A small molecule composition for partial reprogramming of pig somatic cells, a partially reprogrammed nuclear donor cell and application thereof

Abstract

The application provides a small molecule composition for partial reprogramming of pig somatic cells, a partially reprogrammed nuclear donor cell and application thereof, and belongs to the field of animal biotechnology and genetic engineering. The small molecule composition comprises CHIR99021, Repsox-616452, TTNPB and SAG. The small molecule composition provided by the application can safely and significantly reverse the epigenetic and functional aging state of pig aging somatic cells edited by "3+X", restore the nucleo-cytoplasm compartmentalization integrity, and improve the cell cloning expansion capacity by more than 200%. The early abortion rate can be reduced from more than 70% to less than 10% by using the obtained partially reprogrammed nuclear donor cell as a nuclear donor for SCNT, reaching the industry limit level. The application provides an efficient key technology for the large-scale and economic production of "gold standard" donor pigs.

Description

Technical Field

[0001] This invention belongs to the field of animal biotechnology and genetic engineering, and particularly relates to a small molecule composition for partial reprogramming of porcine somatic cells, partially reprogrammed nuclear donor cells, and their applications. Background Technology

[0002] Xenotransplantation is a key strategic direction for addressing the clinical organ shortage crisis. Pigs, due to their organ size and physiological function being highly compatible with humans, short reproductive cycles, and lack of significant ethical controversies, have become the most promising xenotransplant donor species. Currently, the development of xenotransplant donor pigs shows a clear trend towards stratified technology: on the one hand, for long-term, multi-organ transplant needs, it is necessary to construct "super donor pigs" that have undergone extreme multiple gene editing (e.g., ≥22 genes) to comprehensively mitigate immune rejection and biosafety risks; on the other hand, for specific organ transplants (e.g., kidneys, islets) or short-term supportive transplant scenarios, the industry generally recognizes the "gold standard" donor pig protocol of "3+X" key gene modifications—the core of which is the knockout of GGTA1, CMAH, and B4GALNT2 genes that trigger hyperacute immune rejection, followed by the insertion of human complement regulator genes such as CD46, CD55, and CD59 as needed. These "gold standard" donor pigs have significant advantages such as a short construction cycle, low R&D cost, and controllable off-target risk due to their moderate number of gene edits. They represent the most promising technical approach for clinical translation and market application at present.

[0003] However, even with a moderate "3+X" gene editing strategy, the preparation of donor pigs still faces a significant technological bottleneck: donor pig preparation relies on somatic cell nuclear transfer (SCNT) technology, which requires obtaining high-quality porcine somatic cells (such as porcine fetal fibroblasts) as nuclear donors. In actual research and development, donor cells undergo complex procedures including in vitro isolation, multiple amplifications, gene editing (knockout / knock-in), and monoclonal screening. This process inevitably leads to replicative senescence and stress damage, manifesting as disordered epigenetic states (such as the accumulation of repressive modifications like H3K9me3), telomere shortening, decreased proliferation capacity, and increased apoptosis rates. When these degraded donor cells are used for SCNT, incomplete nuclear reprogramming results in embryo implantation failure and a persistently high early miscarriage rate (generally >40% in the industry), severely hindering the efficiency of "gold standard" donor pig preparation and clinical translation.

[0004] To address the challenges of somatic cell aging and reprogramming, existing technologies have identified combinations of small molecule compounds that can regulate epigenetic modifications (such as DNA methylation and histone modifications) to achieve epigenetic reprogramming in somatic cells of mammals like mice and humans, reversing aging-related phenotypes and even inducing pluripotent stem cells (CiPS cells). Theoretically, this type of small molecule reprogramming technology holds promise as an ideal solution for improving the quality of porcine nuclear donor cells due to its advantages of ease of operation, low cost, and lack of risk of exogenous gene integration. However, in practice, applying existing small molecule reprogramming technologies to optimize nuclear donor cells in "3+X" gene-edited pigs faces three fundamental technical challenges, resulting in a long-standing technological vacuum in this field: 1. Significant Species- and Cell State-Specific Barriers: The sensing and response mechanisms of cells from different species to reprogramming signals differ fundamentally. Existing highly effective small molecule compositions (such as specific inhibitor combinations) are all developed based on mouse or human somatic cells, and their active ingredients, optimal concentrations, and combination ratios cannot be directly transferred to porcine somatic cells. Especially for porcine somatic cells that have undergone "3+X" gene editing, are under stress, or are in an aging stage, their intrinsic metabolic microenvironment differs significantly from that of normal somatic cells, and their sensitivity and tolerance to small molecules are altered. Blindly applying existing protocols not only results in extremely low reprogramming efficiency but may also lead to the death of a large number of donor cells due to cytotoxicity, further exacerbating the shortage of nuclear donors.

