Reprogramming vector for blood cells and use thereof

Abstract

The application provides a reprogramming vector for blood cells and application thereof. Through the technical scheme in the disclosure, a low-cost and high-benefit solution can be provided for efficient reprogramming and application of iPSCs, and the wide application of iPSCs in clinical and scientific research fields is significantly promoted, and the iPSCs have important application value and development potential in research and practice in aspects such as disease models, drug screening, cell therapy and the like.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biomedicine, in particular, the present disclosure relates to a reprogramming vector for blood cells and application thereof. BACKGROUND

[0002] Reprogramming is a technical process of reversing a differentiated cell to a more primitive state (such as pluripotency) through human intervention. The current reprogramming technology relies on the introduction of specific transcription factors (such as OCT4, SOX2, KLF4, c-MYC), which activate the pluripotency-related gene network, allowing somatic cells to be reprogrammed into pluripotent cells with stem cell characteristics. However, the existing technology still has certain limitations, mainly including vector type selection, transfection efficiency and genome safety, etc. In the traditional method of obtaining iPSCs using reprogramming technology, the same promoter is usually used to drive the expression of all reprogramming factors. However, in blood cells, if all factors are highly expressed, especially KLF4, high-level expression often has a strong inhibitory effect on cell proliferation, resulting in low reprogramming efficiency or even failure to reprogram. In addition, the expression levels of different promoters in different types of cells are not the same; therefore, how to inhibit the overexpression of KLF4 in the early stage of hematopoietic cell reprogramming, while moderately increasing its expression in the later stage, to ensure the reprogramming efficiency and the quality of the final iPSCs, has become a technical difficulty.

[0003] In addition, the current commonly used reprogramming strategies can be divided into two categories: viral vector systems and non-viral vector systems. Viral vectors (such as lentivirus and retrovirus) can induce efficiently, but their genomic integration characteristics may lead to genomic mutations and potential oncogenic risks, limiting their clinical application. In contrast, non-viral vectors (such as circular DNA plasmid systems) avoid the safety problems caused by genomic integration, and are theoretically more suitable for clinical application. However, traditional non-viral reprogramming methods usually require multiple plasmids to be co-transfected, which not only increases the complexity of operation, but also may lead to uneven expression levels of reprogramming factors, thereby affecting the reprogramming efficiency and cell quality. In addition, other non-integration strategies such as mRNA or small molecule compounds can further reduce the genetic safety risk, but due to low transfection efficiency, cumbersome operation or limited induction efficiency, they have not yet become mainstream methods.

[0004] Episomal Vector is a non-integrated gene delivery system based on extrachromosomal autonomous replication, which is widely used in cell reprogramming, gene therapy and gene editing fields. Its core advantage is to avoid host genome integration, reduce the risk of insertion mutation, and provide stable transgene expression. However, the multi-plasmid co-transfection in the existing Episomal Vector system brings the problems of operation complexity, inefficiency and uneven plasmid copy number. In order to solve the above-mentioned problems existing in the prior art, the reprogramming system needs to be improved. SUMMARY

[0005] Technical problems solved:

[0006] An aspect of the present application is to provide a new reprogramming vector system to solve the problems of operation complexity, inefficiency and genome instability in the traditional reprogramming vector system, improve the generation efficiency of iPSC, avoid the residual of exogenous DNA, and ensure the clinical safety.

[0007] Technical solutions:

[0008] A reprogramming vector for blood cells, comprising:

[0009] (1) an OCT4 gene, a SOX2 gene, a KLF4 gene and a c-MYC gene;

[0010] (2) a first expression frame comprising a first gene transcription regulatory element operably linked; and

[0011] (3) a second expression frame comprising a second gene transcription regulatory element operably linked;

[0012] Wherein, the first gene transcription regulatory element is used to regulate the expression of the OCT4 gene, the SOX2 gene and the c-MYC gene, and the second gene transcription regulatory element is used to regulate the expression of the KLF4 gene, and the second gene transcription regulatory element comprises a regulatory element for inhibiting the expression of the KLF4 gene before reprogramming induction.

[0013] In some embodiments, the above-mentioned first gene transcription regulatory element or the above-mentioned second gene transcription regulatory element can comprise a promoter, an enhancer or a silencer. In other embodiments, a person skilled in the art can add other suitable gene transcription regulatory elements according to the needs, and the technical solutions are also considered to be included in the protection scope of the present application.

[0014] The technical scheme of the present disclosure utilizes different expression frames to express Yamanaka factors, and meanwhile, a regulatory element for inhibiting the expression of KLF4 gene is combined, so that the high-level expression of KLF4 has a relatively strong inhibitory effect on cell proliferation, and thus the problem of low reprogramming efficiency or even unsuccessful reprogramming is avoided. In some embodiments, the promoters contained in the above-mentioned first gene transcription regulatory element or the above-mentioned second gene transcription regulatory element can be the same or different, and are preferably different promoters. Further, a person skilled in the art can select a suitable promoter, but in order to better achieve the purpose of the present disclosure, in some embodiments, the above-mentioned promoter can be selected from SFFV, CMV, EF1a, Sox2, Nanog, RUNX1, GATA2, PU.1. Further, in one embodiment, the promoter contained in the above-mentioned first expression frame can be an SFFV promoter, and the promoter contained in the above-mentioned second expression frame can be an EF1a promoter.

[0015] In some embodiments, the above-mentioned enhancer can be selected from WPRE, EBNA1, SV40, CMV, CAG.

[0016] In some embodiments, the above-mentioned regulatory element for inhibiting the expression of KLF4 gene before reprogramming induction is a miRNA regulatory element. Further, in some embodiments, the above-mentioned miRNA regulatory element can be at least one selected from miRNA-302 family, miRNA-367 family, miR-200 family, miR-34 family, miR-371-373 family, miR-17-92 family, miR-21, miR-199a-3p, miR-142-3pT (2c). In order to better achieve the purpose of the present disclosure, in one embodiment, the above-mentioned miRNA regulatory element can be miR-142-3pT (2c).

