A quantitative epigenetic reprogramming method, reagent and application thereof in preparation of a drug for reversing organ fibrosis

Abstract

The application provides a quantitative epigenetic reprogramming method, reagents and application thereof in reversing organ fibrosis drugs, and belongs to the technical field of biological medicines.The epigenetic age of cells is reduced by 57%-77% by a gene reprogramming method; the gene reprogramming method is that mRNA sequences coding OCT4, SOX2, KLF4 and GLIS1 are delivered to cells for 2-3 weeks, and expression is induced by doxycycline for 1-2 weeks. The application solves the industry problem that reprogramming cannot be carried out in vivo by a quantitative epigenetic partial reprogramming strategy, reverses pathological cells to functional or resting state cells without changing the cell identity, and is not a pluripotent stem cell, thereby solving the industry problem that reprogramming cannot be carried out in vivo, realizing safe reversal of fibrosis in an animal model, and significantly reducing the epigenetic age, and reversing a disease that was once incurable.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a quantitative epigenetic reprogramming method, reagents, and their application in drugs for reversing organ fibrosis. Background Technology

[0002] In vitro induced pluripotent stem cell (iPSC) technology is a typical application of cell reprogramming. This technology completely reprograms somatic cells into a pluripotent stem cell state by overexpressing specific transcription factors (such as OCT4, SOX2, KLF4, and c-MYC), thereby achieving a complete reset of epigenetic state and cell fate. However, this process has an "all-or-nothing" characteristic: it starts with somatic cells with a specific epigenetic age and ends with pluripotent stem cells with an epigenetic age of zero. Initiating such a reprogramming process in vivo often induces tumors or even leads to animal death due to the fundamental change in cell identity. The root cause is that this process is difficult to stably maintain in any intermediate state—that is, to achieve a reduction in epigenetic age without changing the cell identity (e.g., lung cells remain lung cells).

[0003] Furthermore, while this complete reprogramming approach has shown potential to reverse cellular senescence and disease in in vitro laboratory studies, its in vivo application has significant drawbacks: First, complete reprogramming to a pluripotent state carries an extremely high risk of tumorigenesis, as pluripotent stem cells may differentiate into tumor cells, leading to cancer, which limits its clinical safety. Second, current technologies cannot effectively reverse the pathological state of fibrosis; there are currently no approved therapies that can substantially improve fibrosis scores, collagen deposition, or organ failure, leaving fibrosis treatment at the stage of symptom relief rather than fundamental reversal. These shortcomings severely hinder the practical application of iPSC technology in fibrosis treatment, highlighting the urgent need for safer and more targeted intervention methods. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a quantitative epigenetic reprogramming method, reagent, and its application in drugs for reversing fibrosis. This invention avoids the above-mentioned risks through a "quantitative epigenetic partial reprogramming" strategy, quantitatively reversing only pathological cells into functional or resting cells, rather than pluripotent stem cells, thereby solving the industry problem of not being able to reprogram in vivo. It has achieved safe reversal of fibrosis in animal models and significantly reduced epigenetic age, reversing a previously incurable disease.

[0005] The first objective of this invention is to provide a quantitative epigenetic reprogramming method that reduces the epigenetic age of cells by 57% to 77% through gene reprogramming.

[0006] Preferably, the gene reprogramming method involves delivering mRNA sequences encoding OCT4, SOX2, KLF4, and GLIS1 into cells for 2-3 weeks, followed by doxycycline-induced expression for 1-2 weeks.

[0007] Preferably, the delivery is achieved via adeno-associated virus carrying mRNA or mRNA encapsulated by lipid nanoparticles.

[0008] A second object of the present invention is to provide a reagent for quantitative epigenetic reprogramming, comprising a delivery system for delivering mRNA of OCT4, SOX2, KLF4 and GLIS1 and an inducing agent doxycycline.

[0009] Preferably, the delivery system includes: (1) AAV1-inducer that delivers third-generation reverse tetracycline transactivator protein rtTA3 expresses viral pAAV-rtTA3; (2) AAV2-core reprogramming factor virus pAAV-TRE-tight-OSK that delivers mRNA of OCT4, SOX2 and KLF4; (3) AAV3-key regulator virus pAAV-TRE-tight-OG that delivers OCT4, GLIS1 mRNA.

[0010] (4) AAV3-key regulator of the virus pAAV-TRE-tight-OL that delivers OCT4,Lin28 mRNA.

[0011] Preferably, the promoter of the AAV2-core reprogramming factor virus pAAV-TRE-tight-OSK is the TRE3G promoter, and the inserted target gene is a fusion gene sequence encoding OCT4-P2A-SOX2-P2A-KLF4.

[0012] Preferably, the delivery system comprises OCT4, SOX2, KLF4, GLIS and Lin28 mRNA encapsulated in lipid nanoparticles, and the molar ratio of OCT4, SOX2, KLF4, GLIS and Lin28 is 3:1:1:1:1.

[0013] Preferably, the mRNA sequence encapsulated by the lipid nanoparticles consists of the following sequentially linked sequences: OCT4-(P2A)-OCT4-(T2A)-OCT4-(E2A)-SOX2-(F2A)-KLF4-(GSAG)-GLIS1-(P2A)-LIN28.

[0014] A third objective of this invention is to provide the use of the described reagent in the preparation of medicaments for treating organ fibrosis.

[0015] Preferably, the organ fibrosis includes pulmonary fibrosis and liver fibrosis.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a quantitative epigenetic reprogramming method, which reduces the epigenetic age of cells by 57% to 77% through gene reprogramming; the present invention has found that reducing the epigenetic age of cells by 57% to 77% can achieve the effect of "maintaining the cell identity unchanged while significantly reversing its epigenetic age and restoring the cell regeneration potential", providing a new treatment approach for diseases that were previously incurable.

[0017] Furthermore, the present invention provides a reagent for quantitative epigenetic reprogramming, including a delivery system for delivering mRNAs of OCT4, SOX2, KLF4, GLIS1 and Lin28 and an inducing agent doxycycline; the present invention uses GLIS1 to replace c-MYC, which has a high carcinogenic risk in traditional reprogramming, thus significantly improving safety.

[0018] High safety is the core advantage of this invention. Existing iPSC technology reverses somatic cells into pluripotent stem cells through complete reprogramming, but these cells have an extremely high risk of tumorigenesis in vivo. This invention employs a "partial reprogramming" strategy, precisely controlling the combination of reprogramming factors (replacing c-MYC with GLIS1 and forming a novel combination with OCT4, SOX2, and KLF4), expression dosage, and duration of action to reverse pathological cells to a functional or resting state, rather than inducing them into the pluripotent stem cell stage. This achieves precise control over the reprogramming process, and no tumor formation was observed during the experiment, providing a crucial safety guarantee for clinical translation.

[0019] This invention has demonstrated the powerful therapeutic efficacy of its "quantitative epigenetic reprogramming method" in animal models. By partially reprogramming and resetting the epigenetic state of cells, the core pathological features of fibrosis can be significantly reversed. As shown in the example data, the treatment significantly reduced the fibrosis score (Ashcroft / Ishak score), decreased collagen deposition (hydroxyproline content), and the number of activated myofibroblasts (α-SMA-positive cells). Simultaneously, organ function indicators such as lung compliance, airway resistance, and serum ALT / AST also improved, demonstrating that the treatment not only repairs tissue structure but also restores organ physiological function.

