Reprogramming vector for blood cells and application thereof

Abstract

The invention provides a reprogramming vector for blood cells and application of the reprogramming vector. According to the technical scheme, a low-cost and high-benefit solution can be provided for efficient reprogramming and application of the iPSC, wide application of the iPSC in the clinical and scientific research field is remarkably promoted, and the iPSC has important application value and development potential especially in research and practice of disease models, drug screening, cell therapy and the like.

Description

Technical Field

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

[0002] Reprogramming is a technical process that reverses a differentiated cell to a more primitive state (such as pluripotency) through human intervention. Reprogramming technology currently relies on the introduction of specific transcription factors (such as OCT4, SOX2, KLF4, c-MYC), which reprogram somatic cells into pluripotent cells with stem cell characteristics by activating pluripotency-related gene networks. However, in the existing technology, reprogramming technology still faces certain limitations, mainly including problems such as vector type selection, transfection efficiency and genomic safety. Most of the traditional methods of obtaining iPSCs using reprogramming technology use the same promoter 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 successfully reprogram. KLF4 is crucial to the quality of reprogrammed cells. In addition, different promoters have different expression levels in different types of cells; therefore, how to inhibit the excessive expression of KLF4 in the early reprogramming stage of hematopoietic cells and moderately increase its expression in the later stage to ensure the reprogramming efficiency and the quality of the final iPSC has become a technical difficulty.

[0003] In addition, the 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 be induced efficiently, but their genome integration characteristics may lead to genome mutations and potential tumorigenic risks, limiting their clinical application. In contrast, non-viral vectors (such as circular DNA plasmid systems) avoid the safety issues caused by genome integration and are theoretically more suitable for clinical applications. However, traditional non-viral reprogramming methods usually require co-transfection of multiple plasmids, which not only increases the complexity of the operation, but also may lead to uneven expression levels of reprogramming factors, thereby affecting reprogramming efficiency and cell quality. In addition, although other non-integration strategies such as mRNA or small molecule compounds can further reduce genetic safety risks, they have not yet become mainstream methods due to low transfection efficiency, cumbersome operations or limited induction efficiency.

[0004] Episomal Vector is a non-integrating gene delivery system based on extrachromosomal autonomous replication, which is widely used in the fields of cell reprogramming, gene therapy and gene editing. Its core advantage is to avoid host genome integration, reduce the risk of insertion mutation, and provide stable transgenic expression. However, the co-transfection of multiple plasmids in the existing Episomal Vector system brings about problems such as operational complexity, inefficiency and uneven plasmid copy number. In order to solve the problems existing in the prior art mentioned above, it is necessary to improve the reprogramming system. Summary of the invention

[0005] Technical issues solved:

[0006] One aspect of the present invention is to provide a new reprogramming vector system to address the problems of complex operation, low efficiency, genomic instability, etc. in traditional reprogramming vector systems, thereby improving the efficiency of iPSC generation, avoiding exogenous DNA residues, and ensuring its clinical safety.

[0007] Technical solution:

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

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

[0010] (2) a first expression frame, wherein the first expression frame comprises an operably linked first gene transcription regulatory element; and

[0011] (3) a second expression frame, wherein the second expression frame comprises an operably linked second gene transcription regulatory element;

[0012] Among them, the first gene transcription regulatory element is used to regulate the expression of the OCT4 gene, SOX2 gene and 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 includes a regulatory element that inhibits the expression of the KLF4 gene before reprogramming induction.

[0013] In some embodiments, the first gene transcription regulatory element or the second gene transcription regulatory element may include a promoter, an enhancer or a silencer. In other embodiments, those skilled in the art may add other suitable gene transcription regulatory elements as needed, and the technical solutions thereof are also considered to be included in the protection scope of the present invention.

[0014] The technical solution disclosed in the present invention utilizes different expression frames to express Yamanaka factors respectively, and can also be combined with regulatory elements that inhibit KLF4 gene expression, so that the high-level expression of KLF4 has a strong inhibitory effect on cell proliferation, thereby avoiding the problem of low reprogramming efficiency or even failure to successfully reprogram. In some embodiments, the promoters contained in the above-mentioned first gene transcription regulatory element or the above-mentioned second gene transcription regulatory element may be the same or different, preferably different promoters. Further, those 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, EF1α, Sox2, Nanog, RUNX1, GATA2, PU.1. Furthermore, in one embodiment, the promoter contained in the above-mentioned first expression frame may be an SFFV promoter, and the promoter contained in the above-mentioned second expression frame may be an EF1α promoter.

