Use of a quantitative partial reprogramming system in the preparation of a drug for reversing skin aging

Abstract

This invention provides an application of a quantitative partial reprogramming system in the preparation of drugs to reverse skin aging, belonging to the fields of regenerative medicine and medical aesthetics. The quantitative partial reprogramming system includes a nucleic acid delivery system or a small molecule induction system; skin aging includes photoaging-induced skin aging; reversing skin aging includes increasing the thickness of the dermis, increasing the density of type I collagen, and improving skin elasticity. The quantitative partial reprogramming system provided by this invention can fundamentally reverse the cellular aging clock and achieve structural regeneration, and can be applied to the preparation of drugs to reverse skin aging.

Description

Technical Field

[0001] This invention belongs to the fields of regenerative medicine and medical aesthetics, and particularly relates to the application of a quantitative partial reprogramming system in the preparation of drugs to reverse skin aging. Background Technology

[0002] Clinically, skin aging is mainly manifested as deepening wrinkles, skin laxity, and decreased elasticity. Its essence lies in the aging and reduced synthetic capacity of key functional cells such as dermal fibroblasts, leading to the gradual loss of extracellular matrix components such as collagen and elastin. Current mainstream clinical treatments, such as botulinum toxin type A injections, primarily reduce dynamic wrinkles by reversibly blocking neuromuscular transmission. However, they only temporarily alleviate surface symptoms and cannot restore the lost matrix or fundamentally reverse the aging process of cells. Furthermore, long-term use carries the risk of diminishing efficacy and drug resistance.

[0003] Partial reprogramming technology offers new theoretical and technical pathways for reversing cellular senescence. By transiently expressing reprogramming factors such as Oct4, Sox2, and Klf4 at low levels, the epigenetic state of cells can be moderately reset without inducing complete pluripotency, thereby improving cellular function. However, the clinical translation of this technology still faces the following core bottlenecks: Safety risks: c-Myc in the classic OSKM combination is a potent proto-oncogene, and its expression significantly increases the risk of tumorigenesis; Single delivery and realization pathway: Existing studies mostly focus on viral or other nucleic acid delivery pathways, lacking systematic parallel comparisons and efficacy evaluations with small molecule drug induction pathways that are more easily applied in clinical practice; Insufficient controllability of expression: Traditional induction systems (such as Tet-On) have a certain background leakage expression, and the inducer doxycycline has antibiotic activity, which may affect the gut microbiota.

[0004] Therefore, there is an urgent need in this field for a comprehensive anti-aging solution that integrates a combination of safety factors, a highly controllable induction system, diversified implementation paths, and a quantitative verification system. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an application of a quantitative partial reprogramming system in the preparation of a drug for reversing skin aging.

[0006] This invention provides the application of a quantitative partial reprogramming system in the preparation of drugs to reverse skin aging, wherein the quantitative partial reprogramming system includes a nucleic acid delivery system or a small molecule induction system.

[0007] Preferably, skin aging includes skin aging caused by photoaging.

[0008] Preferably, the reversal of skin aging includes at least one of increasing the thickness of the dermis, increasing the density of type I collagen, improving skin elasticity, reducing the depth of wrinkles, and reducing the area of ​​scars.

[0009] Preferably, the nucleic acid delivery system comprises a gene expression system induced by the rapamycin analog AP21967; the gene expression system comprises: (a) The nucleic acid encoding the first fusion protein, the first fusion protein comprising an FKBP12 variant triplet repeat domain and a DNA-binding domain; (b) The nucleic acid encoding the second fusion protein, which contains an FRB variant domain and a transcription activation domain; (c) Effect elements encoding five reprogramming factors: Oct4, Sox2, Klf4, Glis1, and Lin28; said effect elements being operatively linked to an inducible promoter containing a cis-regulatory element specifically recognized by said DNA-binding domain; (d) Inducer: rapamycin analog AP21967.

[0010] Preferably, the FKBP12 variant is FKBP12 (F36V), the FRB variant is FRB (T2098L), and the inducer is AP21967. The DNA binding domain is GAL4-DBD at amino acid positions 1-147 of the yeast GAL4 transcription factor, and the cis-regulatory element comprises at least four tandem GAL4 upstream activation sequences UAS(GAL4). The transcriptional activation domain is the VP64-p65-Rta triple activation domain VPR.

[0011] Preferably, the nucleic acid delivery system includes a recombinant adeno-associated virus vector carrying: (a) a first AAV vector containing nucleic acid encoding the first fusion protein operably linked to a promoter; (b) a second AAV vector containing nucleic acid encoding the second fusion protein operably linked to a promoter; and (c) a third AAV vector containing effector elements encoding five reprogramming factors, Oct4, Sox2, Klf4, Glis1, and Lin28, operably linked to an inducible promoter.

[0012] Preferably, the nucleic acid delivery system comprises mRNA encapsulated by lipid nanoparticles, wherein the mRNA comprises mRNA-1 encoding the first fusion protein, mRNA-2 encoding the second fusion protein, and mRNA-3 encoding five reprogramming factors: Oct4, Sox2, Klf4, Glis1, and Lin28. Alternatively, the mRNA may be a single polycistronic mRNA whose coding sequence is linked in the following order: OCT4-(P2A)-OCT4-(T2A)-OCT4-(E2A)-SOX2-(F2A)-KLF4-(GSAG)-GLIS1-(P2A)-LIN28.