[0005] 2. Lack of dedicated optimization solutions for "3+X" moderate editing load: Current reported technologies for improving porcine SCNT efficiency either target unedited somatic cells or focus on "super donor" cells with extreme multiplex gene editing. No solutions have yet been developed specifically for cells with the "3+X" editing load. The gene expression profiles, epigenetic characteristics, and stress levels of "3+X" gene-edited somatic cells differ from both unedited normal somatic cells and "highly damaged" somatic cells resulting from extreme editing. Their reprogramming requirements (such as the degree of reprogramming and regulatory targets) are unique, and existing solutions, due to insufficient targeting, cannot effectively improve the quality of their nuclear donors.

[0006] 3. Insufficient technical safety and predictability of effects: Existing small molecule reprogramming schemes often pursue "complete reprogramming" (such as inducing pluripotency). Simply applying combinations of such schemes to porcine nuclear donor cells can easily lead to over-reprogramming, resulting in increased genomic instability and a higher rate of karyotype abnormalities, which in turn reduces the developmental potential of SCNT embryos. Simultaneously, non-targeted combinations of small molecules may produce synergistic toxicity due to component redundancy, further deteriorating the cell state. Therefore, current technologies cannot stably produce high-quality porcine nuclear donor cells while ensuring cell health and genomic stability.

[0007] In summary, existing small molecule reprogramming technologies cannot meet the core bottleneck of deterioration in nuclear donor cell quality during the preparation of "3+X" and "gold standard" donor pigs due to species-specific barriers, lack of targeted approaches, and uncontrollable safety issues. Developing a partially reprogrammed small molecule composition that can precisely adapt to "3+X" gene-edited pig somatic cells, efficiently reverse aging phenotypes, and ensure cell safety has become crucial for overcoming the clinical translation bottleneck of "gold standard" donor pigs, possessing urgent technological needs and significant application value. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a small molecule composition for partial reprogramming of porcine somatic cells, partially reprogrammed nuclear donor cells, and their applications. This invention screens and validates a specific small molecule composition suitable for porcine "3+X" gene-edited cells. This small molecule composition can safely and efficiently induce partial reprogramming in porcine "3+X" gene-edited cells, significantly reversing their aging-related phenotypic and functional states.

[0009] This invention provides a small molecule composition for partial reprogramming of porcine somatic cells, comprising CHIR99021, Repsox-616452, TTNPB, and SAG, wherein the concentration of CHIR99021 is 0.3–3 μM; the concentration of Repsox-616452 is 1–5 μM; the concentration of TTNPB is 1–20 nM; and the concentration of SAG is 0.1–2 μM.

[0010] Preferably, the concentration of CHIR99021 is 0.5~2.5μM; the concentration of Repsox-616452 is 2~4μM; the concentration of TTNPB is 3~18nM; and the concentration of SAG is 0.2~1.8μM.

[0011] Preferably, the molar ratio of CHIR99021:Repsox-616452:TTNPB:SAG is 1:(2~4):(0.01~0.05):(0.2~0.6).

[0012] This invention provides a method for preparing partially reprogrammed nuclear donor cells, comprising the following steps: 1) Gene-edited somatic cells were obtained by gene editing of pig somatic cells; 2) Partially reprogramming the gene-edited somatic cells obtained in step 1) using the small molecule composition described above to obtain partially reprogrammed nuclear donor cells; Step 1) involves gene editing by knocking out the GGTA1, CMAH, and B4GALNT2 genes, and simultaneously knocking in 1 to 7 of the following human genes: CD46, CD55, CD59, THBD, TFPI, CD47, and PD-L1.

[0013] Preferably, the partial reprogramming involves co-culturing the small molecule composition with the gene-edited somatic cells for 2-7 days.

[0014] Preferably, the porcine somatic cells include fibroblasts.

[0015] This invention provides partially reprogrammed nuclear donor cells prepared using the aforementioned preparation method.

[0016] This invention provides the application of the partially reprogrammed nuclear donor cells in somatic cell nuclear transfer.

