepared. When mixed at a 2:1 aqueous solution-to-ethanol flow rate ratio (FRR), the RNA concentration was 0.025 mg / mL (for 0.5 mg samRNA scale) or 0.050 mg / mL (for 1.5 mg samRNA scale). When mixed at a 3:1 aqueous solution-to-ethanol flow rate ratio (FRR), the RNA concentration was 0.017 mg / mL (for 0.5 mg samRNA scale). The lipid solution in ethanol was then rapidly mixed with the samRNA solution using a Nanoassemblyr benchtop instrument (Precision Nanosystems) with a zigzag herringbone micromixer tip at a total flow rate (TFR) of 12 mL / min and a flow rate ratio (FRR) of either 2:1 or 3:1 buffered aqueous solution to ethanol. This mixing ratio resulted in an 8:1 molar ratio of ionizable cationic lipid (see Table 1) to samRNA phosphate groups and a 37:1 mass ratio of total lipid to samRNA. The resulting mixture was then diluted 10-fold into 50 mM citrate buffer with 10 mM TCEP at pH 6 and subjected to tangential flow filtration (TFF) using a 300 kJ molecular weight cut-off membrane (mPES) until concentrated to the original volume. The citrate buffer was then replaced with a buffer containing 20 mM Tris buffer, 80 mM sodium chloride, and 3% sucrose at pH 7.5 using diafiltration at 10 diavolumes.The LNP solution was concentrated to a volume of 4–10 mL, filtered using a 0.2 micron PES syringe filter, aliquoted into vials, and frozen at 1°C / min using a Corning® CoolCell® LX cell freezing container until the sample reached -80°C. Samples were stored at -80°C and thawed on wet ice before analysis or use. Total RNA concentration, percent of input RNA recovered (% recovery), and encapsulation efficiency (%EE) were determined using the Ribogreen assay as described elsewhere. Z-average diameter (nm) and polydispersity index (PDI) were measured using dynamic light scattering (Malvern Zetasizer) after a 1:100 dilution in phosphate-buffered saline (PBS). [Table 1]

[0209] Biophysical characterization of lipid nanoparticles Messenger RNA-containing LNP compositions were characterized using analytical methods to determine messenger RNA loading, the percentage of encapsulated messenger RNA, and particle size. The total amount of messenger RNA contained in a sample and the percentage of that messenger RNA that was encapsulated were determined using a fluorescent assay with Ribogreen, a dye that emits light upon binding of messenger RNA. The total amount of messenger RNA was determined by disrupting the LNPs with 1% Triton-X100 (wt%) to expose the encapsulated messenger RNA, adding the dye, and comparing the emission intensity to a standard curve prepared using ribosomal RNA. The amount of unencapsulated messenger RNA was measured in a similar manner to the detergent disruption of omitted LNPs. Given a known total amount of messenger RNA and a known amount of unencapsulated messenger RNA, the percent encapsulated messenger RNA was calculated as follows: Percent encapsulation (%) = ((RNA 合計 -RNA カプセル化されていない ) / RNA 合計 ) x 100

[0210] In the formula, RNA 合計 and RNA カプセル化されていない are the concentrations of total messenger RNA and unencapsulated messenger RNA, respectively. Total messenger RNA content varied based on formulation but generally fell within the range of 0.030–0.200 mg / mL. LNP size was measured using dynamic light scattering of samples diluted 1:100 in PBS buffer.

[0211] pKa Protocol In a black 96-well plate, prepare solutions of samRNA-LNP (final assay concentration 2 μg / mL total RNA) in a series of buffers ranging from pH 4 to 9.5. Buffers from pH 4 to 7.6 are prepared from disodium phosphate and citric acid. Buffers from 7.8 to 9.5 are prepared by titrating Tris buffer with 10 N sodium hydroxide. To each well, add 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS) in water to a final assay concentration of 6 μM. Fluorescence is read on a plate reader at 25 °C with an excitation setting of 321 nm and an emission setting of 445 nm. Intensity values ​​are plotted as a function of pH using GraphPad Prism and fitted with a sigmoidal dose-response curve. The apparent pKa is determined as the EC50 of this curve, where half of the ionizable amines are expected to be protonated.

