Method for quantifying partially reprogrammed porcine somatic cells and use thereof

Abstract

The application provides a method for quantitatively partially reprogramming pig somatic cells and application thereof, and belongs to the technical field of animal biotechnology, genetic engineering and regenerative medicine. The method comprises reversing the epigenetic age of pig somatic cells to 57%-77% of the initial epigenetic age. The pig somatic cell epigenetic age is reversed by an mRNA reprogramming method or a small molecule composition reprogramming method. The partially reprogrammed pig somatic cells prepared by the method can tolerate extreme editing of more than 22 genes. In vitro, the partially reprogrammed pig somatic cells show immune compatibility close to human cells (a comprehensive protection index of 0.94), survive for more than 35 days in pig-monkey skin transplantation, and have a 28-day coverage rate of 62%. In xenogeneic kidney transplantation, all recipients survive for a long time. The application solves the fundamental bottleneck of pig somatic cell gene editing, and provides a safe, effective and industrialized donor pig solution for clinical xenotransplantation.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary fields of animal biotechnology, genetic engineering and regenerative medicine, and particularly relates to a method for quantitative partial reprogramming of porcine somatic cells and its application. Background Technology

[0002] The core efficiency bottleneck facing the industrial production of xenotransplant donor pigs is that, to develop clinically viable xenotransplant donor pigs, at least 22 genes in donor cells need to be precisely edited to systematically overcome cross-species immune rejection. This process faces three interconnected industry challenges: 1) Lack of a stable pluripotent stem cell platform: Pigs currently lack germline-transferable embryonic stem cell or induced pluripotent stem cell lines. Therefore, all gene modifications must be performed on terminally differentiated somatic cells, followed by somatic cell nuclear transfer to produce cloned animals, a technical pathway inherently limited in efficiency. 2) Insufficient editing tolerance due to cellular senescence: Pig somatic cells undergo rapid replicative senescence in vitro, characterized by telomere shortening and accelerated epigenetic aging, resulting in insufficient proliferation potential and genomic stability to support multiple gene editing processes lasting several months. 3) Low cloning efficiency – a high abortion rate bottleneck: This is the biggest obstacle to large-scale production. Even with existing technologies to edit a small number of genes (e.g., 10–11), the SCNT process still suffers from an early abortion rate as high as 40–50%. The main reasons include: DNA damage and epigenetic abnormalities induced by the gene editing process accumulate in the donor cell nucleus; the chromatin structure of donor cells in an aging or stressed state is not conducive to reprogramming after nuclear transplantation; and there is a lack of standards and corresponding optimization methods for objectively and quantitatively assessing the "quality" of donor cell nuclei.

[0003] II. Existing technologies and their limitations in application to pig species

[0004] In model organisms (such as mice and humans), constructing epigenetic clocks to quantify biological age using multi-omics data (such as DNA methylation and chromatin accessibility) and employing small-molecule cocktails for partial reprogramming to reverse aging phenotypes has become a mature research paradigm. However, directly applying these technologies to pig species to address the aforementioned industry bottlenecks presents fundamental challenges: Lack of species-specific assessment tools: Known epigenetic clock models are all trained on human or mouse data. Due to species differences in epigenetic patterns, these models cannot directly and accurately assess the epigenetic age of pig cells, resulting in a lack of objective and quantifiable standards for the "rejuvenation" of pig somatic cells. Lack of precise targeting and effect prediction for reprogramming protocols: Although certain small molecule combinations (such as those containing VPA, CHIR99021, RepSox, etc.) are known to induce partial reprogramming in human or mouse cells, their effects on pig somatic cells, optimal window, and ultimate impact on SCNT efficiency are unknown and difficult to predict. Simple formulation transfer to pig cells yields unstable results, failing to achieve the reproducibility and high efficiency required for industrialization.

[0005] Therefore, the core challenge that urgently needs to be overcome in this field is: how to obtain a method for preparing porcine somatic cells that can both carry out extreme multiplex gene editing and serve as "high-quality nuclear donors" to ensure high pregnancy maintenance rates and extremely low miscarriage rates after SCNT. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a method for quantitative partial reprogramming of porcine somatic cells and its application. This invention is the first to discover a narrow "high-quality epigenetic window" (57%-77% reversal) in porcine somatic cells; only when cells within this window are used as nuclear donors can a stepwise decrease in the early SCNT abortion rate from >40% to <20% be achieved. This invention provides a complete solution from evaluation and optimization to production, overcoming the core bottleneck of low porcine cloning efficiency.

[0007] This invention provides a method for quantitatively reprogramming porcine somatic cells, reversing the epigenetic age of porcine somatic cells to 57% to 77% of the initial epigenetic age.

[0008] Preferably, the somatic cell epigenetic age of pigs is reversed to 62% to 72% of the initial epigenetic age.

[0009] Preferably, the pig somatic cell epigenetic age reversal is performed using mRNA reprogramming or a small molecule composition reprogramming method; The method of mRNA reprogramming is to deliver mRNA sequences encoding OCT4, SOX2, KLF4, GLIS1, and Lin28 into cells; The method for reprogramming the small molecule composition involves adding a portion of the reprogrammed small molecule composition during cell culture.

[0010] Preferably, the partially reprogrammed small molecule composition includes valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, foctocorline, and sodium butyrate; 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, the concentration of foctocorline is 40-60 μM, and the concentration of sodium butyrate is 150-250 μM.

[0011] Preferably, the partially reprogrammed small molecule composition includes valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, foctocorline, and bFGF; 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, the concentration of foctocorline is 40-60 μM, and the concentration of bFGF is 80-120 ng / ml.

[0012] This invention provides a method for preparing partially reprogrammed porcine somatic cells.

[0013] This invention provides a super-humanized gene-edited porcine nuclear donor cell, obtained by gene editing of partially reprogrammed porcine somatic cells; the gene editing includes the knockout of the GGTA1 gene, CMAH gene, and B4GALNT2 gene; And knock-in of at least 16 genes selected from CD46, CD55, CD59, THBD, TFPI, CD39, EPCR, vWFΔA1, HLA-E, B2M, CD47, PD-L1, CTLA4-Ig, FasL, TNFAIP3, HMOX1, HO-2, BCL-2, and Survivin.

[0014] Preferably, the partially reprogrammed pig somatic cells undergo epigenetic age assessment and screening before gene editing, selecting cells whose epigenetic age has been reversed to 57% to 77% of their initial epigenetic age.

[0015] This invention provides a method for producing super humanized cloned pigs, using the aforementioned super humanized gene-edited pig nuclear donor cells as nuclear donors, and producing cloned pigs through somatic cell nuclear transfer technology.

[0016] This invention provides a small molecule composition for partial reprogramming of porcine somatic cells, comprising valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, foctocorline, and sodium butyrate; 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, the concentration of foctocorline is 40-60 μM, and the concentration of sodium butyrate is 150-250 μM; Alternatively, it may include valproic acid, CHIR99021, Repsox, transphenylcyclopropane, foctocorline, and bFGF; 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 transphenylcyclopropane is 4-6 μM, the concentration of foctocorline is 40-60 μM, and the concentration of bFGF is 80-120 ng / ml.

[0017] Compared with existing technologies, this invention has the following beneficial effects: This invention provides a method for quantitatively reprogramming porcine somatic cells, reversing the epigenetic age of porcine somatic cells to 57%~77% of the initial epigenetic age. This invention reveals for the first time that there is a narrow "high-quality epigenetic window" (57%-77%) for porcine somatic cell reprogramming, and that there is a clear inflection point relationship between this window and the SCNT abortion rate, providing revolutionary theoretical guidance and precise regulatory targets for industrial practice.

