Nucleic acid molecule encoding collagen and use thereof

Abstract

The invention provides an mRNA (messenger Ribonucleic Acid) containing a nucleotide sequence for coding collagen, and a composition containing the mRNA and a delivery carrier. Introduction of the mRNA molecule into a subject by the composition may be used to improve skin conditions.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511216833.6 (22) Application Date 2025.08.28 (66) Domestic Priority Data PCT / CN2024 / 115583 2024.08.29 CN (71) Applicant Renjing (Suzhou) Biotechnology Co., Ltd. Address 215000, Suzhou Industrial Park, Suzhou Area, China (Jiangsu) Pilot Free Trade Zone, Suzhou City, Jiangsu Province, China. Applicant Renjing International (Hong Kong) Limited (72) Inventor Dong Yijie Cen Shan (74) Patent Agency Beijing Jingtian & Gongcheng Law Firm 11770 Patent Attorney Li Yingqi Yang Yingyue (51) Int.Cl. C12N 15 / 12 (2006.01) C12N 15 / 866(2006.01) C12N 15 / 864(2006.01) C12N 15 / 861(2006.01) C12N 15 / 867(2006.01) A61K 8 / 60(2006.01) A61K 8 / 02(2006.01) A61Q 19 / 08(2006.01) A61Q 17 / 04(2006.01) A61Q 19 / 00(2006.01) A61K 48 / 00(2006.01) A61K 9 / 51(2006.01) A61P 17 / 18(2006.01) A61P 17 / 16(2006.01) A61P 17 / 02(2006.01) (54) Invention Title: Nucleic Acid Molecule Encoding Collagen and Its Use Thereof (57) Abstract: This application provides an mRNA comprising a nucleotide sequence encoding collagen, and a composition comprising the above mRNA and a delivery vector. The mRNA molecule is introduced into a subject through the composition and can be used to improve skin condition. Claims: 3 pages Description: 58 pages Sequence Listing (Electronic Publication) Drawings: 3 pages CN 121628912 A 2026.03.10 CN 1 21 62 89 12 A 1. An isolated mRNA, wherein the mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 6-20, or comprises 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: 6-20. 2. The mRNA according to claim 1, further comprising the components shown in any one of SEQ ID NO: 6-20The nucleotide sequences are operatively linked to a 5'-UTR, a 3'-UTR, and / or a poly-A tail. 3. The mRNA according to claim 2, wherein 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; 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; and the poly-A tail comprises a polynucleotide sequence as shown in SEQ ID NO:23, or comprises 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 comprises 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 comprises at least one chemically modified nucleoside; optionally, the chemically modified nucleoside is selected from chemically modified uridine; optionally, part 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 further comprises 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 mRNAs 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 vector 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, structural lipids, and PEG lipids; preferably, the neutral lipids are selected from phospholipids, and the structural lipids are 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 of 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; and the fourth mRNA comprises a nucleotide sequence as shown in SEQ ID NO: 6-10. NO: The nucleotide sequence shown in any one of SEQ ID NO:The nucleotide sequence shown in any one of SEQ ID NO: 15-18 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity; 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 the nucleotide sequence shown in any one of SEQ ID NO: 19-20. 15. An isolated DNA encoding the mRNA as described in any one of claims 1-7. 16. A recombinant vector comprising the DNA as described in 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, 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 cell is selected from any of insect cells, mammalian cells, avian cells, and yeast cells, preferably mammalian cells, more preferably human cells; optionally, the prokaryotic cell is selected from bacteria, preferably *Escherichia coli*. 18. A cosmetic composition or pharmaceutical composition comprising mRNA as described in any one of claims 1-7, a composition as described in any one of claims 8-14, the DNA as described in claim 15, or the recombinant vector as described in claim 16. 19. A method of supplementing collagen in a subject in need, comprising administering to the subject the mRNA as described in any one of claims 1-7, the composition as described in any one of claims 8-14, the DNA as described in claim 15, the recombinant vector as described in claim 16, or the cosmetic composition or pharmaceutical composition as described in 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 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, said 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 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. 23. The method of claim 20, wherein the skin aging is selected from skin aging caused by aging due to increased age or skin aging caused by exposure to ultraviolet light or sunlight. 24. The method of claim 21, wherein the increase 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 compared to before application; (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 transparency; (f) improvement of procollagen and / or collagen production; (g) improvement of skin texture and / or promotion of retexturization; (h) improvement of the appearance of skin contour; (i) restoration of skin luster and / or radiance; (j) improvement of skin appearance diminished due to aging and / or menopause; (k) improvement of skin hydration; (l) increase of 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) Improved skin 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 / midfaces, 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 a subject in 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 recombinant vector of claim 16, or the cosmetic or pharmaceutical composition of claim 18.27. A method of treating a disease associated with collagen loss in a subject in 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 carrier of claim 16, or the pharmaceutical composition of claim 18. 28. The method of any one of claims 19-27, wherein the subject is a human. 29. The method of any one of claims 19-27, wherein the administration is selected from percutaneous, subcutaneous, intradermal, and / or superficial injection methods. Claims 3 / 3 Page 4 CN 121628912 A Nucleic Acid Molecules Encoding Collagen and Their Use 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 above mRNAs or methods of using them; and articles thereof or kits. Background Art

