Nucleic acid molecule encoding collagen and use thereof

Abstract

Provided are an mRNA comprising a nucleotide sequence encoding collagen, and a composition comprising the mRNA and a delivery vector. An mRNA molecule is introduced into a subject by means of the composition, which can be used for improving the skin condition.

Description

Nucleic acid molecules encoding collagen and their uses Technical Field

[0001] This disclosure relates in part to mRNAs comprising one or more nucleotide sequences encoding collagen (e.g., one or more human collagen proteins) and compositions comprising the mRNAs thereof or methods of using thereof; and articles thereof or kits thereof. Background Technology

[0002] Like all organs in the human body, skin undergoes continuous and often cumulative changes over time. Skin aging occurs due to numerous factors, including inherent changes within the skin, the effects of gravity and the action of facial muscles on the skin, loss or displacement of soft tissue, and loss of tissue elasticity. Of concern, the “aging” phenotype of the skin can be accelerated by environmental factors, most notably long-term exposure to ultraviolet radiation (e.g., from the sun). Clinically, the aging phenotype of the skin can be described as wrinkling, sagging, and / or, compared to its younger counterpart, typically exhibiting less elasticity and resilience; however, the changes within this phenotype exist between natural chronological aging and photoaging.

[0003] Numerous skincare products have been developed to improve the appearance of human skin. Wrinkles and skin folds are typically treated with cosmetic facial fillers injected into the dermis and subdermis. Dermal atrophy caused by irreversible collagen loss is a significant characteristic of skin aging. There remains a need in this field to improve skin condition (e.g., to improve skin aging) by replenishing the skin with collagen. Summary of the Invention

[0004] Compared to traditional methods of directly filling collagen through in vitro purification, using in vivo mRNA delivery technology (especially LNP) to replenish collagen in the skin has several advantages. This technology delivers collagen mRNA, rather than collagen itself, directly into the skin, promoting the skin cells' own production of collagen lost due to aging. This preserves the protein's natural spatial structure and function, resulting in high protein activity. Because it is a homologous substance, it has fewer side effects and allows for long-term endogenous collagen replenishment to maintain skin strength, firmness, and elasticity, leading to more natural skin tissue regeneration.

[0005] definition

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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-reve rse’ cap analogs with superior translational properties”, RNA, 9:1108-1122 (2003).

[0012] As used herein, “chemically modified nucleosides” include modifications to nucleosides 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.

[0013] 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). 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.

[0014] 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.

[0015] The first aspect of this application provides an isolated mRNA.

[0016] The mRNA molecule encodes an amino acid sequence represented by any one of SEQ ID NO: 1-5.

[0017] The mRNA molecule comprises a nucleotide sequence as shown in any one of SEQ ID NO: 6-20, 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: 6-20.

[0018] In some embodiments, the mRNA molecule further comprises a 5'-UTR, a 3'-UTR, and / or a polyA tail.

[0019] In some embodiments, the mRNA further comprises a 5'-UTR, a 3'-UTR, and / or a polyA tail operably linked to a nucleotide sequence as shown in any of SEQ ID NO: 6-20. In some preferred embodiments, the 5'-UTR comprises a nucleotide sequence as shown in SEQ ID NO: 21, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO: 21.

[0020] In some other preferred embodiments, the 3'-UTR comprises a nucleotide sequence as shown in SEQ ID NO:22, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:22.

[0021] In some other preferred embodiments, the polyA tail comprises a nucleotide sequence as shown in SEQ ID NO:23, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:23.

[0022] In some embodiments, the mRNA of the present invention comprises the following elements from the 5' end to the 3' end: (1) a 5' cap; (2) a 5'-UTR region; (3) a collagen coding region; (4) a 3'-UTR region; and (5) a polyA tail, the sequence of which is shown in SEQ ID NO: 23.

[0023] In some preferred embodiments, the collagen is selected from the amino acid sequences shown in any one of SEQ ID NO: 1-5. More preferably, the collagen is COL17A1 collagen or selected from the amino acid sequence shown in SEQ ID NO: 1.

[0024] In some preferred embodiments, the collagen coding region is selected from the nucleotide sequence shown in any one of SEQ ID NO: 6-20. More preferably, the collagen coding region is selected from the nucleotide sequence shown in SEQ ID NO: 8.

[0025] In some preferred embodiments of this application, the mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 24-26, 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: 24-26.

[0026] In some embodiments, the mRNA described in this application comprises at least one chemically modified nucleoside.

[0027] 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-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-methoxy-uridine. More preferably, it is pseudouridine or N1-methyl-pseudouridine.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Furthermore, the first aspect of this application also provides a combination of mRNAs, the combination comprising one or more of the aforementioned mRNAs.

[0032] In some implementations, the combination of mRNAs includes (i) a first mRNA; and (ii) a second mRNA, a third mRNA, a fourth mRNA, and / or a fifth mRNA.

[0033] In some preferred embodiments, the combination of mRNAs comprises (i) a first mRNA; and (ii) a fourth mRNA.

[0034] In some embodiments, the first mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 6-10, 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: 6-10.

[0035] In some embodiments, the second mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 11-12, 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: 11-12.

[0036] In some embodiments, the third mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 13-14, 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: 13-14.

[0037] In some embodiments, the fourth mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 15-18, 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: 15-18.

