Nucleic acids encoding therapeutic polypeptides and lipid nanoparticle compositions comprising same

Abstract

The present disclosure provides lipid nanoparticle compositions comprising a nucleic acid encoding an RSV antigenic polypeptide. The invention also provides novel antigenic RSV-F polypeptides as well as nucleic acids encoding the antigenic RSV-F polypeptides.

Description

Technical Field

[0001] The present disclosure provides lipid nanoparticle compositions comprising nucleic acids encoding respiratory syncytial virus (RSV) antigenic polypeptides or variants thereof. The present disclosure also provides novel RSV polypeptides and variants thereof, and nucleic acids encoding the RSV polypeptides and variants.

[0002] Also provided are the purposes of RSV polypeptides and variants thereof, nucleic acids encoding the RSV polypeptides and variants thereof, and lipid nanoparticle compositions comprising the nucleic acids in the production of medicines (e.g., vaccines). Also provided are the purposes of the medicines (e.g., vaccines) in treating or preventing RSV infections. Also provided are the purposes of the nucleic acids encoding the RSV polypeptides and variants thereof in treating or preventing RSV infections. Background Art

[0003] RSV is a common cause of acute lower respiratory tract infections (ALRIs) in infants and young children who are outpatients. For example, in the United States, over 60% of infants will contract RSV during their first RSV season, and nearly all infants will have contracted RSV by the time they are 2-3 years old. Approximately 2.1 million US children under the age of 5 are treated annually for RSV infection, including 3% hospitalizations, 25% emergency department visits, and 73% pediatric admissions. Globally, RSV causes an estimated 33.8 million ALRIs in children under the age of 5 years annually (over 22% of all ALRIs), resulting in 66,000 to 199,000 deaths, 99% of which occur in developing countries. RSV is also a common cause of respiratory illness in the elderly, resulting in hospitalization rates comparable to those caused by influenza in people who are immune to severe influenza. RSV is transmitted through respiratory droplets and close contact with infected individuals or fomites. In temperate climates, RSV is prevalent each winter. Infants are at highest risk for severe RSV-related illness during their first 6 months of life, with hospitalizations peaking at 2-3 months of age. Prematurity and cardiopulmonary disease are risk factors for severe RSV-related illness. RSV infection in infants induces partial protective immunity, which appears to decline more rapidly than immunity against other respiratory viruses. Most children who are infected with RSV in their first year will be reinfected the following year, usually with less severe disease. Reinfection can persist throughout life, often with upper respiratory tract symptoms and sometimes with involvement of the lower respiratory tract or sinuses. Recommended treatment for RSV bronchiolitis primarily consists of respiratory support and hydration. There is no commonly used specific antiviral therapy. Palivizumab, a neutralizing monoclonal antibody, is used for prophylaxis in infants at highest risk of severe infection, but its high cost makes it difficult to use widely. There is currently no licensed RSV vaccine, and the development of a safe and effective RSV vaccine is a global public health priority.

[0004] The RSV virion consists of an inner nucleocapsid, which contains the viral RNA associated with a nucleoprotein (N), a phosphoprotein (P), and a large polymerase protein (L). The nucleocapsid is surrounded by a matrix protein (M) and encapsulated by a lipid bilayer containing the viral fusion (F) protein ("RSV-F protein") and the attachment protein (G), as well as a small hydrophobic protein (SH). RSV has two subtypes, A and B. The main difference is in the G glycoprotein, while the F glycoprotein sequence is more conserved. Neutralizing antibodies targeting the RSV-F protein have been shown to limit viral replication and reduce the severity of the disease.

[0005] Therefore, there is still a need to provide mRNA sequences encoding RSV-related proteins and use them for the treatment or prevention of RSV infection, especially in infants, the elderly and immunocompromised patients, and to develop compositions and methods that promote the delivery of mRNA sequences and / or reduce adverse reactions caused by RSV-based nucleic acid sequences. Summary of the Invention

[0006] Disclosed herein are lipid nanoparticle compositions comprising nucleic acids encoding respiratory syncytial virus (RSV) fusion proteins (F proteins) or variants thereof. Also disclosed herein are novel RSV polypeptides, variants thereof, and nucleic acids encoding the same. The disclosure further provides uses of the lipid nanoparticle compositions, the antigenic polypeptides, and the nucleic acids encoding the antigenic polypeptides in inducing an antigen-specific immune response or in producing a drug (e.g., a vaccine).

[0007] In one aspect, the present disclosure provides a lipid nanoparticle composition comprising:

[0008] a target polynucleotide comprising a nucleic acid encoding a respiratory syncytial virus antigenic polypeptide or a variant thereof, and

[0009] A lipid nanoparticle comprising a compound having the following formula (I):

[0010]

[0011] or a pharmaceutically acceptable salt thereof, wherein

[0012] R a Selected from the group consisting of hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl and R 6 ;

[0013] R1 for

[0014] R 2 for

[0015] R 3 for

[0016] R 4 for

[0017] R 5 If it exists,

[0018] R 6 If it exists,

[0019] Each W is independently selected from O, S or NR b , and each R b independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl;

[0020] Each Y is independently selected from O, S, NR c 、N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W), and each R c independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl;

[0021] Each Z is independently selected from C, S or S(O);

[0022] Each n is independently 0, 1, 2, 3, 4 or 5;

[0023] Each m is independently 0, 1, 2 or 3;

[0024] Each p is independently 1, 2, 3 or 4; and

[0025] R 1c 、R 2c 、R 3c and R 4cEach is independently selected from the group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl groups are optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, oxo, cyano, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl groups are optionally interrupted by one or more groups independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0026] In one aspect, the present disclosure provides a respiratory syncytial virus polypeptide mutant, wherein the mutant has respiratory syncytial virus antigen activity.

[0027] In one aspect, the present disclosure provides an isolated nucleic acid sequence encoding a respiratory syncytial virus polypeptide comprising an amino acid sequence of the present disclosure, or a respiratory syncytial virus polypeptide mutant of the present disclosure.

[0028] In one aspect, the present disclosure provides a respiratory syncytial virus vaccine comprising the lipid nanoparticle composition of the present disclosure, the respiratory syncytial virus polypeptide mutant of the present disclosure, or the isolated nucleic acid sequence of the present disclosure.

[0029] In one aspect, the present disclosure provides a method of inducing an antigen-specific immune response in a subject, comprising: administering to the subject a respiratory syncytial virus vaccine of the present disclosure in an amount effective to produce an antigen-specific immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figures 1A to 1E Shown is the in vitro expression of RSV mRNA detected by Western blotting and ELISA.

[0031] Figures 2A to 2B Shown is the in vitro expression of RSV mRNA detected by FACS.

[0032] Figure 3A It was shown that FLuc mRNA contained in the different tested lipid nanoparticles was efficiently expressed. Figure 3B Shown are RSV-binding antibody titers generated in serum following immunization with RSV mRNA contained in different tested lipid nanoparticles.

[0033] Figures 4A to 4C Shown are the levels of RSV F protein-binding antibody titers in serum following immunization with RSV mRNA contained in different tested lipid nanoparticles. Figure 4D Shown are F protein-specific T cell responses (IFNγ levels) in splenocytes of mice following immunization with RSV mRNA contained in different tested lipid nanoparticles.

[0034] Figure 5A Shown are RSV neutralizing antibody titers generated in sera following immunization with RSV mRNA contained in different tested lipid nanoparticles. Figure 5B Shown are the RSV viral loads in the right lungs of the test mice. Figure 5C Microscopic examination results are shown.

[0035] Figure 6 Shown are the RSV F protein binding antibody titer levels in serum following immunization with RSV mRNA contained in different tested lipid nanoparticles.

[0036] Figure 7 Shown are RSV F protein binding antibody titer levels in serum after immunization with RSV mRNA vaccines.

[0037] Figure 8 Shown are RSV F protein binding antibody titer levels in serum after immunization with RSV mRNA vaccines.

[0038] Figure 9 Shown are RSV F protein binding antibody titer levels in serum after immunization with RSV mRNA vaccines.

[0039] FIG. 10A to FIG. 10B Shown are RSV F protein binding antibody titer levels in serum after immunization with RSV mRNA vaccines. Figure 10C and Figure 10D Shown are the prefusion RSV F protein binding antibody titer levels and postfusion RSV F protein binding antibody titer levels in serum after immunization with RSV mRNA vaccines, respectively.

[0040] Figure 11A and Figure 11B Shown are the body weights and temperatures of the tested mice. Figure 11C The levels of IL-2, TNF-α, and IFN-γ produced by the tested mice are shown. Figure 11D The coagulation function levels of the test mice are shown. Figures 11E to 11K The blood routine detection levels of the test mice are shown. DETAILED DESCRIPTION

[0041] Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are illustrated in the accompanying structures and formulas. Although the present disclosure will be described in conjunction with the enumerated embodiments, it should be understood that these embodiments are not intended to limit the present disclosure to these embodiments. On the contrary, the present disclosure is intended to encompass all alternatives, modifications, and equivalents that may be included within the scope of the present disclosure defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to the methods and materials described herein may be used to implement the present disclosure. The present disclosure is in no way limited to the methods and materials described. In the event that one or more of the incorporated references and similar materials (including but not limited to defined terms, term usage, described technology, etc.) are different from or contradictory to the present application, the present disclosure shall prevail. All references, patents, and patent applications cited in the present disclosure are incorporated herein by reference in their entirety.

[0042] definition

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.As used herein, the following terms are intended to have the following meanings.

[0044] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes both a single compound and a plurality of different compounds.

[0045] As used herein, the term "about" is intended to indicate that the recited value is not to be interpreted as an absolute value, and that measurement errors, batch-to-batch variations, and / or device-to-device variations should also be taken into account.

[0046] The words “comprise,” “comprising,” and “include,” “including,” and “includes” as used in the present description and claims are intended to indicate the presence of stated features, integers, components, or steps, but they do not exclude the presence or addition of one or more other features, integers, components, steps, or groups thereof.

[0047] It should be understood that the "compounds" of the present disclosure can exist in solvated and unsolvated forms, such as hydrated forms, solid forms, and that the present disclosure is intended to encompass all such solvated and unsolvated forms. It should also be understood that the "compounds" of the present disclosure can exist in the form of pharmaceutically acceptable salts. In some embodiments, the "compounds" of the present disclosure are ionizable lipids. In some embodiments, the "compounds" of the present disclosure can exist as cationic lipids at physiological pH.

[0048] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, in the 75th edition of the Handbook of Chemistry and Physics, and specific functional groups are generally defined as described herein. In addition, general principles of organic chemistry as well as specific functional moieties and reactivities are described in the following literature: Organic Chemistry, Thomas Sorrell, 2nd edition, University Science Books, Sausalito, 2006; Smith and March, March's Advanced Organic Chemistry, 6th edition, John Wiley & Sons,, Inc., New York, 2007; Larock, Comprehensive Organic Transformations, 3rd edition, VCH Publishers, Inc., New York, 2018; Carruthers, Some Modern Methods of Organic Synthesis, 4th edition, Cambridge University Press, Cambridge, 2004; the entire contents of which are incorporated herein by reference.

[0049] Throughout this disclosure, linking substituents are described. Where a structure clearly requires a linking group, the Markush variable listed for that group is understood to be the linking group. For example, if a structure requires a linking group and the Markush group definition for that variable lists "alkyl," it is understood that "alkyl" means a linking alkylene group.

[0050] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then the substituent may be bonded to any atom in the ring. When a substituent is listed without specifying the atom via which the substituent is bonded to the rest of the compound of a given formula, then the substituent may be bonded via any atom in the formula. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0051] In any variable (e.g., R i ) occurs more than one time in any constituent or formula of a compound, its definition on each occurrence is independent of its definition at every other occurrence. Thus, for example, if the group shown is represented by 0 to 2 R i Partially substituted, the group may optionally be replaced by up to two R i Partially substituted, and R i independently selected at each occurrence from R i Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0052] As used herein, the term "C i-j " indicates a range of carbon atoms, where i and j are integers, and the range of carbon atoms includes the endpoints (i.e., i and j) and every integer point therebetween, and where j is greater than i. For example, C 1-6 Indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, and six carbon atoms. In some embodiments, the term "C 1-24 ” indicates 1 to 24 carbon atoms, in particular 2 to 24 carbon atoms, in particular 4 to 24 carbon atoms, in particular 6 to 24 carbon atoms, in particular 8 to 22 carbon atoms, in particular 10 to 20 carbon atoms, in particular 10 to 18 carbon atoms or in particular 12 to 18 carbon atoms.

[0053] As used herein, the term "alkyl", whether used as part of another term or by itself, refers to a saturated straight or branched chain hydrocarbon group that may be optionally substituted independently with one or more substituents as described below. i-jIn some embodiments, the alkyl group contains 1 to 24 carbon atoms. In some embodiments, the alkyl group contains 1 to 23 carbon atoms. In some embodiments, the alkyl group contains 1 to 22 carbon atoms. In some embodiments, the alkyl group contains 1 to 21 carbon atoms. In some embodiments, the alkyl group contains 1 to 20 carbon atoms, 1 to 19 carbon atoms, 1 to 18 carbon atoms, 1 to 17 carbon atoms, 1 to 16 carbon atoms, 1 to 15 carbon atoms, 1 to In some embodiments, the alkyl group contains 12 to 14 carbon atoms, 1 to 13 carbon atoms, or 1 to 12 carbon atoms. In some embodiments, the alkyl group contains 12 to 18 carbon atoms. In some embodiments, the alkyl group contains 12 to 17 carbon atoms, 12 to 16 carbon atoms, 12 to 15 carbon atoms, or 12 to 14 carbon atoms. In some embodiments, the alkyl group contains 12 to 19 carbon atoms, 12 to 20 carbon atoms, 12 to 21 carbon atoms, 12 to 22 carbon atoms, 12 to 23 carbon atoms, or 12 to 24 carbon atoms. 1-10 Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. 1-6 Examples of “alkyl” are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

[0054] The alkyl group may be further substituted with substituents which independently replace one or more hydrogen atoms on one or more carbon atoms of the alkyl group. Examples of such substituents can include, but are not limited to, acyl, alkyl, alkenyl, alkynyl, oxo, halogen, hydroxy, alkoxy, haloalkyl, haloalkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfmyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, nitro, azido, heterocyclyl, alkaryl, or an aromatic or heteroaromatic moiety. The alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups described below may also be similarly substituted.

[0055] As used herein, the term "alkenyl", whether used as part of another term or independently, refers to a straight or branched hydrocarbon group with at least one carbon-carbon double bond that can be optionally independently substituted by one or more substituents described herein, and includes groups with "cis" orientation and "trans" orientation or alternatively "E" orientation and "Z" orientation. In some embodiments, the alkenyl group contains 2 to 24 carbon atoms. In some embodiments, the alkenyl group contains 2 to 23 carbon atoms. In some embodiments, the alkenyl group contains 2 to 22 carbon atoms, 2 to 21 carbon atoms, 2 to 20 carbon atoms, 2 to 19 carbon atoms, 2 to 18 carbon atoms, 2 to 17 carbon atoms, 2 to 16 carbon atoms, 2 to 15 carbon atoms, 2 to 14 carbon atoms, 2 to 13 carbon atoms, 2 to 12 carbon atoms, 2 to 11 carbon atoms, and in some embodiments, the alkenyl group contains 2 carbon atoms. In some embodiments, the alkenyl group contains 12 to 18 carbon atoms. In some embodiments, the alkenyl group contains 12 to 17 carbon atoms, 12 to 16 carbon atoms, 12 to 15 carbon atoms, or 12 to 14 carbon atoms. In some embodiments, the alkenyl group contains 12 to 19 carbon atoms, 12 to 20 carbon atoms, 12 to 21 carbon atoms, 12 to 22 carbon atoms, 12 to 23 carbon atoms, or 12 to 24 carbon atoms. In some embodiments, the alkenyl group contains one or more "Z" carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, ethylenyl or vinyl, propenyl, butenyl, pentenyl, 1-methyl-2-butene-1-yl, 5-hexenyl, etc. In some embodiments, the alkenyl group has at least one carbon-carbon double bond. In some embodiments, the alkenyl group has at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten carbon-carbon double bonds. In some embodiments, two or more carbon-carbon double bonds in the alkenyl group are conjugated. In some embodiments, two or more carbon-carbon double bonds in an alkenyl group are unconjugated. In some embodiments, two or more carbon-carbon double bonds in an alkenyl group are separated, cumulative, or conjugated. The term "alkenyl," whether used as part of another term or independently, is also intended to include straight or branched chain hydrocarbon groups having at least one carbon-carbon double bond and at least one carbon-carbon triple bond.

[0056] As used herein, the term "alkynyl", whether used as part of another term or independently, refers to a straight or branched chain hydrocarbon group with at least one carbon-carbon triple bond that can be optionally independently substituted by one or more substituents described herein. In some embodiments, the alkynyl group contains 2 to 24 carbon atoms. In some embodiments, the alkynyl group contains 2 to 23 carbon atoms. In some embodiments, the alkynyl group contains 2 to 22 carbon atoms, 2 to 21 carbon atoms, 2 to 20 carbon atoms, 2 to 19 carbon atoms, 2 to 18 carbon atoms, 2 to 17 carbon atoms, 2 to 16 carbon atoms, 2 to 15 carbon atoms, 2 to 14 carbon atoms, 2 to 13 carbon atoms, 2 to 12 carbon atoms, 2 to 10 carbon atoms, and in some embodiments, the alkynyl group contains 2 carbon atoms. In some embodiments, the alkynyl group contains 12 to 18 carbon atoms. In some embodiments, the alkynyl group contains 12 to 17 carbon atoms, 12 to 16 carbon atoms, 12 to 15 carbon atoms, or 12 to 14 carbon atoms. In some embodiments, the alkynyl group contains 12 to 19 carbon atoms, 12 to 20 carbon atoms, 12 to 21 carbon atoms, 12 to 22 carbon atoms, 12 to 23 carbon atoms, or 12 to 24 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like. In some embodiments, the alkynyl group has at least one carbon-carbon triple bond. In some embodiments, the alkynyl group has at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten carbon-carbon triple bonds. In some embodiments, two or more carbon-carbon triple bonds in the alkynyl group are conjugated. In some embodiments, two or more carbon-carbon triple bonds in the alkynyl group are not conjugated. The term "alkynyl," whether used as part of another term or by itself, is also meant to include straight or branched chain hydrocarbon groups having at least one carbon-carbon triple bond and at least one carbon-carbon double bond.

[0057] As used herein, the term "alkoxy", whether used as part of another term or by itself, refers to an alkyl group as previously defined attached to the parent molecular group through an oxygen atom. i-j "Alkoxy" means that the alkyl portion of the alkoxy group has 1 to 1 carbon atoms. In some embodiments, the alkoxy group contains 1 to 10 carbon atoms. In some embodiments, the alkoxy group contains 1 to 9 carbon atoms. In some embodiments, the alkoxy group contains 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. "C 1-6 Examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, neopentoxy, n-hexoxy, and the like.

[0058] As used herein, the term "amino" refers to -NH 2. In some embodiments, the amino group may be substituted with any possible substituent on nitrogen.

[0059] As used herein, the term "aryl", whether used as part of another term or independently, refers to monocyclic and polycyclic ring systems with a total of 5 to 20 ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 12 ring members. Examples of "aryl" include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, etc., which may carry one or more substituents. As used herein, the scope of the term "aryl" also includes groups in which an aromatic ring is fused to one or more additional rings. In the case of a polycyclic system, only one ring needs to be aromatic (e.g., 2,3-dihydroindole), but all rings can be aromatic (e.g., quinoline). The second ring can also be fused, bridged, or spiro. Examples of polycyclic aryl groups include, but are not limited to, benzofuranyl, indanyl, phthalimido, naphthalimido, phenanthridinyl, or tetrahydronaphthyl, etc. Aryl can be substituted by substituents as described above at one or more ring positions.

[0060] As used herein, the term "cycloalkyl", whether used as part of another term or independently, refers to a monovalent non-aromatic saturated or partially unsaturated monocyclic and polycyclic system, wherein all ring atoms are carbon, and the system contains at least three ring carbon atoms. In some embodiments, cycloalkyl can contain 3 to 12 ring carbon atoms, 3 to 11 ring carbon atoms, 3 to 10 ring carbon atoms, 3 to 9 ring carbon atoms, 3 to 8 ring carbon atoms, 3 to 7 ring carbon atoms, 3 to 6 ring carbon atoms, 3 to 5 ring carbon atoms, 3 to 4 ring carbon atoms, 4 to 12 ring carbon atoms, 4 to 11 ring carbon atoms, 4 to 10 ring carbon atoms, 4 to 9 ring carbon atoms, 4 to 8 ring carbon atoms, 4 to 7 ring carbon atoms, 4 to 6 ring carbon atoms, 4 to 5 ring carbon atoms. Cycloalkyl can be saturated or partially unsaturated. Cycloalkyl can be substituted. In some embodiments, cycloalkyl can be a saturated cyclic alkyl. In some embodiments, a cycloalkyl group can be a partially unsaturated cyclic alkyl group containing at least one double or triple bond in the ring system.

[0061] In some embodiments, cycloalkyl can be monocyclic or polycyclic. Examples of monocyclic cycloalkyl include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl.

[0062] In some embodiments, cycloalkyl can be a saturated or partially unsaturated polycyclic (for example, bicyclic and tricyclic) carbocyclic ring system, which can be arranged as a condensed ring, spirocyclic or bridged ring system. As used herein, the term "condensed ring" refers to a ring system in which two rings share two adjacent atoms, the term "spirocyclic" refers to a ring system in which two rings are connected by a single common atom, and "bridged ring" refers to a ring system in which two rings share three or more atoms. The example of condensed carbocyclic group includes but is not limited to naphthyl, benzopyrenyl, anthracenyl, acenaphthenyl, fluorenyl, etc. The example of spirocarbocyclic group includes but is not limited to spiral [5.5] undecyl, spiral-pentadienyl, spiral [3.6]-decyl, etc. The example of bridged carbocyclic group includes but is not limited to bicyclo [1,1,1] pentenyl, bicyclo [2,2,1] heptenyl, bicyclo [2,2,1] heptyl, bicyclo [2,2,2] octyl, bicyclo [3,3,1] nonyl, bicyclo [3,3,3] undecyl, etc.

[0063] As used herein, the term "cyano" refers to -CN.

[0064] As used herein, the term "oxo" refers to =0, which replaces two hydrogen atoms attached to one atom. For example, an oxo-substituted ethyl group is CH3C(=O)- or -C(=O)CH2-.

[0065] As used herein, the term "halogen" refers to an atom selected from the group consisting of fluorine (fluoro), chlorine (chloro), bromine (bromo), and iodine (iodo).