[0002] Like all organs in the human body, the 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, soft tissue loss or displacement, and loss of tissue elasticity. Of concern is that the “aging” phenotype of the skin can be accelerated by environmental factors, most notably prolonged 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, generally exhibiting less elasticity and resilience; however, the changes within this phenotype exist between natural chronological aging and photoaging.

[0003] Numerous skin care products have been developed to improve the appearance of human skin. Wrinkles and skin folds are commonly 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 the art to improve skin condition (e.g., improve skin aging) by replenishing collagen in the skin.

[0004] Compared to traditional methods of directly filling collagen through in vitro purification, using in vivo mRNA delivery technology (especially LNP) to supplement collagen in the skin has many advantages. This technology delivers collagen mRNA, rather than collagen itself, directly to the skin, promoting the skin cells to produce collagen lost due to aging. 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. Simultaneously, it allows for long-term endogenous collagen supplementation to maintain skin strength, firmness, and elasticity, making skin tissue regeneration more natural.

[0005] Definitions

[0006] To facilitate understanding of the invention, certain terms are defined below. Further definitions of the following 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 herein are presented in a 5' to 3' orientation.

[0008] An open reading frame (ORF) refers to a continuous segment of DNA or RNA encoding a protein or polypeptide. Typically, an ORF contains a translation initiation signal or start codon such as ATG or AUG, and a stop codon.

[0009] Untranslated regions (UTRs) refer to untranslated nucleic acids located at the 5' end (5'-UTR) and / or the 3' end (3'-UTR) of an open reading frame, i.e., those that 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, as described on page 1 / 58 of CN 121628912 A. Additionally, viral 5' and 3' UTRs, including orthopoxvirus and cytomegalovirus UTR sequences, can also be used.

[0010] The poly-A tail refers to a polyadenine nucleotide, which is usually located at the 3' end of a polynucleotide (e.g., mRNA) and can increase the stability of the polynucleotide molecule.

[0011] The 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 (“m7G”), which is connected 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 (which can also be represented as m7G(5')ppp(5')(2'-OMeN)pN), it is called "CAP".Type 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 Type II". For example, a cap of 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” includes nucleosides with properties different from those in naturally occurring mRNA. The modifications of the nucleosides seen in the literature include, for example, chemically modified nucleosides including, but not limited to, 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-aza-uridine, 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). 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 molecules 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., nicks can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment and, for non-comparison purposes, can be ignored).(Identical sequences). A molecule is considered identical at a 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 identical positions shared by the sequences, 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 analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). Another example is a global alignment algorithm used to determine the percentage of sequence identity, such as the Needleman-Wunsch algorithm, page 2 / 58 of the specification for aligning protein or nucleotide (e.g., RNA) sequences.

[0014] The nucleotide sequence in this application may represent either a DNA sequence or an RNA sequence, where “T” represents uridine when it represents an RNA sequence.

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

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

[0017] The mRNA molecule 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.

[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 one 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 comprises 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, from the 5' end to the 3' end, the following elements in sequence: (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 sequences 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 more preferred embodiments of this application, the mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO: 24-26, or comprises 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 of 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 comprising one or more of the above-described mRNAs.

[0032] In some embodiments, the combination of mRNAs comprises (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: 11-12.

[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 the 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 the 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 one 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 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 comprises a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with SEQ ID NO:23. Specification 4 / 58 pages 8 CN 121628912 A

[0043] In some more preferred embodiments of this application, the mRNA comprises a nucleotide sequence as shown in any one of SEQ ID NO:24-26, or comprises a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity with any one of SEQ ID NO:24-26.

[0044] In some embodiments, the mRNA of 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 comprises 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 the nucleotide sequence shown in any one of SEQ ID NO: 13-14. Specification 5 / 58 pages 9 CN 121628912 A

[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 the 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 shown in any one 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 operably linked to a nucleotide sequence shown in SEQ ID NO: 6-20.NO:21 has a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity.

