Polynucleotide composition for improving hair conditions

Abstract

A nucleic acid molecule encoding collagen and the use thereof for improving hair conditions. Compared with a traditional direct collagen supplementation, the use of an in-vivo mRNA delivery technique (especially LNPs) enables the delivery of collagen mRNA, instead of the direct delivery of collagen, into the body, thereby promoting the production of collagen by human cells. Such a method not only maintains the natural spatial structure and function of the protein, resulting in high protein activity, but also minimizes side effects due to the homologous nature of the produced substance, and further realizes long-term endogenous collagen supplementation.

Description

Polynucleotide compositions for improving hair condition Technical Field

[0001] The present invention comprises one or more mRNAs encoding nucleotide sequences of collagen (e.g., one or more human collagen proteins), compositions comprising the above mRNAs, or methods of using the present invention; as well as articles thereof, kits, or uses thereof. Background Technology

[0002] Collagen is a major structural protein in the extracellular matrix (ECM) of animals, accounting for 25% to 30% of the total protein in the human body. It is widely found in skin, tissues, bones, intercellular matrix, ligaments, joints, and hair; the cornea is almost entirely composed of collagen. Collagen has a stable structure and unique biological properties such as hemostasis, intercellular interactions, low immunogenicity, biocompatibility, and biodegradability, and has been widely used in the biopharmaceutical, cosmetic, and food industries.

[0003] Traditional methods for extracting collagen mainly involve hot water extraction, acid-base hydrolysis, and enzymatic hydrolysis from terrestrial animal connective tissues and aquatic processing byproducts. However, the separation and purification processes for animal-derived collagen are complex, and monomer separation is difficult. Furthermore, it may carry viruses, posing safety risks and limiting its application and development to some extent.

[0004] Therefore, there is an urgent need for an effective way to provide collagen to meet the needs of various application fields.

[0005] Compared to traditional direct collagen supplementation, supplementing the body's collagen using in vivo mRNA delivery technology (especially LNP) has several advantages. This technology delivers collagen mRNA, rather than collagen itself, directly into the body, promoting the body's own collagen production. This preserves the protein's natural spatial structure and function, resulting in high protein activity. Furthermore, because it is a homologous substance, it has fewer side effects and allows for long-term endogenous collagen supplementation.

[0006] definition

[0007] To facilitate understanding of this invention, certain terms are defined below. Further definitions of the following terms and other terms are set forth throughout the specification.

[0008] As used herein, the term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. Typically, mRNA consists of ribonucleotides. mRNA may contain one or more coding and noncoding regions. mRNA may be purified from natural sources, or generated using recombinant expression systems and optionally purified, chemically synthesized, etc. Unless otherwise stated, mRNA sequences in this document are presented in a 5' to 3' orientation.

[0009] An open reading frame (ORF) is a continuous segment of DNA or RNA that encodes a protein or polypeptide. Typically, an ORF contains a translation start signal or start codon such as ATG or AUG, and a stop codon.

[0010] The untranslated region (UTR) refers to the non-translated nucleic acid at the 5' end (5'-UTR) and / or 3' end (3'-UTR) of the open reading frame, meaning it will be transcribed but not translated into an amino acid sequence. The 5'-UTR begins at the transcription start site (+1 position) and continues to the start codon (but does not include the start codon). Typically, the 5'-UTR often has characteristics like the Kozak sequence. The 3'-UTR begins immediately after the stop codon and continues until the transcription termination signal. Exemplary 3'- and 5'-UTRs include α- and β-globin, albumin, HSD17B4, and eukaryotic elongation factor 1α. Additionally, viral 5' and 3'-UTRs can also be used, including orthopoxvirus and cytomegalovirus UTR sequences.

[0011] The poly-A tail refers to polyadenine nucleotides, which are usually located at the 3' end of polynucleotides (e.g., mRNA) and can increase the stability of polynucleotide molecules.

[0012] A 5' cap structure refers to the structure formed by modifying the 5' end of eukaryotic mRNA. In some embodiments, a suitable cap is 7-methylguanosine monophosphate (“m7G”), which is linked to the 5' end of the first nucleotide via a triphosphate bridge, resulting in m7G(5')ppp(5')N, where N refers to the first transcribed nucleotide. Depending on the degree of methylation, three types of caps can be formed: CAP O, CAP I, and CAP II. Guanosine is linked to the 5' end of mRNA by a 5'-5 pyrophosphate bond. When the 7th nitrogen atom in guanosine is methylated to form m7G(5')ppp(5')N, the cap is called “CAP O”. If the 2'-O position of the first nucleotide of the mRNA is also methylated, forming m7GpppNm (also represented as m7G(5')ppp(5')(2-OMeN)pN), it is called “CAP I”. If the 2'-O positions of the first and second nucleotides of the mRNA are both methylated, becoming m7G-pppNmNm (also represented as m7G(5')ppp(5')(2'-OMeN)p(2'-OMeN)), it is called "CAP II". For example, a cap on mRNA produced by in vitro transcription is m7G(5')ppp(5')G, which has been used as a dinucleotide cap in transcription using T7 or SP6 RNA polymerase in vitro to obtain mRNA with a cap structure at its 5' end. Alternatively, methods for synthesizing capped mRNA in vitro can use a pre-formed dinucleotide form m7G(5')ppp(5')G ("m7GpppG") as a transcription initiator. A pre-formed trinucleotide form m7G(5')ppp(5')(2'-OMeA)pG can also be used as a transcription initiator. Other cap analogues can be found, for example, Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs with superior translational properties”, RNA, 9:1108-1122 (2003).

[0013] As used herein, "chemically modified nucleosides" include modifications introduced onto the nucleoside that differ from those chemical substances found in naturally occurring mRNA. For example, chemically modified nucleosides include, but are not limited to, any one or more of pseudouridine, N1-methyl-pseuuridine, 2-thiouridine, 4-thiouridine, 2-thio-1-methyl-1-diaza-pseuuridine, 2-thio-1-methyl-pseuuridine, 2-thio-5-azauridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-pseuuridine, 5-azauridine, dihydropseuuridine, 5-methoxyuridine, 5-methyluridine, and 2-methoxyuridine. Preferred chemically modified nucleosides are pseudouridine, which can enhance mRNA stability and translational ability, as well as reduce immunogenicity in vivo. See, for example, Molecular Therapy 16(11): 1833-1840 (2008). The methods and techniques for modifying nucleotides are well known to those skilled in the art.