[0020] This invention transcends existing symptom-relieving treatment models, achieving for the first time a significant reduction in the epigenetic age of organs while reversing fibrosis. Genome-wide DNA methylation analysis (based on the Horvath / Hannum clock algorithm) shows that the biological age of organs regresses to a younger state after treatment. The degree of epigenetic age reversal is strongly positively correlated with the improvement in fibrosis indicators (r=0.91), indicating that this invention achieves a fundamental intervention in age-related fibrotic diseases by resetting the disease's "cellular memory" and "biological clock."

[0021] In summary, this invention, through its unique "quantitative partial reprogramming" strategy, not only effectively reverses the pathological structure and function of fibrosis while ensuring safety, but also innovatively reverses its epigenetic age, thereby achieving a comprehensive therapeutic effect that is unattainable by existing technologies. Attached Figure Description

[0022] Figure 1 This diagram illustrates the difference between the quantitative epigenetic reprogramming method provided by this invention and iPS cell induction.

[0023] Figure 2 This is a schematic diagram of the quantitative epigenetic reprogramming method of the present invention. Detailed Implementation

[0024] This invention provides a quantitative epigenetic reprogramming method that reduces the epigenetic age of cells by 57% to 77% through gene reprogramming. This invention does not specifically limit the gene reprogramming method; any gene reprogramming method that can reduce the epigenetic age of cells by 57% to 77% is acceptable. This invention has found that when the epigenetic age of cells is reduced by 57% to 77%, it precisely achieves the effect of "maintaining the cell's identity while significantly reversing its epigenetic age and restoring the cell's regenerative potential." That is, within the above range, only pathological cells are reversed into functional or quiescent cells, rather than pluripotent stem cells; this avoids the situation where fundamental changes in cell identity induce tumors or even lead to individual death.

[0025] In this invention, the gene reprogramming method involves delivering mRNA sequences encoding OCT4, SOX2, KLF4, GLIS1, and Lin28 into cells for 2-3 weeks, followed by doxycycline induction for 1-2 weeks. Preferably, the delivery is achieved via adeno-associated virus carrying mRNA or mRNA encapsulated in lipid nanoparticles. The preferred concentration of doxycycline used is 0.1-2.0 mg / mL; the concentration and duration of doxycycline administration determine the expression levels of OSK and GLIS1. By adjusting the dosage and duration of doxycycline, the reprogramming process can be precisely controlled within the range of "partial reprogramming," thereby achieving 57%-77% epigenetic age reversal while preventing the cell identity from transforming into pluripotent stem cells.

[0026] The present invention also provides a reagent for quantitative epigenetic reprogramming, including a delivery system for delivering mRNA of OCT4, SOX2, KLF4, GLIS1 and Lin28 and an inducing agent doxycycline.

[0027] In this invention, the delivery system comprises: (1) an AAV1-inducer expression virus pAAV-rtTA3 that delivers the third-generation reverse tetracycline transactivator protein rtTA3; (2) an AAV2-core reprogramming factor virus pAAV-TRE-tight-OSK that delivers the mRNA of OCT4, SOX2, and KLF4; (3) an AAV3-key regulator virus pAAV-TRE-tight-OG that delivers the mRNA of OCT4 and GLIS1; and (4) an AAV3-key regulator virus pAAV-TRE-tight-OL that delivers the mRNA of OCT4 and Lin28.

[0028] In this invention, the function of the AAV1-inducer expressing virus pAAV-rtTA3 is constitutive expression of a highly sensitive third-generation inverse tetracycline transactivator protein. A broad-spectrum, potent CMV promoter is used, or a tissue-specific promoter (such as hSP-C in the lungs or TBG in the liver) is selected to achieve organ-specific regulation. The target gene carried by pAAV-rtTA3 is a cDNA sequence encoding the rtTA3 protein. Compared with earlier versions, rtTA3 has higher doxycycline sensitivity and lower background leakage activity. Preferably, the AAV1-inducer expressing virus pAAV-rtTA3 also includes a selection marker, preferably a puromycin resistance gene. The AAV1-inducer expressing virus pAAV-rtTA3 is linked to the target gene via a P2A sequence for screening stable cell lines. If used in vivo, the selection marker should be omitted. Preferably, the AAV1-inducer expressing virus pAAV-rtTA3 carries a polyA signal: bGH polyA.

[0029] In this invention, the aforementioned delivery system is capable of expressing five core reprogramming factors—OCT4, SOX2, KLF4, Glis1, and Lin28—in the presence of an inducer. In this invention, the promoter of the AAV2-core reprogramming factor virus pAAV-TRE-tight-OSK is preferably the TRE3G promoter. TRE3G is an optimized, minimal promoter that is efficiently activated only when bound to the rtTA3 protein and in the presence of doxycycline, exhibiting extremely low background activity and high inducibility. The target gene inserted by the AAV2-core reprogramming factor virus pAAV-TRE-tight-OSK is a fusion gene sequence encoding OCT4-P2A-SOX2-P2A-KLF4. This fusion gene sequence ensures that the three core reprogramming factors, OCT4, SOX2, and KLF4, are expressed in a near stoichiometric ratio of 1:1:1. The AAV2-core reprogramming factor virus pAAV-TRE-tight-OSK preferably carries the polyA signal: SV40 polyA. The AAV2-core reprogramming factor virus pAAV-TRE-tight-OG (Oct4-P2A-Glis1) preferably carries the polyA signal: SV40 polyA. The AAV2-core reprogramming factor virus pAAV-TRE-tight-OL (OCt4-P2A-Glis1) preferably carries the polyA signal: SV40 polyA. The aforementioned delivery system ensures that the molar ratio of OCT4, SOX2, KLF4, GLIS, and Lin28 is 3:1:1:1:1.

[0030] The amino acid sequence of OCT4 is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2; the amino acid sequence of SOX2 is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 4; the amino acid sequence of KLF4 is shown in SEQ ID NO: 5, and the nucleotide sequence is shown in SEQ ID NO: 6; the amino acid sequence of Lin28 is shown in SEQ ID NO: 7, and the nucleotide sequence is shown in SEQ ID NO: 8.

[0031] In this invention, the function of the AAV3-key regulatory factor virus pAAV-TRE-tight-OG is to express the key regulatory factors OCT4 and GLIS1 in the presence of an inducer. The promoter of the AAV3-key regulatory factor virus pAAV-TRE-tight-OG is preferably the TRE3G promoter, and the inserted target gene is a cDNA sequence encoding the proteins OCT4 and GLIS1; preferably, it carries a polyA signal: SV40 polyA.

[0032] The amino acid sequence of the GLIS1 protein is shown in SEQ ID NO: 9, and the nucleotide sequence is shown in SEQ ID NO: 10.

[0033] In this invention, the function of the AAV3-key regulatory factor virus pAAV-TRE-tight-OL is to express the key regulatory factors OCT4 and Lin28 in the presence of an inducer. The promoter of the AAV3-key regulatory factor virus pAAV-TRE-tight-OL is preferably the TRE3G promoter, and the inserted target gene is a cDNA sequence encoding the proteins OCT4 and Lin28; preferably, it carries a polyA signal: SV40 polyA.

[0034] The delivery system described in this invention packages reprogramming factors under independent, strictly controlled TRE3G promoters, avoiding potential risks caused by excessively large capacity of a single vector or expression imbalance.