[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 comprises 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 comprises 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 of 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 a chemically modified uridine, preferably pseudouridine or N1-methyl-pseudouridine. In some other preferred embodiments, all uridines in the mRNA are chemically modified uridines, preferably pseudouridines or N1-methyl-pseudouridines.

[0068] In some embodiments, the mRNA further comprises a 5' cap structure. The 5' cap of the mRNA is selected from m7G(5') ppp(5')(2'-OMeA)pG, m7(3'OMeG)(5')ppp(5')m6(2'OMeA)pG, m7(3'AcmG)(5')ppp(5')(2'Any one of m7G(5')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 lipid nanoparticles (LNPs), which contain 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-dilinyloxy-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-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl- 5-Dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (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-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleothio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dichlorodimethylaminopropaneLinoleyloxy-3-trimethylaminopropane (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearateyloxy-N,N- Dimethylaminopropane (DSDMA), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearate-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-dioleoyloxy-N-[2-(spermine-formylamino)ethyl]-N,N-dimethyl-1-propanediamine 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-oxy)-3'-oxaproxy)-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 not limited to, one or more of, the following: dilauroyl lecithin (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), and 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 (C16Lyso 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-divinyl-sn- Glycerol-3-phosphate ethanolamine, 1,2-diaryl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-glycerol) 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 other preferred embodiments, the neutral lipid is DSPC and DOPE.

[0075] In some embodiments, the structural lipid is selected from, but not limited to, cholesterol, coprosterol, sitosterol, ergosterol, stigmasterol, corticosteroids, or combinations thereof. In some preferred embodiments, the structural lipid is cholesterol. In other embodiments, the structural lipid is cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisolone, 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, the neutral lipids, the structural lipids, and the PEG lipids is 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 more 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 embodiments, an LNP molecule may encapsulate multiple different mRNAs. In other embodiments, an LNP molecule encapsulates only one type of mRNA.

[0081] A third aspect of this application provides a DNA encoding 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 the 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 causes 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 that can replicate 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 viral vectors and non-viral vectors. In other embodiments, the viral vector includes baculovirus vectors, adeno-associated virus vectors, adenovirus vectors, lentivirus vectors, and the non-viral vector includes plasmids.

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

[0086] In some embodiments, the cell is selected from prokaryotic 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] A 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 for administration via transdermal, subcutaneous, and / or intradermal or superficial injection.

[0090] The sixth aspect of this application provides for:

[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 of 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 mRNA as described in the first aspect of this application, a composition as described in the second aspect of this application, DNA or 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 appearance of one or more superficial depressions in the skin in a subject in need, the 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 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 aging due to increased 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.

[0097] In some embodiments, the increase and / or improvement of skin texture, smoothness, elasticity 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) skin thickness, fullness and / or firmness is improved; (d) skin smoothness, suppleness and / or softness is improved; (e) skin color, luster and / or transparency is improved; (f) procollagen and / or collagen production is improved; (g) skin texture is improved and / or retexturization is promoted; (h) the appearance of skin contour is improved; (i) skin luster and / or... Or, brightness is restored; (j) the appearance of skin that has declined due to aging and / or menopause is improved; (k) skin hydration is improved; (l) skin elasticity and / or resilience is increased; (m) skin sagging is treated, reduced, and / or prevented; (n) skin firmness is improved; (o) pigmentation spots, freckled skin, and / or scars are reduced; (p) the optical properties of the skin in terms of light diffraction or reflection are improved; or (q) any combination thereof.

[0098] Furthermore, this application also provides a method for promoting scar healing, tissue repair, or promoting hair follicle repair in a subject in need, comprising administering to a 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 of treating a disease associated with 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.

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

[0102] The seventh aspect of this application provides:

[0103] (1) mRNA as described in the first aspect of this application, 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 cosmetic or pharmaceutical composition as described in the fifth aspect of this application in the preparation ofUse in preparations for supplementing collagen.

[0104] (2) 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 preparations for improving 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 preparations for increasing and / or improving skin texture, smoothness, elasticity and / or tension.

[0106] (4) 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 preparations for reducing the appearance of one or more superficial depressions in the skin.

[0107] Furthermore, 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 embodiments, skin aging is selected from skin aging caused by aging or skin aging caused by ultraviolet light or sunlight exposure.

[0110] In some embodiments, 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, brow drooping, tear troughs, nasolabial folds, bunny lines, cheek / midface drooping, marionette lines, poppy dimpling, smile lines, laugh lines, chin wrinkles, neck wrinkles, platysma bands, and any combination thereof.

