Compounds, lipid nanoparticles comprising the same and uses thereof

Abstract

The present disclosure is related to a compound of Formula (I), the lipid nanoparticle and pharmaceutical composition comprising the same, and the use thereof.

Description

Attorney Docket No. 4404-0141PW01COMPOUNDS, LIPID NANOPARTICLES COMPRISING THE SAME AND USES THEREOFRELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S Provisional Application 63 / 745,019 filed on January 14, 2025, the entire content of which is incorporated herein by reference.FIELD OF INVENTION

[0002] The present disclosure is related to a compound of Formula (I), the lipid nanoparticle and pharmaceutical composition comprising the same, and the use thereof.BACKGROUND OF THE INVENTION

[0003] Increasing advances in nanotechnology and nanoscience have raised great hope in the field of biomedicine. Being a physiological analog to the cellular membrane, lipid nanoparticles have superior biocompatibility compared to polymeric-based nanoparticles, leading to more acceptable biomedical application choices. Therapeutic drugs are incorporated into the nanoparticles (NPs) mainly by physical interactions including, entrapment, surface attachment, or encapsulation. Considered as an attractive carrier for therapeutic agents and remain subject to continued development efforts, there remains a great need for improvement of lipid NP delivery systems.

[0004] With the emergence of pandemics, lipid nanoparticles have been thoroughly investigated and successfully entered the clinic for the delivery of small molecules, siRNA drugs and mRNA. A variety of lipid nanoparticles have been explored and optimized for mRNA delivery, providing valuable information for the future design of mRNA therapeutics. While not limiting, many other lipid nanoparticle-mRNA formulations have been developed and are under clinical evaluation for the prevention and treatment of virus infections, cancer and genetic diseases. The development of novel lipid nanoparticles and other types of delivery formulation will further enable mRNA-based therapies for a broad range of diseases and improve health care in the future.SUMMARY OF THE INVENTION

[0005] An aspect provided herein is a compound of Formula (I):Attorney Docket No. 4404-0141PW01whereinW is hydroxy, Ci-6 alkoxy, or -NR2R3;Ri is linear or branched C6-20 alkyl or alkenyl;R2 and R3 are independently linear or branched C1-6 alkyl unsubstituted or substituted by hydroxy;Zi is absent or -A-C(=O)-;A is O or NH;Z2 is C6-C20 alkyl or ;Ra is hydrogen or Ci-Ce alkyl;Rb and Rc are independently hydrogen or -Rd-OC(=O)Re;Rd is Ci-Ce alkylene;Re is linear or branched C6-C20 alkyl or alkenyl;X is -CH- or -N-; Y is -O- or -NH-;m is 1 to 5; the sum of m and is 1 to 6; o is 1 to 7; p is 1 to 7;provided that Rb and Rc are not both hydrogen; andprovided that when Zi is absent, Z2 is not alkyl.or a pharmaceutically acceptable salt thereof.

[0006] Another aspect provided herein is a lipid nanoparticle, comprising the compound of Formula (I) as defined in the present disclosure.

[0007] Another aspect provided herein is a pharmaceutical composition, comprising the compound of Formula (I) or the lipid nanoparticle as defined in the present disclosure, and a pharmaceutically acceptable excipient.

[0008] Another aspect provided herein is a method of treatment of a disease, comprising administering an effective amount of the lipid nanoparticles or theAttorney Docket No. 4404-0141PW01pharmaceutical composition as defined in the present disclosure to a subject in need thereof.

[0009] Another aspect provided herein is a method of vaccinating a subject in need thereof, comprising administering an effective amount of the lipid nanoparticles or the pharmaceutical composition as defined in the present disclosure to the subject.

[0010] Another aspect provided herein is a method of inducing or eliciting an immune response in a subject in need thereof, comprising administering an effective amount of the lipid nanoparticles or the pharmaceutical composition as defined in the present disclosure to the subject.BRIEF DESCRIPTION OF DRAWING

[0011] Fig. 1 illustrates the functional delivery efficiency (relative light units (RLU / mg protein)) of the ionizable lipids according to Table 4.DETAILED DESCRIPTION OF THE INVENTION

[0012] Various aspects of the disclosure are described in further detail in the following subsections:I. Definition

[0013] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below.

[0014] Unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed.

[0015] The terms “a”, “an” and “the” and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0016] As used herein, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples orAttorney Docket No. 4404-0141PW01elsewhere in the Specification in the context of a particular assay, result or embodiment, the term “about” means within one standard deviation per the practice in the art, or a range of up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0017] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has”, “having”, “contains”, “containing”, or any other variation thereof, are intended to cover a non-exclusive inclusion, subject to any limitation explicitly indicated. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.

[0018] The transitional phrase “consisting of’ excludes any elements, steps, or ingredients not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0019] Where applicants have defined an invention or a portion thereof with an open-ended term such as “comprising” it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the term “consisting of’.

[0020] Where used herein, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “X and / or Y” is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

[0021] The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0022] The expression “in one embodiment” “in some embodiment” and the like generally mean that the particular feature, structure, or characteristic following the phrase may be included in at least one embodiment of the present disclosure, and may be included in more than one embodiment of the present disclosure (importantly, such phrases do not necessarily refer to the same embodiment).Attorney Docket No. 4404-0141PW01

[0023] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context.

[0024] The term “alkyl” refers to a monoradical of a saturated straight or branched hydrocarbon. Specifically, the alkyl group comprises, for example, from 1 to 20 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, which can be abbreviated as C1-20 alkyl, (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, abbreviated as C1-10 alkyl), such as 1 to 17 carbon atoms, 1 to 14 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, 10 to 20 carbon atoms, 12 to 20 carbon atoms, 14 to 20 carbon atoms, 16 to 20 carbon atoms, 12 to 18 carbon atoms, 14 to 18 carbon atoms, or 15 to 20 carbon atoms. Exemplary alkyl groups include but are not limited to methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1 -methylethyl), butyl, iso-butyl, tertbutyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, tridecyl, tetradecyl or a alkyl group having more carbon atoms, or the like. Unless specified otherwise, the term “alkyl” used herein encompasses linear or branched alkyl.

[0025] The term “alkoxy” refers to an alkyl group singularly bonded to oxygen. Specifically, the term “alkoxy” refers to a group of -O-alkyl, wherein the alkyl is as defined herein. More specifically, the term “(Cx-Cy)alkoxy” as used herein refers to a -O-(Cx-Cy)alkyl, for example, “Ci-Ce alkoxy” designates a Ci-Ce alkyl group singularly bonded to an oxygen atom. Exemplary alkoxy group include but are not limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, .scc-butoxy and / c / V-butoxy. In some context, the alkoxy group may be designated with a suffix “-yloxy”. For example, the term “methyloxy” is used interchangeably with methoxy.

[0026] The term “alkylene” as used herein refers to a diradical of a saturated straight or branched hydrocarbon. Specifically, the alkylene comprises, for example, from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more specifically 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1 -propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3 -propylene), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1,1 -iso-butylene, 1,2-iso-Attorney Docket No. 4404-0141PW01butylene, and 1,3-iso-butylene), the pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5 -pentylene, 1,1-iso-pentylene, 1,1-sec-pentyl, 1,1-neo-pentyl), the hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1 -isohexylene), the heptylene isomers (e.g., 1,1 -heptylene, 1,2-heptylene, 1,3 -heptylene, 1,4-heptylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1 -isoheptylene), the octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1 -isooctylene), and the like. The straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene).

[0027] The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon chain having at least one pi (TI) bond between carbon atoms. In general, the number of pi bond(s) in a hydrocarbon chain is limited within the integer calculated by dividing the number of carbon atoms in the alkenyl group by 2. For example, in a case which an alkenyl group has 11 carbon atoms, the number of pi bond is selected from 1, 2, 3, 4, or 5. Specifically, the alkenyl group comprises, for example, from 2 to 20 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, which can be abbreviated as C2-20 alkenyl, (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, abbreviated as C2-10 alkenyl), such as 2 to 17 carbon atoms, 2 to 14 carbon atoms, 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 4 carbon atoms, 10 to 20 carbon atoms, 12 to 20 carbon atoms, 14 to 20 carbon atoms, 16 to 20 carbon atoms, 12 to 18 carbon atoms, 14 to 18 carbon atoms, or 15 to 20 carbon atoms. Although having a limit as described above, the number of pi bond(s) in an alkenyl group is independent of the number of carbon atoms. The alkenyl group may comprise from 2 to 20, abbreviated as C2-20 alkenyl, (e.g., 2 to 20) carbon atoms and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pi bond(s). The alkenyl group may comprise from 5 to 20, abbreviated as C2-20 alkenyl, (e.g., 2 to 20), carbon atoms and 3, 4, 5, 6, 7, 8, 9, or 10 pi bond(s). The alkenyl group may comprise from 10 to 20, abbreviated as C10-20 alkenyl, (e.g., 10 to 20) carbon atoms and 5, 6, 7, 8, 9, or 10 pi bond(s). The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1 -propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-Attorney Docket No. 4404-0141PW01heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like.

[0028] As used herein, the term “cationic lipid” refers to lipid species that have a net positive charge at a selected pH, such as physiological pH. “Physiological pH” as used herein refers to a pH of about 7.5. The ionizable property of a cationic lipid is capable of enhancing efficacy through helping with endosomal escape and reducing toxicity. Depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral. The cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated under certain condition, for example, under physiological conditions.

[0029] The term “helper lipid” as used herein refers to any neutral or zwitterionic lipid material. Without wishing to be held to a particular theory, helper lipids may add stability, rigidity, and / or fluidity within lipid bilayers / nanoparticles.

[0030] The term “amphiphilic”, as used herein, refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. The amphiphilic compound includes, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

[0031] The term “cryoprotectant” relates to a substance that is added to a preparation (e.g., formulation or composition) in order to protect the active ingredients of the preparation during the freezing stages.

[0032] The term “average diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Zaverage with the dimension of a length, and the poly dispersity index (PI), which is dimensionless. HereAttorney Docket No. 4404-0141PW01“average diameter”, “diameter” or “size” for particles is used synonymously with this Value of the average.

[0033] The term “poly dispersity index” as used herein is a measure of the size distribution of an ensemble of particles, e.g., nanoparticles. The poly dispersity index is calculated based on dynamic light scattering measurements by the so-called cumulant analysis.

[0034] The term “nucleic acid” comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. Nucleic acid can be isolated. In the context of the present disclosure, the “isolated nucleic acid” means that the nucleic acid was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, or was produced recombinantly by cloning, or was purified, for example, by cleavage and separation by gel electrophoresis, or was synthesized, for example, by chemical synthesis.

[0035] According to the present disclosure, the term “peptide” or “therapeutic peptide” comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds.

[0036] The term “protein” refers to large peptides, in particular peptides having at least about 151 amino acids, but as the definition of (poly)peptide and protein is not mutually exclusive, the terms “peptide”, “polypeptide”, and “protein” are used herein usually as synonyms, depending on the context.

[0037] A “therapeutic protein” has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. The use of therapeutic protein includes treatment and prevention of a condition or disease. A therapeutic protein has curative or palliative properties and may be administered to prevent, ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term “therapeuticAttorney Docket No. 4404-0141PW01protein” includes entire proteins or peptides and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a protein. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.

[0038] Examples of therapeutic peptides and proteins include, but are not limited to, cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins.

[0039] According to the present disclosure, a nucleic acid such as RNA (e.g., mRNA) encoding a peptide or therapeutic protein once taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject results in expression of said peptide or protein. The cell may express the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or in the nucleus), may secrete the encoded peptide or protein, or may express it on the surface.

[0040] The term “encoding”, “encode”, or the like, as used herein, refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (for example, mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes (e.g., transcription or translation) having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, the final product encoded from a nucleic acid is a protein if transcription and translation of RNA (e.g., mRNA) corresponding to that nucleic acid produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the RNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0041] As used herein, the term “transfect” or “transfection” refers to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and / or polynucleotides) into a cell (e.g., into a target cell). The introduced polynucleotide may be stably or transiently maintained in the target cell.

[0042] The term “pharmaceutical composition” refers to a formulation comprising a therapeutically active agent, optionally with pharmaceutically acceptable excipients. InAttorney Docket No. 4404-0141PW01the context of the present disclosure, the terms “composition” and “formulation” can be interchangeably used. A pharmaceutical composition is utilized for treating, preventing, or reducing the severity of a disease or condition by administration of the pharmaceutical composition to a subject in need thereof. In the context of the present disclosure, the pharmaceutical composition comprises the compound or lipid nanoparticle as described herein. The pharmaceutical compositions of the present disclosure may be in a storable form (e.g., frozen form) or in a “ready-to-use form” (i.e., in a form, e.g., a liquid form, which can be immediately administered to a subject without any processing such as thawing, reconstituting, or diluting), whereas the durability of a storable form is superior to that of a ready-to-use form. Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form. For example, a frozen pharmaceutical composition has to be thawed. Ready to use injectables can be presented in containers such as vials, ampoules or syringes wherein the container may contain one or more doses.

[0043] The term “excipient” as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Non-limiting examples of excipients include carriers, binders, lubricants, preservatives, stabilizers, diluents, buffers, flavoring agents, emulsifiers, surface active agents, thickeners, or colorants.

[0044] The term “diluent”, as used herein, refers to a diluting and / or thinning agent, and includes any one or more of fluid, liquid, or solid suspension and / or mixing media. Non-limiting examples of suitable diluents include ethanol and water.

[0045] The term “carrier” refers to a component which may be natural, synthetic, organic, inorganic in which a therapeutic agent is combined in order to facilitate, assist, improve, or enhance administration, uptake, and / or efficacy of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, but are not limited to, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxy ethylene / polyoxy-propylene copolymers.

[0046] The term “pharmaceutically acceptable” as used herein refers to substances that, within the scope of sound medical judgment, are suitable for use, while being administered, in contact with the tissues of a subject without conferring intolerable orAttorney Docket No. 4404-0141PW01excessive toxicity, irritation, allergic response, or other adverse condition or complication. The acceptability of a pharmaceutical substance varies according to individual case, depending on a reasonable benefit / risk ratio.

[0047] Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts 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 with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, 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 salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(CI-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like.

[0048] Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quartemized alkylated amino salt.

[0049] Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium salts, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate, and aryl sulfonate.

[0050] As used herein, the term “subject” refers to a human or any non-human animal such as mammals (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. A subject may be a human. A subject can be a patient, which refers to a human presenting to a clinician or physician for diagnosis, prevention, and / or treatment of a condition or disease. The term “subject”Attorney Docket No. 4404-0141PW01is used herein interchangeably with “individual” or “patient”. A subject can be afflicted with or is susceptible to a disease or disorder but may or may not, either visually or microscopically, display one or more sign or symptom of the disease or disorder.

[0051] The term “therapeutically effective amount” used herein refers to an amount, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, that is sufficiently capable of treating, diagnosing, preventing, and / or delaying the onset of one or more symptoms of disease, disorder, and / or condition. A person of ordinary skill in the art can understand that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose and at least one cycle of treatment.

[0052] The term “aqueous phase” as used herein in connection with a composition or formulation comprising particles, in particular lipid nanoparticles, refers to the mobile or liquid phase, i.e., the continuous water phase including all components dissolved therein but (formally) excluding the particles.

[0053] The term “protonated form” as used herein in connection to a base (e.g., an organic primary amine such as Tris) refers to the conjugate acid, wherein the conjugate acid contains a proton which is removable by deprotonation resulting in the base. In chemistry, protonation is to add a proton (or hydron, or hydrogen cation), usually denoted by H+, to a molecule or an ion, to form a conjugate acid. A buffer substance is usually constituted of a protonated substance. For example, the protonated form of Tris has the formula [H3N(CH2CH2OH)3]+. A “buffer substance” as used herein refers to a mixture of the base and its protonated form (e.g., a mixture of Tris and [H3N(CH2CH2OH)3]+) in a stationary and equilibrium condition. Consequently, the amount of a buffer substance contained in a composition is the total amounts of both the base and the conjugate acid in the composition that maintain the equilibrium.

[0054] The term “treat,” “treatment,” “treating”, or the like used herein refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce occurrence or incidence of one or more sign or symptoms or features of a particular disease, disorder, and / or condition. Treatment may be provided to a subject who does not exhibit, either visually or microscopically, one or more sign or symptom of disease or condition and / or exhibits only early sign or symptom of the disease for the purpose of decreasing the risk of developing pathological consequence associated with the disease and / or reducing orAttorney Docket No. 4404-0141PW01preventing the mortality resulted from such disease and / or prolonging or extending the survival of a subject.

[0055] In the context of the present disclosure, the treatment is a therapeutic treatment or prophylactic treatment.

[0056] The term “therapeutic treatment” refers to any treatment which improves the health status and / or prolongs (extend) the lifespan of a subject. A treatment may eliminate the disease in a subject, arrest or slow the development of symptoms and / or the progression of disease in a subject, inhibit the development of a disease in a subject, decrease the frequency or severity of symptoms in a subject, and / or decrease the recurrence in a subject who currently has or who previously has had a disease and recovered from said disease.

[0057] The term “prophylactic treatment” or “preventive treatment” refers to any treatment that is intended to prevent a disease from occurring in a subject. The terms “prophylactic treatment” and “preventive treatment” are usually used interchangeably.

[0058] The term “disease” (also referred to as and used interchangeably with “disorder” or “condition” herein) refers to an abnormal and usually pathological condition that afflicts the body of a subject. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as wound or infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases, metabolic malfunction, or cancer. In a human subject, “disease” is often used more broadly in plain definition, referring to any condition that causes pain, dysfunction, distress, social problems, or even death to the individual afflicted, or similar problems for those in contact with the individual. In this sense, the subject is a patient of such disease. In a broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of cell, tissue, organ, physiological system, and / or body of the subject as well as the function thereof, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one’s perspective on life, and one’s personality, which is also included in the target of treatment and prevention as described herein.

[0059] The term “vaccination” or “immunization” as used herein describes the process of treating a subject for therapeutic or prophylactic reasons and relate to the procedure of administering one or more immunogen(s) or antigen(s) or the nucleic acid(s) (e.g.,Attorney Docket No. 4404-0141PW01RNA, especially in a form of mRNA) coding the same to a subject and stimulating an immune response against said one or more immunogen(s) or antigen(s) or cells characterized by presentation of said one or more immunogen(s) or antigen(s). The goal of vaccination is to elicit long-lasting immune memory, in order to mediate protection from infection, or at least to prevent disease in case of exposure to the pathological risk, such as pathogen.

[0060] The term “immune response” as used herein refers to an integrated and systemic bodily response to an antigen administered or encoded by a nucleic acid administered to a subject and particularly refers to a cellular immune response and / or a humoral immune response. According to the present disclosure, the term “immune response to” or “immune response against” with respect to an agent such as an antigen, cell or tissue presenting the same, refers to an immune response such as a cellular response directed against said antigen. An immune response may comprise one or more reactions selected from the group consisting of developing antibodies against one or more antigens and expansion of antigen-specific T-lymphocytes such as CD4+ and CD8+ T-lymphocytes, for example CD8+ T-lymphocytes, which may be detected in various proliferation or cytokine production tests in vitro.

[0061] The terms “inducing an immune response” and “eliciting an immune response” and similar terms in the context of the present disclosure refer to the induction of an immune response, particularly the induction of a cellular immune response and / or a humoral immune response. The immune response may be protective, preventive, prophylactic, and / or therapeutic. The immune response may be directed against any immunogen or antigen or antigenic peptide, for example, against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (such as influenza virus, hepatitis virus, or coronavirus). It is desired that after inducing an immune response in a subject in need thereof, said subject is protected from being afflicted with a disease such as an infectious disease or a cancerous disease or progression of such disease, or the disease condition is ameliorated by inducing an immune response.