[0014] Sequence identity refers to the overall correlation between polymer molecules, such as the overall correlation between oligonucleotide molecules (e.g., DNA and / or RNA molecules) and / or polypeptide molecules. The percentage of identity between two polynucleotide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and identical sequences can be ignored for non-comparison purposes). The molecules are identical at that position when a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of shared identical positions, taking into account the number of gaps and the length of each gap, which need to be introduced to achieve optimal alignment of the two sequences. Sequence comparison and determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. Non-limiting examples of algorithms suitable for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc Acids Res., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol., 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). Another example is global alignment algorithms for determining the percentage of sequence identity, such as the Needleman-Wunsch algorithm for aligning protein or nucleotide (e.g., RNA) sequences.

[0015] The nucleotide sequence in this application can represent either a DNA sequence or an RNA sequence. When it represents an RNA sequence, the "T" represents uridine. Summary of the Invention

[0016] The first aspect of this application provides the use of isolated mRNA in the preparation of a medicament for the prevention and / or treatment of diseases related to collagen loss, or the use of isolated mRNA for the prevention and / or treatment of diseases related to collagen loss, wherein the diseases related to collagen loss are selected from hair loss, gray hair, hair follicle aging, and hair follicle atrophy.

[0017] The mRNA encodes COL17A1 and / or COL3A1, the amino acid sequence of the COL17A1 collagen is shown in SEQ ID NO: 1; the amino acid sequence of the COL3A1 collagen is shown in SEQ ID NO: 2.

[0018] In a preferred embodiment, the mRNA encodes COL17A1.

[0019] The second aspect of this application provides the use of a composition in the preparation of a medicament for the prevention and / or treatment of diseases related to collagen loss, or the use in the prevention and / or treatment of diseases related to collagen loss, wherein the diseases related to collagen loss are selected from hair loss, gray hair, hair follicle aging, and hair follicle atrophy.

[0020] The composition comprises (i) mRNA encoding COL17A1; and / or ii) mRNA encoding COL3A1; the amino acid sequence of the COL17A1 collagen is shown in SEQ ID NO: 1; and the amino acid sequence of the COL3A1 collagen is shown in SEQ ID NO: 2.

[0021] In a preferred embodiment, the composition comprises mRNA encoding COL17A1.

[0022] In a preferred embodiment, the composition comprises mRNA encoding COL17A1 and mRNA encoding COL3A1.

[0023] A third aspect of this application provides the use of isolated mRNA in the preparation of products for improving hair condition, or in improving hair condition, wherein improving hair condition refers to improving hair loss, gray hair, hair follicle aging, and hair follicle atrophy; wherein the mRNA encodes COL17A1 and / or COL3A1, wherein the amino acid sequence of COL17A1 collagen is shown in SEQ ID NO: 1; and the amino acid sequence of COL3A1 collagen is shown in SEQ ID NO: 2.

[0024] In a preferred embodiment, the improvement of hair condition is for non-therapeutic purposes.

[0025] In a preferred embodiment, the product is not a drug. In some preferred embodiments, the product is a medical device.

[0026] In some preferred embodiments, the product is a cosmetic.

[0027] In a preferred embodiment, the mRNA encodes COL17A1.

[0028] The fourth aspect of this application provides the use of a composition in the preparation of a product for improving hair condition, or the use of improving hair condition, wherein improving hair condition refers to improving hair loss, gray hair, hair follicle aging, and hair follicle atrophy.

[0029] The composition comprises (i) mRNA encoding COL17A1; and / or ii) mRNA encoding COL3A1; the amino acid sequence of the COL17A1 collagen is shown in SEQ ID NO: 1; and the amino acid sequence of the COL3A1 collagen is shown in SEQ ID NO: 2.

[0030] In a preferred embodiment, the improvement of hair condition is for non-therapeutic purposes.

[0031] In some preferred embodiments, the product is not a drug.

[0032] In some preferred embodiments, the product is a medical device.

[0033] In some preferred embodiments, the product is a cosmetic.

[0034] In a preferred embodiment, the composition comprises mRNA encoding COL17A1.

[0035] In a preferred embodiment, the composition comprises mRNA encoding COL17A1 and mRNA encoding COL3A1.

[0036] In any of the first, second, third, and fourth aspects of the present invention,

[0037] In some implementations, the collagen deficiency-related diseases are selected from one or more of the following: hair loss, gray hair, hair follicle aging, and hair follicle atrophy.

[0038] In some implementations, the hair loss and graying are hair loss and graying caused by COL17A1 deficiency.

[0039] In some implementations, the hair loss and graying are caused by the loss of hair follicle stem cells.

[0040] In some embodiments, the hair loss is selected from one or more of the following: seborrheic alopecia, neurogenic alopecia, endocrine-related alopecia, nutritional alopecia, physical alopecia, chemical alopecia, infectious alopecia, symptomatic alopecia, congenital alopecia, seasonal alopecia, alopecia areata, senile alopecia, male pattern baldness, diffuse alopecia, female pattern baldness, drug-induced alopecia, and scarring alopecia.

[0041] In some implementations, the gray hair is selected from one or more of the following: congenital generalized gray hair, congenital localized gray hair, senile gray hair, adolescent gray hair, premature gray hair, drug-induced gray hair, and neurogenic gray hair.

[0042] In some embodiments, the mRNA encoding COL17A1 comprises a nucleotide sequence as shown in any one of SEQ ID NO: 3-5, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 3-5.

[0043] In some embodiments, the mRNA encoding COL3A1 comprises a nucleotide sequence as shown in any one of SEQ ID NO: 6-9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 6-9.