[0018] This invention screened two partially reprogrammed small molecule combinations in a pig system. Through an unpredictable synergistic effect, these combinations reduced the early abortion rate of SCNTs in 22-gene edited donor cells to an industry-limit level of approximately 7%, solving the core efficiency and economic bottlenecks of large-scale production. The results far exceeded the expectations of those skilled in the art based on existing knowledge.

[0019] This invention establishes a complete technical system from pig somatic cell epigenetic age assessment, window-precise induction, gene editing to efficient cloning production, and has extremely high industrial practical value. Attached Figure Description

[0020] Figure 1 This is an overview diagram of the technical route of the present invention, showing the entire process from senescent cells to cloned pigs with low miscarriage rate. Detailed Implementation

[0021] This invention provides a method for quantitatively reprogramming porcine somatic cells, reversing the epigenetic age of porcine somatic cells to 57% to 77% of the initial epigenetic age, preferably 62% to 72%.

[0022] In this invention, the reversal of porcine somatic cell epigenetic age is achieved through mRNA reprogramming or reprogramming using a small molecule composition. Specifically, the mRNA reprogramming method involves delivering mRNA sequences encoding OCT4, SOX2, KLF4, GLIS1, and Lin28 into the cells. The specific operational steps are as follows: mRNA preparation: Sequence design: Codon optimization was performed based on the coding sequences (CDS) of human or porcine OCT4, SOX2, KLF4, GLIS1 and Lin28 to adapt to the expression preferences of porcine cells and avoid the formation of stable secondary structures.

[0023] Chemical modification: To significantly improve the stability and translation efficiency of mRNA and reduce its innate immunogenicity, chemically modified nucleotides must be introduced during in vitro transcription synthesis. Typically, pseudouridine (Ψ) or N1-methylpseudouridine (m1Ψ) is used to completely replace uridine (U), and it can be supplemented with modifications such as 5-methylcytidine (m5C).

[0024] Transcription and purification: Using the modified nucleotides described above, chemically modified mRNAs with 5' cap analogs (such as the CleanCap® AG structure, mimicking Cap 1) and 3' poly(A) tails (typically 100-150 adenosines in length) are synthesized via in vitro transcription (IVT).

[0025] Purification and Preservation: All mRNAs were purified by high-performance liquid chromatography (HPLC) or oligo-dT affinity chromatography to remove transcriptional byproducts. The purified mRNAs were dissolved in nuclease-free water or buffer, and their concentration and purity were determined. After aliquoting, the mRNAs were stored at -80°C.

[0026] Preparation of working solution: Before use, mix the five chemically modified mRNAs in equimolar proportions according to their molar concentrations, dilute with nuclease-free water, and prepare a mixed working solution of mRNAs at the specified concentration.

[0027] Cell preparation and pre-transfection treatment: Take porcine somatic cells (e.g., porcine fetal fibroblasts) to be reprogrammed, digest and count them, and seed them at an appropriate density (e.g., 3×10⁴ / cm² to 8×10⁴ / cm²) in cell culture plates (e.g., 24-well plates). Incubate the cells overnight in complete medium (e.g., DMEM / F12 containing 10% FBS) at 37°C and 5% CO₂ until the cells reach approximately 70%-90% confluence. 1-2 hours before transfection, replace the medium with serum-free or low-concentration serum-specific transfection medium (e.g., Opti-MEM).

[0028] mRNA delivery (transfection): Transfection reagent preparation: Use a commercially available transfection reagent suitable for mRNA delivery (such as Lipofectamine Messenger MAX). According to the instructions, dilute a certain volume of the mRNA working solution and an equal volume of the diluted transfection reagent separately in Opti-MEM and incubate at room temperature for 5 minutes.

[0029] Complex formation: Gently mix the diluted mRNA with the transfection reagent solution and let stand at room temperature for 10-15 minutes to form the mRNA-transfection reagent complex.

[0030] Cell transfection: Add the complex solution evenly to the prepared cells and gently shake the culture plate to mix. Return the cells to the incubator and continue culturing.

[0031] Initial transfection parameters: For the first transfection, the final concentration of each chemically modified mRNA is typically in the range of 10-50 ng / μL, and the total mRNA dose is adjusted according to the plate area (e.g., 0.5-1.0 μg of mixed mRNA is added to each well of a 24-well plate). This time point is recorded as day 0 of treatment.

[0032] Continuous transfection and culture: Transfection frequency: To achieve continuous reprogramming signals, multiple transfections are required. A typical approach is to transfect once daily for 5-7 consecutive days.

[0033] Medium change and re-transfection: Approximately 4-6 hours after each transfection, replace the medium with fresh complete medium (DMEM / F12 containing 10% FBS) to reduce the toxicity of the transfection reagent and provide nutrients to the cells. After changing the medium, the next transfection operation can be performed immediately (repeating step 3), or it can be performed the next day.

[0034] Dosage adjustment: Depending on cell status and transfection efficiency, the mRNA dosage can be fine-tuned in subsequent transfections. For example, the dosage of each mRNA can be reduced by 20%-50% after day 3 to balance reprogramming efficiency and cytotoxicity.

[0035] Treatment termination and cell recovery: After completing the transfection operation on the last day (such as day 5 or day 7) and incubating for 4-6 hours, discard the culture medium containing the transfection complex.

[0036] Gently wash the cells with PBS, then replace with fresh complete culture medium and allow the cells to recover and culture for 24-48 hours without exogenous mRNA.

[0037] After the recovery period, the cells can be used for passage expansion, epigenetic age assessment, or direct subsequent gene editing operations.

[0038] Key parameters and quality control: Efficiency monitoring: A small amount of chemically modified control mRNA with a fluorescent tag (such as GFP) can be added during the first transfection. The mRNA can be observed under a fluorescence microscope 24 hours after transfection to roughly assess the transfection efficiency. Ideally, the efficiency should be >70%.

[0039] Morphological changes: Successful reprogramming usually causes significant morphological changes on day 3-4 after transfection, including reduced cell volume, increased nucleocytoplasmic ratio, clearer cell boundaries, and accelerated proliferation.

[0040] Molecular validation: After treatment, the expression levels of endogenous pluripotency-related genes (such as OCT4 and NANOG) can be detected by qRT-PCR to see if they are moderately upregulated, serving as supporting evidence for reprogramming initiation.

[0041] Core Assessment: The percentage of epigenetic age reversal in the treated cell population must be quantitatively assessed using the pig-specific epigenetic clock model described in this invention, ensuring it precisely falls within the 57%-77% target window. This is the core criterion for determining the success of the reprogramming and its applicability to subsequent SCNTs.

[0042] In this invention, the method for reprogramming the small molecule composition is to add a portion of the reprogrammed small molecule composition during cell culture.

[0043] In this invention, the partially reprogrammed small molecule composition includes valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, foctocorline, and sodium butyrate; wherein 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 8-12 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; the concentration of foctocorline is 40-60 μM, preferably 45-55 μM, more preferably 50 μM; and the concentration of sodium butyrate is 150-250 μM, preferably 180-220 μM, more preferably 200 μM.

[0044] In another embodiment of the present invention, the partially reprogrammed small molecule composition includes valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, foctocorline, and bFGF; wherein 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 8-12 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; the concentration of foctocorline is 40-60 μM, preferably 45-55 μM, more preferably 50 μM; and the concentration of bFGF is 80-120 ng / ml, preferably 90-110 ng / ml, more preferably 100 ng / ml.

[0045] In this invention, the method for reprogramming the small molecule composition involves adding a portion of the reprogrammed small molecule composition during cell culture. The specific steps are as follows: Cell preparation and seeding: Porcine somatic cells to be reprogrammed are taken, preferably porcine fetal fibroblasts or skin fibroblasts. Cells are digested using standard trypsin digestion, counted, and seeded at (1-5) × 10⁻⁶. 4 Cells were seeded in cell culture plates at a density of cells / cm². Cells were cultured in complete medium (DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin) at 37°C in a 5% CO2 incubator. Small molecule treatment could begin when the cells reached approximately 60%-80% confluence.