[0017] In some embodiments, the above-mentioned first expression frame can further contain an anti-apoptotic gene, and the expression of the anti-apoptotic gene is regulated by the first gene transcription regulatory element. In some embodiments, a person skilled in the art can select a suitable anti-apoptotic gene to enhance the survival rate of cells. Further, in order to better achieve the purpose of the present disclosure, the above-mentioned anti-apoptotic gene can be at least one selected from BCL-2 family, IAP family, FLIP, Akt / PKB, HSPs, NF-κB, ARC, DAD1, SODD. Further, in one embodiment, the above-mentioned anti-apoptotic gene can be a BCL-XL gene.

[0018] To better achieve the purpose of the present disclosure, a sequence encoding a self-cleavage peptide can also be included between the gene sequences in the above-mentioned first expression frame. A person skilled in the art can select any suitable self-cleavage peptide, but as a preferred, in some embodiments, the above-mentioned self-cleavage peptide can be at least one selected from the 2A peptide family, an intein, and Sortase A.

[0019] In one embodiment, the above-mentioned reprogramming vector is SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-EF1s-KLF4-polyA-142-3pT (2c).

[0020] Another aspect of the present disclosure is to provide a product for blood cell reprogramming, which is a plasmid or a virus containing an OriP / EBNA1 replication system and the above-mentioned reprogramming vector. The carried OriP / EBNA1 replication system can be amplified for a short period in mammalian cells and gradually diluted with cell division, thereby achieving reprogramming without genomic integration. In some embodiments, any suitable virus or plasmid carrying the OriP / EBNA1 replication system can be selected, for example, the above-mentioned virus can be a lentivirus, an adenovirus, an adeno-associated virus, or a retrovirus, and the above-mentioned plasmid can be selected from pCEP4, pCEP5, pREP4, pREP9, pREP7, pREP10, pMEP4, pEB-C5, pCXLE-hOCT3 / 4, pCXLE-hSK, pEBNA-DEST, pEBVHisA, pEBVHisB, pEBVHisC, pEP4 EO2S EN2K, pEP4 EO2SET2K, epiCRISPR, pEB-TRE-Cas9, pOriP-Hygro, pEB-Multi, pEB-CAG-GOI-IRES-Hygro, HEK293-EBNA, CHO-EBNA, or pCMV-EBNA1.

[0021] In the present disclosure, the above-mentioned single-plasmid / virus carrier system is provided, which can solve the problems of operation complexity, low efficiency, and uneven plasmid copy number caused by multi-plasmid co-transfection of the Episomal Vector system in the prior art. By optimizing the single-plasmid design, all reprogramming factors are integrated into one plasmid, thereby simplifying the operation, improving the reprogramming efficiency, and avoiding the potential risk of genomic mutation.

[0022] Meanwhile, in another embodiment, a dual-plasmid / viral vector system is also provided, i.e., a composition of plasmids or viruses for reprogramming blood cells, which comprises plasmid or virus A and plasmid or virus B; wherein the plasmid or virus A comprises the above-mentioned reprogramming vector, and the OCT4 gene, SOX2 gene and c-MYC gene in the reprogramming vector are located downstream of the anti-apoptotic gene; and the plasmid or virus B comprises the above-mentioned reprogramming vector, and the OCT4 gene, SOX2 gene and c-MYC gene in the reprogramming vector are located upstream of the anti-apoptotic gene.

[0023] In another embodiment, a triple-plasmid / viral vector system is also provided, i.e., a composition of plasmids or viruses for reprogramming blood cells, which comprises plasmid or virus D, plasmid or virus E and plasmid or virus F; wherein the plasmid or virus D comprises the first expression cassette in the above-mentioned reprogramming vector, the plasmid or virus E comprises the anti-apoptotic gene and the first gene transcription regulatory element in the above-mentioned reprogramming vector, and the plasmid or virus F comprises the second expression cassette in the above-mentioned reprogramming vector.

[0024] Another aspect of the present disclosure is to provide a method for reprogramming blood cells into induced pluripotent stem cells, comprising:

[0025] Step 1) preparing the above-mentioned product, or the composition of plasmids or viruses, wherein the product is a plasmid or virus;

[0026] Step 2) introducing the product obtained in step 1) into the blood cells;

[0027] Step 3) culturing the blood cells obtained in step 2) to induce reprogramming, to obtain iPSC clones;

[0028] Step 4) screening the clones obtained in step 3).

[0029] In the present disclosure, the above-mentioned blood cells are peripheral blood mononuclear cells.

[0030] Another aspect of the present disclosure is to provide the use of the above-mentioned reprogramming vector, the above-mentioned product, or the composition of plasmids or viruses in the preparation of induced pluripotent stem cells.

[0031] Advantages:

[0032] The improved multi-factor reprogramming vector structure and use method in the technical solution of the present disclosure can effectively reduce the expression level of KLF4 at the stage of hematopoietic cells, and realize higher expression level of KLF4 after the cells enter partial reprogramming. This strategy not only solves the problem of inhibition of cell proliferation caused by overexpression of KLF4, but also significantly improves the efficiency and quality of reprogramming hematopoietic cells into iPS.