[0035] This invention allows for precise regulation of OSK and GLIS1 expression levels through controlled doxycycline dosage, thereby achieving quantitative control over the "depth" of reprogramming. This is crucial for achieving "partial reprogramming" rather than "complete reprogramming." By administering doxycycline within a specific time window, this invention precisely controls the initiation and duration of the reprogramming process, preventing indefinite reprogramming.

[0036] In this invention, the delivery system can also be used for the mRNA of OCT4, SOX2, KLF4 and GLIS encapsulated in lipid nanoparticles.

[0037] In this invention, the mRNA sequence encapsulated by the lipid nanoparticles consists of the following sequentially linked sequences: OCT4-(P2A)-OCT4-(T2A)-OCT4-(E2A)-SOX2-(F2A)-KLF4-(GSAG)-GLIS1-(P2A)-LIN28.

[0038] The sequence information for OCT4, SOX2, KLF4, and GLIS is as described in the delivery system above.

[0039] In this invention, P2A, T2A, E2A, F2A, and GSAG in the mRNA sequence are the mRNA sequences of linking peptides. This invention achieves the synergistic expression of five reprogramming factors (OCT4, SOX2, KLF4, GLIS1, LIN28) through a single mRNA molecule, precisely controlling their internal expression ratio to OCT4 : SOX2 : KLF4 : GLIS1 : LIN28 = 3 : 1 : 1 : 1 : 1. This design, based on reprogramming mechanism research, aims to more efficiently initiate the reprogramming process by increasing the dosage of the dominant factor OCT4, while potentially reducing the overall mRNA dosage and treatment frequency, further improving safety and efficacy.

[0040] Specifically, the mRNA sequence in this invention is designed as a non-equimolar coding sequence: to achieve triploid expression of OCT4, the core coding region is designed as: OCT4-(P2A)-OCT4-(T2A)-OCT4-(E2A)-SOX2-(F2A)-KLF4-(GSAG)-GLIS1-(P2A)-LIN28. During translation, this sequence first generates a "polyprotein" containing all the proteins.

[0041] P2A, T2A, E2A, and F2A are highly efficient self-cleaving peptides. When the ribosome translates to these sequences, it introduces a "break" between the peptide chains, thereby releasing the independent protein post-translation. By tandemly linking three OCT4 coding units (each followed by a 2A peptide) at the front, it is ensured that three OCT4 protein molecules are generated from a single mRNA, while SOX2, KLF4, GLIS1, and LIN28 each generate one.

[0042] A short, flexible GSAG linker peptide is used between KLF4 and GLIS1 to allow the necessary spatial separation of the two domains while maintaining a certain degree of correlation to optimize folding or functional synergy.

[0043] Furthermore, the present invention has performed deep molecular optimization on the mRNA sequence: (1) Species-specific (e.g. mouse) codon optimization was performed on the coding sequences of OCT4, SOX2, KLF4, GLIS1, and LIN28, and repetitive sequences were avoided to maximize translation efficiency and prevent homologous recombination.

[0044] (2) UTR enhancement: 5' UTR: Uses a UTR from the human HBB gene and inserts an internal ribosome entry site-like sequence to further enhance translation initiation. 3' UTR: Uses a hybrid stable UTR from the human ALB and APOE genes and adds a poly(A)150 tail to provide excellent stability for the mRNA.

[0045] (3) Comprehensive nucleotide modification: Use 100% N1-methylpseudouridine to replace uridine, and 5-methylcytidine can be used to partially replace cytidine to synergistically reduce immune recognition.

[0046] In this invention, the preferred method for synthesizing the mRNA sequence is as follows: Using a linearized plasmid as a template, in vitro transcription is performed using T7 polymerase. Co-transcriptional capping is performed using CleanCap AG to ensure a Cap1 structure generation rate of >95%. The mRNA product is then purified by HPLC to obtain a single-peak, highly intact product.

[0047] In this invention, after synthesizing the mRNA sequence, LNP encapsulation is performed. The preferred lipid formulation follows a liver-targeting formulation, such as MC3 or SM-102 / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 mol%. This invention preferably uses microfluidic technology to encapsulate the mRNA into LNPs. The preferred particle size of the lipid nanoparticles prepared in this invention is 80 ± 5 nm; PDI < 0.15; encapsulation efficiency > 98%. This invention preferably verifies the integrity of the mRNA sequence and the absence of degradation by gel electrophoresis.

[0048] This invention also provides the use of the described reagent in the preparation of medicaments for treating organ fibrosis. In this invention, the organ fibrosis preferably includes pulmonary fibrosis and liver fibrosis. This invention does not impose any particular limitation on the dosage form and method of administration of the medicament; conventional dosage forms and methods of administration in the art can be used, such as nasal drops or intravenous injection.

[0049] 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.

[0050] Example 1

[0051] Recombinant AAV Delivery System for Quantitative Induction of Partial Reprogramming and Its Preparation Method

[0052] I. System Design and Plasmid Construction

[0053] This system consists of three different recombinant AAV viruses, which together form a complete inducible, repackaged, and reprogrammable system.

[0054] 1. AAV1 - inducer to express virus (pAAV-rtTA3)

[0055] Function: Constitutive expression of a highly sensitive third-generation reverse tetracycline transactivator protein.

[0056] Carrier structure: Promoters: Use broad-spectrum, potent CMV promoters, or select tissue-specific promoters (such as hSP-C in the lungs or TBG in the liver) to achieve organ-specific regulation.

[0057] Target gene: cDNA sequence encoding the rtTA3 protein. Compared to earlier versions, rtTA3 exhibits higher doxycycline sensitivity and lower background leakage activity.

[0058] Selection marker: May include a puromycin resistance gene linked to the target gene via a P2A sequence, used for screening stable cell lines (omitted for in vivo application).

[0059] polyA signal: bGH polyA.

[0060] 2. AAV2 - Core Reprogramming Factor Virus (pAAV-TRE-tight-OSK)

[0061] Function: In the presence of an inducer, it expresses three core reprogramming factors: OCT4, SOX2, and KLF4.

[0062] Carrier structure: Promoter: TRE3G (Tetracycline Response Element) promoter. This is an optimized, minimal promoter that is efficiently activated only when bound to the rtTA3 protein and in the presence of doxycycline, exhibiting extremely low background activity and high inducibility.

[0063] Target gene: The fusion gene sequence encoding OCT4-P2A-SOX2-P2A-KLF4 ensures that the three factors are expressed in a near 1:1:1 stoichiometric ratio.

[0064] polyA signal: SV40 polyA.

[0065] 3. AAV3 - Key Regulatory Factor Virus (pAAV-TRE-tight-OG)

[0066] Function: In the presence of an inducer, it expresses the key regulatory factors OCT4 and GLIS1.

[0067] Carrier structure: Promoter: The same TRE3G promoter as AAV2.

[0068] Target genes: cDNA sequences encoding OCT4 and GLIS1 proteins.

[0069] polyA signal: SV40 polyA.

[0070] 4. AAV3 - Key Regulatory Factor Virus (pAAV-TRE-tight-OL)

[0071] Function: In the presence of an inducer, it expresses the key regulatory factors OCT4 and LIN28.

[0072] Carrier structure: Promoter: The same TRE3G promoter as AAV2.

[0073] Target genes: cDNA sequences encoding OCT4 and LIN28 proteins.

[0074] polyA signal: SV40 polyA.

[0075] Advantages of repackaging strategy: Safety: By disassembling the reprogramming factor into two independent, strictly controlled TRE3G promoters, potential risks caused by excessive capacity of a single vector or expression imbalance are avoided.