[0066] As used herein, the term "haloalkyl", whether used as part of another term or independently, refers to an alkyl group having one or more halogen substituents. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl (-CF3), pentafluoroethyl (-C2F5), difluoromethyl (-CHF2), trichloromethyl (-CCl3), dichloromethyl (-CHCl2), pentachloroethyl (-C2Cl5), etc.

[0067] As used herein, the term "haloalkoxy", whether used as part of another term or by itself, refers to an alkoxy group having one or more halogen substituents. Thus, the term "halo-C i-j "Alkoxy", whether used as part of another term or independently, refers to a C i-j Alkoxy. Examples of haloalkoxy include, but are not limited to, -O-CF3, -O-C2F5, -O-CHF2, -O-CCl3, -O-CHCl2, -O-C2Cl5, and the like.

[0068] As used herein, the term "heteroatom" refers to nitrogen (N), oxygen (O), sulfur (S), and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen (including nitrogen oxides).

[0069] As used herein, the term "heteroalkyl," "heteroalkenyl," or "heteroalkynyl," whether used as part of another term or by itself, refers to an alkyl, alkenyl, or alkynyl group that contains one or more heteroatoms. Thus, the term "hetero-C i-j Alkyl", "hetero-C i-j Alkenyl" or "hetero-C i-j "Alkynyl", whether used as part of another term or independently, refers to a C i-j Alkyl, C i-j Alkenyl or C i-j For example, the term "hetero-C 1-6 "Alkyl", whether used as part of another term or independently, refers to a C 1-6 In some embodiments, the heteroalkyl, heteroalkenyl, or heteroalkynyl radicals contain at least one heteroatom. In some embodiments, the heteroalkyl, heteroalkenyl, or heteroalkynyl radicals contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten heteroatoms. In some embodiments, two or more heteroatoms in the heteroalkyl, heteroalkenyl, or heteroalkynyl radicals are the same. In some embodiments, two or more heteroatoms in the heteroalkyl, heteroalkenyl, or heteroalkynyl radicals are different. In some embodiments, two or more heteroatoms in the heteroalkyl, heteroalkenyl, or heteroalkynyl radicals are directly bonded. In some embodiments, two or more heteroatoms in the heteroalkyl, heteroalkenyl, or heteroalkynyl radicals are not directly bonded.

[0070] As used herein, the term "heteroaryl", whether used as part of another term or independently, refers to an aryl group containing one or more heteroatoms in addition to carbon atoms. The heteroaryl group can be monocyclic. Examples of monocyclic heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl, and pteridinyl. Heteroaryl also includes a polycyclic group in which a heteroaromatic ring is fused to one or more aryl, heteroaryl, alicyclic, or heterocyclic rings, wherein a linking group or point of attachment is located on the heteroaromatic ring or on another ring. Examples of polycyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, benzothiophenyl, benzofuranyl, benzo[1,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, dihydroquinolyl, dihydroisoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, and the like.

[0071] As used herein, the term "heterocyclic group" refers to a saturated or partially unsaturated carbocyclic group, wherein one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, etc., and the remaining ring atoms are carbon, wherein one or more ring atoms can optionally be substituted independently by one or more substituents. In some embodiments, the heterocyclic group is a saturated heterocyclic group. In some embodiments, the heterocyclic group is a partially unsaturated heterocyclic group with one or more double bonds in its ring system. In some embodiments, the heterocyclic group can contain any oxidized form of carbon, nitrogen or sulfur and any quaternized form of basic nitrogen. Where possible, the heterocyclic group can be carbon-connected or nitrogen-connected. In some embodiments, the heterocycle is carbon-connected. In some embodiments, the heterocycle is nitrogen-connected. For example, the group derived from pyrrole can be pyrrole-1-yl (nitrogen-connected) or pyrrole-3-yl (carbon-connected). Further, the group derived from imidazole can be imidazole-1-yl (nitrogen-connected) or imidazole-3-yl (carbon-connected).

[0072] The heterocyclic group may be monocyclic. Examples of monocyclic heterocyclic groups include, but are not limited to, oxetanyl, 1,1-dioxothietane, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, azetidinyl, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidinyl, piperazinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyrazazolonyl, pyrrolidinonyl, triazinonyl, and the like.

[0073] The heterocyclic group can be polycyclic, including fused rings, spiro rings, and bridged ring systems. Fused heterocyclic groups include groups in which a heterocyclic group is fused to a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. Examples of fused heterocyclic groups include, but are not limited to, phenyl fused rings or pyridyl fused rings, such as quinolyl, isoquinolyl, quinoxalinyl, quinolizinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothiophenyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo[1,2-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, [1,2,3]triazolo[4,3-a]pyridinyl, and the like. Examples of spiroheterocyclyl groups include, but are not limited to, spiropyranyl, spirooxazinyl, 5-aza-spiro[2.4]heptyl, 6-aza-spiro[2.5]octanyl, 6-aza-spiro[3.4]octanyl, 2-oxa-6-aza-spiro[3.3]heptyl, 2-oxa-6-aza-spiro[3.4]octanyl, 6-aza-spiro[3.5]nonanyl, 7-aza-spiro[3.5]nonanyl, 1-oxa-7-aza-spiro[3.5]nonanyl, etc. Examples of bridged heterocyclyl groups include, but are not limited to, 3-aza-bicyclo[3,1,0]hexanyl, 8-aza-bicyclo[3,2,1]octanyl, 1-aza-bicyclo[2,2,2]octanyl, 2-aza-bicyclo[2,2,1]heptyl, 1,4-diazabicyclo[2,2,2]octanyl, etc.

[0074] As used herein, the term "hydroxyl" or "hydroxy" refers to -OH.

[0075] As used herein, the term "alkoxycarbonyl" refers to an alkyl-OC(=O)- group. In some embodiments, the alkoxycarbonyl group may be further substituted on the alkyl group with any of the possible substituents described above. Examples of alkoxycarbonyl groups include, but are not limited to, tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, and 9-fluorenylmethoxycarbonyl.

[0076] As used herein, the term "sulfonyl" refers to R-SO2-, wherein R is hydrogen or any possible substituent on sulfur. Examples of sulfonyl include, but are not limited to, p-toluenesulfonyl, p-bromophenylsulfonyl, 2- or 4-nitrobenzenesulfonyl, trifluoromethanesulfonyl, methanesulfonyl, and 5-(dimethylamino)naphthalene-1-sulfonyl.

[0077] As used herein, the term "acyl", whether used as part of another term or independently, refers to R-C(=O)-, where R is hydrogen or any possible substituent on carbon. Examples of acyl include, but are not limited to, formyl, acetyl, trifluoroacetyl, and benzoyl. The term "alkylacyl" refers to alkyl-C(=O)-, where the alkyl group is optionally substituted with any of the possible substituents described above. The term "arylacyl" refers to aryl-C(O=)-, where the aryl group is optionally substituted with any of the possible substituents described above.

[0078] As used herein, the term "partially unsaturated" refers to a group containing at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to encompass aromatic (ie, fully unsaturated) moieties.

[0079] As used herein, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of a specified portion are replaced by a suitable substituent. It should be understood that "substituted", "substituted by ... " or "substituted by ... " include implicit prerequisites, i.e., such substitutions are consistent with the allowed valence of the substituted atom, and substitutions produce stable or chemically feasible compounds, for example, compounds that do not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc. Unless otherwise stated, "optionally substituted" groups can have appropriate substituents at each substitutable position of the group, and when more than one position in any given structure can be substituted by more than one substituent selected from a specified group, at each position, the substituents can be the same or different. It will be understood by those skilled in the art that, if appropriate, the substituent itself can be substituted. Unless specifically stated as "unsubstituted", reference to a chemical moiety herein should be understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted variants and unsubstituted variants.

[0080] As used herein, the term "interrupted", whether or not preceded by the term "optionally", means that one or more covalent bonds of the specified moiety are replaced by a suitable linking group, but not at the terminal end. In some embodiments, the bond replaced is a carbon-carbon bond. In some embodiments, the bond replaced is a carbon-heteroatom bond. Unless otherwise stated, an "optionally interrupted" group can have a suitable linking group at each replaceable position of the group, and when more than one position in any given structure can be interrupted by more than one linking group selected from the specified group, the linking group at each position can be the same or different. In some embodiments, an alkyl group "interrupted" by a cycloalkyl group refers to an alkyl-cycloalkyl-alkyl group. In some embodiments, an alkenyl group "interrupted" by a cycloalkyl group refers to an alkenyl-cycloalkyl-alkyl group, an alkenyl-cycloalkyl-alkenyl group, an alkenyl-cycloalkyl-alkynyl group, an alkyl-cycloalkyl-alkenyl group, or an alkynyl-cycloalkyl-alkenyl group. In some embodiments, an alkynyl group "interrupted" by a cycloalkyl group refers to an alkynyl-cycloalkyl-alkyl, alkynyl-cycloalkyl-alkenyl, alkynyl-cycloalkyl-alkynyl, alkyl-cycloalkyl-alkynyl, or alkenyl-cycloalkyl-alkynyl. In some embodiments, an alkyl group "interrupted" by a heterocyclyl group refers to an alkyl-heterocyclyl-alkyl group. In some embodiments, an alkenyl group "interrupted" by a heterocyclyl group refers to an alkenyl-heterocyclyl-alkyl group, an alkenyl-heterocyclyl-alkenyl, an alkenyl-heterocyclyl-alkynyl, an alkyl-heterocyclyl-alkenyl, or an alkynyl-heterocyclyl-alkenyl group. In some embodiments, an alkynyl group "interrupted" by a heterocyclyl group refers to an alkynyl-heterocyclyl-alkyl group, an alkynyl-heterocyclyl-alkenyl, an alkynyl-heterocyclyl-alkynyl, an alkyl-heterocyclyl-alkynyl, or an alkenyl-heterocyclyl-alkynyl group. In some embodiments, an alkyl group "interrupted" by an aryl group refers to an alkyl-aryl-alkyl group. In some embodiments, an alkenyl group "interrupted" by an aryl group refers to an alkenyl-aryl-alkyl, alkenyl-aryl-alkenyl, alkenyl-aryl-alkynyl, alkyl-aryl-alkenyl, or alkynyl-aryl-alkenyl. In some embodiments, an alkynyl group "interrupted" by an aryl group refers to an alkynyl-aryl-alkyl, alkynyl-aryl-alkenyl, alkynyl-aryl-alkynyl, alkyl-aryl-alkynyl, or alkenyl-aryl-alkynyl. In some embodiments, an alkyl group "interrupted" by a heteroaryl group refers to an alkyl-heteroaryl-alkyl group. In some embodiments, an alkenyl group "interrupted" by a heteroaryl group refers to an alkenyl-heteroaryl-alkyl, alkenyl-heteroaryl-alkenyl, alkenyl-heteroaryl-alkynyl, alkyl-heteroaryl-alkenyl, or alkynyl-heteroaryl-alkenyl. In some embodiments, an alkynyl group "interrupted" by a heteroaryl group refers to an alkynyl-heteroaryl-alkyl, alkynyl-heteroaryl-alkenyl, alkynyl-heteroaryl-alkynyl, alkyl-heteroaryl-alkynyl, or alkenyl-heteroaryl-alkynyl.

[0081] As used herein, the term "pharmaceutically acceptable" refers to compounds, lipid nanoparticles, lipid nanoparticle compositions, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio. In some embodiments, pharmaceutically acceptable compounds, lipid nanoparticles, lipid nanoparticle compositions, materials, compositions, and / or dosage forms are those approved by regulatory agencies (such as the U.S. Food and Drug Administration, the National Medical Products Administration, or the European Medicines Agency) or listed in generally recognized pharmacopeias (such as the United States Pharmacopoeia, the Chinese Pharmacopoeia, or the European Pharmacopoeia) for use in animals, more particularly in humans.

[0082] As used herein, "pharmaceutically acceptable salts" or "pharmaceutically acceptable salts" refer to compound derivatives in which the parent compound is modified by converting an existing acidic moiety (e.g., carboxyl, etc.) or basic moiety (e.g., amine, base, etc.) into its salt form. In many cases, the compounds of the present invention are capable of forming acid addition salts and / or base salts by virtue of the presence of amino groups, bases, or groups similar thereto. And "pharmaceutically acceptable salts" include acid addition salts or base salts that retain the biological effectiveness and properties of the parent compound, which are generally not biologically or otherwise undesirable. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977), Vol. 66: 1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of an amino group formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid) or organic acids (such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, lactic acid, trifluoroacetic acid, benzoic acid, cinnamic acid, mandelic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malonic acid, fumaric acid, citric acid, malic acid, maleic acid, tartaric acid, succinic acid or methanesulfonic acid), or salts formed using other methods used in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. In some embodiments, inorganic acids from which salts can be derived include, for example, hydrochlorides, sulfates, phosphates, etc. In some embodiments, organic acids from which salts can be derived include, for example, maleates, fumarates, oxalates, p-toluenesulfonates, succinates, L-(+)-tartrates, monoadipates, hemiadipates, etc.

[0083] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or one or more lipid nanoparticles or lipid nanoparticle compositions of the present disclosure with other chemical components (such as pharmaceutically acceptable diluents, excipients or carriers). The purpose of a pharmaceutical composition is to facilitate administration of the compound, lipid nanoparticle or lipid nanoparticle composition to a subject.

[0084] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that participates in carrying or transporting the compounds provided herein from one location, fluid, tissue, organ (internal or external) or part of the body to another location, fluid, tissue, organ or part of the body, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A pharmaceutically acceptable excipient or carrier can be a solvent, diluent, excipient, or other material that can be used to contact animal tissue without producing excessive toxicity or side effects. Non-limiting examples of pharmaceutically acceptable excipients or carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as polyethylene glycol and propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; release agents; Coating agents; sweeteners, flavorings, and fragrances; preservatives; antioxidants; ion exchangers; alumina; aluminum stearate; lecithin; self-emulsifying drug delivery systems (SEDDS), such as d-tocopheryl polyethylene glycol 1000 succinate; surfactants for pharmaceutical dosage forms, such as Tween or other similar polymer delivery matrices; serum proteins, such as human serum albumin; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts; colloidal silicon dioxide; magnesium trisilicate; polyvinyl pyrrolidone; cellulose-based materials; polyacrylates; waxes; and polyethylene-polyoxypropylene-block polymers. Cyclodextrins (such as α-cyclodextrin, β-cyclodextrin, and cyclodextrin), or chemically modified derivatives (such as hydroxyalkyl cyclodextrins, including 2-hydroxypropyl cyclodextrin and 3-hydroxypropyl cyclodextrin), or other solubility derivatives can also be used to enhance the delivery of the compounds described herein. Pharmaceutically acceptable excipients or carriers useful in the present disclosure include those generally known in the art, such as those disclosed in Remington Pharmaceutical Sciences (Mack Pub. Co., New Jersey (1991)), which is incorporated herein by reference.

[0085] As used herein, "administration" of a disclosed compound, lipid nanoparticle, or lipid nanoparticle composition encompasses delivering a compound, lipid nanoparticle, or lipid nanoparticle composition described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, to a subject using any suitable formulation or route of administration as discussed herein.

[0086] As used herein, the term "delivering" means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject can involve administering a lipid nanoparticle composition comprising the therapeutic and / or prophylactic agent to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous route). Administering a lipid particle or a composition comprising a lipid particle to a mammal or mammalian cell can involve contacting one or more cells with the lipid particle or composition.

[0087] As used herein, the term "enhanced delivery" means that the lipid particles deliver more (e.g., at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold or at least 100-fold) of the therapeutic and / or prophylactic agent to the target tissue (e.g., liver, lung, spleen or muscle) or target cell (e.g., hepatocyte, lung cell, spleen cell or muscle cell) as compared to the level of delivery of the therapeutic and / or prophylactic agent by a control lipid particle (e.g., a lipid particle comprising DLin-MC3-DMA) to the target tissue (e.g., liver, lung, spleen or muscle) or target cell (e.g., hepatocyte, lung cell, spleen cell or muscle cell). Delivery therapeutic agent and / or preventive level to specific tissue or cell can be measured in the following manner: the amount of therapeutic agent and / or preventive and the amount of total therapeutic agent and / or preventive in the tissue or cell in comparison tissue or cell, the amount of therapeutic agent and / or preventive and the weight of the tissue in comparison tissue or cell, the amount of protein produced in comparison tissue or cell and the amount of total protein in the tissue or cell, the amount of protein produced in comparison tissue and the weight of the tissue or cell, or the amount of the therapeutic agent and / or preventive delivered to tissue or cell and the total therapeutic agent and / or preventive used. It should be understood that therapeutic agent and / or preventive need not be determined in the subject being treated to the enhancement delivery of target tissue or cell, but can be determined in a substitute such as an animal model (for example, mouse or rat model). In certain embodiments, no matter how route of administration, the lipid granule composition comprising formula (I) compound all has substantially identical delivery enhancement level. For example, some compounds disclosed herein show similar delivery enhancement when being used for intravenous or intramuscular delivery therapeutic agent and / or preventive.

[0088] As used herein, the term "selective delivery," "selectively deliver," or "selectively delivering" means delivering more (e.g., at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold or at least 10-fold) of the therapeutic and / or prophylactic agent to a target tissue (e.g., liver, lung, spleen or muscle) or a target non-target cell (e.g., liver cell, lung cell, spleen cell or muscle cell) by a lipid particle than to a non-target tissue (e.g., liver, lung cell, spleen or muscle) or a target non-target cell (e.g., liver cell, lung cell, spleen cell or muscle cell). Delivery of therapeutic and / or prophylactic levels to specific tissues or cells can be measured by comparing the amount of therapeutic and / or prophylactic in a tissue or cell with the amount of total therapeutic and / or prophylactic in the tissue or cell, comparing the amount of therapeutic and / or prophylactic in a tissue or cell with the weight of the tissue or cell, comparing the amount of protein produced in a tissue or cell with the amount of total protein in the tissue or cell, or comparing the amount of protein produced in a tissue or cell with the weight of the tissue or cell. It should be understood that the ability of lipid particles to specifically deliver therapeutic and / or prophylactic agents to target tissues or cells need not be determined in a treated subject, but can be determined in a surrogate such as an animal model (e.g., mouse or rat model).

[0089] The term "effective amount", "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic and / or prophylactic agent or a compound or pharmaceutical composition described herein delivered to a tissue or cell that is sufficient to prevent, treat, alleviate and / or improve the symptoms and / or underlying causes of any condition or disease in a subject, or an amount of an agent sufficient to produce the desired effect on the target cell, for example, reduced cell migration, increased or inhibited expression of a target nucleic acid in a cell, compared to the normal expression level of the nucleic acid detected when the therapeutic and / or prophylactic agent or a compound or pharmaceutical composition described herein is not delivered to the tissue or cell. In one embodiment, a "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount sufficient to alleviate or eliminate the symptoms of a disease. In another embodiment, a "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount sufficient to overcome the disease itself. In some specific embodiments, a "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount effective for detectably killing cancer cells or inhibiting the growth or spread of cancer cells, reducing the size or number of tumors; or an amount that achieves other indicators of the level, stage, progression or severity of cancer. The pharmaceutically effective amount or therapeutically effective amount will vary according to the subject and the condition being treated, the subject's weight and age, the severity of the condition, the specific composition or excipient selected, the dosing regimen to be followed, the time of administration, the mode of administration, etc., all of which can be easily determined by a person of ordinary skill in the art. A complete therapeutic effect is not necessarily achieved by administering a single dose, but may only occur after administering a series of doses. The specific dosage will vary according to, for example, the specific compound selected, the type of subject and their age / existing health status or health status risk, the dosing regimen to be followed, the severity of the disease, whether used in combination with other agents, the time of administration, the tissue to be administered, and the physical delivery system carrying the drug. Therefore, a pharmaceutically effective amount or therapeutically effective amount can be administered in one or more administrations. For example, but not limited to, in the context of treating cancer, a pharmaceutically effective amount or therapeutically effective amount of a medicament refers to the amount of the medicament that alleviates, improves, alleviates, or eliminates one or more cancer symptoms in a patient.

[0090] The term "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from the DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0091] The polypeptide or polynucleotide molecules of the present disclosure can have a certain degree of sequence similarity or identity with a reference molecule (e.g., a reference polypeptide or a reference polynucleotide). As known in the art, the term "identity" refers to the relationship between two or more polypeptide or polynucleotide sequences determined by comparing sequences. In the art, identity also means the degree of sequence association between them determined by the number of matches between two or more amino acid residue strings or nucleic acid residue strings. Identity measures the percentage of identical matches between the smaller sequences in two or more sequences, where the gap comparison (if any) is processed by a specific mathematical model or computer program (e.g., "algorithm"). When applied to polypeptide or polynucleotide sequences, "% identity" is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid sequence or nucleic acid sequence of the second sequence after comparing sequences and introducing (if necessary) gaps to achieve maximum percentage identity. Methods and computer programs for comparison are well known in the art. It should be understood that identity depends on the calculation of percentage identity, but its value may be different due to the gaps and penalties introduced in the calculation. In some embodiments, variants of a particular polynucleotide or polypeptide have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those of skill in the art.

[0092] "Subjects" contemplated for administration include, but are not limited to, humans (i.e., male or female, of any age, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young, middle-aged, or elderly) and / or other primates (e.g., rhesus monkeys, macaques); mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, goats, rabbits, hamsters, mice, cats, and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and / or turkeys. In some embodiments, the subject has been diagnosed with or may be diagnosed with a disease or condition. In some embodiments, the subject has not yet been diagnosed with a disease or condition.

[0093] lipids

[0094] In one aspect, the present disclosure provides novel compounds useful as lipids.

[0095] The lipids have at least one of the following features: a hydrophilic head group with a different pKa, a cationic amine, a monoamine, a diamine, a triamine, an oligoamine / polyamine, an imidazole, a pyridine, a guanidinium salt, and a hydrophobic tail. In some embodiments, the lipids are ionizable lipids. In some embodiments, the lipids are cationic lipids.

[0096] As used herein, the term "cationic lipid" includes lipids having an amino head and one or more aliphatic chains that can be protonated to form cationic lipids at physiological pH. In some embodiments, the cationic lipid is an amino lipid. Also included are lipids having one or more protonatable or deprotonatable groups, or zwitterionic lipids. In some embodiments, the lipids of the present disclosure have at least one protonatable group such that the lipid is positively charged at a first pH value equal to or lower than physiological pH (e.g., equal to or lower than pH 7.4) and is neutral at a second pH value (e.g., equal to or higher than physiological pH). In some embodiments, the lipids of the present disclosure have at least two or at least three protonatable groups. It should be understood that adding or removing protons according to pH is an equilibrium process, and reference to charged (e.g., protonated) or neutral lipids refers to the properties of the major species (e.g., more than 50%, 60%, 70%, 80%, 90%, 95% or 99%), and does not require that all lipids exist in a charged or neutral form.

