Nano-liposome particle for expressing carnotoxin and application of nano-liposome particle

Abstract

The invention relates to nano-liposome particles for expressing carnotoxin and application of the nano-liposome particles, and particularly provides a nucleic acid molecule containing a sequence as shown in SEQ ID NO: 4. The nucleic acid molecule disclosed by the invention is an optimized sequence for expressing the carnotoxin.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene therapy drugs, and particularly relates to a nano-liposome particle expressing botulinum toxin protein and its application. Background Art

[0002] Botulinum toxin, the full name is botulinum toxin (BTX), is a neurotoxin containing high molecular weight protein produced by Clostridium botulinum. It is the most potent biological toxin among known natural toxins and synthetic agents. According to different toxin antigen types, it is divided into 7 subtypes: A, B, C, D, E, F, and G. Among them, Clostridium botulinum type C is widely distributed in nature, and the botulinum toxins that can cause human poisoning are mainly types A, B, E, and F toxins. Botulinum toxin was first discovered in pickled sausages and later purified for use as a biochemical weapon. It can damage the nervous system of organisms, causing symptoms such as dizziness, difficulty breathing, and muscle weakness. The inhalation lethal dose of botulinum toxin for humans is 0.03 μg.

[0003] The relative molecular mass of botulinum toxin is 150 KD, and it consists of a light chain with a relative molecular mass of 50 KD and a heavy chain of 100 KD. The heavy chain can recognize and bind to specific receptors on the presynaptic membrane of nerve endings, and the light chain specifically cleaves the SNAP25 protein in the SNARE complex, affecting the fusion of synaptic vesicles with the presynaptic membrane and blocking the release of neurotransmitters such as acetylcholine, thereby producing chemical denervation effects such as muscle relaxation and glandular secretion disorders.

[0004] Currently, the innovation breakthroughs in botulinum toxin products are basically in the optimization of the production process and the optimization of drug stability and duration. In terms of patent protection, the patents related to the production, preparation, and use of botulinum toxin have been steadily increasing year by year, and the barriers are very high. The existing botulinum toxin products have also stopped upgrading for many years, mainly because the therapeutic index (safety window) of natural botulinum toxin is relatively low, which is not sufficient to support the broad spectrum of its indications. Botulinum toxins of different products have relative differences due to the production processes of different manufacturers, and the addition of excipients leads to certain adverse reactions of botulinum toxin products.

[0005] Lipid materials and their derivatives have been widely studied and applied in the in vivo delivery of RNA due to their excellent biocompatibility and good encapsulation efficiency for RNA. RNA is safe and effective and has good application prospects in the development of new therapies for genetic diseases, expression of functional proteins and antibodies, vaccines, or gene editing. Currently, commercially available lipid delivery vectors (BNT162b2 of BioNTech and mRNA-1273 of Moderna) express the target protein abundantly in the liver through various administration routes (intravenous, subcutaneous, intramuscular injection), which has potential toxic side effects on the application of extrahepatic lesions and is not conducive to clinical translation and application.

[0006] Therefore, the development of a botulinum toxin LNP delivery vector with local expression specificity will greatly expand the clinical application of botulinum toxin. Summary of the Invention

[0007] In view of the deficiencies of existing products, the present invention provides a nano-liposome particle expressing botulinum toxin type A, which can extend the drug efficacy period and expand the safety window. The nano-liposome particle provided by the present invention can effectively deliver a nucleic acid molecule expressing botulinum toxin type A to the local injection site, and there is no expression in other organs such as the liver, spleen, and kidney.

[0008] In the first aspect of the present invention, there is provided a nucleic acid molecule comprising:

[0009] (1) The sequence expressing botulinum toxin type A shown in SEQ ID NO: 4 or the DNA sequence encoding the same,

[0010] (2) The complementary sequence of (1).

[0011] In one or more embodiments, the nucleic acid molecule is a DNA molecule.

[0012] In one or more embodiments, the nucleic acid molecule is an RNA molecule (such as an mRNA molecule), and the uracil in the RNA molecule is replaced by N1-methylpseudouracil.

[0013] In one or more embodiments, the 5'-end and / or 3'-end of the RNA molecule has a modifying group.

[0014] In one or more embodiments, the 5'-end modifying group of the RNA molecule is selected from ARCA, m7G(5”)ppp(5”)(2”OMeA)pG, m7G(5”)ppp(5”)(2”OMeG)pG, m7(3”OMeG)(5”)ppp(5”)(2”OMeG)pG, m7(3”OMeG)(5”)ppp(5”)(2”OMeA)pG, mCAP, dmCAP, tmCAP or dmCAP.

[0015] In one or more embodiments, the RNA molecule contains a 5'UTR.

[0016] In one or more embodiments, the length of the 5'UTR of the RNA molecule is 10-200 nucleotides, preferably 15-100 nucleotides.

[0017] In one or more embodiments, the 5'UTR sequence of the RNA molecule is as shown in SEQ ID NO.1.

[0018] In one or more embodiments, the 5'UTR of the RNA molecule includes a KOZAK sequence.

[0019] In one or more embodiments, the nucleotide sequence of the Kozak sequence is as shown in SEQ ID NO: 2, wherein SEQ ID NO: 2 is GCCACC.

[0020] In one or more embodiments, the RNA molecule contains a 3'UTR.

[0021] In one or more embodiments, the 3'UTR sequence of the RNA molecule is as shown in SEQ ID NO: 3.

[0022] In one or more embodiments, the 3'-terminal protective modification group of the RNA molecule is poly(A), and the length of the poly(A) is 50 - 200, preferably 80 - 150.

[0023] In one or more embodiments, the 5'-end to 3'-end of the RNA includes a 5' cap, a 5'UTR, a coding sequence of botulinum toxin type A, a 3'UTR, and a 3' poly(A) tail.

[0024] The present invention also provides a nucleic acid delivery reagent, which contains the nucleic acid molecule according to any one of the embodiments of the first aspect of the present invention and a carrier for delivering the nucleic acid.

[0025] In one or more embodiments, the carrier is a nano-lipid particle.

[0026] In one or more embodiments, the nucleic acid molecule is encapsulated by nano-liposome particles.

[0027] In one or more embodiments, the nano-liposome particles contain phospholipids (such as polyunsaturated lipids), PEG lipids, structural lipids, and a compound of formula (I) or its salt or isomer,

[0028]

[0029] wherein,

[0030] R1 is H or C1 - C8 alkyl,

[0031] R2 is C1 - 10 alkyl, C3 - C18 diene group

[0032] R3 is H or R1 - R8 alkyl,

[0033] R4 is C1 - 10 alkyl, C3 - C18 diene group,

[0034] R5 is C1 - C14 alkyl, C2 - C14 alkenyl, or

[0035] M is O or N,

[0036] t is an integer from 3 to 7,

[0037] R6 is H or C1-10 alkyl,

[0038] R7 is C1-10 alkyl, C3-C18 diene group.

[0039] In one or more embodiments, the nano-liposome particles comprise 3% to 10% of PEG2000-DMG, 20% to 50% of the compound of formula (I), 20% to 50% of DSPC, and 5% to 20% of cholesterol by mole percentage.

[0040] The present invention also provides a pharmaceutical composition for treating diseases or symptoms caused by muscle continuous rigidity, comprising the nucleic acid delivery reagent described in any embodiment herein and a pharmaceutically acceptable excipient.

[0041] In one or more embodiments, the pharmaceutical composition is a locally acting pharmaceutical composition.

[0042] In one or more embodiments, the diseases or symptoms caused by muscle continuous rigidity are muscle diseases or muscle continuous rigidity-related symptoms in the field of medical aesthetics.

[0043] In one or more embodiments, the muscle diseases are selected from blepharospasm, strabismus, hemifacial spasm, cervical dystonia, upper limb dystonia, lower limb dystonia, axillary hyperhidrosis, palmar hyperhidrosis, chronic migraine, neurogenic urinary incontinence, overactive bladder syndrome, local spasm after cerebral palsy, sialorrhea, etc. The muscle continuous rigidity-related symptoms in the field of medical aesthetics are selected from glabellar lines, crow's feet, forehead lines, etc.

[0044] The present invention also provides a method for treating diseases or symptoms caused by muscle continuous rigidity, the method comprising the step of administering the nucleic acid delivery reagent described herein to a subject.

[0045] In one or more embodiments, the subject is a mammal, such as a human.

[0046] In one or more embodiments, the diseases or symptoms caused by muscle continuous rigidity are muscle diseases or muscle continuous rigidity-related symptoms in the field of medical aesthetics.

[0047] In one or more embodiments, the muscle diseases are selected from blepharospasm, strabismus, hemifacial spasm, cervical dystonia, upper limb dystonia, lower limb dystonia, axillary hyperhidrosis, palmar hyperhidrosis, chronic migraine, neurogenic urinary incontinence, overactive bladder syndrome, local spasm after cerebral palsy, sialorrhea, etc. The muscle continuous rigidity-related symptoms in the field of medical aesthetics are selected from glabellar lines, crow's feet, forehead lines, etc.

[0048] The present invention also provides a method for producing botulinum toxin type A protein in cells, the method comprising the step of contacting the cells with the nucleic acid delivery reagent described herein, whereby the nucleic acid in the nucleic acid delivery reagent can be translated in the cells to produce a polypeptide.