[0046] Preparation of working solutions for small molecule compositions: Before the treatment begins, each component is dissolved directly in freshly prepared complete culture medium or culture medium containing specific supplements (such as adding 1× N2 / B27 supplement DMEM / F12) within its preferred concentration range, and prepared immediately to form a small molecule treatment working solution.

[0047] For example, for the composition containing sodium butyrate (C2), the working solution comprises: 250 μM valproic acid, 10 μM CHIR99021, 10 μM Repsox, 5 μM transphenylcyclopropane, 50 μM foctocorline, and 200 μM sodium butyrate. For the composition containing bFGF (C3), sodium butyrate is replaced with 100 ng / mL bFGF.

[0048] Small molecule treatment: Timing of addition: When cells reach the appropriate confluence as described in step 1, discard the original culture medium. Initial treatment: Gently aspirate the old culture medium and wash the cells once with phosphate-buffered saline (PBS) preheated to 37°C. Then add sufficient volume (e.g., 2 mL per well) of the above-mentioned small molecule treatment working solution, ensuring complete coverage of the cell layer. This time point is recorded as day 0 of treatment. Treatment conditions: Return the cells to an incubator at 37°C and 5% CO2 for continued culture. Continuous culture and medium change: Medium change protocol: To maintain the activity of small molecules and good cell condition, fresh small molecule treatment working solution needs to be changed daily. The procedure is as follows: Gently aspirate the old culture medium, gently wash the cells once with preheated PBS, and then add freshly prepared small molecule treatment working solution. Treatment cycle: Continuous treatment for 7 days. During the treatment period, cell morphological changes can be observed under a microscope daily. Successful reprogramming is usually accompanied by a "rejuvenated" phenotype such as reduced cell volume, compact morphology, and increased refractive index. Treatment termination and cell collection: After the 7th day of treatment, complete the final medium change and incubate for the appropriate time, then discard the culture medium containing small molecules. Gently wash the cells 1-2 times with PBS to thoroughly remove any residual small molecules. Subsequently, the following procedures can be performed according to the requirements of downstream experiments.

[0049] Direct cell collection: for epigenetic age assessment (core step).

[0050] Passaging and amplification: Cells are digested with trypsin and passaged using conventional methods for subsequent gene editing or amplification culture.

[0051] Key quality control steps: Epigenetic age assessment (required): Following the method described in this invention, the constructed porcine-specific multi-omics epigenetic clock model is used to examine the treated cells and calculate the percentage reversal of their epigenetic age relative to their initial age before treatment. Screening criteria: Only when the reversal percentage falls within the target window of 57% to 77% (preferably 62%-72%) is the batch of cells considered to have successfully completed quantitative partial reprogramming, suitable for subsequent extreme gene editing and somatic cell nuclear transfer (SCNT) as nuclear donors. Optional auxiliary indicators: The correlation coefficient of protein spatial distribution can be measured using a nucleoplasmic compartmentalization (NCC) fluorescence reporter system as an auxiliary assessment of nuclear membrane integrity and functional status.

[0052] This invention provides a method for preparing partially reprogrammed porcine somatic cells. In this invention, the epigenetic age of the partially reprogrammed porcine somatic cells is reversed to 57% to 77% of the initial epigenetic age, preferably 62% to 72%. The partially reprogrammed porcine somatic cells can tolerate extreme multiplex gene editing capabilities and achieve an early abortion rate of less than 10% in somatic cell nuclear transfer (SCNT).

[0053] This invention provides a super-humanized gene-edited porcine nuclear donor cell, obtained by gene editing of partially reprogrammed porcine somatic cells; the gene editing includes the knockout of the GGTA1 gene, CMAH gene, and B4GALNT2 gene; and the knock-in of at least 16 genes selected from CD46, CD55, CD59, THBD, TFPI, CD39, EPCR, vWFΔA1, HLA-E, B2M, CD47, PD-L1, CTLA4-Ig, FasL, TNFAIP3, HMOX1, HO-2, BCL-2, and Survivin.

[0054] The present invention does not specifically limit the gene editing method described herein; conventional gene editing methods in the art can be used.

[0055] In this invention, partially reprogrammed porcine somatic cells undergo epigenetic age assessment and screening before gene editing. Cells whose epigenetic age is reversed to 57% to 77% of their initial epigenetic age and whose protein spatial distribution correlation coefficient is less than 0.25 are selected for subsequent operations.

[0056] This invention is the first to construct and validate a high-precision epigenetic clock specific to pigs: addressing the lack of assessment tools for the pig species, this invention is the first to systematically collect multi-omics data from multiple tissues and developmental stages in pigs, and successfully construct a clock model that can accurately quantify the somatic epigenetic age of pigs, providing a quantifiable benchmark tool for subsequent research.

[0057] The initial construction and evaluation system for a specific pig-specific epigenetic clock includes the following steps: Samples of various tissues (skin fibroblasts, liver, muscle, etc.) from multiple pig breeds (such as Bama Miniature Pig and Large White Pig) from the embryonic stage (0 months) to adulthood (36 months) were collected and whole-genome bisulfite sequencing (WGBS) and chromatin accessibility sequencing (ATAC-seq) were performed.

[0058] Feature selection and model training: CpG sites exhibiting linear changes during pig development and aging (approximately 500-800 sites) were selected from WGBS data. Combined with chromatin open region features identified by ATAC-seq, a pig-specific multi-omics epigenetic age prediction model was trained using the Elastic Net machine learning algorithm. The prediction formula for this model is as follows:

[0059] in: β 0 represents the intercept term. Mr. For the first j methylation β value of each CpG site; AndFor the first k Standardized signal intensity of each chromatin accessibility region; βj , γk These are the regression coefficient weights for the corresponding features.

[0060] Model Validation: On the independent validation set, the model performed excellently: the mean absolute error (MAE) between predicted age and actual chronological age was 2.4 months, and the coefficient of determination R² > 0.92. This indicates that the model provides a reliable tool for quantifying epigenetic age in pigs for the first time.

[0061] Meanwhile, this invention constructs a porcine fetal fibroblast cell line that stably expresses NLS-mCherry and NES-eGFP. The nuclear membrane integrity is quantitatively assessed using the correlation coefficient of protein spatial distribution based on the nucleoplasmic compartmentalization (NCC) fluorescence reporter system, serving as an auxiliary indicator of cell function and viability.

[0062] Based on the above, the present invention also provides a porcine somatic cell nuclear donor quality assessment and screening system. This system integrates the detection results of the following two indicators to predict or screen low-abortion-rate nuclear donor cells suitable for SCNT: (i) Correlation coefficients of protein spatial distribution based on nucleocytoplasmic compartmentalization (NCC) fluorescence reporter system; (ii) Percentage of epigenetic age reversal calculated based on a pig-specific multi-omics epigenetic clock model; Specifically, when the correlation coefficient is below 0.25 and the reversal percentage is in the range of 57% to 77%, the cell is assessed as having low miscarriage potential.

[0063] This invention also provides a method for producing super-humanized cloned pigs, using the aforementioned super-humanized gene-edited pig nuclear donor cells as the nuclear donor, and producing cloned pigs through somatic cell nuclear transfer technology. This invention does not impose any special limitations on the specific operation of the somatic cell nuclear transfer technology; conventional operations in the art can be used.

[0064] This invention provides a partially reprogrammed small molecule composition comprising valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, foctocorline, and sodium butyrate; 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, the concentration of foctocorline is 40-60 μM, and the concentration of sodium butyrate is 150-250 μM; Alternatively, it may include valproic acid, CHIR99021, Repsox, transphenylcyclopropane, foctocorline, and bFGF; 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 transphenylcyclopropane is 4-6 μM, the concentration of foctocorline is 40-60 μM, and the concentration of bFGF is 80-120 ng / ml.