[0097] In some embodiments, the pKa of the protonatable group of the cationic lipids of the present disclosure is in the range of about 4 to about 11. In some embodiments, when incorporated into lipid particles, the pKa of the lipid is about 4 to about 7, about 5 to about 7, or about 5.5 to about 6.8. In some embodiments, lipids with such pKa will be cationic at lower pH values, while the particles will be largely (but not completely) surface neutralized at physiological pH values ​​(e.g., pH 7.4). In some embodiments, at least some nucleic acids associated with the outer surface of particles comprising lipids with such pKa will lose their electrostatic interactions at physiological pH values ​​and be removed by simple dialysis; thereby greatly reducing the susceptibility of the particles to clearance. For example, the pKa of lipids within lipid particles can be measured using the method described by Cullis et al. (1986) Chem Phys Lipids (40, 127-144) using the fluorescent probe 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS).

[0098] In some embodiments, the lipids of the present disclosure are advantageously used in lipid nanoparticles. In some embodiments, the lipid nanoparticles are used to deliver therapeutic agents to cells in vivo. In some embodiments, the lipid nanoparticles are used to deliver therapeutic agents to tissues in vivo.

[0099] In another aspect, the lipid of the present disclosure is a compound having the following formula (I):

[0100]

[0101] or a pharmaceutically acceptable salt thereof, wherein

[0102] R a Selected from the group consisting of hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl and R 6 ;

[0103] R 1 for

[0104] R 2 for

[0105] R 3 for

[0106] R 4 for

[0107] R 5 If it exists,

[0108] R 6 If it exists,

[0109] Each W is independently selected from O, S or NR b , and each R b independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl;

[0110] Each Y is independently selected from O, S, NR c 、N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W), and each R c independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl;

[0111] Each Z is independently selected from C, S or S(O);

[0112] Each n is independently 0, 1, 2, 3, 4 or 5;

[0113] Each m is independently 0, 1, 2 or 3;

[0114] Each p is independently 1, 2, 3 or 4; and

[0115] R 1c 、R 2c 、R 3c and R 4c Each is independently selected from the group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl groups are optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, oxo, cyano, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl groups are optionally interrupted by one or more groups independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0116] In some embodiments, the lipids of the present disclosure are compounds having the following formula (A) or formula (B):

[0117]

[0118] In some embodiments, R 2 、R 3 and R 4 In some embodiments, R 2 、R 3 and R 4 At least three of them are the same.

[0119] In some embodiments, one or more of W is O.

[0120] In some embodiments, one or more of Y is O, NR c or N(R c )N(R c )Z(W).

[0121] In some embodiments, one or more of Z is C or S(O).

[0122] In some embodiments, R 2c 、R 3c and R 4c One or more of them is an alkyl group or an alkenyl group.

[0123] In some embodiments, R 2c 、R 3c and R 4c One or more of them is C 8-24 Alkyl or alkenyl.

[0124] In some embodiments, R 2c 、R 3c and R 4c One or more of them is C 10-24 Alkyl or alkenyl.

[0125] In some embodiments, R 2c 、R 3c and R 4c One or more of the is an alkenyl group containing one, two or three C=C double bonds.

[0126] In some embodiments, wherein R 2c 、R 3c and R 4c One or more of the is an alkenyl group containing one or more Z-olefins.

[0127] In some embodiments, wherein R 1c It is an alkyl group.

[0128] In some embodiments, R 1c C 1-12 alkyl.

[0129] In some embodiments, R 1c C 4-10 alkyl.

[0130] In some embodiments, R a C 1-6 Alkyl, optionally substituted with one or more groups independently selected from the group consisting of hydroxy, cycloalkyl, and heteroaryl.

[0131] In some embodiments, R a is methyl, ethyl, propyl, butyl or pentyl.

[0132] In some embodiments, One or more of are independently selected from the following group:

[0133] In some embodiments, R 1c 、R 2c 、R 3c and R 4c One or more of are independently selected from the following group:

[0134] In some embodiments, R 1c 、R 2c 、R 3c 、R 4c 、R5c and R 6c If present, each does not contain two heteroatoms directly bonded to each other.

[0135] In some embodiments, R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c If present, each contains -N(R c )-N(R c )-or-S(O)2-N(R c )-.

[0136] In some embodiments, the compound of formula (I) is a compound listed in Table 1.

[0137] For illustrative purposes, exemplary compounds of the present disclosure and their structure codes are listed in Table 1 below.

[0138] Table 1. Exemplary compounds

[0139]

[0140]

[0141]

[0142]

[0143] The compounds provided herein are described with reference to general formulae and specific compounds. In addition, the compounds of the present disclosure may exist in a variety of different forms or derivatives, including but not limited to stereoisomers, racemic mixtures, positional isomers, tautomers, salts, prodrugs, soft drugs, active metabolic derivatives (active metabolites), solvated forms, different crystalline forms or polymorphs, all of which are within the scope of the present disclosure.

[0144] The compounds of the present disclosure may contain one or more asymmetric centers and therefore may exist in various stereoisomeric forms, such as enantiomers and / or diastereomers. Therefore, the compounds of the present disclosure and their compositions may be in the form of single enantiomers, diastereomers, or geometric isomers, or may be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the present disclosure are enantiomeric compounds. In certain embodiments, a mixture of enantiomers or diastereomers is provided.

[0145] The term "enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another. The term "diastereomers" refers to a pair of optical isomers that are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities.

[0146] In addition, unless otherwise indicated, certain compounds as described herein may have one or more double bonds that are capable of existing as Z or E isomers. The present disclosure further encompasses compounds that exist as single isomers that are substantially free of other isomers, and alternatively, compounds that exist as mixtures of various isomers (e.g., racemic mixtures of enantiomers). In addition to the compounds described above per se, the present disclosure also encompasses compositions comprising one or more compounds.

[0147] As used herein, the term "isomer" includes any and all geometric isomers and stereoisomers. For example, "isomer" includes cis and trans isomers, E and Z isomers, R and S enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, all of which fall within the scope of the present invention. For example, in some embodiments, a stereoisomer can be provided substantially free of one or more corresponding stereoisomers and can also be referred to as "stereochemically enriched."

[0148] When a particular enantiomer is preferred, in some embodiments, that enantiomer can be provided substantially free of the opposite enantiomer, and can also be referred to as "optically enriched." As used herein, "optically enriched" means that the compound consists of a significantly greater proportion of one enantiomer. In certain embodiments, the compound consists of at least about 90% (by weight) of the preferred enantiomer. In other embodiments, the compound consists of at least about 95%, 98% or 99% (by weight) of the preferred enantiomer. The preferred enantiomer can be separated from the racemic mixture by any method known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts, or prepared by asymmetric synthesis. See, e.g., Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGraw-Hill, New York, 1962); Wilen, SH, Tables of Resolving Agents and Optical Resolutions, p. 268 (E.L. Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0149] The compounds of the present disclosure can also exist in different tautomeric forms, and all such forms are included in the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be converted to each other via a low energy barrier. The presence and concentration of isomeric forms depend on the environment in which the compound is located, and can be different due to, for example, whether the compound is solid or in an organic solution or aqueous solution. By way of example, proton tautomers (also referred to as prototransfer tautomers) include interconversions that occur via proton migration, such as keto-enol, amide-imidic acid, lactam-lactim, imine-enamine isomerization and cyclic forms, in which protons can occupy two or more positions of a heterocyclic ring system. Valence tautomers include interconversions that occur by reorganizing some of the bonding electrons in the bonding electrons. Tautomers can maintain equilibrium or be spatially locked into a form by appropriate substitution. Unless otherwise stated, compounds identified as a specific tautomeric form by name or structure in the present disclosure are intended to include other tautomeric forms.

[0150] The present disclosure is also intended to include all isotopes of atoms in the compounds. Isotopes of atoms include atoms having the same atomic number but different mass numbers. For example, unless otherwise specified, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, or iodine in the compounds of the present disclosure are also intended to include their isotopes, such as, but not limited to, 1 H. 2 H. 3 H. 11 C. 12 C. 13 C. 14 C. 14 N. 15 N. 16 O. 17 O. 18 O. 31 P. 32 P. 32 S. 33 S. 34 S. 36 S. 17 F. 18 F. 19 F. 35 Cl, 37 Cl, 79 Br, 81 Br, 124 I. 127 I and 131 1. In some embodiments, hydrogen includes protium, deuterium, and tritium. In some embodiments, carbon includes 12 C and 13 C.

[0151] Synthesis of compounds

[0152] The synthesis of the compounds provided herein (including pharmaceutically acceptable salts thereof) is described in the synthesis schemes in the examples. The compounds provided herein can be prepared using any known organic synthesis technique and can be synthesized according to any of the many possible synthetic routes, so these schemes are only illustrative and are not intended to limit other possible methods for preparing the compounds provided herein. In addition, the steps in the scheme are for better illustration and can be modified as appropriate. The embodiments of the compounds in the examples are synthesized for research purposes and for possible submission to regulatory agencies.

[0153] The reaction of preparing the disclosed compounds can be carried out in a suitable solvent, which can be easily selected by those skilled in the art of organic synthesis. Under the temperature of the reaction (for example, the temperature can be in the range between the freezing point of the solvent and the boiling point of the solvent), the suitable solvent can substantially not react with the starting material (reactant), intermediate or product. A given reaction can be carried out in a solvent or a mixture of more than one solvent. According to specific reactions steps, those skilled in the art can select a suitable solvent for a particular reaction step.

[0154] The preparation of compounds of the present disclosure may involve the protection and deprotection of various chemical groups. Those skilled in the art can easily determine whether protection and deprotection are needed, as well as the selection of suitable blocking groups. The chemical properties of blocking groups can be found in, for example, TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd edition, Wiley & Sons, Inc., New York (1999), which are incorporated herein by reference in their entirety.

[0155] The reaction can be monitored by any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or chromatography (such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS) or thin layer chromatography (TLC). Compounds can be purified by various methods by those skilled in the art, including high performance liquid chromatography (HPLC) ("Preparative LC-MS Purification: Improved Compound Specific Method Optimization", Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs, "J. Combi. Chem.," 2004, 6(6), 874-883, the entirety of which is incorporated herein by reference) and normal phase silica gel chromatography.

[0156] The structures of the compounds in the examples were characterized by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). The unit of NMR chemical shift (δ) is 10 -6 (ppm). 1 H-NMR spectra were recorded on a Bruker instrument (400 MHz or 500 MHz) in CDCl3, CD3OD or DMSO-d6 solutions (reported in ppm) using tetramethylsilane (TMS) as a reference standard (0.0 ppm).

[0157] Unless otherwise stated, the reactions of the present disclosure were typically performed under a positive pressure of nitrogen or argon or in anhydrous solvents using a drying tube, and the reaction flasks were typically fitted with rubber septa for the introduction of substrates and reagents via syringes. Glassware was oven-dried and / or heat-dried.

[0158] lipid nanoparticles

[0159] The present disclosure also provides lipid particles comprising one or more of the lipids described above. Lipid particles include but are not limited to lipid nanoparticles (LNPs), liposomes, lipid complexes, and lipid multimer complexes (LPPs). In some embodiments, the lipid particles are lipid nanoparticles. The present disclosure also provides a method for preparing lipid particles.

[0160] Lipid nanoparticles of the present disclosure can also comprise one or more extra lipids and / or other compositions (such as sterols). Other lipids can be comprised in lipid nanoparticles of the present disclosure, for various purposes, such as preventing lipid oxidation or attaching a part to the particle surface. Any lipid can be present in lipid nanoparticles of the present disclosure, including amphipathic lipids, neutral lipids, cationic lipids and anionic lipids, which can be used alone or in combination. The example of other lipid components that may exist is described below.

[0161] In some embodiments, the lipid nanoparticles comprise a lipid of the present disclosure (e.g., a compound of formula (I) or any compound in Table 1). In some embodiments, the lipid nanoparticles comprise two or more lipids of the present disclosure. In some embodiments, the mole fraction of the lipid of the present disclosure (e.g., a compound of formula (I) or any compound in Table 1) is about 5% to 75%, about 10% to 75%, about 10% to 70%, about 10% to 65%, about 15% to 65%, about 20% to 65%, about 25% to 65%, about 30% to 65%, about 35% to 65%, about 40% to 65%, about 45% to 65%, about 40% to 60%, about 40% to 55%, or about 40% to 50% of the total lipids present in the lipid nanoparticles. In some embodiments, the mole fraction of the lipid of the present disclosure (e.g., a compound of formula (I) or any compound in Table 1) is about 40% to 50% of the total lipids present in the lipid nanoparticles. In some embodiments, the mole fraction of the lipid of the present disclosure (e.g., a compound of Formula (I) or any compound in Table 1) is about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% of the total lipid present in the lipid nanoparticle.

[0162] In some embodiments, the lipid nanoparticles comprise neutral lipids. The term "neutral lipid" refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. In some embodiments, the neutral lipid is a phospholipid. Examples of phospholipids include, but are not limited to: 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-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2 -Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diondecanoyl-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-cholestyhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (Cl6Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diaramidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and any mixture thereof. In some embodiments, the lipid nanoparticles include a neutral lipid. In some embodiments, the lipid nanoparticles include two or more neutral lipids. In some embodiments, the neutral lipids are selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and any mixture thereof.In some embodiments, the mole fraction of the neutral lipid is about 1% to 40%. In some embodiments, the mole fraction of the neutral lipid is about 1% to 35%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, or about 5% to 10% of the total lipids present in the lipid nanoparticle. In some embodiments, the mole fraction of the neutral lipid is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, 25%, or 30% of the total lipids present in the lipid nanoparticle.

[0163] In some embodiments, the lipid nanoparticles include structural lipids. In some embodiments, the structural lipids are sterols, sterol derivatives, or any mixture thereof. Examples of sterols or sterol derivatives include, but are not limited to, cholesterol, coprosterol, sitosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structural lipids are cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof. In some embodiments, the lipid nanoparticles include cholesterol. In some embodiments, the mole fraction of the structural lipids is about 5% to 50% of the total lipids present in the lipid nanoparticles. In some embodiments, the mole fraction of the structural lipids is about 10% to 50%, about 15% to 50%, about 25% to 50%, or about 40% to 50% of the total lipids present in the lipid nanoparticles. In some embodiments, the mole fraction of the structural lipid is about 10% to 45%, about 15% to 45%, about 25% to 45%, about 30% to 45%, or about 35% to 45% of the total lipid present in the lipid nanoparticle.In some embodiments, the mole fraction of the structural lipid is about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 40.1%, 40.2%, 40.3%, 40.4%, 40.5%, 40.6%, 40.7%, 40.8%, 40.9%, 41%, 41.1%, 41.2%, 41.3%, 41.4%, 41.5%, 41.6%, 41.7%, 41.8%, 41.9%, 42%, 42.1%, 42.2%, 42.3%, 42.4%, 42.5%, 42.6%, 42.7%, 42.8%, 42.9%, 43.0%, 43.1%, 43.2%, 43.3%, 43.4%, 43.5%, 43.6%, 43.7%, 43.8%, 43.9%, 44.1%, 44.2%, 44.3%, 44.4%, 44.5%, 44.6%, 44.7%, 44.8%, 44.9%, 45. .5%, 42.6%, 42.7%, 42.8%, 42.9%, 43%, 43.1%, 43.2%, 43.3%, 43.4%, 43.5%, 43.6%, 43.7%, 43.8%, 43.9%, 44%, 44.1%, 44.2%, 44.3%, 44.4%, 44.5%, 44.6%, 44.7%, 44.8%, 44.9%, 45%, 45.1%, 45.2%, 45.3%, 45.4%, 45.5%, 45.6%, 45.7%, 45.8%, 45.9%, 46%, 46.1%, 46.2%, 46.3%, 46.4%, 46.5%, 46.6%, 4 6.7%, 46.8%, 46.9%, 47%, 47.1%, 47.2%, 47.3%, 47.4%, 47.5%, 47.6%, 47.7%, 47.8%, 47.9%, 48%, 48.1%, 48.2%, 48.3%, 48.4%, 48.5%, 48.6%, 48.7%, 48.8%, 48.9%, 49%, 49.1%, 49.2%, 49.3%, 49.4%, 49.5%, 49.6%, 49.7%, 49.8%, 49.9%, 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8% , 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9%, 54%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9% or 55%.

[0164] In some embodiments, the lipid nanoparticles include lipids selected to reduce lipid nanoparticle aggregation during lipid nanoparticle formation, and this aggregation may be due to the steric stabilization of the particles, and the lipids prevent charge-induced aggregation during formation. Examples of lipids that reduce particle aggregation during formation include, but are not limited to, polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gm1, and polyamide oligomers (PAO). Other compounds (such as PEG or Gm1) with no charge, hydrophilicity, and spatial barrier parts that prevent aggregation during formulation can also be coupled to lipids for use in the methods and compositions of the present disclosure. In some embodiments, the lipid that reduces particle aggregation during formation is a surfactant. In some embodiments, the surfactant is a PEG-modified lipid. Examples of PEG-modified lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some embodiments, the PEG-modified lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol (DSG-PEG), N-(methylpolyoxyethylenecarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)] (DOPE-PEG), or any mixture thereof. Typically, the mole fraction of the lipid component selected to reduce aggregation is about 1% to 15% of the total lipids present in the lipid nanoparticles. In some embodiments, the mole fraction of the lipid component selected to reduce aggregation is about 1% to 10%, about 1% to 7%, about 1% to 5%, or about 0.5% to 5% of the total lipids present in the lipid nanoparticles. In some embodiments, the mole fraction of the lipid component selected to reduce aggregation is about 1% to 5%, about 1% to 2.5%, or about 1.5% to 2% of the total lipid present in the lipid nanoparticle.In some embodiments, the mole fraction of the lipid component selected to reduce aggregation is about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9% or 4% of the total lipid present in the lipid nanoparticles.

[0165] As used herein, the term "PEG", whether used as part of another term or independently, refers to polyethylene glycol. The term "PEGm" or "PEG-m" (wherein m is an integer) refers to a polyethylene glycol molecule or portion with a molecular weight of m. For example, "PEG2000" or "PEG-2000" refers to a polyethylene glycol molecule or portion with a molecular weight of 2000. In some embodiments, PEG is PEG700, PEG800, PEG900, PEG1000, PEG1100, PEG1200, PEG1300, PEG1400, PEG1500, PEG1600, PEG1700, PEG1800, PEG1900, PEG2000, PEG2100, PEG2200, PEG2300, PEG2400, PEG2500, PEG2600, PEG2700, PEG2800, PEG2900, or PEG3000. In some embodiments, the PEG is PEG2000.

[0166] In some embodiments, the lipid nanoparticles of the present disclosure comprise, based on the total lipid present in the lipid nanoparticles, a mole fraction of about 10%-65% of a compound of formula (I); a mole fraction of about 5%-30% of a neutral lipid of the present disclosure; a mole fraction of about 15%-50% of a structural lipid of the present disclosure; and a mole fraction of about 0.5%-5% of a lipid component selected to reduce aggregation of the present disclosure.

[0167] In some embodiments, the lipid nanoparticles of the present disclosure comprise, based on the total lipid present in the lipid nanoparticles, a mole fraction of about 20%-65% of a compound of formula (I); a mole fraction of about 5%-25% of a neutral lipid of the present disclosure; a mole fraction of about 25%-50% of a structural lipid of the present disclosure; and a mole fraction of about 1%-5% of a lipid component selected to reduce aggregation of the present disclosure.

[0168] In some embodiments, the lipid nanoparticles of the present disclosure comprise, based on the total lipid present in the lipid nanoparticles, a mole fraction of about 40%-65% of a compound of formula (I); a mole fraction of about 5%-15% of a neutral lipid of the present disclosure; a mole fraction of about 25%-50% of a structural lipid of the present disclosure; and a mole fraction of about 1%-2.5% of a lipid component selected to reduce aggregation of the present disclosure.

[0169] In some embodiments, the lipid nanoparticles of the present disclosure comprise, based on the total lipid present in the lipid nanoparticles, a mole fraction of about 40%-50% of a compound of formula (I); a mole fraction of about 5%-15% of a neutral lipid of the present disclosure; a mole fraction of about 40%-50% of a structural lipid of the present disclosure; and a mole fraction of about 1.5%-2% of a lipid component selected to reduce aggregation of the present disclosure.

[0170] In some embodiments, the lipid nanoparticles of the present disclosure comprise, based on the total lipid present in the lipid nanoparticle:

[0171] a compound of formula (I) having a mole fraction of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65%;

[0172] a neutral lipid of the present disclosure having a mole fraction of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, 25%, or 30%;

[0173] The structured lipids disclosed herein have a mole fraction of about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 40.1%, 40.2%, 40.3%, 40.4%, 40.5%, 40.6%, 40.7%, 40.8%, 40.9%, 41%, 41.1%, 41.2%, 41.3%, 41.4%, 41.5%, 41.6%, 41.7%, 41.8%, 41.9%, 42%, 42.1%, 42.2%, 42.3%, 42.4%, 42.5%, 42.6%, 42.7%, 42.8%, 42.9%, 43%, 43.1%, 43.2%, 43.3%, 43.4%, 43.5%, 43.6%, 43.7%, 43.8%, 43.9%, 44%, 44.1%, 44.2%, 44.3%, 44.4%, 44.5%, 44.6%, 44.7%, 44.8%, 44.9%, 45%, 45.1%, 45.2%, 45.3%, 45.4%, 45.5%, 45.6%, 45.7%, 45.8%, 45.9%, 46%, 46.1%, 46.2%, 46.3%, 46.4%, 46.5%, 46.6%, 46.7%, 46.8%, 46.9%, 47%, 47.1%, 47.2%, 47.3%, 47.4%, 47.5%, 47.6%, 47.7%, 47.8%, 47.9%, 48%, 48.1%, 48.2%, 48.3%, 48.4%, 48.5%, 48.6%, 48.7%, 48.8%, 48.9%, 49%, 49.1%, 49.2%, 49.3%, 49.4%, 49.5%, 49.6%, 49.7%, 49.8%, 49.9%, 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9%, 54%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9% or 55%; and

[0174] The lipid component selected for reducing aggregation in the present disclosure has a mole fraction of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9% or 4%.

[0175] In some embodiments, the lipid nanoparticles of the present disclosure comprise the components in the mole fractions listed in the table below.