[0044] In some embodiments, the mRNA further comprises a 5'-UTR, a 3'UTR, and / or a polyA tail.

[0045] In some embodiments, the mRNA contains nucleotide sequences such as those shown in any of SEQ ID NO: 3-9, operatively linked to a 5'-UTR, a 3'-UTR, and / or a polyA tail.

[0046] In some preferred embodiments, the 5'-UTR comprises a nucleotide sequence as shown in SEQ ID NO: 10, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 10.

[0047] In some preferred embodiments, the 3'-UTR comprises a nucleotide sequence as shown in SEQ ID NO: 11, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 11.

[0048] In some preferred embodiments, the polyA tail comprises a nucleotide sequence as shown in SEQ ID NO:12, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:12.

[0049] In a more preferred embodiment, the mRNA encoding COL17A1 comprises or is a nucleotide sequence as shown in any one of SEQ ID NO: 13-15, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 13-15.

[0050] In a more preferred embodiment, the mRNA encoding COL3A1 comprises a nucleotide sequence as shown in any one of SEQ ID NO: 16-19, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 16-19.

[0051] In some embodiments, the mRNA described in this application comprises at least one chemically modified nucleoside.

[0052] In some embodiments, the chemically modified nucleoside is selected from chemically modified uridine. In some preferred embodiments, the chemically modified uridine is selected from any one or more of pseudouridine, N1-methyl-pseudouridine, 2-thiouridine, 4-thiouridine, 2-thio-1-methyl-1-diaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 5-methyluridine, and 2-methoxy-uridine. More preferably, it is pseudouridine or N1-methylpseudouridine.

[0053] In some preferred embodiments, a portion of the uridine in the mRNA is chemically modified uridine, preferably pseudouridine or N1-methyl-pseudouridine. In other preferred embodiments, all the uridine in the mRNA is chemically modified uridine, preferably pseudouridine or N1-methyl-pseudouridine.

[0054] In some embodiments, the mRNA further comprises a 5' cap structure. The 5' cap of the mRNA is selected from any one of m7G(5')ppp(5')(2'-OMeA)pG, m7(3'OMeG)(5')ppp(5')m6(2'OMeA)pG, m7(3'AcmG)(5')ppp(5')(2'OMeA)pG and m7G(5')vppp(5')(2'OMeA)pG, preferably m7G(5')ppp(5')(2'-OMeA)pG.

[0055] In some preferred embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeN)pN, wherein the N is a natural or modified nucleoside. In some more preferred embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

[0056] In any one of the first, second, third, and fourth aspects of the present invention, the mRNA is encapsulated by a delivery vector.

[0057] In some embodiments, the delivery carrier is selected from liposomes, liposome nanoparticles (LNPs), sol-gels, and nanogels.

[0058] In some preferred embodiments, the delivery carrier is a lipid nanoparticle (LNP).

[0059] In some preferred embodiments, the lipid nanoparticles comprise ionizable lipids, neutral lipids, structural lipids, and PEG lipids.

[0060] In some embodiments, the ionizable lipid is selected from, but not limited to, one or more of, the following: 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-( 3-Dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinyl-4-N-methylpepiazino-[1,3]-dioxane Cyclopentane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-di... Linoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleothio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), and 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP).Cl), 1,2-dioleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dioleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dioleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylamino Propane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1 2-Dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-carboxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-formylamino)ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), bis(octadecylaminoglycyl)spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA) (3-β-oxy)-3'-oxaproloxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), MC3, SM-102, ALC-0315. Preferably, the ionizable lipid is selected from SM-102 or ALC-0315.

[0061] Preferably, the ionizable lipid is selected from compound 5 and its stereoisomers.

[0062] In some embodiments, the neutral lipid is a phospholipid. Preferably, the phospholipid is selected from, but is not limited to, one or more of, the following: dilauroyl lecithin (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DUPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycerol-3-phosphate choline (18:0Diether PC), 1-oleoyl-2-cholestyldimethylsuccinate-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-divinyl-sn-glycerol-3-phosphate choline, 1,2-diarylyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarylyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt (DOPG) or sphingomyelin.

[0063] In some preferred embodiments, the neutral lipid is DSPC. In other preferred embodiments, the neutral lipid is DOPE. In still other preferred embodiments, the neutral lipid is both DSPC and DOPE.

[0064] In some embodiments, the structured lipid is selected from, but not limited to, cholesterol, coprosterol, sitosterol, ergosterol, stigmasterol, corticosteroids, or combinations thereof. In some preferred embodiments, the structured lipid is cholesterol. In other embodiments, the structured lipid is cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

[0065] In some embodiments, the PEG lipid is a lipid modified with polyethylene glycol (PEG).

[0066] In some embodiments, the PEG lipid is selected from, but not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some preferred embodiments, the PEG lipid is selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. In some more preferred embodiments, the PEG lipid is DMG-PEG2000.

[0067] In some preferred embodiments, the LNP comprises ionizable lipids, neutral lipids, structural lipids, and PEG lipids. Preferably, the molar ratio of the ionizable lipids, neutral lipids, structural lipids, and PEG lipids is (20-60):(5-25):(25-55):(0.5-5). In some more preferred embodiments, the molar ratio of the ionizable lipids, neutral lipids, structural lipids, and PEG lipids is (40-55):(10-15):(35-45):(0.5-2.5).

[0068] In some preferred embodiments, the LNP comprises ionizable lipids, phospholipids, cholesterol, and PEG lipids. Preferably, the molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG lipids is (20–60):(5–25):(25–55):(0.5–5). In some more preferred embodiments, the molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG lipids is (40–55):(10–15):(35–45):(0.5–2.5).

[0069] In some embodiments of this application, the content of ionizable lipids in the lipid nanoparticles is 35 mol%-65 mol%, for example, it can be 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, etc. 42.5mol%, 43mol%, 43.5mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5m ol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7mol%, 48.8mol%, 48. 9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mol%, 49.9mol%, 50mol%, 50.5mol%, 51mol%, 51.5mol%, 52mol%, 52.5mol%, 53mol%, 53.5mol%, 54mol%, 54.5m ol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59.5mol%, 6 0 mol%, 60.5 mol%, 61 mol%, 61.5 mol%, 62 mol%, 62.5 mol%, 63 mol%, 63.5 mol%, 64 mol%, 64.5 mol%, 65 mol%.