[0049] In one or more embodiments, the cells are mammalian cells.

[0050] The present invention provides the use of the nucleic acid molecule, nucleic acid delivery reagent or pharmaceutical composition described in any of the embodiments herein in the preparation of a reagent for diseases or symptoms caused by persistent muscle rigidity.

[0051] In one or more embodiments, the diseases or symptoms caused by persistent muscle rigidity are muscle diseases or symptoms related to persistent muscle rigidity in the field of medical aesthetics.

[0052] In one or more embodiments, the muscle diseases are selected from blepharospasm, strabismus, hemifacial spasm, cervical dystonia, upper limb dystonia, lower limb dystonia, axillary hyperhidrosis, palmar hyperhidrosis, chronic migraine, neurogenic urinary incontinence, overactive bladder syndrome, local spasm after cerebral palsy, drooling, etc. The symptoms related to persistent muscle rigidity in the field of medical aesthetics are selected from glabellar lines, crow's feet, forehead lines, etc.

[0053] The present invention also provides a method for preparing the nucleic acid delivery reagent described herein, wherein the carrier of the nucleic acid delivery reagent is a nano-lipid particle. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 : Schematic diagram of the mechanism of action of botulinum toxin type A;

[0055] Figure 2 : ELISA experimental results of mRNA molecules expressing botulinum toxin type A protein;

[0056] Figure 3 : Pharmacodynamic graph of different mRNA molecules in mice;

[0057] Figure 4 : Pharmacodynamic graph of different nano-lipid particles in mice. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0058] The inventors have repeatedly optimized the codons of the coding sequence of botulinum toxin type A protein and conducted experiments to develop the optimal optimized sequence. After repeated verification, the mRNA molecules used in the present invention are superior to other mRNA molecules expressing the same protein.

[0059] Nucleic acid expressing botulinum toxin type A protein

[0060] The present invention first provides a nucleic acid encoding botulinum neurotoxin type A protein, which comprises the sequence shown in SEQ ID NO: 4. Herein, the nucleic acid can be a DNA or RNA molecule.

[0061] As used herein, "RNA" refers to ribonucleic acid that can be naturally or non-naturally occurring. For example, RNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA can include a cap structure, chain-terminating nucleosides, stem-loops, polyA sequences, and / or polyadenylation signals. Translation of an mRNA encoding a specific polypeptide, such as in vivo translation of mRNA in mammalian cells, can produce the encoded polypeptide.

[0062] The nucleic acids and polynucleotides used in the present disclosure generally include a first region (e.g., a coding region) of linked nucleosides encoding a polypeptide of interest, a first flanking region (e.g., 5-UTR) located at the 5'-end of the first region, a second flanking region (e.g., 3-UTR) located at the 3'-end of the first region, at least one 5'-cap region, and a 3'-stabilizing region. In some embodiments, the nucleic acid or polynucleotide further includes a poly-A region or a Kozak sequence (e.g., in the 5-UTR). In particular, the 5'UTR sequence of the nucleic acid molecule herein is as shown in SEQ ID NO: 1, and the 3'UTR sequence is as shown in SEQ ID NO: 3.

[0063] As used herein, "modified" means non-natural. For example, RNA can be modified RNA. That is, RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers.

[0064] In some cases, the polynucleotide may contain one or more intron nucleotide sequences that can be excised from the polynucleotide. In some embodiments, the polynucleotide or nucleic acid (e.g., mRNA) can include a 5' cap structure, chain-terminating nucleotides, stem-loops, polyadenylate sequences, and / or polyadenylation signals. In one or more embodiments, the 5'-end modification group of the mRNA molecule includes ARCA, m7G(5")ppp(5")(2"OMeA)pG, m7G(5")ppp(5")(2"OMeG)pG, m7(3"OMeG)(5")ppp(5")(2"OMeG)pG, m7(3"OMeG)(5")ppp(5")(2"OMeA)pG, mCAP, dmCAP, tmCAP, or dmCAP. In one or more embodiments, the 3'-end modification group of the mRNA molecule can be poly(A), and the length of the poly(A) is 50-200, preferably 80-150.

[0065] Any region of the nucleic acid can include one or more alternative components (such as alternative nucleosides). In one or more embodiments, the modification of the mRNA molecules described herein includes replacing uracil with N1-methylpseudouracil.

[0066] In a specific embodiment, the 5' end to 3' end of the mRNA for expressing botulinum toxin type A protein includes a 5' cap, a 5' UTR, a coding sequence of botulinum toxin type A protein, a 3' UTR, and a 3' poly(A) tail.

[0067] The RNA herein includes sequence optimization of the RNA. The mRNA molecules used in the present invention are superior to other mRNA molecules expressing the same protein.

[0068] Nucleic acid delivery reagent

[0069] The present invention also provides a nucleic acid delivery reagent, which contains the nucleic acid for expressing botulinum toxin type A protein described herein and a carrier for delivering the nucleic acid. Preferably, the carrier is a nano-lipid particle, and the nucleic acid molecule is encapsulated by the nano-liposome particle to form a nanoparticle composition.

[0070] The present disclosure also provides a method for delivering the nucleic acid for expressing botulinum toxin type A protein to mammalian cells through the nucleic acid delivery reagent, particularly delivering the nucleic acid to a local part of a mammal, producing botulinum toxin type A protein in local cells of the mammal, and a method for treating diseases or symptoms caused by muscle persistent rigidity in a mammal.

[0071] As used herein, "local" refers to cells, tissues, or organs within a certain range of the administration site. The nucleic acid delivery reagent prepared using the compound of formula (I) described herein can achieve local administration and local efficacy within an organ, tissue, or cell. In some embodiments, the nanoparticle composition of the present invention is suitable for preparing locally applied, locally acting products (LALAP). As used herein, "locally acting product", "locally acting product composition", "locally applied, locally acting product", or "locally applied, locally acting" refers to a drug that is applied locally and acts at the application site. If such a drug exhibits systemic effects (such as liver targeting), it is considered an unintended drug effect. Local can include only cells, only organs (such as the heart, liver, spleen, kidney, or lung), or only tissues (such as muscle, skin, or bone). In some embodiments of the present invention, local refers to only the cells, tissues, or organs at the administration site. In some embodiments, when locally administering to an organ (such as the heart, liver, spleen, kidney, or lung), the drug is only delivered into the organ where the administration site is located; when locally administering to a tissue (such as a certain muscle), the drug is only delivered into the tissue (such as the muscle) where the administration site is located; when locally administering to cells, the drug is only delivered into the cells within the organ or tissue (such as the muscle where the cells are located) where the administration site is located. In some embodiments, when administering to the gastrocnemius muscle, the drug is only delivered into the gastrocnemius muscle.

[0072] In some embodiments, local can refer to within 4 - 8 hours (such as 6 hours) after administering the LNP composition described herein to an organ (such as the heart, liver, spleen, kidney, or lung), the LNP composition and the nucleic acid (mRNA) encoding botulinum toxin type A protein are mainly maintained within the organ. In some embodiments, local can refer to within 4 - 8 hours (such as 4 hours) after administering the LNP composition described herein to a tissue (such as muscle or bone), the LNP composition and the nucleic acid (mRNA) encoding botulinum toxin type A protein are mainly maintained within the tissue; in particular, it is not delivered to the liver within 4 - 8 hours (such as 4 hours) after administration.

[0073] Lipids and lipid nanoparticle compositions

[0074] The lipids described herein can be advantageously used in nucleic acid delivery reagents to deliver the nucleic acid encoding botulinum toxin type A protein locally to mammals. The lipids described herein have little or no immunogenicity.

[0075] In some aspects, the compounds described herein have the formula (I) or its salts or isomers. The lipids according to formula (I) may have a positive charge or a partial positive charge at physiological pH. Such lipids can be referred to as cationic lipids.

[0076]

[0077] Wherein, R1 is H or a C1-C8 alkyl group, R2 is a C1-10 alkyl group, a C3-C18 diene group, R3 is H or an R1-R8 alkyl group, R4 is a C1-10 alkyl group, a C3-C18 diene group, R5 is a C1-C14 alkyl group, a C2-C14 alkenyl group, or M is O or N, t is an integer from 3 to 7, R6 is H or a C1-10 alkyl group, R7 is a C1-10 alkyl group, a C3-C18 diene group.

[0078] In one or more embodiments, R1 is the same as R3, and R2 is the same as R4.

[0079] In one or more embodiments, R1 is H, and R2 is a C6-10 alkyl group or a C14-C18 diene group.

[0080] In one or more embodiments, R1 is a C4-C8 alkyl group, and R2 is a C4-C8 alkyl group.

[0081] In one or more embodiments, R3 is H, and R4 is a C6-10 alkyl group or a C14-C18 diene group.

[0082] In one or more embodiments, R3 is a C4-C8 alkyl group, and R4 is a C4-C8 alkyl group.

[0083] In one or more embodiments, R5 is a C1-C4 alkyl group, a C10-C14 alkyl group, a C10-C14 alkenyl group, or M is O or N, t is 3 or 7, R6 is a C4-C8 alkyl group and R7 is a C4-C8 alkyl group, or R6 is H and R7 is a C14-C18 diene group.

