A chimeric polypeptide, related biomaterials and their application in induced cardiomyocytes

Abstract

This invention belongs to the field of biomedicine and regenerative medicine technology. It discloses a chimeric polypeptide, related biomaterials, and their application in inducing cardiomyocytes. The chimeric polypeptide is obtained through family homologous domain substitution and is selected from the following groups: (1) a MEF2 chimeric polypeptide with MEF2C protein as the backbone, wherein the N-terminal and C-terminal transcriptional activation domains are replaced by homologous regions of MEF2B and MEF2D, respectively; (2) a GATA chimeric polypeptide with GATA4 protein as the backbone, wherein the N-terminal transcriptional activation domain is replaced by homologous regions of GATA1 and GATA6. This invention also provides the nucleic acid encoding the polypeptide, the construct, and a method for efficiently inducing fibroblast reprogramming into cardiomyocytes under in vitro conditions. When the chimeric polypeptide of this invention is used alone or in combination, the induced cells exhibit calcium ion oscillations earlier and have higher maturity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a chimeric polypeptide, related biomaterials, and their application in induced cardiomyocytes. Background Technology

[0002] Cardiovascular disease is one of the leading causes of death worldwide, with heart failure caused by myocardial infarction being particularly severe. The regenerative capacity of the human adult heart is extremely weak. After a myocardial infarction, a large number of cardiomyocytes undergo irreversible death and are subsequently replaced by non-contractile fibroblasts and scar tissue, ultimately leading to cardiac pumping failure. Therefore, obtaining sufficient quantities of functional human cardiomyocytes to replenish damaged heart tissue is a core problem that urgently needs to be solved for regenerative medicine to achieve clinical application.

[0003] In 2010, Ieda et al. first reported that mouse fibroblasts could be directly reprogrammed into induced cardiomyocytes (iCMs) using three key transcription factors for heart development—GATA4, MEF2C, and TBX5 (GMT combination) (Cell, 2010, 142(3):375-386). Compared with induced pluripotent stem cell (iPSC) differentiation strategies, this direct reprogramming strategy avoids the potential tumorigenic risks of iPSCs (such as teratoma formation) and has a shorter transformation cycle.

[0004] However, despite laying the foundation for direct reprogramming, the translation of GMT combinations into human clinical applications has encountered significant obstacles. Existing research indicates that the reprogramming barrier for human cells is much higher than that for mouse cells (Stem Cell Reports, 2013, 1(3):235-247). Whether in human skin fibroblasts or cardiac fibroblasts, the efficiency of inducing pulsatile cardiomyocytes solely through overexpression of wild-type GMT combinations is extremely low, and the induced cells are often insufficiently mature, failing to meet the needs of clinical treatment. Although subsequent studies have attempted to add additional transcription factors (such as MESP1, MYOCD, etc.) (Circulation, 2022, 146(20): 1518-1536; Proc Natl Acad Sci USA, 2013, 110(31): 12667-12672), use microRNAs (miRNAs) (Proc Natl Acad Sci USA, 2013, 110(14): 5588-5593) or small molecule compounds to assist reprogramming (Circulation, 2017, 135(10): 978-995), these methods often increase the complexity of the operation, and some strategies (such as the off-target effects of certain small molecules or the safety issues of viral vectors) limit their safety in clinical application.

[0005] Furthermore, induced cardiomyocytes obtained by existing technologies often fail to meet the standards of mature cardiomyocytes in terms of electrophysiological characteristics and structure, frequently exhibiting high resting membrane potential, slow action potential, and disordered sarcomere arrangement (Stem Cell Reports, 2013, 1(3): 235-247; Circulation, 2017, 135(10): 978-995; Circulation, 2022, 146(20): 1518-1536). Therefore, there is an urgent need in this field to develop a more efficient and safer reprogramming strategy that can obtain highly mature cardiomyocytes, especially through rational engineering of core transcription factors, to overcome the efficiency bottleneck of human fibroblast reprogramming. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a chimeric polypeptide, related biomaterials and their application in induced cardiomyocytes, which overcomes the efficiency bottleneck of human fibroblast reprogramming by rationally engineering the core transcription factors.