[0065] The partially reprogrammed small molecule composition provided by this invention can reverse the epigenetic age of cells to 57%–77% of the initial epigenetic age with almost 100% accuracy, and the 22-gene edited donor cells prepared therefrom achieved an extremely low early miscarriage rate of approximately 7%. This effect is significantly superior to the well-known and highly effective OSKGL mRNA regimen (10.7%) and the basic regimen cocktail small molecule composition consisting only of known components (15.4%). Notably, the addition of sodium butyrate or bFGF alone was ineffective in reversing porcine cell senescence and improving SCNT, indicating that the extreme effect achieved by the partially reprogrammed small molecule composition described in this invention is an unpredictable synergistic effect produced by the components in the porcine cell system, which cannot be easily deduced from existing knowledge.

[0066] 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.

[0067] Example 1

[0068] The first construction and evaluation system of a pig-specific epigenetic clock.

[0069] 1. Transformation of cross-species principles and model building

[0070] Drawing upon established principles for constructing epigenetic clocks in humans and mice (e.g., age regression models based on DNA methylation arrays or sequencing data), this system was applied to pigs for the first time. Specific steps included: Data collection: Samples of various tissues (skin fibroblasts, liver, muscle, etc.) from multiple pig breeds (such as Bama Miniature Pig and Large White Pig) from the embryonic stage (0 months) to adulthood (36 months) were collected for whole-genome bisulfite sequencing (WGBS) and chromatin accessibility sequencing (ATAC-seq).

[0071] Feature selection and model training: CpG sites (e.g., about 500-800) that show linear changes during pig development and aging were selected from WGBS data. Combined with chromatin open region features identified by ATAC-seq, machine learning algorithms such as Elastic Net regression were used to train a pig-specific multi-omics epigenetic age prediction model.

[0072] Model Validation: On the independent validation set, the model performed excellently: the mean absolute error (MAE) between the predicted age and the actual chronological age was 2.4 months, and the coefficient of determination R² > 0.92. This indicates that the model provides a reliable tool for quantifying epigenetic age in pigs for the first time.

[0073] Construction and Characterizable Forms of Pig-Specific Epigenetic Clock Models

[0074] 1. Model construction methods and input features

[0075] This invention presents the first pig-specific high-precision epigenetic clock model, trained using the Elastic Net Regression machine learning algorithm by integrating whole-genome DNA methylation data and chromatin accessibility data. The specific steps are as follows: (1) Data acquisition and preprocessing

[0076] Multiple pig breeds were collected from the embryonic stage (0 months) to adulthood (36 months of age), including: Bama Miniature Pig, Large White Pig, Yunnan Small-eared Pig, Xishuangbanna Miniature Pig, Wuzhishan Pig, Tibetan Pig, Jinhua Two-headed Black Pig, Göttingen Miniature Pig, Yucatan Miniature Pig, Hanford Miniature Pig, Pittman-Moore Miniature Pig, and Yorkshire Pig.

[0077] Multiple tissue samples were collected from the above-mentioned varieties, including: skin fibroblasts, peripheral blood mononuclear cells, liver tissue, and skeletal muscle tissue.

[0078] The above samples underwent: whole-genome bisulfite sequencing (WGBS) to obtain DNA methylation levels at single-base resolution; and chromatin accessibility sequencing (ATAC-seq) to obtain information on open chromatin regions. The sequencing data were standardized and preprocessed, including alignment (using tools such as Bismark and Bowtie2), deduplication, normalization, and the methylation β value (range 0–1) for each CpG site and the signal intensity for each open chromatin region were calculated.

[0079] (2) Feature Filtering

[0080] CpG sites exhibiting linear or quasi-linear changes during pig development and aging were screened from WGBS data. The screening method is as follows: Use the R packages “MethylKit” or “SeSAMe” to calculate the slope and significance of a linear regression of methylation level at each CpG site versus age in months. The screening criteria were: regression p-value < 0.001, and methylation change > 10% every 10 months; A total of 612 CpG sites were finally obtained through screening, forming a DNA methylation feature vector M=[M1,M2,...,M612] M 1, M 2,..., M 612].

[0081] Screening for age-significantly age-related chromatin accessibility regions from ATAC-seq data: Open regions were identified using the software "MACS2" and their correlation with age was analyzed using DiffBind or DESeq2. The screening criteria were: FDR < 0.01, and the signal change was significantly correlated with age. The final selection yielded a total of 218 chromatin accessibility regions, forming a feature vector A=[A1,A2,...,A218]. A 1, A 2,..., A 218].

[0082] (3) Model training

[0083] The aforementioned feature vectors are used to train an elastic network regression with the actual chronological age of the samples.

[0084] Objective function:

[0085] in:

[0086] yi represents the actual age in months of the i-th sample;

[0087] β 0 represents the intercept term. β=[β1,β2,…,β 612 ] T This is the coefficient vector of DNA methylation characteristics; γ = [γ1, γ2, ..., γ218] T is the coefficient vector of chromatin accessibility features; λ1 and λ2 are regularization hyperparameters, which are selected through cross-validation optimization.

[0088] 2. The representative mathematical form of the model

[0089] After training, the model can be expressed as the following linear prediction formula:

[0090] in: β 0 represents the intercept term. Mr. For the first jmethylation β value of each CpG site; And For the first k Standardized signal intensity of each chromatin accessibility region; βj , γk These are the regression coefficient weights for the corresponding features.

[0091] Model Validation: To objectively evaluate the model's generalization ability and prediction accuracy, we constructed an independent validation sample set. This validation set contains the following samples: Breeds: Bama miniature pig, Large White pig, and Yunnan miniature pig. These breeds were not included in the model training phase but were used to test the model's cross-breed applicability.

[0092] Tissue types: skin fibroblasts, peripheral blood mononuclear cells, liver tissue, skeletal muscle tissue, covering the main tissue types targeted in the model design.

[0093] Sample size and age distribution: A total of 45 independent samples were collected, with ages ranging from 4 to 32 months, and the samples were evenly distributed across different age groups.

[0094] Data independence: All individual samples in the validation set and their corresponding WGBS and ATAC-seq data were not used in the training and feature selection process of the aforementioned model.

[0095] On this independent validation set, the pig-specific epigenetic clock model performed exceptionally well: the mean absolute error (MAE) between predicted age and actual chronological age was 2.4 months, and the coefficient of determination R² > 0.92. This result demonstrates that the model provides, for the first time, a highly accurate, reliable, and generalizable tool for quantifying epigenetic age in pigs.

[0096] 2. Nucleoplasmic compartmentalization integrity as a functional auxiliary indicator

[0097] Meanwhile, a porcine fetal fibroblast cell line stably expressing NLS-mCherry and NES-eGFP was constructed. The integrity of the nuclear membrane was quantitatively assessed by analyzing the fluorescence colocalization Pearson correlation coefficient through imaging, serving as an auxiliary indicator of cell function and viability.

[0098] Table 1. Evaluation system's ability to distinguish between different states of pig cells.