[0084] In one or more embodiments, R1 is H, R2 is a C6-10 alkyl group, R1 is the same as R3, R2 is the same as R4, R5 is a C10-C14 alkyl group, a C10-C14 alkenyl group or M is O or N, t is 7, R6 is a C4-C8 alkyl group and R7 is a C4-C8 alkyl group.

[0085] In one or more embodiments, R1 is H, R2 is a C14-C18 diene group, R1 is the same as R3, R2 is the same as R4, R5 is M is O, t is 3, R6 is H, R7 is a C14-C18 diene group. Preferably, R7 is the same as R2.

[0086] In one or more embodiments, R1 is a C4-C8 alkyl group, R2 is a C4-C8 alkyl group, R1 is the same as R3, R2 is the same as R4, R5 is a C1-C4 alkyl group, M is O, t is 3, R6 is H, and R7 is a C14-C18 dienyl group.

[0087] In one or more embodiments, when R2, R4, and R7 are C14-C18 dienyl groups, the alkenyl groups of the C14-C18 dienyl groups are located on any two of the carbons numbered 7-14. In one or more embodiments, the alkenyl groups are located on the carbons numbered P and P+3, where P is 7-11. In one or more embodiments, the C14-C18 dienyl group is a 9,12-dienyl group having 14-18 carbons.

[0088] As used herein, the term "alkyl" or "alkyl group" refers to a straight-chain or branched-chain saturated hydrocarbon containing one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms), which is optionally substituted. The symbol "C1-14 alkyl" refers to an optionally substituted straight-chain or branched-chain saturated hydrocarbon containing 1-14 carbon atoms. Unless otherwise specified, the alkyl groups described herein refer to unsubstituted and substituted alkyl groups. In one or more embodiments, each alkyl group described herein is a normal alkyl group.

[0089] As used herein, the term "alkenyl" or "alkenyl group" refers to a straight-chain or branched-chain hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one carbon-carbon double bond, which is optionally substituted. The symbol "C2-14 alkenyl" refers to an optionally substituted straight-chain or branched-chain hydrocarbon containing 2-14 carbon atoms and at least one carbon-carbon double bond. The alkenyl group may include 1, 2, 3, 4 or more carbon-carbon double bonds. Unless otherwise specified, the alkenyl groups described herein refer to unsubstituted and substituted alkenyl groups.

[0090] As used herein, the term "alkynyl" or "alkynyl group" refers to a straight-chain or branched-chain hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted. The symbol "C2-14 alkynyl" refers to an optionally substituted straight-chain or branched-chain hydrocarbon containing 2-14 carbon atoms and at least one carbon-carbon triple bond. The alkynyl group may include 1, 2, 3, 5 or more carbon-carbon triple bonds. Unless otherwise specified, the alkynyl groups described herein refer to unsubstituted and substituted alkynyl groups.

[0091] As used herein, the term "carbocyclic" or "carbocyclic group" refers to an optionally substituted monocyclic or polycyclic system comprising one or more carbon atom rings. The ring can be a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20- or higher-membered ring. The symbol "C3-6 carbocyclic" refers to a carbocyclic comprising a monocyclic ring having 3 to 6 carbon atoms. The carbocyclic can comprise one or more carbon-carbon double or triple bonds and can be non-aromatic or aromatic (e.g., cycloalkyl or aryl). Examples of carbocyclics include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl.

[0092] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic and may or may not comprise any double or triple bonds. Unless otherwise specified, the carbocyclics described herein refer to unsubstituted and substituted carbocyclic groups, i.e., optionally substituted carbocyclics.

[0093] As used herein, the term "heterocyclic" or "heterocyclic group" refers to an optionally substituted monocyclic or polycyclic system comprising one or more rings, wherein at least one ring comprises at least one heteroatom. The heteroatom can be, for example, a nitrogen, oxygen or sulfur atom. The ring can be a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14- or higher-membered ring. The heterocyclic can comprise one or more double or triple bonds and can be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl). Examples of heterocyclics include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thienyl, pyridyl, piperidinyl alkynyl, quinolinyl and isoquinolinyl.

[0094] As used herein, the term "heterocycloalkyl" refers to a non-aromatic heterocyclic and may or may not contain any double or triple bonds. Unless otherwise specified, the heterocyclics described herein refer to unsubstituted and substituted heterocyclic groups, i.e., optionally substituted heterocyclics.

[0095] As used herein, "aryl" is an optionally substituted carbocyclic group comprising one or more aromatic rings. Examples of aryls include phenyl and naphthyl.

[0096] As used herein, "heteroaryl" is an optionally substituted heterocyclic group comprising one or more aromatic rings. Examples of heteroaryls include pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl and thiazolyl. Both aryl and heteroaryl can be optionally substituted. Unless otherwise specified, the aryl or heteroaryl described herein refers to unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl.

[0097] Unless otherwise specified, alkyl, alkenyl, and cyclo groups (such as carbocyclic and heterocyclic groups) may be optionally substituted. Optional substituents are selected from, but not limited to, halogen atoms (such as chlorine, bromine, fluorine, or iodine groups), carboxylic acids (such as -C(O)OH), and alcohols (such as hydroxy, -OH), esters (such as -C(O)OR or -OC(O)R), aldehydes (such as -C(O)H), carbonyls (such as -C(O)R, or represented by C=O), acyl halides (such as -C(O)X, where X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (such as -OC(O)OR), alkoxy groups (such as -OR), acetals (such as -C(OR)2R’, where each OR is an alkoxy group that may be the same or different, and R’ is an alkyl or alkenyl), phosphates (such as P(O)4 3+ ), thiols (such as -SH), sulfoxides (such as -S(O)R), sulfinic acids (such as -S(O)OH), sulfonic acids (such as -S(O)2OH), thiocarbonyls (such as -C(S)H), sulfates (such as S(O)4 2+ ), sulfonyl groups (such as -S(O)2-), amides (such as -C(O)NR2 or -N(R)C(O)R), azido groups (such as -N3), nitro groups (such as -NO2), cyano groups (such as -CN), isocyano groups (such as -NC), acyloxy groups (such as -OC(O)R), amino groups (such as -NR2, -NRH, or -NH2), carbamoyl groups (such as -OC(O)NR2, -OC(O)NRH, or -OC(O)NH2), sulfonamides (such as -S(O)2NR2, -S(O)2NRH, -S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H, or -N(H)S(O)2H), alkyl, alkenyl, and cyclo groups (such as carbocyclic or heterocyclic groups). In any of the foregoing, R is an alkyl or alkenyl as defined herein.

[0098] In some embodiments, the substituent itself may be further substituted by, for example, 1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, a C1-6 alkyl may be further substituted by 1, 2, 3, 4, 5, or 6 substituents as described herein.

[0099] In one or more embodiments, the compound of formula (I) is 2-{3-[4-(dimethylamino)hexahydropyridin-1-yl]-3-oxopropyl}-10-(heptadecan-9-yloxy)-10-oxo-2-[(undecyloxy)carbonyl]decanoic acid undecyl ester (Compound 1).

[0100] As used herein, when the terms "about" and "approximately" are applied to one or more values of interest, they refer to values that are similar to a specified reference value. In certain embodiments, the term "about" or "approximately" means falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) the reference value, unless otherwise stated or apparent from the context (unless the number exceeds 100% of the possible value). For example, when used in the context of the amount of a given compound in the lipid component of a nanoparticle composition, "about" may mean + / - 10% of the value. For example, a nanoparticle composition comprising a lipid component having about 40% of a given compound may comprise 30 - 50% of the compound.

[0101] As used herein, the term "compound" is intended to include all isomers and isotopes of the indicated structure. "Isotope" refers to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the atomic nucleus. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts or complexes of the present disclosure can be prepared by conventional methods of combining with solvent or water molecules to form solvates and hydrates.

[0102] As used herein, the term "contact" means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means physically connecting the mammalian cell and the nanoparticle. Methods for contacting cells with external entities in vivo and in vitro are well known in the biological arts. For example, a nanoparticle composition can be contacted with a mammalian cell by different routes of administration (e.g., intravenous, intramuscular, intradermal and subcutaneous), and can involve different amounts of the nanoparticle composition. In addition, a nanoparticle composition can contact more than one mammalian cell.

[0103] As used herein, the term "delivery" means providing an entity to a destination. For example, delivering a nucleic acid encoding botulinum toxin type A protein to a subject may involve administering to the subject a nanoparticle composition comprising a nucleic acid encoding botulinum toxin type A protein (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering the nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition. "Local delivery" means delivering a drug primarily to a partial location of a subject, such as an organ, a tissue (e.g., a muscle), or inside a cell (e.g., the organ to which the drug is administered, the muscle to which the drug is administered, the cell to which the drug is administered), and no delivery or substantially no delivery to organs, tissues, or cells other than the said organ, tissue, or cell. For example, when a drug is administered into the kidney, the drug is locally delivered to other parts of the kidney, and not delivered to organs, tissues, or cells outside the kidney (e.g., through the circulatory system). When a drug is administered into a muscle (e.g., the gastrocnemius muscle), the drug is locally delivered to other parts of the muscle, and not delivered to organs, tissues, or cells outside the muscle (e.g., through the circulatory system).