[0076] Quantitatively controllable: By controlling the dosage of doxycycline, the expression levels of OSK and GLIS1 can be precisely adjusted, thereby achieving quantitative control over the "depth" of reprogramming, which is the key to achieving "partial reprogramming" rather than "complete reprogramming".

[0077] Timing controllable: By administering the drug (doxycycline) within a specific time window, the initiation and duration of the reprogramming process can be precisely controlled, avoiding indefinite reprogramming.

[0078] II. Packaging and Preparation of Viruses

[0079] 1. Plasmid preparation: Construct the three types of plasmids described above, each containing AAV2 ITR sequences on both sides. The capsid protein plasmid is selected based on the target organ (AAV6 for lung, AAV8 for liver).

[0080] 2. Virus Packaging: The three AAV viruses were packaged in HEK293T cells using a three-plasmid co-transfection method. The packaging system for each virus was independent.

[0081] Packaging pAAV-rtTA3 virus.

[0082] Packaging the pAAV-TRE-tight-OSK virus.

[0083] Packaging the pAAV-TRE-tight-OG virus.

[0084] Packaging pAAV-TRE-tight-OL virus.

[0085] 3. Purification and Titration: Following standard methods (iodixanol gradient centrifugation and ultrafiltration concentration), the three viruses were purified and concentrated separately. The genomic titer (vg / mL) of each virus was accurately determined using qPCR.

[0086] Example 2

[0087] Design, preparation and application scheme of single-stranded non-equimolar expression mRNA (3×OCT4-SKGL)

[0088] I. mRNA sequence design

[0089] 1. Design of non-equimolar coding sequences: To achieve a threefold representation of OCT4, the core coding region is designed as: OCT4-(P2A)-OCT4-(T2A)-OCT4-(E2A)-SOX2-(F2A)-KLF4-(GSAG)-GLIS1-(P2A)-LIN28.

[0090] Design principle: During translation, the sequence first produces a "polyprotein" containing all the proteins.

[0091] P2A, T2A, E2A, and F2A are highly efficient self-cleaving peptides. When the ribosome translates to these sequences, it introduces a "break" between the peptide chains, thereby releasing the independent protein post-translation. By tandemly linking three OCT4 coding units (each followed by a 2A peptide) at the front, it is ensured that three OCT4 protein molecules are generated from a single mRNA, while SOX2, KLF4, GLIS1, and LIN28 each generate one.

[0092] Linker peptide optimization: A short, flexible GSAG linker peptide is used between KLF4 and GLIS1 to allow the necessary spatial separation of the two domains while maintaining a certain degree of correlation to optimize folding or functional synergy.

[0093] 2. Deep molecular optimization: Codon optimization: Species-specific (e.g., mouse) codon optimization was performed on the coding sequences of OCT4, SOX2, KLF4, GLIS1, and LIN28, and repetitive sequences were avoided to maximize translation efficiency and prevent homologous recombination.

[0094] UTR Enhancement: 5'UTR: Employs a UTR derived from the human HBB gene and may embed an internal ribosome entry site-like sequence to further enhance translation initiation.

[0095] 3' UTR: A hybrid stable UTR derived from the human ALB and APOE genes is used, with an additional poly(A)150 tail to provide excellent stability for the mRNA.

[0096] Comprehensive nucleotide modification: 100% N1-methylpseudouridine was used to replace uridine, and 5-methylcytidine was used to partially replace cytidine, in order to synergistically reduce immune recognition.

[0097] II. Preparation, Packaging and Quality Control

[0098] 1. In vitro transcription and modification of mRNA

[0099] Template plasmid linearization: Using the above pAAV-Transfer Plasmid as a template, or constructing an in vitro transcription-specific plasmid containing the same OCT4-SOX2-KLF4-GLIS1-P2A expression cassette, and linearizing it with restriction endonucleases.

[0100] In vitro transcription: T7 or SP6 RNA polymerase was used for in vitro transcription reaction.

[0101] 5' Capping: Co-transcriptional capping using CleanCap® Reagent (or similar) during or after transcription to generate Cap 1 structures is crucial for improving translation efficiency and reducing immunogenicity.

[0102] Nucleotide modification: Replacing UTP with N1-methylpseudouridine in the transcription reaction can significantly reduce the innate immune response of mRNA and enhance its stability and translation efficiency.

[0103] 3' tailing: A long tail of about 100-150 adenosine nucleotides is added to the 3' end of the mRNA using polyA polymerase to stabilize the mRNA.

[0104] Purification: mRNA was purified using LiCl precipitation or cellulose column chromatography to remove byproducts and unreacted nucleotides, and its integrity and concentration were verified by agarose gel electrophoresis and ultraviolet spectrophotometry.

[0105] 2. Fabrication and Packaging of LNPs

[0106] Using microfluidic technology to achieve efficient and uniform nanoparticle encapsulation is currently the gold standard method in the industry.

[0107] Composition of lipid mixture: Ionizable cationic lipids: comprising 50%, these are the core components of LNPs. They are positively charged under acidic conditions and are used to encapsulate negatively charged mRNA, promoting endosome escape in vivo. This invention uses SM-102 or ALC-0315.

[0108] Assisted phospholipids: accounting for 10%, such as DSPC, are used to stabilize the LNP bilayer structure.

[0109] Cholesterol: accounts for approximately 38.5%, and is used to regulate the fluidity and stability of LNP.

[0110] PEGylated lipids: accounting for 1.5%, such as DMG-PEG2000, are used to reduce LNP aggregation, prolong in vivo circulation time and reduce immunogenicity.

[0111] Microfluidic mixing: Ethanol phase: The above four lipids are dissolved in ethanol.

[0112] Aqueous phase: The purified mRNA was dissolved in citrate buffer.

[0113] Using a microfluidic mixing device, the ethanol and aqueous phases were mixed at a volume ratio of 3:1 and a total flow rate of 12 mL / min. During mixing, the lipid solubility changed, leading to spontaneous assembly and encapsulation of mRNA, forming uniformly sized LNPs.

[0114] Dialysis and filtration: The initially formed LNP suspension was dialyzed against PBS buffer to remove ethanol and replace the buffer.

[0115] Sterilization was achieved by using a 0.22 μm sterile filter membrane.

[0116] Quality control: Particle size and polydispersity index: According to dynamic light scattering detection, the qualified LNP particle size should be between 70 and 100 nm and the polydispersity index should be less than 0.2.

[0117] Encapsulation efficiency: Measured using the RIBOGreen fluorescent dye method, the encapsulation efficiency should be > 95%.

[0118] mRNA integrity was verified again by agarose gel electrophoresis.

[0119] III. In vivo drug delivery regimen and theoretical advantages

[0120] 1. Dosing regimen: Route: Injection via tail vein.

[0121] Dosage: Based on the higher efficiency of non-equimolar design, the test dose can be set to 0.2~0.5 mg / kg (below equimolar design).

[0122] Treatment course: Starting in the middle stage of the CCl4-induced liver fibrosis model, administer the drug once every 7-10 days for a total of 2-3 times.

[0123] 2. Expected Mechanism of Action and Advantages: Mechanism: After LNP delivers a single mRNA to hepatocytes, its translation product undergoes self-cleavage, instantaneously producing a protein ratio of a predetermined 3:1:1:1:1. Higher OCT4 levels may more effectively open chromatin, synergistically with other factors, and more rapidly and synchronously initiate partial reprogramming.