[0015] In some embodiments, the enhancer may be selected from WPRE, EBNA1, SV40, CMV, and CAG.

[0016] In some embodiments, the regulatory element that inhibits the expression of the KLF4 gene before reprogramming induction is a miRNA regulatory element. Further, in some embodiments, the miRNA regulatory element can be at least one selected from the 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 miRNA regulatory element can be miR-142-3pT (2c).

[0017] In some embodiments, the first expression cassette may further comprise an anti-apoptotic gene, the expression of which is regulated by the first gene transcription regulatory element. In some embodiments, a person skilled in the art may 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 anti-apoptotic gene may be at least one selected from the group consisting of BCL-2 family, IAP family, FLIP, Akt / PKB, HSPs, NF-κB, ARC, DAD1, and SODD. Furthermore, in one embodiment, the anti-apoptotic gene may be a BCL-XL gene.

[0018] In order to better achieve the purpose of the present disclosure, the gene sequence in the first expression frame may further include a sequence encoding a self-cleaving peptide. Those skilled in the art may select any suitable self-cleaving peptide, but preferably, in some embodiments, the self-cleaving peptide may be at least one selected from the 2A peptide family, an intein, and Sortase A.

[0019] In one embodiment, the 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, wherein the product is a plasmid or a virus, wherein the plasmid or the virus contains the OriP / EBNA1 replication system and the above-mentioned reprogramming vector. The OriP / EBNA1 replication system carried can be amplified in mammalian cells for a short period of time and gradually diluted as the cells divide, thereby achieving reprogramming without genome 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 / viral vector system is provided, which can solve the problems of operational complexity, inefficiency and uneven plasmid copy number caused by co-transfection of multiple plasmids in 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] At the same time, in other embodiments, a dual plasmid / virus vector system is also provided, that is, a composition of plasmids or viruses for blood cell reprogramming, the composition comprising plasmid or virus A and plasmid or virus B; wherein, the plasmid or virus A comprises the above-mentioned reprogramming vector, 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, the OCT4 gene, SOX2 gene and c-MYC gene in the reprogramming vector are located upstream of the anti-apoptotic gene.

[0023] In other embodiments, a three-plasmid / virus vector system is also provided, that is, a plasmid or virus composition for blood cell reprogramming, the composition comprising plasmid or virus D, plasmid or virus E and plasmid or virus F; wherein the plasmid or virus D comprises the first expression frame described in the above-mentioned reprogramming vector, the plasmid or virus E comprises the anti-apoptosis gene and the first gene transcription regulatory element described in the above-mentioned reprogramming vector, and the plasmid or virus F comprises the second expression frame described 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 product, or a composition of the above plasmid or virus, 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) reprogramming and inducing the blood cells obtained in step 2) to obtain iPSC clones;

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

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

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

[0031] Beneficial effects:

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

[0033] At the same time, the single plasmid system in the disclosed technical solution significantly improves the generation efficiency of iPSC, making it highly similar to embryonic stem cells (ESC) in morphology and functional characteristics. These iPSCs present 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 showed that these iPSCs successfully expressed pluripotency markers (such as TRA-1-60 and SSEA4) at different time points, and their expression levels exceeded 90%, which was not significantly different from iPSCs induced by the traditional four-plasmid system, and met the expected standards of pluripotent cells. In addition, the safety assessment showed that after PCR detection of plasmid-specific genes such as EBNA1 and WPRE, no exogenous plasmid residues were detected in iPSCs that were passaged 5 times, indicating that all exogenous DNA has been gradually eliminated with cell division. This feature ensures the genetic stability of iPSCs, avoids immune responses or genomic instability that may be induced by exogenous DNA, and makes them more in line with clinical application standards.