[0104] In certain embodiments, the nanoparticle composition comprising a compound according to formula (I) has substantially the same level of local delivery effect regardless of the administration route. For example, when certain compounds disclosed herein are used for intravenous or intramuscular delivery of therapeutic and / or prophylactic drugs, they exhibit similar local delivery. In some embodiments, intramuscular injection is preferred over intravenous injection.

[0105] As used herein, the terms "specific delivery" and "local delivery" mean that, compared with non-target tissues (e.g., mammalian liver), the target cell, tissue, or organ of interest (e.g., mammalian muscle) is delivered with more (e.g., at least 1.5-fold or more, at least 2-fold or more, at least 3-fold or more, at least 3-fold or more, at least 4-fold or more, at least 5-fold or more, at least 6-fold or more, at least 7-fold or more, at least 8-fold or more, at least 9-fold or more, at least 10-fold or more) nucleic acid encoding botulinum toxin type A protein by nanoparticles. The delivery level of nanoparticles to a specific tissue can be identified by the following comparisons: comparing the amount of protein produced in the tissue with the weight of the tissue, comparing the amount of therapeutic and / or prophylactic in the tissue with the weight of the tissue, comparing the amount of protein produced in the tissue with the total amount of protein in the tissue, or comparing the amount of therapeutic and / or prophylactic in the tissue with the total amount of therapeutic and / or prophylactic in the tissue. It should be understood that the ability of nanoparticles to specifically deliver to a target tissue does not need to be determined in a subject receiving treatment, and it can be determined in an alternative such as an animal model (e.g., a rat model).

[0106] As used herein, "encapsulation efficiency" refers to the amount of nucleic acid encoding botulinum toxin type A protein that becomes part of the nanoparticle composition relative to the total initial amount of nucleic acid encoding botulinum toxin type A protein used to prepare the nanoparticle composition. For example, if 97 mg of nucleic acid out of a total of 100 mg of nucleic acid initially provided to the composition is encapsulated in the nanoparticle composition, the encapsulation efficiency can be 97%.

[0107] As used herein, "expression" of a nucleic acid sequence includes translation of mRNA into a polypeptide or protein and / or post-translational modification of the polypeptide or protein.

[0108] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within a living organism (e.g., an animal, plant, or microorganism). As used herein, the term "in vivo" refers to events that occur within a living organism (e.g., an animal, plant, or microorganism or its cells or tissues). As used herein, the term "ex vivo" refers to events that occur outside of a living organism (e.g., an animal, plant, or microorganism or its cells or tissues). Ex vivo events may occur in an environment that is minimally changed compared to the natural (e.g., in vivo) environment.

[0109] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. A compound may include one or more chiral centers and / or double bonds and thus may exist as stereoisomers. The present disclosure encompasses any and all isomers of the compounds described herein, including stereoisomerically pure forms as well as mixtures of enantiomers and stereoisomers, such as racemates. Methods for separating enantiomeric and stereoisomeric mixtures of compounds and separating them into their enantiomeric or stereoisomeric components are well known.

[0110] As used herein, "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component may include one or more cationic / ionizable, polyethylene glycolylated, structural, or other lipids, such as phospholipids.

[0111] As used herein, "administration" may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering the composition to a subject. The method of administration can be selected to target delivery (e.g., specific delivery) to a particular region or system of the body.

[0112] As used herein, a "nanoparticle composition" is a composition comprising one or more lipids. Nanoparticle compositions are typically on the micron scale or smaller in size and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs, also known as nanolipid particles), liposomes (such as lipid vesicles), and lipid complexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less. In one or more embodiments, the lipid nanoparticles herein are as described in Chinese Patent Application No. 202311039860.1, which is incorporated herein by reference in its entirety.

[0113] As used herein, a "patient" refers to a subject who may seek or require treatment, is in need of treatment, is receiving treatment, will receive treatment, or has been under the care of a trained professional due to a specific disease or condition.

[0114] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that contains a polyethylene glycol component. For example, PEG2000.

[0115] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0116] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a carrier capable of suspending, complexing, or dissolving an active compound) and having substantially non-toxic and non-toxic properties. Excipients may include, for example: anti-adhesives, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, perfumes, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, cross-linked carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0117] In this specification, the structural formula of a compound represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc. It should be understood that not all isomers may have the same level of activity.

[0118] For compounds represented by the chemical formulas herein, there may exist crystal polymorphs. It should be noted that any crystal form, a mixture of crystal forms, or its anhydride or hydrate is included within the scope of the present disclosure. The terms "crystal polymorph", "polymorph", or "crystal form" refer to crystal structures in which the compound (or its salt or solvate) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability, and solubility. The recrystallization solvent, crystallization rate, storage temperature, and other factors may cause one crystal form to predominate. Crystal polymorphs of a compound can be prepared by crystallization under different conditions.

[0119] The composition may also include salts of one or more compounds. The salts can be pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound in which the existing acidic or basic moiety is modified by converting it to its salt form (e.g., by reacting the free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic salts of basic residues such as amino groups; alkali metal salts or organic salts of acidic residues such as carboxylic acids, etc.

[0120] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, dodecyl 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, toluenesulfonate, undecanoate, valerate, etc.

[0121] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc.

[0122] The pharmaceutically acceptable salts of the present disclosure include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic acids or organic acids.

[0123] The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or in a mixture of both; generally, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is preferred. A list of suitable salts can be found in any edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference in its entirety.

[0124] As used herein, "phospholipid" is a lipid that includes a phosphate moiety and one or more carbon chains such as unsaturated fatty acid chains. Phospholipids can include one or more multiple (e.g., double or triple) bonds (e.g., one or more degrees of unsaturation). Certain phospholipids can facilitate fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). Fusion of the phospholipid with the membrane can allow one or more components of the lipid-containing composition to cross the membrane, thereby allowing, for example, delivery of one or more components to the cell.

[0125] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues that are typically linked by peptide bonds and can be produced naturally (e.g., isolated or purified) or synthetically.

[0126] As used herein, a "single unit dose" is the dose of any therapeutic agent administered in one dose / one time / one route / one point of contact, i.e., a single administration event. As used herein, a "divided dose" is the splitting of a single unit dose or the total daily dose into two or more doses. As used herein, the "total daily dose" is the amount given or prescribed within 24 hours. It can be administered as a single unit dose.

[0127] As used herein, "size" or "average size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0128] As used herein, the terms "subject", "subject individual" or "patient" refer to any living organism to which a composition according to the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Preferably, the subject described herein suffers from a disease that benefits from local delivery of a drug, for example, a lesion of an organ or tissue where it is desired to deliver the drug only to that organ or tissue.

[0129] As used herein, "target cell" refers to any one or more cells of interest. Cells can be found in vitro, in vivo, in situ, or in a tissue or organ of a living organism. The living organism can be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0130] As used herein, "target tissue" refers to any one or more tissue types of interest, where delivery of treatment and / or prophylaxis will result in a desired biological and / or pharmacological effect. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. In a particular application, the target tissue can be the kidney, lung, spleen, vascular endothelium in a blood vessel (e.g., within a coronary artery or within the femur), or tumor tissue (e.g., by intratumoral injection) or muscle (e.g., by intramuscular injection).

[0131] As used herein, the term "therapeutically effective amount" means that when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, it treats, ameliorates, improves, relieves, delays the onset of, inhibits the progression of, reduces the severity of, and / or reduces the incidence of the infection, disease, disorder, and / or condition.

[0132] As used herein, "transfection" refers to the introduction of a substance (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0133] As used herein, the term "treatment" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of a particular infection, inhibiting its progression, reducing its severity, and / or reducing its incidence, disease, disorder, and / or condition. For example, "treating" cancer can refer to inhibiting the survival, growth, and / or spread of a tumor. For risk reduction, treatment can be developed for subjects who do not exhibit signs of a disease, disorder, and / or condition and / or subjects who exhibit only early signs of a disease, disorder, and / or condition to develop the pathology associated with the disease, disorder, and / or condition.

[0134] Another aspect of the present disclosure encompasses a nanoparticle composition of a lipid component, the lipid component comprising a compound of formula (I) described herein.

[0135] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition includes two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayers can include one or more ligands, proteins, or channels.

[0136] The nanoparticle composition comprises a lipid component that includes at least one compound according to formula (I). For example, the lipid component of the nanoparticle composition can include one or more of Compounds 1-10. The nanoparticle composition can also include a variety of other components. For example, in addition to the lipids of formula (I), the nanoparticle composition can include one or more cationic lipids.

[0137] The lipid component of the nanoparticle composition can include one or more PEGs or PEG-modified lipids. Such substances may alternatively be referred to as polyethylene glycolated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids can be selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0138] The lipid component of the nanoparticle composition can include one or more structural lipids. The structural lipids can be selected from, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0139] The lipid component of the nanoparticle composition can include one or more phospholipids, such as one or more unsaturated lipids. The phospholipids can be assembled into one or more lipid bilayers. Generally, the phospholipids can include a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid can be a lipid according to formula (II):

[0140]

[0141] Wherein Rp represents a phospholipid moiety, and R1 and R2 represent fatty acid moieties with or without unsaturation, which may be the same or different. The phospholipid moiety is optionally selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysolecithin, and sphingomyelin. The fatty acid moiety is optionally selected from lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0142] Phospholipids useful in the compositions and methods described herein are optionally selected from DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, DUPC, POPC, OChemsPC, DOPG, and sphingomyelin. In some embodiments, the nanoparticle composition comprises DSPC. In some embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises DSPC and / or DOPE.