[0099] Example 2

[0100] Screening and Validation of the Best Reprogramming Solution for Achieving Extremely Low Miscarriage Rate

[0101] Experimental Design

[0102] For senescent porcine fibroblasts (PD>40, epigenetic age ~48.5 months) from the same batch, the following reprogramming protocols were applied for 7 days: Basic culture medium: DMEM / F12 + 10% FBS + 1× N2 / B27 supplement Treatment time: 7 consecutive days, with different treatment groups receiving fresh culture medium containing the following corresponding components daily. Group A: OS (Oct4, Sox2 mRNA) – Reversal of Insufficient Control Group B: OSKM (Oct4, Sox2, Klf4, c-Myc mRNA) – Reversal Overload Control Group C: OSKGL (Oct4, Sox2, Klf4, Glis1, Lin28 mRNA) Group D: Small molecule C1 (basic known combinations): VPA 250μM, CHIR99021 10μM, Repsox-616452 10μM, Tranylcypromine 5μM, Forskolin 50μM Group E: Small molecule C2 (C1 + sodium butyrate 200μM) Group F: Small molecule C3 (C1 + bFGF 100 ng / mL) Group G: Sodium butyrate only 200 μM (control) Group H: bFGF 100 ng / mL only (control)

[0103] [Specific operating methods for each reprogramming processing group]

[0104] All treatments used aging porcine fibroblasts (PD > 40, epigenetic age approximately 48.5 months) from the same batch as the starting material, with cells pre-seeded in culture plates at appropriate densities. The basal medium was DMEM / F12 + 10% fetal bovine serum (FBS) + 1× N2 / B27 supplement. Treatments were performed for 7 consecutive days, with fresh medium containing the corresponding components replaced daily.

[0105] Group A: OS (Oct4, Sox2 mRNA) – Reversal of Insufficient Control

[0106] mRNA preparation: Chemically modified (pseudouridine substitution) porcine codon-optimized Oct4 and Sox2 mRNAs with 5' caps and poly(A) tails were synthesized by in vitro transcription, purified by HPLC, and dissolved in nuclease-free water.

[0107] Preparation of working solution: Before use, mix Oct4 and Sox2 mRNA in an equimolar ratio, dilute with nuclease-free water, and prepare a mixed working solution with a final concentration of 30 ng / μL for each mRNA.

[0108] Transfection procedure: After switching cells to Opti-MEM, Lipofectamine MessengerMAX transfection reagent was used to form a complex with the mRNA working solution according to the reagent instructions. Cells were transfected daily, with a final concentration of approximately 15 ng / μL for each mRNA.

[0109] Culture and medium change: Replace with fresh basal medium (without mRNA) 4-6 hours after each transfection. Repeat transfection the next day for 7 consecutive days.

[0110] Group B: OSKM (Oct4, Sox2, Klf4, c-Myc mRNA) – Reversal Overload Control

[0111] mRNA preparation: Chemically modified porcine codon-optimized Oct4, Sox2, Klf4 and c-Myc mRNAs were prepared.

[0112] Working solution preparation: Mix the four mRNAs in equimolar proportions to prepare a mixed working solution with a final concentration of 25 ng / μL for each mRNA.

[0113] Transfection procedure: The daily transfection method is the same as that of Group A, and the final concentration of each mRNA is approximately 12.5 ng / μL.

[0114] Culture and medium change: Same as Group A, continuous treatment for 7 days.

[0115] Group C: OSKGL (Oct4, Sox2, Klf4, Glis1, Lin28 mRNA)

[0116] mRNA preparation: Chemically modified porcine codon-optimized Oct4, Sox2, Klf4, Glis1, and Lin28 mRNAs were prepared.

[0117] Working solution preparation: Mix the five mRNAs in equimolar proportions to prepare a mixed working solution with a final concentration of 20 ng / μL for each mRNA.

[0118] Transfection procedure: The daily transfection method is the same as that of Group A, and the final concentration of each mRNA is approximately 10 ng / μL.

[0119] Culture and medium change: Same as Group A, continuous treatment for 7 days.

[0120] Group D: Small molecule C1 (basic known combinations)

[0121] Small molecule stock solutions: Prepare DMSO stock solutions of VPA (valproic acid, 500 mM), CHIR99021 (10 mM), Repsox-616452 (10 mM), Tranylcypromine (10 mM), and Forskolin (10 mM).

[0122] Working solution preparation: Prepare fresh solutions daily. Add each stock solution to the pre-warmed basal medium in the specified proportions to achieve the following final concentrations: VPA 250 μM, CHIR99021 10 μM, Repsox 10 μM, transphenylcyclopropane 5 μM, and foctocorline 50 μM. Ensure the final DMSO concentration does not exceed 0.1% (v / v).

[0123] Processing procedure: Discard the old culture medium daily, gently wash the cells once with PBS, and add freshly prepared small molecule C1 working solution.

[0124] Incubation: Incubate at 37°C in a 5% CO2 incubator for 24 hours, then replace with fresh working solution for 7 consecutive days.

[0125] Group E: Small molecule C2 (C1 + sodium butyrate)

[0126] Small molecule stock solution: In addition to all the components in group D, prepare sodium butyrate (1 M aqueous solution or PBS solution).

[0127] Preparation of working solution: Add sodium butyrate stock solution to the C1 working solution daily to bring the final concentration to 200 μM.

[0128] Treatment procedure: Same as group D, but change to C2 working solution containing sodium butyrate daily for 7 consecutive days.

[0129] Group F: Small molecule C3 (C1 + bFGF)

[0130] Small molecule and factor stock solution: In addition to all components of group D, recombinant human bFGF (100 μg / mL PBS solution containing 0.1% BSA) was prepared separately.

[0131] Preparation of working solution: Add bFGF stock solution to the C1 working solution daily to achieve a final concentration of 100 ng / mL.

[0132] Treatment procedure: Same as group D, but change to C3 working solution containing bFGF daily for 7 consecutive days.

[0133] Group G: Sodium butyrate only (control)

[0134] Stock solution: Same as Group E, using sodium butyrate stock solution.

[0135] Preparation of working solution: Add sodium butyrate stock solution to the basal culture medium daily to achieve a final concentration of 200 μM.

[0136] Treatment procedure: Replace the working solution with sodium butyrate-only solution daily for 7 consecutive days. Other procedures are the same as for Group D.

[0137] Group H: bFGF only (control)

[0138] Stock solution: Same as group F, using bFGF stock solution.

[0139] Preparation of working solution: Add bFGF stock solution to the basal culture medium daily to achieve a final concentration of 100 ng / mL.

[0140] Treatment procedure: Replace the working solution with bFGF-only solution daily for 7 consecutive days. Other procedures are the same as for group D.

[0141] Window definition and effect evaluation

[0142] Window entry rate statistics: After treatment, the percentage of cell reversal under each protocol was calculated using the pig-specific epigenetic clock, and the proportion of cells falling into the 57-77% window was statistically analyzed.

[0143] 22-gene editing and SCNT: Standardized 22-gene editing was performed on each group of cells (using the CRISPR-Cas9 system, targeting sites such as GGTA1 and CMAH, and knocking in human genes), according to the method described in Chinese patent CN 108486152 B "Breeding Method and Application of Transgenic Pigs".

[0144] At least five successfully edited monoclonal lines were obtained in each group. After expansion, each monoclonal line served as a nuclear donor, and SCNT was performed using a standardized procedure of transferring 30-35 cloned embryos to each recipient sow. Landrace pigs aged 6-8 months were used as recipients. Pregnancy was confirmed by ultrasound at 30 days of gestation, and abortion events were strictly recorded within 30-60 days. Chi-square test was used to analyze the significance of differences between groups.

[0145] Table 2. Data on the relationship between core performance and miscarriage rate of different reprogramming schemes (mean ± SD)

[0146] Note 1: The OSKM group had a low NCC coefficient but was accompanied by high Nanog expression and abnormal karyotype. Note 2: The results indicate that the abortion rates in groups C2 and C3 were significantly lower than those in groups OSKGL and C1 (p < 0.05). The 11 gene-edited pigs were constructed according to the method described in patent CN 108486152 B, "Breeding and Application of Transgenic Pigs". Based on the above data, the abortion rates of the OS and OSKM protocols, which failed to reach the 57%–77% window, were >55%, while the abortion rates of protocols that successfully reached the window dropped sharply to below 20%. This is the first quantitative demonstration in a pig system of a narrow threshold window crucial for low abortion rates in epigenetic age reversal.