[0124] Theoretical advantages: Efficiency Improvement: OCT4 is a "pioneer factor" for reprogramming. Increasing its dosage is expected to lower the reprogramming threshold, enabling more cells to reach the target epigenetic reversal state (57-77%) in a shorter time / with lower total RNA exposure.

[0125] Improved safety: Due to increased efficiency, the number of administrations and total mRNA dose may be reduced, thereby lowering the risk of immune activation and potential side effects.

[0126] Enhanced controllability: The fixed internal ratio avoids uneven expression caused by differences in transfection among different cells, making the treatment response more consistent and predictable.

[0127] Example 3

[0128] AAV virus intranasal instillation for mouse pulmonary fibrosis model

[0129] I. Virus Preparation

[0130] Viral preparation: AAV virus prepared using the method of Example 1. Serotype AAV6 was selected due to its high transduction efficiency in lung epithelial cells.

[0131] Viral titer: The genomic titer of the viral stock solution was accurately determined by qPCR and adjusted to a working concentration of 1.0 × 10¹² to 1.0 × 10¹³ viral genome copies / mL (vg / mL) before use.

[0132] Diluent: Use sterile, endotoxin-free phosphate-buffered saline (PBS, pH 7.4) as the solvent for dilution and infusion.

[0133] II. Establishment of Animal Models

[0134] Bleomycin (BLM)-induced mouse pulmonary fibrosis model is an internationally recognized and widely used classic model that can well simulate the key pathological features of human idiopathic pulmonary fibrosis (IPF), including alveolar epithelial damage, inflammatory cell infiltration, fibroblast proliferation, and excessive collagen deposition.

[0135] Animal strain: C57BL / 6J mice. This strain has moderate sensitivity to bleomycin, can form a stable fibrosis model, and has good reproducibility.

[0136] Age and weight: 8-10 weeks old, weight 20-25 grams.

[0137] Husbandry environment: Husbands were housed in a standard SPF-grade environment with free access to food and water. The experimental protocol was reviewed and approved by the institution's animal ethics committee.

[0138] Main reagents and instruments

[0139] Modeling reagent: Bleomycin, which should be prepared to the required concentration with sterile physiological saline before use.

[0140] Anesthetic: Sodium pentobarbital (1%) or isoflurane inhalation anesthesia system.

[0141] Main instruments: small animal operating table, laryngoscope, miniature pipette, animal heating pad, surgical sutures, etc.

[0142] Modeling steps

[0143] 1. Grouping and Anesthesia: Mice were randomly divided into a control group (Sham group) and a model group (BLM group).

[0144] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital or inhalation of isoflurane. After complete anesthesia (as indicated by no response to toe pinching), the mice were fixed to the operating board in a supine position.

[0145] 2. Tracheal exposure and infusion: Gently pull the mouse's incisors with tape to straighten its neck. Disinfect the skin on the front of the neck with alcohol.

[0146] Make a longitudinal incision of about 0.5-1 cm along the midline of the neck, bluntly dissect the subcutaneous tissue and glands, and expose the trachea.

[0147] Using an insulin needle or microinjector, insert the needle into the tracheal lumen at a 30-45° angle to the body, between the tracheal cartilage rings. Control group mice were infused with 50 μL of sterile saline, while model group mice were infused with 50 μL of saline solution containing bleomycin.

[0148] Key modeling parameters: Bleomycin dosage: 3.0 mg / kg body weight. This dosage range has been determined in preliminary experiments to ensure animal survival while inducing significant fibrosis.

[0149] Infusion volume: 50 μL / unit.

[0150] Quickly remove the needle and immediately rotate the mouse upright to ensure the medication is evenly distributed in both lungs.

[0151] 3. Suturing and resuscitation: The incision was closed layer by layer using surgical sutures.

[0152] The mice were placed on a 37°C constant temperature mat in a lateral position until they were fully awakened from anesthesia and then returned to their cages.

[0153] Model Validation and Evaluation Metrics

[0154] The peak period of fibrosis formation was from day 21 to 28 after modeling. At this time, the animals were sacrificed for the following analysis to verify whether the model was successfully established.

[0155] 1. General observation: The model group mice exhibited typical symptoms such as weight loss, disheveled fur, reduced activity, and rapid breathing.

[0156] 2. Gross observation of lung tissue: After euthanasia, the lungs were harvested. The lung tissue in the model group was found to be reduced in volume, lacked elasticity, and had a rough and uneven surface, with diffuse pale white nodules visible.

[0157] 3. Histopathological analysis (core indicators): Hematoxylin-eosin (H&E) staining: to observe the overall structural damage, inflammatory cell infiltration and fibrosis of the lungs.

[0158] Masson trichrome staining or Sirius Red staining: specifically displays blue collagen fibers (Masson) or red collagen fibers (Sirius Red), visually reflecting the degree of collagen deposition.

[0159] Ashcroft score: A double-blind, semi-quantitative scoring method (0-8 points) is used to assess the severity of fibrosis in Sirius red-stained lung tissue sections. The Ashcroft score in the model group should be significantly higher than that in the control group (usually >5 points).

[0160] 4. Hydroxyproline content determination (biochemical quantification of collagen deposition): Hydroxyproline is a characteristic amino acid of collagen, and its content can accurately reflect the total collagen content in the lungs.

[0161] Hydroxyproline was extracted from lung tissue using alkaline hydrolysis and quantified using a kit.

[0162] The hydroxyproline content in the lung tissue of the model group should be significantly higher than that of the control group (usually 1.5 to 2 times higher).

[0163] 5. Molecular biological indicators: α-Smooth muscle actin (α-SMA): Localized and semi-quantitatively analyzed by immunohistochemical staining, it is a marker of activated myofibroblasts. The number of α-SMA-positive cells should be significantly increased in the model group.

[0164] Fibrosis-related gene expression: The mRNA expression levels of fibrosis-related genes (such as Col1a1, Acta2, Tgfb1) were detected by real-time quantitative PCR. The expression levels in the model group were significantly upregulated.

[0165] Model success criteria

[0166] A pulmonary fibrosis model is considered successfully established if all of the following conditions are met: Ashcroft score: The model group score was significantly higher than that of the control group (p < 0.01), and the mean was > 5.

[0167] Hydroxyproline content: The content in the model group was significantly higher than that in the control group (p < 0.01).

[0168] Histological evidence: H&E and Masson / Sirius red staining showed severe damage to alveolar structure and extensive collagen deposition in the model group.

[0169] Table 1. Histological evidence data of the pulmonary fibrosis model

[0170] III. Animal Preparation and Anesthesia

[0171] Animals: The established pulmonary fibrosis model mice described above.

[0172] Anesthesia: To ensure that the viral fluid is fully inhaled into the lungs and not actively exhaled or swallowed by the mice, mild anesthesia must be administered. Isoflurane inhalation anesthesia should be used, and the mice should be unconscious but retain a cough reflex.

[0173] IV. Infusion Procedure

[0174] Position: Hold the mouse with one hand, making its body lie on its back with its head slightly upward.

[0175] Infusion: Using a pipette and sterile, low-absorption micropipette tips, slowly and steadily drip the viral fluid into one nostril of the mouse, allowing the mouse to inhale the fluid into its lungs.

[0176] Key parameters: Single infusion volume: 45 μL per mouse per infusion. This volume represents the standard safe upper limit for effective absorption by a single nasal infusion in adult mice.

[0177] Total infusion volume: 5.0 × 10¹ per mouse per infusion. 0 vg (ie: 50 μL of 1.0 × 10¹²vg / mL).