[0143] In some embodiments, the nanoparticle compositions described herein comprise a compound of formula (I), a PEG lipid, DSPC, and cholesterol.

[0144] In some embodiments, the nanoparticle composition comprising one or more lipids described herein may further comprise one or more adjuvants, such as aluminum hydroxide.

[0145] In addition to the nucleic acid encoding botulinum neurotoxin type A protein, the nanoparticle compositions of the present invention may further comprise other therapeutic / prophylactic agents, including but not limited to anti-tumor agents, anti-infective agents, local anesthetics, β-adrenergic blockers, antihypertensive agents, antidepressants, antihistamines, antibiotics / antimicrobials, antifungals, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, anesthetics, imaging agents, cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that elicit an immune response, antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, proteins, other polynucleotides or nucleic acids. Exemplary polynucleotides used according to the present disclosure include but are not limited to DNA, RNA including messenger mRNA, their hybrids, one or more of RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and the like.

[0146] Other components

[0147] In addition to those described in the foregoing section, the nanoparticle composition may include one or more components. For example, the nanoparticle composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0148] The nanoparticle composition may also include one or more permeation enhancer molecules, carbohydrates, polymers, surface modifiers, or other components. The permeation enhancer molecules can be, for example, the molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs). Polymers may be included in and / or used for encapsulating or partially encapsulating the nanoparticle composition. The polymers can be biodegradable and / or biocompatible. The polymers can be selected from, but not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The surface modifiers can include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants, such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, n-acetylcysteine, erdosteine), and DNase (e.g., rhDNase). The surface modifiers can be arranged within the nanoparticles and / or on the surface of the nanoparticle composition (e.g., by coating, adsorption, covalent attachment, or other processes).

[0149] The nanoparticle composition may also contain one or more functionalized lipids. For example, the lipids can be functionalized with alkynyl groups, which can undergo a cycloaddition reaction when exposed to azides under appropriate reaction conditions. In particular, the lipid bilayer can be functionalized in this way with one or more groups that can be used to facilitate membrane penetration, cell recognition, or imaging.

[0150] The surface of the nanoparticle composition can also be conjugated with one or more useful antibodies. Functional groups and conjugates for targeted cell delivery, imaging, and membrane penetration are well known in the art.

[0151] In addition to these components, the nanoparticle composition can include any substance that can be used in a pharmaceutical composition. For example, the nanoparticle composition can include one or more pharmaceutically acceptable excipients or auxiliary components, such as but not limited to one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid carriers, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other substances. Excipients such as waxes, butters, colorants, coating agents, flavoring agents, and fragrances can also be included. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington′s The Science and Practice of Pharmacy, 21st edition).

[0152] The lipid component of the nanoparticle composition can include, for example, lipids according to formula (I), phospholipids (such as unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. Each lipid component can be provided in a specific fraction. In certain embodiments, the lipid component of the nanoparticle composition includes from about 30 mole % to about 60 mole % of the compound of formula (I), from about 0 mole % to about 30 mole % phospholipid, from about 18.5 mole % to about 48.5 mole % structural lipid, and from about 0 mole % to about 10 mole % PEG lipid. In some embodiments, the lipid component of the nanoparticle composition includes from about 35 mole % to about 55 mole % of the compound of formula (I), from about 5 mole % to about 25 mole % phospholipid, from about 30 mole % to about 40 mole % structural lipid, and from about 0 mole % to about 10 mole % PEG lipid. In a particular embodiment, the lipid component includes about 50 mole % of the compound of formula (I), about 10 mole % phospholipid, about 38.5 mole % structural lipid, and about 1.5 mole % PEG lipid. In another embodiment, the lipid component includes about 50 mole % of the compound of formula (I), about 10 mole % phospholipid, about 38.5 mole % structural lipid, and about 3.05 mole % PEG lipid. In another particular embodiment, the lipid component includes about 40 mole % of the compound of formula (I), about 20 mole % phospholipid, about 38.5 mole % structural lipid, and about 1.5 mole % PEG lipid. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-DMG (such as PEG2000-DMG) and / or the structural lipid can be cholesterol.

[0153] Alternatively, the lipid component may be in terms of mole fractions. For example, the lipid component of the nanoparticle composition includes from about 30 to about 60 mole fractions of the compound of formula (I), from about 0 to about 30 mole fractions of phospholipids, from about 18.5 to about 48.5 mole fractions of structural lipids, and from about 0 to about 10 mole fractions of PEG lipids. In some embodiments, the lipid component of the nanoparticle composition includes from about 35 to about 55 mole fractions of the compound of formula (I), from about 5 to about 25 mole fractions of phospholipids, from about 30 to about 40 mole fractions of structural lipids, and from about 0 to about 10 mole percent of PEG lipids. In some embodiments, it includes about 50 mole fractions of the compound of formula (I), about 10 mole fractions of phospholipids, about 38.5 mole fractions of structural lipids, and about 1.5 mole fractions of PEG lipids. In another embodiment, the lipid component includes about 50 mole fractions of the compound of formula (I), about 10 mole fractions of phospholipids, about 38.5 mole fractions of structural lipids, and about 3.05 mole fractions of PEG lipids.

[0154] The amount of the nucleic acid expressing botulinum neurotoxin type A protein in the nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition, as well as the properties of the nucleic acid expressing botulinum neurotoxin type A protein. For example, the amount of RNA that can be used in the nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of the therapeutic and / or prophylactic and other components (such as lipids) in the nanoparticle composition may also vary. In some embodiments, the weight / weight ratio of the lipid component to the nucleic acid expressing botulinum neurotoxin type A protein in the nanoparticle composition may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1. For example, the weight / weight ratio of the lipid component to the nucleic acid expressing botulinum neurotoxin type A protein may be from about 10:1 to about 40:1. In certain embodiments, the weight / weight ratio is about 20:1. The amount of the nucleic acid expressing botulinum neurotoxin type A protein in the nanoparticle composition can be measured, for example, using absorption spectroscopy (such as ultraviolet-visible spectroscopy).

[0155] The average size of the nanoparticle composition can be between 10 nanometers and 100 nanometers, for example, measured by methods well known in the art. For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm. In some embodiments, the average size of the nm particle composition can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm or from about 90 nm to about 100 nm. In certain embodiments, the average size of the nm particle composition can be from about 70 nm to about 100 nm. In a particular embodiment, the average size can be about 70 nm.

[0156] The nanoparticle composition can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be from about 0.05 to about 0.10.

[0157] The encapsulation efficiency of a nucleic acid expressing botulinum neurotoxin type A protein describes the amount of the nucleic acid expressing botulinum neurotoxin type A protein that is encapsulated or otherwise associated with the nanoparticle composition after preparation relative to the initial amount provided. The encapsulation efficiency can be measured, for example, by comparing the therapeutic and / or prophylactic amounts in a solution containing the nanoparticle composition before and after disrupting the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free nucleic acid expressing botulinum neurotoxin type A protein (e.g., RNA) in the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the nucleic acid expressing botulinum neurotoxin type A protein can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0158] The nanoparticle composition can optionally include one or more coatings. For example, the nanoparticle composition can be formulated in a capsule, film, or tablet having a coating. The capsules, films, or tablets of the compositions described herein can have any useful size, tensile strength, hardness, or density.

[0159] The nanoparticle compositions of the present invention can be prepared in a variety of forms suitable for various routes of administration, such as liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical (including buccal and sublingual), transdermal, and / or percutaneous administration (e.g., creams, ointments, pastes, lotions, gels, powders, solutions, sprays, inhalants, and patches), dosage forms for vaginal administration (e.g., vaginal suppositories, tampons, creams, gels, pastes, foams, and sprays), dosage forms for implant administration (e.g., solids, semi-solids, gels), suspensions, powders, and other dosage forms.

[0160] Pharmaceutical composition

[0161] The nanoparticle composition can be formulated, in whole or in part, as a pharmaceutical composition. The pharmaceutical composition can include one or more nanoparticle compositions.

[0162] For example, a pharmaceutical composition may comprise one or more nanoparticle compositions, including one or more different nucleic acids expressing botulinum toxin type A protein. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients, such as those described herein. General guidelines for formulating and manufacturing pharmaceutical compositions and medicaments can be found in Remington's The Science and Practice of Pharmacy, 21st Edition. Conventional excipients and adjuvants can be used in any pharmaceutical composition, unless any conventional excipient or adjuvant may be incompatible with one or more components of the nanoparticle composition. The amount of excipient in the pharmaceutical composition can be determined by those skilled in the art according to need.

[0163] The relative amounts of one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical composition according to the present disclosure will vary depending on the properties, size, and / or condition of the subject being treated, and further depending on the route of administration of the composition. By way of example, the pharmaceutical composition may comprise from 0.1% to 100% (wt / wt) of one or more nanoparticle compositions.