[0038] In some embodiments, the fifth mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 19-20, 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: 19-20.

[0039] In some embodiments, the mRNA further comprises a 5'-UTR, a 3'-UTR, and / or a polyA tail operably linked to a nucleotide sequence as shown in any of SEQ ID NO: 6-20.

[0040] In some preferred embodiments, the 5'-UTR comprises a nucleotide sequence as shown in SEQ ID NO:21, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:21.

[0041] In some other preferred embodiments, the 3'-UTR comprises a nucleotide sequence as shown in SEQ ID NO:22, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:22.

[0042] In some other preferred embodiments, the polyA tail comprises a nucleotide sequence as shown in SEQ ID NO:23, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:23.

[0043] In some preferred embodiments of this application, the mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 24-26, 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: 24-26.

[0044] In some embodiments, the mRNA described in this application comprises at least one chemically modified nucleoside.

[0045] 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-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-methoxy-uridine. More preferably, it is pseudouridine or N1-methyl-pseudouridine.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] A second aspect of this application provides a composition comprising mRNA and lipid nanoparticles (LNPs), wherein the mRNA contains a nucleotide sequence encoding collagen, and the amino acid sequence of the collagen is shown in any one of SEQ ID NO: 1-5. Specifically, the mRNA is encapsulated by lipid nanoparticles (LNPs).

[0050] In some preferred embodiments, the mRNA comprises the mRNA described in the first aspect of this application.

[0051] This application also provides a composition comprising mRNA and a delivery vector, wherein the mRNA comprises the mRNA described in the first aspect of this application.

[0052] This application also provides a composition comprising mRNA and a delivery vector, wherein the mRNA comprises one or more of the mRNAs described in the first aspect of this application.

[0053] In some embodiments, the composition comprises (i) a first mRNA; and (ii) a second mRNA, a third mRNA, a fourth mRNA, and / or a fifth mRNA.

[0054] In some preferred embodiments, the composition comprises (i) a first mRNA; and (ii) a fourth mRNA.

[0055] In some embodiments, the first mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 6-10, 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: 6-10.

[0056] In some embodiments, the second mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 11-12, 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: 11-12.

[0057] In some embodiments, the third mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 13-14, 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: 13-14.

[0058] In some embodiments, the fourth mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 15-18, 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: 15-18.

[0059] In some embodiments, the fifth mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 19-20, 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: 19-20.

[0060] In some embodiments, the mRNA further comprises a 5'-UTR, a 3'-UTR, and / or a polyA tail operably linked to a nucleotide sequence as shown in any of SEQ ID NO: 6-20.

[0061] In some preferred embodiments, the 5'-UTR comprises a nucleotide sequence as shown in SEQ ID NO:21, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:21.

[0062] In some other preferred embodiments, the 3'-UTR comprises a nucleotide sequence as shown in SEQ ID NO:22, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:22.

[0063] In some other preferred embodiments, the polyA tail comprises a nucleotide sequence as shown in SEQ ID NO:23, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:23.

[0064] In some preferred embodiments of this application, the mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 24-26, 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: 24-26.

[0065] In some embodiments, the mRNA described in this application comprises at least one chemically modified nucleoside.

[0066] 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-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-methoxy-uridine. More preferably, it is pseudouridine or N1-methyl-pseudouridine.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In some embodiments, the delivery carrier is selected from liposomes, liposome nanoparticles (LNPs), sol-gels, and nanogels.

[0071] In some preferred embodiments, the delivery carrier is a lipid nanoparticle (LNP) comprising ionizable lipids, neutral lipids, structural lipids, and PEG lipids.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] In some embodiments, the PEG lipid is a lipid modified with polyethylene glycol (PEG).

[0077] 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.

[0078] 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).

[0079] 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).

[0080] In some implementations, an LNP molecule may encapsulate multiple different mRNAs. In other implementations, an LNP molecule encapsulates only one type of mRNA.

[0081] A third aspect of this application provides a DNA that encodes the mRNA described in the first aspect of this application.

[0082] In some embodiments, the DNA comprises a nucleotide sequence shown in any one of SEQ ID NO: 6-20, 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: 6-20.

[0083] In some embodiments, this application also provides a recombinant vector comprising the DNA described above. The term "recombinant vector" as used herein refers to a vector for introducing heterologous nucleic acids into cells to achieve their expression, transcription, or replication. Recombinant vectors include vectors capable of transcribing and expressing nucleic acids operatively linked to regulatory sequences such as promoter regions, which enable the transcription and expression of said nucleic acids. Therefore, a recombinant vector can refer to a DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, that results in the transcription and expression of nucleic acids upon introduction into a suitable host cell. Suitable recombinant vectors are well known to those skilled in the art and include those capable of replicating in eukaryotic cells, as well as those that remain free or are integrated into the host cell genome.

[0084] In some embodiments, the recombinant vector includes a viral vector and a non-viral vector. In other embodiments, the viral vector includes a baculovirus vector, an adeno-associated virus vector, an adenovirus vector, or a lentiviral vector, and the non-viral vector includes a plasmid.

[0085] The fourth aspect of this application provides an isolated cell comprising DNA or a recombinant vector as described in the third aspect of this application.

[0086] In some embodiments, the cells are selected from prokaryotic cells or eukaryotic cells.