[0033] Meanwhile, the single-plasmid system in the technical solution of the present disclosure significantly improves the generation efficiency of iPSCs, making them highly similar to embryonic stem cells (ESCs) in morphological and functional characteristics. These iPSCs exhibit typical pluripotent stem cell morphology, including large cell volume, clear nucleus, neat plasma membrane edge, and cluster growth pattern, reflecting the self-renewal ability of stem cells. In terms of functional verification, flow cytometry analysis shows that these iPSCs successfully express pluripotency markers (such as TRA-1-60 and SSEA4) at different time points, with expression levels exceeding 90%, showing no significant difference from iPSCs induced by traditional four-plasmid systems, meeting the expected standards of pluripotent cells. In addition, safety evaluation shows that after PCR detection of plasmid-specific genes such as EBNA1 and WPRE, no exogenous plasmid residues were detected in iPSCs after 5 passages, indicating that all exogenous DNA has been gradually eliminated with cell division. This feature ensures the genetic stability of iPSCs, avoiding immune responses or genomic instability that may be triggered by exogenous DNA, making them more suitable for clinical application standards.

[0034] Through the technical solution in the present disclosure, a low-cost and high-benefit solution can be provided for efficient reprogramming and application of iPSCs, significantly promoting the widespread application of iPSCs in clinical and scientific research fields, especially in the research and practice of disease models, drug screening, cell therapy, etc., which has important application value and development potential. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 A flowchart for generating induced pluripotent stem cells from peripheral blood mononuclear cells using a single-plasmid system in the embodiments of the present disclosure, PBMNCs are isolated from peripheral blood and pre-cultured for 6 days. On day 0, nuclear transformation is performed to introduce the reprogramming plasmid into PBMNCs. On day 2, the culture medium is replaced with iPSC culture medium to support reprogramming to pluripotency. After 14 days, alkaline phosphatase staining is used to confirm the number of iPSCs. iPSC clones are selected and their pluripotency is verified by various detection methods: qPCR is used to detect plasmid residues, and flow cytometry is used to evaluate cell surface markers;

[0036] Figure 2Figure 1 shows the design of the single plasmid for peripheral blood cell reprogramming in the embodiments of the present disclosure. The plasmid sequence SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-polyA-EF1a-KLF4-polyA-142-3pT (2c) contains the essential reprogramming factors (OCT4, SOX2, MYC, KLF4) linked by self-cleaving peptides (P2A / E2A), contains BCL-XL for enhanced cell survival, SFFV promoter for strong expression, and miRNA 142-3pT (2c) for KLF4 expression modulation for safety. The plasmid backbone contains OriP / EBNA1 system for non-integrating amplification, and EF1a for regulatory elements for optimized expression and Wpre for post-transcriptional regulation.

[0037] Figure 3 Figure 2 shows the number of iPSCs generated from the single plasmid system vector combination in the embodiments of the present disclosure. The bar chart shows the number of iPSC clones obtained at day 14.

[0038] Figure 4 Figure 3 shows the colony morphology of iPSCs generated from the single plasmid system vector combination in the embodiments of the present disclosure. The figure shows the characteristic colony morphology of iPSCs at day 14 selected from two vector combinations (OS+B+M+K and BOSM.Kmir).

[0039] Figure 5 Figure 4 shows the functional verification results of iPSCs generated from the single plasmid system vector combination in the embodiments of the present disclosure. A is the alkaline phosphatase (AP) staining result chart at day 14, which shows that the iPSCs generated from the two vector combinations have the characteristics of pluripotency. B is the flow cytometry analysis of iPSCs at day 14, showing the expression of pluripotency markers TRA-1-60, TRA-1-81 and SSEA4.

[0040] Figure 6 Figure 5 shows the in vivo tumorigenicity experiment results in the embodiments of the present disclosure.

[0041] Sequence description.

[0042] SEQ ID No. 1 is the plasmid template sequence of the reprogramming vector in the embodiments of the present disclosure. DETAILED DESCRIPTION

[0043] The application discloses a reprogramming vector for blood cells and application thereof, and those skilled in the art can refer to the content herein, and appropriately improve process parameters to realize. It needs to be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as being included in the application, and relevant personnel can obviously change or appropriately change and combine the content described herein without departing from the content, spirit and scope of the application, to realize and apply the technical field of the application.

[0044] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Unless otherwise explicitly stated, throughout the specification and claims, the term "comprise" or its variants such as "contain" or "include" and the like are to be construed as including the stated elements or components, without excluding other elements or components. The terms "a" and "an" include plural referents. The term "plurality" means two or more. The terms "such as", "for example", and the like are intended to indicate exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0045] In the present disclosure, when a range of values is provided, it should be understood that, unless the context clearly dictates otherwise, the range includes the endpoints and each individual value between the upper and lower limits of the range and any other specified or intervening value within the stated range and smaller ranges within that specified range.

[0046] In the present disclosure, the term "about" generally means a variation of 0.5-10% above or below the specified value, for example, a variation of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0047] In the present disclosure, "one implementation", "one embodiment", "some implementations", "a particular embodiment", "related embodiments", "certain embodiments", "additional embodiments", or "further embodiments", "further implementations", or "another embodiment", "other embodiments", means that at least in the relevance of the embodiments, a feature or characteristic description is included. Therefore, the above phrases are not necessarily all referring to the same embodiment at various places in the present disclosure. In addition, specific features can be combined in any suitable manner in one or more embodiments.

[0048] In the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Definitions of common terms in molecular biology can be found in Lewin’s GENES, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. Definitions of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: W. H. Freeman. Definitions of common terms in cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. Definitions of common terms in genetics can be found in Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0049] Unless otherwise indicated, the experimental techniques in this text adopt the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook; and the like.