[0013] Preferably, the mRNA is a chemically modified mRNA, in which uracil is wholly or partially replaced by N1-methylpseudouracil.

[0014] Preferably, the small molecule induction system includes valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, and foctocorline; wherein the concentration of valproic acid is 200-300 μM, the concentration of CHIR99021 is 5-15 μM, the concentration of Repsox is 5-15 μM, the concentration of transphenylcyclopropylamine is 4-6 μM, and the concentration of foctocorline is 40-60 μM.

[0015] Compared with existing technologies, this invention has the following beneficial effects: This invention provides the application of a quantitative partial reprogramming system in the preparation of drugs to reverse skin aging. The quantitative partial reprogramming system includes a nucleic acid delivery system or a small molecule induction system. The rapamycin-inducible nucleic acid delivery system used in this invention is based on the FKBP-FRB orthogonal induction mechanism, completely avoiding interference with endogenous mTOR, exhibiting "zero leakage" characteristics (no expression without the inducer), and the inducer rapamycin / AP21967 has no antibiotic activity and does not interfere with the gut microbiota. The nucleic acid delivery system provided by this invention, based on the OSKGL combination, completely avoids the carcinogenic risk of c-Myc. The small molecule induction system used is optimized to maximize reprogramming efficiency while controlling off-target effects. Validation data show that all intervention modalities, while achieving excellent anti-aging effects, exhibited no difference in local and systemic safety compared to the natural aging control group and the traditional therapy control group, resolving the core safety concerns in the clinical translation of reprogramming technology.

[0016] By setting up a botulinum toxin control group, this invention reveals that the quantitative partial reprogramming system provided by this invention can fundamentally reverse the cellular aging clock and achieve structural regeneration, while traditional therapies have no effect on this. Detailed Implementation

[0017] This invention provides the application of a quantitative partial reprogramming system in the preparation of drugs to reverse skin aging, wherein the quantitative partial reprogramming system includes a nucleic acid delivery system or a small molecule induction system.

[0018] In this invention, skin aging includes skin aging caused by photoaging, and also includes skin aging caused by other reasons. This invention takes skin aging caused by photoaging as an example for experimental purposes.

[0019] In this invention, reversing skin aging includes at least one of increasing the thickness of the dermis, increasing the density of type I collagen, improving skin elasticity, reducing the depth of wrinkles, and reducing the area of ​​scars.

[0020] In this invention, the nucleic acid delivery system includes a gene expression system induced by rapamycin or the rapamycin analog AP21967; the nucleic acid delivery system of this invention uses a gene expression system induced by rapamycin or the rapamycin analog AP21967, which is based on the chemical dimerization mechanism of FKBP-FRB and has extremely high induction specificity and extremely low basal leakage expression.

[0021] In this invention, the gene expression system includes: (a) The nucleic acid encoding the first fusion protein, the first fusion protein comprising an FKBP12 variant triplet repeat domain and a DNA-binding domain; (b) The nucleic acid encoding the second fusion protein, which contains an FRB variant domain and a transcription activation domain; (c) Effect elements encoding five reprogramming factors: Oct4, Sox2, Klf4, Glis1, and Lin28; said effect elements being operatively linked to an inducible promoter containing a cis-regulatory element specifically recognized by said DNA-binding domain; (d) Inducer: rapamycin or rapamycin analog AP21967.

[0022] The FKBP12 variant is FKBP12(F36V), the FRB variant is FRB(T2098L), the inducer is AP21967, the DNA binding domain is yeast GAL4 transcription factor amino acid 1-147 GAL4-DBD, the cis-regulatory element comprises at least four tandem GAL4 upstream activation sequences UAS(GAL4); the transcriptional activation domain is the VP64-p65-Rta triple activation domain VPR.

[0023] Preferably, the nucleic acid delivery system includes a recombinant adeno-associated virus vector carrying: (a) a first AAV vector containing nucleic acid encoding the first fusion protein operably linked to a promoter; (b) a second AAV vector containing nucleic acid encoding the second fusion protein operably linked to a promoter; and (c) a third AAV vector containing effector elements encoding five reprogramming factors, Oct4, Sox2, Klf4, Glis1, and Lin28, operably linked to an inducible promoter.

[0024] The specific system components are as follows: (1) First AAV vector (regulator A): encodes FKBP(F36V)×3-GAL4-DBD-NLS fusion protein.

[0025] FKBP(F36V): The F36V mutant derived from human FKBP12 has a high affinity for AP21967 (Kd ≈ 1nM) but a reduced affinity for natural rapamycin.

[0026] GAL4-DBD: The DNA-binding domain (amino acids 1-147) of the yeast GAL4 transcription factor, which specifically recognizes the UAS (GAL4) sequence.

[0027] Triple tandem repeat design: Three FKBPs (F36V) in series can significantly enhance the affinity and stability for binding to the inducer.