[0176]

[0177]

[0178] Lipid nanoparticles can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscope (TEM) or scanning electron microscope (SEM)) can be used to check the morphology and particle size distribution of lipid nanoparticle compositions. Dynamic light scattering (DLS) or potentiometric methods (e.g., potentiometric titration) can be used to measure zeta potential. DLS can also be used to measure particle diameter. Instruments such as Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, England) can also be used to measure the various characteristics of lipid nanoparticles, such as particle diameter, polydispersity index, and zeta potential. The error in DLS measurements may depend on a variety of factors, such as scattering angle and multiple scattering. The typical error in DLS measurements is 5%.

[0179] In some embodiments, the lipid nanoparticles can be from about 40nm to about 150nm, such as about 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm or 150nm. In some embodiments, the average particle size of the lipid nanoparticles can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average particle size of the lipid nanoparticles can be from about 70 nm to about 100 nm. In some embodiments, the average particle size of the lipid nanoparticles can be about 80 nm. In some embodiments, the average particle size of the lipid nanoparticles can be about 100 nm.

[0180] In some embodiments, the lipid nanoparticles may be relatively uniform. A polydispersity index (PDI) can be used to indicate the uniformity of the lipid nanoparticles, for example, the particle size distribution of the lipid nanoparticles. A smaller PDI (e.g., less than 0.3) typically indicates a narrower particle size distribution. In some embodiments, the PDI of lipid nanoparticles of the present disclosure can be from about 0 to about 0.30, such as 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, 0.25, 0.26, 0.27, 0.28, 0.29 or 0.30. In some embodiments, the PDI of the lipid nanoparticles of the present disclosure may be from about 0.05 to about 0.20.

[0181] The zeta potential of lipid nanoparticles can be used to indicate electrokinetic potential. For example, the zeta potential can describe the surface charge of lipid nanoparticles. It is generally desirable to have lipid nanoparticles with relatively low charge (positive or negative charge) because more charged materials may interact adversely with cells, tissues and other elements in the body. In some embodiments, the zeta potential of lipid nanoparticles of the present disclosure can be from about -10mV to about +25mV, from about -10mV to about +20mV, from about -10mV to about +15mV, from about -10mV to about +10mV, from about -10mV to about +5mV, from about -10mV to about 0mV, from about -10mV to about -5mV, from about -5mV to about +25mV, from about -5mV to about +20mV, from about -5mV to about + From about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +25 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +25 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0182] Lipid nanoparticle compositions

[0183] On the other hand, the present disclosure also provides a kind of lipid nanoparticle composition, it refers to the composition comprising one or more lipid particles and target polynucleotides as discussed above, and the target polynucleotides include nucleic acids encoding respiratory syncytial virus antigenic polypeptides or their variants. In some embodiments, the respiratory syncytial virus antigenic polypeptides are respiratory syncytial virus fusion proteins ("RSV-F proteins"). In some embodiments, the respiratory syncytial virus antigenic polypeptides are respiratory syncytial virus attachment proteins ("RSV-G proteins"). In some embodiments, the RSV-F protein is RSV-F protein before fusion. In some embodiments, the RSV-F protein is RSV-F protein after fusion.

[0184] As used herein, the term "polynucleotide" refers to a polymer of nucleotides or nucleoside monomers consisting of natural bases, sugars, and inter-sugar (skeleton) bonds. The term "polynucleotide" also includes polymers comprising non-natural monomers or similar functional parts thereof. Such modified or substituted polynucleotides are generally more preferred than natural forms because they have properties such as enhanced cellular uptake and improved stability in the presence of nucleases. As used herein, the term "nucleotide" comprises sugar (deoxyribose (DNA) or ribose (RNA)), bases, and phosphate groups. Nucleotides are linked together by phosphate groups. "Base" includes purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include but are not limited to modifications introducing new reactive groups (such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides). Typically, the polynucleotides comprise more than 50 nucleotides or nucleoside monomers.

[0185] The polynucleotides present in the compositions of the present disclosure (e.g., lipid nanoparticle compositions comprising one or more target polynucleotides) include any known nucleic acid form. The polynucleotides used herein include, but are not limited to, single-stranded DNA or RNA, or double-stranded DNA or RNA, and DNA-RNA hybrids. Examples of double-stranded DNA include, but are not limited to, structural genes, genes comprising control regions and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include, but are not limited to, siRNA and other RNA interference agents. Single-stranded nucleic acids include, but are not limited to, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, microRNAs, and triplex-forming oligonucleotides. The polynucleotides used herein also include nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, or non-naturally occurring and have binding properties similar to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise limited, the term encompasses nucleic acids containing known natural nucleotide analogs that have binding properties similar to a reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the sequence explicitly indicated. The nucleic acids present in the compositions of the present disclosure may comprise one or more modifications.

[0186] Nucleic acids of the present disclosure can have various lengths, generally depending on the concrete form of the nucleic acid. In some embodiments, the length of the nucleic acid is 10-5000 nucleotides. In some embodiments, the length of the nucleic acid is about 4000 nucleotides. In some embodiments, the length of the nucleic acid is about 3000 nucleotides. In some embodiments, the length of the nucleic acid is about 2500 nucleotides. In some embodiments, the nucleic acid is about 2400, 2300, 2200, 2100, 2000, 1900, 1850, 1800, 1750, 1700, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, or 50 in length.

[0187] As used herein, the term "variant" of a polypeptide refers to a molecule whose amino acid sequence is different from a native or reference sequence. Compared to a native or reference sequence, the amino acid sequence variant may have a substitution, mutation, deletion, and / or insertion at certain positions within the amino acid sequence. In some embodiments, the variant has at least 50%, or at least 60%, or at least 70% sequence identity to the native or reference sequence. In some embodiments, the variant has at least 80% identity, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98% sequence identity to the native or reference sequence.

[0188] In some embodiments, the target polynucleotide is encapsulated in the interior of a lipid nanoparticle. In some embodiments, the target polynucleotide is present in one or more lipid layers of a lipid nanoparticle. In some embodiments, the target polynucleotide is bound to the exterior or interior lipid surface of a lipid nanoparticle.

[0189] As used herein, "encapsulation," "encapsulated," "encapsulating," and "loading," as well as "association," can refer to complete, substantial, or partial enclosure, restriction, surrounding, or wrapping. As used herein, "encapsulation" or "association" can refer to the process of confining a single nucleic acid within a lipid nanoparticle and / or establishing a physicochemical relationship between a single nucleic acid and a lipid nanoparticle.

[0190] The "encapsulation efficiency" of a target polynucleotide refers to the amount of target polynucleotide encapsulated or otherwise associated with the lipid nanoparticles after preparation, relative to the initial amount provided. The encapsulation efficiency is preferably high (e.g., greater than 80%, greater than 85%, greater than 90%, or greater than 95%). The encapsulation efficiency can be measured, for example, by comparing the amount of target polynucleotide in a solution containing the lipid nanoparticles before and after disintegration of the lipid nanoparticles with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free target polynucleotide (e.g., RNA) in the solution. In some embodiments, the encapsulation efficiency of the target polynucleotide can be at least 50%, e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0191] In the lipid nanoparticle, the amount of target polynucleotide can depend on particle diameter, composition, required target and / or application or other characteristics of lipid nanoparticle, and the characteristic of target polynucleotide.For example, the amount of available RNA can depend on size, sequence and other characteristics of RNA in the lipid nanoparticle.The relative amount of target polynucleotide and other compositions (for example, lipid) in the lipid nanoparticle also can change. In some embodiments, the mass ratio of lipids (e.g., cationic lipids, neutral lipids, structural lipids (e.g., sterols), and lipids selected to reduce aggregation (such as surfactants)) in the lipid nanoparticle composition to the target polynucleotide (e.g., RNA) can be about 5:1 to about 60:1, such as about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the mass ratio of lipids to target polynucleotides can be about 10:1 to about 50:1. In some embodiments, the mass ratio of lipids to target polynucleotides is about 40:1. In some embodiments, the mass ratio of lipids to target polynucleotides is about 20:1. In some embodiments, the mass ratio of lipid to target polynucleotide is calculated by dividing the total mass of cationic lipid, neutral lipid, structural lipid (such as sterol) and surfactant without solvent by the mass of anhydrous target polynucleotide (e.g., RNA). For example, the amount of target polynucleotide in the lipid nanoparticle composition can be measured using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0192] In some embodiments, the target polynucleotide is RNA. In some embodiments, the target polynucleotide is messenger RNA (mRNA). mRNA can be natural, or can include modified and / or non-naturally occurring components, such as one or more core bases, nucleosides, nucleotides or linkers. In some embodiments, the target polynucleotide is mRNA, which includes encoding RSV antigenic polypeptides or variants thereof. In some embodiments, the RSV antigenic polypeptide is RSV-F protein. In some embodiments, the RSV-F protein is RSV-F protein before fusion. In some embodiments, the RSV-F protein is RSV-F protein after fusion.

[0193] "Messenger RNA" (mRNA) refers to any polynucleotide that encodes at least one polypeptide (e.g., a naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide. Translation of mRNA (e.g., in vivo translation of mRNA in mammalian cells) can produce a polypeptide. It will be understood by those skilled in the art that, unless otherwise indicated, the polynucleotide sequences set forth in this application will be marked with "T" in the representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" will be replaced by a "U". Therefore, any RNA polynucleotide encoded by a DNA identified by a particular sequence identification number can also include a corresponding RNA (e.g., mRNA) sequence encoded by the DNA, wherein each "T" in the DNA sequence is replaced by a "U".

[0194] The mRNA of the present disclosure can be transcribed in vitro from template DNA. In vitro transcription of RNA is known in the art, and those skilled in the art can easily and certainly obtain mRNA sequences based on the template DNA sequence provided. In some embodiments, RNA transcripts are synthesized by an in vitro enzymatic transcription reaction encoded by a non-amplified linear DNA template for the target gene, which utilizes T7 phage, RNA polymerase, and nucleotide triphosphates with desired chemical properties.

[0195] The mRNA molecule may comprise a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. In some embodiments, the essential components of the mRNA molecule include at least a coding region, a 5'-untranslated region (5'-UTR), a 3'UTR, and a polyA sequence. In some embodiments, the 5'-untranslated region (UTR) comprises a nucleic acid selected from the group consisting of SEQ ID NOs: 34, 37, and 70-71. In some embodiments, the 3'-untranslated region (UTR) comprises a nucleic acid selected from the group consisting of SEQ ID NOs: 35 and 72-74. In some embodiments, the poly-A region is 50-120 nucleotides in length. For example, the polyA region may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 adenosine monophosphates. In some embodiments, the mRNA of the present disclosure further comprises a 5'-terminal cap. In some embodiments, the 5'-terminal cap may be selected from the group consisting of capOGG, cap1GG, and cap1AG. In some embodiments, the mRNA of the present disclosure includes one or more modified nucleotides selected from the group consisting of pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methyluridine (m5U), 2-thiouridine (s2U), 5-methylcytidine (m5C), and 5-methoxyuridine (5moU).

[0196] In some embodiments, the target polynucleotide encodes the respiratory syncytial virus antigenic polypeptide, comprising: a) an amino acid sequence having amino acid residues 26-574 of SEQ ID NO: 38, or b) a mutant of (a) having at least one amino acid substitution selected from the group consisting of: NO:38's S55C, P102A, T103C, R106K, R109Q, R133Q, K134Q, R135Q, R136Q, L142Q, I148C, A149C, S155C, L188C, S190F, V207L, K209P, Q210P, Q210C, S211P, C212P, S290C, I379V, M447V, Y458C, L481P, V482P, Q501G and L512K, wherein the mutant has respiratory syncytial virus antigen activity.

[0197] In some embodiments, the target polynucleotide further comprises a nucleic acid encoding a signal peptide, which is connected to the respiratory syncytial virus antigenic polypeptide. In some embodiments, the target polynucleotide further comprises a termination sequence and a linker coding sequence. The term "signal peptide" as used herein refers to a peptide comprising 15-60 amino acids at the N-terminus of a protein, which is generally required for transmembrane transport in the secretory pathway and therefore generally controls the process by which most proteins in eukaryotes and prokaryotes enter the secretory pathway. Signal peptides typically include three regions: an N-terminal region of varying lengths, which typically comprises positively charged amino acids; a hydrophobic region; and a short carboxyl-terminal peptide region.

[0198] In some embodiments, the signal peptide used herein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 185, 187, 189, 191, 193, 195, 197, 199, and 201.

[0199] Table 2. Signal peptides

[0200]

[0201]

[0202] In some embodiments, the signal peptide is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 184, 186, 188, 190, 192, 194, 196, 198 and 200.

[0203] Table 3. Signal peptide coding sequence

[0204]

[0205] In some embodiments, the target polynucleotide (eg, mRNA) encodes the respiratory syncytial virus antigenic polypeptide comprising SEQ ID NO: 38 or SEQ ID NO: NO:39-69, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 179, 181, 183, and 287-370, or the amino acid sequence of SEQ ID NO:38 or SEQ ID NO:39-69, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 11 5, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 179, 181, 183 and 287-370 have amino acid sequences that are at least 95% (e.g., 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%) identical.

[0206] In some embodiments, the target polynucleotide (e.g., mRNA) comprises a nucleic acid sequence of SEQ ID NO: 1-33, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 182, 174, 176, 178, 180, 182, and 202-286, or a nucleic acid sequence of SEQ ID NO: 1-33, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, NO:1-33, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122 ,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,1 66, 168, 170, 182, 174, 176, 178, 180, 182 and 202-286 have a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%) identical, said sequence encoding a polypeptide having respiratory syncytial virus antigenic activity.

[0207] In some embodiments, the target polynucleotide (e.g., mRNA) comprises a nucleic acid sequence of SEQ ID NO: 2, 4, 14, 16, 18, 24, 26, 30, 31, 32, or 33, or a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 2, 4, 14, 16, 18, 24, 26, 30, 31, 32, or 33, which encodes a polypeptide having respiratory syncytial virus antigenic activity.

[0208] In some embodiments, the target polynucleotide (e.g., mRNA) comprises a nucleic acid sequence of SEQ ID NO: 2, 4, 14, 16, 18, 24, 26, 30, 31, 32, 33, 203, 205, 215, 217, 219, 225, 227, 231, 232, 233, or 234, or a nucleic acid sequence identical to SEQ ID NO: NO:2, 4, 14, 16, 18, 24, 26, 30, 31, 32, 33, 203, 205, 215, 217, 219, 225, 227, 231, 232, 233 or 234 has a nucleic acid sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%) identical, wherein the nucleic acid sequence encodes a polypeptide having respiratory syncytial virus antigenic activity.

[0209] In some embodiments, the target polynucleotide (e.g., mRNA) comprises SEQ ID NO:

[0210] (i) a 5' untranslated region (UTR) comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 34, 37, and 70-71; and / or

[0211] (ii) a 3' untranslated region (UTR) comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 35 and 72-74; and / or

[0212] (iii) a poly-A region having a length of 50-120 nucleotides, or a poly-A region comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 36 and 75-79.

[0213] Table 4. UTR and PolyA

[0214]

[0215]

[0216] Respiratory syncytial virus polypeptide mutant

[0217] In another aspect, the present disclosure provides respiratory syncytial virus polypeptide mutants for inducing antigen-specific immune responses.

[0218] In some embodiments, the respiratory syncytial virus polypeptide mutant comprises an amino acid sequence having amino acid residues 26-574 of SEQ ID NO: 38, wherein the amino acid sequence having amino acid residues 26-574 of SEQ ID NO: 38 has at least one amino acid substitution, wherein the at least one amino acid substitution is selected from the group consisting of: NO:38's S55C, P102A, T103C, R106K, R109Q, R133Q, K134Q, R135Q, R136Q, L142Q, I148C, A149C, S155C, L188C, S190F, V207L, K209P, Q210P, Q210C, S211P, C212P, S290C, I379V, M447V, Y458C, L481P, V482P, Q501G and L512K, wherein the mutant has respiratory syncytial virus antigen activity.

[0219] In some embodiments, the respiratory syncytial virus polypeptide mutant comprises at least two amino acid substitutions of an amino acid sequence having amino acid residues 26-574 of SEQ ID NO:38, wherein at least one amino acid substitution is selected from the group consisting of S55C, P102A, T103C, R106K, R109Q, R133Q, K134Q, R135Q, R136Q, L142Q, I148C, A149C, S155C, L188C, S190F, V207L, K209P, Q210P, Q210C, S211P, C212P, S290C, I379V, M447V, Y458C, L481P, V482P, Q501G, and L512K according to SEQ ID NO:38, wherein the mutant has respiratory syncytial virus antigenic activity.

[0220] In some embodiments, the respiratory syncytial virus polypeptide mutant comprises an amino acid sequence having amino acid residues 26-574 of SEQ ID NO: 38, wherein the amino acid sequence comprises one or more of the following amino acid residue substitutions and / or one or more amino acid residue deletions:

[0221] a) a 27-amino acid residue stretch from E110 to R136 is substituted by -GS- residues;

[0222] b) a 45-amino acid residue stretch from E110 to V154 substituted by -GS- residues;

[0223] c) a 50 amino acid residue stretch from I525 to N574 substituted by residues GYIPEAPRDGQAYVRKDGEWVLLSTFL;

[0224] d) a deletion of an 18-amino acid residue segment from F137 to V154;

[0225] e) a deletion of a 24-amino acid residue segment from K551 to N574;

[0226] f) A fragment of 50 amino acid residues from I525 to N574 is deleted.

[0227] In some embodiments, the respiratory syncytial virus polypeptide mutant comprises at least one amino acid residue substitution of the amino acid sequence of amino acid residues 26-574 in SEQ ID NO: 38, including a combination of amino acid substitutions selected from the group consisting of:

[0228] i) K209P and Q210P;

[0229] ii) Q210P and S211P;

[0230] iii) S211P and C212P;

[0231] iv) L481P;

[0232] v) L481P and V482P;

[0233] vi) K209P, Q210P, L481P and V482P;

[0234] vii) Q210P, S211P, L481P and V482P;

[0235] viii) S211P, C212P, L481P and V482P;

[0236] ix) K209P, Q210P and L481P;

[0237] x) Q210P, S211P and L481P;

[0238] xi) S211P, C212P and L481P;

[0239] xii) S55C, P102A, T103C, I148C, L188C, V207L, Q210P, I379V, and M447V;

[0240] xiii) S55C, P102A, T103C, I148C, L188C, Q210P, I379V, and M447V;

[0241] xiv) P102A, T103C, I148C, V207L, Q210P, I379V, and M447V;

[0242] xv) S55C, P102A, L188C, V207L ​​Q210P, I379V, and M447V;

[0243] xvi) P102A, T103C, I148C, Q210P, I379V, and M447V;

[0244] xvii) S55C, P102A, L188C, Q210P, I379V, and M447V;

[0245] xviii) P102A, V207L, Q210P, I379V, and M447V;

[0246] xix) V207L ​​and Q210P;

[0247] xx) S55C, P102A, T103C, I148C, L188C, V207L, Q210C, S213C, I379V, and M447V;

[0248] xxi) S55C, P102A, T103C, I148C, L188C, Q210C, S213C, I379V, and M447V;

[0249] xxii) P102A, T103C, I148C, V207L, Q210C, S213C, I379V, and M447V;

[0250] xxiii) S55C, P102A, L188C, V207L, Q210C, S213C, I379V, and M447V;

[0251] xxiv) P102A, T103C, I148C, Q210C, S213C, I379V, and M447V;

[0252] xxv) S55C, P102A, L188C, Q210C, S213C, I379V, and M447V;

[0253] xxvi) P102A, V207L, Q210C, S213C, I379V, and M447V;

[0254] xxvii) V207L, Q210C, and S213C;

[0255] xxviii) S55C, P102A, L188C, I379V, and M447V;

[0256] xxix) P102A, T103C, I148C, I379V, and M447V;

[0257] xxx)P102A, I379V, and M447V

[0258] xxxi) P102A and M447V;

[0259] xxxii) E30V, P102A, I379V, and M447V;

[0260] xxxiii) T54H, P102A, I379V, and M447V;

[0261] xxxiv) N88S, P102A, I379V, and M447V;

[0262] xxxv) P102A, T103A, I379V, and M447V;

[0263] xxxvi) P102A, A122T, I379V, and M447V;

[0264] xxxvii) P102A, K124N, I379V, and M447V;

[0265] xxxviii) P102A, T125N, I379V, and M447V;

[0266] xxxix) P102A, R136K, I379V, and M447V;

[0267] xxxx)P102A, V152I, I379V, and M447V;

[0268] xxxxi) P102A, S190I, I379V, and M447V;

[0269] xxxxii) P102A, N227S, I379V, and M447V;

[0270] xxxxiii) P102A, V296I, I379V, and M447V;

[0271] xxxxiv) P102A, Q354L, I379V, and M447V;

[0272] xxxxv)P102A, L373R, I379V, and M447V;

[0273] xxxxvi) P102A, I379V, M447V, and D486S;

[0274] xxxxvii) P102A, I379V, M447V, and S540L;

[0275] xxxxviii) P102A, I379V, M447V, and L547F;

[0276] xxxxix) S155C, P102A, S290C, I379V, and M447V;

[0277] xxxxx) P102A, A149C, I379V, M447V, and Y458C;

[0278] xxxxxi) a 27 amino acid residue stretch from E110 to R136 is replaced by residues -GS-, and a 50 amino acid residue stretch from I525 to N574 is replaced by residues GYIPEAPRDGQAYVRKDGEWVLLSTFL;

[0279] xxxxxii) a 27 amino acid residue stretch from E110 to R136 is substituted with residues -GS-, and a 24 amino acid residue stretch from K551 to N574 is deleted;

[0280] xxxxxiii) an 18 amino acid residue stretch from F137 to V154 is deleted, and a 50 amino acid residue stretch from I525 to N574 is replaced by residues GYIPEAPRDGQAYVRKDGEWVLLSTFL;

[0281] xxxxxiv) deletion of an 18 amino acid residue segment from F137 to V154, and deletion of a 50 amino acid residue segment from I525 to N574;

[0282] xxxxxv) deletion of an 18 amino acid residue segment from F137 to V154;

[0283] xxxxxvi) a 45 amino acid residue stretch from E110 to V154 is substituted by residues -GS-, and a 50 amino acid residue stretch from I525 to N574 is substituted by residues GYIPEAPRDGQAYVRKDGEWVLLSTFL;

[0284] xxxxxvii) a 45 amino acid residue stretch from E110 to V154 is substituted with residues -GS-, and a 24 amino acid residue stretch from K551 to N574 is deleted; and

[0285] xxxxxviii) A stretch of 45 amino acid residues from E110 to V154 is substituted by the residues -GS-.

[0286] In some embodiments, the respiratory syncytial virus polypeptide mutant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 287-370.