[0062] “Inducing” or “eliciting” in the context of the present disclosure refers to that there was no immune response against a particular antigen or pathogen prior to induction, but it may also mean that there was a certain level of immune response against a particular antigen or pathogen before induction and after induction said immune response is enhanced and elevated. Thus, “inducing the immune response” in this context also includes “enhancing the immune response”.Attorney Docket No. 4404-0141PW01

[0063] Accordingly, including inducing and enhancing the immune response, the “vaccination” and the subsequent immune response is broadly contemplated to encompass “prime vaccination” and “booster vaccination”. In this context, the term “prime vaccination” refers to the very first vaccine dose, or the first vaccine injections close in time to each other, which activates naive T cells, which undergo proliferation, contraction and a differentiation program to develop into primary memory T cells. As soon as the second vaccine dose is administered, when the primary effector response has started to contract, it can actually be called a boost. In this context, the term “booster vaccination” refers to the subsequent vaccination that restimulates primary memory T cells, and also primes new naive T cells, although primary memory T cells have an advantage to respond over naive T cells.

[0064] Multiple immunizations are required for most vaccine strategies, to induce efficient protection. Repeated vaccinations can be necessary to increase the frequency of responders among vaccinees, and to ensure potent individual and herd immunity. Thus, in the context of the present disclosure, the vaccination or immunization is not limited to single dose or single cycle of administration.

[0065] The term “delivery”, as used herein, encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient’s circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery”).

[0066] As used herein, “expression” of a nucleic acid sequence (e.g., DNA and / or RNA) refers to one or more steps of transcription of DNA into RNA, translation of an mRNA into a polypeptide, assemble multiple polypeptides into an intact protein (e.g., enzyme) and / or post-translational modification of a polypeptide or fully assembled protein (e.g., enzyme). The terms “expression” and “production,” and grammatical equivalents thereof, are used interchangeably.

[0067] In the context of the present disclosure, the term “transcription” relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA (especially mRNA) may be translated into peptide or protein. With respect to RNA, the term “translation” relates to the process inAttorney Docket No. 4404-0141PW01the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.IL Compound

[0068] One aspect of the present disclosure is a compound of Formula (I):whereinW is hydroxy, Ci-Ce alkoxy, or -NR2R3;Ri is linear or branched C6-C20 alkyl or alkenyl;R2 and R3 are independently linear or branched Ci-Ce alkyl unsubstituted or substituted by hydroxy;Zi is absent or -A-C(=O)-;A is O or NH;Z2 is linear or branched C6-C20 alkyl or ;Ra is hydrogen or Ci-Ce alkyl;Rb and Rc are independently hydrogen or -Rd-OC(=O)Re;Rd is Ci-Ce alkylene;Re is linear or branched C6-C20 alkyl or alkenyl;X is -CH- or -N-; Y is -O- or -NH-;m is 1 to 5; the sum of m and is 1 to 6; o is 1 to 7; p is 1 to 7;provided that Rb and Rc are not both hydrogen;provided that when Zi is absent, Z2 is not alkyl;or a pharmaceutically acceptable salt thereof.

[0069] In one embodiment,W is hydroxy, Ci-Ce alkoxy, or -NR2R3;Attorney Docket No. 4404-0141PW01Ri is linear or branched C6-C20 alkyl;R2 and R3 are independently linear or branched Ci-Ce alkyl unsubstituted or substituted by hydroxy;Zi is absent or -A-C(=O)-;A is O or NH;Z2 is linear or branched C6-C20 alkyl or ;Ra is hydrogen or Ci-Ce alkyl;Rb and Rc are independently hydrogen or -Rd-OC(=O)Re;Rd is Ci-Ce alkylene;Re is linear or branched C6-C20 alkyl;X is -CH-; Y is -O- or -NH-;m is 1 to 5; the sum of m and is 1 to 4; o is 1 to 7; p is 1 to 7.

[0070] In one embodiment, Ri is linear C6-C20 alkyl. In another embodiment, Ri is branched C6-C20 alkyl. In another embodiment, Ri is linear C6-C20 alkenyl. In still another embodiment, Ri is branched C6-C20 alkenyl. In some embodiment, Ri is a alkenyl having 1, 2, 3, or 4 pi bond(s). In some embodiment, Ri is alkenyl having one or more pi bond(s) independently in cis (Z) or trans (E) configuration.

[0071] In one embodiment, W is hydroxy. In one embodiment, W is methoxy. In one embodiment, W is -NR2R3, wherein R2 and R3 are independently linear or branched Ci-Ce alkyl. In a particular embodiment, W is -NR2R3, wherein R2 and R3 are independently methyl.

[0072] In one embodiment, X is -CH-. In another embodiment, X is -N-.

[0073] In one embodiment, R2 is linear or branched Ci-Ce alkyl. In one embodiment, R2 is linear or branched Ci-Ce alkyl substituted by hydroxy. In one embodiment, R2 is methyl. In one embodiment, R2 is hydroxymethyl.

[0074] In one embodiment, R3 is linear or branched Ci-Ce alkyl. In one embodiment, R3 is linear or branched Ci-Ce alkyl substituted by hydroxy. In one embodiment, R3 is methyl. In one embodiment, R3 is hydroxymethyl.Attorney Docket No. 4404-0141PW01

[0075] In one embodiment, R2 and R3 are identical. In another embodiment, R2 and R3 are different. In one embodiment, R2 and R3 are both methyl. In another embodiment, R2 and R3 are both hydroxymethyl.

[0076] In one embodiment, Zi is absent. In another embodiment, Zi is -OC(=O)-. In still another embodiment, Zi is -NC(=O)-.

[0077] In one embodiment, Z2 is linear C6-C20 alkyl. In one embodiment, Z2 isZi is absent and Z2 is . In one embodiment, Zi is -OC(=O)- and Z2 is C6-C20 alkyl. In one embodiment, Zi is -NC(=O)- and Z2 is C6-C20 alkyl. In one embodiment,

[0078] In one embodiment, Ra is hydrogen. In another embodiment, Ra is Ci-Ce alkyl. In a particular embodiment, Ra is methyl.

[0079] In one embodiment, Rb and Rc are independently -Rd-OC(=O)Re. In another embodiment, one of Rb and Rc is hydrogen and the other is -Rd-OC(=O)Re. In one embodiment, Rb and Rc are identical. In another embodiment, Rb and Rc are different.Attorney Docket No. 4404-0141PW01

[0080] In one embodiment, Zi is absent, Z2 is , Ra is methyl, and Rb and Rc are independently -Rd-OC(=O)Re. In another embodiment, Zi is -OC(=O)-, Z2 is, Ra is methyl, and Rb and Rc are independently -Rd-OC(=O)Re.

[0081] In a particular embodiment, Zi is absent, Z2 is and Ra is Ci-Cealkyl. In a particular embodiment, Zi is -OC(=O)-, Z2 is , and Ra is Ci-Cealkyl. In another particular embodiment, Zi is -NC(=O)-, Z2 is and Ra is C1-C6 alkyl.Attorney Docket No. 4404-0141PW01

[0082] In a particular embodiment, Zi is absent, Z2 is , and Ra is methyl.In a particular embodiment, Zi is -OC(=O)-, Z2 is , and Ra is methyl. Inanother particular embodiment, Zi is -NC(=O)-, Z2 is and Ra is methyl.

[0083] In one embodiment, Rd is methylene. In another embodiment, Rd is ethylene.

[0084] In one embodiment, Re is linear C6-C20 alkyl. In another embodiment, Re is branched C6-C20 alkyl. In another embodiment, Re is linear C6-C20 alkenyl. In still another embodiment, Re is branched C6-C20 alkenyl. In some embodiment, Re is a alkenyl having 1, 2, 3, or 4 pi bond(s). In some embodiment, Ri is a alkenyl having one or more pi bond(s) independently in cis (Z) or trans (E) configuration.

[0085] In one embodiment, Re is linear or branched Ce-Cio alkyl; for example, Re is selected from octyl, nonyl, or decyl, more specifically, selected from n-octyl, 2-ethyl-hexyl, n-nonyl, or n-decyl.

[0086] In one embodiment, both Ri and Re are alkyl. However, according to the present disclosure, any or both of the tail(s) of the lipid may comprise an alkenyl group. Thus, in one embodiment, Ri is alkyl and Re is alkenyl. In another embodiment, Ri is alkenyl and Re is alkyl. In still another embodiment, Ri and Re are both alkenyl.

[0087] In one embodiment, Y is -O-. In another embodiment, Y is -NH-.

[0088] In one embodiment, A and Y are both O. In another embodiment, A and Y are both NH. In still another embodiment, one of A and Y are NH and the other is O.

[0089] In some embodiments, m is 1 to 5, for example, m is 1, 2, 3, 4, or 5. In some embodiment, o is 1 to 7, for example, o is 1, 2, 3, 4, 5, 6, or 7. In some embodiments, p is 1 to 7, for example, p is 1, 2, 3, 4, 5, 6, or 7. In one embodiment, o and p are identical. In another embodiment, o and p are different.Attorney Docket No. 4404-0141PW01

[0090] In some embodiments, the sum of m and is 1 to 6 (i.e., m + n2=l-6), for example, the sum of m and is 1, 2, 3, 4, 5, or 6, such that the Formula (I) comprises a 3 to 8-membered heterocyclyl ring comprising one or two heteroatoms of nitrogen(s). In one embodiment, m and m are identical. In another embodiment, m and are different. In some embodiments, m and are both 1. In some embodiment, m and are both 2. In some embodiments, m and are both 3.

[0091] In some embodiments, one of m and is 0, such that the other is 6 at most, for example, 6, 5, 4, or 3. In some embodiments, one of m and is at least 1, such that the other is 5 at most, for example, 5, 4, 3, or 2.

[0092] In some embodiments, X is -CH- and the sum of m and is 2, 3, 4, 5, or 6. In some embodiments, X is -N- and the sum of m and is 4. In a particular embodiment, X is -N- and m and are both 2.

[0093] In one embodiment, X is -CH-, Y is -O-, and Ri is Cis alkyl. In one embodiment, W is hydroxy, X is -CH-, Y is -O-, and Ri is Cis alkyl. In one embodiment, W is -NR2R3, X is -CH-, Y is -O-, and Ri is Cis alkyl, wherein R2 and R3 are independently methyl.

[0094] In one embodiment, X is -CH-, Y is -O-, Ri is Cis alkyl, Zi is -OC(=O)-, and Z2 is selected from C11 alkyl, C13 alkyl, Cis alkyl, and C17 alkyl. In one embodiment, W is hydroxy, X is -CH-, Y is -O-, Ri is Cis alkyl, Zi is -OC(=O)-, and Z2 is selected from C11 alkyl, C13 alkyl, Cis alkyl, and C17 alkyl. In one embodiment, W is -NR2R3, X is -CH-, Y is -O-, Ri is Cis alkyl, Zi is -OC(=O)-, and Z2 is selected from C11 alkyl, C13 alkyl, Cis alkyl, and C17 alkyl, wherein R2 and R3 are independently methyl.

[0095] Another aspect of the present disclosure is a compound of Formula (I):whereinW, Ri, R2, R3, Re, X, Y, m, m, n2, o, and p are as defined above;Attorney Docket No. 4404-0141PW01Z1 is absent and Z2 is ;Ra is methyl; Rb and Rc are independently -Rd-OC(=O)Re; Rd is methylene.

[0096] Another aspect of the present disclosure is a compound of Formula (I):whereinW, Ri, R2, R3, Re, X, Y, m, m, n2, o, and p are as defined above;Z1 is -O-C(=O)- and Z2 is ;Ra is methyl; Rb and Rc are independently -Rd-OC(=O)Re; Rd is methylene.

[0097] In some embodiments, the compound of Formula (I) is selected from the group consisting of:Attorney Docket No. 4404-0141PW01Attorney Docket No. 4404-0141PW01Attorney Docket No. 4404-0141PW01Attorney Docket No. 4404-0141PW01Attorney Docket No. 4404-0141PW01

[0098] Compounds as described herein may be used in the preparation of compositions (e.g., to construct pharmaceutical compositions) that facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).III. Lipid particle

[0099] In the context of the present disclosure, the term “particle” refers to a spherically structured entity formed by molecules complexes, such as particle forming compounds, e.g., phospholipid. The particle is a micro- or nano-sized structure, such asAttorney Docket No. 4404-0141PW01a micro- or nano-sized compact structure, which is referred to as “microparticle” or “nanoparticle”. The particle contains a shell made of one or more types of amphiphilic substances (e.g., amphiphilic lipids, amphiphilic polymers, and / or amphiphilic proteins / polypeptides), in which the shell may be one or more layer(s) or lamella(s). Lipid nanoparticles (abbreviated herein as “LNP”) are artificially prepared spherical vesicles made of a lipid bilayer. To deliver the molecules to sites of action, the lipid bilayer can fuse with other bilayers such as the cell membrane, thus delivering the liposome contents inside the cell. The lipid nanoparticle comprises in its structure, a hydrophilic core; and an outer lipid bilayer shell formed by the compound of the present disclosure. Further, the lipid nanoparticle of the present disclosure further comprises a therapeutic agent disposed in the hydrophilic core or optionally in the outer lipid bilayer shell of the nanoparticle. The therapeutic agent will be in the hydrophilic core if hydrophilic or in the lipid shell if hydrophobic. The therapeutic agent may be a nucleic acid of a target protein.

[0100] In one aspect, the present disclosure provides a lipid nanoparticle comprising the compound according to the present disclosure.

[0101] The term “nanoparticle” as used herein refers to a particle and at least one cationic lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 2000 nm (e.g., between 10 and 990 nm, such as between 15 and 900 nm, between 20 and 800 nm, between 30 and 700 nm, between 40 and 600 nm, or between 50 and 500 nm). The longest and shortest axes of the nanoparticle do not differ significantly, i.e., rendering the nanoparticle spherical. The size of a particle is its diameter. In certain embodiments, the nanoparticle encapsulates a therapeutic agent, e.g., a therapeutic nucleic acid (such as RNA, for example, mRNA) or a therapeutic peptide or protein.

[0102] The term “lipid (nano-)particle” as used herein, which may be also known as “liposome”, or the like refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, a lipid nanoparticle as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and additional polymer.

[0103] In some embodiments, the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids, amphiphilic polymers, and / or amphiphilic proteins / polypeptides; more particularly, amphiphilic lipids) optionally in combinationAttorney Docket No. 4404-0141PW01with additional amphiphilic or non-amphiphilic substances (e.g., additional lipids and / or additional polymers).

[0104] Unless specified otherwise, the amount of a compound of the present disclosure in a particle is described as a percentage (i.e., molar ratio (“mol%”)) of the combined molar amounts of total components of a particle (e.g., the combined molar amounts of all lipids present in a liposomal delivery vehicle).

[0105] In some embodiments, a lipid nanoparticle according to the present disclosure comprises the compound according to the present disclosure (as a cationic lipid) and optionally further comprises:(i) non-cationic lipid(s),(ii) steroid(s), and / or(iii) polymer-conjugated lipid(s).

[0106] In some embodiments, the lipid nanoparticle according to the present disclosure further comprises a therapeutic agent. In one embodiment, the therapeutic agent is a nucleic acid, a peptide, or a protein.

[0107] The molar ratio of the cationic lipid, the non-cationic lipid, the steroid, and the polymer-conjugated lipid is A: B: C: D, where A + B + C + D = 100%. Unless specified otherwise, the percentage of components of the LNP is presented as molar ratio of a component being referred to.

[0108] In certain embodiments, the lipid nanoparticle according to the present disclosure comprises: - a cationic lipid at a molar ratio of 35% to 55%; - a non-cationic lipid at a molar ratio of 5% to 35%; - a steroid at a molar ratio of 20% to 50%; and - a polymer-conjugated lipid at a molar ratio of 0.25% to 2.75%, wherein all of the molar ratios are relative to the total content of the LNP.

[0109] In some embodiments, the molar ratio of the cationic lipid in the LNP relative to the total lipids (i.e., A) is about 35% to about 55%. In a particular embodiment, the molar ratio of the cationic lipid is about 40% to about 55%. In a particular embodiment, the molar ratio of the cationic lipid is about 45% to about 50%. In a particular embodiment, the molar ratio of the cationic lipid is about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%.

[0110] In some embodiments, the molar ratio of the non-cationic lipid in the LNP relative to the total lipids (i.e., B) is about 5% to about 35%. In a particular embodiment, the molar ratio of the non-cationic lipid is about 5% to about 30%. In a particular embodiment, the molar ratio of the non-cationic lipid is about 5% to about 20%. In aAttorney Docket No. 4404-0141PW01particular embodiment, the molar ratio of the non-cationic lipid is about 5% to about 10%. In a particular embodiment, the molar ratio of the non-cationic lipid is about 10% to about 25%. In a particular embodiment, the molar ratio of the non-cationic lipid is about 15% to about 20%. In a particular embodiment, the molar ratio of the cationic lipid is about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%.[OHl] In some embodiments, the molar ratio of the cationic lipid to the one or more non-cationic lipid(s) is from about 11:1 to about 5:1, about 4:1 to about 2:1, or about 1:1. In some embodiments, the molar ratio of the cationic lipid to the one or more noncationic lipid(s) is from about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, or about 11:1.

[0112] In some embodiments, the molar ratio of the steroid in the LNP relative to the total lipids (i.e., C) is about 20% to about 50%. In a particular embodiment, the molar ratio of the steroid is about 25% to about 45%. In a particular embodiment, the molar ratio of the steroid is about 30% to about 40%. In a particular embodiment, the molar ratio of the steroid is about 37% to about 43%. In a particular embodiment, the molar ratio of the steroid is about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, or about 43%.

[0113] In some embodiments, the molar ratio of the polymer-conjugated lipid relative to the total lipids (i.e., D) is about 0.25% to about 2.75%. In a particular embodiment, the molar ratio of the polymer-conjugated lipid is about 0.5% to about 2.5%. In a particular embodiment, the molar ratio of the polymer-conjugated lipid is about 0.75% to about 2.25%. In a particular embodiment, the molar ratio of the polymer-conjugated lipid is about 1.0% to about 2.0%. In a particular embodiment, the molar ratio of the polymer-conjugated lipid is about 1.25% to about 1.75%.

[0114] In some embodiments, the amount of steroid and the polymer-conjugated lipid has approximately the same molar amount as the non-cationic lipid. In some embodiments, the amount of steroid and the polymer-conjugated lipid has less molar amount than the non-cationic lipid.

[0115] In certain embodiments, the lipid nanoparticle of the present disclosure comprises:a cationic lipid in a molar ratio of 35% to 55% or 40% to 50% (e.g., a cationic lipid at a molar ratio of 35%, 36%, 37%, 38%, 39%, 40%, 41% 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%);Attorney Docket No. 4404-0141PW01a non-cationic lipid in a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., a helper lipid at a molar ratio of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%),a steroid in a molar ratio of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., a steroid at a molar ratio of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41% 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%); anda polymer-conjugated lipid in a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., a polymer-conjugated lipid at a molar ratio of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%);wherein all of the molar ratios are relative to the total content of the LNP.