[0028] Promoter: Use a broad-spectrum, potent CMV promoter, or select a tissue-specific promoter (such as a skin-specific K14 promoter) to achieve skin-specific regulation.

[0029] (2) Second AAV vector (regulator B): encodes FRB(T2098L)-VPR-NLS fusion protein.

[0030] FRB(T2098L): The T2098L mutant of the FRB domain of human mTOR. This mutation prevents it from binding to natural rapamycin and specifically binds to AP21967 (bump-hole strategy), completely avoiding interference with the endogenous mTOR signaling pathway.

[0031] VPR: Triple transcription activation domain, composed of VP64, p65 and Rta in tandem, has extremely strong transcriptional activation activity.

[0032] Promoter: Same as regulator A.

[0033] (3) Third AAV vector (effector): encodes the OSKGL five factors and is controlled by the UAS(GAL4) inducible promoter.

[0034] Inducible promoter: 5×UAS(GAL4)-hsp68 minimal promoter, which is efficiently activated only when GAL4-DBD is bound and AP21967 is present.

[0035] Target genes: Encoding five reprogramming factors—Oct4, Sox2, Klf4, Glis1, and Lin28—are linked via 2A peptides to achieve an expression ratio of 3:1:1:1:1. Specific design: OCT4-P2A-OCT4-T2A-OCT4-E2A-SOX2-F2A-KLF4-GSAG-GLIS1-P2A-LIN28.

[0036] In this invention, the nucleic acid delivery system comprises mRNA encapsulated by lipid nanoparticles, wherein the mRNA includes mRNA-1 encoding the first fusion protein, mRNA-2 encoding the second fusion protein, and mRNA-3 encoding five reprogramming factors: Oct4, Sox2, Klf4, Glis1, and Lin28; or the mRNA is a single polycistronic mRNA whose coding sequence is sequentially linked as follows: OCT4-(P2A)-OCT4-(T2A)-OCT4-(E2A)-SOX2-(F2A)-KLF4-(GSAG)-GLIS1-(P2A)-LIN28. In this invention, the mRNA is a chemically modified mRNA, in which uracil is wholly or partially replaced by N1-methylpseudouracil.

[0037] Specifically, it includes the following components: (1) mRNA-1: Encodes FKBP(F36V)×3-GAL4-DBD-NLS fusion protein.

[0038] (2) mRNA-2: encodes FRB(T2098L)-VPR-NLS fusion protein.

[0039] (3) mRNA-3: Encodes OSKGL factor 5, a single polycistronic mRNA with the following sequence: OCT4-(P2A)-OCT4-(T2A)-OCT4-(E2A)-SOX2-(F2A)-KLF4-(GSAG)-GLIS1-(P2A)-LIN28.

[0040] mRNA molecule optimization: Chemical modification: 100% N1-methylpseuuridine (m1Ψ) replaces uridine, reducing immunogenicity and improving translation efficiency.

[0041] UTR optimization: The 5' UTR uses a human β-globin UTR; the 3' UTR uses a stable UTR composed of human ALB and APOE, with an additional poly(A)150 tail.

[0042] Capping: CleanCap AG co-transcriptional capping was used to ensure a Cap1 structure generation rate of >95%.

[0043] Purification: HPLC purification was used to remove dsRNA byproducts.

[0044] The LNP formulation is as follows:

[0045] Quality control parameters: particle size 80~120 nm, PDI <0.2, encapsulation efficiency >90%, zeta potential -5 to +5 mV.

[0046] This system uses rapamycin or its analogue AP21967 as an inducer: Rapamycin: CAS No. 53123-88-9, applicable to FKBP12 wild-type and FRB wild-type systems, recommended concentration 1~10 nM.

[0047] AP21967 (A / C Heterodimerizer): CAS No. 195514-79-5, specifically binds FKBP (F36V) and FRB (T2098L) to form an orthogonal induction system, completely avoiding interference with endogenous mTOR, recommended concentration 10~100 nM.

[0048] In this invention, the small molecule induction system includes valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, and foctocorline. In this invention, the concentration of valproic acid is 200-300 μM, preferably 220-280 μM, more preferably 250 μM; the concentration of CHIR99021 is 5-15 μM, preferably 8-13 μM, more preferably 10 μM; the concentration of Repsox is 5-15 μM, preferably 8-12 μM, more preferably 10 μM; the concentration of transphenylcyclopropylamine is 4-6 μM, preferably 4.5-5.5 μM, more preferably 5 μM; and the concentration of foctocorline is 40-60 μM, preferably 45-55 μM, more preferably 50 μM.

[0049] In this invention, the nucleic acid sequence information of each key functional component is as follows. The specific nucleic acid sequence and protein sequence are marked with sequence number (SEQ ID NO) and included in the sequence listing at the end of this application.

[0050] (a) Sequence of FKBP12 (F36V) monomer

[0051] The FKBP12(F36V) monomer is the core inducible response module of the first fusion protein. It is derived from the human FKBP12 protein (UniProt P62942). According to the numbering rules of Clackson et al. (1998, PNAS), the phenylalanine (F) at position 36 is mutated to valine (V), which greatly increases its affinity for AP21967 (Kd≈1 nM) while completely eliminating its binding activity to natural rapamycin.