[0050] Terms:

[0051] The term "reprogramming" herein refers to a process of changing or reversing the differentiation state of a differentiated cell (e.g., a somatic cell). In other words, reprogramming refers to driving a cell differentiation process back to a more undifferentiated or more primitive cell type. The core is to reset the gene expression pattern of a cell through epigenetic modification, restoring the differentiation potential. In embodiments of the present disclosure, the form of reprogramming involved is induced pluripotent stem cell (iPS) technology. In addition to this, other approaches or forms of reprogramming include, for example, somatic cell nuclear transfer (SCNT), i.e., transplanting a somatic cell nucleus into an enucleated oocyte, reprogramming into a totipotent cell (e.g., the cloned animal "Dolly the sheep"); transdifferentiation, i.e., directly converting one differentiated cell into another without passing through a pluripotent state (e.g., directly converting fibroblasts into neurons), etc.

[0052] The term "induced pluripotent stem cell (iPS)" herein is a form of cell reprogramming. It is a pluripotent stem cell with the characteristics of embryonic stem cells (ESC) by reprogramming technology, converting somatic cells (e.g., fibroblasts) into somatic cells. iPS cells can self-renew and differentiate into almost all types of somatic cells.

[0053] The term "vector" herein has a general meaning, which is capable of introducing the nucleic acid into prokaryotic and / or eukaryotic host cells. In certain embodiments, the vector can be a linear vector, or a circular vector. It can be a non-viral vector such as a plasmid, or a viral vector, or a vector using a transposon. The vector can contain regulatory sequences such as promoters, terminators, and marker sequences such as drug resistance genes, reporter genes, etc. In addition, the above-mentioned vector can also contain a sequence encoding a suicide gene, which can be activated by administering a substance to activate the suicide gene during the treatment process. As the above-mentioned viral vector, it can be a plasmid vector, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, etc. In some embodiments, the vector is a plasmid vector.

[0054] The term "Yamanaka factors" herein, i.e., Yamakana factors, generally refers to the combination of four transcription factors, i.e., the OCT4 gene, the SOX2 gene, the KLF4 gene, and the c-MYC gene, which were first screened by Shinya Yamanaka for reprogramming fibroblasts into iPSCs (see Yamanaka, 2009, Cell 137:13-17).

[0055] The term "expression cassette" herein generally refers to an artificially constructed functional DNA sequence module for driving expression of a specific gene in a host cell. It is generally composed of three core elements: a promoter (such as CMV or EF1a promoter, controlling the initiation of transcription), a coding sequence of a gene of interest (such as an open reading frame of a protein), and a terminator (marking the end of transcription), and can be supplemented with enhancers, regulatory sequences or marker genes as needed.

[0056] The term "operably linked" or "operably connected" herein refers to the functional linkage of polynucleotide sequence elements. The functionality of the linked elements is intended to be maintained. For example, a polynucleotide sequence is operably linked with another polynucleotide sequence when the elements are in a functional relationship with each other. In some embodiments, a transcriptional regulatory polynucleotide sequence (e.g., a promoter, enhancer, or other expression control element) is operably linked to a polynucleotide sequence encoding a protein if the transcriptional regulatory polynucleotide sequence affects the transcription of the polynucleotide sequence encoding the protein. The elements that are operably linked need not be contiguous.

[0057] The term "reprogramming induction" herein refers to the step of initiating reprogramming, which is placing the transduced cells in pluripotency culture conditions, with specific growth factors (such as LIF, bFGF) in the medium, and using feeder cells (such as mouse embryonic fibroblasts) or Matrigel for support. The cells then gradually lose their original characteristics and form clonal colonies similar to embryonic stem cells (about 2-4 weeks). In some embodiments, the second gene transcriptional regulatory element is capable of regulating the down-regulation of KLF4 gene expression in the time period from the introduction of the cells to the reprogramming induction step.

[0058] Chemical induction techniques have been used in conjunction with reprogramming. In some embodiments, the following exemplary small molecule compounds can be used: valproic acid (VPA) and trichostatin A (TSA) in epigenetic modifiers can increase the openness of chromatin by inhibiting histone deacetylase (HDAC), activating pluripotency genes (such as Oct4); and 5-azacytidine (5-Azacytidine) can promote the initiation of reprogramming by inhibiting DNA methyltransferase, relieving gene silencing. In terms of signal pathway regulators, CHIR99021 as a GSK-3β inhibitor can activate the Wnt / β-catenin pathway, synergize with the mountain factor to enhance pluripotency gene expression; SB431542 can inhibit epithelial-mesenchymal transition (EMT) by blocking the TGF-β signal pathway, indirectly improving reprogramming efficiency. Metabolic regulators such as vitamin C not only reduce the damage of reactive oxygen species (ROS) to cells through antioxidant effect, but also activate histone demethylase, further remodeling epigenetic characteristics; and PS48 provides energy support to cells by enhancing glycolysis metabolism.

[0059] The reprogramming plasmid / virus system in the embodiments of the present disclosure:

[0060] 1. BOSM single-plasmid system.

[0061] The BOSM single-plasmid system integrates key reprogramming factors (OCT4, SOX2, MYC, KLF4) and auxiliary elements (BCL-XL) and regulatory elements (miRNA) into the same plasmid, simplifying the operation process, reducing costs, and reducing the risk of residual foreign plasmids, especially suitable for clinical-level iPSC preparation. Compared with the multi-plasmid system, the BOSM single-plasmid system ensures high transfection efficiency while improving the reliability and stability of iPSC clone formation.

[0062] 2. Double-plasmid system (OSMB+K, BOSM+K).

[0063] If the researchers or production process need to flexibly regulate the expression level of different factors, the reprogramming factors can be split into 2-3 vectors, for example, one vector carries only the core factors (OCT4, SOX2, KLF4, MYC), and the other vector carries auxiliary factors (BCL-XL), regulatory elements (miRNA) or reporter genes. In this way, the promoters, cleavage peptides and regulatory sequences of each vector can be optimized independently, thereby obtaining higher expression controllability or more flexible satisfaction of different experimental needs.