[0212] Characterization of in vitro efficacy of LNPs The ability of samRNA-LNPs to transfect cultured cells is characterized using an in vitro assay based on the percentage of cells expressing the antigen of interest. Specifically, for each LNP, 1 million BHK-21 cells are co-incubated with various concentrations of samRNA-LNPs in 0.3 mL of medium at 37 °C and 5% CO for 17–19 h. Cells are then detached from the dish by treatment with TrypLE (Gibco) to form a single-cell suspension, fixed and permeabilized (BD Cytofix / Perm kit), and then stained with fluorophore-conjugated antigen-specific antibodies against H5 and N1. The percentage of double-antigen-positive cells is quantified using a BD Accuri flow cytometer.

[0213] Characterization of the in vivo efficacy of LNPs The ability of mRNA-LNPs to act as vaccines was assessed by measuring antibody and cellular immune responses following a prime-boost vaccination schedule. Balb / c mice were given a priming vaccination by intramuscular injection (i.m.) on day 0, followed by a booster vaccination 21 days later. After another 21 days (day 42 of the experiment), the mice were sacrificed, and serum and splenocytes were collected for further analysis.

[0214] Sera will be analyzed for vaccine-specific antibody responses using IgG ELISA, pseudovirus microneutralization assay, hemagglutination inhibition (HAI) assay, and enzyme-linked lectin assay (ELLA). Results will be compared with adjuvanted inactivated virus vaccines (aH5N1) tested in the same experiment using one-way ANOVA statistical tests.

[0215] Splenocytes from the above experiments were analyzed for antigen-specific cytokine production using intracellular cytokine staining by flow cytometry after in vitro peptide stimulation. Splenocytes were pooled (n=5 / group) and stimulated ex vivo in duplicate in the absence or presence of H5 or N1 peptide. Mean levels of response, as measured by interferon-gamma, interleukin-2, and / or tumor necrosis factor-alpha production, are shown, with error bars indicating measurement precision.

[0216] Taken together, these in vivo results are expected to demonstrate that LNPs prepared using lipids SL26-SL33 are immunogenic in this preclinical model and are effective as influenza vaccines.

[0217] Characterization of in vivo transfection efficiency of LNPs The in vivo efficacy of mRNA-LNPs was assessed using samRNA expressing the reporter protein firefly luciferase to quantify the location, relative amount, and duration of protein expression. LNPs formulated with luciferase-expressing mRNA were injected intramuscularly into mice, for example, in the hind leg. At defined time points, such as daily, the mice were administered luciferin and bioluminescence was imaged and quantified.

[0218] Characterization of genotoxic potential, tolerability, biodistribution, and biodegradability Because no single test can detect all genotoxic mechanisms leading to tumorigenicity, a standard test battery to predict genotoxic potential (DNA damage) is used. As an example, if a positive result is seen in an in vitro mammalian cell assay, clearly negative results in two in vivo assays, with appropriate tissues and sufficient test substance exposure as indicated, are considered evidence of the lack of genotoxic potential in vivo. Relevant guidelines, such as S2(R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, are followed. • Testing for genetic mutations in bacteria. • In vitro cytogenetic testing for chromosomal damage (metaphase chromosomal aberrations or micronucleus test) or mouse lymphoma Tk gene mutation assay. • In vivo genotoxicity testing of chromosomal damage using rodent hematopoietic cells for either micronuclei or chromosomal aberrations in metaphase cells.

[0219] In silico screening of novel lipids for toxicity Using commercially available computer-assisted toxicity assessment products and / or services, compounds of Formula I are screened for potential toxicity and mutagenicity to meet the international "Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) M7(R1) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals To Limit Potential Carcinogenic Risk" standard. For example, two complementary quantitative (Q)SAR methods, the expert-rule-based Derek Nexus and the statistically-based Sarah Nexus (or Leadscope), are used to predict bacterial mutagenicity outcomes. Classification is based on Classes 1–5, ranging from known mutagenic carcinogens (Class 1) to the absence of mutagenicity or carcinogenicity (Class 5). Results of the computer-based analysis are outlined, along with the relevance of positive, negative, contradictory, or inconclusive predictions and the rationale for the provided conclusions.