[0017] This invention provides a method for producing cloned pigs, which uses partially reprogrammed nuclear donor cells as nuclear donors and produces cloned pigs through somatic cell nuclear transfer technology.

[0018] This invention provides the application of cloned pigs prepared by the method in the preparation of xenograft biomaterials.

[0019] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a small molecule composition for partial reprogramming of porcine somatic cells, comprising CHIR99021, Repsox-616452, TTNPB, and SAG, wherein the concentration of CHIR99021 is 0.3~3 μM; the concentration of Repsox-616452 is 1~5 μM; the concentration of TTNPB is 1~20 nM; and the concentration of SAG is 0.1~2 μM. Through combination screening and dosage optimization of various known small molecules with epigenetic regulatory functions, the present invention unexpectedly discovered that the combination of the above small molecules: CHIR99021 (GSK-3β inhibitor), Repsox-616452 (TGF-β receptor inhibitor), TTNPB (retinoic acid receptor agonist), and Smoothened Agonist (SAG, Hedgehog pathway agonist) is effective. At the concentrations described above, partial reprogramming of porcine "3+X" gene-edited cells can be induced most safely and efficiently, significantly reversing their aging-related phenotypic and functional states. Crucially, adding other common reprogramming aids (such as Y-27632, ABT-869, etc.) to this small molecule composition may reduce its effectiveness in porcine cell systems or increase its toxicity.

[0020] Treating aged porcine fibroblasts with "3+X" gene editing using the aforementioned small molecule composition, and then using them as SCNT nuclear donors, yields an early abortion rate consistently below 10% (preferably <8%). This achievement represents a leading level in current porcine cloning technology and provides a reliable guarantee for the large-scale production of a moderate number of gene-modified donor pigs.

[0021] The "3+X" edited cells treated with the aforementioned small molecule composition have an in vitro clonal expansion potential (passage number) that is increased by more than 200%, effectively solving the bottleneck of single-clonal line expansion caused by cell senescence in the gene editing process, and significantly improving the experimental success rate and production throughput.

[0022] Compared to complex solutions designed for higher editing loads, the small molecule composition described in this invention exhibits superior kinetic properties (such as shorter treatment cycles) and a better safety window when reversing senescence in "3+X" edited cells. This indicates that there exists a specific "optimal reprogramming stimulation spectrum" for cells under different gene editing stresses, and the small molecule composition described in this invention is an optimized solution tailored for cells with moderate editing loads. Attached Figure Description

[0023] Figure 1 3+X gene-edited pig somatic cells were constructed for chemical reprogramming. Detailed Implementation

[0024] This invention provides a small molecule composition for partial reprogramming of porcine somatic cells, comprising CHIR99021, Repsox-616452, TTNPB, and SAG, wherein the concentration of CHIR99021 is 0.3–3 μM; the concentration of Repsox-616452 is 1–5 μM; the concentration of TTNPB is 1–20 nM; and the concentration of SAG is 0.1–2 μM.

[0025] In this invention, CHIR99021 is a GSK-3β inhibitor, preferably used at a concentration of 0.5~2.5 μM, more preferably 1.0 μM; Repsox-616452 is a TGF-β receptor inhibitor, preferably used at a concentration of 2~4 μM, more preferably 3.0 μM; TTNPB is a retinoic acid receptor agonist, preferably used at a concentration of 3~18 nM, more preferably 10 nM; and SAG is a Hedgehog pathway agonist, preferably used at a concentration of 0.2~1.8 μM, more preferably 0.5 μM.

[0026] In this invention, the molar ratio of CHIR99021:Repsox-616452:TTNPB:SAG is preferably 1:(2~4):(0.01~0.05):(0.2~0.6), more preferably 1:3:0.01:0.5.

[0027] The present invention also provides a method for preparing partially reprogrammed nuclear donor cells, comprising the following steps: 1) gene editing of porcine somatic cells to obtain gene-edited somatic cells; 2) partially reprogramming the gene-edited somatic cells obtained in step 1) using the small molecule composition to obtain partially reprogrammed nuclear donor cells.

[0028] In this invention, pig somatic cells are first genetically edited to obtain gene-edited somatic cells. The gene editing involves knocking out the GGTA1, CMAH, and B4GALNT2 genes, while simultaneously knocking in 1 to 7 of the following human genes: CD46, CD55, CD59, THBD, TFPI, CD47, and PD-L1. This invention is referred to as "3+X" editing. The specific method of gene editing is not particularly limited in this invention; conventional gene editing methods in the art can be used.