[0087] In some embodiments of this application, the eukaryotic cells are selected from any of insect cells, mammalian cells, avian cells, and yeast cells, preferably mammalian cells, and more preferably human cells. In other preferred embodiments of this application, the prokaryotic cells are selected from bacteria, preferably Escherichia coli.

[0088] The fifth aspect of this application provides a cosmetic composition or pharmaceutical composition comprising mRNA as described in the first aspect of this application, or a composition as described in the second aspect of this application, or DNA or a recombinant vector as described in the third aspect of this application.

[0089] In some embodiments of this application, the pharmaceutical composition is formulated to be administered via transdermal, subcutaneous, and / or intradermal or superficial injection.

[0090] The sixth aspect of this application provides:

[0091] (1) A method of supplementing collagen in a subject in need, comprising administering to the subject mRNA as described in the first aspect of this application, a composition as described in the second aspect of this application, DNA or recombinant vector as described in the third aspect of this application, or a cosmetic or pharmaceutical composition as described in the fifth aspect of this application.

[0092] (2) A method of improving skin aging in a subject in need, comprising administering to the subject mRNA as described in the first aspect of this application, a composition as described in the second aspect of this application, DNA or recombinant vector as described in the third aspect of this application, or a cosmetic or pharmaceutical composition as described in the fifth aspect of this application.

[0093] (3) A method for increasing and / or improving at least one of the texture, smoothness, elasticity or tension of the skin of a subject in need, said method comprising administering to the subject mRNA as described in the first aspect of this application, a composition as described in the second aspect of this application, DNA or a vector as described in the third aspect of this application, or a cosmetic or pharmaceutical composition as described in the fifth aspect of this application.

[0094] (4) A method for reducing the occurrence of one or more superficial pits in the skin in a subject in need, the method comprising administering to the subject an mRNA as described in the first aspect of this application, a composition as described in the second aspect of this application, a DNA or recombinant vector as described in the third aspect of this application, or a cosmetic or pharmaceutical composition as described in the fifth aspect of this application.

[0095] In some embodiments of this application, skin aging is selected from skin aging caused by increasing age or skin aging caused by ultraviolet light or sunlight exposure.

[0096] In some embodiments of this application, one or more superficial depressions in the skin are selected from the group consisting of: nasolabial folds, crow's feet, frown lines, worry lines, scars, frown lines, drooping eyebrows, tear troughs, nasolabial folds, bunny lines, drooping cheeks / midfaces, marionette lines, poppy dimpling, smile lines, laugh lines, chin wrinkles, neck wrinkles, platysma bands, and any combination thereof.

[0097] In some implementations, the increase and / or improvement in skin texture, smoothness, elasticity, or tension is selected from: (a) treatment, reduction, and / or prevention of fine lines and / or wrinkles compared to before application; (b) reduction in skin pore size; (c) improvement in skin thickness, fullness, and / or firmness; (d) improvement in skin smoothness, suppleness, and / or softness; (e) improvement in skin color, radiance, and / or translucency; (f) improvement in procollagen and / or collagen production; and (g) improvement and / or retexturization of skin texture. (h) Improved appearance of skin contour; (i) Restoration of skin luster and / or brightness; (j) Improvement of skin appearance diminished due to aging and / or menopause; (k) Improved skin hydration; (l) Increased skin elasticity and / or resilience; (m) Treatment, reduction and / or prevention of skin sagging; (n) Improved skin firmness; (o) Reduction of pigmentation spots, freckled skin and / or scars; (p) Improvement of skin optical properties in terms of light diffraction or reflection; or (q) Any combination thereof.

[0098] In addition, this application also provides a method for promoting scar healing, tissue repair or promoting hair follicle repair in subjects in need, comprising administering to the subject mRNA as described in the first aspect of this application, or a composition as described in the second aspect of this application, DNA or recombinant vector as described in the third aspect of this application, or a cosmetic or pharmaceutical composition as described in the fifth aspect of this application.

[0099] This application also provides a method for treating a disease related to collagen loss in a subject in need, comprising administering to the subject mRNA as described in the first aspect of this application, or a composition as described in the second aspect of this application, DNA or a recombinant vector as described in the third aspect of this application, or a pharmaceutical composition as described in the fifth aspect of this application.

[0100] In some embodiments of this application, the subject is a human being.

[0101] In some embodiments of this application, the administration is performed percutaneously, subcutaneously, and / or intradermally to the subject or via superficial injection.

[0102] The seventh aspect of this application provides:

[0103] (1) Use of mRNA as described in the first aspect of this application, composition as described in the second aspect of this application, DNA or recombinant vector as described in the third aspect of this application, or cosmetic or pharmaceutical composition as described in the fifth aspect of this application in the preparation of a collagen-supplementing formulation.

[0104] (2) Use of mRNA as described in the first aspect of this application, composition as described in the second aspect of this application, DNA or recombinant vector as described in the third aspect of this application, or cosmetic or pharmaceutical composition as described in the fifth aspect of this application in the preparation of an agent to improve skin aging.

[0105] (3) Use of mRNA as described in the first aspect of this application, compositions as described in the second aspect of this application, DNA or recombinant vectors as described in the third aspect of this application, or cosmetic or pharmaceutical compositions as described in the fifth aspect of this application in the preparation of formulations that increase and / or improve the texture, smoothness, elasticity and / or tension of the skin.