[0220] Because most structural alerts are based on bacterial mutagenicity, compounds with structural alerts can be detected in standard test batteries. In addition, some chemical classes are more easily detected in mammalian cell chromosomal damage assays than in bacterial mutation assays. A negative result from a compound with a structural alert in either test battery should be considered non-genotoxic.

[0221] In vitro screening for genotoxicity Compounds of Formula I are evaluated first for mutagenicity in a bacterial reverse mutation test (Ames), which detects relevant genetic alterations and most genotoxic rodent and human carcinogens, and then for genotoxicity in an in vitro micronucleus (MN) assay using mammalian cells.

[0222] The Ames assay follows the Organization for Economic Cooperation and Development (OECD) Guideline for Testing of Chemicals No. 471, Bacterial Reverse Mutation Test, and the S2 (R1) Guidance for Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, to assess mutagenic potential. At least five strains of bacteria, Salmonella typhimurium TA98, TA100, TA1537, TA1535, and TA102, or E. coli WP2, are exposed to the test substance in the presence and absence of a metabolic activation system, the rat liver metabolic system (S-9).

[0223] The in vitro mammalian cell micronucleus genotoxicity test follows OECD Guideline for Testing Chemicals No. 487. The test should detect MN and potential chromosomal damage (aneuploidogens and aberrants) in the cytoplasm of interphase cells. Well-validated and relevant assays are the mouse lymphoma L5178Y Tk (thymidine kinase) gene mutation assay (MLA) and the human lymphocyte micronucleus assay (HLM) in treated with rat liver metabolizer (S-9) in the absence and presence of the enzyme.

[0224] In vivo screening for genotoxicity Because some drugs are mutagenic in vivo but not in vitro, compounds of Formula I are evaluated in vivo in either acute or repeated dose rat studies. The assays of choice are micronuclei in erythrocytes (in blood or bone marrow) or chromosomal aberrations in metaphase cells in bone marrow.

[0225] Biodistribution and biodegradation evaluation Ex vivo biodegradability evaluation: Compounds of Formula I are screened for enzymatic biodegradation using enzyme-containing solutions prepared from relevant species, such as humans, mice, and rats. Lipids are screened both in their neat (unformulated) form and when incorporated into LNPs with or without RNA. For example, novel ionizable cationic lipids are diluted in aqueous solutions of human or rat liver microsomes to approximate concentrations of 1.0–0.001 mg / mL and incubated at temperatures ranging from 25–37°C for periods of 0.1–24 hours. The amount of intact lipid remaining is then measured using liquid chromatography (LC) with evaporative light scattering (ELS) or mass spectrometry (MS) detection and compared to control samples that were not treated with enzyme or incubated at 2–8°C to inhibit enzyme activity. Additionally, the appearance of new peaks in the chromatogram, possibly representing degradation products, is investigated using MS to confirm their identity. Without wishing to be bound by theory, it is expected that degradation may occur via ester hydrolysis, and that the structure of the lipid influences the rate of ester hydrolysis.

[0226] In vivo biodistribution, pharmacokinetics, and biodegradability evaluation: Biodistribution and pharmacokinetics [absorption, distribution, metabolism, and excretion (ADME)] evaluations will be performed in rats, which will receive the novel lipids (in their neat form and / or incorporated into LNPs with or without RNA) intramuscularly or intravenously.

[0227] Biodistribution assessment will be based on the principles of ICH-M3(R2) and the World Health Organization (WHO) guidelines for nonclinical evaluation of vaccines, WHO Technical Report Series No. 927, Annex 1. Blood samples will be collected multiple times for lipid, mRNA, and immunogenicity analysis. Tissues will be collected on designated days for lipid analysis using a qualified liquid chromatography-mass spectrometry (LC-MS / MS) method. RT-qPCR analysis of nucleic acid payloads will be performed, if necessary.