[0052] FKBP12(F36V) monomeric amino acid sequence (108 aa, SEQ ID NO:1): MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE Optimized nucleotide sequence of human codon encoding FKBP12(F36V) monomer (324 bp, SEQ ID NO:2, without stop codon): ATGGGCGTGCAGGTGGAGACCATCTCCCCCGGCGACGGCAGGACATTCCCCAAGCGGGGC CAGACATGCGTGGTGCACTACACAGGCATGCTGGAGGACGGCAAGAAGGTGGACAGCAGC AGGGACCGGAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAGGAGGTGATCAGGGGCTGG GAGGAGGGCGTGGCCCAGATGAGCGTGGGCCAGAGGGCCAAGCTGACCATCAGCCCCGAC TACGCCTACGGCGCCACGGGCCACCCAGGCATCATCCCCCCTCACGCCACCCTGGTGTTT GACGTGGAGCTGCTGAAGCTGGAG

[0053] (ii) Sequence of the first fusion protein [FKBP12(F36V)×3-GAL4(1-147)DBD-NLS]

[0054] The first fusion protein consists of the following domains tandemly linked from N to C: ① Three FKBP12 (F36V) monomers (each 108 aa), connected by a (GGSGGS) flexible linker; ② A yeast GAL4 transcription factor DNA-binding domain (aa 1-147, derived from the Saccharomyces cerevisiae GAL4 gene, NCBI Gene ID: 856219, S288C strain), which specifically recognizes the UAS (GAL4) cis-regulatory element; ③ The SV40 large T antigen nuclear localization signal (NLS), with the amino acid sequence PKKKRKV, ensuring the fusion protein is localized to the cell nucleus. The total length of the first fusion protein is approximately 496 aa (including the linker sequence).

[0055] The GGSGGS flexible linker (6 aa, SEQ ID NO:3) encodes a nucleotide sequence (18 bp, SEQ ID NO:4): SEQ ID NO:3 (AA): GGSGGS SEQ ID NO:4 (NA): GGTGGCAGCGGTGGCAGC The SV40 nuclear localization signal (7 aa, SEQ ID NO:5) encodes a nucleotide sequence (21 bp, SEQ ID NO:6): SEQ ID NO:5(AA): PKKKRKV SEQ ID NO:6 (NA): CCCAAGAAGAAGAGGAAGGTG The nucleotide coding sequence of GAL4-DBD (aa 1-147) was optimized using human codons based on the GAL4 gene of Saccharomyces cerevisiae (NCBI Gene ID: 856219, reference sequence NP_009839.3, amino acids 1-147) to enhance translation efficiency in mammalian cells. The amino acid sequence of GAL4-DBD (1-147) can be found in the NCBI database at positions 1-147 of NP_009839.3.

[0056] The overall structure of the nucleotide sequence encoded by the first fusion protein is as follows: [FKBP12(F36V) CDS]×3 – [GGSGGS]×3 – [GAL4-DBD(1-147) CDS] – [SV40NLS CDS] The total length is approximately 1,488 bp (human codon optimization).

[0057] (iii) Sequence of the second fusion protein [FRB(T2098L)-VPR-NLS]

[0058] The second fusion protein consists of the following domains tandemly linked in the N→C direction: ① The FRB domain (amino acid positions 2025-2114, a total of 90 aa) of the human mTOR protein (NCBI NP_004949.1), carrying the T2098L point mutation—this mutation eliminates the binding to natural rapamycin, allowing FRB (T2098L) to specifically form a ternary complex (bump-hole strategy) with AP21967 and FKBP12 (F36V), completely isolating it from interference with endogenous mTOR signaling; ② The VP64-p65-Rta triplet transcriptional activation domain (VPR), consisting of VP64 (quadruple tandem VP16 minimal activation domain DALDDFDLDML), the human NF-κB p65 subunit activation domain (RelA aa285-551, NCBI NP_068810.3), and the EBV Rta transcriptional activation domain (UniProt P03211, aa (1-415) tandemly composed; ③SV40 NLS (SEQ ID NO:5). The total length of the second fusion protein is approximately 560 aa.

[0059] The nucleotide coding sequence of the FRB(T2098L) domain (90 aa) is based on amino acid sequences 2025-2114 of the human mTOR (NP_004949.1), with a threonine (T) mutation to leucine (L) at position 2098 (T2098L). After human codon optimization, the length is 270 bp (excluding the stop codon). The amino acid sequence of FRB(T2098L) can be found in NCBI NP_004949.1, positions 2025-2114 (including the T2098L mutation).

[0060] The monomeric amino acid sequence of the minimum activation domain of VP64 (11 aa, SEQ ID NO:7) is as follows: SEQ ID NO:7(AA):DALDFDFDLDML The VP64 consists of four of the above-mentioned units connected in series via a GSSG short connector, with a total length of approximately 59 aa.

[0061] The overall structure of the nucleotide sequence encoded by the second fusion protein is as follows: [FRB(T2098L) CDS] – [VP64 CDS] – [p65-AD CDS] – [Rta-AD CDS] – [SV40NLS CDS] The second fusion protein is approximately 1,680 bp in length (human codon optimized).