[0116] With respect to lipid nanoparticles (especially RNA LNPs such as mRNA LNPs), the N / P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N / P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N / P ratios is from about 4 to about 12, for the reason that positively charged nanoparticles are considered favorable for transfection. In such case, RNA is considered to be completely bound to nanoparticles.

[0117] In some embodiments, the lipid nanoparticle according to the present disclosure has a N / P ratio of between 1 and 10. In some embodiments, the lipid nanoparticle according to the present disclosure has a N / P ratio above 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, the lipid nanoparticle according to the present disclosure has an N / P ratio of 4.

[0118] In one embodiment, the lipid nanoparticle according to the present disclosure have a size (specifically, a diameter, i.e., double the radius such as double the radius of gyration (Rg) value or double the hydrodynamic radius) in the range of about 10 to about 2000 nm, such as at least about 15 nm (specifically at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or at most 2000 nm (specifically at most about 1900 nm, at most about 1800 nm, at most aboutAttorney Docket No. 4404-0141PW011700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), for example, in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to 800 nm, about 80 to 700 nm, about 90 to 600 nm, or about 50 to 500 nm or about 100 to 500 nm, such as in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, 150 to 250 nm, or 100 to 150 nm.

[0119] Without wishing to be bound by any theory, the selection of cationic lipids, non-cationic lipids, steroids and / or polymer-conjugated lipids which constitute the lipid nanoparticle, as well as the relative molar ratio of such components to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the therapeutic agent to be delivered. Additional considerations include, but are not limited to, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted based on the above considerations.

[0120] In some embodiments, the lipid nanoparticle according to the present disclosure encapsulates a nucleic acid, optionally an mRNA which encodes a peptide or protein after entering a target cell. In some embodiments, the lipid nanoparticle encapsulates an mRNA encoding a peptide or protein, optionally for use in a vaccine. In some embodiments, the peptide or a protein is an antigen or a therapeutic peptide.

[0121] As used herein, the phrase “encapsulation efficiency” refers to the fraction of therapeutic agent (e.g. mRNA) that is effectively encapsulated within a liposomal-based vehicle (e.g. a lipid nanoparticle) relative to the initial fraction of therapeutic agent present in the lipid phase. In one embodiment, the lipid nanoparticles have an encapsulation efficiency for mRNA of at least 50%. In one embodiment, the lipid nanoparticles have an encapsulation efficiency for mRNA of at least 55%. In one embodiment, the lipid nanoparticles have an encapsulation efficiency for mRNA of at least 60%. In one embodiment, the lipid nanoparticles have an encapsulation efficiency for mRNA of at least 65%. In one embodiment, the lipid nanoparticles have an encapsulation efficiency for mRNA of at least 70%. In one embodiment, the lipidAttorney Docket No. 4404-0141PW01nanoparticles have an encapsulation efficiency for mRNA of at least 75%. In one embodiment, the lipid nanoparticles have an encapsulation efficiency for mRNA of at least 80%. In one embodiment, the lipid nanoparticles have an encapsulation efficiency for mRNA of at least 85%. In one embodiment, the lipid nanoparticles have an efficiency percentage for mRNA of at least 90%. In one embodiment, the lipid nanoparticles have an encapsulation efficiency for mRNA of at least 95%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency for mRNA of from about 95% to about 99%.

[0122] Accordingly, in some embodiments, the compounds of the present disclosure may be used as a component of a lipid nanoparticle to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).

[0123] In one embodiment, the lipid nanoparticle according to the present disclosure comprises an additional cationic lipid. In some embodiments, the additional cationic lipid comprised in the lipid nanoparticle is selected from the group consisting of N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), l,2-dioleoyl-3 -dimethylammoniumpropane (DODAP), heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino)butanoate (DLin-MC3-DMA), and 4-((di((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-l -amine (DPL-14).

[0124] Further examples of additional cationic lipids include, but are not limited to, 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-diacyl oxy-3 -dimethylammonium propanes, l,2-dialkyloxy-3 -dimethylammonium propanes, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9, 12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-di oleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA), 2,3-Dilinoleoyloxy-N,N-Attorney Docket No. 4404-0141PW01dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2, 2-dilinoleyl-4-dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2, 2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-di oxolane (DLin-KC2-DMA), [(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl] 4- (dimethylamino)butanoate (DLin-MC3-DMA), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), di((Z)-non-2-en-l-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan- 1 -amine (DMDMA), di((Z)-non-2-en- 1 -yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N12-5), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200) tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate (3060il0), decyl (2-(dioctylammonio)ethyl) phosphate (9 A 1 P9), ethyl 5 , 5 -di((Z)-heptadec-8-en- 1 -yl)- 1 -(3 -(pyrrolidin- 1 -yl)propyl)-2, 5 -dihydro- lH-imidazole-2-carboxylate (A2-Iso5-2DC18), bis(2- (dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate (BAME-O16B), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (cKK-El 2), hexa(octan-3 -yl) 9,9',9'',9"',9'''',9"'"-((((benzene-l,3,5-tricarbonyl)yris(azanediyl)) tris (propane-3, 1-diyl)) tris(azanetriyl))hexanonanoate (FTT5), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4, 1 -diyl))bis(azanetriyl))tetrakis(ethane-2, 1 -diyl) (9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12"Z,12"'Z)-tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin).

[0125] In some embodiments, the steroid is a sterol-based lipid, for example, a cholesterol -based lipid such as cholesterol.Attorney Docket No. 4404-0141PW01

[0126] Contemplated examples of cholesterol-based lipids include but are not limited to DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine, imidazole cholesterol ester, sitosterol (22,23 -dihydrostigmasterol), P-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol, desmosterol (3P-hydroxy-5,24-cholestadiene), lanosterol (8,24-lanostadien-3P-ol), 7-dehydrocholesterol (A5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5a-cholesta-8,24-dien-3P-ol), lathosterol (5a-cholest-7-en-3P-ol), diosgenin ((3p,25R)-spirost-5-en-3-ol), campesterol (campest-5-en-3P-ol), campestanol (5a-campestan-3P-ol), 24-methylene cholesterol (5,24(28)-cholestadien-24-methylen-3P-ol), cholesteryl margarate (cholest-5-en-3P-yl heptadecanoate), cholesteryl oleate, cholesteryl stearate, and other modified forms of cholesterol.

[0127] In some embodiments, the polymer-conjugated lipid is a PEG-conjugated (PEGylated) lipid.

[0128] Contemplated examples of PEGylated lipids include but are not limited to a polyethylene glycol (PEG) chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 (e.g., Cs, C10, C12, C14, Ci6, or Cis) length, such as a derivatized ceramide (e.g., N-octanoyl-sphingosine-l-[succinyl(m ethoxypoly ethylene glycol)] (C8 PEG ceramide)).

[0129] In some embodiments, the PEGylated lipid is a PEG-dialky oxypropylcarbamate; 1,2-distearoyl-rac-glycero-polyethelene glycol (DSG-PEG); 1.2-dilauroyl-sn-glycero-3 -phosphoethanolamine-poly ethylene glycol (DLPE-PEG); 1.2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1.2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0130] In some embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In certain embodiments, the PEG is PEG2000 (PEG-2K). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159. In certain embodiments, the PEGylated lipid is DMG-PEG2000.

[0131] Also contemplated herein is the derivatized lipids such as derivatized ceramides (PEG-CER), for example, N-octanoyl-sphingosine-l-[succinyl(methoxy polyethylene glycol)-2000] (C8 PEG-2000 ceramide), which can be used in combination with one or more of the compounds of the present disclosure and, in someAttorney Docket No. 4404-0141PW01embodiments, other lipids which together constitutes the lipid nanoparticle. In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., (Cw) or (Cis)). The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the nucleic acid composition to the target cell.

[0132] Contemplated example of the polymer-conjugated lipid also includes a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material, i.e., a lipid or lipid-like material which comprises polysarcosine (poly(N-m ethyl glycine)). The polysarcosine may comprise acetylated (neutral end group) or other functionalized end groups. In one embodiment, the polysarcosine is conjugated to or covalently bound to a non-cationic lipid or lipid-like material comprised in the particles.

[0133] In some embodiments, the polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material is selected from the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate, and any combination thereof.

[0134] According to the present disclosure, the term “non-cationic lipid” is meant to include any neutral, zwitterionic or anionic lipid. As used herein, the term “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. In some context, the non-cationic lipid is referred to as a “helper lipid” of a lipid particle.

[0135] In one embodiment, the neutral lipid is a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Particular examples of phospholipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 LysoAttorney Docket No. 4404-0141PW01PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl-phosphatidylethanolamine (DPyPE). In a particularly embodiment, the neutral lipid is DSPC.IV. Nucleic acid

[0136] The term “nucleic acid”, used herein in its broadest sense, refers to any compound and / or substance that is or can form or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In one embodiment, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In one embodiment, the “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In other embodiments, the “nucleic acid” refers to a oligonucleotide or a polynucleotide.

[0137] According to the present disclosure, the term “RNA” means a nucleic acid molecule which includes ribonucleotide residues. In some embodiments, the RNA contains all or a majority of natural ribonucleotide residues. As used herein, the term “ribonucleotide” refers to the canonical definition that a nucleotide with a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. RNA encompasses, without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard or non-natural nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, the modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides (i.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains “a majority of ribonucleotide residues” if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The totalAttorney Docket No. 4404-0141PW01number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally or non-naturally occurring) nucleotide residues or analogs thereof).

[0138] In some embodiments, the “nucleic acid” encompasses single and / or doublestranded DNA (including cDNA) and / or RNA. In some embodiments, the “nucleic acid” encompasses both RNA and DNA. In some embodiments, “nucleic acid” encompasses deoxyribonucleic acid (DNA), including but not limited to any one or more of singlestranded DNA (ssDNA), double-stranded DNA (dsDNA) and complementary DNA (cDNA). In embodiments, DNA may be in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. In some embodiments, the “nucleic acid” encompasses ribonucleic acids (RNA), including but not limited to any one or more of interference RNAs (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (IncRNA), micro-RNA (miRNA) multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA) and CRISPRRNA (crRNA). In embodiments, RNA may be in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfermessenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), micro-RNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), 73K RNA, retrotransposons, a viral genome, a viroid, satellite RNA, or derivatives of these groups.

[0139] In some embodiments, the RNA (such as mRNA) contains one or more modifications, e.g., in order to increase its stability and / or increase translation efficiency and / or decrease immunogenicity and / or decrease cytotoxicity. In some embodiments, the RNA is modified within a coding region. In other embodiments, the RNA is modified within a non-coding region.Attorney Docket No. 4404-0141PW01

[0140] For example, in order to increase expression of the RNA (such as mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, without altering the sequence of the peptide or protein intended to be expressed. The modifications of RNA include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5'-and / or 3 '-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to decrease or increase the GC content of the RNA). The term “modification” in the context of modified mRNA according to the present disclosure also encompasses any modification of an mRNA which is not naturally present in RNA (such as mRNA).

[0141] In some embodiments, mRNA according to the present disclosure may be synthesized as unmodified or modified mRNA. A modified mRNA according to the disclosure can thus include nucleotide modification that are, for example, backbone modifications, sugar modifications or base modifications.

[0142] In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g., 1-methyl-adenine, 2-m ethyl -adenine, 2-methylthio-N-6-isopentenyl-adenine, N-6-methyl-adenine, N-6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queuosine, beta-D-mannosyl-queuosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.

[0143] In some embodiments, an RNA (such as mRNA) is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-Attorney Docket No. 4404-0141PW01aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C5-propynyl-cytidine, C5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2’ -fluororibose, ribose, 2’ -deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5’-N-phosphoramidite linkages).

[0144] In some embodiments, the RNA (such as mRNA) comprises a 5 ’-cap structure. In a specific embodiment, the mRNA does not have uncapped 5 ’-triphosphates. In one embodiment, the RNA (such as mRNA) may comprise a conventional 5’-cap and / or a 5’-cap analog. The term “conventional 5’-cap” refers to a cap structure found on the 5’-end of an mRNA molecule and generally consists of a guanosine 5 ’-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5’-end of the next nucleotide of the mRNA (i.e., the guanosine is connected via a 5’ to 5’ triphosphate linkage to the rest of the mRNA). The guanosine may be methylated at position N7(resulting in the cap structure m7Gppp). The term “5’-cap analog” refers to a 5’-cap which is based on a conventional 5’-cap but which has been modified at either the 2’- or 3’-position of the m7guanosine structure in order to avoid an integration of the 5 ’-cap analog in the reverse orientation (such 5 ’-cap analogs are also called anti-reverse cap analogs (ARC As)). Contemplated examples of 5 ’-cap analogs include those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5 ’-cap analogs at the P-phosphate (such as m27’2OG(5’)ppSp(5’)G (referred to as beta-S-ARCA or P-S-ARCA)). Providing an RNA (such as mRNA) with a 5 ’-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5 ’-cap compound, wherein said 5 ’-cap structure is co-transcriptionally incorporated into the generated RNA (such as mRNA) strand, or the RNA (such as mRNA) may be generated, for example, by in vitro transcription, and the 5 ’-cap structure may be attached to the mRNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.Attorney Docket No. 4404-0141PW01

[0145] In some embodiments, the RNA (such as mRNA) comprises a 5 ’-cap structure selected from the group consisting of m27’2OG(5’)ppSp(5')G (in particular its DI diastereomer), m27’3 OG(5')ppp(5')G, and m27’3'oGppp(mi2'o)ApG.

[0146] As used herein, the term “poly-A tail” or “poly-A sequence” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3 ’-end of an RNA (such as mRNA) molecule.

[0147] Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. RNAs (such as mRNAs) disclosed herein can have a poly-A tail attached to the free 3 ’-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0148] The poly-A tail (abbreviated as polyA) may be of any length. In some embodiments, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, “essentially consists of’ means that majority of nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but additionally permits some interstitial nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).

[0149] In some embodiments, the RNA (such as mRNA) used in present disclosure comprises a 5’-UTR and / or a 3 ’-UTR. The term “untranslated region” or “UTR” refers to a non-coding region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region thereof in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5’ (upstream) of an open reading frame (5 ’-UTR) and / or 3’ (downstream) of an open reading frame (3 ’-UTR). A 5 ’-UTR, if present, is located at the 5 ’-end, upstream of the start codon of a protein-encoding region. A 5’-UTR is downstream of the 5’-cap (if present), e.g., directly adjacent to the 5’-cap. A 3’-UTR, if present, is located at the 3’-end, downstream of the termination codon of a protein-encoding region, but the term “3’-UTR” does not include the poly-A sequence in most of the context unless specifiedAttorney Docket No. 4404-0141PW01otherwise. Thus, the 3’-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3’-UTR into the 3 ’-non translated region of an RNA (e.g., mRNA) molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3’-UTRs (which may be arranged in a head-to-tail orientation). The 3’-UTRs may be autologous or heterologous to the RNA into which they are introduced. In one embodiment, the 3’-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2-globin, alphal -globin, or beta-globin, particularly human betaglobin. For example, the RNA may be modified by the replacement of the existing 3’-UTR with or the insertion of one or more, e.g., two copies of a 3’-UTR derived from a globin gene, such as alpha2 -globin, alphal -globin, beta-globin, particularly human betaglobin.

[0150] In some embodiments, the RNA (e.g., mRNA) described herein may comprise a 5 ’ UTR that includes one or more elements that enhance mRNA stability or translation. In some embodiments, a 5’ UTR may be about 10 to 5,000 nucleotides in length. In some embodiments, a 5’ UTR may be about 50 to 500 nucleotides in length. In some embodiments, the 5’ UTR is at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length or about 5,000 nucleotides in length.

[0151] In some embodiments, the mRNA disclosed herein may comprise a 3’ UTR comprising a binding site for proteins that affect an mRNA’s stability of location in a cell, one or more binding sites for miRNAs, and / or one or more of polyadenylation signal sequence. In some embodiments, a 3’ UTR may be 50 to 5,000 nucleotides in length or longer. In some embodiments, a 3’ UTR may be 50 to 1,000 nucleotides inAttorney Docket No. 4404-0141PW01length or longer. In some embodiments, the 3’ UTR is at least about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or about 5,000 nucleotides in length.

[0152] In some embodiments, the mRNA disclosed herein may comprise a 5’ or 3’ UTR that is derived from a gene distinct from the one encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0153] In one embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide or protein.

[0154] In one embodiment, the RNA (specifically, mRNA) which encodes a peptide or protein has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0155] Nucleic acids according to the present disclosure may be synthesized according to any known methods. For example, mRNAs as described herein may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular isolated DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, mutated T7 or SP6 RNA polymerase), optional DNAse I, pyrophosphatase, and / or optional RNAse inhibitor. The exact conditions will vary according to the specific application.Attorney Docket No. 4404-0141PW01

[0156] In some embodiments, for the preparation of mRNA as described herein, a DNA template is transcribed in vitro (i.e., synthesized via IVT). A suitable DNA template typically has a promoter, for example a T3, T7, mutated T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and an optional termination signal.

[0157] Desired mRNA sequence as described herein may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G / C content can be optimized to achieve the highest possible G / C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.V. Composition

[0158] In one aspect, the present disclosure provides a pharmaceutical composition, comprising the compound or the lipid nanoparticle of the present disclosure, and a pharmaceutically acceptable excipient.

[0159] In some embodiments, a composition (e.g., a pharmaceutical composition) comprises an mRNA encapsulated within a lipid nanoparticle. In some embodiments, a composition comprises a non-coding RNA encapsulated within a lipid nanoparticle. In some embodiments, a composition comprises a DNA encapsulated within a lipid nanoparticle. In some embodiments, a composition comprises a DNA or mRNA encoding a peptide. In some embodiments, a composition comprises a DNA or mRNA encoding a protein.

[0160] In some embodiments, an mRNA encodes a peptide or protein for use in the treatment of a disease or condition, as described in the present disclosure. In some embodiments, an mRNA encodes a peptide or protein for use in the vaccination or immunization. In some embodiments, an mRNA encodes a peptide or protein for use in the treatment of an immune cell.

[0161] In some embodiments, the composition according to the present disclosure is prepared in a form for storage (e.g., for long-term storage). In one embodiment, theAttorney Docket No. 4404-0141PW01composition according to the present disclosure is in lyophilized (frozen) form. In one embodiment, the composition is in lyophilized form and can be stored at a temperature of about -90°C or higher, such as about -90°C to about -10°C. For example, the lyophilized composition described herein can be stored at a temperature ranging from about -90°C to about -10°C, such as from about -80°C to about -40°C or from about -40°C to about -25°C, or from about -25°C to about -10°C, or a temperature of about -20°C.

[0162] In one embodiment of the pharmaceutical compositions in lyophilized form, and the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months at, for example, about -20°C. In one embodiment of the pharmaceutical compositions in lyophilized form, and the pharmaceutical composition can be stored for at least 4 weeks at, for example, about -20°C.

[0163] In a particular embodiment, after thawing the frozen composition and compared to the RNA integrity of the initial composition, the integrity of RNA in the lipid nanoparticle in the composition according to the present disclosure is at least 50%, such as at least 52%, at least 54%, at least 55%, at least 56%, at least 58%, or at least 60%, e.g., after thawing the lyophilized composition which has been stored at about -20°C or less.