[0034] The technical solutions disclosed in the present invention can provide a low-cost, high-efficiency solution for the 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., and have important application value and development potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Flow chart of generating induced pluripotent stem cells from peripheral blood mononuclear cells using a single plasmid system in the disclosed embodiments, 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 the PBMNCs, on day 2, the culture medium is replaced with iPSC culture medium to support reprogramming to pluripotency, and after 14 days, alkaline phosphatase staining is used to confirm the number of iPSCs, iPSC clones are selected, and their pluripotency is verified by multiple detection methods: plasmid residues are detected by qPCR, and cell surface markers are evaluated by flow cytometry;

[0036] Figure 2The single plasmid design diagram for peripheral blood cell reprogramming in the disclosed embodiment is shown in FIG. The plasmid sequence SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-polyA-EF1a-KLF4-polyA-142-3pT (2c) contains basic reprogramming factors (OCT4, SOX2, MYC, KLF4) connected by self-cleaving peptides (P2A / E2A), BCL-XL to enhance cell survival, SFFV promoter for strong expression, and miRNA 142-3pT (2c) for regulating KLF4 expression to improve safety. The plasmid backbone contains the OriP / EBNA1 system for non-integration amplification, as well as EF1α regulatory elements for optimizing expression and Wpre for post-transcriptional regulation;

[0037] Figure 3 A result graph of the number of iPSCs generated by the single plasmid system vector combination in the disclosed embodiment, the bar graph shows the number of iPSC clones obtained on day 14;

[0038] Figure 4 The clonal morphology diagram of iPSCs generated for the single plasmid system vector combination in the disclosed embodiment, which respectively shows the characteristic clonal morphology of iPSCs on day 14 selected from two vector combinations (OS+B+M+K and BOSM.Kmir);

[0039] Figure 5 The functional verification result diagram of iPSC generated by the single plasmid system vector combination in the embodiment of the present disclosure, wherein A is the alkaline phosphatase (AP) staining result diagram on day 14, which shows that the iPSC generated from the two vector combinations has pluripotency characteristics, and B is the flow cytometry analysis of iPSCs on day 14, showing the expression of pluripotency markers TRA-1-60, TRA-1-81 and SSEA4;

[0040] Figure 6 This is a diagram showing the results of an in vivo tumorigenic experiment in an embodiment 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 present invention discloses a reprogramming vector for blood cells and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] In the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "including" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The term "a", "an" and "the" include plural indicators. The term "multiple" refers to two or more. The terms "such as", "for example", etc. are intended to refer to 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 the endpoints are included in the range and each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the specified range and any value in the smaller range between the specified values ​​are included unless the context clearly dictates otherwise.

[0046] In this disclosure, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 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 a specified value.

[0047] In the present disclosure, "one embodiment", "an example", "some embodiments", "specific embodiments", "related embodiments", "an example", "some examples", "additional examples", or "further examples", "further implementations", or "another example", "other examples" mean that at least one feature or characteristic description is included in the relevance to the embodiment. Therefore, the above phrases do not necessarily refer to the same embodiment in 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 specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The definition 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. The definition of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. The definition 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. The definition 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 specified, the experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in standard books such as the following: Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0050] the term:

[0051] The term "reprogramming" herein refers to the process of changing or reversing the differentiation state of differentiated cells (such as somatic cells). In other words, reprogramming refers to the process of driving cell differentiation, so that it regresses to a more undifferentiated or more primitive cell type. Its core is to reset the gene expression pattern of the cell through epigenetic modification and restore the differentiation potential. In the embodiment of the present disclosure, the form involved in reprogramming is induced pluripotent stem cell (iPS) technology. In addition, the pathways or forms of reprogramming also include, for example, somatic cell nuclear transplantation (SCNT), that is, transplanting somatic cell nuclei into enucleated oocytes, reprogramming into omnipotent cells (such as cloned animals "Dolly the sheep"); transdifferentiation, that is, directly converting one differentiated cell to another without going through a pluripotent state (such as directly converting fibroblasts into neurons), etc.

[0052] The term "induced pluripotent stem cells (iPS)" in this article is a way of cell reprogramming. It is a reprogramming technology that converts somatic cells (such as fibroblasts) into pluripotent stem cells with the characteristics of embryonic stem cells (ESC). iPS cells can self-renew and differentiate into almost all types of somatic cells.

[0053] The term "vector" as used herein has a general meaning, which is capable of introducing the nucleic acid into a prokaryotic and / or eukaryotic host cell. In certain embodiments, the vector may be a linear vector or a circular vector. It may be a non-viral vector such as a plasmid, a viral vector, or a vector utilizing a transposon. The vector may contain regulatory sequences such as a promoter and a terminator, as well as marker sequences such as drug-resistant genes and reporter genes. In addition, the above-mentioned vector may also contain a sequence encoding a suicide gene, and a substance that activates the suicide gene may be administered according to the course of treatment. As the above-mentioned viral vector, it may 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 vector in the form of a plasmid.