[0228] Plasma lipid pharmacokinetics will be assessed by qualified LS-MS / MS.

[0229] In vitro biodegradation evaluation: Formula I compounds are screened by testing the stability of the test compounds in the presence of cryopreserved hepatocytes. For example, a cryopreserved hepatocyte suspension (final cell density 0.5 x 10 viable cells / mL in Williams E medium supplemented with 2 mM L-glutamine and 25 mM HEPES) is pre-incubated at 37°C to initiate the reaction before adding the test compound (final substrate concentration 1 μM, final DMSO concentration 0.25%). Each species includes two control compounds. Each compound is incubated at 37°C for 0, 5, 10, 20, 40, and 60 minutes. The reaction is terminated at the appropriate time point by transferring the mixture to acetonitrile in a 1:3 ratio. The terminal plate is centrifuged at 3,000 rpm for 30 minutes at 4°C to precipitate the proteins. After protein precipitation, sample supernatants are combined into cassettes of up to four compounds, internal standards are added, and the samples are analyzed using Cyprotex generic LC MS / MS conditions.

[0230] In vivo tolerability assessment The tolerability of the compounds of Formula I is evaluated in an appropriate species, such as mice or rats. For example, groups of Sprague-Dawley rats are administered the novel lipid-containing RNA LNP vaccine via intramuscular injection at doses ranging from 0.1 to 30 μg of total RNA per animal. Each animal receives one to three injections, and the effects of treatment on weight loss, food intake, and body temperature are measured. In addition, injection sites are monitored using the Draize skin irritation scoring system. In addition, serum is collected to evaluate the vaccine's immune response using ELISA and functional antibody assays. In addition, complete blood counts, blood chemistries, and blood coagulation are evaluated at one or more time points during the study. At the end of the study during necropsy, selected organs can be macroscopically evaluated and weighed. Novel lipids associated with RNA LNP vaccines that maintain high immunogenicity while causing minimal undesirable responses such as weight loss, fever, or injection site reactogenicity are considered more promising candidates than lipids with less attractive tolerability profiles.

[0231] Toxicity evaluation Repeated-dose general toxicity study: The toxicity of candidate compounds of Formula I is evaluated in pharmacologically relevant non-clinical species (e.g., rats). The novel lipid-containing RNA / LNP vaccine is administered intramuscularly to Sprague-Dawley rats once every three weeks (a total of three doses). The reversibility or persistence of any effects is evaluated after a three-week recovery period.

[0232] Toxicity assessments are based on mortality, clinical observations, body weight, food intake, ophthalmological observations, administration site (skin) observations, body temperature, as well as clinical and anatomic pathology, micronucleus analysis. Blood samples are collected for immunogenicity analysis.

[0233] Itemized List of Embodiments 1. A compound of formula I, [ka] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: R1 is selected from the group consisting of formulas IA-II; [ka] Z, A, and B are each independently selected from the group consisting of -C(H)2-, -N(H)-, -O-, and -S-, and at least one of Z, A, and B is -N(H)-, -O-, or -S-; n and q are each independently an integer of 0 to 8, t, u, v, and w are each independently an integer of 0 to 10; D, E, F, and G are each independently a straight chain or branched C 1-16 -alkyl, [ka] represents a single or double bond, R 2 and R 3 However, each independently, C 1-8 -Alkyl and C 1-8 -alkanols, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0234] 2.R 2 But C 1-8 Item 1. The compound according to item 1, wherein -alkyl.

[0235] 3.R 2 But C 1-3 The compound according to item 1 or 2, wherein - is alkyl.

[0236] 4. If present, R 3 But C 1-8 -alkyl.

[0237] 5. If present, R 3 But C 1-3 -alkyl.

[0238] 6.R 1 is formula IA.

[0239] 7.R 1 but, [ka] 7. The compound according to any one of items 1 to 6, which is of formula IA selected from the group consisting of:

[0240] 8.R 1 is formula IB.