[0106] (4) Use of the mRNA as described in the first aspect of this application, the composition as described in the second aspect of this application, the DNA or recombinant vector as described in the third aspect of this application, or the cosmetic or pharmaceutical composition as described in the fifth aspect of this application in the preparation of an agent that reduces the appearance of one or more superficial pits in the skin.

[0107] In addition, this application also provides the use of mRNA as described in the first aspect of this application, compositions as described in the second aspect of this application, DNA or recombinant vectors as described in the third aspect of this application, or cosmetic or pharmaceutical compositions as described in the fifth aspect of this application in the preparation of formulations that promote scar healing, tissue repair, or hair follicle repair.

[0108] This application also provides the use of mRNA as described in the first aspect of this application, compositions as described in the second aspect of this application, DNA or recombinant vectors as described in the third aspect of this application, or pharmaceutical compositions as described in the fifth aspect of this application in the preparation of formulations for the prevention and / or treatment of diseases related to collagen loss.

[0109] In some implementations, skin aging is selected from skin aging caused by increasing age or skin aging caused by ultraviolet light or sunlight exposure.

[0110] In some implementations, one or more superficial depressions in the skin are selected from the group consisting of: nasolabial folds, crow's feet, frown lines, worry lines, scars, frown lines, drooping eyebrows, tear troughs, nasolabial folds, bunny lines, drooping cheeks / midfaces, marionette lines, poppy dimpling, smile lines, laugh lines, chin wrinkles, neck wrinkles, platysma bands, and any combination thereof.

[0111] In some implementations, the increase and / or improvement of skin texture, smoothness, elasticity, and / or tension is selected from: (a) treatment, reduction, and / or prevention of fine lines and / or wrinkles; (b) reduction of skin pore size; (c) improvement of skin thickness, fullness, and / or firmness; (d) improvement of skin smoothness, suppleness, and / or softness; (e) improvement of skin color, radiance, and / or translucency; (f) improvement of procollagen and / or collagen production; and (g) improvement of skin texture and / or promotion of retexturation. (h) Improved appearance of skin contour; (i) Restoration of skin luster and / or brightness; (j) Improvement of skin appearance diminished due to aging and / or menopause; (k) Improved skin hydration; (l) Increased skin elasticity and / or resilience; (m) Treatment, reduction and / or prevention of skin sagging; (n) Improved skin firmness; (o) Reduction of pigmentation spots, freckled skin and / or scars; (p) Improvement of the skin's optical properties in terms of light diffraction or reflection; or (q) Any combination thereof.

[0112] In some embodiments, the dosage form of the product or drug includes, but is not limited to, gel, emulsion, cream, ointment, injectable dosage form, microneedle, etc., preferably an injectable solution.

[0113] In some implementations, the dosage form is a transdermal dosage form, a subcutaneous dosage form, and / or an intradermal dosage form.

[0114] In some implementations, the product or drug is administered via superficial injection. Attached Figure Description

[0115] Figure 1 shows the comparison of PolyA integrity results for plasmids containing the COL17A1 coding sequence when using two different PolyA sequences.

[0116] Figure 2 shows the HE and Masson staining results of a mouse model of skin photoaging.

[0117] Figure 3 shows the effect of mRNA encoding collagen on dermal collagen fiber content in a mouse model of skin photoaging.

[0118] Figure 4 shows the effect of mRNA encoding collagen on pathological scores in a mouse model of skin photoaging.

[0119] Figure 5 shows the expression level of COL17A1 protein in cells after transfection with mRNAs encoding different sequences of collagen.

[0120] Figure 6 shows the effect of mRNA encoding collagen on dermal collagen fiber content in a mouse model of skin photoaging.

[0121] Figure 7 shows the effect of mRNA encoding collagen on dermal thickness in a mouse model of skin photoaging. Specific Implementation

[0122] 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.

[0123] 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. All animal experiments in this application were performed by Beijing Langke Biotechnology Co., Ltd. Unless otherwise specified, the nucleotide sequences described in the specification 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 sequence in the specification and the sequence listing, the sequence described in the specification shall prevail.

[0124] Example 1: Design of DNA sequence encoding collagen and preparation of mRNA

[0125] 1.1 Synthesis of collagen sequences and construction of recombinant vectors

[0126] A DNA fragment containing an ORF sequence encoding collagen was designed and synthesized for transcription to obtain the corresponding mRNA. In this application, a DNA fragment for mRNA transcription was constructed and prepared, wherein the fragment, from its 5' to 3' end, sequentially comprises: an XbaI restriction site sequence (TCTAGA), a T7 promoter sequence, a transcription start site sequence (AGG), a 5' UTR sequence (SEQ ID NO: 21), an ORF sequence encoding collagen (any one of SEQ ID NO: 6-20), a 3' UTR sequence (SEQ ID NO: 22), a sequence encoding a polyA tail (SEQ ID NO: 23), a SapI restriction site sequence (CGAAGAGC), and a NotI restriction site sequence (GCGGCCGC).