[0062] (iv) Nucleic acid sequences of the five reprogramming factors

[0063] The five reprogramming factors used in this invention are all human proteins, and their encoding nucleotide sequences are based on the following NCBI reference sequences (CDS region), with selective codon optimization to achieve optimal expression efficiency in target cells such as skin fibroblasts. None of the factor encoding sequences contain a stop codon (to achieve in-frame fusion of the 2A peptide or adapter sequence), except for the last factor (Lin28A) at the end of the effector element, which carries a stop codon (TGA).

[0064] The nucleotide sequence (1,083 bp) encoding Oct4 (POU5F1, human subtype 1) is based on the NCBI reference sequence NM_002701.6 (CDS region), encoding a protein of 360 aa. The human codon optimized version is synonymously replaced with the reference sequence, and the protein amino acid sequence remains unchanged. See the corresponding entry in the NCBI NP.

[0065] The encoding nucleotide sequence (954 bp) of Sox2 (SOX2, human) is based on the NCBI reference sequence NM_003106.4 (CDS region), encoding protein 317 aa; the human codon optimized version is synonymously replaced with the reference sequence, and the protein amino acid sequence remains unchanged. See the corresponding entry in NCBI NP.

[0066] The coding nucleotide sequence (1,440 bp) of Klf4 (KLF4, human) is based on the NCBI reference sequence NM_004235.6 (CDS region), encoding protein 479 aa; the human codon optimized version is synonymously replaced with the reference sequence, and the protein amino acid sequence remains unchanged. See the corresponding entry in NCBI NP.

[0067] The coding nucleotide sequence (1,866 bp) of Glis1 (GLIS1, human) is based on the NCBI reference sequence NM_147193.3 (CDS region), encoding protein 621 aa; the human codon optimized version has synonymous substitutions with the reference sequence, and the protein amino acid sequence remains unchanged. See the corresponding entry in NCBI NP.

[0068] The encoding nucleotide sequence (630 bp) of Lin28A (LIN28A, human) is based on the NCBI reference sequence NM_024674.6 (CDS region), encoding protein 209 aa; the human codon optimized version is synonymously replaced with the reference sequence, and the protein amino acid sequence remains unchanged. See the corresponding entry in NCBI NP.

[0069] (v) Self-cleaving 2A peptide sequence

[0070] In the OSKGL multicistronic effector element, the various factors are separated by different 2A self-cleaving peptides to achieve independent expression of multiple proteins. The coding sequences of each 2A peptide (including the N-terminal GSG linker to improve cleavage efficiency) are as follows: P2A (Porcine Chancell Virus-1 2A, 22 aa, SEQ ID NO:8) Amino acid sequence: GSGATNFSLLKQCGDVEENPGP Nucleotide sequence (SEQ ID NO:9 (66 bp), human codon optimized): GGTAGCGGAGCCACCAACTTCTCCCTGCTGAAGCAGTGCGGCGACGTGGAGGAAAACCCCGGCCCC T2A (Lice-moth virus 2A, 21 aa, SEQ ID NO:10) Amino acid sequence: GSGEGRGSLLTCGDVEENPGP Nucleotide sequence (SEQ ID NO:11 (63 bp), human codon optimized): GGTAGCGGAGAGGGCAGGGGCAGCCTGCTGACATGCGGCGACGTGGAGGAAAATCCCGGCCCC E2A (Equine rhinitis virus A2A, 23aa, SEQ ID NO:12) Amino acid sequence: GSGQCTNYALLKLAGDVESNPGP Nucleotide sequence (SEQ ID NO:13 (69 bp), human codon optimized): GGTAGCGGACAGTGCACCAATTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC F2A (Foot-and-mouth disease virus 2A, 25 aa, SEQ ID NO:14) Amino acid sequence: GSGVKQTLNFDLLKLAGDVESNPGP Nucleotide sequence (SEQ ID NO:15 (75 bp), human codon optimized): GGTAGCGGAGTCAAGCAGACCCTGAATTTTGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC The GSAG short linker (4 aa, SEQ ID NO:16) between KLF4 and GLIS1 encodes a nucleotide sequence (12 bp, SEQ ID NO:17): SEQ ID NO:16(AA): GSAG SEQ ID NO:17 (NA): GGTAGCGCCGGC

[0071] (vi) OSKGL five-factor multicistronic effector overall nucleic acid sequence