[0164] The size (Zaverage) (and / or size distribution and / or poly dispersity index (PDI)) of the lipid nanoparticle(s) after thawing the lyophilized composition is equal to the size (and / or size distribution and / or PDI) of the lipid nanoparticle(s) before the composition has been lyophilized. In one embodiment, the size of the lipid nanoparticle(s) after thawing the lyophilized composition is between about 50 nm and about 500 nm, particularly between about 40 nm and about 200 nm, more particularly between about 40 nm and about 120 nm. In one embodiment, the PDI of the lipid nanoparti cle(s) after thawing the lyophilized composition is less than 0.3, particularly less than 0.2, more particularly less than 0.1. In one embodiment, the size of the lipid nanoparti cle(s) after thawing the lyophilized composition is between about 50 nm and about 500 nm, particularly between about 40 nm and about 200 nm, more particularly between about 40 nm and about 120 nm, and the size (and / or size distribution and / or PDI) of the lipid nanoparticle(s) after thawing the lyophilized composition is equal to the size (and / or size distribution and / or PDI) of the lipid nanoparticle(s) before lyophilizing. In oneAttorney Docket No. 4404-0141PW01embodiment, the size of the lipid nanoparticle(s) after thawing the lyophilized composition is between about 50 nm and about 500 nm, particularly between about 40 nm and about 200 nm, more particularly between about 40 nm and about 120 nm, and the PDI of the lipid nanoparticle(s) after thawing the lyophilized composition is less than 0.3 (particularly less than 0.2, more particularly less than 0.1).

[0165] In an alternative embodiment, the composition is in liquid form. In one embodiment of the pharmaceutical composition in liquid form, the RNA integrity of the liquid composition, when stored, e.g., at 0°C or higher for at least one week, is sufficient to produce the desired effect, such as immunization or vaccination.

[0166] In one embodiment, the pharmaceutical composition according to the present disclosure is in liquid form and can be stored at a temperature ranging from about 0°C to about 20°C. For example, the liquid pharmaceutical compositions according to the present disclosure can be stored at a temperature ranging from about 1°C to about 15°C, such as from about 2°C to about 10°C, or from about 2°C to about 8°C, or at a temperature of about 5°C.

[0167] In one embodiment of the pharmaceutical compositions in liquid form, the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 6 months, particularly at least 4 weeks at, for example, about 5°C.

[0168] As such, the RNA integrity of the liquid composition, when stored, e.g., at 0°C or higher for at least one week, is sufficient to produce the desired therapeutic or immunizing effect, e.g., to induce an immune response. For example, the RNA integrity of the liquid composition, when stored, e.g., at 0°C or higher for at least one week, may be at least 50%, such as at least 52%, at least 54%, at least 55%, at least 56%, at least 58%, or at least 60%, compared to the RNA integrity of the initial composition. The size (Zaverage) (and / or size distribution and / or poly dispersity index (PDI)) of the lipid nanoparticles of the liquid composition, when stored, e.g., at 0°C or higher for at least one week, is sufficient to produce the desired therapeutic or immunizing effect, e.g., to induce an immune response. For example, the size (and / or size distribution and / or poly dispersity index (PDI)) of the lipid nanoparticles of the liquid composition, when stored, e.g., at 0°C or higher for at least one week, is equal to the size (and / or size distribution and / or PDI) of the lipid nanoparticles of the initial composition, i.e., before storage. In one embodiment, the size of the lipid nanoparticles after storage of the liquid composition e.g., at 0°C or higher for at least one week is between about 50 nm andAttorney Docket No. 4404-0141PW01about 500 nm, particularly between about 40 nm and about 200 nm, more particularly between about 40 nm and about 120 nm. In one embodiment, the PDI of the lipid nanoparticles after storage of the liquid composition e.g., at 0°C or higher for at least one week is less than 0.3, particularly less than 0.2, more particularly less than 0.1. In one embodiment, the size of the lipid nanoparticles after storage of the liquid composition e.g., at 0°C or higher for at least one week is between about 50 nm and about 500 nm, particularly between about 40 nm and about 200 nm, more particularly between about 40 nm and about 120 nm, and the size (and / or size distribution and / or PDI) of the lipid nanoparticles after storage of the liquid composition e.g., at 0°C or higher for at least one week is equal to the size (and / or size distribution and / or PDI) of the lipid nanoparticles before storage. In one embodiment, the size (Zaverage) of the lipid nanoparticles after storage of the liquid composition e.g., at 0°C or higher for at least one week is between about 50 nm and about 500 nm, particularly between about 40 nm and about 200 nm, more particularly between about 40 nm and about 120 nm, and the PDI of the lipid nanoparticles after storage of the liquid composition e.g., at 0°C or higher for at least one week is less than 0.3 (particularly less than 0.2, more particularly less than 0.1).

[0169] In an aspect, the present disclosure provides a method of preparing a composition (e.g., pharmaceutical composition) comprising lipid nanoparticles dispersed in a final aqueous phase, comprising providing a lipid nanoparticle and a suitable buffer, wherein the lipid nanoparticles comprise the compound of the present disclosure and RNA, and optionally a non-cationic lipid, a steroid, and a polymer-conjugated lipid. The final aqueous phase comprises a buffer system comprising a final buffer substance and a final monovalent anion, the final buffer substance being selected from the group consisting of Tris and its protonated form, Bis-Tris-methane and its protonated form, and TEA and its protonated form, and the final monovalent anion being selected from the group consisting of chloride, acetate, glycolate, lactate, the anion of MES, the anion of MOPS, and the anion of HEPES.

[0170] In some embodiments, the pharmaceutical composition according to the present disclosure further comprises a buffer. In some embodiments, the buffer is suitable for maintaining the pH of the composition during manufacturing, storage and use of the composition. In some embodiments, the buffer may be a solution of salt selected from citric acid salt, boric acid salt, phosphoric acid salt (e.g., sodium bicarbonate, monosodium phosphate, disodium phosphate, monopotassium phosphate,Attorney Docket No. 4404-0141PW01dipotassium phosphate), [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(Bis(2-hydroxyethyl)amino)acetic acid (Bicine), 2-Amino-2-(hydroxymethyl)propane-l,3-diol (Tris), N-(2 -Hydroxy- 1,1-bis(hydroxymethyl)ethyl)glycine (Tricine), 3-[[l,3-dihydroxy-2- (hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-l -sulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), 2- [[1,3 -dihydroxy -2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 1,4-piperazinediethanesulfonic acid (PIPES), dimethylarsinic acid, 2-morpholin-4-ylethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), or phosphate buffered saline (PBS).

[0171] In some embodiments, the pharmaceutical composition according to the present disclosure further comprises a chelating agent. In some embodiments, the chelating agent is capable of forming at least two coordinate covalent bonds with a metal ion, thereby generating a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions, which may otherwise induce accelerated RNA degradation in the present disclosure. Examples of suitable chelating agents include, but are not limited to, citric acid, transdiaminocyclohexanetetraacetic acid (DCTA), ethylenediaminetetraacetic acid (EDTA), desferrioxamine B, deferoxamine, dithiocarb sodium, fumaric acid, iminodiacetic acid, nitrilotriacetic acid, penicillamine, pentetate calcium, a sodium salt of pentetic acid, succimer, trientine, diethylenetriaminepentaacetic acid (DTPA), bis(aminoethyl)glycolether-N,N,N',N' -tetraacetic acid, tartaric acid, or a salt thereof. In one embodiment, the chelating agent is EDTA or a salt of EDTA. In one embodiment, the chelating agent is EDTA disodium dihydrate.

[0172] In some embodiments, the pharmaceutical composition according to the present disclosure further comprises a cryoprotectant as stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of RNA activity during storage and / or freezing, for example to reduce or prevent aggregation, particle collapse, RNA degradation and / or other types of damage. In some embodiments, the cryoprotectant is selected from saccharides, sorbitol, amino acids, peptides, polymers and proteins such as albumins (e.g., bovine serum albumin, human serum albumin) or gelatins. In one embodiment, the cryoprotectant is a carbohydrate.

[0173] The term “carbohydrate”, as used herein, refers to and encompasses monosaccharides, di saccharides, trisaccharides, oligosaccharides and polysaccharides.Attorney Docket No. 4404-0141PW01In one embodiment, the cryoprotectant is a carbohydrate selected from monosaccharide, di saccharide, tri saccharide, sugar alcohol, oligosaccharide or its corresponding sugar alcohol, and a straight chain polyalcohol. In one embodiment, the cryoprotectant may be a polyol. In some embodiments, a cryoprotectant may be selected from mannose, sucrose, lactose, trehalose, maltose, sorbitol, mannitol, glycerol, inositol, glucose, fructose, arginine, glycerin, dextran, and mixtures thereof.

[0174] In an embodiment, the cryoprotectant is an oligosaccharide formed by 3 to about 15, or 3 to about 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure. Exemplary oligosaccharide cryoprotectants include cyclodextrins, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. An oligosaccharide can be oxidized or reduced.

[0175] In an embodiment, the cryoprotectant is a cyclic oligosaccharide formed by 3 to about 15, for example, 6, 7, 8, 9, or 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a cyclic structure. Exemplary cyclic oligosaccharide cryoprotectants include cyclic oligosaccharides that are discrete compounds, such as a cyclodextrin, p cyclodextrin, or y cyclodextrin.

[0176] An exemplary cryoprotectant is a polysaccharide formed by at least 16 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure, and includes polymers that comprise polysaccharides as part of their backbone structure. In backbones, the polysaccharide can be linear or cyclic. Exemplary polysaccharide cryoprotectants include glycogen, amylose, cellulose, dextran, maltodextrin and the like.

[0177] In an embodiment, the cryoprotectant is a sugar alcohol containing at least two carbon atoms and one hydroxyl group attached to each carbon atom. Typically, sugar alcohols are derived from sugars (e.g., by hydrogenation of sugars) and are water-soluble solids. The term “sugar”, as used herein, refers to sweet-tasting, soluble carbohydrates. Examples of sugar alcohols include ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, and polyglycitol. In one embodiment, the sugar alcohol has the formula HOCH2(CHOH)nCH2OH, wherein n is 0 to 22 (e.g., 0, 1, 2, 3, or 4), or is a cyclic variant thereof (which can formally be derivedAttorney Docket No. 4404-0141PW01by dehydration of the sugar alcohol to give cyclic ether; e.g., isosorbide is the cyclic dehydrated variant of sorbitol).

[0178] In one embodiment, the cryoprotectant is selected from sucrose, glucose, glycerol, sorbitol, and any combination thereof. In one embodiment, the cryoprotectant comprises sucrose and / or glycerol. In one embodiment, the cryoprotectant is sucrose.

[0179] In one embodiment, the pharmaceutical composition according to the present disclosure comprises the cryoprotectant in a concentration of at least 1% w / v, such as at least 2% w / v, at least 3% w / v, at least 4% w / v, at least 5% w / v, at least 6% w / v, at least 7% w / v, at least 8% w / v or at least 9% w / v. In one embodiment, the concentration of the cryoprotectant in the pharmaceutical composition is less than 25% w / v, such as less than 20% w / v, less than 19% w / v, less than 18% w / v, less than 17% w / v, less than 16% w / v, less than 15% w / v, less than 14% w / v, less than 13% w / v, less than 12% w / v, or less than 11% w / v. In one embodiment, the concentration of the cryoprotectant in the pharmaceutical composition is 1% w / v to 20% w / v, such as 2% w / v to 19% w / v, 3% w / v to 18% w / v, 4% w / v to 17% w / v, 5% w / v to 16% w / v, 5% w / v to 15% w / v, 6% w / v to 14% w / v, 7% w / v to 13% w / v, 8% w / v to 12% w / v, 9% w / v to 11% w / v, or about 10% w / v. In one embodiment, the pharmaceutical composition according to the present disclosure comprises a cryoprotectant (in particular, sucrose and / or glycerol) in a concentration of from 5% w / v to 15% w / v, such as from 6% w / v to 14% w / v, from 7% w / v to 13% w / v, from 8% w / v to 12% w / v, or from 9% w / v to 11% w / v, or in a concentration of about 10% w / v.

[0180] The present LNPs can be prepared by various techniques presently known in the art. For example, multilam ellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion that results in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0181] Therefore, in one aspect, the present disclosure provides a method of preparing a lyophilized pharmaceutical composition comprising lipid nanoparticles (LNPs) comprising a cationic lipid, a non-cationic (e.g., neutral) lipid, and a steroid, and / or aAttorney Docket No. 4404-0141PW01polymer-conjugated lipid, and at least one therapeutic agent (e.g., nucleic acid, peptide, and / or protein), wherein the method comprises:providing a liquid composition comprising said LNPs,spraying the liquid composition in conditions suitable to obtain liquid droplets, and freezing the liquid droplets obtained to obtain lyophilized LNPs.

[0182] The spraying of liquid composition, as described herein, may be carried out with an electromagnetic droplet stream generator, a piezoelectric droplets stream generator, a hydraulic droplets aerosol generator, a pneumatic nozzle, a ultrasonic spray nozzle, a thermal droplets stream generator, or an electrohydrodynamic droplets (EHD) generator.

[0183] In some embodiment, the method of preparing a lyophilized pharmaceutical composition further comprises drying the lyophilized LNPs obtained under conditions suitable to obtain lyophilized-dried LNPs.

[0184] In some embodiments, the drying is carried out by rotary drum vacuum lyophilization, atmospheric drying with a flow of cold air, vacuum chamber lyophilization, or vacuum tunnel lyophilization.

[0185] In some embodiments, the pharmaceutical composition according to the present disclosure comprises a single dose of the mRNA vaccine contains 1-50 pg of mRNA (e.g., monovalent or multivalent). For example, a single dose may contain about 2.5 pg, about 5 pg, about 7.5 pg, about 10 pg, about 12.5 pg, or about 15 pg of the mRNA. In further embodiments, a multi-valent single dose of an lipid nanoparticle vaccine contains multiple (e.g., 2, 3, or 4) kinds of lipid nanoparticles, each for a different antigen, and each kind of liquid nanoparticles has an mRNA amount of, e.g., 2.5 pg, about 5 pg, about 7.5 pg, about 10 pg, about 12.5 pg, or about 15 pg.

[0186] In one aspect, the present disclosure provides an article of manufacture, such as a kit, that provides the pharmaceutical composition of the present disclosure (e.g., as a vaccine) in a single container, or provides the pharmaceutical composition (e.g., as a vaccine) in one container and a physiological buffer for reconstitution in another container. The one or more containers may contain a single-use dosage or multi-use dosage. Then one or more containers may be pre-treated glass vials or ampules. The article of manufacture may include instructions for use as well.VI. Treatment

[0187] In one aspect, the present disclosure provides a lipid nanoparticle of the present disclosure for the use in the treatment of disease.Attorney Docket No. 4404-0141PW01

[0188] In another aspect, the present disclosure provides a method of treatment of disease, comprising administering an effective amount of the lipid nanoparticles of the present disclosure to a subject in need thereof.

[0189] In still another aspect, the present disclosure provides a use of the lipid nanoparticle of the present disclosure for the manufacture of a medicament for treating disease in a subject in need thereof.

[0190] In one aspect, the present disclosure provides a lipid nanoparticle of the present disclosure for the use in inducing or eliciting an immune response in a subject in need thereof.

[0191] In another aspect, the present disclosure provides a method of inducing or eliciting an immune response in a subject in need thereof, comprising administering an effective amount of the lipid nanoparticle of the present disclosure to the subject.

[0192] In still another aspect, the present disclosure provides use of the lipid nanoparticle of the present disclosure for the manufacture of a medicament for inducing or eliciting an immune response in a subject in need thereof.

[0193] In one aspect, the present disclosure provides a lipid nanoparticle of the present disclosure for use in vaccination.

[0194] In another aspect, the present disclosure provides a method of vaccinating a subject in need thereof, comprising administering an effective amount of the lipid nanoparticle of the present disclosure to the subject.

[0195] In still another aspect, the present disclosure provides use of the lipid nanoparticle of the present disclosure for the manufacture of a medicament for vaccination.

[0196] The compound as disclosed herein may be prepared as a vaccine (for example, tissue(organ)-targeted lipid nanoparticle). The vaccine according to the present disclosure may be a preventive vaccine against coronavirus or therapeutic vaccine against cancer, dengue, or other disease.

[0197] According to the embodiments of the present disclosure, diseases treatable by the present method may be cancers, infectious diseases, inflammatory diseases, transplant and / or tissue rejection, autoimmune diseases, and the like.

[0198] Examples of the infectious diseases that can be treated or prevented by administering formulations comprising the LNPs of the present disclosure include, but are not limited to, bacterial, viral, and fungal infections, or combinations thereof. In one embodiment, the present LNP encapsulating mRNA encoding the spike protein ofAttorney Docket No. 4404-0141PW01SARS-CoV-2 is administered to a subject to prophylax against SARS-CoV-2 viral infection.

[0199] In some embodiments, the lipid nanoparticle of the present disclosure is administered intramuscularly, intranasally, intravenously, subcutaneously, or intradermally to a subject in need thereof.

[0200] In certain embodiments, additional treatments may be provided to a subject in combination with the lipid nanoparticle of the present disclosure. Such additional treatments include one or more therapies selected from, e.g., radiation therapy, surgery, hyperthermia therapy and administration of a further therapeutic agent other than the lipid nanoparticle of the present disclosure.

[0201] In some embodiment, the further therapeutic agent is a checkpoint inhibitor, which, without wishing to be bound by any theory, is a molecule that totally or partially reduces, inhibits, interferes with or negatively modulates one or more checkpoint proteins or that totally or partially reduces, inhibits, interferes with or negatively modulates expression of one or more checkpoint proteins. In some embodiments, the checkpoint inhibitor binds to one or more molecules regulating checkpoint proteins. In certain embodiments, the checkpoint inhibitor binds to precursors of one or more checkpoint proteins e.g., on DNA- or RNA-level. In certain embodiments, the checkpoint inhibitor prevents inhibitory signals associated with the immune checkpoint.

[0202] In some embodiment, the further therapeutic agent is a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.

[0203] In a certain embodiment, the lipid nanoparticle of the present disclosure includes an antigenic molecule such as a peptide and / or a protein, or a nucleic acid encoding said peptide and / or protein. A peptide and protein antigen, i.e., a vaccine antigen, optionally a recombinant antigen, described herein when provided to a subject by administration of, for example, RNA encoding the antigen results in stimulation, priming and / or expansion of T cells in the subject. Said stimulated, primed and / or expanded T cells are directed against the target antigen, in particular the target antigen expressed by diseased cells, tissues and / or organs, i.e., the disease-associated antigen.

[0204] Thus, in a particular embodiment, a vaccine antigen may comprise the disease-associated antigen, or a fragment or variant thereof. In one embodiment, the fragment or variant is immunologically equivalent to the disease-associated antigen, i.e., which isAttorney Docket No. 4404-0141PW01an equivalent capable of successfully vaccinating a subject with desired effect comparable to a disease-associated antigen. In the context of the present disclosure, the term “fragment of an antigen” or “variant of an antigen” means an agent which results in stimulation, priming, and / or expansion of T cells which stimulated, primed and / or expanded T cells target the disease-associated antigen, in particular when expressed on the surface of diseased cells, tissues and / or organs. Thus, the vaccine antigen administered as described herein may correspond to or may comprise the disease-associated antigen, may correspond to or may comprise a fragment of the disease-associated antigen or may correspond to or may comprise an antigen which is homologous to the disease-associated antigen or a fragment thereof. If the vaccine antigen administered as described herein comprises a fragment of the disease-associated antigen or an amino acid sequence which is homologous to a fragment of the disease-associated antigen said fragment or amino acid sequence may comprise an epitope of the disease-associated antigen or a sequence which is homologous to an epitope of the disease-associated antigen, wherein the T cells bind to said epitope. Thus, according to the disclosure, an antigen may comprise an immunogenic fragment of the disease-associated antigen or an amino acid sequence being homologous to an immunogenic fragment of the disease-associated antigen. An “immunogenic fragment of an antigen” according to the disclosure refers to a fragment of an antigen which is capable of stimulating, priming and / or expanding T cells, wherein the vaccine antigen (equivalent to the disease-associated antigen) provides the relevant epitope for binding by T cells. In some embodiments, the vaccine antigen is expressed on the surface of a cell such as an antigen-presenting cell so as to provide the relevant epitope for binding by the T cells.VII. Delivery

[0205] In one aspect, the present disclosure is related to target delivery of the lipid nanoparticle of the present disclosure to certain cells or tissues. In some embodiments, the lipid nanoparticle of the present disclosure targets the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. The “lymphatic system” is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and the circulating lymph. The primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and the bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, which include lymph nodes and theAttorney Docket No. 4404-0141PW01spleen, maintain mature naive lymphocytes and initiate an adaptive immune response. Targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen is contemplated herein if the lipid nanoparticle (for example, the LNP comprising RNA) being administered contains an antigen or a nucleic acid encoding an antigen that induces an immune response.