[0127] The collagen used in the embodiments of this application are various subtypes of collagen, and their sequences are shown in SEQ ID NO: 1-5 (hereinafter referred to as COL17A1, COL1A1, COL1A2, COL3A1, and COL6A1, respectively). The ORF sequences encoding the above collagen are as follows:

[0128] COL17A1: SEQ ID NO: 6 - 10;

[0129] COL1A1: SEQ ID NO: 11 - 12;

[0130] COL1A2: SEQ ID NO: 13 - 14;

[0131] COL3A1: SEQ ID NO: 15 - 18; and

[0132] COL6A1: SEQ ID NO: 19 - 20.

[0133] Exemplarily, the full - length sequence of the mRNA used to translate COL17A1 in the embodiments of the present application is shown as any one of SEQ ID NO: 24 - 26 (starting from the transcription start site sequence AGG to the polyA tail). For the full - length sequence of the mRNA used to translate other subtypes of collagen, its structure and composition are the same as any one of SEQ ID NO: 24 - 26, except that the ORF sequence therein is replaced with any one of SEQ ID NO: 9 - 20.

[0134] The above DNA fragments were digested with XbaI and NotI double enzymes, and ligated with the pUC57 - GW - Kan (Genewiz) vector backbone fragment digested with XbaI and NotI to construct respective recombinant plasmids containing the coding sequences of each subtype of collagen.

[0135] 1.2 Screening of the polyA element of the mRNA encoding collagen COL17A1

[0136] During the previous research, the inventors found that using the known 120A tail (120 consecutive A's) in the collagen - encoding mRNA would lead to the defect of base deletion during DNA plasmid replication, resulting in instability. Therefore, the inventors developed a polyA element, RG2 (SEQ ID NO: 23), which can greatly improve the replication stability of DNA plasmids.

[0137] For example, the inventors used DNA plasmids containing ORF sequences (SEQ ID NO: 8), 5'UTR sequences (SEQ ID NO: 21), and 3'UTR sequences (SEQ ID NO: 22) as the basis for studying the effect of different poly(A) variants on plasmid stability. After the constructed vector plasmid was confirmed to be correct by sequencing, it was transformed into *E. coli* DH5α. The transformed plates were grown at 30°C, and plasmid extraction and sequencing were completed. After sequencing, the stability and base deletion of different poly(A) variants were analyzed and calculated based on the sequencing results. Replication stability was expressed as the percentage of clones that did not undergo any base changes; a higher percentage indicated higher replication stability of the plasmid in *E. coli*. Finally, the chi-square test was used for data statistics. The results showed that the replication stability of COL17A1-RG2 was significantly higher than that of COL17A1-A120 (89.0% vs. 70.8%, p < 0.01, χ² test).

[0138] Table 1. Evaluation of the replication stability of different polyA values ​​for collagen-encoding DNA plasmids.

[0139] 1.3 mRNA preparation

[0140] 1.3.1 Plasmid linearization

[0141] 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 2.

[0142] Table 2. Plasmid linearization enzyme digestion system

[0143] 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).

[0144] 1.3.2 In vitro transcription and purification of mRNA products

[0145] Using the linearized recombinant plasmid obtained in Example 1.3.1 as a template, in vitro transcription was performed using the High Yield T7 RNA Synthesis Kit (Shanghai Zhaowei Technology Development Co., Ltd., product catalog number ON-040) according to the instructions, and the reaction was carried out at 37°C for 3 hours. The transcription system is shown in Table 3.

[0146] Table 3. In vitro transcription system

[0147] Among them, ΨUTP (100mM) is pseudouridine triphosphate, also known as N1-Me-pUTP (Shanghai Zhaowei Technology Development Co., Ltd., product catalog number R5-027).

[0148] CleanCap AG is m7G(5')ppp(5')(2'-OMeA)pG (catalog number ON-134, Shanghai Zhaowei), used as a transcription initiation primer.

[0149] 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 12000g 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 12000g 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.

[0150] 1.4 Lipid nanoparticles (LNPs) encapsulating mRNA

[0151] The mRNAs obtained in step 1.3.2 were dispersed separately in 20 mM acetic acid solution (pH 5.0) and adjusted to a final concentration of 200 μg / mL. They were mixed according to a molar ratio of ionizable lipids:cholesterol:DSPC:DMG-PEG2000 = 40–55:35–45:10–15:0.5–2.5 to form a lipid mixture. The flow rates of the aqueous and oil phases were controlled by T-junction to ensure each mRNA was individually mixed with the lipid mixture. The syringe pump was started to mix the mRNA solution with the lipid mixture to form an LNP solution. Subsequently, the above solution was diluted 10-fold with diluent, concentrated by ultrafiltration centrifugation, and subjected to three solution replacements. The pH of the obtained solution was adjusted to 7.0–8.0 with Tris aqueous solution to obtain the LNP-loaded mRNA solution. In addition, empty LNPs without mRNA were prepared as a control using the same procedure.

[0152] The concentration of mRNA loaded in LNPs and the particle size of LNPs were determined using a Ribogreen RNA quantification kit (Invitrogen, R11490) and a Darwin ZetaSizer particle size analyzer. Characterization data for exemplary LNPs are shown in Table 4.