[0287] Also provided herein are respiratory syncytial virus polypeptides comprising a suitable signal peptide, as well as respiratory syncytial virus polypeptide mutants. The signal peptide can be fused to the N-terminus or C-terminus of the respiratory syncytial virus polypeptide mutant. In some embodiments, the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 185, 187, 189, 191, 193, 195, 197, 199, and 201. In some embodiments, the respiratory syncytial virus polypeptide mutant comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39-69, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 179, 181, 183.

[0288] Nucleic Acids

[0289] In another aspect, the present disclosure provides nucleic acids for inducing an antigen-specific immune response. In some embodiments, the nucleic acids of the present disclosure are isolated nucleic acid sequences.

[0290] In another aspect, the present disclosure provides an isolated nucleic acid sequence encoding a respiratory syncytial virus polypeptide, the isolated nucleic acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-69, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 179, 181, 183, and 287-370.

[0291] In some embodiments, the isolated nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 202-286. In some embodiments, the isolated nucleic acid sequence comprises a nucleic acid sequence that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 202-286.

[0292] In some embodiments, the isolated nucleic acid further comprises a nucleic acid encoding a signal peptide linked to a respiratory syncytial virus polypeptide. In some embodiments, the isolated nucleic acid further comprises a termination sequence and a linker encoding sequence. In some embodiments, the nucleic acid sequence encoding the signal peptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 184, 186, 188, 190, 192, 194, 196, 198, and 200. In some embodiments, the isolated nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-33, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 182, 174, 176, 178, 180, and 182.

[0293] sequence

[0294] Table 5. Nucleic acid sequence and amino acid sequence of RSV

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391] Table 6. Nucleic acid and amino acid sequences of RSV containing the underlined signal peptide

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496] Pharmaceutical composition

[0497] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the lipid nanoparticle composition provided herein and a pharmaceutically acceptable excipient.

[0498] The pharmaceutically acceptable excipients are conventional pharmaceutical excipients in the art, which can be prepared in a manner known in the pharmaceutical field. Some examples of materials that can be used as pharmaceutically acceptable excipients or carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and horse potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter or suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil ; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) alcohols such as ethanol and propanol; (20) phosphate buffer; and (21) other non-toxic compatible substances used in pharmaceutical formulations, such as acetone.

[0499] The pharmaceutical composition may contain pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, toxicity regulators, etc., as needed to approximate physiological conditions, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0500] The form of the pharmaceutical composition depends on many criteria including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0501] The pharmaceutical composition can be formulated for oral, nasal, rectal, transdermal, intravenous, intradermal, intramuscular, intranasal and / or subcutaneous administration. Depending on the desired route of administration, the pharmaceutical composition can be formulated in the form of tablets, capsules, pills, dragees, powders, granules, sachets, cachets, lozenges, suspensions, emulsions, solutions, syrups, aerosols (in the form of solid or liquid media), sprays, ointments, pastes, creams, lotions, gels, patches, inhalants or suppositories.

[0502] The pharmaceutical composition can be formulated using flow processes known in the art to provide an active ingredient that is quick, continuous or delayed in release after being administered to the patient. In some embodiments, the pharmaceutical composition is formulated into a sustained release form. In some embodiments, the time extended can be about 1 hour to 24 hours, 2 hours to 12 hours, 3 hours to 8 hours, 4 hours to 6 hours, 1 to 2 days or longer. In certain embodiments, the time extended is at least about 4 hours, at least about 8 hours, at least about 12 hours or at least about 24 hours. The pharmaceutical composition can be formulated into the form of a tablet. For example, not only can the dissolution of the activating agent in the gastrointestinal fluid and subsequent diffusion from the tablet or pill be independent of the pH value, the release rate of the activating agent is controlled, the release rate of the activating agent may also be subject to the influence of the physical process of tablet disintegration and dissolution. In some embodiments, polymeric materials such as those disclosed in "Medical Applications of Controlled Release", Langerh and Wise (eds.), CRC Press, Boca Raton, FL (1974); "Controlled Drug Bioavailability", in "Drug Product Design and Performance", Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, "Journal of Polymer Science: Reviews of Polymer Chemistry", Journal of Polymer Science: Reviews of Polymer Chemistry, 1992; and Ranger and Peppas, 1993. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105. The above references are incorporated herein by reference in their entirety.

[0503] In certain embodiments, the pharmaceutical compositions of the present disclosure may be administered in an amount sufficient to deliver from about 0.0001 mg / kg to about 10 mg / kg in a given dose (e.g., from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg). , from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 2.5 mg / kg, from about 0.001 mg / kg to about 2.5 mg / kg g, from about 0.005 mg / kg to about 2.5 mg / kg, from about 0.01 mg / kg to about 2.5 mg / kg, from about 0.05 mg / kg to about 2.5 mg / kg, from about 0.1 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about 2.5 mg / kg, from about 2 mg / kg to about 2.5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.05 mg

[0015] In some embodiments, the present invention provides a dosage level of the therapeutic agent of from about 0.1 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.25 mg / kg, from about 0.001 mg / kg to about 0.25 mg / kg, from about 0.005 mg / kg to about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg, or from about 0.1 mg / kg to about 0.25 mg / kg), wherein a dose of 1 mg / kg provides 1 mg of the therapeutic and / or prophylactic agent per 1 kg of subject body weight.

[0504] In certain embodiments, the pharmaceutical composition can be formulated into a unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for human subjects and other mammals, each unit containing a predetermined amount of active substance, which is calculated to produce the desired therapeutic effect in combination with a suitable pharmaceutical carrier. In some embodiments, the dosage of the therapeutic agent that can be applied is about 0.001 mg / kg to about 10 mg / kg. In other embodiments, the dosage of the therapeutic agent that can be applied is about 0.005 mg / kg to about 2.5 mg / kg. In certain embodiments, the dosage of the therapeutic agent that can be applied is about 0.1 mg / kg to about 1 mg / kg. In other embodiments, the dosage of the therapeutic agent that can be applied is about 0.05 mg / kg to about 0.25 mg / kg. The dosage can be applied once or multiple times per day with the same or different amounts to obtain the desired level of treatment, diagnosis, prevention or imaging effect. The desired dosage can be delivered, for example, three times a day, twice a day, once a day, every other day, every three days, every week, every two weeks, every three weeks or every four weeks.

[0505] Respiratory syncytial virus vaccine

[0506] In another aspect, the present disclosure provides a respiratory syncytial virus vaccine ("RSV vaccine") comprising the lipid nanoparticle composition of the present disclosure, the respiratory syncytial virus polypeptide mutant of the present disclosure, or the isolated nucleic acid sequence of the present disclosure.

[0507] RSV vaccine of the present disclosure can be used as a therapeutic agent or prophylactic. It can be used for preventing and / or treating infectious diseases. In some embodiments, RSV vaccine of the present disclosure is used to provide preventive protection for RSV. After administering RSV vaccine of the present disclosure, preventive protection for RSV can be achieved. The vaccine can be administered once, twice, three times, four times or more times, but administering a vaccine may be sufficient (optionally followed by a single booster). Although less than ideal, it is possible to administer the vaccine to infected individuals to achieve therapeutic response. Dosage may need to be adjusted accordingly.

[0508] Methods for inducing antigen-specific immune responses

[0509] In another aspect, the present disclosure provides a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject an effective amount of a lipid nanoparticle composition of the present disclosure, an effective amount of an RSV polypeptide or variant thereof of the present disclosure, an effective amount of an isolated nucleic acid sequence of the present disclosure, or a respiratory syncytial virus vaccine of the present disclosure. The method of the present disclosure elicits an immune response against RSV infection in the subject, wherein the subject's anti-antigenic polypeptide antibody titer increases.

[0510] In another aspect, the present disclosure provides uses of the lipid nanoparticle compositions of the present disclosure, the RSV polypeptides or variants thereof of the present disclosure, or the isolated nucleic acid sequences of the present disclosure, or for producing a vaccine.

[0511] In some embodiments, antigen-specific immune response includes T cell response or B cell response or both.In some embodiments, antigen-specific immune response includes the following situation: compared with the subject that is immunized or not immunized with a control (for example, a control vaccine), the titer of the anti-RSV antibodies (for example, anti-RSV-F antibodies) produced by the subject that is immunized with the RSV vaccine of the present disclosure increases.In some embodiments, compared with a control group, the anti-RSV antibody titer produced by the subject increases by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10 times.

[0512] In some embodiments, the method for inducing antigen-specific immune response relates to the respiratory syncytial virus vaccine of single administration. In some embodiments, the method for inducing antigen-specific immune response also includes: administering the respiratory syncytial virus vaccine of booster dose. Booster vaccine used herein can comprise any RSV vaccine of the present disclosure, and can be identical with the RSV vaccine initially administered. In some embodiments, the same RSV vaccine is administered annually in each RSV season.

[0513] Example

[0514] The following examples are included for illustrative purposes. However, it should be understood that these examples do not limit the present disclosure and are only intended to illustrate methods for implementing the present disclosure. Those skilled in the art will recognize that the chemical reactions described can be easily adapted to prepare a variety of other compounds of the present disclosure, and alternative methods for preparing compounds of the present disclosure are considered to be within the scope of the present disclosure. For example, non-exemplary compounds according to the present disclosure can be successfully synthesized by modifications apparent to those skilled in the art, such as by appropriately protecting interfering groups, by utilizing other suitable reagents known in the art in addition to the described reagents and building blocks, and / or by making conventional modifications to reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be considered to be applicable to the preparation of other compounds of the present disclosure.

[0515] For illustrative purposes, the general synthetic scheme for preparing the compounds of the present disclosure and key intermediates is shown below. It will be appreciated by those skilled in the art that other synthetic schemes can be used to synthesize the compounds of the present invention. Although specific starting materials and reagents are described in the general scheme and discussed below, other starting materials and reagents can be easily replaced to provide various derivatives and / or reaction conditions. In addition, many compounds prepared by the methods described below can be further modified according to the present disclosure using conventional chemical methods well known to those skilled in the art.

[0516] Example 1: General Chemical Synthesis Scheme 1

[0517]

[0518] in,

[0519] Each W is independently selected from O, S and NH;

[0520] Each Y is independently selected from O, S and NH;

[0521] Each n is independently 1, 2 or 3;

[0522] Each m is 0 or 1;

[0523] Each p is 1 or 2.

[0524] General Process

[0525] General procedure 1: Heat an ethanol solution of compound S1-1 to 60-80°C. Add compound S1-2 to the mixture. After the reaction is complete, cool the mixture to room temperature, then wash and dry to obtain the selective monosubstituted intermediate S1-3.

[0526] General Procedure 2: A mixture of intermediate S1-3, compound S1-4 (along with ACN and / or acetic acid) and BHT was added to a sealed container. The mixture was heated at 60-80°C for 12-24 hours and then cooled to room temperature. The mixture was purified by column chromatography to give compound S1-5.

[0527] Example 1.1: Synthesis of Compound 1

[0528]

[0529] Step 1: At room temperature, compound 1-4-1 (280 mg, 1.0 mmol), anhydrous DCM (6 mL) and compound 1-4-2 (104 mg, 1.1 mmol) were added to a container in sequence. The mixture was stirred for 5 minutes and then cooled in an ice bath. Triethylamine (211 mg, 2.1 mmol) was added to the mixture. The mixture was allowed to warm to room temperature and stirred at the same temperature for 5 hours. Thin layer chromatography (TLC) (eluent: petroleum ether / DCM = 3 / 1, KMnO4 staining) indicated that the reaction was essentially complete. The mixture was purified by silica gel column chromatography (eluent: petroleum ether with 0-33% DCM, v / v) and concentrated under reduced pressure to obtain compound 11-4 as a colorless oil (220 mg, yield 65%).

[0530] Compounds 1-4 1 H NMR: (400MHz, chloroform-d) δ6.33(dd,J=17.3,1.6Hz,1H),6.05(dd,J=17.4,10.4Hz,1H),5.74(dd,J=10.4,1.5Hz,1H),5.34–5.20(m ,2H),4.08(t,J=6.8Hz,2H),1.94(q,J=6.4Hz,4H),1.59(p,J=6.8Hz,2H),1.22(q,J=8.0,5.5Hz,24H),0.81(t,J=6.7Hz,3H).

[0531] Step 2: A solution of compound 1-1 (9.46 g, 65.13 mmol) in ethanol (600 mL) was heated at 65 ° C (internal temperature) and compound 1-2 (2.0 g, 12.80 mmol) was added. The mixture was heated at 70-75 ° C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (500 mL) and brine (400 mL) was added thereto under stirring. The mixture was heated and stirred at 35 ° C (internal temperature) for 10 minutes, then allowed to stand and separated. The aqueous layer was extracted again with DCM (500 mL) and repeated 4-5 times. TLC (eluent: DCM / MeOH=5:1, 3 drops of NH4OH were added) indicated that the extraction was complete. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give compound 1-3 (3.47 g, yield 97%).

[0532] Step 3: Compound 1-3 (80 mg, 0.27 mmol) was placed in a 4 mL container and, under a N2 atmosphere, compound 1-4 (308 mg, 0.95 mmol) and BHT (5 mg) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to afford compound 1 (250 mg, 74% yield).

[0533] Compound 1 1 H NMR: (400MHz, chloroform-d) δ5.44–5.26(m,5H),4.05(q,J=6.4Hz,6H),3.58(dd,J=6.9,3.4Hz,1H),2.97–2.83(m,1H),2.72(dt,J=24.8,7.0Hz ,5H),2.57(dt,J=14.3,7.3Hz,1H),2.01(q,J=6.4Hz,12H),1.61(t,J=7.0Hz,10H),1.28(q,J=8.2,4.7Hz,91H),0.87(t,J=6.6Hz,14H).

[0534] Example 1.2: Synthesis of Compound 2

[0535]

[0536] Step 1: Anhydrous DCM (200 mL), NaHCO (7.43 g, 88.5 mmol), compound 2-4-1 (9.6 g, 35.7 mmol) and compound 2-4-2 (4 g, 44.2 mmol) were added to a container in an ice bath. The mixture was allowed to warm to room temperature and stirred at the same temperature for 2 hours. TLC (I staining) indicated that the reaction was complete. The reaction was quenched with water (200 mL) and stirred for 10 minutes. The organic layer was washed twice with saturated NaHCO aqueous solution (200 mL) and then washed with water (200 mL). The organic layer was dried and concentrated to give a crude compound 2-4 (11.4 g, 87%).

[0537] Compounds 2-4 1 H NMR: (400 MHz, CHLOROFORM-d) δ 6.29 (dd, J = 17.0, 1.5 Hz, 1H), 6.10 (dd, J = 17.0, 10.3 Hz, 1H), 5.68–5.48 (m, 2H), 3.39–3.31 (m, 2H), 1.56 (p, J = 7.1 Hz, 2H), 1.28 (s, 29H), 0.90 (t, J = 6.7 Hz, 3H).

[0538] Step 2: A solution of compound 2-1 (9.46 g, 65.13 mmol) in ethanol (600 mL) was heated at 65 ° C (internal temperature) and compound 2-2 (2.0 g, 12.97 mmol) was added. The mixture was heated at 70-75 ° C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (500 mL) and brine (400 mL) was added thereto under stirring. The mixture was heated and stirred at 35 ° C (internal temperature) for 10 minutes, then allowed to stand and separated. The aqueous layer was extracted again with DCM (500 mL) and repeated 4-5 times. TLC (eluent: DCM / MeOH=5:1, 3 drops of NH4OH were added) indicated that the extraction was complete. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give compound 2-3 (3.4 g, yield 95%).

[0539] Step 3: Compound 2-4 (384 mg, 1.19 mmol) and BHT (5 mg) were added to compound 2-3 (100 mg, 0.33 mmol). The mixture was heated for 48 hours. TLC indicated that the reaction was complete. The mixture was diluted with DCM (10 mL) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 2 as a colorless oil (140 mg, 45% yield).

[0540] Compound 2 1 H NMR: (400 MHz, chloroform-d) δ 5.88–5.71 (m, 1H), 5.49–5.25 (m, 6H), 5.05–4.81 (m, 2H), 4.05 (q, J = 6.4 Hz, 7H), 3.58 (td, J = 7.0, 3.6 Hz, 1H), 2.90 (dt, J = 14.5, 7.4 Hz, 1H), 2.73 (dt, J = 25.2 ,6.9Hz,6H),2.57(dt,J=14.4,7.3Hz,2H),2.43(q,J=7.0,6.4Hz,13H),2.36–1.91(m,27 H), 1.61 (t, J = 7.1Hz, 12H), 1.30 (ddd, J = 17.8, 10.8, 5.4Hz, 96H), 0.88 (t, J = 6.7Hz, 12H).

[0541] Example 1.3: Synthesis of Compound 3

[0542]

[0543] Step 1: A solution of compound 3-1 (9.46 g, 65.13 mmol) in ethanol (600 mL) was heated at 65 ° C (internal temperature) and compound 3-2 (2.2 g, 11.94 mmol) was added. The mixture was heated at 70-75 ° C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (500 mL) and brine (400 mL) was added thereto under stirring. The mixture was heated and stirred at 35 ° C (internal temperature) for 10 minutes, then allowed to stand and separated. The aqueous layer was extracted again with DCM (500 mL) and repeated 4-5 times. TLC (eluent: DCM / MeOH=5:1, 3 drops of NH4OH were added) indicated that the extraction was complete. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give compound 3-3 (3.5 g, yield 97%).

[0544] Step 2: Compound 3-4 (350 mg, 1.08 mmol) and BHT (5 mg) were added to compound 3-3 (100 mg, 0.30 mmol). The mixture was heated for 48 hours. TLC indicated that the reaction was complete. The mixture was diluted with DCM (10 mL) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM with 0-5% methanol, v / v) and concentrated under reduced pressure to obtain compound 3 as a colorless oil (198 mg, 70% yield).

[0545] Compound 3 1 H NMR: (400MHz, chloroform-d) δ4.05(q,J=6.6Hz,6H),3.58(s,1H),2.91(dt,J=14.3,7.5Hz,1H),2.72(dt,J=23.3,6.7Hz ,7H),2.42(h,J=6.4Hz,10H),2.24(d,J=48.6Hz,6H),1.68–1.52(m,9H),1.25(s,112H),0.88(t,J=6.8Hz,12H).

[0546] Example 1.4: Synthesis of Compound 4

[0547]

[0548] Step 1: In an ice bath, anhydrous DCM (300 mL), NaHCO 3 (11.14 g, 132.6 mmol), compound 4-4-1 (14.15 g, 76.3 mmol) and compound 4-4-2 (6.0 g, 66.3 mmol) were added to the container in sequence. The mixture was allowed to warm to room temperature and stirred at the same temperature for 2 hours. TLC (I 2 staining) indicated that the reaction was complete. The reaction was quenched with water (300 mL) and stirred for 10 minutes. The organic layer was washed twice with saturated NaHCO 3 aqueous solution (300 mL) and then washed with water (300 mL). The organic layer was dried and concentrated to give crude compound 4-4 (8.4 g, 47%).

[0549] Compound 4-4 1 H NMR: (400 MHz, chloroform-d) δ 6.27 (d, J = 16.9 Hz, 1H), 6.09 (dd, J = 17.0, 10.2 Hz, 1H), 5.64 (t, J = 11.1 Hz, 2H), 3.32 (q, J = 6.8 Hz, 2H), 1.53 (p, J = 7.1 Hz, 2H), 1.26 (s, 24H), 0.88 (t, J = 6.6 Hz, 3H).

[0550] Step 2: A solution of compound 4-1 (1.145 g, 9.77 mmol) in ethanol (100 mL) was heated at 65 ° C (internal temperature) and compound 4-2 (0.30 g, 1.63 mmol) was added. The mixture was heated at 70-75 ° C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (300 mL) and brine (300 mL) was added thereto under stirring. The mixture was heated and stirred at 35 ° C (internal temperature) for 10 minutes, then allowed to stand and separated. The extraction was repeated 3-4 times. TLC (eluent: DCM / MeOH=5:1, containing 2 drops of NH4OH) indicated that the extraction was complete. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give compound 4-3 (0.47 g, yield 95%).

[0551] Step 3: Compound 4-3 (20 mg, 0.07 mmol) was placed in a 2 mL container, and compound 4-4 (61 mg, 0.25 mmol), BHT (15 mg), and acetic acid (3 μL) were added. The mixture was heated at 80° C. for 105 hours. TLC (eluent: DCM / MeOH = 10 / 1, containing NH 4 OH) indicated that the reaction was essentially complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 1-1.3% methanol and 0.5% NH 4 OH, v / v) and concentrated under reduced pressure to obtain compound 4 (45 mg, 66% yield).

[0552] Compound 41 H NMR: (400MHz, chloroform-d) δ7.12–6.96(m,1H),6.93(t,J=5.5Hz,1H),3.67–3.48(m,1H),3.10(dq,J=26.9,6.6Hz,6H),2.91–2.81(m,1H),2.54(d,J=37. 5Hz,9H),2.44–2.22(m,11H),2.16(dd,J=14.0,6.4Hz,2H),2.08–1.85(m ,2H),1.49–1.30(m,9H),1.20(d,J=11.0Hz,80H),0.81(t,J=6.8Hz,14H).

[0553] Example 1.5: Synthesis of Compound 5

[0554]

[0555] Step 1: Compound 5-3 was prepared according to the method of step 1 in Example 1.4.

[0556] Step 2: Compound 5-3 (19.6 mg, 0.07 mmol) was placed in a 2 mL container, and compound 5-4 (78.6 mg, 0.24 mmol) and BHT (17.2 mg) were added thereto. The mixture was heated at 70° C. for 67 hours and then at 90° C. for 23 hours. TLC (eluent: DCM / MeOH=20 / 1) indicated that the reaction was essentially complete. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-5% methanol, v / v) and concentrated under reduced pressure to give compound 5 (40.8 mg, 49% yield).

[0557] Compound 5 1 H NMR: (400MHz, chloroform-d) δ4.05(q,J=7.2Hz,5H),3.62(dt,J=11.9,6.5Hz,1H),2.94(dt,J=14.2,7.3Hz,2H),2.84(q,J=6.6Hz,2H),2.77(t,J=6. 9Hz, 5H), 2.65–2.58 (m, 1H), 2.47 (dq, J = 13.8, 7.5, 7.0Hz, 7H), 2.40–2.17 (m, 2H), 1.71–1.53 (m, 6H), 1.25 (s, 106H), 0.88 (t, J = 6.8Hz, 12H).

[0558] Example 1.6: Synthesis of Compound 6

[0559]

[0560] Step 1: Compound 6-3 was prepared according to the method of step 2 of Example 1.2.