[0206] In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in the spleen.

[0207] The term “antigen” according to the present disclosure encompasses any substance that will elicit or induce an immune response and / or any substance against which an immune response or an immune mechanism such as a cellular response is directed, and is processed into antigenic peptides. An immune response or an immune mechanism is directed against one or more antigenic peptides, in particular if presented in the context of MHC molecules. In particular, an “antigen” refers to any substance, such as a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T-cells). According to the present disclosure, the term “antigen” encompasses any molecule which comprises at least one epitope, such as a T cell epitope. An antigen in the context of the present disclosure is a molecule which, optionally after processing, induces an immune reaction, specific for the antigen (including cells expressing the antigen). In some embodiments, an antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such antigen.

[0208] The term “epitope” refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies of T cells or B cells, in particular when presented in the context of MHC molecules. An epitope of a protein comprises a continuous or discontinuous portion of said protein and is particularly between about 5 and about 100, between about 5 and about 50, between about 8 and about 0, between about 10 and about 25 amino acids in length, for example, the epitope may be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, the epitope in the context of the present disclosure is a T cell epitope.

[0209] In some embodiments, the lipid nanoparticle of the present disclosure is prepared to preferentially distribute to other target tissues, cells or organs, including but not limited to heart, lungs, kidneys, spleen. In some embodiments, the lipid nanoparticleAttorney Docket No. 4404-0141PW01of the present disclosure is prepared to achieve enhanced delivery to the target cells and tissues. For example, the nucleic acid (e.g., mRNA) encapsulated in the lipid nanoparticle of the present disclosure can be delivered to and / or transfect targeted cells or tissues.

[0210] In some embodiments, the encapsulated nucleic acid (e.g., mRNA) are capable of being expressed and functional polypeptide products produced (and in some circumstances excreted) by the target cell, thereby providing a beneficial property to the target cells or tissues. Such encapsulated nucleic acid (e.g., mRNA) may encode therapeutic agents such as peptides and proteins. Non-limiting examples of the therapeutic agents include cytokines, hormones, adhesion molecules, immunoglobulins, immunostimulatory agents, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressing proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins. Although various therapeutic agents are contemplated, the definitions of the aforesaid terms are not mutually exclusive; for example, a therapeutic agent can be both cytokine and immunostimulatory agent.

[0211] The term “cytokines” refers to proteins which have a molecular weight of about 5 to 20 kDa and which participate in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). In particular, when released, cytokines exert an effect on the behavior of cells around the place of their release. Cytokines contemplated herein include but are not limited to lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). Particular examples of cytokines include but are not limited to erythropoietin (EPO), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon alfa (IFNa), interferon beta (IFNP), interferon gamma (INFy), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), and interleukin 21 (IL-21).

[0212] The term “hormone” as used herein is a peptide or protein hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormones (such as human grown hormone or bovine somatotropin), oxytocin, atrial -natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptins.Attorney Docket No. 4404-0141PW01

[0213] The term “immunostimulatory agents” as used herein is any compound altering an immune response, by inducing and / or suppressing maturation of immune cells, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells.

[0214] Immunostimulatory agents include but are not limited to antiviral and antitumor compounds, and can also down-regulate other aspects of the immune response, for example shifting the immune response away from a TH2 immune response, which is useful for treating a wide range of TH2 mediated diseases. Immunostimulatory agents can be useful as vaccine adjuvants. Particular examples of immunostimulatory agents include interleukins, colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, and antigens, in particular tumor-associated antigens, pathogen-associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens.

[0215] The term “growth factors” refers to molecules which can stimulate cellular growth, proliferation, healing, and / or cellular differentiation. Typically, growth factors act as signaling molecules between cells. The term “growth factors” include particular cytokines and hormones which bind to specific receptors on the surface of their target cells. Examples of growth factors include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), such as VEGFA, epidermal growth factor (EGF), insulin-like growth factor, ephrins, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, neuregulins, neurotrophins (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (anti-apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-P)), and tumor necrosis factor-alpha (TNF-a). In some cases, a “growth factor” is a peptide or protein growth factor.

[0216] The term “tumor suppressing proteins” refers to the molecules, such as peptides or proteins, which protect a cell from carcinogenesis (i.e., progression to cancer). Tumor-suppressing proteins exhibit a weakening or repressive effect on the regulation of the cell cycle and / or promote apoptosis. Without wishing to be bound by any theory,Attorney Docket No. 4404-0141PW01the functions of tumor suppressing protein include repressing gene(s) essential for the continuing of the cell cycle; causing damage of DNA; arresting cell cycle; initiating apoptosis, provided that the damaged DNA is irreparable; suppressing metastasis (e.g., impeding cancer cells from migrating and / or invading); and DNA repair. Particular and non-limiting examples of tumor-suppressing proteins include p53, adenomatous polyposis coli (APC), BRCA, phosphatase and tensin homolog (PTEN), SWI / SNF (SWItch / Sucrose Non-Fermentable), von Hippel-Lindau tumor suppressor (pVHL), Neurofibromin (NF-1), Protein patched homolog (PTCH), retinoblastoma protein (Rb), CD95, suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 14 (STM), and Yippee-like 3 (YPEL3).

[0217] The term “apoptosis regulators” refers to the molecules, such as peptides or proteins, which modulate apoptosis, i.e., which either activate or inhibit apoptosis. Apoptosis regulators can be grouped into two broad classes: those which modulate mitochondrial function and those which regulate caspases. The first class includes proteins (e.g., BCL-2, BCL-xL) which act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and / or release of pro-apoptotic proteins such as cytochrome C into the cytosol. Also to this first class belong proapoptotic proteins (e.g., BAX, BAK, BIM) which promote release of cytochrome C. The second class includes proteins such as the inhibitors of apoptosis proteins (e.g., XIAP) or FLIP which block the activation of caspases.

[0218] The term “transcription factors” refers to the molecules containing at least one DNA-binding domain which binds to a specific DNA sequence, usually adjacent to the genes regulated by the transcription factors, which regulate the rate of transcription of certain gene(s) from DNA to mRNA, in particular by binding to a specific DNA sequence. Transcription factors may regulate, for example, cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and / or in response to signals from outside the cell, such as a hormone. Particular and non-limiting examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.

[0219] An “adjuvant”, as described herein, refers to a substance or vehicle that enhances the immune response to an antigen. The pharmaceutical compositions of the present disclosure optionally comprise one or more adjuvants, or may be administered with one or more adjuvants so as to enhance the immune response to the antigen administered. Examples of suitable adjuvants include, but are not limited to, aAttorney Docket No. 4404-0141PW01suspension of minerals (e.g., alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; a water-in-oil or oil-in-water emulsion in which antigen solution is emulsified in mineral oil or in water (e.g., Freund’s incomplete adjuvant). Adjuvants may comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund’s adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes.

[0220] Non-limiting examples of adjuvants include LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cyctokines, such as monokines, lymphokines, interleukins, chemokines. The chemokines may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF a, INF-y, GM-CSF, LT-a. Further known adjuvants are aluminium hydroxide, Freund’s adjuvant or mineral oil. Other adjuvants contemplated herein for use in the present disclosure include lipopeptides, such as Pam3Cys.

[0221] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intradermal, transdermal, topical, intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal. In one embodiment, the route of administration is selected from intravenous, intrathecal, intramuscular, intranasal, sublingual, or by pulmonary delivery. In one embodiment, the pharmaceutical composition of the present disclosure is intramuscularly administered. In some particular embodiments, the intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In some embodiments, the pharmaceutical composition of the present disclosure is administered for delivering the nucleic acids to a target muscle cell.

[0222] AbbreviationsACN AcetonitrileBOC tert-Butyloxycarbonyl protecting groupDCM DichloromethaneDIPEA N,N-DiisopropylethylamineDMAP 4-DimethylaminopyridineDMF DimethylformamideEDCI l-Ethyl-3-(3-dimethylaminopropyl)carbodiimideAttorney Docket No. 4404-0141PW01HBTU Hexafluorophosphate Benzotriazole Tetramethyl UroniumMe MethylnPr n-PropylnBu n-ButylRT Room temperatureTFA Trifluoroacetic AcidTHF TetrahydrofuranGeneral procedure of Compound Preparation

[0223] The compounds of formula (I) may be prepared using the methods disclosed herein and routine modifications thereof, which will be apparent given the disclosure herein and methods well known in the art.

[0224] General Scheme:"6a, R-Me, R' -Me 6b, R'=nPr, R"=nPr 6c, R'=nBu, R"=nBuAttorney Docket No. 4404-0141PW01" " "Examples

[0225] The following working examples illustrate the embodiments of the disclosure that are presently best known. However, it is to be understood that the following are only exemplary or illustrative of the application of the principles of the present disclosure. Numerous modifications and alternative compositions, methods, and systems may be devised by those skilled in the art without departing from the spirit and scope of the present disclosure. Thus, while the present disclosure has been described above with particularity, the following examples provide further detail in connection with what are presently deemed to be the most practical embodiments of the disclosure.

[0226] Synthesis of intermediate compounds

[0227] Scheme of Synthesis of compounds Al, A2, and A3A2 A3

[0228] Example Al: Synthesis of compound AlAttorney Docket No. 4404-0141PW01A1Compound Al: Methyl l-(3-(benzyloxy)propyl)piperidine-4-carboxylate

[0229] A mixture solution of l-Methylpiperidine-4-carboxylic acid (5 g, 34.9 mmol) and Benzyl 3-bromopropyl ether (4 g, 17.5 mmol) in ACN / CHCI3 was added with DIPEA (6.1 mL, 35 mmol) and the mixture was heated to 90 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (30 / 1) to yield the compound Al (4.35 g, 14.9 mmol) as a colorless oil. 1H NMR (600 MHz, CDC13) 6 1.85 (brs., 4H), 1.95 (brs., 2H), 2.10 (brs., 2H), 2.33 (brs., 1H), 2.50 (br. s., 2H), 2.91 (brs., 2H) 3.52 (t, J= 6.44 Hz, 2H), 3.68 (s, 3H), 4.50 (s, 2H), 7.2 -7.38 (m, 5H). MS (M+l): 292

[0230] Example A2: Synthesis of compound A2Compound A2: l-(3-(Benzyloxy)propyl)piperidine-4-carboxylic acid

[0231] A mixture solution of compound Al (4.35 g, 14.9 mmol) in MeOH / THF was added with 2M LiOH (14.9 mL, 30 mmol). After stirring at RT for 19 hours, the reaction solution was evaporated and then MeOH was added to dissolve the residue. The salt was filtered and the filtrate was evaporated to afford the compound A2 (5.4 g, 19.5 mmol) as a yellow solid. 'H NMR (600 MHz, CDCh) 6 1.87-2.07 (m, 6 H), 2.29 (brs., 1H), 2.78 (brs., 2H), 2.89 - 3.09 (m, 2H), 3.22-3.42 (m, 2H), 3.42-3.53 (m, 2H), 4.41 (s, 2H), 7.1-7.30 (m, 5H). MS (M+l): 278

[0232] Example A3: Synthesis of compound A3Attorney Docket No. 4404-0141PW01A3Compound A3: l-(3-Hydroxypropyl)piperidine-4-carboxylic acid

[0233] A mixture solution of compound A2 (3 g, 10.80 mmol) and Pd / C (3 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the desired product A3 (1.5 g, 8 mmol, 79%) as a colorless oil was obtained without further purification. 'H NMR (600 MHz, CD3OD) 6 1.88-2.04 (m, 4H), 2.13 (s, 2H), 2.11 (s, 1H), 2.45 (brs., 2H), 3.05 (brs., 2H), 3.13-3.21 (m, 2H), 3.48 (brs., 2H), 3.66 (t, J= 5.72 Hz, 2H). MS (M+l): 188

[0234] Scheme of Synthesis of compounds Bl, B2, and B3

[0235] Example B 1 : Synthesis of compound B 1B1Compound Bl: Methyl l-(4-(benzyloxy)butyl)piperidine-4-carboxylate

[0236] A mixture solution of l-methylpiperidine-4-carboxylic acid (5.0 g, 35.0 mmol) and benzyl 4-bromobutyl ether (4.25 g, 17.5 mmol) in ACN / CHCI3 was added withAttorney Docket No. 4404-0141PW01DIPEA (6.0 mL, 35.0 mmol) and the mixture was heated to 90 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (30 / 1) to yield the compound Bl (4.9 g, 16.0 mmol) as a colorless oil. 'H NMR (600 MHz, CDCh) 51.54-1.66 (m, 4H), 1.71-1.81 (m, 2H), 1.85-1.95 (m, 2H), 1.95-2.05 (m, 2H), 2.26-2.38 (m, 3H) 2.86-2.88 (m, 2H), 3.43-3.50 (m, 2H), 3.63-3.70 (s, 3H), 4.49 (s, 2H), 7.25-7.29 (m, 1H), 7.29 - 7.35 (m, 4H). MS (M+l): 306

[0237] Example B2: Synthesis of compound B2Compound B2: l-(4-(Benzyloxy)butyl)piperidine-4-carboxylic acid

[0238] A mixture solution of compound Bl (4.9 g, 16.0 mmol) in MeOH / THF was added with 2M Li OH (16 mL, 32.0 mmol). After stirring at RT for 19 hours, the reaction solution was evaporated and then MeOH was added to dissolve the residue. The salt was filtered and the filtrate was evaporated to afford the compound B2 (6.7 g, 22.9 mmol) as a yellow solid. 'H NMR (600 MHz, CD3OD) 6 1.62-1.74 (m, 2H), 1.78-1.89 (m, 3H), 1.92 (brs., 1H), 2.11 (brs., 2H), 2.53 (brs., 1H), 3.02 (br. s., 2H) ,3.07-3.13 (m, 2H), 3.34-3.53 (m, 2H), 3.55 (t, J= 5.96 Hz, 2H), 4.51 (s, 2H), 7.21-7.40 (m, 5H). MS (M+l): 292

[0239] Example B3: Synthesis of compound B3B3Compound B3: l-(4-Hydroxybutyl)Piperidine-4-Carboxylic Acid

[0240] A mixture solution of compound B2 (3 g, 10.3 mmol) and Pd / C (3 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, theAttorney Docket No. 4404-0141PW01desired product B3 (2 g, 10.0 mmol, 97%) as a colorless oil was obtained without further purification. MS (M+l): 202

[0241] Scheme of Synthesis of compound Cl, C2, and C3

[0242] Example Cl: Synthesis of compound ClC1Compound Cl: Methyl l-(5-(benzyloxy)pentyl)piperidine-4-carboxylate

[0243] A mixture solution of l-Methylpiperidine-4-carboxylic acid (5 g, 34.9 mmol) and Benzyl 5 -bromoamyl ether (4.5 g, 17.5 mmol) in ACN / CHCI3 was added with DIPEA (6.1 mL, 34.9 mmol) and the mixture was heated to 90 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (30 / 1) to yield the compound Cl (4.7 g, 14.6 mmol) as a colorless oil. 'H NMR (600 MHz, CDCh) 61.32-1.46 (m, 2H), 1.56 (brs., 2H), 1.58-1.72 (m, 2H), 1.84-2.07 (m, 6H), 2.38 (brs., 3H), 2.91 (brs., 2H), 3.46 (t, J = 6.44 Hz, 2H), 3.68 (s, 3H), 4.49 (s, 2H), 7.23-7.37 (m, 5H). MS (M+l): 320

[0244] Example C2: Synthesis of compound C2Compound C2: l-(5-(Benzyloxy)Pentyl)Piperidine-4-Carboxylic AcidAttorney Docket No. 4404-0141PW01

[0245] A mixture solution of compound Cl (4.7 g, 14.6 mmol) in MeOH / THF was added with 2M LiOH (14.7 mL, 29 mmol). After stirring at RT for 19 hours, the reaction solution was evaporated and then MeOH was added to dissolve the residue. The salt was filtered and the filtrate was evaporated to afford the compound C2 (5.39 g, 17.6 mmol) as a yellow solid.XH NMR (600 MHz, CDCh) 5 1.40-1.51 (m, 2H), 1.62-1.69 (m, 2H), 1.69-1.78 (m, 2H), 1.92 (brs., 2H), 2.07-2.09 (m, 2H), 2.41 (brs., 1H), 2.97 (brs., 1H), 3.00-3.08 (m, 2H), 3.42 (br. s., 1H) ,3.52 (td, J=6.20, 1.91 Hz, 2H), 4.48 (s, 2H), 4.85-4.87 (m, 2H), 7.24-7.30 (m, 1H), 7.30-7.35 (m, 4H). MS (M+l): 306

[0246] Example C3: Synthesis of compound C3Compound C3: l-(5-Hydroxypentyl)Piperidine-4-Carboxylic Acid

[0247] A mixture solution of compound C2 (3.26 g, 10.7 mmol) and Pd / C (3 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the desired product C3 (2 g, 9.3 mmol, 87%) as a colorless oil was obtained without further purification.1H NMR (600 MHz, CD3OD) 61.43 (m, 2H), 1.52-1.64 (m, 2H), 1.70-1.85 (m, 2H), 1.98 (brs., 2H,) 2.12-2.14 (m, 2H), 2.48 (br. s., 1H), 3.00-3.14 (m, 4H), 3.48 (s, 1H), 3.56 (t, J=6.44 Hz, 2H), 4.87-4.9 (m, 1H). MS (M+l): 216

[0248] Scheme of Synthesis of compounds DI and D2Attorney Docket No. 4404-0141PW01

[0249] Example DI : Synthesis of compound DID1Compound DI: Methyl l-(6-(Benzyloxy)Hexyl)Piperidine-4-Carboxylate

[0250] A mixture solution of l-Methylpiperidine-4-carboxylic acid (5.00 g, 34.90 mmol) and Benzyl 6-bromohexyl ether (4.75 g, 17.50 mmol) in ACN / CHCh was added with DIPEA (6.00 mL, 34.90 mmol) and the mixture was heated to 90 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (30 / 1) to yield the compound DI (3.95 g, 11.86 mmol) as a colorless oil.JH NMR (600 MHz, CDCI3) 8 I.25-1.31 (m, 2H), 1.31-1.39 (m, 2H), 1.39-1.51 (m, 2H), 1.53-1.65 (m, 2H), 1.68-1.80 (m, 2H), 1.83-1.91 (m, 2H), 1.95 (t, J= 10.97 Hz, 2H), 2.21-2.31 (m, 3H), 2.84 (d, J = II.44 Hz, 2H), 3.43 (t, J = 6.44 Hz, 2H), 3.64 (s, 3H), 4.46 (s, 2H), 7.18-7.28 (m, 1H), 7.28-7.39 (m, 4H). MS (M+1): 334.