[0064] The double-plasmid system uses two plasmids to carry different reprogramming factors and auxiliary factors, so as to optimize the expression ratio of factors, improve the reprogramming efficiency and enhance the cell survival rate. Experiments show that the system can obtain a large number of iPSC clones and has high flexibility, and is suitable for cell therapy and scientific research applications.

[0065] 3. Three-plasmid system (OSM+B+K).

[0066] The three-plasmid system further refines the loading mode of reprogramming factors, and allocates OCT4, SOX2, MYC, BCL-XL and KLF4 to three plasmids, so as to accurately control the gene expression level. The system optimizes the cell reprogramming process, improves the transfection efficiency and the pluripotency stability of iPSC, and is suitable for applications requiring fine gene regulation.

[0067] Embodiment:

[0068] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below in combination with specific embodiments.

[0069] In this embodiment, an improved multi-factor reprogramming vector structure and use method based on the technical concept of the present application are disclosed. By highly expressing key factors such as BCL-XL, OCT4, SOX2 and MYC in hematopoietic cells (driven by SFFV promoter), and using EF1α (or PGK, CMV, CAG, etc.) to drive KLF4, while adding double-copy target sequences (142-3pT (2c)) of miR-142-3p behind the KLF4 gene, the expression level of KLF4 is effectively reduced in the hematopoietic cell stage, and after the cell enters partial reprogramming and gradually loses a large amount of miRNA142-3p expression, higher level expression of KLF4 is realized. This strategy not only solves the problem of inhibition of cell proliferation caused by excessive expression of KLF4, but also significantly improves the efficiency and quality of hematopoietic cell reprogramming into iPS.

[0070] The vector structure and elements include:

[0071] (1) High-efficiency reprogramming factor region:

[0072] BCL-XL, P2A-OCT4, E2A-SOX2 and E2A-MYC are driven by SFFV promoter, followed by wpre and polyA signal. The SFFV promoter has high expression activity in hematopoietic cells, ensuring sufficient expression of factors such as BCL-XL, OCT4, SOX2 and MYC to support the rapid start and progress of early reprogramming.

[0073] (2) KLF4 expression regulation region:

[0074] KLF4 gene driven by EF1a (or PGK, CMV, CAG, etc.) promoter, followed by polyA signal and double copy target sequence 142-3pT (2c) of miRNA142-3p.

[0075] Due to the high expression of miRNA142-3p in hematopoietic cells, the 142-3pT (2c) element can effectively weaken the translation and stability of KLF4 at an early stage, thereby weakening its inhibitory effect on cell proliferation.

[0076] When the cells enter partial reprogramming or approach complete reprogramming, the level of miRNA142-3p decreases, KLF4 is fully expressed, and the quality of iPS cells is improved.

[0077] Meanwhile, a single-plasmid reprogramming vector system based on the technical concept of the present application is disclosed in the present embodiment. The system integrates multiple key reprogramming factors (OCT4, SOX2, KLF4, c-MYC), anti-apoptotic genes (BCL-XL), and regulatory factors (such as miRNA), significantly simplifying the traditional iPSC reprogramming process. The traditional Episomal Vector system usually requires multiple plasmids to be co-transfected, resulting in uneven copy numbers, affecting the synergistic expression of reprogramming factors and reprogramming efficiency. The present application ensures the synchronous and balanced expression of all key factors in the cell through a single-plasmid system, effectively solving the problem of uneven plasmid copy numbers, allowing the cell to efficiently complete reprogramming after a single transfection, greatly improving the repeatability and transfection success rate of the experiment. In addition, the introduction of BCL-XL enhances cell survival rate and optimizes reprogramming efficiency. The miRNA regulatory factor (such as miRNA 142-3pT (2c)) can accurately regulate the expression level of KLF4, avoiding the safety hazards caused by its overexpression, further reducing the cell stress response, and improving the stability of the reprogramming process. The system not only improves the induction efficiency of iPSC, but also reduces the risk of residual exogenous plasmid, ensuring its genetic stability, thereby providing a safer and more efficient solution for the preparation of clinical-grade iPSC.

[0078] Example 1: Isolation and culture of peripheral blood mononuclear cells (PBMNC).

[0079] 1. Collection of peripheral blood from subjects.

[0080] Blood samples were collected from healthy donors or target patients. Freshly collected peripheral blood was anticoagulated with EDTA or heparin. The volume of blood collected was 20-50 mL. All blood samples were collected and used in accordance with the ethical requirements. The research protocol was approved by the ethics committee, and the donors were informed of the purpose of the study, the operation process, and the possible effects before signing the informed consent form. The collection, transportation, and storage of samples must comply with biosafety standards to ensure the repeatability of the experiment and the reliability of the data.

[0081] Blood collection was performed using aseptic technique with vacuum blood collection tubes or blood bags to reduce the risk of contamination and ensure sample quality. If cell separation cannot be performed immediately, the blood sample should be stored at 4°C and the separation process should be completed within 2-4 hours to maintain cell activity and experimental repeatability. During transportation and temporary storage, avoid violent shaking and follow standard biosafety procedures to ensure sample stability and reliability.

[0082] 2. Isolation of PBMNC.

[0083] Density gradient centrifugation was used to isolate PBMNC to obtain high-purity mononuclear cells. First, mix the peripheral blood with PBS at a ratio of 1:1, mix thoroughly, and then slowly layer it on top of the pre-prepared Ficoll-Hypaque (1.077 g / mL) separation solution. Ensure that the interface between the layers is clear and avoid mixing the liquids. Then, centrifuge at 400 x g for 30 minutes (room temperature). After centrifugation, a clear layering is formed: the upper layer is plasma, the middle white membrane layer is PBMNC, and the lower layer is Ficoll solution and red blood cells. Use a pipette or pipette to carefully aspirate the white membrane layer (PBMNC) and transfer it to a new centrifuge tube. Wash 1-2 times with PBS (300 x g, 10 minutes) to remove platelets and Ficoll residues and improve cell purity.