[0164] In certain embodiments, the nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or transportation (e.g., stored at a temperature of 4°C or lower, such as a temperature between about -150°C) and between about 0°C or about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C).

[0165] For example, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein can be stable for at least about 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months, such as at a temperature of 4 °C or lower (e.g., between about 4 °C and -20 °C). In one embodiment, the formulation is stable for at least 4 weeks at 4 °C. In certain embodiments, the pharmaceutical compositions disclosed herein comprise the nanoparticle compositions disclosed herein and a pharmaceutically acceptable excipient selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pharmaceutical compositions disclosed herein have a pH value between about 7 and 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7 and 7.8).

[0166] In the context of the present disclosure, "stability" and "stable" refer to the nanoparticle compositions and / or pharmaceutical compositions disclosed herein being resistant to chemical or physical changes (e.g., degradation, particle size changes, aggregation, encapsulation changes, etc.) under given manufacturing, preparation, transportation, storage, and / or use conditions, such as when stresses such as shear forces, freeze / thaw stresses, etc. are applied.

[0167] The nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including those patients or subjects that can benefit from the therapeutic effects provided by delivering nucleic acids encoding botulinum neurotoxin type A to one or more specific cells, tissues, organs, or systems, or groups thereof.

[0168] Although "compositions" primarily refers to compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other mammal.

[0169] Modifying compositions suitable for administration to humans to make them suitable for administration to various animals is well known, and an ordinarily skilled veterinary pharmacologist can design and / or perform such modifications with only ordinary (if any) experimentation. The subjects expected to receive the compositions include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0170] A pharmaceutical composition comprising one or more nanoparticle compositions can be prepared by any method known in the pharmacological art or developed later. Generally, such preparation methods include combining the active ingredient with excipients and / or one or more other auxiliary ingredients, and then, if necessary or desired, dividing, shaping, and / or packaging the product into the desired single or multiple forms. Multiple-dose units.

[0171] The pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of the active ingredient (e.g., the nanoparticle composition). The amount of the active ingredient is usually equal to the dose of the active ingredient to be administered to a subject and / or a convenient fraction of that dose, such as one-half or one-third of that dose.

[0172] The pharmaceutical composition can be prepared in a variety of forms suitable for a variety of administration routes and methods. For example, the pharmaceutical composition can be prepared as a liquid dosage form (e.g., emulsion, microemulsion, nanoemulsion, solution, suspension, syrup, and elixir), an injectable dosage form, a solid dosage form (e.g., capsule, tablet, pill, powder, and granule), a dosage form for topical (including buccal and sublingual), transdermal, and / or percutaneous administration (e.g., cream, ointment, paste, lotion, gel, powder, solution, spray, inhalant, and patch), a dosage form for vaginal administration (e.g., vaginal suppository, tampon, cream, gel, paste, foam, and spray), a dosage form for implant administration (e.g., solid, semi-solid, gel), suspension, powder, and other dosage forms.

[0173] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof. In addition to the inert diluent, the oral composition may include additional nucleic acids expressing botulinum toxin type A protein, additional reagents such as wetting agents, emulsifiers, and suspending agents, sweetening agents, flavoring agents, and / or aromatic agents. In certain embodiments of parenteral administration, the composition is mixed with a solubilizer, such as, alcohol, oil, modified oil, ethylene glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof.

[0174] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersing, wetting and / or suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension and / or emulsion in a non-toxic parenterally acceptable diluent and / or solvent. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Sterile oils can be used as solvents or suspending media. For this purpose, any mild, non-volatile oil can be used, including synthetic mono- or di-glycerides of fatty acids. Fatty acids such as oleic acid can be used in the preparation of injectables. The injectable preparation can be sterilized, for example, by filtration through a bacteria-retaining filter, and / or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0175] Compositions for rectal or vaginal administration are generally suppositories, which can be prepared by mixing the composition with a suitable non-irritating excipient that is solid at ambient temperature but liquid at body temperature and thus melts in the rectum or vaginal cavity and releases the active ingredient.

[0176] The present disclosure also contemplates the use of transdermal patches, and such dosage forms can be prepared, for example, by dissolving and / or dispersing the compound in a suitable medium. Alternatively or additionally, the rate can be controlled by providing a rate-controlling membrane and / or by dispersing the compound in a polymeric matrix and / or gel.

[0177] Suitable devices for delivering the intradermal pharmaceutical compositions described herein include any type of liquid or solid injection device, such as conventional syringes, fine-gauge syringes, short-needle syringes, liquid jet injectors, compressed gas-accelerated powder injectors, ballistic powder delivery devices, etc.

[0178] The pharmaceutical composition can be prepared, packaged and / or sold in a formulation suitable for buccal pulmonary administration. Such formulations can contain dry granules having the active ingredient. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir and / or using a self-propelling solvent / powder dispensing container. Such formulations can also be prepared, packaged and / or sold as an aqueous solution and / or dilute alcoholic solution and / or suspension (optionally sterile, containing the active ingredient), and can be conveniently administered using any nebulizing and / or atomizing device. Such formulations can also contain one or more additional ingredients, including but not limited to flavoring agents such as sodium saccharin, volatile oils, buffering agents, surfactants and / or preservatives such as methylparaben.

[0179] The formulations described herein that are useful for pulmonary delivery can also be used for intranasal delivery of pharmaceutical compositions. Such formulations are administered by sniffing, i.e., rapidly inhaling through the nasal passages from a powder container close to the nose. The pharmaceutical compositions can be prepared, packaged, and / or sold in a formulation suitable for oral administration. Such formulations can be, for example, in the form of tablets and / or lozenges prepared using conventional methods.

[0180] Methods and Uses

[0181] The present disclosure provides a method for producing botulinum toxin type A protein in mammalian cells, which involves contacting the cells with a nanoparticle composition comprising a nucleic acid (e.g., mRNA) described herein. When the cells are contacted with the nanoparticle composition, the nucleic acid can be taken up and translated in the cells to produce botulinum toxin type A protein.

[0182] Generally, the step of contacting mammalian cells with a nanoparticle composition comprising mRNA encoding a polypeptide of interest can be carried out in vivo, ex vivo, in culture, or in vitro. The amount of the nanoparticle composition contacted with the cells and / or the amount of mRNA therein can depend on the type of cells or tissues contacted, the mode of administration, the physiochemical characteristics (e.g., size, charge, and chemical composition) of the nanoparticle composition and mRNA, and other factors. Efficiency indicators may include polypeptide translation (represented by polypeptide expression), mRNA degradation level, and immune response indicators.

[0183] The step of contacting a nanoparticle composition comprising mRNA with cells can involve or cause transfection. The phospholipids contained in the lipid component of the nanoparticle composition can promote transfection and / or improve transfection efficiency, for example, by interacting and / or fusing with cell membranes or intracellular membranes. Transfection can allow mRNA to be translated inside the cells.

[0184] Methods for Treating Diseases and Disorders

[0185] The nanoparticle compositions herein can be used to treat diseases or conditions. Subsequent translation of the mRNA can produce botulinum toxin type A protein, thereby reducing or eliminating diseases or conditions caused by persistent muscle rigidity.

[0186] Diseases or conditions caused by persistent muscle rigidity are mainly anti-muscle diseases or aesthetic medicine-related symptoms. The muscle diseases include blepharospasm, strabismus, and cervical dystonia; the aesthetic medicine-related symptoms include glabellar lines, outer canthal lines, and forehead lines, etc. In the above-mentioned indications, the pathology is local symptoms caused by persistent muscle rigidity at the local site, and the nucleic acid molecules, nucleic acid delivery reagents, or pharmaceutical compositions described in any embodiment herein can block the release of acetylcholine in neurotransmitters at the injection site, inhibit neuromuscular conduction, and thus relieve the clinical symptoms of the above diseases.

[0187] The present disclosure provides methods related to administering a nanoparticle composition comprising one or more nucleic acids expressing botulinum neurotoxin type A protein, and a therapeutic or prophylactic composition comprising the same. The therapeutic or prophylactic composition can be administered to a subject in an effective amount for preventing, treating, or by any reasonable amount and any route of administration. The specific amount administered to a subject can vary depending on the species, age, and general condition of the subject, the particular composition, and the mode of administration. The compositions according to the present disclosure can be formulated in unit dosage form for ease of administration and uniformity of dosage. However, it should be understood that the specific dosage of the compositions of the present disclosure will be determined by the attending physician within the scope of reasonable medical judgment.

[0188] The nanoparticle composition comprising one or more nucleic acids expressing botulinum neurotoxin type A protein can be administered by any route. In some embodiments, the nanoparticle compositions described herein are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal or intradermal, interdermal, rectal, intravaginal, intraperitoneal, intraocular, subretinal, intravitreal, mucosal, nasal, oral, enteral, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation, as an oral spray and / or powder, nasal spray and / or aerosol, and / or via a portal vein catheter. Local administration is preferred, such as intramuscular, subcutaneous, transdermal or intradermal, interdermal, intraperitoneal, intraocular, subretinal, intravitreal, mucosal, nasal, oral, intratumoral, intranasal. The appropriate route of administration will depend on a variety of factors, including the nature of the nanoparticle composition, the nucleic acid expressing botulinum neurotoxin type A protein, the condition of the patient, etc.