[0007] In a first aspect, the present invention provides a chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising an amino acid sequence with MEF2C protein as its backbone, wherein the N-terminal transcriptional activation domain of the MEF2C protein is replaced by a corresponding homologous region of MEF2B, and the C-terminal domain is replaced by a corresponding homologous region of MEF2D; and (b) a second chimeric polypeptide comprising an amino acid sequence with GATA4 protein as its backbone, wherein the N-terminal transcriptional activation domain of the GATA4 protein is replaced by corresponding homologous regions of GATA1 and GATA6.

[0008] More preferably, the N-terminal segment replaced in the first polypeptide is derived from SEQ ID NO:14 (MEF2B), and the C-terminal segment replaced is derived from SEQ ID NO:15 (MEF2D); the N-terminal segment replaced in the second polypeptide is derived from SEQ ID NO:16 (GATA1) and SEQ ID NO:17 (GATA6).

[0009] The substitution region in the chimeric polypeptide of this invention is derived from the following naturally occurring wild-type proteins:

[0010] SEQ ID NO:14: The full-length amino acid sequence of human MEF2B protein. The N-terminal transcriptional activation domain (positions 1-73) of the MEF2 chimeric polypeptide of this invention (such as SEQ ID NO:3) is derived from the corresponding homologous segment of this sequence.

[0011] SEQ ID NO:15: The full-length amino acid sequence of human MEF2D protein. The C-terminal domain (positions 385-463) of the MEF2 chimeric polypeptide of this invention (such as SEQ ID NO:3) is derived from the corresponding homologous region of this sequence.

[0012] SEQ ID NO:16: The full-length amino acid sequence of human GATA1 protein. The N-terminal transcriptional activation domain (positions 1-214) of the GATA chimeric polypeptide of this invention (such as SEQ ID NO:4) is derived from the corresponding homologous segment of this sequence.

[0013] SEQ ID NO:17: The full-length amino acid sequence of the human GATA6 protein, which was used as one of the basic homologous sequences for constructing the GATA family chimeric mutant library (LibGATA). Specifically, GATA family members (GATA1-GATA6), including SEQ ID NO:17, were fragmented, and their homologous regions were used for random recombination (DNA shuffling) to generate a diverse chimeric library for subsequent screening of highly active reprogramming factors.

[0014] Preferably, the amino acid sequence of the chimeric polypeptide has one of the following characteristics: (1) compared with SEQ ID NO:1, amino acids 1-73 are replaced with the sequence shown in SEQ ID NO:5, and amino acids 385-463 are replaced with the sequence shown in SEQ ID NO:6; (2) compared with SEQ ID NO:2, amino acids 1-214 are replaced with the sequence shown in SEQ ID NO:7, and amino acids 215-289 are replaced with the amino acid sequence shown in SEQ ID NO:8.

[0015] Preferably, the chimeric polypeptide has the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

[0016] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the chimeric polypeptide, the nucleic acid molecule comprising the nucleotide sequences shown in SEQ ID NO:18-21.

[0017] Those skilled in the art will understand that the nucleic acid includes any one or both of the complementary double strands, and covers variants that encode the same amino acid sequence due to codon degeneracy.

[0018] Thirdly, the present invention provides a nucleic acid construct comprising the aforementioned nucleic acid molecule, wherein the construct is an expression vector, and the expression vector is a viral vector or a plasmid vector.

[0019] The construct is capable of transferring the target nucleic acid sequence into host cells to obtain recombinant cells.

[0020] Fourthly, the present invention provides an engineered host cell, wherein the host cell contains the aforementioned nucleic acid molecule or the aforementioned nucleic acid construct, or wherein the aforementioned nucleic acid molecule is integrated into its genome; the host cell is a fibroblast.

[0021] Preferably, the present invention provides a method for preparing the chimeric polypeptide, comprising the following steps:

[0022] Homologous amino acid segments of GATA family members were selected, including the amino acid sequences shown in SEQ ID NO:9 and SEQ ID NO:10, and GATA chimeric mutant libraries were constructed by recombination.