[0111] In some embodiments, the increase and / 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; (b) skin pore size is reduced; (c) skin thickness,(d) Improved fullness and / or firmness; (e) Improved skin smoothness, suppleness and / or softness; (f) Improved skin tone, radiance and / or transparency; (g) Improved procollagen and / or collagen production; (h) Improved appearance of skin contour; (i) Restoration of skin luster and / or radiance; (j) Improved appearance of skin 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) Improved optical properties of the skin 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, lotion, cream, ointment, injectable dosage form, microneedle, etc., preferably an injectable solution.

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

[0114] In some embodiments, the product or drug is administered by superficial injection. Brief Description of the Drawings

[0115] Figure 1 shows the comparison of the integrity of PolyA in a plasmid 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 mRNAs encoding collagen on dermal collagen fiber content in a mouse model of skin photoaging.

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

[0122] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some embodiments of the present invention, but 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 in the following embodiments are conventional methods, in accordance with the literature in the art.The described techniques or conditions are carried out in accordance with the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available. All animal experiments in this application were commissioned to Beijing Langke Biotechnology Co., Ltd. Unless otherwise specified, the nucleotide sequences recorded 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. When the sequences in the instructions are inconsistent with those in the sequence listing, the sequences recorded in the instructions shall prevail.

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

[0125] 1.1 Synthesis of collagen sequence and construction of recombinant vector

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

[0127] The collagen used in this application embodiment is 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-mentioned 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 this application is shown in any one of SEQ ID NO: 24-26 (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, the only difference being that SEQ ID NO is used.Replace the ORF sequence in any of NO: 9-20.

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

[0135] 1.2 Screening of polyA elements for collagen COL17A1 mRNA

[0136] In the previous research, the inventors found that if the known 120A tail (120 consecutive A's) is used in the collagen mRNA, it will cause the DNA plasmid to have a base deletion defect during replication, resulting in instability. Therefore, the inventors developed a polyA element that can greatly improve the replication stability of DNA plasmids: RG2 (SEQ ID NO: 23).

[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 effects 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 to complete plasmid extraction and sequencing. 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 indicates higher plasmid replication stability 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, χ²test)

[0138] Table 1 Evaluation of replication stability of different polyA sequences for collagen DNA plasmids

[0139] PolyA sequence COL17A1-A120 COL17A1-RG2 Correct clone number 68 89 Deletion clone number 28 11 Stability 70.8% 89.0% chi^2vs120A - p<0.01

[0140] 1.3 Preparation of mRNA

[0141] 1.3.1 Plasmid linearization Instructions 12 / 58 pages 16 CN 121628912 A

[0142] The plasmid linearized in Example 1 was linearized with restriction endonuclease SapⅠ. The recombinant plasmid prepared in Section 1 was digested with enzymes to linearize the above recombinant plasmid at 37°C for 3 hours. The reaction system is shown in Table 2:

[0143] Table 2. Plasmid linearization enzyme digestion system

[0144] 10×Cutsmart buffer 5μLSapI enzyme (10000U / mL) 1μL, plasmid 10μg, ddH2O to bring to 50μL

[0145] After the reaction, 2μL of the enzyme digestion product was subjected to 1% agarose gel electrophoresis to detect the linearization of the plasmid. Subsequently, each linearized recombinant plasmid was purified using a PCR product recovery kit (Comway Century).

[0146] 1.3.2 In vitro transcription and purification of mRNA products

[0147] Using the linearized recombinant plasmid obtained in Example 1.3.1 as a template, in vitro transcription was performed using a high-yield T7RNA 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℃ for 3h. The transcription system is shown in Table 3.

[0148] Table 3. In vitro transcription system

[0149] 5×ReactionBuffer 4μL ATP (100mM) 2μL ΨTP (100mM) 2μL CTP (100mM) 2μL GTP (100mM) 2μL Enzymemix 1μL Linearized DNA template 500ng-1μg CleanCapAG (100mM) 1μL Nuclease-free H2O Add to 20μL

[0150] Wherein, ΨUTP (100mM) is pseudouridine triphosphate, also represented as N1-Me-pUTP (Shanghai Zhaowei Technology Development Co., Ltd., product catalog number R5-027).

[0151] CleanCap AG is m7G(5')ppp(5')(2'-OMeA)pG (catalog number ON-134, Shanghai Zhaowei), used as transcription initiation primer.

[0152] After the transcription reaction was completed, 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 collected at the bottom. The supernatant was discarded. 1 mL of 70% ethanol was added to wash the RNA, and the mixture was centrifuged at 12000 g for 5 min. The supernatant was discarded. 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.