[0561] Step 2: Compound 6-3 (80 mg, 0.27 mmol) was placed in a 4 mL container. Under a N2 atmosphere, compound 6-4 (310 mg, 0.96 mmol, prepared according to the method of Example 1.1, Step 1) and BHT (5 mg) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 6 (230 mg, 68% yield).

[0562] Compound 6 1 H NMR: (400 MHz, chloroform-d) δ 5.88–5.71 (m, 1H), 5.49–5.25 (m, 6H), 5.05–4.81 (m, 2H), 4.05 (q, J = 6.4 Hz, 7H), 3.58 (td, J = 7.0, 3.6 Hz, 1H), 2.90 (dt, J = 14.5, 7.4 Hz, 1H), 2.73 (dt, J = 25.2 ,6.9Hz,6H),2.57(dt,J=14.4,7.3Hz,2H),2.43(q,J=7.0,6.4Hz,13H),2.36–1.91(m,27 H), 1.61 (t, J = 7.1Hz, 12H), 1.30 (ddd, J = 17.8, 10.8, 5.4Hz, 96H), 0.88 (t, J = 6.7Hz, 12H).

[0563] Example 1.7: Synthesis of Compound 7

[0564]

[0565] Step 1: A solution of compound 7-1 (1.44 g, 9.91 mmol) in ethanol (100 mL) was heated at 65 ° C (internal temperature) and compound 7-2 (0.40 g, 1.75 mmol) was added. The mixture was heated at 70-75 ° C (external temperature) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (200 mL) and water (200 mL) was added thereto under stirring. The mixture was heated and stirred at 35 ° C (internal temperature) for 10 minutes, then allowed to stand and separated. The extraction was repeated 3-4 times. TLC (eluent: DCM / MeOH=5:1, containing 3 drops of NH4OH) indicated that the extraction was complete. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give compound 7-3 (0.43 g, yield 66%).

[0566] Step 2: Compound 7-3 (100 mg, 0.27 mmol) was placed in a 4 mL container. Under a N2 atmosphere, compound 7-4 (270 mg, 0.94 mmol, prepared according to the method of Example 1.1, Step 1) and BHT (5 mg) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 7 (220 mg, 67% yield).

[0567] Compound 7 1 H NMR: (400MHz, chloroform-d) δ5.45–5.21(m,5H),4.05(td,J=6.8,4.3Hz,6H),3.89–3.74(m,1H),3.53–3.34(m,4H),2.98–2. 82(m,1H),2.74(td,J=7.0,4.5Hz,6H),2.62–2.35(m,16H),2.00(hept,J=5.9,5.2Hz,12H),1.58(dt,J=18.9,7.0Hz, 12H ),1.47–1.07(m,90H),0.87(t,J=6.7Hz,13H).

[0568] Example 1.8: Synthesis of Compound 8

[0569]

[0570] Step 1: Compound 8-3 was prepared according to the method of step 2 in Example 1.1.

[0571] Step 2: Compound 8-3 (100 mg, 0.33 mmol) was placed in a 4 mL container and, under a N2 atmosphere, compound 8-4 (350 mg, 1.09 mmol) and BHT (10 mg) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to give compound 8 (230 mg, 55% yield).

[0572] Compound 8 1H NMR: (400 MHz, chloroform-d) δ 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.46–5.23 (m, 13H), 5.05–4.88 (m, 2H), 4.17–3.94 (m, 6H), 3.58 (ddt, J = 10.4, 6.9, 3.4 Hz, 1H), 3.02–2.83 (m, 1H ),2.82–2.65(m,12H),2.56(q,J=7.4,6.8Hz,2H),2.51–2.11(m,21H),2.05(q,J=6.7 Hz, 16H), 1.61 (dd, J=10.6, 4.3Hz, 12H), 1.49–1.10 (m, 71H), 0.89 (t, J=6.8Hz, 12H).

[0573] Example 1.9: Synthesis of Compound 9

[0574]

[0575] Step 1: Compound 9-3 was prepared according to the method of step 2 in Example 1.2.

[0576] Step 2: Compound 9-3 (100 mg, 0.33 mmol) was placed in a 4 mL container and, under a N2 atmosphere, compound 9-4 (360 mg, 1.12 mmol) and BHT (10 mg) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to afford compound 9 (221 mg, 56% yield).

[0577] Compound 9 1 H NMR: (400 MHz, CHLOROFORM-d) δ 5.45–5.26 (m, 12H), 4.05 (q, J = 7.1 Hz, 6H), 3.60 (s, 1H), 2.93 (dt, J = 13.9, 7.5 Hz, 3H), 2.75 (dt, J = 17.7, 6.7 Hz, 15H), 2.54–2.20 (m, 14H), 2.05 (q, J = 6.8 Hz, 14H), 1.88 (s, 5H), 1.62 (p, J = 6.7 Hz, 9H), 1.46–1.19 (m, 72H), 0.88 (td, J = 6.8, 4.5 Hz, 14H).

[0578] Example 1.10: Synthesis of Compound 10

[0579]

[0580] Step 1: Compound 10-3 was prepared according to the method of step 1 in Example 1.7.

[0581] Step 2: Compound 10-3 (120 mg, 0.32 mmol) was placed in a 4 mL container and, under a N2 atmosphere, compound 10-4 (350 mg, 1.09 mmol) and BHT (10 mg) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to afford compound 10 (205 mg, 50% yield).

[0582] Compound 10 1 H NMR: (400 MHz, chloroform-d) δ 5.48–5.24 (m, 14H), 4.06 (q, J = 7.2 Hz, 6H), 3.79 (dq, J = 9.9, 5.1 Hz, 1H), 2.87 (dq, J = 17.0, 9.7, 8.5 Hz, 3H), 2.77 (t, J = 6.4 Hz, 7H), 2.68 (dt, J = 13.0 ,6.3Hz,5H),2.44(dt,J=13.2,7.7Hz,7H),2.29(d,J=6.9Hz,4H),2.13–2.03(m,12H ),1.59(dt,J=17.9,6.9Hz,18H),1.43–1.20(m,88H),0.88(td,J=6.8,4.1Hz,16H).

[0583] Example 1.11: Synthesis of Compound 11

[0584]

[0585] Step 1: DCM (100 mL) solution of compound 11-4-1 (10 g, 57.1 mmol) and compound 11-4-2 (10.3 g, 59.9 mmol) was added DMAP (698 mg, 5.7 mmol). Under N2 atmosphere, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 21.9 g, 114.2 mmol) was added to the mixture in portions at a temperature below 10 ° C. The mixture was allowed to warm to room temperature and stirred at the same temperature for 16 hours. The reaction was quenched with saturated NaHCO3 aqueous solution (100 mL). The aqueous layer was extracted with ethyl acetate (200 mL). The combined organic layers were washed with 5% citric acid aqueous solution (100 mL) and brine (100 mL), and then washed with brine (100 mL). The organic layer was dried over Na2SO4 and concentrated to give 18.5 g of crude compound 11-4-3. Compound 11-4-3 was used directly without further purification.

[0586] Compound 11-4-3 1 H NMR: (400MHz, DMSO-d6) δ7.15(t,J=6.2Hz,1H),4.02(t,J=6.5Hz,2H),3.64(d,J=6.2Hz,2 H), 1.55 (t, J = 6.9Hz, 2H), 1.38 (s, 8H), 1.34 (s, 2H), 1.24 (s, 19H), 0.85 (t, J = 6.5Hz, 4H).

[0587] Step 2: Under N2 atmosphere, HCl (4M in ethyl acetate, 93 mL) was added to a solution of compound 111-4-3 (18.5 g, 56.1 mmol, crude product) in ethyl acetate (93 mL) at a temperature below 10 ° C. The mixture was allowed to warm to room temperature and stirred at the same temperature for 16 hours. TLC indicated that the starting material was consumed. The mixture was cooled to 5 ° C and stirred at the same temperature for 2 hours. The mixture was filtered, and the residue was washed with ethyl acetate and dried to give 12.5 g of crude compound 11-4-4. Compound 11-4-4 was used directly without further purification.

[0588] Compound 11-4-4 1 H NMR: (400MHz, DMSO-d6) δ8.63(s,3H),4.11(t,J=6.6Hz,2H),3.73(s,2H),1.57(p,J=6.7Hz,2H),1.23(s,17H),0.84(t,J=6.6Hz,3H).

[0589] Step 3: Under N2 atmosphere, DCM (50 mL) solution of compound 11-4-4 (5 g, 18.9 mmol) and compound 11-4-5 (1.9 g, 20.7 mmol) was added to DMAP (698 mg, 5.7 mmol) at a temperature lower than 10 ° C. At the same temperature, a DCM (5 mL) solution of N, N-diisopropylethylamine (DIEA, 4.9 g, 37.8 mmol) was added dropwise to the mixture. The mixture was allowed to warm to room temperature and stirred at the same temperature for 3 hours. TCL indicated that the starting material was consumed. The reaction was quenched with saturated NaHCO3 aqueous solution (50 mL). The aqueous layer was extracted with ethyl acetate (100 mL). The combined organic layer was washed with 5% citric acid aqueous solution (50 mL) and brine (50 mL), and then washed with brine (100 mL). The organic layer was dried over Na2SO4 and concentrated to give a crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether and 20% ethyl acetate, v / v) and concentrated under reduced pressure to give compound 11-4 (4.4 g, 27% yield over 3 steps).

[0590] Compound 11-4 1 H NMR: (400MHz, DMSO-d6) δ8.51(t,J=6.0Hz,1H),6.29(dd,J=17.1,10.2Hz,1H),6.11(dd,J=17.1,2.1Hz,1H),5.63(dd ,J=10.2,2.1Hz,1H),4.04(t,J=6.6Hz,2H),3.90(d,J=5.9Hz,2H),1.57(s,2H),1.24(s,16H),0.85(t,J=6.6Hz,3H).

[0591] Step 4: Compound 11-3 was prepared according to the method of step 1 in Example 1.3.

[0592] Step 5: Compound 11-3 (400 mg, 1.2 mmol), BHT (53.4 mg), and compound 11-4 (1.24 g, 4.4 mmol) were placed in a 2 mL container and acetic acid (14.6 mg) was added. The mixture was heated at 80°C for 42 hours. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 1) indicated that the reaction was essentially complete. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 1.4-24% methanol and 0.5% NH4OH, v / v) and concentrated under reduced pressure to obtain compound 11 (390 mg, 27% yield).

[0593] Compound 11 1 H NMR: (400MHz, chloroform-d)δ7.87(t,J=5.5Hz,1H),7.68(t,J=5.5Hz,2H),4.15-4.01(m,6H ),4.01-3.88(m,4H),3.68(dt,J=17.8,8.6Hz,1H),3.03-2.91(m,1H),2.74(t,J=6. 0Hz,4H),2.66(dt,J=14.1,7.3Hz,2H),2.59-2.39(m,9H),2.40–2.19(m,5H),1.81- 1.66(m,4H),1.62(p,J=6.9Hz,6H),1.52-1.04(m,68H),0.87(q,J=5.9,5.5Hz,12H). HR-MS:[M+H] + =1180.00680.

[0594] Example 1.12: Synthesis of Compound 12

[0595]

[0596] Step 1: Compound 12-3 was prepared in a similar manner to that in Example 1.1, Step 2.

[0597] Step 2: Compound 12-3 (80 mg, 0.29 mmol) was placed in a 4 mL container. Under a N2 atmosphere, BHT (5 mg) and compound 12-4 (340 mg, 1.05 mmol, prepared according to the method of Example 1.1, Step 1) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 12 (295 mg, 81% yield).

[0598] Compound 12 1 H NMR: (400MHz, chloroform-d) δ5.44–5.26(m,5H),4.05(q,J=6.4Hz,6H),3.58(dd,J=6.9,3.4Hz,1H),2.97–2.83(m,1H),2.72(dt,J=24.8,7.0Hz ,5H),2.57(dt,J=14.3,7.3Hz,1H),2.01(q,J=6.4Hz,12H),1.61(t,J=7.0Hz,10H),1.28(q,J=8.2,4.7Hz,91H),0.87(t,J=6.6Hz,14H). HR-MS:[M+H] + =1240.15427.

[0599] Example 1.13: Synthesis of Compound 13

[0600]

[0601] Step 1: Compound 13-3 was prepared according to the method of step 1 in Example 1.3.

[0602] Step 2: Compound 13-3 (80 mg, 0.24 mmol) was placed in a 4 mL container. Under a N2 atmosphere, BHT (5 mg) and compound 13-4 (308 mg, 0.95 mmol, prepared according to the method of Example 1.1, Step 1) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 13 (250 mg, 74% yield).

[0603] Compound 13 1H NMR: (400MHz, chloroform-d) δ5.49–5.22(m,6H),4.05(q,J=6.5Hz,6H),3.58(td,J=6.8,3.4Hz,1H),2.91(dt,J=14.3,7.5Hz,1H),2.72(dt,J=23.7,6.9Hz, 6H),2.43(q,J=7.6Hz,13H),2.34–2.12(m,6H),2.00(dh,J=11.6,6.5Hz,1 1H), 1.63 (s, 10H), 1.30 (dt, J = 16.3, 9.4Hz, 91H), 0.88 (t, J = 6.7Hz, 13H). [M+H] + =1297.21294.

[0604] Example 1.14: Synthesis of Compound 14

[0605]

[0606] Step 1: Compound 14-3 was prepared in a similar manner to that of Example 1.3, Step 1.

[0607] Step 2: Compound 14-3 (80 mg, 0.22 mmol) was placed in a 4 mL container. Under a N2 atmosphere, BHT (5 mg) and compound 14-4 (259 mg, 0.80 mmol, prepared according to the method of Example 1.1, Step 1) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 14 (220 mg, 74% yield).

[0608] Compound 14 1 H NMR: (400MHz, chloroform-d) δ5.45–5.23(m,6H),4.05(q,J=6.5Hz,6H),3.58(td,J=6.7,3.4Hz,1H),2.90(dt,J=14.3,7.5Hz ,1H),2.80–2.36(m,19H),2.33–1.87(m,20H),1.60(q,J=7.0Hz,11H),1.51–1.06(m,102H),0.88(t,J=6.7Hz,14H). [M+H] + =1325.24099.

[0609] Example 1.15: Synthesis of Compound 15

[0610]

[0611] Step 1: Compound 15-3 was prepared in a similar manner to Example 1.3, Step 1.

[0612] Step 2: Compound 15-3 (100 mg, 0.26 mmol) was placed in a 4 mL container. Under a N2 atmosphere, BHT (5 mg) and compound 15-4 (301 mg, 0.93 mmol, prepared according to the method of Example 1.1, Step 1) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 15 (250 mg, 69% yield).

[0613] Compound 15 1 H NMR: (400MHz, chloroform-d) δ5.49–5.12(m,5H),4.05(q,J=6.6Hz,6H),3.58(td,J=6.9,3.5Hz,1H),2.91(dt,J=14.3,7.5Hz,1H),2.72(dt,J=23.2,6.8Hz, 6H),2.64–2.52(m,2H),2.52–2.19(m,17H),2.01(q,J=6.4Hz,14H),1.61 (t, J=7.0Hz, 11H), 1.28 (dd, J=18.7, 7.3Hz, 99H), 0.88 (t, J=6.7Hz, 13H). [M+H] + =1297.21294.

[0614] Example 1.16: Synthesis of Compound 16

[0615]

[0616] Step 1: Compound 16-3 was prepared in a similar manner to Example 1.3 Step 1.

[0617] Step 2: Compound 16-3 (100 mg, 0.24 mmol) was placed in a 4 mL container. Under a N2 atmosphere, BHT (5 mg) and compound 16-4 (280 mg, 0.87 mmol, prepared according to the method of Example 1.1, Step 1) were added. The mixture was heated at 70°C for 48 hours. TLC indicated the reaction was complete. The mixture was purified by silica gel flash column chromatography (eluent: DCM with 0-10% methanol, v / v) and concentrated under reduced pressure to obtain compound 16 (197 mg, 59% yield).

[0618] Compound 16 1H NMR: (400MHz, chloroform-d) δ5.48–5.23(m,5H),4.05(q,J=6.6Hz,6H),3.58(td,J=7.5,7.1,3.7Hz,1H),2.91(dt,J=14.5,7.5Hz,1 H), 2.83–2.14 (m, 26H), 2.01 (q, J = 6.4Hz, 12H), 1.80–1.53 (m, 10H), 1.28 (dd, J = 20.0, 7.6Hz, 109H), 0.87 (t, J = 6.7Hz, 14H). [M+H] + =1381.31640.

[0619] Example 1.17: Synthesis of Compound 17

[0620]

[0621] Step 1: Compound 17-3 was prepared in a similar manner to that of Example 1.3, Step 1.

[0622] Step 2: Compound 17-3 (30 mg, 0.09 mmol) was placed in a vial, and BHT (1 mg), compound 17-4 (137 mg, 0.32 mmol) and ACN (0.3 mL) were added thereto. The mixture was ventilated and decanted three times under a N2 atmosphere. The mixture was heated at 70°C for 2-3 days. TLC indicated that the reaction was complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-3% methanol, v / v) and concentrated under reduced pressure to give compound 17 (110 mg, 76% yield).

[0623] Compound 17 1 H NMR: (400 MHz, CHLOROFORM-d) δ 4.37 (q, J = 6.3 Hz, 6H), 3.57 (s, 1H), 2.91 (dt, J = 14.3, 7.4 Hz, 1H), 2.83–2.63 (m, 5H), 2.63–2.08 (m, 25H), 1.25 (s, 23H), 0.87 (t, J = 6.8 Hz, 3H).

[0624] Example 1.18: Synthesis of Compound 18

[0625]

[0626] Step 1: Compound 18-3 was prepared in a similar manner to that of Example 1.3, Step 1.

[0627] Step 2: Compound 18-3 (30 mg, 0.09 mmol) was placed in a vial, and BHT (1 mg), compound 18-4 (170 mg, 0.33 mmol) and ACN (0.3 mL) were added thereto. The mixture was ventilated and decanted three times under a N2 atmosphere. The mixture was heated at 70°C for 2-3 days. TLC indicated that the reaction was complete. The mixture was purified by silica gel column chromatography (eluent: DCM with 0-3% methanol, v / v) and concentrated under reduced pressure to give compound 18 (100 mg, 58% yield).

[0628] Compound 18 1 H NMR: (400 MHz, CHLOROFORM-d) δ 4.37 (q, J = 6.4 Hz, 6H), 3.57 (s, 1H), 2.91 (dt, J = 14.0, 7.4 Hz, 1H), 2.78–2.66 (m, 5H), 2.56–2.15 (m, 25H), 1.25 (s, 24H), 0.88–0.84 (m, 3H).

[0629] Example 1.19: Synthesis of Compound 19

[0630]

[0631] Step 1: Under N2 atmosphere, compound 19-4-1 (1.6 g, 7.3 mmol) and triethylamine (1.86 g, 18.4 mmol) were added to DCM (20 mL). The mixture was cooled to 5 ° C. Compound 19-4-2 (1.0 g, 6.1 mmol) was added dropwise. The mixture was allowed to warm to room temperature and stirred at the same temperature for 16 hours. TLC (eluent: DCM) indicated product formation. The mixture was purified by silica gel column chromatography to obtain compound 19-4 (0.7 g, yield 37%).

[0632] Compound 19-4 1 H NMR: (400MHz, chloroform-d) δ6.51(dd,J=16.5,9.9Hz,1H),6.23(d,J=16.6Hz,1H),5.93(d,J=9.9Hz,1H) ,4.44(s,1H),3.00(q,J=6.8Hz,2H),1.53(t,J=7.2Hz,2H),1.25(s,24H),0.87(t,J=6.7Hz,3H).

[0633] Step 2: Compound 19-3 was prepared according to the method of step 2 in Example 1.1.

[0634] Step 3: Under N2 atmosphere, BHT (15 mg) and acetic acid (7 mg) were added to a solution of compound 19-3 (30 mg, 0.096 mmol) and compound 19-4 (117.3 mg, 0.386 mmol) in ACN (0.2 mL) at room temperature. The mixture was heated at 75°C for 40 hours. TLC (eluent: DCM / MeOH=10 / 1) indicated that the starting material was consumed in large quantities. The mixture was purified by silica gel column chromatography and concentrated under reduced pressure to give compound 19 (67 mg, 55% yield).

[0635] Compound 19 1 H NMR: (400 MHz, chloroform-d) δ 5.78 (t, J = 6.1 Hz, 1H), 3.65 (td, J = 8.7, 7.5, 4.0 Hz, 1H), 3.44–3.26 (m, 2H), 3.19 (dt, J = 19.3, 5.2 Hz, 7H), 3.07 (q, J = 7.1 Hz, 7H), 2.99–2.81 (m, 4H) ,2.75–2.63(m,5H),2.55(dt,J=28.9,7.4Hz,3H),2.46–2.20(m,2H),2.00(d,J=11. 9Hz, 5H), 1.58 (dt, J = 12.5, 4.7Hz, 6H), 1.26 (d, J = 5.9Hz, 81H), 0.94–0.73 (m, 12H). [M+H] + =1212.00789.

[0636] Example 1.20: Synthesis of Compound 20

[0637]

[0638] Step 1: Under N2 atmosphere, compound 20-4-1 (1 g, 4.38 mmol) and compound 20-4-2 (2.8 g, 43.8 mmol) were added to methanol (3 mL). The mixture was stirred at room temperature for 40 hours. TLC (eluent: DCM / MeOH=10 / 1) indicated that the starting material was not completely consumed. Compound 20-4-2 (2.8 g, 43.8 mmol) was added to the mixture. The mixture was heated at 60 ° C for 16 hours. TLC (eluent: PE) indicated that the starting material was consumed. The mixture was concentrated and diluted with water (10 mL). The mixture was stirred for 30 minutes and filtered. The residue was washed and dried to obtain compound 20-4-3 (540 mg).

[0639] Compound 20-4-3 1H NMR: (400 MHz, chloroform-d) δ 6.82 (s, 1H), 3.90 (s, 2H), 2.14 (t, J = 7.6 Hz, 2H), 1.62 (t, J = 7.3 Hz, 2H), 1.26 (d, J = 10.9 Hz, 18H), 0.87 (t, J = 6.7 Hz, 3H). HRMS: [M+H] + 229.22922.

[0640] Step 2: Under N2 atmosphere, compound 20-4-3 (0.54 g, 2.4 mmol) and DIEA (0.62 g, 4.8 mmol) were added to DCM (20 mL). The mixture was cooled to 5 ° C. Compound 20-4-4 (0.26 g, 2.9 mmol) was added dropwise at 5-10 ° C. The mixture was allowed to warm to room temperature and stirred at the same temperature for 2 hours. TLC (eluent: DCM / MeOH=10 / 1) indicated that the starting material was consumed. The mixture was purified by silica gel column chromatography to obtain a crude product. The crude product was added to water (10 mL), stirred for 30 minutes, and dried to give compound 20-4 (510 mg). HRMS: [M+H] + 283.24978.