[0072] The full-length nucleotide sequence of the OSKGL polycistronic effect element (SEQ ID NO:35) is approximately 8,496 bp, linked by the following framework structure. None of the elements are preceded by a stop codon, except for the Lin28A coding sequence which carries a stop codon (TGA) at the end: [OCT4 CDS]—[P2A]—[OCT4 CDS]—[T2A]—[OCT4 CDS]—[E2A]—[SOX2 CDS]—[F2A]— [KLF4 CDS]—[GSAG]—[GLIS1 CDS]—[P2A]—[LIN28A CDS]—[TGA] OCT4 CDS ×3 3 × 1,080 bp = 3,240 bp Three copies achieve a 3:1:1:1:1 protein ratio P2A + T2A + E2A 66+63+69 = 198 bp Intercopylic fragmentation of OCT4 and OCT4-SOX2 SOX2 CDS 951 bp F2A 75 bp SOX2-KLF4 inter-fraction KLF4 CDS 1,437 bp GSAG connector 12 bp KLF4-GLIS1 fusion expression GLIS1 CDS 1,863 bp P2A 66 bp GLIS1-LIN28A inter-fraction LIN28A CDS + Termination 627 + 3 = 630 bp Total: ~8,472 bp (including stop codon) The complete nucleotide sequence described above is assembled from the component sequences in the order presented. In practical applications, each coding sequence can be optimized using human codons as needed, without altering the protein sequence itself.

[0073] Note: The GAL4-DBD coding sequence, FRB(T2098L) coding sequence, VP64 coding sequence, full-length coding sequence of the second fusion protein, and full-length coding sequence of the first fusion protein have all been optimized with human codons.

[0074] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0075] Example 1

[0076] 1. Experimental Methods

[0077] (1) Model establishment: 105 eight-week-old female SKH-1 hairless mice, except for the G1 group (n=15), the remaining 90 mice were subjected to UVB irradiation for 10 weeks (dose: 150 mJ / cm² each time, 3 times a week) to induce photoaging.

[0078] (2) Grouping and intervention: n=15, intervention lasted for 6 weeks.

[0079] The grouping is as follows:

[0080] (3) All interventions lasted for 6 weeks, and the endpoint assessment was conducted 24 hours after the end of the intervention.

[0081] 2. Experimental Results

[0082] 2.1 Evidence for the core mechanism: Quantitative reversal of skin epigenetic age

[0083] The experimental endpoint was the isolation of dermal fibroblasts for DNA methylation sequencing analysis (using a mouse-specific DNA methylation clock, following the method established by Thompson et al. 2018, Thompson MJ, Chwia). kowska K, Rubbi L, Lusis AJ, Davis RC, Srivastava A, Korstanje R, Churchill GA, Horvath S, Pellegrini M. A multi-tissue full lifespan epigenetic clock for mice. Aging(Albany NY). 2018 Oct 21; 10(10): 2832–2854. DOI: 10.18632 / aging.101590.PMID: 30348905.).

[0084] Table 1. Epigenetic age acceleration values ​​of dermal fibroblasts in the skin

[0085] Note: SD represents standard deviation; NS indicates no statistical difference.

[0086] Conclusion: All three OSKGL reprogramming interventions based on the rapamycin-induced aging system significantly reversed the aging clock of skin cells, with effects far superior to the positive-positive drug retinoic acid. Traditional botulinum toxin therapy had no significant effect on epigenetic age, confirming that its effects do not address the fundamental mechanisms of aging.

[0087] 2.2 Evidence for Skin Tissue Structure Regeneration

[0088] Table 2 Quantitative analysis results of skin tissue structure

[0089] Conclusion: Both the rapamycin-inducible nucleic acid delivery system and the novel small molecule composition significantly promoted dermal structural reconstruction, while the botulinum toxin mimic group had no promoting effect on collagen synthesis and caused a slight decrease in dermal thickness due to the inhibition of the inflammatory response, highlighting the limitations of traditional therapies in structural repair.

[0090] 2.3 Evidence of Improved Skin Biomechanical Function

[0091] Table 3. Changes in skin elasticity recovery rate (R5 value)

[0092] Conclusion: All rapamycin-induced reprogramming interventions significantly improved skin elasticity, with improvements far exceeding those of traditional therapies.

[0093] 2.4 Verification of Expression Controllability

[0094] After the intervention in group G5 (AAV-rapamycin-induced type), skin samples were collected at different time points (0h, 12h, 24h, 48h, 72h) after the withdrawal of AP21967 to detect the expression level of OSKGL protein.

[0095] Results: 12 hours after AP21967 withdrawal, OSKGL protein levels decreased to below 50% of peak value; 24 hours to below 20%; and 48 hours to baseline levels. LC-MS / MS analysis showed that 24 hours after withdrawal, the concentration of AP21967 in skin tissue was below 1 nM, far below the induction threshold (10-50 nM). This confirms that gene expression can be rapidly and effectively shut down by withdrawing the inducer, achieving the design goal of "partial reprogramming" rather than "continuous reprogramming".

[0096] 2.5 Systemic Security Assessment

[0097] Table 4 Security Incident Statistics

[0098] Note: There were no statistically significant differences in the incidence of any safety indicators among the groups.

[0099] Conclusion: All intervention modalities based on the rapamycin-inducible OSKGL system and novel small molecule compositions of this invention did not introduce any additional risk of local or systemic tumor development during the experimental observation period, demonstrating good safety. The unique "zero leakage" characteristic of the rapamycin system (no expression without inducer) further enhances the safety of clinical translation.

[0100] Example 2

[0101] Validation of wrinkle-improving effects based on rapamycin-induced mRNA system

[0102] 1. Experimental Design

[0103] (1) Model establishment: 72 eight-week-old female SKH-1 hairless mice were exposed to UVB for 12 weeks (dose: 150mJ / cm² each time, 3 times a week) to induce skin photoaging and wrinkle formation.