[0153] 1.4 Lipid nanoparticles (LNP) encapsulating mRNA

[0154] The mRNAs obtained in step 1.3.2 were dispersed in 20 mM acetic acid solution (pH 5.0) and their final concentrations were adjusted.The concentration was 200 μg / mL. The mixture was prepared by mixing ionizable lipids, cholesterol, DSPC, and DMG-PEG2000 at a molar ratio of 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 then 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 using Tris aqueous solution (page 13 / 58, CN 121628912 A) to obtain the LNP-loaded mRNA solution. In addition, empty LNPs without mRNA loading were prepared as a control using the same procedure.

[0155] The concentration of mRNA encapsulated 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, respectively. Exemplary LNP characterization data are shown in Table 4.

[0156] Table 4. Characterization data of collagen mRNA-LNPs

[0157]

[0158] Example 2: In vivo effect study of mRNA encoding collagen

[0159] 2.1 Construction of a mouse model of skin photoaging

[0160] 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 ultraviolet irradiation of their back skin every other day for a total of 8 weeks to construct 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 30cm away from the back of the mice for irradiation. The UV irradiation dose was monitored using a UV illuminometer. The specific irradiation doses were: 60mJ / cm2 per irradiation for the first two weeks, 120mJ / cm2 per irradiation for the third week, 180mJ / cm2 per irradiation for the fourth week, and 240mJ / cm2 per irradiation for the fifth to eighth weeks.

[0161] After the irradiation treatment, the skin tissue of the mice 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 whether the photoaging mouse model was successfully constructed.As shown in Figure 2, compared with the skin of normal mice that had not been exposed to ultraviolet light, the mice treated with ultraviolet light exhibited typical skin photoaging tissue structure characteristics. The epidermis thickened, the wavy connection between the epidermis and dermis was smoothed, the papillae and dermal papillae disappeared, and the dermal collagen fibers often showed abnormal changes such as disordered arrangement, uneven distribution, degeneration, and breakage. The above results indicate that the skin photoaging mouse model in this embodiment was successfully constructed and produced the expected pathological structure, which can be used for in vivo effect evaluation.

[0162] 2.2 Evaluation of the anti-aging effect of mRNA encoding collagen

[0163] Twenty mice that were successfully modeled in Section 2.1 were randomly divided into 4 groups of 5 mice each. At the same time, 5 normal mice that were not modeled were selected as negative controls for the aging model. According to the grouping situation shown in Table 5, the LNPs prepared in Table 4 of Section 1.4 of Example were injected intradermally on days 0, 4, 7, 14, and 21. On day 28, mouse skin samples were collected and stained with HE and Masson.

[0164] Table 5. Mouse grouping for anti-aging effect evaluation, page 14 / 58, CN 121628912 A

[0165]

[0166] Digital images were collected by scanning sections to observe histopathological structural changes:

[0167] Masson staining of skin collagen fiber percentage: 10X fields of view were randomly selected, and collagen fibers were selected using image analysis software ImageJ to calculate the percentage of collagen fiber area in the total skin tissue area. The statistical method of two-tailed unpaired t-test in GraphPad was used to analyze the significance between data groups (*p<0.05, **p<0.01).

[0168] The analysis results are shown in Figure 3. Compared with normal mice, the proportion of collagen fibers in the empty LNP control group was significantly reduced, which confirms the successful construction of the mouse model. Compared with the empty LNP control group, the collagen fiber content of COL3A1 and COL6A1 in the drug treatment group was slightly increased, but there was no statistical difference. However, in the COL17A1 group, the content of dermal collagen fibers in mice was significantly increased compared with that in the empty LNP group. The above results show that COL17A1 can effectively alleviate the loss of collagen fibers caused by ultraviolet radiation in vivo, but the alleviating effect of COL3A1 and COL6A1 is not significant.

[0169] The skin was scored according to the degree of epidermal thickening, keratinization and reduction of dermal collagen fibers. No lesion 0 points, mild 1 point, mild 2 points, moderate 3 points and severe 4 points. The average value of each pathological score was taken. The statistical method of two-tailed unpaired t test in GraphPad was used to analyze the significance between the data groups (*p<0.05, **p<0.01, ***p<0.001).

[0170] The analysis results are shown in Figure 4. The pathological scores of the empty LNP control group and normal mice were significantly different.This further confirms that the model mice have obvious photoaging pathological features. Compared with the empty LNP control group, the pathological scores of each treatment group were significantly reduced, which indicates that the mRNA of each group showed a mitigating effect on photoaging pathological tissues in the in vivo disease model. In addition, it is worth noting that among the treatment groups, the mitigating effect of COL17A1 was higher than that of COL3A1 and COL6A1.