[0084] The isolated PBMNC were evaluated for cell viability by trypan blue staining and counted using a hemocytometer or an automatic cell counter. Finally, the cell concentration was adjusted to the required range (1-5 x 10 6 cells / mL) to ensure the stability and repeatability of subsequent experiments.

[0085] 3. Preliminary expansion / pre-culture of PBMNC.

[0086] To improve cell survival rate and optimize reprogramming efficiency, PBMNCs are usually subjected to initial expansion under specific culture conditions. Culture medium can be Stem-line II Hematopoietic Stem Cell Expansion Medium (Sigma, S0192) supplemented with key cytokines, including 100 ng / mL Stem Cell Factor (SCF), 10 ng / mL Interleukin-3 (IL-3), 2 U / mL Erythropoietin (EPO), 20 ng / mL Insulin-like Growth Factor-1 (IGF-1), 1 mM Dexamethasone, and 0.2 mM 1-Thioglycerol. Cell culture is performed in an incubator at 37°C, 5% CO2, 95% relative humidity, usually for 6 days of pre-culture to facilitate monocyte activation and improve subsequent transfection efficiency.

[0087] During pre-culture, cell status should be monitored daily, including morphological changes and proliferation. If cell density is too high, culture medium can be added appropriately to dilute, to maintain an appropriate culture environment. In addition, cytokines can be supplemented as appropriate according to cell growth to optimize cell expansion and ensure the stability of the experiment.

[0088] Example 2: Construction and preparation of single-plasmid vector.

[0089] The schematic diagram of the construction of the single-plasmid vector is shown in Figure 2 .

[0090] 1. Plasmid vector backbone and key elements.

[0091] The reprogramming vector in this embodiment uses pCEP4 or other backbone containing OriP / EBNA1 sequence to ensure non-integrated replication of plasmid in eukaryotic cells, while gradually diluted during cell division to avoid the safety risk of genomic integration. The reprogramming factors (OCT4, SOX2, KLF4, c-MYC, BCL-XL) and the miRNA structure that can be used (such as miRNA 142-3pT (2c)) are integrated into the same open reading frame and connected by P2A, E2A or T2A self-cleavage peptide to achieve synchronous expression of multiple proteins, ensuring stable co-expression of reprogramming factors. The transcription of target genes is driven by strong promoters such as SFFV, CMV or EF1a to enhance expression efficiency. In addition, 3' regulatory elements such as PolyA tail signal and WPRE are introduced to improve mRNA stability and transcription efficiency, thereby optimizing the success rate and safety of cell reprogramming. The sequence of its single plasmid template is as follows: SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-ployA-EF1a-KLF4-polyA-142-3pT (2c). The sequence is shown in SEQ ID No. 1.

[0092] 2. Plasmid preparation.

[0093] After small-scale testing confirms the success of construction, large-scale plasmid preparation is carried out to obtain high-purity, low-endotoxin plasmid DNA. Special endotoxin removal kits (such as Zymo Midi Kit) are used for extraction to ensure compliance with experimental or clinical application requirements. During preparation, 50-200 μL of bacterial solution is inoculated into 50 mL of culture medium containing the corresponding antibiotic, and shaken for 16-18 hours. Part of the bacterial solution is reserved for glycerol bacterial sample preparation for long-term storage. After bacterial solution culture, the bacterial pellet is collected, and P1, P2, and P3 solutions are added in sequence to lyse the cells. Cell debris and chromosomal DNA are removed by centrifugation, followed by the addition of Binding Buffer for filtration. Plasmid DNA is collected by affinity column and washed with Wash1 and Wash2 solutions in sequence. Finally, it is eluted with elution buffer and endotoxin is removed.

[0094] The extracted plasmid is measured for concentration and purity by spectrophotometer 、 to ensure that its quality meets the experimental requirements. The target concentration is usually 1000-1400 ng / μL, and if the concentration is too high, it can be appropriately diluted. The correctness of the plasmid sequence is verified by enzyme digestion analysis and Sanger sequencing (NP sequencing). The plasmid is dissolved in sterile TE buffer or appropriate storage buffer and stored at -20°C or -80°C for long-term storage to maintain its stability and ensure the reproducibility of subsequent experiments.

[0095] Example 3: Nucleofection and reprogramming induction.

[0096] The experimental flow chart is shown in Figure 1

[0097] 1. Optimization of electroporation conditions.

[0098] Commercial electroporators with preset programs (such as Lonza, Neon, or other brands) are used, and the corresponding cell transfection buffer is used to improve transfection efficiency and cell survival rate. If the device has no preset program, the electroporation parameters can be optimized according to the cell type, including voltage, pulse time and pulse number, to ensure high transfection efficiency while maximizing cell viability.

[0099] 2. Electroporation steps.

[0100] (1) PBMNCs are isolated from the peripheral blood of healthy donors using Ficoll-Hypaque density gradient centrifugation to obtain high-purity PBMNCs, which are then pre-cultured in erythroid medium for 6 days, and stem cell factor (SCF) and interleukin (IL-3) are added to the medium to improve cell viability and transfection reaction.

[0101] (2) Resuspend the cells with transfection buffer solution, count and adjust the concentration to 1-2 x 10 6 cells / reaction system.

[0102] (3) Add an appropriate amount of single-plasmid DNA (such as 5-10 μg), mix gently to ensure uniform distribution of DNA.

[0103] (4) Transfer the mixture to an electroporation tube or micro-well and follow the optimized electroporation program.

[0104] (5) After electroporation, immediately transfer the cells to preheated (37°C) erythroid medium or serum-free medium to reduce cell damage and promote survival rate.