[0147] Among all the protocols that efficiently induce the window of induction, protocols C2 and C3 further reduced the early abortion rate to a critical level of 7%, significantly outperforming other protocols. Sodium butyrate or bFGF alone were completely ineffective in reversing porcine cell senescence and subsequent processes, but when combined with the basic known combination C1, they produced a far superior effect. This demonstrates that C2 / C3 achieves an unpredictable synergistic effect in the porcine cell system that cannot be extrapolated from the known functions of each component, rather than a simple additive effect.

[0148] Example 3

[0149] Quantitative comparison of the effects of two reprogramming schemes

[0150] Table 3 Comparison of reprogramming effects between mRNA and small molecule protocols Note 1: All data are based on 6 independent donor porcine cell lines, with n=5 for each experiment. Key conclusion: The small molecule approach offers unique advantages such as low cost, simple operation, good reproducibility, and no induction of Nanog expression, while achieving equivalent reprogramming effects (precisely falling within the safety window).

[0151] Note 2: The data for the small molecule combination schemes in this table are based on the C3 (containing bFGF) scheme in Example 2, representing the typical effect of the small molecule composition reprogramming scheme described in this invention. The C2 scheme has equivalent efficacy and similar properties.

[0152] Verification of the boundary effect of the safety window period

[0153] Table 4. Decisive Influence of Epigenetic Reversal Degree on Pig Somatic Cell Fate Note: Editing success rate is the probability of obtaining a complete 22-gene edited monoclonal clone; tumor incidence rate refers to the proportion of piglets that survive more than 1 month after birth and have any tumors found within 6 months; n = 8 independent cell lines / groups.

[0154] As shown above, 55%-75% is the only feasible window for pig somatic cells to safely and efficiently complete 22-gene editing and produce healthy cloned pigs. Within this window, the cells gain "regeneration" ability but do not lose their somatic cell identity; deviations from this window all lead to failure or danger.

[0155] Example 4

[0156] Different gene-edited pigs were prepared, as shown in the table below.

[0157] Table 5 Comparison of gene editing lists for different genetically engineered pigs

[0158] In vitro immunocompatibility system assessment

[0159] Instructions for assessing in vitro immune rejection barrier function

[0160] A. Pre-existing antibody barrier

[0161] Human IgG / IgM binding (MFI): Porcine endothelial cells or fibroblasts were incubated with healthy human serum (containing natural xenoreactive antibodies) at 37°C for 1 hour. After washing, FITC-labeled goat anti-human IgG or IgM secondary antibody was added, and the cells were incubated in the dark for 30 minutes. Flow cytometry was used to detect the fluorescence intensity on the cell surface, and the binding level was expressed as mean fluorescence intensity (MFI).

[0162] B. Complement attack barrier

[0163] CDC kill rate (%): Porcine cells were mixed with human serum (complement source) in a specific ratio and incubated at 37°C for 2 hours. The percentage of cytotoxicity was calculated using the lactate dehydrogenase (LDH) release assay or propidium iodide (PI) staining combined with flow cytometry.

[0164] MAC / C3b deposition (MFI): After complement attack, cells were washed and fluorescently labeled anti-human C5b-9 (membrane attack complex) or anti-C3b antibody was added, and the deposition intensity was detected by flow cytometry.

[0165] C. Coagulation compatibility barrier

[0166] TAT generation (ng / mL): After co-incubating porcine cells with human plasma, the supernatant was collected and detected using a human thrombin-antthrombin complex (TAT) ELISA kit.

[0167] aPC activity (%): The relative activity was calculated using a chromogenic substrate method aPC activity assay kit, with human umbilical vein endothelial cells (HUVEC) as a reference (set as 100%).

[0168] Platelet aggregation inhibition rate (%): Porcine cells were co-incubated with human platelets, and thrombin or ADP was added to induce aggregation. The maximum aggregation rate was recorded using a platelet aggregator, and the inhibition rate was calculated.

[0169] D. Innate immune cell attack

[0170] NK cell killing rate (%): NK cells were isolated from human peripheral blood and co-incubated with CFSE-labeled porcine cells at an effector-target ratio of 10:1 for 4 hours. The killing rate was calculated using the Calcein-AM release method or the LDH method.

[0171] Macrophage phagocytosis rate (%): CFSE-labeled porcine cells were co-cultured with macrophages derived from human monocytes, and the proportion of intracellular fluorescence signals in macrophages was detected by flow cytometry.

[0172] E. Adaptive immune regulation

[0173] T cell activation inhibition (%): porcine cells were co-cultured with human peripheral blood mononuclear cells, and stimulated with anti-CD3 / CD28 monoclonal antibodies for 72 hours. The proportion of CD25+CD69+ T cells was detected by flow cytometry, and the inhibition rate relative to the control without porcine cells was calculated.

[0174] B cell antibody inhibition (%): porcine cells and human B cells were co-cultured and stimulated with CpG or anti-IgM for 5 days. The concentration of human IgG in the culture supernatant was detected by ELISA, and the inhibition rate was calculated.

[0175] F. Comprehensive Protection Index

[0176] The above indicators were normalized to the human endothelial cell reference value (set to 1.0) and then weighted averaged according to predefined weights (complement barrier 30%, coagulation compatibility 25%, cell attack 20%, antibody barrier 15%, immune regulation 10%).

[0177] Table 6 Comparison data of in vitro immune rejection barrier function

[0178] Note: The comprehensive protection index is the weighted average of all indicators after normalization; all data are n=5 animals / groups. The p-value for genome 22 is less than that for genomes 10 and 11; the human endothelial cells are HUVECs.

[0179] It is evident that the 22-gene edited pigs achieved near-perfect compatibility with human cells across all six major types of immune rejection barriers, with a comprehensive protection index of 0.94 (compared to 1.00 for human cells), significantly superior to the 10-gene (0.58) and 11-gene (0.66) pigs.

[0180] Example 5

[0181] 1. Pig-monkey skin graft model

[0182] Experimental methods for pig-monkey skin transplantation model

[0183] 1. Laboratory animals and grouping

[0184] Donors: Wild-type pigs, 10-gene-edited pigs, 11-gene-edited pigs, and the 22-gene-edited pigs described in this invention were selected (all aged 6-8 months, weighing 25-35 kg, regardless of sex). Two weeks before transplantation, a full-thickness skin graft (approximately 0.3-0.4 mm thick) was prepared from the back of each donor pig and preserved in organ preservation solution at 4°C for later use.

[0185] Recipients: Healthy adult cynomolgus monkeys (Macaca fascicularis), weighing 3.5-5.5 kg, of any sex, without immune system diseases. They were divided into the following experimental groups according to donor type (n=10 per group): Allogeneic transplantation control group (monkey skin → monkey) Wild-type pig skin transplant group 10 Gene-Edited Pig Skin Transplant Group 11 Gene-Edited Pig Skin Transplant Group 22-Gene Edited Pig Skin Transplant Group (This Invention)

[0186] 2. Skin grafting surgery

[0187] Recipient preparation: After anesthesia, a full-thickness skin defect (approximately 2 cm × 2 cm in area) was prepared on each side of the back of the recipient monkey, reaching the fascia layer, and hemostasis was achieved.

[0188] Skin grafting: The pre-treated donor skin graft is trimmed to the same size as the wound, placed on the wound, and fixed with interrupted sutures using 5-0 absorbable sutures to ensure close contact between the skin graft and the wound base.

[0189] Bandaging and protection: The transplant area was covered with Vaseline gauze and sterile dressing, and then bandaged with an elastic bandage. Animals were kept individually post-surgery to prevent scratching of the transplant area. No systemic or local immunosuppressants were used in any of the experimental groups during the perioperative period or post-surgery.

[0190] 3. Postoperative observation and evaluation index recording

[0191] General observation: Observe the animal's mental state, activity, diet, and the appearance of the transplanted area daily.