[0171] Example 3 Further optimization of nucleic acid sequence

[0172] Although Example 2 proved that the mRNA encoding COL17A1 has the advantage of the mRNA encoding other types of collagen in improving skin aging, its ORF sequence (corresponding to SEQ ID NO:6) is difficult to detect. Therefore, we conducted an optimization experiment based on the wild-type nucleic acid sequence (corresponding to SEQ ID NO:9). The specific process is as follows:

[0173] (1) 293T cells were seeded in 6-well plates, with 5X105 cells seeded in each well. After 24 hours, when the cell mixing degree was about 70%-80%, transfection was performed.

[0174] (2) 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) was mixed with 4 μl of transfection reagent Lipofectamine™ 3000 and incubated at room temperature for 15 min.

[0175] (3) The mRNA and Lipofectamine™ 3000 were added to 293T cells;

[0176] (4) Cell lysis was performed 24 h after transfection: the culture medium was aspirated, the cells were washed once with pre-cooled PBS, and 200 μl of cell lysis buffer was added to each well.

[0177] (5) Western Blot: according to the protein concentration determination results, 5 μg of each sample was loaded and separated on a 4-12% gradient gel on page 15 / 58 of the instruction manual, CN 121628912 A. After electrophoresis, the protein samples were transferred to a 0.22 μm nitrocellulose membrane using a wet transfer method. They were then co-incubated with a COL17A1 specific antibody (primary antibody) and a fluorescent secondary antibody before detection. Imaging was performed on a Bio-Rad gel imaging system, the ChemiDoc™ MP Imaging System.

[0178] A multi-parameter optimization design method was employed. By optimizing key parameters such as Codon usage bias, GC content, and mRNA secondary structure, sequence stability and gene expression efficiency were improved. Furthermore, during the optimization process, customization requirements for specific patterns were incorporated, including optimization...The expression efficiency was regulated by modifying the Shine-Dalgarno or Kozak sequences to improve gene expression efficiency, as well as by modifying RNA instability motifs. 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 optimized nucleic acid sequences that balance RNA stability and expression efficiency through the above comprehensive optimization strategies.

[0179] 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.

[0180] Furthermore, it was verified that optimized group 1 was easier to detect than the mRNA encoding COL17A1 in Example 2.

[0181] Example 4: In vivo effect study of optimized sequence

[0182] To further explore the anti-skin aging effect of optimized nucleic acid sequence in vivo, a photoaging mouse model was used for further study.

[0183] 4.1 Lipid nanoparticle-encapsulated mRNA

[0184] mRNA and lipid nanoparticle-encapsulated mRNA were prepared according to the methods in Sections 1.3 and 1.4, respectively. The characterization data of mRNA-LNP is shown in Table 6.

[0185] Table 6 Characterization data of collagen mRNA-LNP

[0186]

[0187] 4.2 Construction of skin photoaging mouse model

[0188] The skin photoaging mouse model was constructed using the same method as in Section 2.1.

[0189] 4.3 Evaluation of anti-aging effect of sequence-optimized mRNA

[0190] Ten mice that were successfully modeled in Section 4.2 were randomly divided into two groups of five mice each: a model control group and a COL17A1 group. Five normal mice were used as controls. According to the grouping shown in Table 7, the normal group was not given any medication, and the model control group and the COL17A1 group were injected intradermally with PBS and LNP prepared in Table 6 on days 0, 4, 7, 14 and 21, respectively. On day 28, mouse skin samples were collected and subjected to HE and Masson staining.

[0191] Table 7, Grouping of mice for anti-aging effect evaluation

[0192] Instructions 16 / 58 pages 20 CN 121628912 A

[0193]

[0194] By scanning sections, digital images were acquired and histopathological structural changes were observed:

[0195] Masson staining of skin collagen fiber percentage: 10X field of view was randomly selected, and collagen fibers were selected using image analysis software ImageJ to calculate the percentage of collagen fiber area to the total skin tissue area. Using the two-tailed non-matching in GraphPadThe statistical method of t-test was used to analyze the significance of each data group (*p<0.05, **p<0.01, ***p<0.001).

[0196] The analysis results are shown in Figure 6. Compared with normal mice, the proportion of collagen fibers in the model control group was reduced, which confirms the successful construction of the mouse model. Compared with the model control group, the content of dermal collagen fibers in the COL17A1 group was significantly increased. The above results show that COL17A1 can effectively alleviate the loss of collagen fibers caused by ultraviolet radiation in vivo.