[0023] Homologous amino acid segments from MEF2 family members were selected, including the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and MEF2 chimeric mutant libraries were constructed by recombination.

[0024] The peptides in the mutant library were introduced into fibroblasts, and their efficiency in inducing fibroblasts to reprogram into cardiomyocytes was detected.

[0025] Peptides with higher induction efficiency than wild-type controls were screened out.

[0026] More preferably, SEQ ID NO:9 and SEQ ID NO:10 are specific homologous amino acid segments selected from the GATA family. These segments are used as basic recombination units when constructing GATA chimeric mutant libraries, generating diverse GATA mutants through recombination.

[0027] SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13 are specific homologous amino acid segments selected from the MEF2 family. These segments were used as basic recombination units in the construction of the MEF2 chimeric mutant library (LibMEF2), generating diverse MEF2 mutants through recombination.

[0028] Fifthly, the present invention provides the use of the chimeric polypeptide, the nucleic acid molecule, or the nucleic acid construct described herein in the preparation of a formulation for promoting cell reprogramming.

[0029] Preferably, the formulation is used for: (1) reprogramming fibroblasts into cardiomyocytes; or (2) treating or preventing myocardial infarction, heart failure, or cardiac tissue damage.

[0030] In a sixth aspect, the present invention provides a method for inducing non-myocardial cells to transform into cardiomyocytes under in vitro conditions, comprising overexpressing the chimeric polypeptide in the non-myocardial cells; the method comprising at least one of the following features: (a) the non-myocardial cells individually overexpressing a polypeptide having the sequence shown in SEQ ID NO:3; (b) the non-myocardial cells simultaneously overexpressing a polypeptide having the sequence shown in SEQ ID NO:3, a polypeptide having the sequence shown in SEQ ID NO:4, and TBX5; (c) culturing the cells in a basal medium containing DMEM and M199, followed by culturing in a basal medium containing 1640.

[0031] Preferably, the volume ratio of DMEM to M199 is 4:1;

[0032] Further preferably, on the 10th day of culture, the cells are replaced with a basal medium containing 1640 for further culture;

[0033] More preferably, the total culture time is at least 14 days.

[0034] The beneficial effects of this invention are:

[0035] 1. The MEF2 (BCCD) chimera of this invention exhibits remarkable transcriptional activity. Experimental results show that fibroblast reprogramming can be induced simply by overexpressing MEF2 (BCCD), and its induction efficiency is approximately 7 times that of the classic GATA4+MEF2C+TBX5 (GMT) triad combination. When using the "super combination" (MEF2(BCCD)+GATA(164)+TBX5) of this invention, the efficiency is further improved, significantly outperforming existing technologies.

[0036] 2. This invention constructs GATA(164) by introducing the homologous N-terminal domain of GATA1. This mutant not only has no inhibitory effect, but also produces a significant synergistic effect with MEF2(BCCD) and TBX5, greatly enhancing the induction effect.

[0037] 3. The chimera of this invention is composed entirely of homologous sequences from within the heart-related transcription factor family (MEF2 and GATA family). This "fully homologous / fully human" design minimizes the risk of non-specific activation of heterologous genes (such as skeletal muscle genes), resulting in purer, safer, and more suitable cardiomyocytes for clinical application.

[0038] 4. The induced cardiomyocytes obtained using the chimeric polypeptide or method described in this invention not only have a morphology closer to mature cardiomyocytes, but also exhibit key electrophysiological characteristics such as calcium oscillation earlier. Transcriptome sequencing analysis further confirms that the cells induced by this invention are closer to mature cardiomyocytes in gene expression profiles than those induced by traditional GMT. Attached Figure Description

[0039] Figure 1 This describes the process and verification results for obtaining MEF2 (BCCD) and GATA (164) according to embodiments of the present invention. Specifically, A. α-Actinin-positive cells were screened by flow cytometry and used for sequencing identification; B. Highly enriched mutants were identified after sequencing.

[0040] Figure 2 This is an amino acid sequence comparison between MEF2 (BCCD) and MEF2C.