[0192] Survival time (days): The number of days from the date of transplantation until the graft undergoes complete necrosis, detachment, or histologically confirmed complete rejection (defined as the survival endpoint). If the graft is still alive at the end of the observation period (e.g., 35 days), it is recorded as ">Days at the end of the observation period".

[0193] Coverage (%): On days 7, 14, 21, and 28 post-transplantation, the area of ​​surviving skin grafts (normal color and texture, without necrosis, scabs, or detachment) was measured using the transparent film tracing method or digital image analysis software (such as ImageJ), and the percentage of the surviving skin graft area was calculated. The formula is: Coverage = (Surviving skin graft area / Initial transplant area) × 100%.

[0194] Incision scoring: On day 7 post-transplantation, two researchers, whose groups were unknown, independently assessed the healing of the peripheral suture incision in the transplant area using the following scoring criteria: 3 points: The incision has healed completely, with no redness, swelling, or oozing, and the apposition is good.

[0195] 2 points: The incision is slightly red and swollen or has a small amount of oozing, but the closure is acceptable.

[0196] 1 point: Moderate redness and swelling of the incision, oozing, or partial dehiscence.

[0197] 0 points: Severe infection, necrosis, or complete dehiscence of the incision.

[0198] The average of the two scores is taken as the final cut score.

[0199] Histological rejection grading: On day 28 post-transplantation or at the endpoint of graft rejection, a full-thickness tissue sample (including both graft and part of the recipient's marginal tissue) is harvested, fixed in 10% neutral formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The sample is then evaluated by a pathologist under single-blind conditions according to the Banff Xenograft Pathological Grading Criteria (2016). Banff Level 0: No obvious signs of rejection.

[0200] Banff Grade 1: Very slight rejection, with occasional small amounts of lymphocyte infiltration.

[0201] Banff grade 2: Mild rejection, focal lymphocytic infiltration, with mild vasculitis.

[0202] Banff grade 3: moderate rejection, diffuse inflammatory cell infiltration, moderate vasculitis, with endothelial cell swelling.

[0203] Banff Grade 4: Severe rejection, extensive bleeding, thrombosis, vascular necrosis, or graft necrosis.

[0204] Hyperacute rejection: Characterized by widespread thrombosis, hemorrhage, and neutrophil infiltration within minutes to hours after transplantation.

[0205] 4. Data Analysis

[0206] All continuous data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA or nonparametric tests were used for comparisons between groups, and the Kaplan-Meier method and Log-rank test were used for survival analysis. A p-value < 0.05 was considered statistically significant. This indicates that compared with the allogeneic group, p < 0.05. This indicates that compared with the 10-gene and 11-gene editing groups, p < 0.01.

[0207] Table 7 Quantitative evaluation results of the skin graft model

[0208] Note: n = 10 monkeys / group; Incision score: 3 points = completely normal, 2 points = mild abnormality, 1 point = moderate abnormality, 0 points = necrosis; This indicates that compared with genes 10 and 11, p < 0.001.

[0209] Example 6

[0210] Validation of long-term survival model of xenotransplantation

[0211] This embodiment provides a standardized preclinical model for evaluating the long-term survival and function of donor pig kidneys with different degrees of gene modification in primates. By comparing the transplantation effects of pig kidneys edited with 10 genes, 11 genes, and the 22 genes of this invention, the breakthrough advantage of this invention in achieving long-term survival is confirmed.

[0212] 1. Experimental Design and Animal Preparation

[0213] 1.1 Donor Pigs

[0214] Breeds and genotypes: Three different genetically modified Xishuangbanna miniature pigs were used as donors, all of which were female and had blood type OO.

[0215] 10 gene-edited donor pigs: The gene editing list is the same as Table 5 in the patent text. It includes GGTA1, CMAH, and B4GALNT2 knockout, as well as CD46, CD55, THBD, PROCR, CD47, TNFAIP3, and HMOX1 gene knock-in (a total of 10 edits).

[0216] 11 gene-edited donor pigs: The gene editing list is the same as Table 5 in the patent text. One gene knock-in, such as CD39 or TFPI, is added to the 10-gene list (a total of 11 edits).

[0217] 22 gene-edited donor pigs (this invention): The gene editing list is the same as Table 5 and claim 7 in the patent text. This includes GGTA1, CMAH, and B4GALNT2 knockout, and at least 16 human gene knock-ins selected from CD46, CD55, CD59, THBD, TFPI, CD39, EPCR, vWFΔA1, HLA-E, B2M, CD47, PD-L1, CTLA4-Ig, FasL, TNFAIP3, HMOX1, HO-2, BCL-2, and Survivin (total number of edits ≥ 22).

[0218] Selection criteria: donors must be 6-8 weeks old, weigh 5-12 kg, be confirmed as being free of specific pathogens through quarantine, and have normal kidney function as assessed by biochemical and imaging methods.

[0219] 1.2 Receptors and Grouping

[0220] Recipients: Healthy adult cynomolgus monkeys (Macaca fascicularis), weighing 4-12 kg, regardless of sex. Individuals with low levels of pre-existing anti-pig antibodies are selected after serological screening prior to transplantation.

[0221] Experimental grouping: Based on the donor genotype, the recipient monkeys were divided into three groups, with six recipient monkeys in each group. Group A (10-gene edited donor kidney control group) Group B (11-gene-edited donor kidney control group) Group C (22 gene-edited donor kidney experimental group, this invention)

[0222] 2. Immunosuppression and Surgical Treatment

[0223] All recipient monkeys received the same clinically relevant immunosuppression regimen and standard surgical procedures to ensure a fair comparison of the inherent differences between different donor genotypes.

[0224] Immunosuppressive regimen: Induction therapy: Lymphocyte depletion is achieved before transplantation using anti-thymocyte globulin and anti-CD20 monoclonal antibody.

[0225] Maintenance therapy: Post-transplantation, immunosuppression is maintained by co-stimulation blockade based on anti-CD154 antibody combined with mycophenolate mofetil.

[0226] Adjunctive therapy: short-term use of tacrolimus and corticosteroids during the perioperative period.

[0227] Surgical procedures: A standardized ectopic kidney transplantation model was used. After removing both recipient kidneys, the donor kidney was transplanted into the right iliac fossa. End-to-side anastomoses were performed between the donor kidney's arteries and veins and the recipient's abdominal aorta and inferior vena cava, as well as a ureterovesical anastomosis. Standardized surgical procedures were ensured to minimize technical variations.

[0228] 3. Postoperative monitoring and evaluation

[0229] Postoperative comprehensive monitoring of the recipient was conducted, and the core indicators were consistent with those in Table 8 of the patent: Survival period: The number of days from transplantation to death or the predetermined end of the study.

[0230] Kidney function: Regularly monitor serum creatinine (SCr), blood urea nitrogen (BUN), and urine output.

[0231] Complications: Record events such as thrombotic microangiopathy (TMA), acute antibody-mediated rejection (AMR), infection, and thrombocytopenia.

[0232] Immune response: Monitoring peripheral blood lymphocyte subsets and the production of new donor-specific antibodies (dnDSA).

[0233] Pathological evaluation: At the end of the course, an autopsy was performed. The transplanted kidney tissue was stained with H&E, PAS and C4d and then evaluated by a pathologist according to the revised Banff criteria to diagnose the type of rejection and the pattern of damage.

[0234] 4. Verification Results

[0235] After implementing the above model, the key data are summarized as follows: Table 8. Survival data of pig-nonhuman primate xenotransplantation of kidneys.

[0236] Note: As of the data lockout, all six non-human primates that underwent the 22-gene donor kidney transplant have survived and have normal kidney function. The study is still ongoing.

[0237] Specific survival and endpoint analysis: The 10 gene-edited group had a short survival time, with a median survival of only 26 days. All receptors required enhanced immunosuppression, but TMA (4 / 6) and AMR (5 / 6) were still highly prevalent, and renal function was maintained for a short time (approximately 22 days).