[0241] 9.R 1 but, [ka] 9. The compound according to any one of items 1 to 5 and 8, which is of formula IB selected from the group consisting of:

[0242] 10. The compound according to any one of items 1 to 9, wherein Z is selected from the group consisting of -N(H)-, -O-, or -S-.

[0243] 11. The compound according to any one of items 1 to 10, wherein Z is -O-.

[0244] 12. The compound according to any one of items 1 to 11, wherein n and q are each independently an integer of 0 to 4.

[0245] 13. The compound according to any one of items 1 to 12, wherein n is 2.

[0246] 14. The compound according to any one of items 1 to 13, wherein q is 2.

[0247] 15. The compound according to any one of items 1 to 14, wherein n is 2 and q is 2.

[0248] 16. The compound according to any one of items 1 to 15, wherein A is selected from the group consisting of -N(H)-, -O-, and -S-.

[0249] 17. The compound according to any one of items 1 to 16, wherein A is -O-.

[0250] 18. The compound according to any one of items 1 to 17, wherein B is —N(H)—, —O—, and —S—.

[0251] 19. The compound according to any one of items 1 to 18, wherein B is -O-.

[0252] 20. D, E, F, and G are each independently a linear C 1-16 - alkyl.

[0253] 21. D, E, F, and G are each independently a linear C 1-8 21. The compound according to any one of items 1 to 20, wherein - alkyl.

[0254] 22. At least one of D, E, F, and G is in branch C. 1-16 - alkyl.

[0255] 23. At least one of D, E, F, and G is in branch C. 1-8 23. The compound according to any one of items 1 to 19 and 22, wherein - alkyl.

[0256] twenty four. [ka] is a double bond.

[0257] 25. The compound of formula I is a compound of formula III-A, [ka] In the formula, R 2 , R 3 25. The compound according to any one of items 1 to 24, wherein Z, A, B, D, E, F, and G are as defined herein.

[0258] 26. The compound of formula I is a compound of formula IV-A, [ka] In the formula, R 2 , R 3 26. The compound according to any one of items 1 to 25, wherein A, B, D, E, F, and G are as defined herein.

[0259] 27. The compound of formula I is a compound of formula VA, [ka] In the formula, R 2 , R 3 27. The compound according to any one of items 1 to 26, wherein D, E, F, and G are as defined herein.

[0260] 28. The compound of formula I is [ka] 28. The compound according to any one of items 1 to 27, selected from the group consisting of:

[0261] 29. A lipid nanoparticle (LNP) comprising a lipid component comprising a compound according to any one of items 1 to 28.

[0262] 30. The lipid nanoparticle according to item 29, wherein the lipid component further comprises one or more of a neutral lipid, a structured lipid, and a PEGylated lipid.

[0263] 31. Neutral lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. DPPC, 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine 31. The lipid nanoparticle according to item 30, wherein the lipid nanoparticle is selected from the group consisting of 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), and sphingomyelin.

[0264] 32. The lipid nanoparticles according to item 30, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol.

[0265] 33. The lipid nanoparticle according to item 30, wherein the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, optionally PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

[0266] 34. The lipid nanoparticles according to item 30, wherein the lipid component comprises about 25 mol% to about 60 mol% of the compound according to any one of claims 1 to 6, about 2 mol% to about 25 mol% of a neutral lipid, about 18.5 mol% to about 60 mol% of a structured lipid, and about 0.2 mol% to about 10 mol% of a PEGylated lipid.

[0267] 35. The lipid nanoparticle according to any one of items 29 to 34, wherein the lipid nanoparticle further comprises a polynucleotide.

[0268] 36. The lipid nanoparticle according to item 35, wherein the polynucleotide is selected from the group consisting of messenger RNA (mRNA), self-amplifying mRNA (sa-mRNA), small interfering RNA (siRNA), microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA interference complementary RNA (micRNA), short hairpin RNA (shRNA), polyvalent RNA, Dicer substrate RNA, antisense oligonucleotide, plasmid DNA, DNA, and complementary DNA (cDNA).

[0269] 37. The lipid nanoparticle according to item 35 or 36, wherein the polynucleotide is ribonucleic acid (RNA).