[0178] Infusion frequency and cycle: A short-term, intensive dosing strategy is typically adopted. Frequency: Once daily. Cycle: 2 consecutive days of infusion.

[0179] V. Post-administration management

[0180] After the infusion is complete, keep the mouse in a supine position for 1-2 minutes to ensure complete aspiration of the fluid. Place the mouse in a clean cage, lying on its side on a 37°C heated mat, until it is fully awakened from anesthesia. Closely monitor its respiratory status to ensure no asphyxiation occurs.

[0181] VI. Reasons for and Considerations for the Scheme Design

[0182] Dosage basis: Total dose 5.0 × 10 10  VG represents an effective and safe dose range that has been widely validated in AAV lung gene therapy studies, sufficient to achieve a sufficiently broad range of lung epithelial cell transduction while avoiding the strong immune response that may be triggered by excessively high doses.

[0183] Advantages of fractionated administration: Compared with a single large-volume infusion, continuous fractionated administration over two days (50 μL per day) can: improve the uniformity of virus distribution, reduce the risk of a single operation and the stress response in animals, and increase overall transduction efficiency.

[0184] Timing control: After completing the viral infusion and waiting for 2 weeks (allowing for sufficient viral transduction and basal rtTA3 expression), doxycycline induction via drinking water or injection can be initiated.

[0185] The specific steps are as follows: The AAV6 virus encoding OCT4, SOX2, KLF4, and GLIS1 (titer 1.0 × 10⁻⁶) was used. 12 (vg / mL) once daily, 50 μL each time (i.e., 5.0 × 10) 10The virus was administered intranasally to the lungs of bleomycin-induced pulmonary fibrosis model mice via a regimen of doxycycline (vg / dose / mouse) over two consecutive days. Two weeks after the end of the viral infusion, doxycycline (1 mg / mL) was added to the drinking water to induce reprogramming factor expression, and the expression was analyzed after another two weeks.

[0186] Results analysis: Table 2. Efficacy data of AAV recombinant virus nasal instillation in the treatment of pulmonary fibrosis (n=8, (p<0.01 vs model group)

[0187] Table 3. Epigenetic changes in pulmonary fibrosis (n=6, (p<0.01 vs model group)

[0188] Example 4

[0189] LNP-mRNA treatment in mouse models of liver fibrosis

[0190] By systematically delivering mRNA encoding partial reprogramming factors to the liver, hepatic stellate cells and damaged hepatocytes can be reprogrammed, reversing the fibrosis process and reducing epigenetic age.

[0191] I. LNP-mRNA was prepared according to the method described in Example 2.

[0192] II. Establishment of an animal model of liver fibrosis

[0193] In this embodiment of the invention, a carbon tetrachloride (CCl4)-induced mouse / rat liver fibrosis model is mainly used. This model is a classic and reliable model for studying the pathogenesis of liver fibrosis and evaluating the efficacy of drugs. It can well simulate the core pathological processes of human liver fibrosis, including hepatocyte damage, inflammatory response, hepatic stellate cell activation, and excessive deposition of extracellular matrix.

[0194] laboratory animals

[0195] Animal strain: C57BL / 6J mouse. C57BL / 6J is a standard inbred strain with a well-defined genetic background and is sensitive to fibrosis induction.

[0196] Age and weight: Mice: 6-8 weeks old, weight 18-22 grams.

[0197] Housing environment: Animals are housed in a standard SPF-grade environment with free access to food and water. All animal experimental procedures have been reviewed and approved by the institution's animal ethics committee.

[0198] Main reagents and instruments

[0199] Modeling reagents: carbon tetrachloride (CCl4), olive oil.

[0200] Syringe (1 mL for mice), iodine solution, alcohol swabs.

[0201] Instruments: balance, tissue homogenizer, paraffin sectioner, microscope, etc.

[0202] Modeling steps (intraperitoneal injection method)

[0203] This method is simple to operate, has good repeatability, and can produce diffuse liver fibrosis.

[0204] 1. Solution preparation: The CCl4 stock solution was mixed with olive oil as a dispersant at a certain volume ratio and then thoroughly vortexed.

[0205] Key modeling parameters: CCl4 concentration: The specific concentration of 10%-20% (v / v) CCl4 olive oil solution used for mice needs to be determined through preliminary experiments, so as to produce significant fibrosis while ensuring the survival rate of the animals.

[0206] Injection dose: 5 μL / g body weight (calculated based on CCl4 volume).

[0207] Injection frequency: twice a week (Monday and Thursday).

[0208] Modeling cycle: 6-8 weeks of continuous injections. The degree of fibrosis worsens with prolonged injection cycles.

[0209] 2. Grouping and Injection: Animals were randomly divided into a control group (Control) and a model group (CCl4).

[0210] Control group: Intraperitoneal injection of an equal volume of pure olive oil.

[0211] Model group: Intraperitoneal injection of CCl4 olive oil solution of corresponding concentration.

[0212] The animal must be properly secured during injection to avoid damaging its internal organs.

[0213] Model Validation and Evaluation Metrics

[0214] Animals were sacrificed 48–72 hours after the last injection, and the following analyses were performed to verify the successful establishment of the model.

[0215] 1. General condition and gross observation of the liver: The model group animals exhibited slow or decreased weight gain, sparse and dull fur, and lethargy.

[0216] After euthanasia, the liver was removed. The livers of the model group were found to be enlarged or shrunken, with a rough and uneven surface, yellowish-brown or dark red color, hardened texture, and diffuse granular or nodular changes.

[0217] 2. Serum biochemical indicators: Blood is collected and serum is separated.

[0218] The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. The ALT and AST activities in the model group should be significantly higher than those in the control group, indicating significant hepatocellular damage.

[0219] 3. Histopathological analysis (core indicators): Hematoxylin-eosin (H&E) staining: to observe the destruction of liver lobule structure, fatty degeneration of hepatocytes, ballooning degeneration, necrotic foci and inflammatory cell infiltration.

[0220] Sirius Red staining: Under a polarized light microscope, type I collagen appears as a bright yellow or red birefringence, while type III collagen appears as a green birefringence. This staining is the "gold standard" for evaluating collagen fiber deposition and distribution.

[0221] Semi-quantitative fibrosis scoring: The Ishak scoring system (0-6 points) is more refined and can better distinguish between moderate and severe fibrosis.

[0222] Metavir scoring system (F0-F4): Commonly used in clinical practice.

[0223] The scoring was performed in a double-blind manner by at least two pathologists who were unaware of the experimental group. A successful model group should have an Ishak score of ≥4 (significant fibrosis) or a Metavir score of F3 (severe fibrosis) or higher.

[0224] 4. Determination of hydroxyproline content in liver tissue (biochemical quantification of collagen deposition): Hydroxyproline is a characteristic amino acid of collagen.

[0225] A portion of liver tissue was taken, and hydroxyproline was extracted using acid hydrolysis. The quantification was performed using a kit.

[0226] The hydroxyproline content in the liver tissue of the model group should be significantly higher than that of the control group (usually 2-3 times higher).

[0227] 5. Molecular biological indicators: α-Smooth muscle actin (α-SMA): As shown by immunohistochemistry or immunofluorescence staining, it is a marker of hepatic stellate cells activating into myofibroblasts. The number of α-SMA-positive cells in the portal area and fibrous septa should be significantly increased in the model group.