[0070] In some embodiments of this application, the total content of the phospholipids and the structural lipids in the lipid nanoparticles can be 35 mol%-65 mol%, for example, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 ... 2mol%, 42.5mol%, 43mol%, 43.5mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5mol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7mol%, 48.8mol% , 48.9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mo l%, 49.9mol%, 50mol%, 50.5mol%, 51mol%, 51.5mol%, 52mol%, 52.5mol%, 53mol%, 53.5mol%, 54mol%, 54. 5mol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59.5mol%, 60mol%, 60.5mol%, 61mol%, 61.5mol%, 62mol%, 62.5mol%, 63mol%, 63.5mol%, 64mol%, 64.5mol%, 65mol%.

[0071] In some embodiments of this application, the phospholipid content in the lipid nanoparticles can be 5 mol%-15 mol%, for example, 5 mol%, 5.5 mol%, 6 mol%, 6.5 mol%, 7 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 11.5 mol%, 12 mol%, 12.5 mol%, 13 mol%, 13.5 mol%, 14 mol%, 14.5 mol%, or 15 mol%; for example, it can be 10 mol%-15 mol%.

[0072] In some embodiments of this application, the content of the structural lipids in the lipid nanoparticles can be 30 mol%-50 mol%, for example, 30 mol%, 30.5 mol%, 31 mol%, 31.5 mol%, 32 mol%, 32.5 mol%, 33 mol%, 33.5 mol%, 34 mol%, 34.5 mol%, 35 mol%, 35.5 mol%, 36 mol%, 36.5 mol%, 37 mol%, 37.5 mol%, 38 mol%, 38.5 mol%, 39 mol%, 39.5 mol%, 40 mol%, 40.5 mol%, 41 mol%, 41.5 mol%, 42 mol%, 42.5 mol%, 43 mol%, 43.5 mol%. mol%, 44mol%, 44.5mol%, 45mol%, 45.5mol%, 46mol%, 46.5mol%, 47mol%, 47.5mol%, 48mol%, 48.1mol%, 48.2mol%, 48.3mol%, 48.4mol%, 48.5mol%, 48.6mol%, 48.7m ol%, 48.8mol%, 48.9mol%, 49mol%, 49.1mol%, 49.2mol%, 49.3mol%, 49.4mol%, 49.5mol%, 49.6mol%, 49.7mol%, 49.8mol%, 49.9mol%, 50mol%; for example, it can be 35mol%-45mol%.

[0073] In some embodiments of this application, the content of the PEG lipid in the lipid nanoparticles is 0.5 mol%-5 mol%, for example, it can be 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, etc. l%, 2.5mol%, 2.6mol%, 2.7mol%, 2.8mol%, 2.9mol%, 3mol%, 3.1mol%, 3.2mol%, 3.3mol%, 3.4mol%, 3.5mol%, 3.6mol%, 3.7mo 1%, 3.8mol%, 3.9mol%, 4mol%, 4.1mol%, 4.2mol%, 4.3mol%, 4.4mol%, 4.5mol%, 4.6mol%, 4.7mol%, 4.8mol%, 4.9mol%, 5mol%.

[0074] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 35 mol%-65 mol%, the combined content of phospholipids and cholesterol is 35 mol%-65 mol%, and the content of PEG lipids is 0.5 mol%-5 mol%.

[0075] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the component ratio is: 50 mol% ionizable lipids, 48.5 mol% phospholipids and cholesterol, and 1.5 mol% PEG lipids.

[0076] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids. The content of ionizable lipids in the lipid nanoparticles is 40 mol%-50 mol%, the content of phospholipids is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipids is 1.5 mol%-2.5 mol%.

[0077] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids. The content of ionizable lipids in the lipid nanoparticles is 48 mol%-49 mol%, the content of phospholipids is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of PEG lipids is 1.5 mol%-2.5 mol%.

[0078] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 42.5 mol%, and the content of PEG lipids is 2.5 mol%.

[0079] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0080] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 48.5%, the content of phospholipids is 5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 1.5 mol%.

[0081] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 44.2 mol%, the content of phospholipids is 9.3 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 1.5 mol%.

[0082] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 50 mol%, the content of phospholipids is 5 mol%, the content of cholesterol is 43 mol%, and the content of PEG lipids is 2 mol%.

[0083] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 40 mol%, the content of phospholipids is 12.5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 2.5 mol%.

[0084] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 46 mol%, the content of phospholipids is 11 mol%, the content of cholesterol is 41 mol%, and the content of PEG lipids is 2 mol%.

[0085] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 47.5 mol%, the content of phospholipids is 5 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 2.5 mol%.

[0086] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 40 mol%, the content of phospholipids is 13 mol%, the content of cholesterol is 45 mol%, and the content of PEG lipids is 2 mol%.

[0087] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 50 mol%, the content of phospholipids is 12.5 mol%, the content of cholesterol is 35 mol%, and the content of PEG lipids is 2.5 mol%.

[0088] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 50 mol%, the content of phospholipids is 8 mol%, the content of cholesterol is 39.5 mol%, and the content of PEG lipids is 2.5 mol%.

[0089] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 42.3 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 40.3 mol%, and the content of PEG lipids is 2.5 mol%.

[0090] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 46 mol%, the content of phospholipids is 11 mol%, the content of cholesterol is 41 mol%, and the content of PEG lipids is 2 mol%.

[0091] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 47.5 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 35 mol%, and the content of PEG lipids is 2.5 mol%.

[0092] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 48.5 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 35 mol%, and the content of PEG lipids is 1.5 mol%.

[0093] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids is 50 mol%, the content of phospholipids is 5 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0094] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 40 mol%, the content of phospholipids is 15 mol%, the content of cholesterol is 43.5 mol%, and the content of PEG lipids is 1.5 mol%.