[0041] Figure 3 This is an amino acid sequence comparison of GATA(164) with GATA1, GATA4, and GATA6.

[0042] Figure 4This is a demonstration of the efficiency of MEF2 (BCCD) reprogramming into cardiomyocytes. A is an immunofluorescence image of GATA4, MEF2C, TBX5, and MEF2 (BCCD) after 28 days of culture, according to an embodiment of the present invention; B is a statistical result graph of the reprogramming efficiency of GATA4, MEF2C, TBX5, and MEF2 (BCCD) after 28 days.

[0043] Figure 5 This diagram illustrates the efficiency of GATA4, MEF2C, TBX5; GATA4, MEF2(BCCD), TBX5; MEF2(BCCD), GATA(164), TBX5 according to embodiments of the present invention. Specifically, A. is an immunofluorescence image during 28 days of induction; B. is a statistical result graph of the reprogramming efficiency of GATA4, MEF2C, TBX5; GATA4, MEF2(BCCD), TBX5; MEF2(BCCD), GATA(164), TBX5 after 28 days.

[0044] Figure 6 These are observed and statistically analyzed calcium oscillation diagrams. A. is a diagram showing the calcium oscillations of GATA4, MEF2(BCCD), TBX5; MEF2(BCCD), GATA(164), TBX5; and MEF2(BCCD) after 14 days of culture under a fluorescence microscope, according to an embodiment of the present invention. B. is a diagram showing the changes in fluorescence intensity of calcium ions in GATA4, MEF2(BCCD), TBX5; MEF2(BCCD), GATA(164), TBX5; and MEF2(BCCD) after 14 days.

[0045] Figure 7 This is a graph showing the transcriptome data analysis results of HCF;GATA4, MEF2C, TBX5GATA4, MEF2(BCCD), TBX5;MEF2(BCCD), GATA(164), and TBX5MEF2(BCCD) according to embodiments of the present invention. Detailed Implementation

[0046] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available products, such as those purchased from Illumina.

[0047] The term "cell" as used herein refers to any available cell; in the following examples, human heart fibroblasts (HCF cells) were selected.

[0048] Cells can be transfected or infected by any means known in the art.

[0049] I. Experimental Methods:

[0050] Cell culture

[0051] 293T cell culture medium: 10% FBS, 1X DMEM;

[0052] HCF medium: 10% FBS, 1X DMEM, 1X Glutamax, 1X NEAA;

[0053] hiCMs induction medium: 10% FBS, DMEM:M199 (4:1), 1X Glutamax, 1X NEAA, 1X PIS; on day 10, the medium was changed to 1X 1640, 2% FBS, 1X Glutamax, 1X NEAA, 1X PIS, 0.1% BSA, 100ug / ml L-ascorbic acid, 1X B27.

[0054] Chimeric mutant library construction

[0055] Multiple sequence alignment of the amino acid sequences of MEF2A, MEF2B, MEF2C, and MEF2D proteins was performed to identify homologous regions among family members. Using homologous sequences as boundaries, each member was separated into multiple functional fragments using PCR technology. Then, a homologous recombination strategy was employed to guide random recombination, constructing the MEF2 family chimeric mutant library LibMEF2, which was then cloned into the pLVX-TetOn-Puroe vector.

[0056] Similarly, GATA1–GATA6 were fragmented and randomly recombinated to construct the GATA family chimeric mutant library LibGATA.

[0057] Preparation of retroviruses

[0058] 293T cells were counted after trypsin digestion and divided into groups of 8 × 10⁻⁶. 6 Seed at a density of 10 cells / 10cm culture dish. Incubate for approximately 24 hours, and when the cell density reaches 70–90%, replace with fresh culture medium 1 hour before transfection.