[0251] Example D2: Synthesis of compound D2Attorney Docket No. 4404-0141PW01Compound D2: l-(6-(Benzyloxy)Hexyl)Piperidine-4-Carboxylic Acid

[0252] A mixture solution of compound DI (7.91 g, 23.71 mmol) in MeOH / THF was added with 2M Li OH (23.70 mL, 47.40 mmol). After stirring at RT for 19 hours, the reaction solution was evaporated and then MeOH was added to dissolve the residue. The salt was filtered and the filtrate was evaporated to afford the compound D2 (7.36 g, 23.03 mmol) as a yellow solid.XH NMR (600 MHz, CD3OD) 5 1.34-1.42 (m, 2H), 1.42- 1.50 (m, 2H), 1.58-1.68 (m, 3H), 1.68-1.79 (m, 3H), 1.95 (br. s., 2H), 2.12 (d, J= 12.40 Hz, 2H), 2.48 (br. s.,lH), 2.97-3.09 (m, 3H), 3.50 (t, J= 6.44 Hz, 3H), 4.48 (s, 2H), 7.21-7.37 (m, 5H). MS (M+l): 320.

[0253] Example 1: Synthesis of compound AP07-012>Attorney Docket No. 4404-0141PW01AP07-01Scheme: AP07-01 synthesis

[0254] Step 1: Synthesis of compound 33Compound 3: 6-((tert-Butoxycarbonyl)amino)hexyl 2-hexyldecanoate

[0255] A mixture solution of compound 1 (4.24 g, 19.50 mmol) and compound 2 (10 g, 39.00 mmol) in di chloromethane (DCM) was stirred at 0°C, and then l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (7.5 g, 39.00 mmol) and (4-dimethylaminopyridine) (DMAP) (1 g, 7.80 mmol) were added. After stirring atRT for 19 hours, the reaction solution was washed with 2N HCl(aq) and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with ethyl acetate / hexane (EA / Hex) (1 / 10) to yield the compound 3 (8 g, 17.50 mmol) as a white solid. 'HNMR (600 MHz, CDCh) 60.87 (td, J = 7.03, 1.67 Hz, 6H), 1.19-1.32 (m, 20H), 1.3 -1.40 (m, 4H), 1.4 -1.52 (m, 12H), 1.52-1.66 (m, 4H),Attorney Docket No. 4404-0141PW012.30 (tt, J= 9.06, 5.25 Hz, 1H), 3.10 (brs., 2H), 4.06 (t, J= 6.68 Hz, 2H), 4.49 (brs., 1H). MS (M+l): 456

[0256] Step 2: Synthesis of compound 44Compound 4: 6-Aminohexyl 2-Hexyldecanoate

[0257] A solution of compound 3 (8 g, 17.50 mmol) in dichloromethane (DCM) was stirred at 0°C, and then trifluoroacetic acid (TFA) (13.4 mL, 175.00 mmol) was added. After stirring at RT for 19 hours, the reaction solution was evaporated to remove the TFA, the residue was neutralized with 2N NaOH(aq) until pH = 10-11. Then the residue was extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (5 / 1) to yield the compound 4 (6.2 g, 17.50 mmol) as a colorless oil. 'HNMR (600 MHz, CDC13) 60.87 (td, J= 7.15, 1.43 Hz, 6H), 1.18-1.34 (m, 20H), 1.34-1.51 (m, 6H), 1.51 - 1.60 (m, 2H), 1.60-1.66 (m, 2H), 1.71 (t, J= 7.15 Hz, 2H), 2.30 (s, 1H), 2.97 (d, J= 5.72 Hz, 2H), 4.05 (t, J= 6.68 Hz, 2H) 7.95 (brs., 2 H). MS (M+l): 356

[0258] Step 3: Synthesis of compound 6bCompound 6b: Azanediylbis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0259] A mixture solution of compound 4 (6.2 g, 17.40 mmol) and compound 5b (4.18 g, 11.50 mmol) in ACN / CHCI3 was added with DIPEA (6.4 mL, 36.6 mmol) and the mixture was heated to 90 °C for 4 hours. The solvent was removed and extracted withAttorney Docket No. 4404-0141PW01ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound 6b (2 g, 3.1 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 6 0.87 (t, J = 6.91 Hz, 14H), 1.16-1.32 (m, 47H), 1.32-1.47 (m, 14H), 1.53-1.72 (m, 11H), 1.88 (d, J= 6.20 Hz, 3H), 2.30 (td, J= 9.18, 4.05 Hz, 3H), 2.86-3.00 (m, 3H), 3.97-4.11 (m, 4H), 9.44 (brs., 1H).MS (M+l): 639

[0260] Step 4: Synthesis of compound AP07-01AP07-01Compound AP07-01: ((l-(2-Hydroxyethyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0261] A mixture solution of compound 6b (0.15 g, 0.9 mmol), l-(2-hydroxyethyl)piperidine-4-carboxylic acid (0.72 g, 1 mmol) and HBTU (0.33 g, 0.9 mmol) in DMF was added with DIPEA (0.8 mL, 4.3 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-01 (0.28 g, 0.3 mmol) as a yellow oil.1H NMR (600 MHz, CDCh) 8 0.87 (t, J= 6.91 Hz, 12H), 1.21-1.31 (m, 43 H), 1.32-1.44 (m, 10H), 1.46 - 1.66 (m, 13H),1.83 (brs., 1H) 1.86-1.95 (m, 2H) 2.23-2.34 (m, 2H), 2.36 (brs., 1H), 2.52 (brs., 1H), 2.65 (brs., 2H), 3.10 (brs., 2H), 3.1 -3.24 (m, 2H), 3.24-3.30 (m, 2H), 3.67 (brs., 2H), 4.05 (dt, J= 16.69, 6.68 Hz, 4H). MS (M+l): 850

[0262] Example 2: Synthesis of compound AP07-02Attorney Docket No. 4404-0141PW01AP07-02Compound AP07-02: ((l-(3-Hydroxypropyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0263] Amixture solution of compound 6b (0.67 g, 1.0 mmol), A3 (0.15 g, 0.8 mmol) and HBTU (0.3 g, 0.8 mmol) in DMF was added with DIPEA (0.7 mL, 4.0 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-02 (0.31 g, 0.35 mmol) as a yellow oil.JH NMR (600 MHz, CDCh) 60.87 (t, J= 6.91 Hz, 12H), 1.24-1.66 (m, 67H), 1.78 (brs., 2 H), 1.84-1.93 (m, 2H), 2.24-2.34 (m, 2H), 3.11-3.23 (m, 4H), 3.18-3.28 (m, 6H), 3.80 (t, J= 5.25 Hz, 2H), 4.05 (dt, J= 16.81, 6.85 Hz, 4H). MS (M+1): 864.

[0264] Example 3: Synthesis of compound AP07-03Attorney Docket No. 4404-0141PW01AP07-03Compound AP07-03: ((l-(4-Hydroxybutyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0265] A mixture solution of 6b (0.62 g, 0.9 mmol), B3 (0.15 g, 0.75 mmol) and HBTU (0.28 g, 0.7 mmol) in DMF was added with DIPEA (0.6 mL, 3.7 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-03 (80 mg, 0.09 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 60.87 (td, J= 7.03, 1.67 Hz, 12H), 1.25-1.70 (m, 70H), 1.91 (brs., 3H), 2.26-2.35 (m, 2H), 2.63 (brs., 2H), 2.98 (brs., 1H), 3.07 (br. s., 1H), 3.14-3.23 (m, 2H), 3.23-3.30 (m, 2H), 3.30-3.36 (m, 2H), 3.54-3.71 (m, 2H), 3.96-4.10 (m, 4H). MS (M+1): 878.

[0266] Example 4: Synthesis of compound AP07-04Attorney Docket No. 4404-0141PW01AP07-04Compound AP07-04: ((l-(5-Hydroxypentyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0267] A mixture solution of compound 6b (0.66 g, 0.9 mmol), compound C3 (0.17 g, 0.8 mmol) and HBTU (0.3 g, 0.8 mmol) in DMF was added with DIPEA (0.7 mL, 4.0 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSCU After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-04 (0.3 g, 0.33 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 60.86 (t, J= 6.91 Hz, 12H), 1.20-1.66 (m, 72H), 1.88 (m, 5 H), 2.24-2.34 (m, 2H), 2.41 (brs., 3 H), 3.04 (brs., 2H), 3.16-3.23 (m, 2H), 3.23-3.29 (m, 2H), 3.63 (t, J = 6.44 Hz, 2H), 4.05 (dt, J = 18.48, 6.50 Hz, 4H). MS (M+l): 892.

[0268] Example 5: Synthesis of compound AP07-05Attorney Docket No. 4404-0141PW01AP07-05Scheme: AP07-05 synthesis

[0269] Step 1: Synthesis of compound 6a6aCompound 6a: 6-((6-((2-Butyloctanoyl)oxy)hexyl)amino)hexyl 2-hexyldecanoateAttorney Docket No. 4404-0141PW01

[0270] A mixture solution of compound 4 (2.6 g, 7.3 mmol) and compound 5a (1.6 g, 4.4 mmol) in ACN / CHCI3 was added with DIPEA (2.7 mL, 15.4 mmol) and the mixture was heated to 90 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSCU. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound 6a (1.1 g, 1.7 mmol) as a yellow oil.1H NMR (600 MHz, CDCh) 6 0.87 (td, J= 7.15, 2.86 Hz, 12H), 1.20-1.33 (m, 32H), 1.33-1.49 (m, 12H), 1.52-1.72 (m, 10H), 1.74-1.93 (m, 4H), 2.25 - 2.35 (m, 2H), 2.83-2.95 (m, 3 H), 4.00-4.10 (m, 4H). MS (M+l): 639.

[0271] Step 2: Synthesis of compound AP07-05AP07-05Compound AP07-05: 6-(7V-(6-((2-Butyloctanoyl)oxy)hexyl)-l-(4- hydroxybutyl)piperidine-4-carboxamido)hexyl 2-hexyldecanoate

[0272] A mixture solution of compound 6a (0.57 g, 0.9 mmol), compound B3 (0.15 g, 0.75 mmol) and HBTU (0.28 g, 0.7 mmol) in DMF was added with DIPEA (0.6 mL, 3.7 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSCk After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-05 (48 mg, 0.06 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 6 0.80-0.98 (m, 12H), 1.25-1.62 (m, 62H), 1.89 (brs., 2H), 1.99 (brs., 2H), 2.28-2.33 (m, 2H), 2.61 (brs., 2H), 2.97 (brs., 1H), 3.06 (brs., 1H), 3.17-3.19 (m, 2H), 3.25-3.27 (m, 2H), 3.43 (brs., 3H), 3.72 (brs., 1H), 4.00-4.12 (m, 4H). MS (M+l): 822

[0273] Example 6: Synthesis of compound AP07-06Attorney Docket No. 4404-0141PW01AP07-06Scheme: AP07-06 synthesis

[0274] Step 1: Synthesis of compound 6cCompound 6c: 6-((6-((2-Hexyldecanoyl)oxy)hexyl)amino)hexyl 2- heptylundecanoate

[0275] A mixture solution of compound 4 (2.8 g, 7.9 mmol) and compound 5c (2.1 g, 4.7 mmol) in ACN / CHCh was added with DIPEA (2.9 mL, 16.5 mmol) and the mixtureAttorney Docket No. 4404-0141PW01was heated to 90 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound 6c (1.0 g, 1.4 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 60.79 - 0.95 (m, 12H), 1.17-1.47 (m, 52H), 1.53-1.70 (m, 9H), 1.85 (brs., 3H), 2.25-2.34 (m, 3 H), 2.53 (brs., 4H), 2.81-3.00 (m, 3H), 3.93 - 4.13 (m, 4H), 9.27 (brs., 1H). MS (M+l): 723

[0276] Step 2: Synthesis of compound AP07-06AP07-06Compound AP07-06: 6-(N-(6-((2-Hexyldecanoyl)oxy)hexyl)-l-(4- hydroxybutyl)piperidine-4-carboxamido)hexyl 2-heptylundecanoate

[0277] A mixture solution of compound 6c (0.65 g, 0.90 mmol), compound B3 (0.15 g, 0.75 mmol) and HBTU (0.28 g, 0.70 mmol) in DMF was added with DIPEA (0.6 mL, 3.7 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSCk After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-06 (0.25 g, 0.27 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 6 0.78-0.90 (m, 12H), 1.07 (brs., 2H) 1.19-1.39 (m, 52H) ,1.43 (brs., 1H), 1.49 (m, 3H), 1.53-1.69 (m, 11H), 1.69 - 1.79 (m, 3H), 1.84-2.03 (m, 4H), 2.25-2.35 (m, 4H), 2.94 (m, 1H), 3.04 (brs., 2H), 3.14-3.24 (m, 2H), 3.24-3.32 (m, 3H), 3.45 (brs., 2H), 3.68-3.75 (m, 1H), 3.98 - 4.10 (m, 4H). MS (M+l): 906

[0278] Example 7: Synthesis of compound AP07-07Attorney Docket No. 4404-0141PW01 >>Attorney Docket No. 4404-0141PW01" <" AP07-07Scheme: AP07-07 synthesis

[0279] Step 1: Synthesis of compound 8Compound 8: diethyl 2-(6-(Benzyloxy)hexyl)-2-methylmalonate

[0280] NaH (2.3 g, 58.6 mmol) was added to a solution of compound 7 (5.1 g, 29.3 mmol) in THF at 0°C, and then benzyl 6-bromohexyl ether (6.35 g, 23.4 mmol) was added. After stirring under reflux overnight, the reaction solution was washed with sat. NH4Cl(aq) and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with EA / Hex (1 / 25) to yield the compound 8 (3.0 g, 8.2 mmol, 35%) as a colorless oil. 'H NMR (600 MHz, CDCh) 6 1.20-1.26 (m, 6H), 1.26-1.37 (m, 6H), 1.39 (s, 3H), 1.57-1.63 (m, 2H) 1.81-1.87 (m, 2H), 3.45 (t, J = 6.44 Hz, 2H), 4.17 (d, J= 7.15 Hz, 4H), 4.49 (s, 2H), 7.28 (dt, J = 5.60, 2.68 Hz, 1H), 7.31-7.36 (m, 4H). MS (M+1): 365

[0281] Step 2: Synthesis of compound 99Compound 9: 2-(6-(Benzyloxy)hexyl)-2-methylpropane-l,3-diolAttorney Docket No. 4404-0141PW01

[0282] L1AIH4 (0.63 g, 16.50 mmol) was added to a solution of compound 8 (3.0 g, 8.2 mmol) in THF at 0°C. After stirring under RT overnight, the reaction solution was quenched with IM NaOH(aq) at 0°C and the residue was filtered and the filtrate was evaporated to afford the compound 9 (2.3 g, 8.1 mmol) as a yellow solid. 'H NMR (600 MHz, CDCh) 60.81 (s, 3H), 1.23-1.35 (m, 5H), 1.35-1.42 (m, 2H), 1.57-1.64 (m, 2H), 1.97 (brs., 3H), 3.46 (t, J = 6.68 Hz, 2H), 3.50 - 3.57 (m, 4H), 4.50 (s, 2H) ,7.27-7.30 (m, 1H), 7.32-7.36 (m, 4H). MS (M+l): 281

[0283] Step 3: Synthesis of compound 10Compound 10: 2-(6-(Benzyloxy)hexyl)-2-methylpropane-l,3-diyl bis-(decanoate)

[0284] A mixture solution of compound 9 (2.3 g, 8.1 mmol) and capric acid (5.0 g, 29.0 mmol) in dichloromethane (DCM) was stirred at 0 °C, and then EDCI (5.6 g, 29.0 mmol) and DMAP (0.71 g, 5.85 mmol) were added. After stirring at RT for 19 hours, the reaction solution was washed brine and dried over MgSCh. After evaporation, the residue was purified by silica gel column chromatography with ethyl acetate / hexane (EA / Hex) (1 / 10) to yield the compound 10 (4.3 g, 7.3 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 60.88 (t, J= 7.15 Hz, 6H), 0.91 (s, 3H), 1.16-1.34 (m, 32H), 1.60-1.63 (m, 6H), 2.30 (t, J= 7.63 Hz, 4H), 3.46 (t, J= 6.68 Hz, 2H), 3.84-3.94 (m, 4H), 4.50 (s, 2H), 7.27-7.29 (m, 1H), 7.30-7.37 (m, 4H). MS (M+l): 589

[0285] Step 4: Synthesis of compound 11Compound 11: 2-(6-Hydroxyhexyl)-2-methylpropane-l,3-diyl bis(decanoate)

[0286] A mixture solution of compound 10 (4.3 g, 7.3 mmol) and Pd / C (4 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the desired product 11 (3.7 g, 7.3 mmol, 100%) as a colorless oil was obtained withoutAttorney Docket No. 4404-0141PW01further purification. 'H NMR (600 MHz, CDCh) 6 0.88 (t, J = 7.15 Hz, 6H), 0.91 (s, 3H), 1.21-1.35 (brs., 35H), 1.56 (m, 3H), 2.30 (t, J= 7.63 Hz, 4H), 3.62 - 3.66 (m, 3H), 3.89 (d, J=2.38 Hz, 4H)., MS (M+l): 499

[0287] Step 5: Synthesis of compound 12Compound 12: 2-(6-Iodohexyl)-2-methylpropane-l,3-diyl bis(decanoate)

[0288] A mixture solution of compound 11 (975 mg, 2.202 mmol), PPhi (635 mg, 2.242 mmol) and imidazole (165 mg, 2.242 mmol) in DCM was stirred at 0°C, followed by the addition of I2 (670 mg, 2.643 mmol). The reaction solution was allowed to warm to RT and stirred overnight. After washing with sat. Na2S2Ch(aq) and brine, the residue was dried over MgSC and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography with EA / Hex (1 / 25) to yield the desired product 12 (1.096 g, 90%) as a colorless oil. 'H NMR (600 MHz, CDCh) 60.88 (t, J = 7.15 Hz, 6H), 0.92 (s, 3H), 1.19-1.36 (m, 32H), 1.57-1.66 (m, 4H), 1.7 -1.87 (m, 2H), 2.30 (t, J= 7.39 Hz, 4H), 3.18 (t, J= 6.91 Hz, 2H), 3.81-3.94 (m, 4 H).