[0054] The term "Yamanaka factors" in this article generally refers to a combination of four transcription factors first screened by Shinya Yamanaka for reprogramming fibroblasts into iPSCs. These four transcription factors are OCT4 gene, SOX2 gene, KLF4 gene and c-MYC gene (see Yamanaka, 2009, Cell 137:13-17).

[0055] The term "expression cassette" in this article usually refers to an artificially constructed functional DNA sequence module used to drive the expression of a specific gene in a host cell. It is generally composed of three core elements: a promoter (such as CMV or EF1α promoter, which controls the start of transcription), a coding sequence of the target gene (such as an open reading frame of a protein), and a terminator (which marks the end of transcription). Enhancers, regulatory sequences, or marker genes can also be added as needed.

[0056] The term "operably linked" or "operably connected" herein refers to polynucleotide sequence elements being connected in a functional relationship. It is intended to consider their function after connection, without considering how the elements are connected. For example, when a polynucleotide sequence is in a functional relationship with another polynucleotide sequence, the polynucleotide sequence is operably linked. In some embodiments, if a transcriptional regulatory polynucleotide sequence (e.g., a promoter, enhancer or other expression control element) affects the transcription of a polynucleotide sequence encoding a protein, the transcriptional regulatory polynucleotide sequence is operably linked to a polynucleotide sequence encoding the protein. Operably linked elements can be continuous or non-continuous.

[0057] The term "reprogramming induction" herein refers to the step of initiating reprogramming, which is to place the transduced cells in pluripotency culture conditions, the culture medium contains specific growth factors (such as LIF, bFGF), and uses feeder cells (such as mouse embryonic fibroblasts) or matrix gel to provide support. After that, the cells gradually lose their original characteristics and form clones similar to embryonic stem cells (about 2 to 4 weeks). In some embodiments, the second gene transcription regulatory element can regulate the downregulation of the expression of the KLF4 gene from the time of introduction into the cell to the time before the reprogramming induction step.

[0058] Chemical induction technology has been used in combination 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 and activate pluripotency genes (such as Oct4) by inhibiting histone deacetylase (HDAC); while 5-azacytidine (5-Azacytidine) can promote the initiation of reprogramming by inhibiting DNA methyltransferase, relieving gene silencing. In terms of signaling pathway regulators, CHIR99021, as a GSK-3β inhibitor, can activate the Wnt / β-catenin pathway and cooperate with Yamanaka factors to enhance the expression of pluripotency genes; SB431542 inhibits epithelial-mesenchymal transition (EMT) by blocking the TGF-β signaling 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 effects, but also activate histone demethylases to further reshape epigenetic characteristics; and PS48 provides energy support for cells by enhancing glycolysis metabolism.

[0059] Reprogramming plasmid / virus system in the disclosed embodiments:

[0060] 1. BOSM single plasmid system.

[0061] The BOSM single-plasmid system integrates key reprogramming factors (OCT4, SOX2, MYC, KLF4) with auxiliary elements (BCL-XL) and regulatory elements (miRNA) into the same plasmid, which simplifies the operation process, reduces costs, and reduces the risk of exogenous plasmid residues. It is especially suitable for clinical-grade iPSC preparation. Compared with the multi-plasmid system, the BOSM single-plasmid system improves the reliability and stability of iPSC cloning while ensuring high transfection efficiency.

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

[0063] If researchers or production processes need to flexibly regulate the expression levels of different factors, the reprogramming factors can be split into 2~3 vectors, for example, one vector carries only 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 promoter, splicing peptide and regulatory sequence of each vector can be optimized independently, thereby obtaining higher expression controllability or more flexible to meet different experimental needs.

[0064] The dual-plasmid system uses two plasmids to carry different reprogramming factors and auxiliary factors to optimize the factor expression ratio, improve reprogramming efficiency and enhance cell survival. Experiments have shown that this system can obtain a large number of iPSC clones and has high flexibility, making it 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 method of reprogramming factors, distributing OCT4, SOX2, MYC, BCL-XL and KLF4 to three plasmids to precisely control gene expression levels. This system optimizes the cell reprogramming process, improves transfection efficiency and the pluripotency stability of iPSCs, and is suitable for applications that require fine gene regulation.