[0059] The transfection system was as follows: 10 μg plasmid DNA and 40 μL PEI were added to 1 mL Opti-MEM, mixed thoroughly, and incubated at room temperature for 15 min. The mixture was then slowly added dropwise to 293T cells. Fresh medium was added 12 h after transfection. Viral supernatant was collected at 48 h and 72 h post-transfection, filtered through a 0.4 μm filter, and incubated overnight at 4 °C with 1×PEG8000. Subsequently, the cells were centrifuged at 1600 g for 1 h at 4 °C, the supernatant was discarded, and the viral pellet was resuspended in 1×DPBS at a 10:1 volume ratio. The pellet was then aliquoted and stored at −80 °C.

[0060] Induction of hiCMs

[0061] The medium was changed for the first time 24 hours after HCF resuscitation, and then every 48 hours thereafter. Cells were digested with 0.25% trypsin and then cultured at 3 × 10⁻⁶ cells / mL. 4 Cells were seeded per well in 24-well plates and cultured for 24 h. Before infection, concentrated virus solution was mixed with polybrene (final concentration 8 μg / mL), and 0.125 mL of virus solution was added to each well. In this study, LibGATA, LibMEF2, GATA4, MEF2C, TBX5, MEF2(BCCD), and GATA(164) were all infected with HCF under these conditions. After 24 h of infection, the medium was replaced with hiCMs induction medium, which was recorded as day 0. Thereafter, the medium was changed every 48 h and morphological observations were performed.

[0062] Flow sorting

[0063] On day 14 of induction, the LibGATA+LibMEF2+TBX5 experimental group and the HCF control group were digested with 0.05% trypsin, and digestion was terminated with hiCMs medium. Cells were then centrifuged at 300g, 22℃ for 5 min. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.25% PBST for 10 min, and blocked with 5% BSA for 1 h. Subsequently, α-Actinin primary antibody (Proteintech, 1:200, diluted with 1% BSA) was added, and the cells were incubated overnight at 4℃. The next day, Goat anti-Rabbit Alexa Fluor 488 secondary antibody (Invitrogen, 1:200) was added, and the cells were incubated at room temperature for 1 h, followed by nucleus staining with DAPI for 5 min. Finally, the cells were resuspended in 500 μL of 1% BSA and transferred to flow cytometry tubes for sorting.

[0064] Document authentication

[0065] Genomic DNA was extracted from sorted cells using the EZNA MicroElute Genomic DNA Kit (Omega). Chimeric mutant sequences obtained through screening were amplified using PCR. The PCR reaction volume was 25 μL, using 2×Phanta UniFiMaster Mix (Vazyme), with a final primer concentration of 10 μM, template DNA of 6.3 ng, and a cycle number of 35. The PCR products were purified by agarose gel electrophoresis. The vector pLVX-Tet3G-Map was digested with BamHI and EcoRI (1 μL each) at 37°C for 1 h, purified by gel electrophoresis, and its concentration was determined. In a 10 μL ligation system, the vector and insert were added at a 1:5 molar ratio using 2×CE Mix (Vazyme), and the reaction was carried out at 50°C for 1 h. The ligation product was transformed into Stbl3 competent cells, which were then heat-shocked, recovered, and plated on LB agar plates containing Amp. Single clones were picked for Sanger sequencing to verify the chimeric mutant sequences.

[0066] Immunofluorescence analysis

[0067] Cells were fixed with 4% PFA for 15 min, permeabilized with 0.5% PBST for 7 min, and blocked with 5% BSA for 1 h. Primary antibody α-Actinin (Proteintech, 1:400) was added, and the cells were incubated overnight at 4°C. The next day, secondary antibody Goat anti-Rabbit Alexa Fluor 488 (Invitrogen, 1:400) was added, and the cells were incubated at room temperature for 1 h. Nuclear staining with DAPI was performed for 5 min. Cells were washed three times with 0.01% PBST for 5 min each time.

[0068] Calcium transient staining

[0069] 4 μM Fluo-4 AM working solution was prepared using HBSS, and 0.02% Pluronic F-127 was added. Cells were incubated at 37°C in the dark for 45 min, then washed twice with HBSS and replaced with fresh culture medium, and incubated at 37°C for 20 min.

[0070] Calcium ions were imaged using a fluorescence microscope with an excitation wavelength of 494 nm and an emission wavelength of 516 nm.