[0153] Table 4. Characterization data of collagen mRNA-LNP

[0154] Example 2: In vivo effect study of mRNA encoding collagen

[0155] 2.1 Construction of a mouse model of skin photoaging

[0156] The mouse model of skin photoaging was constructed according to the method described in the reference (Yi You et al., Intradermally delivered mRNA encapsulating extracellular vesicles for collagen-replacement therapy. Nat. Biomed. Eng., 2023). Specifically, 10-12 week old female nude mice were subjected to UVB irradiation of their back skin every other day for a total of 8 weeks to establish the mouse model of skin photoaging. Specifically, mice were anesthetized with 1.5% isoflurane, and a UV lamp (Philips; a 311nm UVB lamp) was placed 30 cm away from the back of the mouse for irradiation. The UV irradiation dose was monitored using a UV illuminometer. The specific irradiation doses were: 60 mJ / cm² per irradiation for the first two weeks, 120 mJ / cm² per irradiation for the third week, 180 mJ / cm² per irradiation for the fourth week, and 240 mJ / cm² per irradiation for the fifth to eighth weeks.

[0157] After irradiation, the mouse skin tissue was stained with HE and Masson staining. Based on the staining results, the skin tissue structure and the distribution and morphology of collagen fibers were evaluated and analyzed to verify the successful establishment of the photoaging mouse model. The results are shown in Figure 2. Compared with normal mouse skin that had not undergone UV irradiation, the irradiated mice exhibited typical photoaging tissue structure characteristics: thickened epidermis, smoothed wavy connections between the epidermis and dermis, disappearance of papillae and dermal papillae, and abnormal changes in dermal collagen fibers such as disordered arrangement, uneven distribution, degeneration, and breakage. These results indicate that the photoaging mouse model of skin in this embodiment was successfully established, producing the expected pathological structure, and can be used for in vivo efficacy evaluation.

[0158] 2.2 Evaluation of the anti-aging effect of mRNA encoding collagen

[0159] Twenty mice that successfully underwent modeling in Section 2.1 were randomly divided into four groups of five. Five normal mice that did not undergo modeling were selected as negative controls for the aging model. According to the grouping shown in Table 5, the LNPs prepared in Table 4 of Example 1.4 were injected intradermally on days 0, 4, 7, 14, and 21. On day 28, skin samples were collected from the mice and stained with HE and Masson's stain.

[0160] Table 5. Grouping of mice for anti-aging effect evaluation

[0161] Digital images are acquired by scanning tissue sections to observe changes in histopathological structure.

[0162] Masson staining of skin collagen fiber percentage: 10X fields of view were randomly selected, and collagen fibers were selected using ImageJ image analysis software. The percentage of collagen fiber area to the total skin tissue area was calculated. Statistical analysis of the significance between data groups was performed using the two-tailed unpaired t-test in GraphPad (*p<0.05, **p<0.01).

[0163] The results are shown in Figure 3. Compared with normal mice, the proportion of collagen fibers in the empty LNP control group was significantly reduced, confirming the successful establishment of the mouse model. Compared with the empty LNP control group, the collagen fiber content in the drug-treated groups COL3A1 and COL6A1 was slightly increased, but the difference was not statistically significant. However, in the COL17A1 group, the dermal collagen fiber content of mice was significantly increased compared with the empty LNP group. These results show that COL17A1 can effectively alleviate collagen fiber loss caused by ultraviolet radiation in vivo, but the allergic effect of COL3A1 and COL6A1 is not significant.

[0164] The pathological scores were based on the degree of epidermal thickening, keratinization, and dermal collagen fiber reduction: 0 points for no lesion, 1 point for mild, 2 points for mild, 3 points for moderate, and 4 points for severe. The average score was taken for each pathological score. Statistical analysis of the differences between data groups was performed using the two-tailed unpaired t-test in GraphPad (*p<0.05, **p<0.01, ***p<0.001).

[0165] The analysis results are shown in Figure 4. The pathological scores of the empty LNP control group were significantly different from those of normal mice, further confirming that this model mouse exhibits obvious photoaging pathological characteristics. Compared with the empty LNP control group, the pathological scores of each treatment group were significantly reduced, indicating that the mRNAs in each group showed a mitigating effect on photoaging pathological tissues in the in vivo disease model. Furthermore, it is noteworthy that among the treatment groups, COL17A1 showed a higher mitigating effect than COL3A1 and COL6A1.

[0166] Example 3: Further optimization of nucleic acid sequences

[0167] Although Example 2 demonstrated the advantages of mRNA encoding COL17A1 in improving skin aging compared to mRNA encoding other types of collagen, its ORF sequence (corresponding to SEQ ID NO:6) presents certain challenges in detection. Therefore, we conducted optimization experiments based on the wild-type nucleic acid sequence (corresponding to SEQ ID NO:9). The specific process is as follows:

[0168] (1) 293T cells were seeded in 6-well plates, with 5 x 10 cells per well. 5 100 cells. Transfection was performed 24 hours later when the cell mixture reached approximately 70%-80%.

[0169] (2) Mix 2 μg mRNA (the plasmid was constructed according to the method in Section 1.1, the difference being the replacement of the corresponding ORF sequence, wherein the ORF sequences of optimized group 1, wild type group and optimized group 2 are SEQ ID NO:8-10 respectively, and the 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.

[0170] (3) Add mRNA and Lipofectamine™ 3000 to 293T cells;

[0171] (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.

[0172] (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.

[0173] 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.

[0174] As shown in Figure 5, the expression level of optimized group 1 (corresponding ORF is SEQ ID NO:8) is more than 1.5 times that of the wild-type group (corresponding ORF is SEQ ID NO:9), while the expression level of optimized group 2 (corresponding ORF is SEQ ID NO:10) is only about 20% of that of the wild-type group.