[0641] Step 3: Compound 20-3 was prepared according to the method of step 2 in Example 1.1.

[0642] Step 1: Under N2 atmosphere, to a solution of compound 20-3 (30 mg, 0.096 mmol) and compound 20-4 (101.2 mg, 0.36 mmol, prepared according to the method of Example 1.16 Steps 1 to 2) in ACN (0.2 mL) were added BHT (15 mg) and acetic acid (10 mg) at room temperature. The mixture was heated at 80°C for 16 hours. TLC (eluent: DCM / MeOH = 10 / 1 + 0.5% NH4OHH) indicated that the starting material was largely consumed. The mixture was purified by silica gel column chromatography and concentrated under reduced pressure to give compound 20 (50 mg, 43% yield).

[0643] Compound 20 1 H NMR: (400MHz, chloroform-d) δ3.70(s,1H),3.07–2.87(m,1H),2.68(t,J=5.6Hz,2H),2.48–2.31(m,6H),2.31–2 .19(m,5H),2.15(d,J=18.3Hz,2H),1.62(d,J=8.0Hz,7H),1.26(d,J=5.9Hz,48H),0.94–0.76(m,12H). HRMS:[M+H] + 1149.01684.

[0644] Other compounds of the present disclosure were synthesized using similar methods, but with modified reaction conditions and different starting materials.

[0645] Example 1.21: Synthesis of Compounds 21 to 39

[0646] Compounds 21 to 39 were synthesized by the methods shown in the following schemes.

[0647]

[0648] in,

[0649] Each W is independently selected from O, S and NH;

[0650] Each Y is independently selected from O, S and NH;

[0651] Each n is independently 1, 2 or 3;

[0652] Each m is 0 or 1;

[0653] Each p is 1 or 2.

[0654] General Process

[0655] A mixture of compounds S2-1, S2-2, 2,6-di-tert-butyl-4-methylphenol (BHT), acetonitrile (ACN), and / or acetic acid is added to a sealed container. The mixture is heated at 60-80°C for 18-36 hours. The mixture is then cooled to room temperature and purified by column chromatography to obtain the desired compound S2-3.

[0656] The following compounds were synthesized using modified reaction conditions and different starting materials.

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663] Example 2: Preparation of mRNA

[0664] Preparation of mRNA

[0665] mRNA can be prepared in vitro by any method known in the art. For example, the mRNA used in the exemplary embodiment can be prepared using the TranscriptAid T7 High Yield Transcription Kit (Thermo K0441). In vitro transcription is performed using the TranscriptAid T7 High Yield Transcription Kit, using linear DNA as a template to generate the target mRNA, to which a certain proportion of pseudouridine and capping reagent are added. In vitro transcription conditions: The reaction system is prepared according to the kit instructions, the reaction is carried out at 37°C for 0.5-2 hours, the transcript is digested with DNase for 30 minutes, and the transcript is purified using the Monarch RNA Cleanup Kit (NEBT2040L).

[0666] Exemplary mRNA constructs are shown in Table 7 below. The signal peptide in each construct can be replaced with an alternative sequence that achieves the same or similar function. The amino acid sequence of an exemplary signal peptide is SEQ ID NO: 185, which is encoded by the nucleic acid sequence of SEQ ID NO: 184.

[0667] Table 7. Description and sequence of test mRNA

[0668]

[0669]

[0670] Example 3: In vitro expression assay

[0671] Western Blot and ELISA

[0672] HEK293 cells were transfected with the test mRNA. HEK293 cells in the logarithmic growth phase were used and the concentration of the cell suspension was adjusted to 5×10 5 cells / ml and seeded them into 6-well plates at 1 ml per well. The cells were cultured overnight in a 5% CO2, 37°C incubator to allow them to adhere. 4 μg of test mRNA and 8 μL of Lipofectamine 2000 (ThermoFisher Scientific) were diluted in 250 μL of serum-free Opti-MEM, respectively, and then incubated at room temperature for 5 minutes. The above mRNA solution was mixed with the above Lipofectamine 2000 and allowed to stand at room temperature for 20 minutes. 500 μL of oligonucleotide-liposome complex was added to the cell culture plate, and 500 μL of serum-free Opti-MEM was added to the 6-well plate to make the transfection system volume 1 mL. 24 hours after transfection, HEK293 cells were lysed using M-PER mammalian protein extraction reagent (Thermo), proteins were extracted, and the resulting proteins were used for Western blotting and ELISA tests. Figures 1A to 1EThe expression of RSV F protein and prefusion RSV-F protein ("pre-F protein") in cell lines was detected by Western blotting and ELISA.

[0673] FACS flow cytometry assay

[0674] COS7 cells were transfected with the tested RSV-F mRNA. COS7 cells in the logarithmic growth phase were used and the concentration of the cell suspension was adjusted to 5×10 5 Cells / ml were plated in 6-well plates at 1 ml per well. The cells were placed in a 5% CO2, 37°C incubator and cultured overnight to allow them to adhere. 4 μg of test mRNA and 8 μL of Lipofectamine 2000 (ThermoFisher Scientific) were diluted in 250 μL of serum-free Opti-MEM, respectively, and then incubated at room temperature for 5 minutes. The above mRNA solution was mixed with the above Lipofectamine 2000 and allowed to stand at room temperature for 20 minutes. 500 μL of oligonucleotide-liposome complexes were added to the cell culture plate, and 500 μL of serum-free Opti-MEM was added to the 6-well plate to make the transfection system 1 mL. RSVF-specific antibodies (sinobiological; No. 11049-R302) and Alexa Fluor 577 were used. 488-labeled goat anti-mouse IgG antibody (abcam, No. ab150113) or FITC-labeled hIgG antibody D25 ("D25", which recognizes the prefusion RSV-F specific antigenic site) ), antibody 4D7 ("4D7", recognizes RSV-F specific antigenic site I after fusion) and palivizumab ("Pali", recognizes the common antigenic site between RSV-F before fusion and RSV-F after fusion) were stained. Analyzed by flow cytometry (FACS). Flow cytometry data were quantitatively evaluated using FlowJo software. Figures 2A to 2B The results shown indicate that the expression of RSV F protein, prefusion RSV-F protein, and postfusion RSV-F protein in the cell lines was detected by FACS.

[0675] Example 4: Preparation of lipid nanoparticle composition

[0676] Step 1: mRNA was dissolved in citrate buffer (pH 4) and the mRNA concentration was adjusted to 0.2 mg / mL to obtain an aqueous layer.

[0677] Step 2: The test compound, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and DMG-PEG2000 were dissolved in anhydrous ethanol at the desired molar fractions (shown in Table 8 below), and the total lipid concentration was adjusted to 10 mg / mL to obtain an organic layer.

[0678] Table 8. Mole fractions of each component in lipid particles

[0679]

[0680] Step 3: Microfluidic device ( Ignite TM ), the aqueous and organic layers were mixed in a 3:1 (v / v) ratio at a total flow rate of 12 mL / min. The mixture was diluted 10-fold with PBS buffer (pH 7.4). The ethanol was separated by tangential flow filtration (Repligen, TFF). The solution was concentrated to 0.1 mg / mL (mRNA concentration) and filtered through a 0.22 μm microporous filter to obtain lipid particles containing mRNA.

[0681] The average particle size and polymer dispersibility index (PDI) of exemplary lipid particles were measured using DLS by Malvern Zetasizer.

[0682] Based on the use Total and free RNA concentrations were measured using fluorescence measurements performed with the ELISA kit (ThermoFisher Scientific). Encapsulation efficiency (EE) was calculated as follows:

[0683]

[0684] The total RNA concentration was measured by appropriately diluting the test lipid particles with 1xTE buffer containing 0.2% Triton-X 100. The free RNA concentration was measured by appropriately diluting the test lipid particles with 1xTE buffer.

[0685] Table 9. Physical properties of lipid particles used in the test

[0686] Recipe Number Particle size (μm) Polydispersity Index (PDI) Encapsulation efficiency (EE, %) 5 83.22 0.085 95 6 77.03 0.0945 92 7 79.67 0.0862 93.7 8 86.66 0.0749 93.1 9 82.52 0.067 93.1 10 76.1 0.0927 94.6 11 84.11 0.0659 94.7 12 77.97 0.0475 93.5 13 81.79 0.0719 96.1 14 81.25 0.0874 96.1 15 87.02 0.0664 95.2 16 103.8 0.0279 97.6 17 106.7 0.0384 97.5 18 96.22 0.0502 97 19 113.3 0.0223 97 20 115.8 0.0482 97.1

[0687] Example 5: In vitro delivery assay

[0688] Lipid nanoparticle compositions containing FLuc mRNA or test RSV mRNA (WT (v2)) were prepared according to the method of Example 4. The components and their molar fractions in the test lipid nanoparticles are shown in Table 10 below. The solution was concentrated to 0.5 mg / mL (mRNA concentration) and filtered through a 0.22 μm microporous filter to obtain the mRNA-containing lipid nanoparticle composition.

[0689] The obtained lipid nanoparticles containing mRNA were injected into C57 mice and Balb / C mice respectively. The expression of fluorescent protein was measured by fluorescence imaging ( Figure 3A ). Two weeks after injection, the titer of RSV binding antibodies in the serum was measured ( Figure 3B ). It can be seen that the lipid nanoparticles used in the test effectively delivered mRNA into mice, and the mRNA was stably expressed in vivo.

[0690] Table 10. Mole fractions of components in lipid nanoparticles

[0691]

[0692] Example 6: In vivo immunogenicity test

[0693] A lipid nanoparticle composition comprising RSV mRNA (WT (v2)) and STAR0116 was prepared according to the method of Example 4. The solution was concentrated to 0.5 mg / mL (mRNA concentration) and filtered with a 0.22 μm microporous filter to obtain mRNA containing the lipid nanoparticle composition. The lipid nanoparticle composition was tested using female Balb / C mice (6 to 8 weeks old). 10 mice were used in each group, and 50 μL was injected per mouse according to Table 11 below. An equal volume of normal saline was injected as a negative control. Immunizations were performed on days 0 and 28. Blood was collected on days 14 (week 2), 21 (week 3), 35 (week 5), and 56 (week 8) and used for serological testing. On day 49, 3 mice were killed in each group, and their spleens were taken for IFN-γ ELISPOT testing.

[0694] Table 11. Immunization doses and schedule

[0695]

[0696]

[0697] RSV-F protein antibody test

[0698] ELISA plates were coated with recombinant human RSV fusion glycoprotein (rec.hu F protein, final concentration: 0.2 μg / mL) (Sino Biological Inc.). The coated plates were incubated with the given serum dilutions and the specific binding of the antibodies to the RSV-F protein was determined using an HRP-labeled anti-mouse antibody using TMB substrate. Figure 4A and Figure 4B It was shown that the lipid nanoparticle composition (RSV mRNA vaccine) induced higher RSV-F antibody titers (total IgG, IgG1, IgG2a, IgG2b and IgG2c).

[0699] RSV neutralizing antibody test

[0700] Before the test, the mouse serum collected on the 42nd day was inactivated in a 56°C water bath for 0.5 hours. The samples were diluted using a gradient. The diluted samples (8 concentrations, double wells and double wells) were incubated with RSV A long virus at 37°C and 5% CO2 for 2 hours. HEp-2 cells were then inoculated into the test wells and cultured at 37°C and 5% CO2 for 5 days. Cell control wells (cells were not treated with samples or infected with viruses) and virus control wells (cells were infected with viruses but not treated with samples) were set up. The viral protein in each well was determined by ELISA, and the neutralizing activity of samples of different concentrations was calculated using the raw data. The serum neutralization titer was then calculated using a 4-parameter curve fit in Graphpad Prism. As shown Figure 4C As shown, the tested lipid nanoparticle compositions induced higher antibody titer levels.

[0701] Mouse spleen IFN-γ ELISPOT assay

[0702] Mouse spleens were placed on a 70 μm cell sieve and ground. Dulbecco's phosphate-buffered saline (DPBS) containing fetal bovine serum (FBS) was added to wash the cells on the sieve. After centrifugation, red blood cell lysis buffer was added. After lysis was complete, DPBS containing 5% FBS was added to terminate the lysis. After centrifugation, the cells were resuspended in RPMI-1640 medium (containing 10% FBS and 1% penicillin-streptomycin (PS)). The cells were counted using a cell counter.

[0703] The mouse IFN-γ ELISpot plate (pre-coated with IFN-γ antibody) was washed with DPBS and sealed with culture medium containing serum and penicillin-streptomycin solution. The culture medium in the sealed plate was discarded, and then spleen cells and stimulation conditions were added to the wells (control group: culture medium; positive control group: concanavalin A (ConA); test group: PepMix TMHRSVB (fusion protein F0). The plate was incubated in a 37°C cell culture incubator for 40 hours. After incubation, the plate was washed with DPBS, and the test antibody was added and incubated for 2 hours. The medium was discarded, the plate was washed with DPBS, and the alkaline phosphatase marker was added and incubated for 1 hour. The medium was discarded, the plate was washed with DPBS, and the BCIP-NBT staining substrate was added. After staining, the plate was washed with water for quenching, and dried. The spots were counted by ELISPOT.

[0704] like Figure 4D As shown in PepMix TM Under stimulation with the HRSVB (fusion protein F0) peptide library, the average number of spots in the 0.5 μg, 10 μg, and 50 μg groups was significantly increased compared to the average number of spots in the PBS group (average number of spots was 860, 715, 1197, and 28, respectively). The results indicate that under RSVB (fusion protein F0) peptide stimulation conditions, significant F protein-specific T cell responses were detected in the splenocytes of mice immunized with the test lipid nanoparticle composition.

[0705] Example 7: In vivo immunogenicity assay and stimulation test

[0706] A lipid nanoparticle composition comprising RSV mRNA (WT (v2)) and STAR0116 was prepared according to the method of Example 4. The solution was concentrated to 0.5 mg / mL (mRNA concentration) and filtered with a 0.22 μm microporous filter to obtain mRNA containing the lipid nanoparticle composition. The test lipid nanoparticle composition was tested using female Balb / C mice (6 to 8 weeks old). Ten mice were used in each group, and each mouse was injected with 50 μL, with a dose gradient set to 0.5 μg, 10 μg and 50 μg. The negative control was an injection of an equal volume of normal saline. The animals were immunized on day 0 and day 21, respectively, and the weight of the mice was monitored after administration. On day 35, blood was collected and used for serological testing. On day 42, 1.7x10 6 Mice were challenged intranasally with 1000 FU of RSV A2 (stain number: Lot20220414, deposited at the State Key Laboratory of Virology) and weighed daily after infection. On day 4 post-infection, mice were euthanized. Viral load was measured in the right lung. The left lung was fixed with 4% paraformaldehyde.

[0707] Clinical observation

[0708] During the immunization phase, mice in all groups displayed normal climbing, foraging, drinking, exercise, and stress responses, with no signs of piloerection, arched backs, or lethargy. Weight gain was observed in all groups. During the challenge phase, all mice in each group experienced weight loss after infection, with no other abnormal clinical manifestations.

[0709] Neutralizing antibody test

[0710] Neutralizing antibody titers were determined as described in Example 6 above. Figure 5A As demonstrated in the previous examples, the results showed that the tested lipid nanoparticle compositions were highly immunogenic and could induce higher antibody titer levels.

[0711] Viral stimulation test

[0712] Add 1 ml of PBS to the tissue grinding tube. Place the tissue grinding tube on a tissue cell disruptor and process it at 5000 rpm for 40 seconds twice. It was observed that the lung tissue was broken into a uniform suspension. Centrifuge the tissue grinding tube at 1,000 g for 1 minute, centrifuge the foam and tissue residue, and transfer it to a biosafety cabinet. Take 200 μL of supernatant and extract RNA using the Vazyme nucleic acid extraction kit (RM201-02 96rxn). After RNA extraction, cDNA was obtained by reverse transcription using the ZOMANBIO reverse transcription kit, and then the virus was quantitatively determined by the Monad qPCR kit. The subsequent experimental system and process were carried out in accordance with the instructions of the selected qPCR kit. The viral load in the sample was calculated using the standard curve external standard method. As Figure 5B As shown, on day 4 after infection, the viral load in the right lung of the vaccine groups (including low-, medium-, and high-dose groups) was lower than that of the control group, and the viral load in the high- and medium-dose groups was significantly different from that of the control group. In addition, the tissue viral load showed a downward trend with increasing vaccine doses. These results indicate that the lipid nanoparticle composition has a protective effect against RSV A2 infection.

[0713] Pathology of lung tissue after stimulation

[0714] The left lung tissue of the mice was fixed with 4% paraformaldehyde and subjected to histopathological examination. The lung tissue was subjected to pathological analysis, mainly evaluating inflammatory cell infiltration, alveolar inflammation, capillary dilation, cross-sectional bronchial counts, and cross-sectional inflammatory cell infiltration bronchial counts. The scoring criteria are shown in Table 12, and the results are shown in Table 13. Some microscopic examination results are shown in Figure 5C The above results show that the immune group and the negative control group showed similar lung tissue pathology. The lipid nanoparticle composition of the present invention does not cause significant vaccine-associated enhanced respiratory disease (VAERD) phenomenon, that is, the situation in which the vaccine-immunized population has worsened lower respiratory tract symptoms after pathogen infection.

[0715] Table 12. Pathological scoring criteria

[0716]

[0717] Table 13. Lung histopathological scores after stimulation

[0718]

[0719]

[0720] Example 8: Immunization protocol optimization

[0721] A lipid nanoparticle composition comprising RSV mRNA (WT (v2)) and STAR0116 was prepared according to the method of Example 4. The solution was concentrated to 0.5 mg / mL (mRNA concentration) and filtered with a 0.22 μm microporous filter to obtain mRNA containing the lipid nanoparticle composition. The test lipid nanoparticle composition was tested using female Balb / C mice (6 to 8 weeks old). Five mice were used in each group, and according to the following Table 14, 50 μL were injected into each mouse at a dose of 10 μg vaccine. An equal volume of normal saline was injected as a negative control. Blood was collected on the 14th day after the second injection for serological testing.

[0722] Table 14. Immunization doses and schedule

[0723]

[0724] RSV F antibody test

[0725] ELISA plates were coated with recombinant human RSV fusion glycoprotein (rec.hu F protein, final concentration: 0.2 μg / mL) (Sino Biological Inc.). The coated plates were incubated with the given serum dilutions and the specific binding of the antibodies to the RSV-F protein was determined using an HRP-labeled anti-mouse antibody using TMB substrate. Figure 6 The results showed that the lipid nanoparticle composition tested induced higher RSV-F antibody titers and that the double-dose vaccination schedule on day 0 / day 21 was more effective, which will be used in the following examples.

[0726] Example 9: In vivo immunogenicity test of RSV mRNA vaccine (1)

[0727] A lipid nanoparticle composition comprising RSV mRNA and STAR0002 was prepared according to the method of Example 4. The solution was concentrated to 0.5 mg / mL (mRNA concentration) and filtered with a 0.22 μm microporous filter to obtain mRNA containing the lipid nanoparticle composition. The test lipid nanoparticle composition was tested using female Balb / C mice (6 to 8 weeks old). The mRNA sequence and the lipid nanoparticles used in the RSV mRNA vaccine are shown in Table 15 below. Five mice were used in each group, and 50 μL was injected into each mouse at a 10 μg vaccine dose. An equal volume of normal saline was injected as a negative control. Immunization was performed on day 0 and day 21. Blood was collected on day 14 ("Week 2"), day 21 ("Week 3"), day 28 ("4W"), day 35 ("Week 5"), day 42 ("Week 6"), day 49 ("Week 7"), day 56 ("Week 8"), and / or day 63 ("Week 9") for serological testing.

[0728] Table 15. RSV vaccines

[0729]

[0730] RSV F antibody test

[0731] ELISA plates were coated with recombinant human RSV fusion glycoprotein (rec.hu F protein, final concentration: 0.2 μg / mL) (Sino Biological Inc.). The coated plates were incubated with the given serum dilutions and the specific binding of the antibodies to the RSV-F protein was determined using an HRP-labeled anti-mouse antibody using TMB substrate. Figure 7 、 Figure 8 and Figure 9 It showed that RSV mRNA vaccine induced higher RSV-F antibody titers.

[0732] Example 10: RSV mRNA vaccine in vivo immunogenicity test (2)

[0733] A lipid nanoparticle composition comprising RSV mRNA and STAR0225 was prepared according to the method of Example 4. The solution was concentrated to 0.5 mg / mL (mRNA concentration) and filtered with a 0.22 μm microporous filter to obtain mRNA containing the lipid nanoparticle composition. The test lipid nanoparticle composition was tested using female Balb / C mice (6 to 8 weeks old). The mRNA sequence and the lipid nanoparticles used in the RSV mRNA vaccine are shown in Table 16 below. Five mice were used in each group, and 50 μL was injected into each mouse at a 10 μg vaccine dose. An equal volume of normal saline was injected as a negative control. Immunization was performed on day 0 and day 21. Blood was collected on day 14 ("week 2") and day 35 ("week 5") and used for serological testing.

[0734] Table 16. RSV vaccines

[0735] RSV vaccine mRNA sequences lipid nanoparticles 210-TSV SEQ ID NO:14 STAR0225 210-TV SEQ ID NO:16 STAR0225 210-T SEQ ID NO: 18 STAR0225 210-213-TV SEQ ID NO:24 STAR0225 210-213-T SEQ ID NO:26 STAR0225 WT(v2) SEQ ID NO:2 STAR0225

[0736] RSV F antibody test

[0737] ELISA plates were coated with recombinant human RSV fusion glycoprotein (rec.hu F protein, final concentration: 0.2 μg / mL) (SinoBiological Inc.). The coated plates were incubated with the given serum dilutions, and specific binding of the antibodies to the RSV-F protein was determined using an HRP-labeled anti-mouse antibody using TMB substrate. FIG. 10A to FIG. 10B It showed that RSV mRNA vaccine induced higher RSV-F antibody titers.

[0738] Prefusion RSV-F antibody and postfusion RSV-F antibody test

[0739] The RSV-F protein before fusion and the RSV-F protein after fusion in serum were detected by the RSV-F antibody (IgG) quantitative and qualitative detection kit (ELISA) (Vazyme, catalog number: DD3910-01) and the RSV-F antibody (IgG) quantitative and qualitative detection kit (ELISA) (Vazyme, catalog number: DD3911-01). The detection was carried out according to the kit instructions, and the results were Figure 10C and Figure 10D As shown in . It can be seen that RSV mRNA vaccine can induce higher RSV prefusion protein antibody titers and higher RSV postfusion protein antibody titers.