[0071] RNA sequencing analysis

[0072] Total RNA was extracted from reprogrammed cells induced on day 28 using TRIzol. Samples were stored at −80℃ before sequencing. RNA-seq followed standard human library preparation procedures, using the T7 amplification strategy and PE150 sequencing. Sequencing results were aligned using STAR, and expression matrices were obtained using FeatureCounts. Differential analysis was performed using DEseq2.

[0073] result:

[0074] 1. Chimeric mutants with high cardiomyocyte induction efficiency were obtained during screening and sequencing.

[0075] Reprogramming was induced in HCF cells by overexpression of LibGATA, LibMEF2, and TBX5, and α-Actinin-positive hiCMs were obtained by flow cytometry sorting on day 14. Third-generation sequencing was then used to identify the chimeric mutants enriched in the sorted cells, and several candidate mutants with potential reprogramming activity were screened. Based on the enrichment of mutants in the sequencing results, MEF2(BCCD) and GATA(164) were finally selected as highly efficient inducing factors (see results). Figure 1 Sequence alignment results show that, compared with MEF2C, amino acids 1–73 of MEF2(BCCD) are replaced by the corresponding sequence of MEF2B, and amino acids 385–463 are replaced by the corresponding sequence of MEF2D, as shown in SEQ ID NO:3.

[0076] MEF2(BCCD) amino acid Sequence:

[0077] MGRKKIQISRILDQRNRQVTFTKRKFGLMKKAYELSVLCDCEIALIIFNSANRLLFQYASTDMDRVLLKYTEYSEPHESRTNSDIVEALNKKENKGCESPDPDSSYALTPRTEEKYK KINEEFDNMIKSHKIPAVPPPNFEMPVSIPVSSHNSLVYSNPVSSLGNPNLLPLAHPSLQRNSMSPGVTHRPPSAGNTGGLMGGDLTSGAGTSAGNGYGNPRNSPGLLVSPGNLNK NMQAKSPPPMNLGMNNRKPDLRVLIPPGSKNTMPSVNQRINNSQSAQSLATPVVSVATPTLPGQGMGGYPSAISTTYGTEYSLSSADLSSLSGFNTASALHLGSVTGWQQQHLHNM PPSALSQLGACTSTHLSQSSNLSLPSTQSLNIKSEPVSPSRERSPAPPPPAVFPAARPEPGDGLSSPAGGSYETGDRDDGRGDFGPTLGLLRPAPEPEAEGSAVKRMRLDTWTLK. And its DNA sequence is shown in SEQ ID NO:22.

[0078] Sequence alignment revealed numerous amino acid mutations in MEF2 compared to BCCD (see results). Figure 2 Compared to GATA4, amino acids 1–214 of GATA(164) are replaced by the corresponding sequence of GATA1, and amino acids 215–289 are replaced by the corresponding sequence of GATA6, as shown in SEQ ID NO:4.

[0079] GATA(164) amino acid Sequence:

[0080] MEFPGLGSLGTSEPLPQFVDPALVSSTPESGVFFPSGPEGLDAAASSTAPSTATAAAAALAYYRDAEAYRHSPVFQVYPLLNCMEGIPGGSPYAGWAYGKTGLYPASTVCPTREDSPPQAVEDLDGKGSTSFLETLKTERLSPDLLTLGPALPSSLPVPNSAYGGPDFSSTFFSPTGSPLNSAAYSSPKLRGTLPLPPCEARECVNCGSIQTPLW RRDGTGHYLCNACGLYSKMNGLSRPLIKPQKRVPSSRRLGLSCANCHTTTTLWRRNAEGEPVCNACGLYMKLHGVPRPLAMRKEGIQTRKRKPKNLNKSKTPAAPS GSESLPPASGASSNSSNATTSSSEEMRPIKTEPGLSSHYGHSSSVSQTFSVSAMSGHGPSIHPVLSALKLSPQGYASPVSQSPQTSSKQDSWNSLVLADSHGDIITA. And its DNA sequence is shown in SEQ ID NO:23.