[0289] Step 6: Synthesis of compound 13Compound 13: 2-(6-((6-((2-Hexyldecanoyl)oxy)hexyl)amino)hexyl)-2- methylpropane-l,3-diyl bis(decanoate)

[0290] A mixture solution of compound 4 (2.0 g, 5.6 mmol) and compound 12 (2.0 g, 3.4 mmol) in ACN / CHCh was added with DIPEA (2.0 mL, 11.8 mmol) and the mixtureAttorney Docket No. 4404-0141PW01was heated to 90 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound 13 (1.05 g, 1.25 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 60.87 (t, J= 6.91 Hz, 12H), 0.91 (s, 3H), 1.12-1.41 (m, 59H), 1.43-1.64 (m, 10H), 1.83 (brs., 3H), 2.30 (t, J= 7.39 Hz, 4H), 2.83-2.95 (m, 3H), 3.84-3.93 (m, 4H), 4.00-4.09 (m, 2H). MS (M+1): 837

[0291] Step 7: Synthesis of compound AP07-07AP07-07Compound AP07-07: 2-(6-(N-(6-((2-Hexyldecanoyl)oxy)hexyl)-l-(4- hydroxybutyl)piperidine-4-carboxamido)hexyl)-2-methylpropane-l,3-diyl bis(decanoate)

[0292] A mixture solution of compound 13 (0.75 g, 0.90 mmol), compound B3 (0.15 g, 0.75 mmol) and HBTU (0.28 g, 0.70 mmol) in DMF was added with DIPEA (0.6 mL, 3.7 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSCk After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-07 (50 mg, 0.05 mmol) as a yellow oil.XH NMR (600 MHz, CDCh) 60.85-0.90 (m, 12H), 1.17-1.36 (m, 58H), 1.60 (qd, J = 14.46, 7.63 Hz, 12H), 1.77-1.87 (m, 4H), 1.91-2.07 (m, 7H), 2.25-2.37 (m, 6H), 3.12 (d, J= 5.72 Hz, 2H), 3.1 -3.26 (m, 1H), 3.26-3.35 (m, 4H), 3.40-3.58 (m, 3H), 3.70-3.83 (m, 2H), 3.83-3.95 (m, 4H), 3.99-4.10 (m, 2H). MS (M+1): 1020

[0293] Example 8: Synthesis of compound AP07-08Attorney Docket No. 4404-0141PW01AP07-08Scheme: AP07-08 synthesis

[0294] Step 1: Synthesis of compound 15Attorney Docket No. 4404-0141PW01Compound 15: 6-((6-((3-(Decanoyloxy)-2-((decyloxy)methyl)-2- methylpropanoyl)oxy)hexyl)amino)hexyl 2-hexyldecanoate

[0295] Amixture solution of compound 4 (3.35 g, 7.14 mmol) and compound 14 (2.50 g, 4.30 mmol) in ACN / Di oxane was added with DIPEA (2.57 mL, 14.80 mmol) and the mixture was heated to 100 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSCk. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound 15 (1.76 g, 2.00 mmol) as a yellow oil.JH NMR(600 MHz, CDCh) 60.87 (td, J= 7.03, 1.19 Hz, 12H), 1.20-1.32 (m, 47H), 1.33- 1.40 (m, 8H), 1.40-1.48 (m, 4H), 1.52-1.66 (m, 8H), 1.73-1.82 (m, 4H), 2.28 (t, J= 7.63 Hz, 5H), 2.85-2.96 (m, 4H), 4.03 (t, J= 6.68 Hz, 2H), 4.08 (t, J= 6.68 Hz, 2H), 4.18 (d, J= 11.44 Hz, 2H), 4.23 (d, J= 10.97 Hz, 2H). MS (M+l): 881.

[0296] Step 2: Synthesis of compound 16Compound 16: 6-((6-((3-(Decanoyloxy)-2-((decyloxy)methyl)-2- methylpropanoyl)oxy)hexyl)amino)hexyl 2-hexyldecanoate

[0297] A mixture solution of compound 15 (3.56 g, 0.40 mmol), compound C3 (0.18 g, 0.60 mmol) and HBTU (0.16 g, 0.40 mmol) in DMF was added with DIPEA (0.35 mL, 2.00 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue wasAttorney Docket No. 4404-0141PW01purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 16 (0.31 g, 0.26 mmol) as a yellow oil. 'H NMR(600 MHz, CDCh) 60.87 (td, J= 6.91, 1.43 Hz, 12H), 1.17-1.32 (m, 52H), 1.32-1.45 (m, 12H), 1.45-1.51 (m, 3H), 1.51-1.70 (m, 17H), 1.89 (d, J= 9.54 Hz, 3H), 2.23-2.33 (m, 5H), 3.13 - 3.23 (m, 2H), 3.23-3.30 (m, 2H), 3.46 (t, J= 6.44 Hz, 2 H), 3.98-4.14 (m, 5H), 4.14-4.27 (m, 4H), 4.48 (s, 2H), 7.26-7.28 (m, 1H), 7.31-7.35 (m, 4H). MS (M+l): 1168.

[0298] Step 3: Synthesis of compound AP07-08AP07-08Compound AP07-08: 6-(7V-(6-((3-(Decanoyloxy)-2-((decyloxy)methyl)-2- methylpropanoyl)oxy)hexyl)-l-(5-hydroxypentyl)piperidine-4carboxamido)hexyl 2-hexyldecanoate

[0299] A mixture solution of compound 16 (0.31 g, 0.26 mmol) and Pd / C (0.31 g) in ethyl acetate was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-08 (0.16 g, 0.14 mmol) as a yellow oil.1HNMR(600 MHz, CDCh) 80.82-0.90 (m, 12H), 1.19- 1.32 (m, 51H), 1.32-1.38 (m, 5H), 1.38-1.45 (m, 3H), 1.45-1.51 (m, 4H), 1.51-1.65 (m, 15H), 1.83 (br. s., 2H), 1.88-1.96 (m, 2H), 2.22-2.30 (m, 6H), 2.83 (br. s., 3H), 3.16-3.20 (m, 2H), 3.21-3.29 (m, 2H), 3.32-3.40 (m, 2H), 3.64 (t, J= 6.20 Hz, 2H), 4.01-4.13 (m, 4H), 4.18 (dd, J= 10.97, 4.29 Hz, 2H), 4.23 (dd, J= 10.97, 6.68 Hz, 2H). MS (M+l): 1078.

[0300] Example 9: Synthesis of AP07-09Attorney Docket No. 4404-0141PW01">AP07-09Scheme: AP07-09 synthesis

[0301] Step 1: Synthesis of compound 17Compound 17: ((l-(6-(Benzyloxy)hexyl)piperidine-4-carbonyl)Attorney Docket No. 4404-0141PW01azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0302] A mixture solution of compound 6b (0.30 g, 0.50 mmol), compound D2 (0.24 g, 0.75 mmol) and HBTU (0.19 g, 0.50 mmol) in DMF was added with DIPEA (0.45 mL, 2.50 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 17 (0.41 g, 0.42 mmol) as a yellow oil.JH NMR (600 MHz, CDCh) 60.89 (td, J= 7.03, 1.67 Hz, 12H), 1.20-1.35 (m, 45H), 1.35-1.40 (m, 6H), 1.43 (dd, J= 14.54, 8.82 Hz, 7H), 1.52 (dd, J= 15.26, 8.11 Hz, 3H), 1.54 - 1.70 (m, 20H), 1.89 (br. s., 2H), 2.25-2.37 (m, 2H), 3.20 (t, J = 7.63 Hz, 2H), 3.23-3.31 (m, 2H), 3.47 (t, J= 6.44 Hz, 2H), 4.00-4.13 (m, 4 H), 4.50 (s, 2 H), 7.29-7.31 (m, 1H), 7.33-7.38 (m, 4H). MS (M+l): 996.

[0303] Step 2: Synthesis of compound AP07-09AP07-09Compound AP07-09: ((l-(6-Hydroxyhexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0304] A mixture solution of compound 17 (0.41 g, 0.42 mmol) and Pd / C (0.41 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-09 (0.30 g, 0.33 mmol) as a yellow oil.XH NMR (600 MHz, CDCh) 60.82 (t, J= 6.91 Hz, 12H), 1.17-1.26 (m, 46H), 1.36 (td, J= 13.59, 4.77 Hz, 15H), 1.43-1.47 (m, 2H), 1.50-1.55 (m, 7H), 1.55-1.61 (m, 5H), 1.74 (br. s., 2H), 1.92 (br. s., 2H), 2.21-2.29 (m, 2H), 2.80 (br. s., 2H), 3.12-3.18 (m, 2H), 3.21 (t, J= 7.63 Hz, 2H), 3.27-3.36 (m, 2H), 3.54-3.61 (m, 2H), 4.01 (dt, J= 12.64, 6.56 Hz, 4H). MS (M+l): 906.

[0305] Example 10: Synthesis of AP07-10Attorney Docket No. 4404-0141PW01AP07-10Scheme: AP07-10 synthesis

[0306] Step 1: Synthesis of compound 6dAttorney Docket No. 4404-0141PW01Compound 6d: 6-((6-(Decanoyloxy)hexyl)amino)hexyl 2-heptylundecanoate

[0307] A mixture solution of compound 4 (2.8 g, 7.9 mmol) and compound 5d (1.6 g, 4.8 mmol) in ACN / CHCh was added with DIPEA (2.9 mL, 16.5 mmol) and the mixture was heated to 90 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSCU. After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound 6d (1.0 g, 1.4 mmol) as a yellow oil. MS (M+l): 611

[0308] Step 2: Synthesis of compound 18Compound 18: ((l-(6-(Benzyloxy)hexyl)piperidine-4-carbonyl) azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate)

[0309] A mixture solution of compound 6d (0.30 g, 0.50 mmol), compound D2 (0.24 g, 0.75 mmol) and HBTU (0.19 g, 0.50 mmol) in DMF was added with DIPEA (0.45 mL, 2.50 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 18 (0.41 g, 0.42 mmol) as a yellow oil. MS (M+l): 996.

[0310] Step 3: Synthesis of AP07-10Attorney Docket No. 4404-0141PW01AP07-10Compound AP07-10: 6-(N-(6-(Decanoyloxy)hexyl)-l-(6-hydroxyhexyl)piperidine- 4-carboxamido)hexyl 2-hexyldecanoate

[0311] A mixture solution of compound 18 (0.42 g, 0.42 mmol) and Pd / C (0.41 g) in MeOH was stirred at RT under Eb atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-10 (0.30 g, 0.33 mmol) as a yellow oil. MS (M+l): 821

[0312] Example 11: Synthesis of AP07- 11Attorney Docket No. 4404-0141PW012AP07-11Attorney Docket No. 4404-0141PW01Scheme: AP07-11 synthesis

[0313] Step 1: Synthesis of compound 2020Compound 20: tert-Butyl (6-(2-hexyldecanamido)hexyl)carbamate

[0314] A mixture solution of compound 19 (3 g, 13.6 mmol) and compound 2 (2.95 g, 13.6 mmol) in THF was stirred at 0°C, and then HBTU (5.7 g, 15.0 mmol) and DIPEA (4,8 g, 27.0 mmol) were added. After stirring at RT for 19 hours, the reaction solution was washed with 2N NaHCC>3(aq) and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with ethyl acetate / hexane (EA / Hex) (1 / 8) to yield the compound 20 (6 g, 13.2 mmol) as a white solid. MS (M+l): 455.

[0315] Step 2: Synthesis of compound 2121Compound 21: N-(6-Aminohexyl)-2-hexyldecanamide A solution of compound 20 (6 g, 13.2 mmol) in di chloromethane (DCM) was stirred at 0°C, and then trifluoroacetic acid (TFA) (10 mL) was added. After stirring at RT for 19 hours, the reaction solution was evaporated to remove the TFA, the residue was neutralized with 2N NaOH(aq) until pH = 10-11. Then the residue was extracted with ethyl acetate and brine and dried over MgSC , and concentrated under reduced pressure to afford compound 21 (4.5 g, 12.7 mmol) a white solid. MS (M+l): 355

[0316] Step 3: Synthesis of compound 23Attorney Docket No. 4404-0141PW01Compound 23: N,N'-(Azanediylbis(hexane-6,l-diyl))bis(2-hexyldecanamide)

[0317] A mixture solution of compound 21 (3.5 g, 9.8 mmol) and compound 22 (3.2 g, 6.9 mmol) in ACN was added with K2CO3 (1.4 g, 9.8 mmol) and the mixture was heated to 90 °C for 4 hours. The solvent was removed and extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the compound 23 (2 g, 2,9 mmol) as a yellow oil. MS (M+l): 693

[0318] Step 4: Synthesis of compound 2424Compound 24: l-(6-(Benzyloxy)hexyl)-N,N-bis(6-(2- hexyldecanamido)hexyl)piperidine-4-carboxamide

[0319] A mixture solution of compound 23 (0.30 g, 1.0 mmol), compound D2 (0.57 g, 0.8 mmol) and HBTU (0.39 g, 1.0 mmol) in DMF was added with DIPEA (0.9 mL, 5.1 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 24 (0.45 g, 0.45 mmol) as a yellow oil. MS (M+l): 994.

[0320] Step 5: Synthesis of compound AP07-11Attorney Docket No. 4404-0141PW01AP07-11Compound AP07-11: N,N-Bis(6-(2-hexyldecanamido)hexyl)-l-(6- hydroxyhexyl)piperidine-4-carboxamide

[0321] Compound AP07-11 was synthesized according to the procedure similar to the scheme of AP07-09 synthesis using 24. The title compound was obtained as a pale yellow oil. 'H NMR (600 MHz, CDCh) 6 5.57-5.51 (m, 2H), 3.64 (t, J = 6.3 Hz, 2H), 3.45-3.40 (m, 2H), 3.34 (d, J= 10.0 Hz, 2H), 3.29-3.22 (m, 6H), 3.16 (t, J= 7.8 Hz, 2H), 2.97-2.93 (m, 2H), 2.69-2.59 (m, 2H), 1.99-1.85 (m, 6H), 1.59-1.24 (m, 71H), 0.88-0.85 (m, 12H). MS (M+l): 904.

[0322] Example 12: Synthesis of AP07-12AP07-12Compound AP07-12: 6-(N-(6-((2-Butyloctanoyl)oxy)hexyl)-l-(6- hydroxyhexyl)piperidine-4-carboxamido)hexyl 2-hexyldecanoate

[0323] Compound AP07-12 was synthesized according to the procedure similar to the scheme of AP07-09 synthesis using D2, except that compound 6a was used instead of compound 6b. The title compound was obtained as a pale yellow oil. 'H NMR (600 MHz, CDCh) 64.08-4.03 (m, 4H), 3.68-3.64 (m, 2H), 3.58 (d, J= 11.0 Hz, 2H), 3.30-3.00 (m, 9H), 2.33-2.07 (m, 9H), 1.97-1.95 (m, 3H), 1.83-1.76 (m, 3H), 1.66 -1.25 (m, 55H), 0.89-0.86 (m, 12H). MS (M+l): 850.

[0324] Example 13: Synthesis of AP07-13Attorney Docket No. 4404-0141PW01AP07-13Scheme: AP07-13 synthesis

[0325] Step 1: Synthesis of compound 25Compound 25: ((l-(6-Iodohexyl)piperidine-4-carbonyl)azanediyl)bis(hexane-6,l- diyl) bis(2-hexyldecanoate)Attorney Docket No. 4404-0141PW01

[0326] A mixture solution of compound AP07-09 (500 mg, 0.6 mmol), PPhi (320 mg, 1.2 mmol) and imidazole (100 mg, 1.2 mmol) in THF was stirred at 0°C, followed by the addition of I2 (280 mg, 1.1 mmol). The reaction solution was allowed to warm to RT and stirred overnight. After washing with sat. Na2S2C>3(aq) and brine, the residue was dried over MgSC and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (20 / 1) to yield the desired product 25 (0.5 g, 89%) as a colorless oil.

[0327] Step 2: Synthesis of compound AP07-13AP07-13Compound AP07-13: 6-(N-(6-((2-Butyloctanoyl)oxy)hexyl)-l-(6- hydroxyhexyl)piperidine-4-carboxamido)hexyl 2-hexyldecanoate

[0328] A mixture solution of compound 25 (0.5 g, 0.5 mmol) and (Me)2NH in 2M MeOH (0.7 mL, 1.4 mmol) were added in ACN (3 mL) and the mixture was heated to 60 °C for 8 hours. The solvent was removed and then purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (10 / 1) to yield the desired product AP07-13 (0.05 g, 11%) as a yellow oil. 'H NMR (600 MHz, CDCI3) 8 4.05 (dt, J= 19.5, 7 Hz, 4H), 3.27 (t, J= 7.6 Hz, 2H), 3.20 (t, J= 7.7 Hz, 2H), 2.41-2.27 (m, 15H), 2.05 (brs, 2H), 2.05 (s, 1H), 1.92-1.85 (m, 2H), 1.66-1.24 (m, 72H), 0.87 (t, J = 6.9 Hz,12H). MS (M+l): 933.

[0329] Example 14: Synthesis of AP07-14Attorney Docket No. 4404-0141PW01Scheme: AP07-14 synthesis

[0330] Step 1: Synthesis of compound 27Attorney Docket No. 4404-0141PW01Compound 27: ((l-(6-(Benzyloxy)hexyl)piperidine-3- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0331] A mixture solution of compound 6b (0.30 g, 0.50 mmol), compound 26 (0.24 g, 0.75 mmol) and HBTU (0.19 g, 0.50 mmol) in DMF was added with DIPEA (0.45 mL, 2.50 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 27 (0.34 g, 0.35 mmol) as a yellow oil. MS (M+l): 996.

[0332] Step 2: Synthesis of compound AP07-14Compound AP07-14: ((l-(6-Hydroxyhexyl)piperidine-3- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0333] A mixture solution of compound 27 (0.34 g, 0.35 mmol) and Pd / C (0.34 g) in MeOH was stirred at RT under Eb atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-14 (0.18 g, 0.20 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 64.09-4.03 (m, 4H), 3.67-3.58 (m, 2H), 3.53 (d, J= 12.1 Hz, 1H), 3.44 (d, J= 11.0 Hz, 1H), 3.40-3.04 (m, 6H),Attorney Docket No. 4404-0141PW012.33-2.28 (m, 2H), 1.98-1.79 (m, 8H), 1.67-1.50 (m, 14H), 1.45-1.25 (m, 57H), 0.87 (t, J= 6.5 Hz, 12H). MS (M+l): 906.

[0334] Example 15: Synthesis of AP07-15Scheme: AP07-15 synthesis

[0335] Step 1: Synthesis of compound 29Attorney Docket No. 4404-0141PW01Compound 29: ((l-(6-(Benzyloxy)hexyl)piperidine-2- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0336] A mixture solution of compound 6b (0.30 g, 0.50 mmol), compound 28 (0.24 g, 0.75 mmol) and HBTU (0.19 g, 0.50 mmol) in DMF was added with DIPEA (0.45 mL, 2.50 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 29 (0.34 g, 0.35 mmol) as a yellow oil. MS (M+l): 996.

[0337] Step 2: Synthesis of compound AP07-15Compound AP07-15: ((l-(6-Hydroxyhexyl)piperidine-2- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0338] A mixture solution of compound 29 (0.34 g, 0.35 mmol) and Pd / C (0.34 g) in MeOH was stirred at RT under Eb atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography withAttorney Docket No. 4404-0141PW01dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-15 (0.18 g, 0.20 mmol) as a yellow oil. MS (M+l): 906.

[0339] Example 16: Synthesis of AP07-16Scheme: AP07-16 synthesis

[0340] Step 1: Synthesis of compound 31Attorney Docket No. 4404-0141PW01Compound 31: ((l-(6-(Benzyloxy)hexyl)pyrrolidine-3- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0341] A mixture solution of compound 6b (1.55 g, 2.2 mmol), compound 30 (1 g, 3.27 mmol) and HBTU (1.3 g, 3.4 mmol) in DMF was added with DIPEA (3.2 mL, 18 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 31 (1.1 g, 1.1 mmol) as a yellow oil. MS (M+l): 982.