[0105] 3. Inoculation and reprogramming culture.

[0106] (1) Preparation of feeder layer or matrix.

[0107] Before culture, choose an appropriate matrix or feeder layer to support cell growth. Common methods include using 0.1% gelatin or Matrigel coating, or pre-seeding feeder cells (such as REF, MEF) treated with mitomycin C. After inoculation, place the culture dish in a 37°C, 5% CO2 incubator for several hours to promote cell adhesion and provide a stable growth environment.

[0108] (2) Reprogramming induction.

[0109] ​a) Day 0-2: Post-electroporation cells are maintained in erythroid medium (same as pre-culture) to promote cell recovery and increase survival rate.

[0110] b) Day 2 onwards: Switch to iPSC induction medium (e.g. KnockOut DMEM / F12 with FGF2, ITS, ascorbic acid, etc.) to officially initiate the reprogramming process.

[0111] c) Continuous culture: Fresh reprogramming medium is replaced every 1-2 days, and ROCK inhibitor (Y-27632) is added as needed based on cell status to improve cell survival and promote colony formation.

[0112] (3) iPSC colony formation and selection.

[0113] iPSC colonies start to appear in the culture dish around 8-10 days post-transfection, and reach a suitable state for selection around 14 days. The number of iPSC colonies is counted and the results are shown in Figure 3 The characteristic colony morphology of iPSC at day 14 is selected from two vector combinations (OS+B+M+K and BOSM.Kmir) and shown in Figure 4 Morphological observation shows that typical iPSC colonies can be observed around 8-10 days post-transfection using the single-plasmid vector system of the present application, and the colony size is sufficient for selection around 14 days. The iPSC colonies formed by reprogramming exhibit a typical "cobblestone" structure, with tightly packed cells, regular morphology, large nuclei occupying the majority of the volume, and clear nucleoli. The overall boundary is distinct. These morphological characteristics are consistent with the typical morphology of pluripotent stem cells, indicating that the iPSC is successfully established and in a stable pluripotent state. Typical iPSC colonies are tightly packed, with large nuclei, clear cell boundaries, regular morphology, and smooth surface.

[0114] When selecting colonies, a glass needle or Pasteur pipette can be used to manually pick the target colonies and transfer them to a new culture dish for expansion to obtain stable and high-quality iPSC cell lines. The entire process must be performed under sterile conditions, and the cell status should be observed regularly to ensure the growth quality and pluripotency characteristics of the colonies.

[0115] Example 4: Expansion and identification of iPSC.

[0116] 1. Passage and cryopreservation of iPSC.

[0117] (1) Cell passage culture.

[0118] During cell passaging, appropriate digestion method should be chosen to maintain cell activity and proliferation ability. Common methods include using enzyme solution (such as Accutase) or EDTA to dissociate cells into small clusters or single cell state. Cells after digestion should be transferred to culture plates pre-coated with Matrigel or feeder layer in time, and fresh culture medium should be replaced every 24 hours to maintain appropriate growth environment. According to the growth of cell clones, passaging should be performed once every 3-5 days to ensure that cells maintain good proliferation state and avoid excessive dense culture affecting reprogramming efficiency.

[0119] (2) Cell cryopreservation.

[0120] After 2-3 times of cell passaging, cells can be cryopreserved to retain their proliferation ability and biological characteristics. After collecting cells, resuspend them in cryopreservation solution containing 10% DMSO + 90% FBS (or serum substitute) to ensure cell survival in low temperature environment. Then, use step-by-step cooling method to slowly cool cells to -80°C, and then transfer them to liquid nitrogen for long-term storage after 24 hours to minimize the damage of cryopreservation process to cell activity.

[0121] 2. Pluripotency marker detection: collect iPSCs and prepare single cell suspension to ensure uniform distribution of cells. Use fluorescently labeled antibodies (such as TRA-1-60, TRA-1-81, SSEA4) to stain cells to detect their pluripotency characteristics. Then, analyze the positive expression proportion of markers by flow cytometry, and generally expect that the proportion of positive cells is more than 80-90% to confirm the quality and pluripotency state of iPSCs. The results are shown in A and B of Figure 5 The results show that the iPSCs obtained by reprogramming show that the positive expression rate of pluripotent cell surface markers (TRA-1-60, TRA-1-81, SSEA4) is more than 90% after flow cytometry analysis, which is comparable to iPSCs generated by traditional multi-plasmid reprogramming method, indicating that the single-plasmid system can efficiently induce pluripotency state. In addition, the immunofluorescence detection results show that the core pluripotency transcription factors such as OCT4, SOX2, NANOG in iPSCs are highly expressed and located in the nucleus, further verifying the pluripotency of reprogrammed cells. These results show that the single-plasmid vector system of the present application can effectively generate iPSCs with stable pluripotency characteristics, providing a safer and more efficient reprogramming strategy for clinical application.

[0122] 4. Functional test.

[0123] (1) In vitro three germ layer differentiation.

[0124] To verify the pluripotency of iPSCs, they can be induced to differentiate into three germ layer-derived cells through embryoid bodies (EBs). Under specific induction conditions, iPSCs can differentiate into ectoderm (such as neural cells), mesoderm (such as cardiomyocytes, skeletal muscle cells), and endoderm (such as hepatocyte-like cells, pancreatic islet cells). After differentiation, corresponding markers (such as βIII-tubulin for ectoderm, Nkx2.5 for mesoderm, and AFP for endoderm) are used for identification to evaluate the differentiation ability of the cells and confirm their pluripotency characteristics.