[0104] (2) Grouping and intervention: n=12 per group, intervention lasts for 8 weeks:

[0105] (3) Endpoint assessment should be conducted 24 hours after the intervention ends.

[0106] 2. Experimental Results

[0107] 2.1 Quantitative Analysis of Wrinkles

[0108] The skin wrinkle impressions on the back of mice were collected using the silicone replication method, and the wrinkle parameters were quantified using a computer image analysis system.

[0109] Table 5 Results of Quantitative Analysis of Wrinkles

[0110] Wrinkle improvement rate (%) = (Wrinkle area in group G2 - Wrinkle area in the intervention group) / Wrinkle area in group G2 × 100%

[0111] Group G2, the photoaging model group (mean wrinkle area 38.5 mm²), served as the pathological baseline for calculating the improvement rate. The improvement rate was calculated using wrinkle area as the core indicator, reflecting the degree of wrinkle area reduction achieved by each intervention relative to the photoaging model.

[0112] 2.2 Histological analysis

[0113] After the intervention, back skin was taken for Masson staining and H&E staining to assess collagen fiber arrangement and epidermal structure.

[0114] result: Group G5 (mRNA-rapamycin-induced type): The dermis is significantly thickened, collagen fibers are neatly and densely arranged in a reticular interwoven structure, elastic fibers proliferate significantly, the thickness of the epidermis returns to normal, and the stratum corneum structure is intact.

[0115] Group G6 (small molecule induced): The dermis thickened significantly, collagen fiber density increased, the arrangement was more orderly, and the number of elastic fibers increased.

[0116] Group G3 (retinoic acid): The epidermis thickened, but the improvement in collagen fibers in the dermis was limited, and a mild inflammatory response was present.

[0117] Group G4 (botulinum toxin): There was no significant difference compared with the model group; the collagen fibers were still loosely arranged.

[0118] 2.3 Validation of Molecular Mechanism

[0119] The expression of collagen synthesis-related genes in dermal fibroblasts was detected by qPCR.

[0120] Table 6. Changes in the expression of collagen synthesis-related genes.

[0121] Conclusion: OSKGL reprogramming intervention based on the rapamycin-inducible mRNA system can significantly reverse the formation of wrinkles caused by photoaging, and its effect is significantly better than that of traditional therapies in terms of wrinkle area, number, and depth. Mechanistically, this intervention significantly upregulates the expression of type I and type III collagen, while downregulating the expression of matrix metalloproteinase-1, thus synergistically promoting dermal matrix reconstruction from both collagen synthesis and degradation directions, achieving fundamental improvement in wrinkles.

[0122] Example 3

[0123] Validation of scar improvement effect based on rapamycin-induced AAV system

[0124] 1. Experimental Design

[0125] (1) Model establishment: 48 eight-week-old female C57BL / 6J mice were used to create a full-thickness skin excision wound with a diameter of 6 mm on their backs. The wound healed naturally for 4 weeks to form a hypertrophic scar model.

[0126] (2) Grouping and intervention: n=8 per group, intervention lasts for 6 weeks (starting after scar formation):

[0127] (3) Endpoint assessment should be conducted 24 hours after the intervention ends.

[0128] 2. Experimental Results

[0129] 2.1 Scar area and appearance assessment

[0130] High-resolution imaging and ImageJ software were used to analyze scar area, color, and elevation. The scar improvement rate was calculated as follows: Scar improvement rate (%) = (Scar area in group G2 - Scar area in intervention group) / Scar area in group G2 × 100% Group G2 was the scar model group (mean scar area 28.5 mm²), serving as the pathological baseline for calculating the improvement rate. The improvement rate was calculated using scar area as the core indicator, reflecting the degree of area reduction achieved by each intervention relative to the scar model.

[0131] Table 7 Results of Scar Quantitative Analysis

[0132] 2.2 Histological analysis

[0133] After the intervention, scar tissue was taken for Masson staining, Sirius red staining, and H&E staining.

[0134] result: Group G4 (AAV-rapamycin-induced type): Scar tissue was significantly remodeled, collagen fiber arrangement was restored from disordered nodular to bundle arrangement parallel to normal skin, the ratio of type I collagen to type III collagen was restored to normal (about 4:1), fibroblast density was reduced, there was no abnormal proliferation, and hair follicle and sebaceous gland structures were partially regenerated.

[0135] Group G5 (mRNA-rapamycin induced type): The scar area was significantly reduced, the collagen fiber arrangement was significantly improved, the number of fibroblasts was reduced, and the epidermal structure tended to be normal.

[0136] Group G6 (small molecule induced): Significant improvement in scar tissue, more orderly arrangement of collagen fibers, and reduced density of fibroblasts.

[0137] Group G3 (Triamcinolone): The scar area has shrunk, but the dermis has thinned, the collagen fibers are still arranged in a disordered manner, and there is local atrophy.

[0138] 2.3 Validation of Molecular Mechanism

[0139] The expression of fibrosis-related genes in scar tissue was detected by qPCR.