[0067] Example:

[0068] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0069] This embodiment discloses an improved multi-factor reprogramming vector structure and use method based on the technical concept of the present invention, by highly expressing key factors such as BCL-XL, OCT4, SOX2, MYC (driven by SFFV promoter) in hematopoietic cells, and using EF1α (or PGK, CMV, CAG, etc.) to drive KLF4, and adding a double copy target sequence of miR-142-3p (142-3pT (2c)) behind the KLF4 gene, thereby effectively reducing the expression level of KLF4 in the hematopoietic cell stage, and achieving a higher level of KLF4 expression after the cells enter partial reprogramming and gradually lose a large amount of miRNA142-3p expression. This strategy not only solves the problem of KLF4 overexpression inhibiting cell proliferation, but also significantly improves the efficiency and quality of hematopoietic cell reprogramming into iPS.

[0070] The carrier structure and components include:

[0071] (1) Highly efficient reprogramming factor region:

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

[0073] (2) KLF4 expression regulatory region:

[0074] The KLF4 gene is driven by the EF1α (or PGK, CMV, CAG, etc.) promoter, followed by a polyA signal and a double copy target sequence 142-3pT of miRNA142-3p (2c).

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

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

[0077] At the same time, this embodiment discloses a single plasmid reprogramming vector system based on the technical concept of the present invention. The system integrates multiple key reprogramming factors (OCT4, SOX2, KLF4, c-MYC), anti-apoptotic genes (BCL-XL) and regulatory factors (such as miRNA), which significantly simplifies the traditional iPSC reprogramming process. The traditional Episomal Vector system usually requires multiple plasmids to be co-transfected, resulting in an unbalanced copy number, affecting the coordinated expression of reprogramming factors and the reprogramming efficiency. The present invention uses a single plasmid system to ensure that all key factors are synchronously and evenly expressed in the cell, effectively solving the problem of unbalanced plasmid copy number, so that cells can efficiently complete reprogramming after one transfection, greatly improving the repeatability of the experiment and the transfection success rate. In addition, the introduction of BCL-XL enhances cell survival and optimizes reprogramming efficiency. MiRNA regulatory factors (such as miRNA 142-3pT (2c)) can accurately regulate the expression level of KLF4, avoid the safety hazards caused by its overexpression, further reduce cell stress response, and improve the stability of the reprogramming process. This system not only improves the induction efficiency of iPSCs, but also reduces the risk of exogenous plasmid residues and ensures their genetic stability, thus providing a safer and more efficient solution for clinical-grade iPSC preparation.

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

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

[0080] Blood samples come from healthy donors or target patients. Fresh peripheral blood collected must be anticoagulated (EDTA or heparin anticoagulation), and the routine blood collection volume is 20~50mL. The collection and use of all blood samples must comply with ethical requirements. The research plan must be approved by the ethics committee, and before collection, it must be ensured that the donor has a detailed understanding of the research purpose, operation procedures and possible impacts, and voluntarily signs an informed consent form. The collection, transportation and storage of samples must follow biosafety regulations to ensure the repeatability of the experiment and the reliability of the data.

[0081] Blood collection is performed under sterile conditions using 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 separated within 2 to 4 hours to maintain cell activity and experimental repeatability. Avoid violent shaking during transportation and temporary storage, and follow standard biosafety procedures to ensure sample stability and reliability.

[0082] 2. Isolation of PBMNCs.

[0083] PBMNCs were separated by density gradient centrifugation to obtain highly pure mononuclear cells. First, peripheral blood was mixed with PBS in a 1:1 ratio, and then slowly layered onto the pre-prepared Ficoll-Hypaque (1.077 g / mL) separation solution after thorough mixing, ensuring a clear interface between the layers and avoiding liquid mixing. Subsequently, the cells were centrifuged at 400 × g for 30 minutes (room temperature), and distinct layers were formed after the centrifugation: the upper layer was plasma, the middle buffy coat layer was PBMNCs, and the lower layer was Ficoll solution and red blood cells. The buffy coat layer (PBMNCs) was carefully aspirated with a pipette or a pipette, transferred to a new centrifuge tube, and washed 1 to 2 times with PBS (300 × g, 10 minutes) to remove platelets and Ficoll residues to improve cell purity.