[0197] The analysis results are shown in Figure 7. Compared with normal mice, the dermal thickness in the model control group was significantly reduced, which once again confirms that the model mice have obvious photoaging pathological characteristics. Compared with the model control group, the dermal thickness in the COL17A1 group was significantly increased. This result shows that COL17A1-mRNA has a mitigating effect on the decrease in dermal thickness due to photoaging in the in vivo disease model.

[0198] The above results demonstrate that the optimized mRNA sequence encoding COL17A1 can also effectively alleviate the symptoms of photoaging of the skin.

[0199] 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 the electronic sequence listing shows DNA sequences, the nucleotide sequences in the sequence listing of this application can represent either DNA sequences or RNA sequences. When representing an RNA sequence, the "T" represents uridine.

[0200] Sequence Description: Specification page 17 / 58, 21 CN 121628912 A

[0201] Specification page 18 / 58, 22 CN 121628912 A

[0202] Specification page 19 / 58, 23 CN 121628912 A

[0203] Specification page 20 / 58, 24 CN 121628912 A

[0204] Specification page 21 / 58, 25 CN 121628912 A

[0205] Specification page 22 / 58, 26 CN 121628912 A

[0206] Specification page 23 / 58, 27 CN 121628912 A

[0207] Specification page 24 / 58, 28 CN 121628912 A

[0208] Specification page 25 / 58, 29 CN 121628912 A

[0209] Specification 26 / 58 pages 30 CN 121628912 A

[0210] Specification 27 / 58 pages 31 CN 121628912 A

[0211] Specification 28 / 58 pages 32 CN 121628912 A

[0212] Description

[0213] Specification 29 / 58 pages 33 CN 121628912 A

[0214] Specification 31 / 58 pages 35 CN 121628912 A

[0215] Specification 32 / 58 pages 36 CN 121628912 A

[0216] Specification 33 / 58 pages 37 CN 121628912 A

[0217] Specification 34 / 58 pages 38 CN 121628912 A

[0218] Specification 35 / 58 pages 39 CN 121628912 A

[0219] Specification 36 / 58 pages 40 CN 121628912 A

[0220] Specification 37 / 58 pages 41 CN 121628912 A

[0221] Specification page 38 / 58, page 42, CN 121628912 A

[0222] Specification page 39 / 58, page 43, CN 121628912 A

[0223] Specification page 40 / 58, page 44, CN 121628912 A

[0224] Specification page 41 / 58, page 45, CN 121628912 A

[0225] Specification page 42 / 58, page 46, CN 121628912 A

[0226] Specification page 43 / 58, page 47, CN 121628912 A

[0227] Specification page 44 / 58, page 48, CN 121628912 A

[0228] Specification page 45 / 58, page 49, CN 121628912 A

[0229] Specification page 46 / 58, page 50, CN 121628912 A

[0230] Specification page 47 / 58 51 CN 121628912 A

[0231] Specification page 48 / 58 52 CN 121628912 A

[0232] Specification page 49 / 58 53 CN 121628912 A

[0233] Specification page 50 / 58 54 CN 121628912 A

[0234] Specification page 51 / 58 55 CN 121628912 A

[0235] Specification page 52 / 58 56 CN 121628912 A

[0236] Specification page 53 / 58 57 CN 121628912 A

[0237] Specification page 54 / 58 58 CN121628912 A

[0238] Specification 55 / 58 pages 59 CN 121628912 A

[0239] Specification 56 / 58 pages 60 CN 121628912 A

[0240] Specification 57 / 58 pages 61 CN 121628912 A

[0241] Specification 58 / 58 pages 62 CN 121628912 A Figure 1 Figure 2 Specification Figure 1 / 3 pages 63 CN 121628912 A Figure 3 Figure 4 Figure 5 Specification Figure 2 / 3 pages 64 CN 121628912 A Figure 6 Figure 7 Specification Figure 3 / 3 pages 65 CN 121628912 A Provided are an mRNA comprising a nucleotide sequence encoding collagen, as well as a composition comprising said mRNA and a delivery carrier. By introducing the mRNA molecule into a subject by means of the composition, the skin conditions could be improved. Abstract

Claims

1. An isolated mRNA, wherein, the mRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 6-20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOs: 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 set forth in any one of SEQ ID NOs: 6-20.

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

23.

4. The mRNA according to any one of claims 1 to 3, wherein, the mRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 24-26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOs: 24-26.