[0189] In certain embodiments, the compositions according to the present disclosure can be administered at levels sufficient to deliver the following doses: from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 2.5 mg / kg, from about 0.001 mg / kg to about 2.5 mg / kg, from about 0.005 mg / kg to about 2.5 mg / kg, from about 0.01 mg / kg to about 2.5 mg / kg, from about 0.05 mg / kg to about 2.5 mg / kg, from about 0.1 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about 2.5 mg / kg, from about 2 mg / kg to about 2.5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.05 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.25 mg / kg, from about 0.001 mg / kg to about 0.25 mg / kg, from about 0.005 mg / kg to about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg, or from about 0.1 mg / kg to about 0.25 mg / kg of a nucleic acid (such as mRNA) encoding botulinum neurotoxin type A protein.

[0190] The dosage can be administered one or more times per day in the same or different amounts to achieve the desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage can be delivered, for example, three times a day, twice a day, once a day, every other day, every three days, weekly, bi-weekly, tri-weekly, or monthly. In certain embodiments, multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations) can be used to deliver the desired dosage.

[0191] Nanoparticle compositions comprising one or more nucleic acids encoding botulinum toxin type A protein can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. Each agent will be administered at the dosage and / or schedule determined for that agent. The therapeutic, prophylactic, diagnostic, or imaging active agents used in combination can be administered together in a single composition or separately in different compositions.

[0192] Those skilled in the art will recognize or be able to ascertain, using only routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather as set forth in the appended claims.

[0193] In the claims, articles such as "a," "an," and "the" may refer to one or more than one, unless there is a contrary indication or it is obvious from the context.

[0194] The term "comprising" is intended to be open and permits, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, the terms "consisting essentially of" and "consisting of" are thus also covered and disclosed. Further, it should be understood that the order of steps or the order of performing certain actions is not important so long as the invention remains operable. Two or more steps or actions can be carried out simultaneously.

[0195] The compounds of the present disclosure can be prepared in a variety of ways by using commercially available starting materials, compounds known in the literature, or intermediates prepared readily by employing standard synthetic methods and procedures known or to be appreciated by those skilled in the art. The synthesis of the compounds of the present disclosure will be apparent to those skilled in the art in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and for functional group transformation and manipulation can be obtained from relevant scientific literature or standard textbooks in the art, such as Smith, M.B., March, J., March′s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 thedition, John Wiley & Sons: New York, 2001. The synthetic methods described in the embodiments of this article are intended to illustrate but not limit the general procedures for preparing the compounds of the present disclosure. Those of ordinary skill in the art will note that during the reaction sequences and synthetic schemes described herein, the order of certain steps can be changed, such as the introduction and removal of protecting groups. In the reaction schemes described herein, multiple stereoisomers can be produced. Those of ordinary skill in the art will recognize that the reaction can be optimized to preferentially produce one isomer, or new schemes can be designed to produce a single isomer. If a mixture is produced, techniques such as preparative thin-layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC can be used to separate the isomers.

[0196] Compounds 1 - 7 described in the embodiments of this article are as follows:

[0197] Table 1

[0198]

[0199]

[0200] Example

[0201] The specific steps of the present invention are illustrated below by examples, but are not limited by the examples.

[0202] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. The present invention is further described in detail below in conjunction with specific examples and with reference to the data. It should be understood that these examples are only for illustrative purposes of the present invention and do not limit the scope of the present invention in any way. In the following examples, various processes and methods not described in detail are conventional methods well known in the art. The content of the present invention is specifically illustrated below by examples:

[0203] Example 1: Detection of the Secretion Level of the Target Protein in the Cell Line

[0204] First, we used two mRNA molecules encoding the same protein but with different codon optimization strategies: the candidate mRNA molecule (BONTA, CDS SEQ ID NO: 4) and the reference mRNA molecule (RefSeq, CDS SEQ ID NO: 5). The UTR of the candidate mRNA molecule was obtained from Kaituo Biotechnology Co., Ltd. The candidate mRNA molecule includes, in sequence from 5' to 3', a 5' cap (m7G(5”)ppp(5”)(2”OMeA)pG), the 5' UTR shown in SEQ ID NO: 1, the botulinum toxin protein coding sequence shown in SEQ ID NO: 4, the 3' UTR shown in SEQ ID NO: 3, and a 3' poly(A) consisting of 100 adenines.

[0205] The CDS part of the reference mRNA molecule was also codon-optimized, but a different codon optimization strategy from the candidate mRNA molecule was used. The UTR parts at both ends used the sequences used by Moderna Inc. of the United States for its COVID-19 vaccine product SpikeVax; the other parts were exactly the same. The reference mRNA molecule includes, in sequence from 5' to 3', a 5' cap (m7(3′OMeG)(5′)ppp(5′)(2′OMeA)pG), a 5' UTR shown in SEQ ID NO.6, a botulinum toxin protein coding sequence shown in SEQ ID NO:5, a 3' UTR shown in SEQ IDNO.7, and a 3' poly(A) consisting of 120 adenines.

[0206] Human kidney epithelial cell line HEK293T in logarithmic growth phase was taken, and 2 mL of cell suspension containing 5×10 5 cells was inoculated into a 6-well plate; 24 hours later, Lipofectamine 2000 was mixed with 3 μg of the candidate mRNA molecule (BONTA, CDS SEQ ID NO:4) or the reference mRNA molecule (Ref Seq, CDS SEQ ID NO:5) and slowly added dropwise into the above wells, and the above medium was replaced with fresh medium 6 hours later; 24 hours after transfection, the above cells were lysed for ELISA experiment. The results are as Figure 2 shown. Both mRNA molecules successfully expressed botulinum toxin type A protein, but the protein expression level in the cells transfected with the candidate mRNA molecule was significantly higher than that of the reference mRNA molecule. The above results indicate that the CDS sequence optimization strategy provided by the present invention is superior to other optimization strategies. It should be particularly noted that the UTRs of Moderna Inc. and Kaituo Biotechnology Co., Ltd. have both been proven to be able to efficiently induce the expression of mRNA, and the 5' caps of both can also induce the expression of the target gene. Therefore, the UTR and 5' cap of Moderna are not the main reasons for the low expression of the reference mRNA molecule.

[0207] Example 2: Pharmacodynamic detection of different mRNA molecules in mice

[0208] LNP preparation

[0209] All four components of the organic-phase lipid were dissolved in absolute ethanol to 10 mg / mL and formulated according to the proportions in Table 2 below. 1 mg / mL of Luc mRNA was formulated into 70 μg / mL with 25 mM sodium acetate buffer as the aqueous phase. After the two-phase channels of the microfluidic instrument (stainless steel chip) were cleaned with absolute ethanol and pure water respectively, the aqueous phase was rinsed twice with 25 mM sodium acetate buffer. At the start of the preparation, 300 μL of the prepared organic-phase component and aqueous-phase component were first used to expel the air in the pipelines of the two phases respectively, and then the preparation solution with an organic-phase to aqueous-phase volume ratio of 1:3 was aspirated. LNP was prepared at the flow rates of the organic phase and the aqueous phase being 4 mL / min and 12 mL / min respectively. The first 0.5 mL of the effluent was discarded, and only the subsequent effluent was collected until the end (the preparation methods of SM102, Compound 1-LNP to Compound 7-LNP were the same).

[0210] Table 2

[0211]

[0212]

[0213] Note: The unit is mol%, and the unit in parentheses is mol part.

[0214] LNP Ultrafiltration

[0215] The prepared LNP stock solution was added to 5 volumes of PBS. Using an ultrafiltration tube, ultrafiltration was carried out at 2000 - 3000 rpm for 10 min until the volume was about 1 mL. Then, 10 volumes of PBS were added to wash the ultrafiltration tube membrane and centrifuged to about 1 mL. Again, 10 volumes of PBS were added to wash the ultrafiltration tube membrane and centrifuged to about 1 mL. After repeatedly rinsing the membrane, the above 1 mL solution was taken out and placed in a non-enzymatic EP tube and stored in a 4°C refrigerator for later use.

[0216] As described above, the nucleic acid delivery reagent, i.e., the nano-lipid particle, encapsulated 3 ng of the candidate mRNA molecule (BONTA, CDS SEQ ID NO: 4) and 150 ng of the reference mRNA molecule (RefSeq, CDS SEQ ID NO: 5). Subsequently, the above nano-lipid particles were diluted to 50 μL with PBS and injected into the right gastrocnemius muscle of ICR female mice. At 72 hours after the injection, the digital abduction score (DAS) of the right foot toes was observed and recorded.

[0217] Figure 3The results of LNPs prepared using compound 1 (2-{3-[4-(dimethylamino)piperidin-1-yl]-3-oxyylidenepropyl}-10-(heptadecan-9-yloxy)-10-oxyylidene-2-[(undecyloxy)carbonyl]decanoate) as a cationic lipid are shown. Figure 3 As shown, only 1 of the 10 mice injected with the reference mRNA molecule had a score of 1, and the other mice had no score; while all 10 mice injected with the candidate mRNA molecule had scores, of which 1 reached the highest score of 4, 7 reached a score of 3, and 2 reached a score of 2. The DAS values ​​of the two showed a statistically significant difference (p < 0.0001). The above results show that the candidate mRNA molecule can produce a significantly better pharmacological effect than the reference mRNA molecule in mice, and further prove that under the premise that UTR can efficiently induce CDS expression, the mRNA molecule used in the present invention is superior to other mRNA molecules expressing the same protein.