[0095] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the content of ionizable lipids in the lipid nanoparticles is 48 mol%, the content of phospholipids is 10.3 mol%, the content of cholesterol is 39.6 mol%, and the content of PEG lipids is 2.1 mol%.

[0096] In one specific embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, cholesterol, and PEG lipids, wherein the component ratio in the lipid nanoparticles is: 50 mol% ionizable lipids, 10 mol% phospholipids, 38.5 mol% cholesterol, and 1.5 mol% PEG lipids.

[0097] In some embodiments of this application, the molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids in the lipid nanoparticles is 35-65:35-65:0.5-5.

[0098] In the lipid nanoparticles of this application, the molar ratio of the sum of the ionizable lipids, the phospholipids, and the structural lipids, and the PEG lipids is (35-65):(35-65):(0.5-5), where 35-65 can take any value between 35 and 65, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39. .5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 5 6.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 0.5-5 can take any value between 0.5 and 5, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.

[0099] In one specific embodiment, the molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids in the lipid nanoparticles is (40-50):(35-65):(1-3).

[0100] In one specific embodiment, the molar ratio of the sum of the ionizable lipids, the phospholipids and the structural lipids, and the PEG lipids in the lipid nanoparticles is (50-65):(35-65):(1-3).

[0101] In one specific embodiment, the molar ratio of the sum of the ionizable lipids, the phospholipids, and the structural lipids, and the PEG lipids is not 50:48.5:1.5.

[0102] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (40-65):(5-15):(30-50):(1-3).

[0103] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (40-65):(10-15):(35-45):(1.5-2.5).

[0104] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (40-50):(5-15):(30-50):(1-3). In the molar ratio (40-50):(5-15):(30-50):(1-3), 40-50 can take any value between 40 and 50, for example, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 5-15 can take any value between 5 and 15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 30-50 can take any value between 30 and 50, for example, The possible values ​​are 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, and 46. 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 1-3 can be any value between 1 and 3, for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.

[0105] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (40-50):(5-15):(35-45):(1.5-2.5). For the molar ratio (40-50):(5-15):(35-45):(1.5-2.5), 40-50 can take any value between 40 and 50, such as 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, and 50; 5-15 can take any value between 5 and 15, such as 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, and 11. 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 35-45 can be any value between 35 and 45, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45; 1.5-2.5 can be any value between 1.5 and 2.5, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5.

[0106] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (50-65):(5-15):(30-50):(1-3). Wherein, for the molar ratio (50-65):(5-15):(30-50):(1-3), 50-65 can take any value between 50 and 65, for example, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60. 5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 5-15 can be any value between 5 and 15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 30-50 can... Choose any value between 30 and 50, for example: 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50; 1-3 can be any value between 1 and 3, for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.

[0107] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is not 50:10:38.5:1.5.

[0108] In one specific embodiment, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid in the lipid nanoparticles is (50-65):(5-15):(35-45):(1.5-2.5). For the molar ratio (50-65):(5-15):(35-45):(1.5-2.5), 50-65 can take any value between 50 and 65, for example, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65; 5-15 can take any value between 5 and 15, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15; 35-45 can be any value between 35 and 45, for example, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45; 1.5-2.5 can be any value between 1.5 and 2.5, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5.

[0109] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, the sum of DSPC and cholesterol, and DMG-PEG2000 is (35-65):(35-65):(0.5-5).

[0110] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(5-15):(30-50):(1-3).

[0111] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(10-15):(35-45):(1.5-2.5).

[0112] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(5-15):(30-50):(1-3).

[0113] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(10-15):(35-45):(1.5-2.5).

[0114] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5 is 35 mol%-65 mol%, the sum of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0115] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5 is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0116] In one specific embodiment, the lipid nanoparticles comprise compound 5, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5 is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0117] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, and cholesterol, and DMG-PEG2000 is (35-65):(35-65):(0.5-5).

[0118] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(5-15):(30-50):(1-3).

[0119] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (40-49):(10-15):(35-45):(1.5-2.5).

[0120] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(5-15):(30-50):(1-3).

[0121] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of compound 5A, DSPC, cholesterol, and DMG-PEG2000 is (51-60):(10-15):(35-45):(1.5-2.5).

[0122] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5A in the lipid nanoparticles is 35 mol%-65 mol%, the combined content of DSPC and cholesterol is 35 mol%-65 mol%, and the content of DMG-PEG2000 is 0.5 mol%-5 mol%.

[0123] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5A is 40 mol%-50 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0124] In one specific embodiment, the lipid nanoparticles comprise compound 5A, DSPC, cholesterol, and DMG-PEG2000, wherein the content of compound 5A is 48 mol%-49 mol%, the content of DSPC is 10 mol%-15 mol%, the content of cholesterol is 35 mol%-45 mol%, and the content of DMG-PEG2000 is 1.5 mol%-2.5 mol%.

[0125] In some embodiments, the molar ratio of the ionizable lipid to the polynucleotide in the polynucleotide complex is 4 to 6:1, preferably 5.2:1.

[0126] In some implementations, an LNP molecule may encapsulate multiple different mRNAs. In other implementations, an LNP molecule encapsulates only one type of mRNA.

[0127] In any one of the first, second, third, or fourth aspects of the present invention, the drug or product is formulated to be administered via transdermal, subcutaneous, and / or intradermal or superficial injection.

[0128] Optionally, the dosage form of the drug or product includes, but is not limited to, gel, emulsion, cream, ointment, injectable dosage form, microneedle, etc., preferably an injectable solution. Attached Figure Description

[0129] Figure 1 shows the expression level of COL17A1 protein in cells after transfection with mRNAs encoding different sequences of collagen.

[0130] Figure 2 shows HE staining of mouse skin tissue after PBS intervention.

[0131] Figure 3 shows the HE staining of mouse skin tissue after hyaluronic acid intervention.

[0132] Figure 4 shows the HE staining of mouse skin tissue after intervention with mRNA encoding COL17A1 protein.