[0270] 38. The lipid nanoparticle according to any one of items 35 to 37, wherein the polynucleotide is a conventional or self-amplifying mRNA.

[0271] 39. The lipid nanoparticle according to any one of items 29 to 38, wherein the lipid nanoparticle has a diameter of about 30 nm to about 160 nm.

[0272] 40. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to any one of items 29 to 39 and a pharmaceutically acceptable carrier.

[0273] 41. A method for delivering a polynucleotide to a mammalian cell, comprising administering to a subject the lipid nanoparticles described in any one of items 29 to 39 or the pharmaceutical composition described in item 40, thereby contacting the cells with the lipid nanoparticles or pharmaceutical composition and delivering the polynucleotide to the cells.

[0274] 42. The method of item 41, wherein the cells are cells of a human subject.

[0275] 43. A method for producing a polypeptide of interest in mammalian cells, comprising contacting the cells with lipid nanoparticles according to any one of items 29 to 39 or the pharmaceutical composition according to item 40, wherein the lipid nanoparticles comprise conventional or self-amplifying mRNA encoding the polypeptide.

[0276] 44. A method for treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject the lipid nanoparticles of any one of items 29 to 39 or the pharmaceutical composition of item 40, thereby treating the disease, disorder, or condition.

[0277] 45. The method of item 44, wherein the disease, disorder, or condition is selected from the group consisting of a rare disease, an infectious disease, a cancer, a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

[0278] 46. ​​A vaccine comprising the lipid nanoparticles according to any one of items 29 to 39 or the pharmaceutical composition according to item 40, and mRNA encoding the polypeptide.

[0279] 47. The vaccine according to item 46, wherein the vaccine is selected from a tumor vaccine, an influenza vaccine, and a SARS vaccine, including SARS-CoV-2.

Claims

1. A compound of formula I, 【Chemistry 1】 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: R １ is selected from the group consisting of formulas IA to II; 【Chemistry 2】 Z, A, and B are each independently -C(H) ２ -, -N(H)-, -O-, and -S-, and at least one of Z, A, and B is -N(H)-, -O-, or -S-; n and q are each independently an integer from 0 to 8; t, u, v, and w are each independently an integer from 0 to 10; D, E, F, and G are each independently a straight-chain or branched C １－１６ - alkyl, 【Transformation 3】 represents a single or double bond, R ２ and R ３ However, each independently, C １－８ -Alkyl and C １－８ -alkanols, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

2. R ２ But C １－８ The compound of claim 1, wherein:

3. R ２ But C １－３ The compound of claim 1, wherein:

4. If present, R ３ But C １－８ The compound of claim 1, wherein:

5. If present, R ３ But C １－３ The compound of claim 1, wherein:

6. R １ The compound of claim 1, wherein: is of formula IA.

7. R １ but, 【Chemistry 4】 2. The compound of claim 1, which is of formula IA selected from the group consisting of:

8. R １ The compound of claim 1, wherein is of formula IB.

9. R １ but, 【Transformation 5】 2. The compound of claim 1, which is of formula IB selected from the group consisting of:

10. 2. The compound of claim 1, wherein Z is selected from the group consisting of -N(H)-, -O-, or -S-.

11. The compound of claim 1, wherein Z is —O—.

12. 2. The compound of claim 1, wherein n and q are each independently an integer from 0 to 4.

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

14. The compound of claim 1 , wherein q is 2.

15. 2. The compound of claim 1, wherein n is 2 and q is 2.

16. 2. The compound of claim 1, wherein A is selected from the group consisting of -N(H)-, -O-, and -S-.

17. The compound of claim 1, wherein A is —O—.

18. The compound of claim 1, wherein B is —N(H)—, —O—, or —S—.

19. The compound of any one of claims 1 to 18, wherein B is -O-.

20. D, E, F, and G are each independently a linear C １－１６ The compound of claim 1, wherein:

21. D, E, F, and G are each independently a linear C １－８ The compound of claim 1, wherein:

22. At least one of D, E, F, and G is a branch C １－１６ The compound of claim 1, wherein:

23. At least one of D, E, F, and G is a branch C １－８ The compound of claim 1, wherein: 【Request Item 24】 【Chemistry 6】 The compound of claim 1 , wherein is a double bond.