[0125] Through in vitro differentiation experiments, it is verified whether the iPSCs obtained by reprogramming with a single plasmid system have pluripotency. These iPSCs can be successfully induced to differentiate into three germ layer-derived cells, including ectoderm (neurons), mesoderm (cardiomyocytes), and endoderm (hepatocyte-like cells), and their differentiation ability is comparable to that of embryonic stem cells (ESCs) and iPSCs obtained by traditional reprogramming methods. In addition, in the teratoma formation assay, after injecting iPSCs into immunodeficient mice, three germ layer-derived tissue structures can be observed, further confirming their pluripotency. These results show that the single plasmid vector system of the present application can efficiently generate functional iPSCs, providing reliable support for their application in regenerative medicine, disease modeling, and cell therapy.

[0126] (2) In vivo tumorigenicity experiment.

[0127] To evaluate the pluripotency of iPSCs, the cells are injected into immunodeficient mice (such as NOD / SCID) to observe whether they can form teratomas containing three germ layer-derived tissues in vivo. After tumor formation, histopathological analysis of different tissue types is performed, and specific markers are detected to confirm whether they are derived from ectoderm, mesoderm, and endoderm, thereby proving the pluripotency of iPSCs. The results are shown in Figure 6 . The results show that the obtained tumors are derived from ectoderm, mesoderm, and endoderm.

[0128] 5. Quality control and precautions.

[0129] Quality control is carried out throughout the entire process of cell reprogramming to ensure the repeatability of the experiment and compliance with GMP standards. First, the transfection efficiency and cell viability should be evaluated regularly, and the transfection parameters should be optimized according to the experimental requirements to improve the success rate of reprogramming. Second, strict aseptic operation should be performed to avoid bacterial, fungal, and mycoplasma contamination, and the cleanliness of the culture environment should be monitored regularly. Finally, a complete record and traceability system should be established, with detailed records of each operation, cell passage, freezing, and thawing of key information to ensure data traceability, in order to meet the requirements of GMP quality management and provide reliable basis for subsequent experiments and clinical applications.

[0130] During cell manipulation, key technical details should be paid attention to ensure the stability of the experiment and the reliability of the data. First, the trypsin digestion time should be strictly controlled before cell passage or immunostaining to avoid excessive digestion leading to cell damage or loss of key surface markers, thereby affecting the experimental results. Second, for in vivo functional verification experiments (such as teratoma experiments), relevant animal ethics and welfare standards must be followed to ensure that the experimental protocol is approved by the ethics committee and appropriate anesthesia, care and euthanasia measures are taken to reduce animal suffering and meet the requirements of experimental animal management.

[0131] 6. Data analysis.

[0132] Graphpad Prism 8.0.1 (Graphpad software, San Diego, CA) was used for plotting and statistical analysis. The results are mean ± SEM. One-way ANOVA was used to determine the significance between experimental and control groups. P < 0.05 is a significant difference; p < 0.05; p < 0.01; p < 0.001.

[0133] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A reprogramming vector for peripheral blood mononuclear cells (PBMNCs), characterized in that, The reprogramming vector comprises: (1) an OCT4 gene, a SOX2 gene, a KLF4 gene, and a c-MYC gene; (2) a first expression cassette comprising a first gene transcription regulatory element operably linked thereto; and (3) a second expression cassette comprising a second gene transcription regulatory element operably linked thereto; wherein the first gene transcription regulatory element is used to regulate the expression of the OCT4 gene, the SOX2 gene, and the c-MYC gene, the second gene transcription regulatory element is used to regulate the expression of the KLF4 gene, and the second gene transcription regulatory element comprises a regulatory element that inhibits the expression of the KLF4 gene before reprogramming induction; the first gene transcription regulatory element or the second gene transcription regulatory element comprises a promoter or an enhancer; wherein the promoter of the first gene transcription regulatory element is SFFV, and the promoter of the second gene transcription regulatory element is EF1a; the regulatory element that inhibits the expression of the KLF4 gene before reprogramming induction is miR-142-3pT(2c); the first expression cassette and the second expression cassette are disposed on the same vector.

2. The reprogramming vector of claim 1, wherein The first expression cassette further comprises an anti-apoptotic gene, and the expression of the anti-apoptotic gene is regulated by the first gene transcription regulatory element.

3. The reprogramming vector of claim 2, wherein The anti-apoptotic gene is at least one selected from the BCL-2 family, the IAP family, FLIP, Akt / PKB, HSPs, and NF-κB.

4. The reprogramming vector of claim 1, wherein The first expression cassette further comprises a sequence encoding a self-cleaving peptide between the gene sequences.

5. The reprogramming vector of claim 4, wherein The self-cleaving peptide is at least one selected from the 2A peptide family.

6. The reprogramming vector of claim 1, wherein The reprogramming vector is SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-EF1a-KLF4-polyA-142-3pT(2c).

7. A product for reprogramming of peripheral blood mononuclear cells, characterized in that, The product is a plasmid or a virus, and the plasmid or the virus comprises an OriP / EBNA1 replication system and the reprogramming vector according to any one of claims 1-6.

8. The product of claim 7, wherein, The virus is an adenovirus, an adeno-associated virus, or a retrovirus, and the plasmid is selected from pCEP4, pEB-C5, pEBNA-DEST, pEBVHisA, pEBVHisB, pEBVHisC, or pEB-Multi.

9. The product of claim 8, wherein, The retrovirus is a lentivirus.

10. A method of reprogramming blood cells into induced pluripotent stem cells (iPSCs) characterized by, The method comprises: Step 1) preparing the product according to claim 7, 8, or 9; Step 2) introducing the product obtained in step 1) into the blood cells; Step 3) culturing the blood cells obtained in step 2) to induce reprogramming to obtain iPSC clones; Step 4) screening the clones obtained in step 3); The blood cells are peripheral blood mononuclear cells.

11. Use of the reprogramming vector according to any one of claims 1-6 or the product according to claim 7, 8, or 9 in the preparation of induced pluripotent stem cells.

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