[0133] Figure 5 shows a comparison of hair growth in mice after treatment with different intervention methods. Detailed Implementation

[0134] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0135] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the nucleotide sequences described in the instructions are written from the 5' to the 3' end, and the amino acid sequences are written from the amino terminus to the carboxyl terminus. In case of discrepancies between the sequences in the instructions and the sequence listing, the sequences described in the instructions shall prevail.

[0136] Example 1: Design of DNA sequence encoding collagen and preparation of mRNA

[0137] 1.1 Synthesis of nucleic acid sequence encoding collagen and construction of recombinant vector

[0138] DNA fragments containing an ORF sequence encoding collagen were designed for transcription to obtain the corresponding mRNA. In this application, seven DNA fragments for mRNA transcription were constructed and prepared. Each fragment, from its 5' to 3' end, sequentially contains: a T7 promoter sequence, a transcription start site sequence (AGG), a 5' UTR sequence (SEQ ID NO: 10), an ORF sequence encoding collagen (any one of SEQ ID NO: 3-9), a 3' UTR sequence (SEQ ID NO: 11), a sequence encoding a polyA tail (SEQ ID NO: 12), and a SapI / BspQ1 restriction site sequence (CGAAGAGC).

[0139] Using Genewiz Biotechnology Co., Ltd.'s gene synthesis service, the above DNA fragments were inserted between the XbaI and XhoI restriction sites on the pUC57-GW-Kan vector backbone to obtain plasmids containing the coding sequences of each collagen protein.

[0140] The collagen used in this application embodiment is various subtypes of collagen, and their sequences are shown in SEQ ID NO: 1-2 (hereinafter referred to as COL17A1 and COL3A1, respectively). The ORF sequences encoding the above collagen are as follows:

[0141] COL17A1: SEQ ID NO: 3-5; and

[0142] COL3A1: SEQ ID NO: 6-9.

[0143] Exemplarily, the encoding DNA sequence for translating the mRNA to produce COL17A1 in this application embodiment is shown in any one of SEQ ID NO: 13-15 (from the transcription start site sequence AGG to the poly A tail). For the encoding DNA sequence for translating the mRNA to produce other collagen subtypes, its structure and composition are the same as any one of SEQ ID NO: 13-15, except that the ORF sequence is replaced by any one of SEQ ID NO: 6-9.

[0144] 1.2 mRNA preparation

[0145] 1.2.1 Plasmid linearization

[0146] The recombinant plasmids prepared in Example 1.1 were linearized by digestion with the restriction endonuclease SapⅠ at 37°C for 3 hours. The reaction system is shown in Table 1.

[0147] Table 1. Plasmid linearization enzyme digestion system

[0148] After the reaction was completed, 2 μL of the enzyme digestion product was subjected to 1% agarose gel electrophoresis to detect the linearization of the plasmids. Subsequently, each linearized recombinant plasmid was purified using a PCR product recovery kit (Comway Century).

[0149] 1.2.2 In vitro transcription and purification of mRNA products

[0150] Using the linearized recombinant plasmid obtained in Example 1.2.1 as a template, in vitro transcription was performed using a High Yield T7 RNA Synthesis Kit (Shanghai Zhaowei Technology Development Co., Ltd., product catalog number ON-040) according to the instructions, with the reaction time being 37°C for 3 hours. The transcription system is shown in Table 2.

[0151] Table 2. In vitro transcription system

[0152] Among them, ΨUTP (100mM) is pseudouridine triphosphate, also represented as N1-Me-pUTP.

[0153] CleanCapAG is m7G(5')ppp(5')(2'-OMeA)pG, used as the transcription initiation primer.

[0154] After transcription, 1 μL of DNase I was added, and the mixture was incubated at 37°C for 15 min. Then, 15 μL of ammonium acetate stop solution was added and mixed well. Next, 1 / 3 volume of 7.5 M Lithium Chloride (LiCl) precipitation solution was added (to a final concentration of 2.5 M), and the mixture was incubated at -20°C for 30 min. The mixture was centrifuged at 12000 g for 15 min, and the RNA precipitate was discarded. 1 mL of 70% ethanol was added to wash the RNA, and the mixture was centrifuged at 12000 g for 5 min, discarding the supernatant. After drying, 50 μL of RNase-free water was added to dissolve the precipitate, and mRNA quantification was performed using a UV spectrophotometer to obtain 5'-capped mRNAs encoding various collagen subtypes.

[0155] Example 2: Study on the in vitro expression effect of mRNA encoding collagen

[0156] Wild-type and optimized mRNAs encoding COL17A1 were selected for in vitro expression evaluation. The specific procedure is as follows:

[0157] (1) 293T cells were seeded in 6-well plates, with 5 x 10⁵ cells per well. After 24 hours, when the cell mixture reached approximately 70%-80%, transfection was performed.

[0158] (2) Mix 2 μg mRNA (plasmids were constructed according to the method in Section 1.1, where the ORF sequences of wild-type group, optimized group 1 and optimized group 2 were SEQ ID NO:3-5, and mRNA was prepared according to Section 1.3) with 4 μl of transfection reagent Lipofectamine™ 3000 and let stand at room temperature for 15 min.

[0159] (3) Add mRNA and Lipofectamine™ 3000 to 293T cells;

[0160] (4) Cell lysis was performed 24 hours after transfection: the culture medium was removed, the cells were washed once with pre-cooled PBS, and 200 μl of cell lysis buffer was added to each well.

[0161] (5) Western Blot: Based on the protein concentration determination results, 5 μg of each sample was loaded and separated on a 4-12% gradient gel. After electrophoresis, the protein samples were transferred to a 0.22 μm nitrocellulose membrane by wet transfer, and then co-incubated with COL17A1 specific antibody (primary antibody) and fluorescent secondary antibody in sequence before detection. Imaging was performed on the Bio-Rad gel imaging system ChemiDoc™ MP Imaging System.