[0029] After obtaining the gene-edited somatic cells, the present invention preferably mixes the small molecule composition with the gene-edited somatic cells and cultures them for 2-7 days to obtain partially reprogrammed nuclear donor cells. The preferred culture time is 5 days.

[0030] In this invention, the porcine somatic cells include fibroblasts, specifically ear fibroblasts, ovarian fibroblasts, kidney fibroblasts, or peripheral blood-derived mononuclear cells.

[0031] This invention provides partially reprogrammed nuclear donor cells prepared using the aforementioned preparation method.

[0032] This invention provides the application of the partially reprogrammed nuclear donor cells in somatic cell nuclear transfer.

[0033] This invention provides a method for producing cloned pigs, using partially reprogrammed nuclear donor cells as nuclear donors and producing cloned pigs through somatic cell nuclear transfer technology. This invention does not have any special limitations on the nuclear transfer procedure; conventional standard procedures in the field can be used.

[0034] This invention provides the application of cloned pigs prepared by the method in the preparation of xenograft biomaterials.

[0035] 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.

[0036] Example 1

[0037] Using aged Bama miniature pig ear fibroblasts (PD>35) that had undergone "3+2 gene editing" (GGTA1 / CMAH / B4GALNT2 knockout, CD46 / CD55 knock-in) as a model, different small molecule combinations were tested. The cells were mixed with the small molecule composition and treated for 5 days.

[0038] The key comparison groups are as follows: Control group: basal culture medium (DMEM, supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin) C4-full group: a comparative combination of six small molecules, with the following components and final concentrations: CHIR99021 (10 μM), Repsox-616452 (10 μM), TTNPB (2 μM), Y-27632 (2 μM), SAG (0.5 μM), and ABT-869 (1 μM).

[0039] The C4-pro group (the present invention) consists of four components: CHIR99021, Repsox-616452, TTNPB and SAG, with the following final concentrations: CHIR99021 (10 μM), Repsox-616452 (10 μM), TTNPB (2 μM) and SAG (0.5 μM).

[0040] Positive control group: A known highly effective complex small molecule regimen was used, comprising the following components at final concentrations: valproic acid 250 μM, CHIR99021 10 μM, E-616452 10 μM, trans-cyclopropylamine 5 μM, and foscocrane 50 μM. Treatment of senescent cells with this composition under low serum culture conditions for approximately 4 days significantly restored the integrity of nucleoplasmic compartmentalization, reversed transcriptomic age, and restored the expression profile of the youthful gene, without loss of cell identity or induction of pluripotency.

[0041] Evaluation indicators: Cell viability and morphology: Viability was detected by CCK-8 assay, and morphology was observed under a microscope.

[0042] Nucleoplasmic compartmentalization (NCC): Pearson correlation coefficients were calculated using a stably expressed NLS-mCherry / NES-eGFP reporter system (lower coefficients indicate better nuclear membrane integrity and "younger" cells).

[0043] Clonal formation and amplification capacity: The limiting dilution method was used to determine the single-cell clonal formation rate and the number of passages (PDs) that amplify a clone.

[0044] The experimental results are shown in Table 1.

[0045] Table 1. Effects of different small molecule regimens on the treatment of "3+2 gene editing" pig cells.

[0046] As shown in Table 1, the C4-full group exhibited significant cytotoxicity to porcine cells. The C4-pro group of this invention, while ensuring high cell viability, achieved NCC recovery and clonal expansion capabilities comparable to known high-efficiency positive control protocols, and the formulation was simpler, demonstrating a better balance between safety and efficacy.

[0047] Example 2

[0048] Standardized somatic cell nuclear transfer (SCNT) was performed using the "3+2 gene-edited" cells treated in each group of Example 1, as well as the "5 gene-edited" senescent cells (Old-Edit group) without any reprogramming treatment, as nuclear donors. At least three biologically independent monoclonal cell lines were used in each group, and each line produced cloned embryos, which were then transferred to at least five recipient sows. Early miscarriage events were tracked and recorded within 30 to 60 days of gestation.