[0228] Fibrosis-related gene expression: The mRNA expression levels of Col1a1 (type I collagen), Acta2 (α-SMA), Timp1 (tissue inhibitor of matrix metalloproteinase 1), and Tgfb1 (transforming growth factor β1) were detected by real-time quantitative PCR. The expression levels were significantly upregulated in the model group.

[0229] Model success criteria

[0230] A liver fibrosis model is considered successfully established if all of the following conditions are met: Histological score: The Ishak score of the model group was significantly higher than that of the control group (p < 0.01), and the mean score was ≥ 4.

[0231] Hydroxyproline content: The content in the model group was significantly higher than that in the control group (p < 0.01).

[0232] Serological markers: ALT / AST levels were significantly elevated in the model group (p < 0.01).

[0233] Molecular markers: α-SMA positive area and / or fibrosis gene expression were significantly upregulated in the model group.

[0234] Table 4. Validation data of molecular markers for liver fibrosis model

[0235] Single-stranded mRNAs can be expressed intracellularly and self-cleaved to produce OCT4, SOX2, KLF4, GLIS1, and LIN28 proteins, with a molar ratio of OCT4 to the other factors of 3:1. The mRNAs were prepared into LNP formulations using ionizable lipids (SM-102) and injected into liver fibrosis model mice via tail vein at a dose of 0.3 mg / kg, once every 10 days for a total of two injections. The results showed that, compared with the group injected with equimolar expression (1:1:1:1:1) OSKGL mRNA, the treatment group of this implementation scheme (3:1:1:1:1) achieved comparable or better fibrosis reversal at a lower total dose (Ishak score decreased by 60% vs 55%, hydroxyproline content decreased by 58% vs 52%); (2) epigenetic age reversal was more significant (reduction of 70% ± 5% vs 62% ± 6%); and (3) serum inflammatory factor (such as IFN-α) levels were significantly lower, indicating a milder immune response. This demonstrates that controlling the internal expression ratio through mRNA sequence design is an effective strategy for optimizing the efficacy and safety of partial reprogramming.

[0236] Table 5. Data on the efficacy of mRNA in treating liver fibrosis (n=8, (p<0.01 vs model group)

[0237] Table 6. Epigenetic changes in liver fibrosis (n=6, (p<0.01 vs model group)

[0238] Table 7 Comparison of the therapeutic effects of equimolar and non-equimolar mRNA designs on liver fibrosis (n=8, p<0.05 (vs. equal molar treatment group)

[0239] Example 5

[0240] Relationship between doxycycline dosage and reprogramming effect

[0241] I. Experimental Objective

[0242] To accurately assess the quantitative effects of doxycycline (DOX) dosage on the magnitude of epigenetic age reversal, cell identity maintenance, and safety (tumor development) in a mouse model of pulmonary fibrosis via an AAV-induced reprogramming system, thereby determining a safe and effective treatment window.

[0243] II. Experimental Design and Grouping

[0244] Animal model: The same bleomycin (BLM)-induced C57BL / 6J mouse pulmonary fibrosis model as in Example 3 was used.

[0245] Treatment method: The same recombinant AAV delivery system as in Example 3 (pAAV-TRE-tight-OSK virus, pAAV-TRE-tight-OG virus, pAAV-TRE-tight-OL virus) was used for targeted lung delivery via nasal instillation. Viral titer, dosage, and instillation regimen (total dose 5.0 × 10¹) 0 (vg / each, administered via two-day drip) is completely consistent with Example 3.

[0246] Experimental groups (n=8 animals / group): Control group (DOX 0 mg / mL): fibrotic model mice that received AAV virus delivery but whose drinking water did not contain added doxycycline.

[0247] Experimental group 1 (DOX 0.2 mg / mL): Two weeks after AAV delivery, participants drank water containing 0.2 mg / mL doxycycline for two weeks.

[0248] Experimental group 2 (DOX 0.5 mg / mL): Two weeks after AAV delivery, participants drank water containing 0.5 mg / mL doxycycline for another two weeks.

[0249] Experimental group 3 (DOX 1.0 mg / mL): Two weeks after AAV delivery, participants drank water containing 1.0 mg / mL doxycycline for two weeks.

[0250] Experimental group 4 (DOX 2.0 mg / mL): Two weeks after AAV delivery, the participants drank water containing 2.0 mg / mL doxycycline for three weeks (the induction time was extended to observe the limit effect because the reversal process may be slower in this dose group).

[0251] Necessity of setting up a control group: A control group with a DOX concentration of 0 is established to eliminate the potential impact of leaked expression from the AAV system itself (even without DOX, rtTA3 may have very low activity activating TRE3G) on epigenetic age. Data should show a very small reversal rate (<5%), demonstrating the rigor of the system.

[0252] III. Key Detection Indicators and Methods

[0253] One week after the end of the DOX induction period, the mice were sacrificed and the following analyses were performed: The extent of decrease in epigenetic age: Samples: Lung tissue was taken from each group of mice.

[0254] Methods: The procedure was strictly performed according to the “Genome DNA Methylation Analysis and Epigenetic Age Assessment Method” described in Example 6.

[0255] Calculation: The reversal magnitude was calculated using the formula ΔEpiAge (%) = [ (EpiAge_model - EpiAge_treated) / (EpiAge_model - EpiAge_control) ] × 100%. Where EpiAge_model is the mean epigenetic age of the untreated model group (BLM group) in Example 3, and EpiAge_control is the mean age of the healthy control group.

[0256] Changes in cell identity (taking lung epithelial cells as an example): Indicator: Percentage of SP-C positive cells in lung tissue sections (SP-C is a marker of type II alveolar epithelial cells).

[0257] method: a. Immunofluorescence staining: SP-C and DAPI co-staining were performed on lung tissue sections.

[0258] b. Quantitative image analysis: Using a confocal microscope, take pictures and automatically count the number of SP-C positive cells and the total number of cell nuclei in at least 5 non-overlapping fields of view using ImageJ or similar software, and calculate the percentage.

[0259] Significance: A stable SP-C positivity rate indicates that the lung epithelial cells maintain their identity well; a significant decrease suggests that the cells may dedifferentiate or transdifferentiate.

[0260] Tumor incidence: Macroscopic observation: After execution, carefully examine the lungs and major organs (liver, spleen, kidneys) for any visible nodules or tumors.

[0261] Histological verification: H&E staining was performed on lung tissue from all experimental groups (especially the high-dose group), and pathologists observed under a microscope in a blind manner for the presence of abnormal proliferation, dysplasia, or neoplastic lesions.

[0262] Long-term observation group (optional): For the high-dose group (e.g., 2.0 mg / mL), an additional group of animals can be set up to continue feeding for 3-6 months after the cessation of DOX induction, with regular monitoring of weight and condition, and finally pathological examination to assess the long-term tumorigenic risk.

[0263] Table 8. Effects of different doxycycline doses on reprogramming Note: The table clearly shows that by controlling the dosage and duration of DOX, the effect can be precisely targeted within a safe and effective window (e.g., 0.5 mg / mL, 2 weeks).

[0264] Example 6

[0265] The extent of reduction in organ epigenetic age under different treatments in Examples 3 and 4 was detected. Epigenetic age was calculated using the Horvath epigenetic clock model based on whole-genome DNA methylation sequencing (Illumina EPIC array) data.

[0266] The specific methods for whole-genome DNA methylation analysis and epigenetic age assessment are as follows: I. Sample Collection and Preprocessing Sample type: Processed fibrotic organ (such as lung and liver) tissues were collected, and a healthy control group, a fibrotic model group and a treatment group were set up.