[0175] Meanwhile, it was verified that the optimized group 1 was easier to detect the sequence compared to the mRNA encoding COL17A1 in Example 2.

[0176] Example 4: In vivo effect study of optimized sequence

[0177] To further investigate the anti-skin aging effects of optimized nucleic acid sequences in vivo, a photoaging mouse model was used for further research.

[0178] 4.1 Lipid nanoparticles encapsulating mRNA

[0179] mRNA and lipid nanoparticle-loaded mRNA were prepared using the methods described in Sections 1.3 and 1.4, respectively. The characterization data of mRNA-LNP are shown in Table 6.

[0180] Table 6 Characterization data of collagen mRNA-LNP

[0181] 4.2 Construction of a mouse model of skin photoaging

[0182] A mouse model of skin photoaging was constructed using the same method as in Section 2.1.

[0183] 4.3 Evaluation of the anti-aging effect of sequence-optimized mRNA

[0184] Ten mice that successfully developed the model as described in Section 4.2 were randomly divided into two groups of five each: a model control group and a COL17A1 group. Five normal mice were used as controls. According to the groupings shown in Table 7, the normal control group received no medication. The model control group and the COL17A1 group received intradermal injections of PBS and LNPs prepared as shown in Table 6 on days 0, 4, 7, 14, and 21, respectively. On day 28, skin samples were collected from the mice and stained with HE and Masson's stain.

[0185] Table 7. Grouping of mice for anti-aging effect evaluation

[0186] Digital images are acquired by scanning tissue sections to observe changes in histopathological structure.

[0187] Masson staining of skin collagen fiber percentage: 10X fields of view were randomly selected, and collagen fibers were selected using ImageJ image analysis software to calculate the percentage of collagen fiber area to the total skin tissue area. Statistical analysis of the significance between data groups was performed using the two-tailed unpaired t-test in GraphPad (*p<0.05, **p<0.01, ***p<0.001).

[0188] The analysis results are shown in Figure 6. Compared with normal mice, the proportion of collagen fibers in the model control group was reduced, confirming the successful establishment of the mouse model. Compared with the model control group, the dermal collagen fiber content of mice in the COL17A1 group was significantly increased. These results show that COL17A1 can effectively alleviate collagen fiber loss caused by ultraviolet radiation in vivo.

[0189] The analysis results are shown in Figure 7. Compared with normal mice, the dermal thickness of the model control group was significantly reduced, further confirming that the model mice have obvious photoaging pathological characteristics. Compared with the model control group, the dermal thickness of the COL17A1 group was significantly increased, indicating that COL17A1-mRNA has a mitigating effect on the decrease in dermal thickness due to photoaging in the in vivo disease model.

[0190] The results above demonstrate that the optimized mRNA sequence encoding COL17A1 can also effectively alleviate the symptoms of photoaging of the skin.

[0191] 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 sequence listing, the nucleotide sequences in the sequence listing of this application may represent either DNA or RNA sequences. When representing an RNA sequence, the "T" represents uridine.

[0192] Sequence description:

Claims

1. An isolated mRNA, wherein, The mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 6-20, 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: 6-20.

2. The mRNA of claim 1, further comprising a 5'-UTR, a 3'-UTR, and / or a poly-A tail operably linked to a nucleotide sequence as shown in any one of SEQ ID NO: 6-20.

3. The mRNA according to claim 2, wherein, The 5'-UTR contains a nucleotide sequence as shown in SEQ ID NO:21, or contains a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:21; The 3'-UTR contains a nucleotide sequence as shown in SEQ ID NO:22, or contains a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:22; The poly-A tail comprises a polynucleotide sequence as shown in SEQ ID NO:23, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:

23.

4. The mRNA according to any one of claims 1-3, wherein, The mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 24-26, 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: 24-26.

5. The mRNA according to any one of claims 1-4, wherein, The mRNA contains at least one chemically modified nucleoside; Optionally, the chemically modified nucleoside is selected from chemically modified uridine; Optionally, some or all of the uridine in the mRNA is chemically modified uridine; 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.

6. The mRNA according to any one of claims 1-5, wherein, The mRNA also contains a 5' cap structure.

7. The mRNA according to claim 6, wherein, The 5' cap structure is m7G(5')ppp(5')(2'-OMeN)pN, where N is a natural or modified nucleoside; Preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

8. A composition comprising one or more mRNAs and lipid nanoparticles (LNPs), said mRNA comprising a nucleotide sequence encoding collagen, preferably, the amino acid sequence of said collagen being as shown in any one of SEQ ID NO: 1-5.

9. The composition of claim 8, wherein the mRNA is selected from the mRNA of any one of claims 1-7.

10. A composition comprising one or more mRNAs and a delivery vector, said mRNA being selected from the mRNAs of any one of claims 1-7.

11. The composition of claim 10, wherein the delivery carrier is selected from liposomes, lipid nanoparticles (LNPs), sol-gels, and nanogels.

12. The composition according to any one of claims 8, 9, and 11, wherein the LNP comprises ionizable lipids, neutral lipids, structured lipids, and PEG lipids. Preferably, the neutral lipid is selected from phospholipids, and the structural lipid is selected from cholesterol; Preferably, the molar ratio of the ionizable lipids, phospholipids, cholesterol, and PEG lipids is (40-55):(10-15):(35-45):(0.5-2.5).