[0081] Sequence alignment revealed numerous amino acid mutations in GATA(164) compared to GATA4 (see results). Figure 3 The above results indicate that chimeric mutants acquire stronger myocardial reprogramming potential by integrating functional domains from different family members.

[0082] 2. MEF2 (BCCD) alone can efficiently induce cardiomyocyte reprogramming.

[0083] Overexpression of MEF2 (BCCD) successfully induced the formation of cardiomyocyte-like cells in HCF, expressing the cardiomyocyte-specific marker α-actin and exhibiting distinct sarcomere characteristics. Its reprogramming efficiency was significantly higher than the combination of GATA4, MEF2C, and TBX5, with an improvement of approximately 7-fold (see results). Figure 4 ).

[0084] Functional analysis showed that MEF2(BCCD)-induced hiCMs exhibited stable calcium ion oscillations. Transcriptome analysis further confirmed that, compared with the classic three-factor combination, MEF2(BCCD)-induced cells were closer to the state of mature cardiomyocytes at the overall transcriptional level (see results). Figure 6 and Figure 7 ).

[0085] 3. The combination of MEF2 can accelerate cardiomyocyte reprogramming and improve reprogramming efficiency.

[0086] Immunofluorescence staining with α-Actinin, a cardiomyocyte-specific marker, revealed that the reprogramming efficiency of the combinations GATA4, MEF2(BCCD), and TBX5, as well as GATA(164), MEF2(BCCD), and TBX5, was 4-5 times higher than that of the combination GATA4, MEF2C, and TBX5 (see results). Figure 5 In addition, the reprogramming factor combinations GATA4, MEF2(BCCD), TBX5 and GATA(164), MEF2(BCCD), TBX5 exhibited calcium ion oscillations and were more similar to mature cardiomyocytes in transcriptome compared to the GATA4, MEF2C, TBX5 combination (see results). Figure 6 and Figure 7 ).

Claims

1. A chimeric polypeptide, characterized in that: The chimeric polypeptide is selected from one of the following: (1) A polypeptide with an amino acid sequence as shown in SEQ ID NO:3; (2) A polypeptide with an amino acid sequence as shown in SEQ ID NO:

4.

2. The chimeric polypeptide according to claim 1, characterized in that: The polypeptide shown in SEQ ID NO:3 is based on the MEF2C protein backbone, wherein: Amino acids 1-73 are replaced by the corresponding homologous region of MEF2B; Amino acids 385-463 are replaced by the corresponding homologous region of MEF2D.

3. The chimeric polypeptide according to claim 1, characterized in that: The polypeptide shown in SEQ ID NO:4 is based on the GATA4 protein backbone, wherein: Amino acids 1-214 are replaced by the homologous region corresponding to GATA1; Amino acids at positions 215-289 are replaced by the homologous region corresponding to GATA6.

4. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric polypeptide according to any one of claims 1-3, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:22 or SEQ ID NO:

23.

5. A nucleic acid construct, characterized in that, The construct comprises the nucleic acid molecule of claim 4, and the construct is an expression vector, which is a viral vector or a plasmid vector.

6. An engineered host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 4 or the nucleic acid construct of claim 5, or its genome integrates the nucleic acid molecule of claim 4; the host cell is a cardiac fibroblast.

7. Use of the chimeric polypeptide of any one of claims 1-3, the nucleic acid molecule of claim 4, or the nucleic acid construct of claim 5 in the preparation of a reagent for inducing the reprogramming of cardiac fibroblasts into cardiomyocytes under in vitro conditions.

8. A method for inducing cardiac fibroblasts to transform into cardiomyocytes under in vitro conditions, characterized in that, The method includes overexpressing the chimeric polypeptide of any one of claims 1-3 in the cardiac fibroblasts; the method includes at least one of the following features: (a) the cardiac fibroblasts overexpressing a polypeptide with the sequence shown in SEQ ID NO:3 alone; (b) the cardiac fibroblasts simultaneously overexpressing a polypeptide with the sequence shown in SEQ ID NO:3, a polypeptide with the sequence shown in SEQ ID NO:4, and TBX5.

Images