[0267] Collection and preservation: After euthanizing the animal, quickly take about 20-30 mg of the target tissue, immediately place it in liquid nitrogen for flash freezing, and then transfer it to -80°C for long-term preservation, avoiding repeated freeze-thaw cycles.

[0268] Sample size: Each group should contain at least 6 biological replicates to ensure statistical power.

[0269] II. Genomic DNA Extraction and Quality Control

[0270] Extraction method: Total DNA was extracted from tissue samples using a commercially available genomic DNA extraction kit based on column chromatography (such as the Qiagen DNeasyBlood & Tissue Kit).

[0271] Quality control standards: Purity: Measured using a UV spectrophotometer, the A260 / A280 ratio should be between 1.8 and 2.0.

[0272] Integrity: Assessed by agarose gel electrophoresis, the main DNA band should be clear and without significant degradation.

[0273] Concentration: Use a quantitative fluorescence method (such as Qubit dsDNA HS Assay) to accurately quantify and ensure sufficient DNA for subsequent analysis.

[0274] III. Whole-genome DNA methylation detection

[0275] Detection platform: The Illumina Infinium MethylationEPIC (850K) BeadChip chip is used. This chip covers more than 850,000 CpG sites, including gene promoter regions, enhancer regions, coding regions, and differentially methylated regions closely related to aging and disease. It is the industry gold standard for epigenetic clock analysis.

[0276] Experimental procedure: Bisulfite conversion: 500 ng of high-quality genomic DNA was bisulfite-treated using kits such as the EZ DNA Methylation Kit to convert unmethylated cytosine to uracil, while methylated cytosine remained unchanged.

[0277] Microarray hybridization and scanning: The transformed DNA was fragmented, precipitated, and resuspended, then hybridized with EPIC microarray probes. After extension and staining, fluorescence signals were scanned using an Illumina iScan series microarray scanner.

[0278] Raw data output: The scanning software generates an IDAT file for each sample, containing methylation signal intensity data for all CpG sites.

[0279] IV. Bioinformatics Analysis and Epigenetic Age Calculation

[0280] Data preprocessing: Use the R language environment and the minfi or SeSAMe package to read IDAT files.

[0281] Background correction, dye deviation correction, probe type correction, and functional normalization are performed.

[0282] Filter out probes that fail to detect signals (p-value > 0.01), are located on sex chromosomes, or are known to have cross-reactivity in all samples.

[0283] β value calculation: For each CpG site, calculate its methylation level (β value) using the following formula: β value = methylated signal intensity / (methylated signal intensity + unmethylated signal intensity + 100) The β value ranges from 0 (completely unmethylated) to 1 (completely methylated).

[0284] Epigenetic age calculation: Horvath Clock Algorithm: This algorithm applies the pan-tissue epigenetic clock model developed by Dr. Steven Horvath. Using the β values ​​of 353 specific CpG sites in the preprocessed data, the algorithm directly calculates the "DNA methylation age" (in years) of a sample through a pre-trained penalized regression model (Elastic Net).

[0285] The Hannum clock algorithm: This algorithm applies a blood-based clock model developed by Dr. Gregory Hannum, which is applicable to a variety of tissues. The model utilizes 71 marker CpG sites for calculation.

[0286] To ensure robustness, this invention preferably employs two algorithms simultaneously, with Horvath clock age as the primary reporting metric.

[0287] Calculation of the degree of age reversal: For each treatment group sample, the epigenetic age reversal magnitude (ΔEpiAge) is calculated using the following formula: ΔEpiAge (%) = [ (EpiAge_model - EpiAge_treated) / (EpiAge_model - EpiAge_control) ] × 100% Where EpiAge_model is the mean epigenetic age of the model group, EpiAge_treated is the individual age of the treatment group, and EpiAge_control is the mean epigenetic age of the healthy control group.

[0288] This formula quantifies the proportion by which treatment reverses the aging process accelerated by fibrosis back to a healthy, youthful state. The safe and effective window defined in this invention is ΔEpiAge = 57% - 77%.

[0289] Statistical analysis: All data are expressed as mean ± standard deviation.

[0290] One-way ANOVA was used to compare differences among multiple groups, followed by post-hoc tests (such as Tukey's HSD test).

[0291] Pearson correlation analysis was used to assess the correlation between epigenetic age reversal magnitude (ΔEpiAge) and fibrosis scores (such as Ashcroft scores).

[0292] p < 0.05 was considered statistically significant. All treatment groups were statistically significant compared to the model group.

[0293] Table 9. Reduction in organ epigenetic age under different treatments

[0294] Through the above-mentioned rigorous and repeatable standard operating procedures, this invention obtained reliable data, empirically demonstrating that the treatment plan based on partial reprogramming can accurately and significantly reverse the epigenetic age of fibrotic organs by 57%-77%, providing key quantitative evidence for achieving organ aging reversal and functional remodeling.

[0295] 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. A quantitative epigenetic reprogramming method, characterized in that, The epigenetic age of cells can be reduced by 57% to 77% through gene reprogramming.

2. The quantitative epigenetic reprogramming method according to claim 1, characterized in that, The gene reprogramming method involves delivering mRNA sequences encoding OCT4, SOX2, KLF4, GLIS1, and Lin28 into cells for 2-3 weeks, followed by doxycycline-induced expression for 1-2 weeks.

3. The quantitative epigenetic reprogramming method according to claim 2, characterized in that, The delivery is achieved via adeno-associated virus carrying mRNA or mRNA encapsulated in lipid nanoparticles.

4. A reagent for quantitative epigenetic reprogramming, characterized in that, This includes delivery systems for delivering mRNAs of OCT4, SOX2, KLF4, GLIS1, and Lin28, and the inducing agent doxycycline.

5. The reagent according to claim 4, characterized in that, The delivery system includes: (1) AAV1-inducer that delivers third-generation reverse tetracycline transactivator protein rtTA3 expresses viral pAAV-rtTA3; (2) AAV2-core reprogramming factor virus pAAV-TRE-tight-OSK that delivers mRNA of OCT4, SOX2, and KLF4; (3) AAV3-key regulator virus pAAV-TRE-tight-OG that delivers OCT4, GLIS1 mRNA. (4) AAV3-key regulator of the virus pAAV-TRE-tight-OL that delivers OCT4,Lin28 mRNA.

6. The reagent according to claim 5, characterized in that, The promoter of the AAV2-core reprogramming factor virus pAAV-TRE-tight-OSK is the TRE3G promoter, and the inserted target gene is the fusion gene sequence encoding OCT4-P2A-SOX2-P2A-KLF4.

7. The reagent according to claim 4, characterized in that, The delivery system comprises lipid nanoparticles encapsulating the mRNAs of OCT4, SOX2, KLF4, GLIS, and Lin28, with a molar ratio of 3:1:1:1:

1.

8. The reagent according to claim 7, characterized in that, The mRNA sequence encapsulated by the lipid nanoparticles consists of the following sequentially linked sequences: OCT4-(P2A)-OCT4-(T2A)-OCT4-(E2A)-SOX2-(F2A)-KLF4-(GSAG)-GLIS1-(P2A)-LIN28.

9. The use of the reagent according to any one of claims 4 to 8 in the preparation of a medicament for treating organ fibrosis.

10. The application according to claim 9, characterized in that, The organ fibrosis includes pulmonary fibrosis and liver fibrosis.

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