25. The compound of formula I is a compound of formula III-A, 【Transformation 7】 In the formula, R ２ , R ３ , Z, A, B, D, E, F, and G are as defined herein.

26. The compound of formula I is a compound of formula IV-A, 【Transformation 8】 In the formula, R ２ , R ３ 10. The compound of claim 1, wherein A, B, D, E, F, and G are as defined herein.

27. The compound of formula I is a compound of formula VA, 【Chemistry 9】 In the formula, R ２ , R ３ 10. The compound of claim 1, wherein D, E, F, and G are as defined herein.

28. The compound of formula I is 【Chemistry 10】 2. The compound of claim 1 selected from the group consisting of:

29. A lipid nanoparticle (LNP) comprising a lipid component comprising the compound of claim 1.

30. 30. The lipid nanoparticle of claim 29, wherein the lipid component further comprises one or more of a neutral lipid, a structured lipid, and a PEGylated lipid.

31. The neutral lipid may be 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine 31. The lipid nanoparticle of claim 30, wherein the lipid nanoparticle is selected from the group consisting of amines, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

32. 31. The lipid nanoparticle of claim 30, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol.

33. 31. The lipid nanoparticle of claim 30, wherein the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, optionally PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

34. The lipid nanoparticle of claim 30, wherein the lipid component comprises from about 25 mol% to about 60 mol% of a compound according to any one of claims 1 to 6, from about 2 mol% to about 25 mol% of a neutral lipid, from about 18.5 mol% to about 60 mol% of a structured lipid, and from about 0.2 mol% to about 10 mol% of a PEGylated lipid.

35. The lipid nanoparticle of claim 29, wherein the lipid nanoparticle further comprises a polynucleotide.

36. The lipid nanoparticle of claim 35, wherein the polynucleotide is selected from the group consisting of messenger RNA (mRNA), self-amplifying mRNA (sa-mRNA), small interfering RNA (siRNA), microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA interference complementary RNA (micRNA), short hairpin RNA (shRNA), polyvalent RNA, dicer substrate RNA, antisense oligonucleotide, plasmid DNA, DNA, and complementary DNA (cDNA).

37. The lipid nanoparticle of claim 35, wherein the polynucleotide is a ribonucleic acid (RNA).

38. The lipid nanoparticle of claim 35, wherein the polynucleotide is a conventional or self-amplifying mRNA.

39. 30. The lipid nanoparticle of claim 29, wherein the lipid nanoparticle has a diameter of about 30 nm to about 160 nm.

40. 30. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to claim 29 and a pharmaceutically acceptable carrier.

41. A method for delivering a polynucleotide to a mammalian cell, comprising administering to a subject a lipid nanoparticle described in claim 29 or a pharmaceutical composition described in claim 40, thereby contacting the cell with the lipid nanoparticle or pharmaceutical composition and delivering the polynucleotide to the cell.

42. 42. The method of claim 41, wherein the cell is a cell of a human subject.

43. A method for producing a polypeptide of interest in mammalian cells, comprising contacting the cells with a lipid nanoparticle described in claim 29 or a pharmaceutical composition described in claim 40, wherein the lipid nanoparticle contains conventional or self-amplifying mRNA encoding the polypeptide.

44. A method for treating a disease, disorder, or condition in a subject in need of such treatment, comprising administering to the subject the lipid nanoparticles of claim 29 or the pharmaceutical composition of claim 40, thereby treating the disease, disorder, or condition.

45. 45. The method of claim 44, wherein the disease, disorder, or condition is selected from the group consisting of a rare disease, an infectious disease, a cancer, a proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

46. A vaccine comprising the lipid nanoparticle of claim 1 or the pharmaceutical composition of claim 40 and mRNA encoding a polypeptide.

47. 47. The vaccine of claim 46, wherein the vaccine is selected from a tumor vaccine, an influenza vaccine, and a SARS vaccine including SARS-CoV-2.

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