[0084] After separation, the viability of PBMNCs was assessed by trypan blue staining, and the cells were counted using a hemocytometer or an automated cell counter. Finally, the cell concentration was adjusted to the required range (1–5 × 10 6 cells / mL) to ensure the stability and repeatability of subsequent experiments.

[0085] 3. Initial expansion / pre-culture of PBMNCs.

[0086] To improve cell survival and optimize reprogramming effects, PBMNCs are usually preliminarily expanded under specific culture conditions. The culture medium can be erythroid culture medium, such as Stem-line II Hematopoietic Stem Cell Expansion Medium (Sigma, S0192), and key cytokines are added, including 100 ng / mL stem cell factor (SCF), 10 ng / mL interleukin-3 (IL-3), 2U / mL erythropoietin (EPO), 20 ng / mL insulin-like growth factor-1 (IGF-1), 1mM dexamethasone, and 0.2mM 1-thioglycerol. Cell culture is carried out in an incubator at 37°C, 5% CO2, and 95% relative humidity, usually pre-cultured for 6 days to promote monocyte activation and improve subsequent transfection efficiency.

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

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

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

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

[0091] The reprogramming vector in this embodiment uses pCEP4 or other backbones containing OriP / EBNA1 sequences to ensure non-integrated replication of the plasmid in eukaryotic cells, and is gradually diluted during cell division to avoid safety risks caused by genomic integration. Reprogramming factors (OCT4, SOX2, KLF4, c-MYC, BCL-XL) and possible miRNA structures (such as miRNA 142-3pT (2c)) are integrated into the same open reading frame and connected by P2A, E2A or T2A self-cleavage peptides to achieve synchronous expression of multiple proteins and ensure stable co-expression of reprogramming factors. Transcription of the target gene is driven by strong promoters such as SFFV, CMV or EF1α to enhance expression efficiency. In addition, 3' regulatory elements such as PolyA tail signals and WPRE are introduced to improve the stability and transcription efficiency of mRNA, thereby optimizing the success rate and safety of cell reprogramming. The single plasmid template sequence is as follows: SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-ployA-EF1a-KLF4-polyA-142-3pT (2c). Its sequence is shown in SEQ ID No.1.

[0092] 2. Plasmid preparation.

[0093] After the small-scale test confirms the successful construction, large-scale plasmid preparation is performed to obtain high-purity, low-endotoxin plasmid DNA. Use a special endotoxin removal kit (such as Zymo Midi Kit) for extraction to ensure that it meets the needs of experimental or clinical applications. During the preparation process, 50~200μL of bacterial solution is inoculated into 50mL of culture medium containing the corresponding antibiotics, shaken and cultured for 16~18 hours, and part of the bacterial solution is retained for glycerol bacterial sample preparation for long-term storage. After the bacterial solution is cultured, the bacterial precipitate is collected, and P1, P2, and P3 solutions are added in sequence to lyse the cells, and the cell fragments and chromosomal DNA are removed by centrifugation, and then Binding Buffer is added for filtration. The plasmid DNA is collected by affinity column, washed with Wash1 and Wash2 solutions in sequence, and finally eluted with eluent to remove endotoxins.

[0094] The concentration and purity of the extracted plasmid were determined by spectrophotometer ( , ), ensure that its quality meets the experimental requirements. The target concentration is usually 1000-1400ng / μL. If the concentration is too high, it can be diluted appropriately. The correctness of the plasmid sequence is verified by restriction 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 a long time to maintain its stability and ensure the repeatability of subsequent experiments.

[0095] Example 3: Nucleofection and reprogramming induction.

[0096] The experimental flow chart is as follows Figure 1 shown.

[0097] 1. Optimization of electroporation conditions.

[0098] Use a commercial electroporator (such as Lonza, Neon, or other brands) with preset programs and the corresponding cell transfection buffer to improve transfection efficiency and cell survival. If the device does not have a preset program, the electroporation parameters, including voltage, pulse time, and number of pulses, can be optimized according to the cell type to ensure high transfection efficiency while maintaining cell viability to the greatest extent.

[0099] 2. Electroporation step.

[0100] (1) PBMNCs were isolated from the peripheral blood of healthy donors and purified by Ficoll-Hypaque density gradient centrifugation. They were then pre-cultured in erythroid culture medium for 6 days, and stem cell factor (SCF) and interleukin (IL-3) were added to the culture medium to improve cell viability and transfection response.