[0740] Example 11: Safety Study (1)

[0741] A lipid nanoparticle composition comprising RSV mRNA (WT (v2)) and STAR0116 was safety tested using cynomolgus monkeys. The lipid nanoparticle composition was prepared according to the method of Example 4. The solution was concentrated to 0.5 mg / mL (mRNA concentration) and filtered with a 0.22 μm microporous filter to obtain mRNA containing the lipid nanoparticle composition. The immunization dose and schedule are shown in Table 17 below. The lipid nanoparticle composition was administered to cynomolgus monkeys at a dose of 125 μg per cynomolgus monkey via upper arm intramuscular injection, with an injection volume of 500 μl per monkey. An equal volume of normal saline was injected as a negative control. Immunization was performed on days 0 and 21. After administration, the injection site was observed once a day until day 49 (observation for tenderness, erythema, swelling, etc.). Body temperature was measured 1 hour before each administration (-1 hour), at the time of inoculation (0 hour), and 0.5 hours, 2 hours, 6 hours, and 24 hours after administration. Body weight was measured once a week. Blood was collected on day 0 (D0), day 1 (D1), and day 22 (D22) for CBA tests to assess vaccine safety. On day 28, blood was collected for biochemical tests to assess vaccine safety.

[0742] Table 17. Immunization doses and schedule

[0743]

[0744] Clinical observation in cynomolgus monkeys

[0745] During the experiment, the test group did not show any adverse clinical manifestations, and there was no significant change in the body weight of the animals in each group. During the experiment, the body temperature of the crab-eating macaques in the RSV mRNA group increased slightly at 2h, 6h, and 24h after the first administration compared with the negative control group, but there was no statistical difference. After the second administration, the RSV mRNA group showed a slight increase in body temperature at 6h and 24h, and the increase in body temperature at 6h was statistically different. Among them, the body temperatures of the negative control group and the RSV mRNA group were 38.80±0.26℃ and 40.24±0.63℃, respectively.

[0746] Determination of cytokine levels in plasma samples by CBA

[0747] Plasma samples were thawed and diluted 4-fold for use according to LEGENDplex TM NHP Th cytokine panel (10-plex) (BioLegend-740387) instructions, to measure cytokines (IL-2, IL-5, IL-6, IL-10, IL-13, TNF-α, IFN-γ, IL-4, IL-17A, IL-21) in plasma samples.

[0748] Date handling:

[0749] Standard curve: The standard concentration is used as the X-axis, the logarithm (standard median) is used as the Y-axis, and a 4-parameter curve fit is used (Y=(AD) / [1+(x / C)^B]+D).

[0750] The sample concentration was calculated using the standard curve, x(sample concentration) = [(AD) / (yD)-1]^(1 / B)×C.

[0751] Cytokine concentration (pg / mL) = IL-2 / IL-5 / IL-6 / IL-10 / IL-13 / IFN-γ / IL-4 / IL-17A / IL-21 / TNF-α concentration × 4 (sample dilution factor)

[0752] Cytokine release curves were fitted using GraphPad Prism.

[0753] The results are shown in Table 18. Compared with the IL-6 level on day 0 before administration, the plasma IL-6 levels of cynomolgus monkeys in the RSV mRNA groups 2F001, 2F002, 2F003, 2M001, and 2M002 were significantly increased on day 1 and day 22 after administration. The overall levels of IL-2, IL-5, IL-10, IL-13, IFN-γ, IL-4, IL-17A, IL-21, and TNF-α did not change significantly.

[0754] Table 18. Cytokine levels in plasma samples

[0755]

[0756] “-” indicates that the cytokine concentration is below the test line.

[0757] Blood biochemistry tests

[0758] The serum collected on day 28 was thawed and 27 blood biochemical parameters in the serum were measured using a Hitachi 7100 automated biochemical analyzer. The results are shown in Table 19. Compared with the negative control group, the serum NEFA (non-esterified fatty acid) level in the RSV mRNA group was significantly increased, while the other parameters showed no significant differences.

[0759] Table 19. Blood biochemical indicators

[0760] Example 12: Safety Study (2)

[0761] A lipid nanoparticle composition comprising RSV mRNA (WT (v2)) and STAR0132 was safety tested in cynomolgus monkeys. The lipid nanoparticle composition was prepared according to the method of Example 4. The solution was concentrated to 0.5 mg / mL (mRNA concentration) and filtered through a 0.22 μm microporous filter to obtain mRNA containing the lipid nanoparticle composition. The immunization dose is shown in Table 20 below. The lipid nanoparticle composition was injected into rats at 300 μL per rat. An equal volume of solvent was used as a negative control. SM102 contained in STAR0002 was used as a positive control.

[0762] Immunizations were performed on day 0. Body temperature and body weight were measured post-immunization. Blood samples were collected on days 3 and 14 for cytokine, blood count, and coagulation tests. On day 14, rats from groups 4, 5, 6, and 7 were sacrificed, and their hearts, livers, lungs, and kidneys were harvested for histopathological analysis.

[0763] Table 20. Rat immunization dose

[0764]

[0765] Clinical observation

[0766] like Figure 11A and Figure 11B As shown, during the experiment, the animals in the RSV mRNA group did not develop adverse clinical symptoms, and compared with the negative control group, the body weight and body temperature of the rats in the RSV mRNA group did not change significantly after administration.

[0767] Cytokine assay

[0768] The rat blood samples were assayed on day 3 after administration using LiankeBio rat IL-2 ELISA kit (EK302), rat TNF-α ELISA kit (EK382), and rat IFN-γ ELISA kit (EK380). Figure 11C As shown in the figure, compared with the negative control group, there was no significant change in the levels of IL-2, TNF-α and IFN-γ in each RSV mRNA group.

[0769] Coagulation function tests

[0770] The blood samples of rats were collected for coagulation function test on the 14th day after administration. Figure 11D Compared with the negative control group, the coagulation function levels of each RSV mRNA group had no significant changes.

[0771] Routine blood test

[0772] Blood samples of rats were collected for routine blood tests on the 14th day after administration. Figures 11E to 11K Compared with the negative control group, there was no significant change in the blood routine test levels of RSV mRNA in each group.

[0773] Organ histopathology test

[0774] The heart, liver, lung and kidney tissues of Groups 4, 5, 6 and 7 were fixed with 4% paraformaldehyde and subjected to histopathological tests. Pathological analysis of the heart, liver, lung and kidney tissues was performed to evaluate the relevant indicators of acute toxicity in rats. A small number of inflammatory cells were observed in the heart tissues of the above four groups. A small number of inflammatory cells and mild vacuolar degeneration of hepatocytes were observed in the liver tissues of the above four groups. Moderate to severe inflammatory cell infiltration and thickening of the pulmonary septum were observed in most of the lung tissues of the above four groups. Mild to moderate degeneration and necrosis were observed in the renal tubular epithelial cells of the above four groups, accompanied by a small amount of inflammatory cell infiltration. No significant differences were found in organ abnormalities between the negative control group 7 and the other three RSV mRNA groups.

[0775] The foregoing description is to be considered merely as an illustration of the principles of the present disclosure. Furthermore, since numerous modifications and variations will be apparent to those skilled in the art, the present invention is not limited to the exact configuration and process shown above. Therefore, all suitable modifications and equivalents are deemed to fall within the scope of the present invention as defined by the appended claims.

Claims

1. A lipid nanoparticle composition comprising: a target polynucleotide comprising a nucleic acid encoding a respiratory syncytial virus antigenic polypeptide or a variant thereof, and Lipid nanoparticles comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R a Selected from the group consisting of hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl and R 6 ; R 1 for R 2 for R 3 for R 4 for R 5 If it exists, R 6 If it exists, Each W is independently selected from O, S or NR b , and each R b independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl; Each Y is independently selected from O, S, NR c 、N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W), and each R c independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl; Each Z is independently selected from C, S or S(O); Each n is independently 0, 1, 2, 3, 4 or 5; Each m is independently 0, 1, 2 or 3; Each p is independently 1, 2, 3 or 4; and R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c Each is independently selected from the group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl groups are optionally substituted with one or more groups independently selected from the group consisting of halogen, hydroxy, oxo, cyano, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl groups are optionally interrupted by one or more groups independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl.

2. The lipid nanoparticle composition according to claim 1, wherein The compound is of formula (A) or formula (B):

3. The lipid nanoparticle composition according to claim 1 or 2, wherein R 2 、R 3 and R 4 At least two of them are the same.

4. The lipid nanoparticle composition according to any one of claims 1 to 3, wherein R 2 、R 3 and R 4 At least three of them are the same.

5. The lipid nanoparticle composition according to any one of claims 1 to 4, wherein One or more of Y is O, NR c or N(R c )N(R c )Z(W).

6. The lipid nanoparticle composition according to any one of claims 1 to 5, wherein One or more of W is O.

7. The lipid nanoparticle composition according to claim 5, wherein R c One or more of them is hydrogen.

8. The lipid nanoparticle composition according to any one of claims 1 to 7, wherein One or more of Z is C or S(O).

9. The lipid nanoparticle composition according to any one of claims 1 to 8, wherein R 2c 、R 3c and R 4c One or more of them is an alkyl group or an alkenyl group.

10. The lipid nanoparticle composition according to any one of claims 1 to 9, wherein R 2c 、R 3c and R 4c One or more of them is C 8-24 Alkyl or alkenyl.

11. The lipid nanoparticle composition according to any one of claims 1 to 10, wherein R 2c 、R 3c and R 4c One or more of them is C 10-24 Alkyl or alkenyl.

12. The lipid nanoparticle composition according to any one of claims 1 to 11, wherein R 2c 、R 3c and R 4c One or more of the is an alkenyl group containing one, two or three C=C double bonds.

13. The lipid nanoparticle composition according to any one of claims 1 to 12, wherein R 2c 、R 3c and R 4c One or more of the is an alkenyl group containing one or more Z-olefins.

14. The lipid nanoparticle composition according to any one of claims 1 to 13, wherein R 1c It is an alkyl group.

15. The lipid nanoparticle composition according to any one of claims 1 to 14, wherein R 1c C 1-12 alkyl.

16. The lipid nanoparticle composition according to any one of claims 1 to 15, wherein R 1c C 4-10 alkyl.

17. The lipid nanoparticle composition according to any one of claims 1 to 16, wherein R a C 1-6 Alkyl, optionally substituted with one or more groups independently selected from the group consisting of hydroxy, cycloalkyl, and heteroaryl.

18. The lipid nanoparticle composition according to any one of claims 1 to 17, wherein R a is methyl, ethyl, propyl, butyl or pentyl.

19. The lipid nanoparticle composition according to any one of claims 1 to 4, wherein One or more of are independently selected from the following group:

20. The lipid nanoparticle composition according to any one of claims 1 to 4, wherein R 1c 、R 2c 、R 3c and R 4c One or more of are independently selected from the following group:

21. The lipid nanoparticle composition according to any one of claims 1 to 20, wherein R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c If present, each does not contain two heteroatoms directly bonded to each other.

22. The lipid nanoparticle composition according to any one of claims 1 to 20, wherein R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c If present, each contains -N(R c )-N(R c )-or-S(O)2-N(R c )-.

23. The lipid nanoparticle composition according to any one of claims 1 to 22, wherein The compounds have the structures listed in the table below:

24. The lipid nanoparticle composition according to any one of claims 1 to 23, wherein The lipid nanoparticles further comprise a neutral lipid, a sterol or a sterol derivative, and a surfactant.

25. The lipid nanoparticle composition according to claim 24, wherein (i) The neutral lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), 1,2-di-O-octadecenyl-5-glycero-3-phosphocholine (18:0 diether PC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1-oleoyl-2-cholestyhemisuccinyl-5-glycero-3-phosphocholine (OChemsPC), and any mixture thereof; (ii) the sterol is cholesterol, β-sitosterol, stigmasterol, ergosterol, brassicasterol, coproposterol or campesterol; and / or (iii) the surfactant is selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol (DSG-PEG), N-(methylpolyoxyethylenecarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)] (DOPE-PEG), and any mixture thereof.

26. The lipid nanoparticle composition according to claim 25, wherein The neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), the sterol is cholesterol, and the surfactant is DMG-PEG.

27. The lipid nanoparticle composition according to any one of claims 24 to 26, wherein Based on the total amount of the lipid nanoparticles, the lipid nanoparticles contain the following molar ratios: (i) 10% to 65% of the compound of formula (I); (ii) 5% to 30% of said neutral lipids; (iii) 15% to 50% of said sterol or said sterol derivative; and (iv) 0.5% to 5% of said surfactant.

28. The lipid nanoparticle composition according to any one of claims 24 to 27, wherein Based on the total amount of the lipid nanoparticles, the lipid nanoparticles contain the following molar ratios: (i) 40-65% of the compound of formula (I); (ii) 5% to 15% of said neutral lipids; (iii) 25% to 50% of said sterol or said sterol derivative; and (iv) 1% to 5% of said surfactant.

29. The lipid nanoparticle composition according to any one of claims 24 to 28, wherein Based on the total amount of the lipid nanoparticles, the lipid nanoparticles contain the following molar ratios: (i) 40-65% of the compound of formula (I); (ii) 5% to 15% of said DSPC; (iii) 25% to 50% of said cholesterol; and (iv) 1%-5% of said DMG-PEG.

30. The lipid nanoparticle composition according to any one of claims 24 to 29, wherein Based on the total amount of the lipid nanoparticles, the lipid nanoparticles contain the following molar ratios: (i) 40-50% of the compound of formula (I); (ii) 5% to 15% of said DSPC; (iii) 25% to 50% of said cholesterol; and (iv) 1.5%-2.55% of said DMG-PEG.

31. The lipid nanoparticle composition according to any one of claims 1 to 30, wherein The nucleic acid encodes the respiratory syncytial virus antigenic polypeptide, which comprises: a) an amino acid sequence having amino acid residues 26-574 of SEQ ID NO: 38, or b) a variant of (a) having at least one amino acid substitution selected from the group consisting of: NO:38, S55C, P102A, T103C, R106K, R109Q, R133Q, K134Q, R135Q, R136Q, L142Q, I148C, A149C, S155C, L188C, S190F, V207L, K209P, Q210P, Q210C, S211P, C212P, S290C, I379V, M447V, Y458C, L481P, V482P, Q501G and L512K, wherein the variant has respiratory syncytial virus antigenic activity.

32. The lipid nanoparticle composition according to any one of claims 1 to 31, wherein The nucleic acid encodes the respiratory syncytial virus antigenic polypeptide, and the respiratory syncytial virus antigenic polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:38-69, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 179, 181, 183, and 287-370, or an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:38-69, 81, 83, 85, 87, 89, 91, NO:38-69, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 13 3, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 179, 181, 183, and 287-370.

33. The lipid nanoparticle composition according to any one of claims 1 to 32, wherein The nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-33, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 182, 174, 176, 178, 180, 182, and 202-286.

34. The lipid nanoparticle composition according to any one of claims 1 to 33, wherein The target polynucleotide comprises a nucleic acid having SEQ ID NO: 2, 4, 14, 16, 18, 24, 26, 30, 31, 32, 33, 203, 205, 215, 217, 219, 225, 227, 231, 232, 233 or 234, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 2, 4, 14, 16, 18, 24, 26, 30, 31, 32, 33, 203, 205, 215, 217, 219, 225, 227, 231, 232, 233 or 234, which encodes a polypeptide having respiratory syncytial virus antigenic activity.

35. The lipid nanoparticle composition according to any one of claims 1 to 34, wherein The target polynucleotide comprises: (i) a 5' untranslated region (UTR) comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 34, 37, and 70-71; and / or (ii) a 3' untranslated region (UTR) comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 35 and 72-74; and / or (iii) a poly-A region, which is 50-120 nucleotides in length.

36. A respiratory syncytial virus polypeptide variant comprising an amino acid sequence having amino acid residues 26-574 of SEQ ID NO: 38, wherein the amino acid sequence having amino acid residues 26-574 of SEQ ID NO: 38 has at least one amino acid substitution, wherein: The at least one amino acid substitution is selected from the group consisting of S55C, P102A, T103C, R106K, R109Q, R133Q, K134Q, R135Q, R136Q, L142Q, I148C, A149C, S155C, L188C, S190F, V207L, K209P, Q210P, Q210C, S211P, C212P, S290C, I379V, M447V, Y458C, L481P, V482P, Q501G and L512K according to SEQ ID NO:38, wherein the variant has respiratory syncytial virus antigenic activity.

37. The respiratory syncytial virus polypeptide variant of claim 36, comprising at least two amino acid substitutions selected from the group consisting of S55C, P102A, T103C, R106K, R109Q, R133Q, K134Q, R135Q, R136Q, L142Q, I148C, A149C, S155C, L188C, S190F, V207L, K209P, Q210P, Q210C, S211P, C212P, S290C, I379V, M447V, Y458C, L481P, V482P, Q501G, and L512K according to SEQ ID NO: 38, wherein The variant has respiratory syncytial virus antigenic activity.

38. The respiratory syncytial virus polypeptide variant of claim 36 or 37, comprising an amino acid substitution selected from the group consisting of: i) K209P and Q210P; ii) Q210P and S211P; iii) S211P and C212P; iv) L481P; v) L481P and V482P; vi) K209P, Q210P, L481P and V482P; vii) Q210P, S211P, L481P and V482P; viii) S211P, C212P, L481P and V482P; ix) K209P, Q210P and L481P; x) Q210P, S211P and L481P; xi) S211P, C212P and L481P; xii) S55C, P102A, T103C, I148C, L188C, V207L, Q210P, I379V, and M447V; xiii) S55C, P102A, T103C, I148C, L188C, Q210P, I379V, and M447V; xiv) P102A, T103C, I148C, V207L, Q210P, I379V, and M447V; xv) S55C, P102A, L188C, V207L, Q210P, I379V, and M447V; xvi) P102A, T103C, I148C, Q210P, I379V, and M447V; xvii) S55C, P102A, L188C, Q210P, I379V, and M447V; xviii) P102A, V207L, Q210P, I379V, and M447V; xix) V207L ​​and Q210P; xx) S55C, P102A, T103C, I148C, L188C, V207L, Q210C, S213C, I379V, and M447V; xxi) S55C, P102A, T103C, I148C, L188C, Q210C, S213C, I379V, and M447V; xxii) P102A, T103C, I148C, V207L, Q210C, S213C, I379V, and M447V; xxiii) S55C, P102A, L188C, V207L, Q210C, S213C, I379V, and M447V; xxiv) P102A, T103C, I148C, Q210C, S213C, I379V, and M447V; xxv) S55C, P102A, L188C, Q210C, S213C, I379V, and M447V; xxvi) P102A, V207L, Q210C, S213C, I379V, and M447V; xxvii) V207L, Q210C, and S213C; xxviii) S55C, P102A, L188C, I379V, and M447V; xxix) P102A, T103C, I148C, I379V, and M447V; xxx) P102A, I379V, and M447V; xxxi) P102A and M447V; xxxii) E30V, P102A, I379V, and M447V; xxxiii) T54H, P102A, I379V, and M447V; xxxiv) N88S, P102A, I379V, and M447V; xxxv) P102A, T103A, I379V, and M447V; xxxvi) P102A, A122T, I379V, and M447V; xxxvii) P102A, K124N, I379V, and M447V; xxxviii) P102A, T125N, I379V, and M447V; xxxix) P102A, R136K, I379V, and M447V; xxxx)P102A, V152I, I379V, and M447V; xxxxi) P102A, S190I, I379V, and M447V; xxxxii) P102A, N227S, I379V, and M447V; xxxxiii) P102A, V296, I379V I, and M447V; xxxxiv) P102A, Q354L, I379V, and M447V; xxxxv)P102A, L373R, I379V, and M447V; xxxxvi) P102A, I379V, M447V, and D486S; xxxxvii) P102A, I379V, M447V, and S540L; xxxxviii) P102A, I379V, M447V, and L547F; xxxxix) S155C, P102A, S290C, I379V, and M447V; xxxxx) P102A, A149C, I379V, M447V, and Y458C; xxxxxi) a stretch of 27 amino acid residues from E110 to R136 is substituted by the residues -GS-, and a 50-amino acid residue stretch from I525 to N574 is replaced by residues GYIPEAPRDGQAYVRKDGEWVLLSTFL; xxxxxii) a stretch of 27 amino acid residues from E110 to R136 is substituted by the residues -GS-, and a 24-amino acid residue segment from K551 to N574 is deleted; xxxxxiii) an 18 amino acid residue stretch from F137 to V154 is deleted, and a 50 amino acid residue stretch from I525 to N574 is replaced by residues GYIPEAPRDGQAYVRKDGEWVLLSTFL; xxxxxiv) deletion of an 18 amino acid residue segment from F137 to V154, and deletion of a 50 amino acid residue segment from I525 to N574; xxxxxv) deletion of an 18 amino acid residue segment from F137 to V154; xxxxxvi) a stretch of 45 amino acid residues from E110 to V154 is substituted by the residues -GS-, and a 50-amino acid residue stretch from I525 to N574 is replaced by residues GYIPEAPRDGQAYVRKDGEWVLLSTFL; xxxxxvii) a stretch of 45 amino acid residues from E110 to V154 is substituted by the residues -GS-, and a 24 amino acid residue segment from K551 to N574 is deleted; and xxxxxviii) A stretch of 45 amino acid residues from E110 to V154 is substituted by the residues -GS-.

39. The respiratory syncytial virus polypeptide variant of any one of claims 36 to 38, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-69, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 179, 181, 183, and 287-370.

40. An isolated nucleic acid sequence encoding a respiratory syncytial virus polypeptide comprising an amino acid sequence of amino acids 26-574 of SEQ ID NO: 38, or a respiratory syncytial virus polypeptide variant according to any one of claims 36 to 39.

41. The isolated nucleic acid sequence of claim 40, wherein The isolated nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-33, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 182, 174, 176, 178, 180, 182, and 202-286.

42. A respiratory syncytial virus vaccine comprising the lipid nanoparticle composition of any one of claims 1 to 35, the respiratory syncytial virus polypeptide variant of any one of claims 36 to 39, or the isolated nucleic acid sequence of claim 40 or 41.

43. A method of inducing an antigen-specific immune response in a subject, comprising: The respiratory syncytial virus vaccine of claim 44 is administered to the subject in an amount effective to generate an antigen-specific immune response.

44. The method according to claim 43, wherein The method of inducing an antigen-specific immune response involves a single administration of the respiratory syncytial virus vaccine.

45. The method according to claim 43 or 44, further comprising: A booster dose of the RSV vaccine was administered.

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