13. The composition according to any one of claims 8-12, wherein the plurality of mRNAs comprises (i) a first mRNA, and (ii) a second mRNA, a third mRNA, a fourth mRNA, and / or a fifth mRNA; preferably, wherein the plurality of mRNAs comprises (i) a first mRNA, and (ii) a fourth mRNA.

14. The composition according to claim 13, wherein: The first mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 6-10, 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: 6-10; The second mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 11-12, 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: 11-12; The third mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 13-14, 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: 12-13; The fourth mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 15-18, 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: 15-18; The fifth mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 19-20, 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: 19-20.

15. An isolated DNA that encodes the mRNA as described in any one of claims 1-7.

16. A recombinant vector comprising the DNA of claim 15. Preferably, the vector is selected from viral vectors or non-viral vectors; More preferably, the viral vector is selected from baculovirus vectors, adeno-associated virus vectors, adenovirus vectors, and lentivirus vectors, and the non-viral vector is a plasmid.

17. An isolated cell comprising the DNA as described in claim 15 or the recombinant vector as described in claim 16; optionally, the cell is selected from prokaryotic cells or eukaryotic cells; Optionally, the eukaryotic cells are selected from any of insect cells, mammalian cells, avian cells, and yeast cells, preferably mammalian cells, and more preferably human cells; Optionally, the prokaryotic cells are selected from bacteria, preferably Escherichia coli.

18. A cosmetic composition or pharmaceutical composition comprising mRNA according to any one of claims 1-7, a composition according to any one of claims 8-14, DNA according to claim 15, or a recombinant vector according to claim 16.

19. A method of supplementing collagen in a subject in need, comprising administering to the subject the mRNA of any one of claims 1-7, the composition of any one of claims 8-14, the DNA of claim 15, the recombinant vector of claim 16, or the cosmetic composition or pharmaceutical composition of claim 18.

20. A method for improving skin aging in a subject in need, comprising administering to the subject the mRNA of any one of claims 1-7, the composition of any one of claims 8-14, the DNA of claim 15, the recombinant vector of claim 16, or the cosmetic composition or pharmaceutical composition of claim 18.

21. A method for increasing and / or improving at least one of the texture, smoothness, elasticity, or tension of the skin of a subject in need, the method comprising administering to the subject the mRNA of any one of claims 1-7, the composition of any one of claims 8-14, the DNA of claim 15, the recombinant vector of claim 16, or the cosmetic or pharmaceutical composition of claim 18.

22. A method for reducing the appearance of one or more superficial pits in the skin in a subject in need, the method comprising administering to the subject the mRNA of any one of claims 1-7, the composition of any one of claims 8-14, the DNA of claim 15, the recombinant vector of claim 16, or the cosmetic composition or pharmaceutical composition of claim 18.

23. The method of claim 20, wherein, The skin aging referred to is skin aging caused by increasing age or skin aging caused by ultraviolet light or sunlight exposure.

24. The method of claim 21, wherein, The increase or improvement in skin texture, smoothness, elasticity and / or tension is selected from: (a) fine lines and / or wrinkles are treated, reduced and / or prevented compared with before application; (b) skin pore size is reduced; (c) Improved skin thickness, fullness, and / or firmness; (d) Improved skin smoothness, suppleness, and / or softness; (e) Improved skin tone, radiance, and / or translucency; (f) Improved procollagen and / or collagen production; (g) Improved skin texture and / or enhanced retexturation; (h) Improved appearance of skin contour. (i) Skin luster and / or radiance are restored; (j) Improvement of skin appearance due to aging and / or menopause; (k) Improvement of skin hydration; (l) Increased skin elasticity and / or resilience; (m) Treatment, reduction and / or prevention of skin sagging; (n) Improvement of skin firmness; (o) Reduction of pigmentation spots, freckled skin and / or scars; (p) Improvements in the skin's optical properties in terms of light diffraction or reflection; or (q) any combination thereof.

25. The method of claim 22, wherein, One or more superficial depressions in the skin are selected from the group consisting of: nasolabial folds, crow's feet, frown lines, worry lines, scars, frown lines, drooping eyebrows, tear troughs, nasolabial folds, bunny lines, drooping cheeks / midface, marionette lines, poppy dimpling, smile lines, laugh lines, chin wrinkles, neck wrinkles, platysma bands, and any combination thereof.

26. A method for promoting scar healing, tissue repair, or hair follicle repair in subjects in need, wherein, The method includes administering to the subject the mRNA of any one of claims 1-7, the composition of any one of claims 8-14, the DNA of claim 15, the recombinant vector of claim 16, or the cosmetic composition or pharmaceutical composition of claim 18.

27. A method for treating a disease associated with collagen loss in subjects of need, wherein, The method comprises administering to the subject the mRNA of any one of claims 1-7, the composition of any one of claims 8-14, the DNA of claim 15, the vector of claim 16, or the pharmaceutical composition of claim 18.

28. The method according to any one of claims 19-27, wherein, The subjects were human.

29. The method according to any one of claims 19-27, wherein, The application method is selected from percutaneous, subcutaneous, intradermal, and / or superficial injection methods.

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