5. The mRNA according to any one of claims 1 to 4, wherein, the mRNA comprises at least one chemically modified nucleoside; optionally, the chemically modified nucleoside is selected from a chemically modified uridine; optionally, some or all of the uridines of the mRNA are chemically modified uridines; 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-diazepin-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-azauridine, dihydropseudouridine, 5-methoxyuridine, 5-methyluridine, and 2-methoxy-uridine, preferably, the chemically modified uridine is pseudouridine or N1-methyl-pseudouridine.

6. The mRNA according to any one of claims 1 to 5, wherein, the mRNA further comprises a 5’ cap structure.

7. The mRNA of claim 6, wherein, the 5’ cap structure is m7G(5’)ppp(5’)(2’-OMeN)pN, wherein 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 a lipid nanoparticle (LNP), the mRNA comprising a nucleotide sequence encoding a collagen protein, preferably the amino acid sequence of the collagen protein is set forth in any one of SEQ ID NOs: 1-5.

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

11. The composition of claim 10, wherein the delivery vehicle is selected from a liposome, a lipid nanoparticle (LNP), a sol-gel, and a nanogel.

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

13. The composition of 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 of claim 13, wherein: the first mRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 6-10, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 6-10; the second mRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 11-12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 11-12; the third mRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 13-14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 12-13; the fourth mRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 15-18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOs: 15-18; the fifth mRNA comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 19-20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOs: 19-20.

15. An isolated DNA encoding the mRNA of any one of claims 1-7.

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

17. An isolated cell comprising the DNA of claim 15 or the recombinant vector of claim 16; optionally, the cell is selected from a prokaryotic cell or a eukaryotic cell; Optionally, the eukaryotic cell is selected from any one of an insect cell, a mammalian cell, an avian cell, and a yeast cell, preferably a mammalian cell, more preferably a human cell; Optionally, the prokaryotic cell is selected from a bacterium, preferably Escherichia coli.

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

19. A method of supplementing collagen in a subject in need thereof, 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.

20. A method of improving skin aging in a subject in need thereof, 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.

21. A method of increasing and / or improving at least one of the texture, smoothness, elasticity, or tone of the skin of a subject in need thereof, 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 of reducing the appearance of one or more superficial depressions in the skin in a subject in need thereof, 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.

23. The method as recited in claim 20, wherein, The skin aging is selected from the group consisting of skin aging caused by age increase or skin aging caused by ultraviolet or solar light exposure.

24. The method as recited in claim 21, wherein, The increasing or improving the texture, smoothness, elasticity, and / or firmness of the skin is selected from the group consisting of: (a) treatment, reduction, and / or prevention of fine lines and / or wrinkles; (b) reduction of skin pore size; (c) improvement of skin thickness, plumpness, and / or tautness; (d) improvement of skin smoothness, suppleness, and / or softness; (e) improvement of skin color, radiance, and / or transparency; (f) improvement of procollagen and / or collagen production; (g) improvement of skin texture and / or promotion of retexturization; (h) improvement of the appearance of skin contours; (i) restoration of skin glow and / or radiance; (j) improvement of the appearance of skin that has diminished due to aging and / or menopause; (k) improvement of skin hydration; (1) increase in skin elasticity and / or resiliency; (m) treatment, reduction, and / or prevention of skin sagging; (n) improvement of skin firmness; (o) reduction of pigment spots, mottled skin, and / or scars; (p) improvement of the optical properties of the skin in terms of light diffraction or reflection; or (q) any combination thereof. The one or more superficial depressions in the skin is selected from the group consisting of nasolabial folds, crow’s feet, frown lines, worry lines, scars, glabellar lines, droopy eyebrows, tear troughs, nasal labial folds, bunny lines, midface jowls, marionette lines, poppy dimpling, smile lines, laugh lines, chin creases, neck lines, platysmal bands, and any combination thereof. 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 recombinant vector of claim 16, or the cosmetic or pharmaceutical composition of claim 18. 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 recombinant vector of claim 16, or the pharmaceutical composition of claim 18. The subject is a human.

25. The method of claim 22, wherein, The administering is selected from the group consisting of transdermally, subcutaneously, intradermally, and / or superficially.

26. A method of promoting scar healing, tissue repair, or promoting hair follicle repair in a subject in need thereof, wherein, The administering is selected from the group consisting of transdermally, subcutaneously, intradermally, and / or superficially.

27. A method of treating a disease associated with collagen loss in a subject in need thereof, wherein, ​ 28. The method of any one of claims 19-27, wherein, ​ 29. The method of any one of claims 19-27, wherein, ​

Images