[0140] Table 8. Changes in the expression of fibrosis-related genes.

[0141] 2.4 Security Assessment

[0142] Table 9 Security Incident Statistics

[0143] Conclusion: OSKGL reprogramming intervention based on the rapamycin-inducible system significantly improves hypertrophic scars, with superior results compared to traditional glucocorticoid treatment in terms of scar area, elevation, and pigmentation. Mechanistically, this intervention significantly inhibits the expression of α-SMA, TGF-β1, and COL1A1, promoting the transformation of collagen fibers from a disordered nodular arrangement to a parallel bundle arrangement, thus achieving structural remodeling of scar tissue. Importantly, unlike glucocorticoids, the rapamycin-inducible system does not cause skin atrophy and has a superior safety profile.

[0144] As can be seen from the above embodiments, the quantitative partial reprogramming system provided by the present invention is safe and effective, and can fundamentally reverse and repair skin aging from three levels: epigenetics, tissue structure and biomechanics, and can effectively reduce wrinkles and scars.

[0145] Rapamycin-inducible nucleic acid delivery systems (AAV and mRNA) are most effective in reversing the aging clock and promoting matrix regeneration. Their "zero leakage" characteristics and rapid shutdown kinetics provide extremely high safety assurance for clinical translation. Although the induction pathway of novel small molecule compositions is slightly inferior, it exhibits a clear and significant "root cause treatment" effect, providing an important and optimized technical path for developing non-invasive and convenient skin anti-aging products. The "quantitative reprogramming" anti-aging paradigm represented by this invention is fundamentally different from traditional therapies such as botulinum toxin and retinoic acid, which only target symptoms, in terms of mechanism of action and ultimate effect, and has comprehensive and significant advantages.

[0146] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a quantitative partial reprogramming system in the preparation of drugs to reverse skin aging, characterized in that, The quantitative partial reprogramming system includes a nucleic acid delivery system or a small molecule induction system.

2. The application according to claim 1, characterized in that, Skin aging includes skin aging caused by photoaging.

3. The application according to claim 2, characterized in that, The reversal of skin aging includes at least one of the following: increasing the thickness of the dermis, increasing the density of type I collagen, improving skin elasticity, reducing the depth of wrinkles, and reducing the area of ​​scars.

4. The application according to claim 1, characterized in that, The nucleic acid delivery system includes a gene expression system induced by rapamycin or its analogue AP21967; the gene expression system includes: (a) The nucleic acid encoding the first fusion protein, the first fusion protein comprising an FKBP12 variant triplet repeat domain and a DNA-binding domain; (b) The nucleic acid encoding the second fusion protein, which contains an FRB variant domain and a transcription activation domain; (c) Effect elements encoding five reprogramming factors: Oct4, Sox2, Klf4, Glis1, and Lin28; said effect elements being operatively linked to an inducible promoter containing a cis-regulatory element specifically recognized by said DNA-binding domain; (d) Inducer: rapamycin or rapamycin analog AP21967.

5. The application according to claim 4, characterized in that, The FKBP12 variant is FKBP12(F36V), the FRB variant is FRB(T2098L), and the inducer is AP21967. The DNA binding domain is GAL4-DBD at amino acid positions 1-147 of the yeast GAL4 transcription factor, and the cis-regulatory element comprises at least four tandem GAL4 upstream activation sequences UAS(GAL4). The transcriptional activation domain is the VP64-p65-Rta triple activation domain VPR.

6. The application according to claim 5, characterized in that, The nucleic acid delivery system includes a recombinant adeno-associated virus vector carrying: (a) a first AAV vector containing nucleic acid encoding the first fusion protein operably linked to a promoter; (b) a second AAV vector containing nucleic acid encoding the second fusion protein operably linked to a promoter; and (c) a third AAV vector containing effector elements encoding five reprogramming factors, Oct4, Sox2, Klf4, Glis1, and Lin28, operably linked to an inducible promoter.

7. The application according to claim 4, characterized in that, The nucleic acid delivery system includes mRNA encapsulated by lipid nanoparticles, wherein the mRNA comprises mRNA-1 encoding the first fusion protein, mRNA-2 encoding the second fusion protein, and mRNA-3 encoding five reprogramming factors: Oct4, Sox2, Klf4, Glis1, and Lin28. Alternatively, the mRNA may be a single polycistronic mRNA whose coding sequence is linked in the following order: OCT4-(P2A)-OCT4-(T2A)-OCT4-(E2A)-SOX2-(F2A)-KLF4-(GSAG)-GLIS1-(P2A)-LIN28.

8. The application according to claim 6, characterized in that, The mRNA is a chemically modified mRNA, in which uracil is wholly or partially replaced by N1-methylpseudouracil.

9. The application according to claim 1, characterized in that, The small molecule induction system includes valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, and foctocorline; wherein the concentration of valproic acid is 200-300 μM, the concentration of CHIR99021 is 5-15 μM, the concentration of Repsox is 5-15 μM, the concentration of transphenylcyclopropylamine is 4-6 μM, and the concentration of foctocorline is 40-60 μM.

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