[0162] A multi-parameter optimization design approach was employed to improve sequence stability and gene expression efficiency by optimizing key parameters such as Codon usage bias, GC content, and mRNA secondary structure. Furthermore, the optimization process incorporated customized requirements for specific patterns, including optimizing Shine-Dalgarno or Kozak sequences to enhance gene expression efficiency, and modifying RNA instability motifs to regulate expression efficiency. Simultaneously, sequence stability was further ensured by eliminating or inserting restriction enzyme sites and reducing repetitive sequences (direct repeats, inverted repeats, and dimer repeats). The aim was to obtain an optimized nucleic acid sequence that balances RNA stability and expression efficiency through this comprehensive optimization strategy.

[0163] As shown in Figure 1, the expression level of optimized group 1 (corresponding ORF is SEQ ID NO:4) is more than 1.5 times that of the wild-type group (corresponding ORF is SEQ ID NO:3), while the expression level of optimized group 2 (corresponding ORF is SEQ ID NO:5) is only about 20% of that of the wild-type group.

[0164] Example 3: In vivo study on the effect of collagen-encoding mRNA on promoting hair follicle hyperplasia

[0165] 3.1 Lipid nanoparticles (LNPs) encapsulating mRNA

[0166] The mRNA obtained in step 1.2.2 was dispersed in 20 mM acetic acid solution and adjusted to a final concentration of 200 μg / mL. The mixture was then prepared according to the molar ratio of compound 5A (synthesized according to CN117777089A): cholesterol: DSPC: DMG-PEG2000 = 48-49: 35-45: 10-15: 1.5-2.5 to form a lipid mixture. The flow rates of the aqueous and oil phases were controlled by T-junction to mix the mRNA solution with the lipid mixture. The syringe pump was started to further mix the mRNA solution and lipid mixture to form an LNP solution. The solution was then diluted with diluent, concentrated by centrifugation through an ultrafiltration tube, and the solution was replaced. The pH of the obtained solution was adjusted to 7.0–8.0 with PBS to obtain the LNP-loaded mRNA solution. The solution was then filtered through a 0.22 μm PES filter. The encapsulation efficiency and particle size of lipid nanoparticles were determined using the Ribogreen RNA quantification kit (Invitrogen, R11490) and the Darwin ZetaSizer particle size analyzer.

[0167] Compound 5A

[0168] Table 3. Characterization data of collagen mRNA-LNP

[0169] 3.2 Animal grouping and administration

[0170] Fifteen SPF-grade, female BalB / C mice, weighing 18-20g and 9 weeks old, were randomly divided into three groups (G1, G2, and G3), with five mice in each group. Hair was removed from the back of the mice, and four injection sites were selected for intradermal administration of the drug. Each injection site received 50 μl of the drug on days 0, 4, 7, 14, and 21. Skin tissue was harvested on day 28 for HE staining to assess hair follicle growth.

[0171] Table 4. Grouping and Dosing Information

[0172] The following explanations are provided regarding the groups of drugs in Table 4:

[0173] Group G1: Commercially available PBS solution;

[0174] Group G2: Weigh sodium hyaluronate and lidocaine hydrochloride and add them to a 20 mmol / L phosphate and 0.6% sodium chloride solution to make the sodium hyaluronate concentration 5 mg / ml and the lidocaine hydrochloride concentration 3 mg / ml. Filter the solution through a 0.22 μPES filter and the pH of the solution is 7.0-8.0.

[0175] Group G3: The mRNA solution obtained in step 3.1 of Example 3 was diluted to 2 μg / ml with 5 mg / ml sodium hyaluronate and 3 mg / ml lidocaine hydrochloride phosphate solution.

[0176] 3.3 Evaluation of the anti-hair loss effect of mRNA encoding collagen

[0177] Digital images were acquired by scanning tissue sections to observe histopathological structural changes. HE-stained images of 10X fields were randomly selected. The results are shown in Figures 2-4. The results indicate that mice treated with the mRNA product of this invention (Figure 4) showed a significant increase in hair follicle cell growth, while mice treated with PBS (Figure 2) or the excipient hyaluronic acid (Figure 3) showed almost no growth promotion in their hair follicle cells. Promoting hair follicle cell growth is key to the prevention and treatment of hair loss; therefore, the mRNA product of this invention has a significant effect on the prevention and treatment of hair loss.

[0178] Example 4: In vivo study of the effects of collagen-encoding mRNA on hair growth

[0179] To further investigate the effect of collagen-encoding mRNA on promoting hair growth in vivo, a mouse model of hair loss was constructed to examine the specific effects. The specific procedures are as follows:

[0180] 4.1 Building the Model

[0181] C57BL / 6J mice were used, and the hair on the back of a 3cm × 2cm area was removed using depilatory cream. A male-dependent alopecia model was established daily by subcutaneous injection of 50mg / kg of testosterone propionate solution (with testosterone dissolved in 50% ethanol) into the shaved area on the back. The testosterone propionate concentration was 10mg / ml, and the injection volume was 100ul. The medication was administered 1 hour after injection.

[0182] 4.2 Grouping and Processing

[0183] G1-model group: 1 hour after modeling, 3 model mice were injected intradermally with physiological saline on their backs.

[0184] G2-Minoxidil treatment group: 1 hour after modeling, 0.2 ml of 5% minoxidil solution was applied to the back of 5 model mice once a day.

[0185] G3-mRNA low-dose group: 1 hour after modeling, 5 model mice were injected intradermally with the drug on days 1, 8, 15 and 22. The drug concentration based on mRNA was 2 μg / mL and the injection volume was 250 μL each time.

[0186] G4-mRNA high-dose group: 1 hour after modeling, 5 model mice were injected intradermally with the drug on days 1, 8, 15 and 22. The drug concentration was 4 μg / mL based on mRNA, and the injection volume was 250 μL each time.

[0187] G5-negative control group: 0.1 ml of physiological saline was applied to the backs of 5 normal mice once a day.

[0188] In groups G3 and G4, LNP-loaded mRNA was used for drug administration, and the preparation was carried out in the same manner as in Example 3.

[0189] Section 3.1.

[0190] Hair growth was observed in all groups of mice.