[0238] Group 11 (gene-edited): Survival was extended, with a median survival of 45 days, but intensive immunosuppression was still required. The incidence of TMA (2 / 6) and AMR (3 / 6) was reduced, but not eradicated.

[0239] 22-gene editing group (this invention): A significant improvement in survival was achieved, with a median survival of over 90 days and a maximum survival of over 120 days, and all receptors were alive at the study cutoff. No enhanced immunosuppression was required for any receptors, and no TMA or AMR events were observed. Renal function remained normal for a long period (>75 days).

[0240] Pathological and mechanistic verification: Histological analysis further confirmed the reliability of the above results. Following transplantation of the 22-gene-edited pig kidneys, no obvious acute rejection or thrombotic damage was observed, and the glomerular and tubular structures remained intact. In contrast, the 10-gene and 11-gene control groups generally exhibited varying degrees of antibody deposition, complement activation (C4d positive), and microvascular thrombosis.

[0241] 5. Conclusion

[0242] This embodiment establishes a standardized pig-nonhuman primate xenotransplantation model to directly compare the effects of different gene modification strategies. Experimental results conclusively demonstrate that while editing only 10 or 11 genes can partially improve compatibility, it cannot overcome coagulation dysfunction and strong immune rejection, leading to short survival, high complication rates, and dependence on intensive immunosuppression. The 22-gene edited pig kidney provided by this invention, through systematic multi-gene combination modification, achieves long-term survival (>90 days) without intensive immunosuppression and completely avoids TMA and AMR, two major complications of xenotransplantation. This result, along with in vitro immunocompatibility data (overall protection index 0.94) and skin transplantation data (survival >35 days), constitutes a complete chain of evidence from the cellular, tissue, to organ levels, fully demonstrating the superiority and clinical translational potential of the "super-humanization" strategy described in this invention.

[0243] As demonstrated by the above embodiments, this invention has discovered and verified that 55%–75% epigenetic reversal is the only safe and effective window for porcine somatic cells to tolerate extreme gene editing, providing a clear quantitative standard for the field. This invention also develops two equivalent partial reprogramming schemes: a small molecule composition scheme, which reduces costs by 88%, simplifies operation, improves reproducibility, and does not induce Nanog expression; and an mRNA scheme, providing alternatives for different application scenarios. This invention achieves a significant breakthrough in porcine somatic cell tolerance to editing by partially reprogramming porcine somatic cells: increasing the number of editable genes from the industry limit of 11–12 to 22, and improving the editing success rate from <1% to 8.5%. The obtained cell in vitro compatibility is close to that of humans: the comprehensive protection index reaches 0.94 (human cells 1.00), significantly better than existing schemes. Excellent in vivo survival performance: skin transplant coverage remains at 62% after 28 days, and all kidney transplant recipients have long-term survival and normal kidney function. This invention also achieves comprehensive safety assurance, precise window period control to avoid tumor risk, and 22-gene editing to comprehensively improve porcine organ-to-human compatibility.

[0244] The present invention provides a partially reprogrammable small molecule composition for the partial reprogramming of porcine somatic cells: the composition is well-defined, the cost is low, the operation is simple, and it is suitable for large-scale production.

[0245] The overall process of this invention has good reproducibility: the coefficient of variation between batches is <10%; the animals are healthy and fertile: the cloned pigs are healthy, can reproduce normally, and have stable genotype inheritance.

[0246] 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 method for quantitative partial reprogramming of porcine somatic cells, characterized in that, The somatic cell epigenetic age of pigs was reversed to 57%–77% of the initial epigenetic age.

2. The method according to claim 1, characterized in that, The somatic epigenetic age of pigs was reversed to 62%–72% of the initial epigenetic age.

3. The method according to claim 1 or 2, characterized in that, The pig somatic cell epigenetic age reversal is achieved by mRNA reprogramming or by reprogramming with a small molecule composition. The method of mRNA reprogramming is to deliver mRNA sequences encoding OCT4, SOX2, KLF4, GLIS1, and Lin28 into cells; The method for reprogramming the small molecule composition involves adding a portion of the reprogrammed small molecule composition during cell culture.

4. The method according to claim 3, characterized in that, The partially reprogrammed small molecule composition includes valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, foctocorline, and sodium butyrate; 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, the concentration of foctocorline is 40-60 μM, and the concentration of sodium butyrate is 150-250 μM.

5. The method according to claim 3, characterized in that, The partially reprogrammed small molecule composition includes valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, foctocorline, and bFGF; 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, the concentration of foctocorline is 40-60 μM, and the concentration of bFGF is 80-120 ng / ml.

6. Partially reprogrammed porcine somatic cells prepared by the method according to any one of claims 1 to 5.

7. A super-humanized gene-edited porcine nuclear donor cell, characterized in that, The partial reprogrammed porcine somatic cells described in claim 6 were obtained through gene editing; the gene editing included the knockout of the GGTA1 gene, the CMAH gene, and the B4GALNT2 gene. And knock-in of at least 16 genes selected from CD46, CD55, CD59, THBD, TFPI, CD39, EPCR, vWFΔA1, HLA-E, B2M, CD47, PD-L1, CTLA4-Ig, FasL, TNFAIP3, HMOX1, HO-2, BCL-2, and Survivin.

8. The super-humanized gene-edited porcine nuclear donor cell according to claim 7, characterized in that, The partially reprogrammed pig somatic cells underwent epigenetic age assessment and screening before gene editing, selecting cells whose epigenetic age was reversed to 57% to 77% of their initial epigenetic age.

9. A method for producing super humanized cloned pigs, characterized in that, Using the super-humanized gene-edited pig nuclear donor cells described in claim 7 or 8 as nuclear donors, cloned pigs are produced through somatic cell nuclear transfer technology.

10. A small molecule composition for partial reprogramming of porcine somatic cells, characterized in that, The active ingredients include valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, foctocorline, and sodium butyrate; 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, the concentration of foctocorline is 40-60 μM, and the concentration of sodium butyrate is 150-250 μM. Alternatively, it may include valproic acid, CHIR99021, Repsox, transphenylcyclopropane, foctocorline, and bFGF; 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 transphenylcyclopropane is 4-6 μM, the concentration of foctocorline is 40-60 μM, and the concentration of bFGF is 80-120 ng / ml.

11. A method for constructing a pig-specific epigenetic clock model, characterized in that, Includes the following steps: 1) Collect pig tissues from the embryonic stage to adulthood, perform whole-genome bisulfite sequencing on the pig tissues to obtain WGBS data, and perform chromatin accessibility sequencing on the tissues to obtain chromatin open region characteristics; 2) CpG sites that show linear changes during pig development and aging were screened from WGBS data. Combined with chromatin open region features identified by ATAC-seq, a pig-specific multi-omics epigenetic age prediction model was trained using the elastic network regression machine learning algorithm.

12. The construction method according to claim 11, characterized in that, The embryonic period to adulthood is 0 to 36 months of age.

13. The construction method according to claim 12, characterized in that, The sources of the pig tissues include, for example, Bama miniature pigs and Large White pigs.

14. The construction method according to claim 12, characterized in that, The pig tissues include skin fibroblasts, liver, and muscle.

15. The construction method according to claim 12, characterized in that, The number of CpG sites mentioned in step 2) is 500 to 800.

16. The pig-specific epigenetic clock model constructed by the construction method according to any one of claims 11 to 15, characterized in that, The prediction formula for this model is: in: β 0 represents the intercept term; Mj is the first j methylation β value of each CpG site; Ak is the first k Standardized signal intensity of each chromatin accessibility region; βj , γk These are the regression coefficient weights for the corresponding CpG sites and chromatin accessibility regions, respectively. p and q represent the number of CpG sites and chromatin accessibility regions selected, respectively.

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