[0049] The results are shown in Table 2. The C4-pro group of this invention reduced the early abortion rate of SCNTs in "3+2 gene editing" donor cells from 71.4% to 6.3%, reaching the industry's top level. The existing unoptimized control group (C4-full group) performed poorly, with an abortion rate as high as 60%, indicating the need for specialized screening and optimization of porcine cells. The C4-pro group provided by this invention achieved the highest healthy litter / pregnancy recipient ratio (0.94), demonstrating the comprehensive advantages of the small molecule composition described in this invention in improving overall clonal production efficiency.

[0050] Table 2. SCNT results for different nuclear donor preparation schemes

[0051] Example 3

[0052] To explore the universality of the present invention's scheme for cells with different gene editing loads, its treatment effects on senescent Bama miniature pig fibroblasts were further tested, involving 6-gene editing (GGTA1 / CMAH / B4GALNT2 knockout and CD46 / CD55 / CD59 knockout, i.e., "3+3") and 7-gene editing (adding THBD knockout to the former, i.e., "3+4"). Specific methods are described in Examples 1 and 2.

[0053] The results are shown in Table 3. As the number of gene edits increased, the replication pressure and epigenetic disorder faced by the untreated senescent edited cells (Old-Edit group) intensified, and the early abortion rate of their SCNTs increased sharply (83.3% in the "3+3" edit group and as high as 100% in the "3+4" edit group), making it almost impossible to obtain healthy piglets.

[0054] Table 3. SCNT results for different nuclear donor preparation schemes

[0055] In contrast, although the early SCNT abortion rate of cells treated with the C4-pro group of this invention showed a slight upward trend with increasing editing load (from 6.3% to 14.3% and 25.0%), it was still controlled at an extremely low level and was significantly better than the unoptimized C4-full control group. Meanwhile, the C4-pro group maintained a high pregnancy rate and a healthy parturition / pregnancy recipient ratio in both "3+3" and "3+4" edited cells.

[0056] As can be seen from the above embodiments, the small molecule composition C4-pro provided by the present invention, when applied to pig somatic cells that have undergone “3+X” gene editing (knocking out 3 key xenogeneic antigen genes and knocking in X human protective genes), enables them to obtain an optimized phenotypic state, thereby achieving a high pregnancy maintenance rate and an extremely low early miscarriage rate in somatic cell nuclear transfer (SCNT).

[0057] 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 small molecule composition for partial reprogramming of porcine somatic cells, characterized in that, The formulation includes CHIR99021, Repsox-616452, TTNPB, and SAG, wherein the concentration of CHIR99021 is 0.3~3μM; the concentration of Repsox-616452 is 1~5μM; the concentration of TTNPB is 1~20nM; and the concentration of SAG is 0.1~2μM.

2. The small molecule composition for partial reprogramming of porcine somatic cells according to claim 1, characterized in that, The concentration of CHIR99021 used is 0.5~2.5μM; the concentration of Repsox-616452 used is 2~4μM; the concentration of TTNPB used is 3~18nM; and the concentration of SAG used is 0.2~1.8μM.

3. The small molecule composition according to claim 2, characterized in that, The molar ratio of CHIR99021:Repsox-616452:TTNPB:SAG is 1:(2~4):(0.01~0.05):(0.2~0.6).

4. A method for preparing partially reprogrammed nuclear donor cells, characterized in that, Includes the following steps: 1) Gene-edited somatic cells were obtained by gene editing of pig somatic cells; 2) Partially reprogramming the gene-edited somatic cells obtained in step 1) using the small molecule composition according to any one of claims 1 to 3 to obtain partially reprogrammed nuclear donor cells; Step 1) involves gene editing by knocking out the GGTA1, CMAH, and B4GALNT2 genes, and simultaneously knocking in 1 to 7 of the following human genes: CD46, CD55, CD59, THBD, TFPI, CD47, and PD-L1.

5. The preparation method according to claim 4, characterized in that, The partial reprogramming involves co-culturing the small molecule composition with the gene-edited somatic cells for 2-7 days.

6. The preparation method according to claim 4, characterized in that, The porcine somatic cells include fibroblasts.

7. Partially reprogrammed nuclear donor cells prepared by the preparation method according to any one of claims 4 to 6.

8. The application of the partially reprogrammed nuclear donor cells as described in claim 7 in somatic cell nuclear transfer.

9. A method for producing cloned pigs, characterized in that, Using the partially reprogrammed nuclear donor cells described in claim 7 as nuclear donors, cloned pigs are produced through somatic cell nuclear transfer technology.

10. The application of the cloned pig prepared by the method of claim 9 in the preparation of xenotransplantation biomaterials.

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