[0342] Step 2: Synthesis of compound AP07-16Compound AP07-16: ((l-(6-hydroxyhexyl)pyrrolidine-3- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0343] A mixture solution of compound 31 (1.1 g, 1.1 mmol) and Pd / C (1 g) in MeOH was stirred at RT under Eb atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-16 (0.3 g, 0.33 mmol) as a yellow oil. ‘HNMR (600 MHz, CDCh) 6 4.07-4.04 (m, 4H), 3.64 (t, J= 6.4 Hz, 2H), 3.52-3.19 (m, 7H), 2.97-2.73 (m, 4H), 2.41-2.27 (m, 3H), 2.05-2.00 (m, 1H), 1.72-1.48 (m, 17H), 1.46-1.25 (m, 55H), 0.88-0.86 (m, 12H). MS (M+l): 892.Attorney Docket No. 4404-0141PW01

[0344] Example 17: Synthesis of AP07-17AP07-17Compound AP07-17: ((l-(6-Methoxyhexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate)

[0345] A mixture solution of compound 6b (0.54 g, 0.8 mmol), compound 32 (0.35 g, 1.1 mmol) and HBTU (0.46 g, 1.2 mmol) in DMF was added with DIPEA (1.1 mL, 6.4 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-17 (0.36 g, 0.4 mmol) as a yellow oil. 'H NMR. (600 MHz, CDCh) 64.05 (m, 4H), 3.36 (t, J= 6.6 Hz, 2H), 3.32 (s, 3H), 3.27 (t, J= 7.6 Hz, 2H), 3.21 (t, J= 7.7 Hz, 2H), 2.98 (d, J= 7.6 Hz, 2H), 2.36-2.27 (m, 5H), 1.93-1.86 (m, 4H), 1.70-1.47 (m, 19H), 1.45-1.25 (m, 55H), 0.87 (t, J= 6.9 Hz, 12H). MS (M+l): 920.Attorney Docket No. 4404-0141PW01

[0346] Example 18: Synthesis of AP07-19Scheme: AP07-19 synthesis

[0347] Step 1: Synthesis of compound 33Attorney Docket No. 4404-0141PW01Compound 33: ((l-(6-(Benzyloxy)hexyl)piperidine-4- carbonyl)azanediyl)bis(pentane-5,l-diyl) bis(2-heptylundecanoate)

[0348] A mixture solution of compound 6e (0.86 g, 1.2 mmol), compound D2 (0.57 g, 1.8 mmol) and HBTU (0.46 g, 1.2 mmol) in DMF was added with DIPEA (1.1 mL, 6 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 33 (0.98 g, 0.96 mmol) as a yellow oil. MS (M+l): 1024.

[0349] Step 2: Synthesis of compound AP07-19AP07-19Compound AP07-19: ((l-(6-Hydroxyhexyl)piperidine-4- carbonyl)azanediyl)bis(pentane-5,l-diyl) bis(2-heptylundecanoate)

[0350] A mixture solution of compound 33 (0.98 g, 0.96 mmol) and Pd / C (0.41 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-19 (0.30 g, 0.33 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 64.05 (m, 4H), 3.64 (t, J = 6.6 Hz, 2H), 3.28 (d, J= 15.2 Hz, 2H), 3.22 (d, J= 15.3 Hz, 2H), 2.98 (d, J= 10.9 Hz, 2H), 2.39-2.26 (m, 7H), 1.96-1.86 (m, 4H), 1.69-1.48 (m, 22H), 1.40-1.08 (m, 54H), 0.89-0.83 (m, 12H). MS (M+l): 934

[0351] Example 19: Synthesis of AP07-20Attorney Docket No. 4404-0141PW01Compound 34: ((l-(6-(Benzyloxy)hexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-heptylundecanoate)

[0353] A mixture solution of compound 6f (0.6 g, 0.9 mmol), compound D2 (0.4 g, 1.2 mmol) and HBTU (0.5 g, 1.3 mmol) in DMF was added with DIPEA (1.2 mL, 7Attorney Docket No. 4404-0141PW01mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 34 (0.74 g, 0.7 mmol) as a yellow oil. MS (M+l): 1052.

[0354] Step 2: Synthesis of compound AP07-20AP07-20Compound AP07-20: ((l-(6-Hydroxyhexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-heptylundecanoate)

[0355] A mixture solution of compound 34 (0.74 g, 0.7 mmol) and Pd / C (0.7 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-20 (0.31 g, 0.33 mmol) as a yellow oil. *HNMR (600 MHz, CDCh) 64.08-4.02 (m, 4H), 3.64 (t, J= 6.6 Hz, 2H), 3.27 (d, J= 15.2 Hz, 2H), 3.21 (d, J= 15.3 Hz, 2H), 3.06 (brs, 2H), 2.43 (brs, 2H), 2.30-2.26 (m, 5H), 1.90-1.47 (m, 25H), 1.40-1.06 (m, 59H), 0.89-0.82 (m, 12H). MS (M+l): 962.

[0356] Example 20: Synthesis of AP07-21Attorney Docket No. 4404-0141PW0135Compound 35: ((l-(6-(Benzyloxy)hexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-heptylnonanoate)Attorney Docket No. 4404-0141PW01

[0358] A mixture solution of compound 6g (0.62 g, 0.9 mmol), compound D2 (0.4 g, 1.2 mmol) and HBTU (0.5 g, 1.3 mmol) in DMF was added with DIPEA (1.2 mL, 7 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 35 (0.64 g, 0.65 mmol) as a yellow oil. MS (M+l): 996.

[0359] Step 2: Synthesis of compound AP07-20AP07-21Compound AP07-21: ((l-(6-Hydroxyhexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-heptylnonanoate)

[0360] A mixture solution of compound 35 (0.64 g, 0.65 mmol) and Pd / C (0.7 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-21 (0.30 g, 0.33 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 64.08-4.03 (m, 4H), 3.64 (t, J= 6.6 Hz, 2H), 3.26 (d, J= 15.2 Hz, 2H), 3.19 (d, J= 15.3 Hz, 2H), 2.67 (brs, 3H), 2.33-2.27 (m, 2H), 1.91-1.48 (m, 24H), 1.44-1.25 (m, 56H), 0.87 (t, J= 6.5 Hz, 12H). MS (M+l): 906.

[0361] Example 21: Synthesis of AP07-22Attorney Docket No. 4404-0141PW0136Compound 36: ((l-(6-(Benzyloxy)hexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-octyldecanoate)

[0363] A mixture solution of compound 6g (0.67 g, 0.9 mmol), compound D2 (0.4 g, 1.2 mmol) and HBTU (0.5 g, 1.3 mmol) in DMF was added with DIPEA (1.2 mL, 7Attorney Docket No. 4404-0141PW01mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 36 (0.73 g, 0.7 mmol) as a yellow oil. MS (M+l): 1052.

[0364] Step 2: Synthesis of compound AP07-20AP07-22Compound AP07-22: ((l-(6-Hydroxyhexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-octyldecanoate)

[0365] A mixture solution of compound 36 (0.73 g, 0.7 mmol) and Pd / C (0.7 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-22 (0.31 g, 0.33 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 64.08-4.03 (m, 4H), 3.64 (t, J= 6.6 Hz, 2H), 3.30-3.25 (m, 4H), 3.21 (d, J= 15.3 Hz, 2H), 2.73 (brs, 4H), 2.33-2.27 (m, 3H), 1.96 (brs, 4H), 1.67-1.47 (m, 17H), 1.43-1.25 (m, 63H), 0.88 (t, J= 6.5 Hz, 12H). MS (M+l): 962.

[0366] Example 22: Synthesis of AP07-23Attorney Docket No. 4404-0141PW01Compound 37: ((l-(6-(Benzyloxy)hexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyloctanoate)Attorney Docket No. 4404-0141PW01

[0368] A mixture solution of compound 6i (0.57 g, 0.9 mmol), compound D2 (0.4 g, 1.2 mmol) and HBTU (0.5 g, 1.3 mmol) in DMF was added with DIPEA (1.2 mL, 7 mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSO-r After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 37 (0.47 g, 0.5 mmol) as a yellow oil. MS (M+1): 940.

[0369] Step 2: Synthesis of compound AP07-23AP07-23Compound AP07-23: ((l-(6-Hydroxyhexyl)piperidine-4- carbonyl)azanediyl)bis(octane-8,l-diyl) bis(2-hexyloctanoate)

[0370] A mixture solution of compound 37 (0.47 g, 0.5 mmol) and Pd / C (0.5 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-23 (0.29 g, 0.33 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 64.08-4.04 (m, 4H), 3.64 (t, J= 6.6 Hz, 2H), 3.26 (d, J= 15.3 Hz, 2H), 3.21 (d, J= 15.3 Hz, 2H), 3.12 (brs, 2H), 2.50 (brs, 3H), 2.33-2.28 (m, 4H), 1.94-1.89 (m, 2H), 1.78 (brs, 2H), 1.64-1.51 (m, 14H), 1.49-1.25 (m, 58H), 0.87 (t, J= 6.5 Hz, 12H). MS (M+1): 906.

[0371] Example 23: Synthesis of AP07-24Attorney Docket No. 4404-0141PW01Compound 38: ((l-(6-(Benzyloxy)hexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(decanoate)

[0373] A mixture solution of compound 6j (0.47 g, 0.9 mmol), compound D2 (0.4 g, 1.2 mmol) and HBTU (0.5 g, 1.3 mmol) in DMF was added with DIPEA (1.2 mL, 7Attorney Docket No. 4404-0141PW01mmol). After stirring at RT for 19 hours, the reaction solution was extracted with ethyl acetate and brine and dried over MgSC . After evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound 38 (0.49 g, 0.6 mmol) as a yellow oil. MS (M+l): 828.AP07-24Compound AP07-24: ((l-(6-Hydroxyhexyl)piperidine-4- carbonyl)azanediyl)bis(hexane-6,l-diyl) bis(decanoate)

[0374] A mixture solution of compound 38 (0.49 g, 0.6 mmol) and Pd / C (0.5 g) in MeOH was stirred at RT under H2 atmosphere overnight. After filtration and evaporation, the residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) (15 / 1) to yield the compound AP07-24 (0.29 g, 0.33 mmol) as a yellow oil. 'H NMR (600 MHz, CDCh) 64.07-4.02 (m, 4H), 3.64 (t, J= 6.6 Hz, 2H), 3.27 (d, J= 15.3 Hz, 2H), 3.20 (d, J= 15.3 Hz, 2H), 3.12 (brs, 1H), 2.49 (brs, 3H), 2.31-2.27 (m, 4H), 1.92-1.87 (m, 2H), 1.78 (brs, 2H), 1.72-1.47 (m, 20H), 1.41-1.26 (m, 37H), 0.87 (t, J= 6.5 Hz, 12H). MS (M+l): 738.

[0375] Preparation Example: Preparation and characterization of nucleic acid loaded lipid nanoparticles

[0376] 1. Preparation of lipid nanoparticles with SARS-CoV-2-WT-FLS mRNA

[0377] LNPs comprised of commercially available ionizable lipids were formulated with a total lipid concentration of 50 mM. SM-102-LNPs were formulated with SM-102 / DSPC / Cholesterol / DMG-PEG2000 in the molar ratio of 50 / 10 / 38.5 / 1.5. ALC-0315-and AP07-04-LNPs were formulated in the molar ratio of 46.3 / 9.4 / 42.7 / 1.6, respectively. Each lipid was dissolved in ethanol and mixed according to the specified molar ratios in the organic phase. The SARS-CoV-2-WT-FLS mRNA was dissolved in 50 mM sodium acetate buffer (pH 4.5), with the NP lipid:mRNA ratio at a constantAttorney Docket No. 4404-0141PW01value of 6.5, prior to mixing in the Ignite NanoAssmblr (Precision NanoSystems). In the Ignite system, a 300-pL aliquot of the organic phase and an 850-pL aliquot of the aqueous phase were mixed, and then dialyzed against PBS.

[0378] Measurement of LNP size and poly dispersity index (PDI)

[0379] LNPs were diluted 100-fold in PBS (pH 7.4) and transferred into a 384-well microplate for size and poly dispersity index (PDI) measurements by Wyatt DynaPro Plate Reader III (Malvern Instruments, UK). Results are summarized in Table 1.

[0380] Table 1. Size and PDI of LNPs

[0381] The data in Table 1 confirmed that the present cationic lipids, AP07-04 could associate with other helper lipids to form nanoparticles. The physical characteristics of AP07-04-LNP, including size and poly dispersity index (PDI), were similar to those of other LNPs composed of commercially available cationic lipids by Ignite. The present cationic AP07-04-LNP can efficiently deliver the mRNA encoding the SARS-CoV-2 spike protein for expression. The SARS-CoV-2 FLS mRNA were individually transfected into 293T cells. Prior to flow cytometry analysis, the cells were collected and incubated with monoclonal antibodies RBD-chAb 75 purified in our lab previously. Furthermore, in terms of mRNA delivery efficiency, AP07-04-LNP outperformed other LNPs composed of lipids from commercial sources (i.e., SM-102 or ALC-0315). Based on these findings, this novel cationic lipid AP07-04 could be deemed suitable for application in LNPs for gene and drug delivery.

[0382] 2. Preparation of lipid nanoparticles with Fluc-mRNA

[0383] LNPs comprised of commercially available ionizable lipids were formulated with a total lipid concentration of 25.92 mM. SM-102-LNPs were formulated with SM-Attorney Docket No. 4404-0141PW01102 / DSPC / Cholesterol / DMG-PEG2000 in the molar ratio of 50 / 10 / 38.5 / 1.5. ALC-0315- and AP07-LNPs were formulated in the molar ratio of 46.3 / 9.4 / 42.7 / 1.6, respectively. Each lipid sample was dissolved in ethanol and mixed according to the specified molar ratios in the organic phase. The Firefly luciferase mRNA (Fluc-mRNA) was dissolved in 25 mM sodium acetate buffer (pH 4), with the NP lipid:mRNA ratio at a constant value of 6.5, prior to mixing in the Ignite NanoAssmblr (Precision NanoSystems). In the Ignite system, a 600-pL aliquot of the organic phase and an 1800-pL aliquot of the aqueous phase were mixed, and then dialyzed and concentrated with Amicon® ultra-15 (MWCO 30 kDa).

[0384] 3. Measurement of LNP size and poly dispersity index (PDI)

[0385] LNPs were diluted 100-fold in PBS (pH 7.4) and transferred into a 384-well microplate for size and poly dispersity index (PDI) measurements by Wyatt DynaPro Plate Reader III (Malvern Instruments, UK). Results are summarized in Table 2.

[0386] Table 2. Size and PDI of LNPs

[0387] 4. Measurement of LNP encapsulation efficiency and concentration

[0388] LNPs were diluted in TE (20-fold) or 1% Triton X-100 / TE (40-fold) for free and total RNA quantification, respectively, and analyzed by RiboGreen assay using a SpectraMax iD3. Results are summarized in Table 3.

[0389] Table 3. Encapsulation Efficiency and Concentration of LNPsAttorney Docket No. 4404-0141PW01

[0390] 5. Fluc-mRNA LNPs Delivery in HEK293T Cells

[0391] HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were seeded into white 96-well plates at a density of approximately 1 * 104cells per well and allowed to adhere overnight. Fluc-mRNA-loaded LNPs were added to the cells at a dose corresponding to approximately 200 ng mRNA per well. The cells were incubated at 37 °C under 5% CO2 for 48 hours. Thereafter, luciferase expression was measured using a luminescence assay reagent (for example, One-Gio or an equivalent reagent). Luminescence was detected using a microplate luminometer, and the results were expressed as relative light units (RLU / mg protein) to evaluate the functional delivery efficiency of selected ionizable lipids in Table 4 and Fig.l.

[0392] Table 4. Delivery efficiency of LNPs

[0393] The experimental results in Table 4 demonstrate that the lipid nanoparticles comprising the cationic lipids of the present invention (e.g., AP07-15) exhibit unexpectedly superior transfection efficiency compared to conventional lipid formulations based on ALC-0315. Accordingly, the present invention provides a delivery vehicle suitable for various therapeutic applications. The enhanced potency of the present lipids renders them particularly suitable for use in compositions for preventing or treating infectious diseases, cancers, and genetic disorders.

[0394] While the embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions willAttorney Docket No. 4404-0141PW01now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Docket No. 4404-0141PW01CLAIMSWhat is claimed is:

1. A compound of F ormula (I) :whereinW is hydroxy, Ci-6 alkoxy, or -NR2R3;Ri is linear or branched C6-20 alkyl or alkenyl;R2 and R3 are independently linear or branched C1-6 alkyl unsubstituted or substituted by hydroxy;Zi is absent or -A-C(=O)-;A is O or NH;Z2 is linear or branched C6-C20 alkyl or ;Ra is hydrogen or Ci-Ce alkyl;Rb and Rc are independently hydrogen or -Rd-OC(=O)Re;Rd is Ci-Ce alkylene;Re is linear or branched C6-C20 alkyl or alkenyl;X is -CH- or -N-; Y is -O- or -NH-;m is 1 to 5; the sum of m and is 1 to 6; o is 1 to 7; p is 1 to 7;provided that Rb and Rc are not both hydrogen; andprovided that when Zi is absent, Z2 is not alkyl.or a pharmaceutically acceptable salt thereof.Attorney Docket No. 4404-0141PW012. The compound of claim 1, wherein Zi is absent, Z2 is , Ra is methyl, and Rb and Rc are independently -Rd-OC(=O)Re.

3. The compound of claim 1, wherein Zi is -OC(=O)-, Z2 is , Ra is methyl, and Rb and Rc are independently -Rd-OC(=O)Re.

4. The compound of claim 1, wherein W is hydroxy or methoxy, and / or wherein Rd is methylene.

5. The compound of claim 1, selected from the group consisting of:Attorney Docket No. 4404-0141PW01Attorney Docket No. 4404-0141PW01Attorney Docket No. 4404-0141PW01Attorney Docket No. 4404-0141PW01Attorney Docket No. 4404-0141PW016. A lipid nanoparticle, comprising the compound of claim 1.

7. The lipid nanoparticle of claim 6, further comprising:a non-cationic lipid;a steroid; anda polymer-conjugated lipid.

8. The lipid nanoparticle of claim 6 or 7, further comprising a therapeutic agent.

9. The lipid nanoparticle of claim 8, wherein the therapeutic agent is a nucleic acid, a peptide, or a protein.

10. The lipid nanoparticle of claim 6, wherein the compound of claim 1 is in an amount of 35 mol% to about 55 mol% of the lipid nanoparticle.

11. The lipid nanoparticle of claim 6, wherein the non-cationic lipid is in an amount of 5 mol% to about 35 mol% of the lipid nanoparticle.

12. The lipid nanoparticle of claim 7, wherein the steroid is in an amount of 20 mol% to about 50 mol% of the lipid nanoparticle.

13. The lipid nanoparticle of claim 7, wherein the polymer-conjugated lipid is in an amount of 0.25 mol% to about 2.75 mol% of the lipid nanoparticle.

14. A pharmaceutical composition, comprising the compound of claim 1 or the lipid nanoparticle of claim 6, and a pharmaceutically acceptable excipient.

15. A method of treatment of a disease, comprising administering an effective amount of the lipid nanoparticles of claim 6 or the pharmaceutical composition of claim 14 to a subject in need thereof.Attorney Docket No. 4404-0141PW0116. A method of vaccinating a subject in need thereof, comprising administering an effective amount of the lipid nanoparticles of claim 6 or the pharmaceutical composition of claim 14 to the subject.

17. A method of inducing or eliciting an immune response a subject in need thereof, comprising administering an effective amount of the lipid nanoparticles of claim 6 or the pharmaceutical composition of claim 14 to the subject.

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