[0191] 4.3 Test Results

[0192] As shown in Figure 5, compared with the model group, the minoxidil treatment group, the low-dose mRNA group, and the high-dose group can all effectively promote hair growth in mice. In particular, the high-dose mRNA group has a hair growth effect that is basically equivalent to that of the minoxidil treatment group.

[0193] The sequences used in the above embodiments of this application are shown in the sequence listing. It should be understood that these sequences are merely exemplary sequences for the embodiments of this application and are not intended to limit the scope of this application. Although DNA sequences are shown in the electronic listing, the nucleotide sequences in the sequence listing of this application may represent either DNA or RNA sequences, and when they represent RNA sequences, "T" represents uridine.

Claims

1. Use of an isolated mRNA in the preparation of a product for improving hair condition, or use in improving hair condition, wherein improving hair condition refers to improving hair loss, gray hair, hair follicle aging, and hair follicle atrophy; the product is not a drug; The mRNA encodes COL17A1 and / or COL3A1. The amino acid sequence of the COL17A1 collagen is shown in SEQ ID NO: 1; the amino acid sequence of the COL3A1 collagen is shown in SEQ ID NO:

2.

2. Use of a composition in the preparation of a product for improving hair condition, or use for improving hair condition, wherein improving hair condition refers to improving hair loss, gray hair, hair follicle aging, and hair follicle atrophy; The composition comprises (i) mRNA encoding COL17A1; and / or ii) mRNA encoding COL3A1; the amino acid sequence of the COL17A1 collagen is shown in SEQ ID NO: 1; the amino acid sequence of the COL3A1 collagen is shown in SEQ ID NO: 2; the product is not a drug.

3. The product is a medical device product according to any one of claims 1-2.

4. The product is a cosmetic product as described in any one of claims 1-2.

5. The use according to any one of claims 1-4, The mRNA encoding COL17A1 comprises a nucleotide sequence as shown in any one of SEQ ID NO: 3-5, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with a nucleotide sequence shown in any one of SEQ ID NO: 3-5; or The mRNA encoding COL3A1 comprises a nucleotide sequence as shown in any one of SEQ ID NO: 6-9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 6-9.

6. The use according to any one of claims 1-5, wherein the mRNA molecule further comprises an operatively linked 5'-UTR, 3'UTR and / or polyA tail.

7. The use according to claim 8, wherein, The 5'-UTR contains a nucleotide sequence as shown in SEQ ID NO: 10, or contains a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 10; The 3'-UTR contains a nucleotide sequence as shown in SEQ ID NO: 11, or contains a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 11; The polyA tail comprises a nucleotide sequence as shown in SEQ ID NO:12, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:

12.

8. The use according to any one of claims 1-7, wherein the mRNA encoding COL17A1 comprises or is a nucleotide sequence as shown in any one of SEQ ID NO: 13-15, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with a nucleotide sequence shown in any one of SEQ ID NO: 13-15; The mRNA encoding COL3A1 comprises or is a nucleotide sequence as shown in any of SEQ ID NO: 16-19, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with the nucleotide sequence shown in any of SEQ ID NO: 16-19.

9. The use according to any one of claims 1-8, wherein the mRNA comprises at least one chemically modified nucleoside; Optionally, a portion of the uridine in the mRNA is a chemically modified uridine, preferably a pseudouridine or an N1-methyl-pseudouridine; Optionally, all uridines in the mRNA are chemically modified uridines, preferably pseudouridines or N1-methyl-pseudouridines; Optionally, the chemically modified uridine is selected from any one or more of pseudouridine, N1-methyl-pseudouridine, 2-thiouridine, 4-thiouridine, 2-thio-1-methyl-1-diaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-azauridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 5-methyluridine, and 2-methoxyuridine. Preferably, the chemically modified uridine is pseudouridine or N1-methyl-pseudouridine.

10. The use according to any one of claims 1-9, wherein the mRNA further comprises a 5' cap structure; Preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeN)pN, wherein, The N is a natural or modified nucleoside; More preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

11. The use according to any one of claims 1-10, wherein the mRNA is encapsulated by a delivery vector, preferably, the delivery vector is selected from liposomes, liposome nanoparticles (LNPs), sol-gels, and nanogels.

12. The use according to claim 11, wherein the delivery carrier is a liposome nanoparticle (LNP).

13. The use according to claim 12, wherein the lipid nanoparticles comprise ionizable lipids, neutral lipids, structural lipids, and PEG lipids.

14. The use according to claim 13, wherein the neutral lipid is a phospholipid, preferably, the neutral lipid is DSPC and / or DOPE.

15. The use according to claim 13, wherein the structural lipid is selected from cholesterol, coccosterol, phytosterol, ergosterol, stigmasterol, corticosteroids or combinations thereof, preferably, the structural lipid is cholesterol.

16. The use according to claim 13, wherein the PEG lipid is selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid, preferably, the PEG lipid is DMG-PEG2000.

17. In the use according to any one of claims 13-16, the molar ratio of the ionizable lipid, the neutral lipid, the structural lipid, and the PEG lipid is (20-60):(5-25):(25-55):(0.5-5), preferably (40-55):(8-15):(35-45):(0.5-2.5).

18. The use according to any one of claims 1-17, wherein the product is formulated to be administered via transdermal, subcutaneous and / or intradermal or superficial injection. Optionally, the product includes, but is not limited to, gels, lotions, creams, ointments, injectable formulations, microneedles, etc., and is preferably an injectable solution.

19. The use according to any one of claims 1-18, wherein the hair loss is selected from one or more of seborrheic alopecia, neurogenic alopecia, endocrine-related alopecia, nutritional alopecia, physical alopecia, chemical alopecia, infectious alopecia, symptomatic alopecia, congenital alopecia, seasonal alopecia, alopecia areata, senile alopecia, male pattern baldness, diffuse alopecia, female pattern baldness, drug-induced alopecia, and scarring alopecia.

20. The use according to any one of claims 1-19, wherein the gray hair is selected from one or more of congenital generalized gray hair, congenital localized gray hair, senile gray hair, adolescent gray hair, premature gray hair, drug-induced gray hair, and neurogenic gray hair.

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