[0218] Example 3: Detection of the efficacy of different nanolipid particles in mice

[0219] First, we used the above-mentioned nanolipid particles and the nanolipid particles SM102 used in Moderna's new crown vaccine product SpikeVax to encapsulate 200ng of the candidate mRNA molecule (BONTA, CDS SEQ ID NO: 4), where the CDS part of the candidate mRNA molecule was consistent with the above, and the UTR parts at both ends used the UTR sequence used by the German BioNTech company for its new crown vaccine product Comirnaty; then, we diluted the above-mentioned nanolipid particles to 50μL with PBS and injected it into the right leg gastrocnemius muscle of ICR female mice. 24 hours after the injection, we observed and recorded the DAS values ​​of the right toes. Figure 4 As shown, among the 10 mice injected with SM102 nanolipid particles, one mouse had a score of 3, three mice had a score of 2, five mice had a score of 1, and one mouse had a score of 0; while all 10 mice injected with compound 1-LNP (#15) achieved the highest score of 4, and the DAS values ​​of the two mice were statistically significantly different (p < 0.0001). The above results show that for the same mRNA molecule, the use of the nanolipid particles of the present invention can produce a significantly better drug effect in mice than the commercially available nanolipid particles.

[0220] It should be noted that, in the indications of the present invention, since there is no reported animal model at present, when applying for marketing, the foreign recombinant botulinum toxin protein product Dysport was injected into the gastrocnemius muscle of rats to observe the changes in muscle action potential to evaluate its efficacy, and finally obtained FDA approval (Pharmacology Review Report:https: / / www.accessdata.fda.gov / drugsatfda docs / nda / 2009 / 125274s000PharmR.pdf , Approval letter: https: / / www.accessdata.fda.gov / drugsatfda_docs / appletter / 2009 / 125274s000, 125274s001ltr.pdf). The use of DAS as a pharmacodynamic evaluation index has the same change trend as the muscle action potential [1], and is widely used in the pharmacodynamic evaluation of botulinum drugs due to its simplicity and accuracy in evaluation [1-3]. Therefore, the results of this experiment can support the application of the nano-lipid particles of the present invention in the said indication.

[0221] References

[0222] 1. Chung ME, et al. Comparative Study of Biological Activity of Four Botulinum Toxin Type A Preparations in Mice. Dermatol Surg. 2013 Jan; 39(1 Pt 2): 155-64. doi: 10.1111 / dsu.12071.

[0223] 2. Miyashita S, et al. Delivery of single-domain antibodies into neurons using a chimeric toxin-based platform is therapeutic in mouse models of botulism. Sci Transl Med. 2021 Jan 6; 13(575): eaaz4197. doi: 10.1126 / scitranslmed.aaz4197.

[0224] 3. Périer C, et al. Recombinant botulinum neurotoxin serotype A1 in vivo characterization. Pharmacol Res Perspect. 2021 Oct; 9(5): e00857. doi: 10.1002 / prp2.857.

Claims

1. A nucleic acid molecule, comprising: (1) The sequence shown in SEQ ID NO: 4 or a DNA sequence encoding the same, (2) The complementary sequence of (1), Preferably, the nucleic acid molecule is an RNA molecule.

2. The nucleic acid molecule according to claim 1, wherein, The nucleic acid molecule has one or more of the following characteristics: Uracil in the RNA molecule is replaced with N1-methylpseudouracil, The 5'-end and / or 3'-end of the RNA molecule has a modifying group, The 5'-end modifying group of the RNA molecule is selected from: ARCA, m7G(5”)ppp(5”)(2”OMeA)pG, m7G(5”)ppp(5”)(2”OMeG)pG, m7(3”OMeG)(5”)ppp(5”)(2”OMeG)pG, m7(3”OMeG)(5”)ppp(5”)(2”OMeA)pG, mCAP, dmCAP, tmCAP or dmCAP, The RNA molecule contains a 5'UTR, The length of the 5'UTR of the RNA molecule is 10 - 200 nucleotides, The 5'UTR of the RNA molecule includes a KOZAK sequence, The RNA molecule contains a 3'UTR, The 3'-end protective modifying group of the RNA molecule is polyA, Preferably, the RNA includes a 5' cap, 5'UTR, the coding sequence of botulinum toxin type A, 3'UTR, and a 3' polyA tail from the 5'-end to the 3'-end.

3. The nucleic acid molecule according to claim 1 or 2, wherein The 5'UTR sequence of the RNA molecule is as shown in SEQ ID NO: 1, The nucleotide sequence of the KOZAK sequence is as shown in SEQ ID NO: 2, The 3'UTR sequence of the mRNA molecule is as shown in SEQ ID NO:

3.

4. A nucleic acid delivery reagent, comprising the nucleic acid molecule according to any one of claims 1 - 3 and a carrier for delivering the nucleic acid, Preferably, the carrier is a nano-lipid particle.

5. The nucleic acid delivery reagent according to claim 4, wherein The nano-liposome particle comprises phospholipids, PEG lipids, structural lipids, and a compound of formula (I) or its salt or isomer, wherein, R1 is H or C1 - C8 alkyl, R2 is C1 - 10 alkyl, C3 - C18 alkenyl R3 is H or R1 - R8 alkyl, R4 is C1 - 10 alkyl, C3 - C18 alkenyl, R5 is C1-C14 alkyl, C2-C14 alkenyl, or M is O or N, t is an integer from 3 to 7, R6 is H or C1 - 10 alkyl, R7 is C1 - 10 alkyl, C3 - C18 alkenyl, Preferably, the compound of formula (I) has one or more of the following characteristics: R1 is the same as R3, R2 is the same as R4, and / or R1 is H, R2 is C6 - 10 alkyl or C14 - C18 alkenyl, and / or R1 is C4 - C8 alkyl, R2 is C4 - C8 alkyl, and / or R3 is H, R4 is C6 - 10 alkyl or C14 - C18 alkenyl, and / or R3 is C4 - C8 alkyl, R4 is C4 - C8 alkyl, and / or R5 is a C1-C4 alkyl group, a C10-C14 alkyl group, a C10-C14 alkenyl group, or R6 is a C4-C8 alkyl group and R7 is a C4-C8 alkyl group, or R6 is H and R7 is a C14-C18 diene group where M is O or N, and t is 3 or 7.

6. The nucleic acid delivery reagent according to claim 5, wherein R1 is H, R2 is C6-10 alkyl, R1 is the same as R3, R2 is the same as R4, R5 is C10-C14 alkyl, C10-C14 alkenyl or M is O or N, t is 7, R6 is C4-C8 alkyl and R7 is C4-C8 alkyl, or, R1 is H, R2 is a C14-C18 dienyl group, R1 is the same as R3, R2 is the same as R4, R5 is M is O, t is 3, R6 is H, R7 is a C14-C18 dienyl group; preferably, R7 is the same as R2, or R1 is a C4-C8 alkyl group, R2 is a C4-C8 alkyl group, R1 is the same as R3, R2 is the same as R4, R5 is a C1-C4 alkyl group, M is O, t is 3, R6 is H, R7 is a C14-C18 dienyl group, Preferably, the compound of formula (I) is 2-{3-[4-(dimethylamino)piperidin-1-yl]-3-oxopropyl}-10-(nonadec-9-yloxy)-10-oxo-2-[(undecyloxy)carbonyl]decanoic acid undecyl ester).

7. A pharmaceutical composition comprising the nucleic acid delivery reagent according to any one of claims 4-6 and a pharmaceutically acceptable excipient.

8. A method for producing botulinum neurotoxin type A protein in a cell, the method comprising the step of contacting the cell with the nucleic acid delivery reagent according to any one of claims 4-6, whereby the nucleic acid in the nucleic acid delivery reagent can be translated in the cell to produce a polypeptide, Preferably, the cell is a mammalian cell.

9. Use of the nucleic acid molecule according to any one of claims 1-3, the nucleic acid delivery reagent according to any one of claims 4-6, and / or the pharmaceutical composition according to claim 7 in the preparation of a reagent for a disease or symptom caused by persistent muscle rigidity, Preferably, the disease or symptom caused by persistent muscle rigidity is a muscle disease or a symptom related to persistent muscle rigidity in the field of medical aesthetics, More preferably, the muscle diseases are selected from blepharospasm, strabismus, hemifacial spasm, cervical dystonia, upper limb dystonia, lower limb dystonia, axillary hyperhidrosis, palmar hyperhidrosis, chronic migraine, neurogenic urinary incontinence, overactive bladder syndrome, local spasm after cerebral palsy, and sialorrhea, and the symptoms related to persistent muscle rigidity in the field of medical aesthetics are selected from glabellar lines, crow's feet, and forehead lines.

10. A method for preparing the nucleic acid delivery reagent according to any one of claims 4-6, wherein the carrier of the nucleic acid delivery reagent is a nano-lipid particle.

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