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

[0102] (3) Add an appropriate amount of single plasmid DNA (e.g. 5-10 µg) and mix gently to ensure that the DNA is evenly distributed.

[0103] (4) Transfer the mixture to an electroporation tube or microwell and process according to the optimized electroporation program.

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

[0105] 3. Inoculation and reprogramming culture.

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

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

[0108] (2) Reprogramming induction.

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

[0110] b) From the second day onwards: Change to iPSC induction medium (such as KnockOut DMEM / F12, supplemented with fibroblast growth factor 2 (FGF2), ITS, ascorbic acid, etc.) to officially start the reprogramming process.

[0111] c) Continuous culture: Replace the reprogramming medium with fresh one to two days and add ROCK inhibitor (Y-27632) as appropriate according to the cell status to improve cell survival rate and promote clone formation.

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

[0113] 8-10 days after transfection, iPSC clones began to appear in the culture dish, and about 14 days later, the clones reached a state suitable for selection. The statistical results of the number of iPSC clones are shown in Figure 2. Figure 3 The characteristic clone morphology of iPSCs on day 14 selected from two vector combinations (OS+B+M+K and BOSM.Kmir) is shown in Figure 2. Figure 4 As shown. Morphological observations showed that after reprogramming using the single plasmid vector system of the present invention, typical iPSC clones can be observed about 8 to 10 days after transfection, and the clone size is sufficient for selection around 14 days. The iPSC clones formed by reprogramming present a typical "paving stone"-like structure, with cells arranged tightly, regular morphology, large nuclei occupying the main volume, clearly visible nucleoli, and clear overall boundaries. These morphological characteristics are consistent with the typical morphology of pluripotent stem cells, indicating that iPSCs were successfully established and are in a stable pluripotent state. Typical iPSC clones are tightly arranged, with large nuclei, clear boundaries between cells, regular morphology and smooth surface.

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

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

[0116] 1. Passaging and cryopreservation of iPSCs.

[0117] (1) Cell subculture.

[0118] During cell passaging, an appropriate digestion method should be selected to maintain cell activity and proliferation ability. Common methods include using enzymatic solutions (such as Accutase) or EDTA to dissociate cells into small clusters or single cells. The digested cells need to be transferred to a culture plate pre-coated with Matrigel or a feeder layer in a timely manner, and fresh culture medium should be replaced every 24 hours to maintain a suitable growth environment. Depending on the growth of cell clones, passaging is generally performed every 3 to 5 days to ensure that the cells maintain a good proliferation state and avoid over-intensive culture that affects reprogramming efficiency.

[0119] (2) Cell cryopreservation.

[0120] Cells can be cryopreserved after 2-3 passages to preserve their proliferation ability and biological characteristics. After collecting the cells, resuspend them in a cryopreservation solution containing 10% DMSO + 90% FBS (or serum substitute) to ensure that the cells survive in a low-temperature environment. Subsequently, the cells are slowly cooled to -80°C using a step-by-step cooling method, and then transferred to liquid nitrogen for long-term storage after 24 hours to minimize the damage to cell activity during the freezing process.

[0121] 2. Detection of pluripotency markers: iPSCs are collected and single-cell suspensions are prepared to ensure uniform distribution of cells. Cells are stained with fluorescently labeled antibodies (such as TRA-1-60, TRA-1-81, SSEA4) to detect their pluripotency characteristics. Subsequently, the positive expression ratio of the markers is analyzed by flow cytometry. It is usually expected that the proportion of positive cells exceeds 80-90% to confirm the quality and pluripotency status of iPSCs. The results are as follows: Figure 5 As shown in A and B. The results showed that after flow cytometry analysis, the iPSCs obtained by reprogramming showed that the positive expression rate of their pluripotency cell surface markers (TRA-1-60, TRA-1-81, SSEA4) exceeded 90%, which was comparable to the iPSCs generated by the traditional multi-plasmid reprogramming method, indicating that the single-plasmid system can efficiently induce the pluripotency state. In addition, the results of immunofluorescence detection showed that core pluripotency transcription factors such as OCT4, SOX2, and NANOG in iPSCs were expressed at high levels and localized in the cell nucleus, further verifying the pluripotency of the reprogrammed cells. These results show that the single-plasmid vector system of the present invention can effectively generate iPSCs with stable pluripotency characteristics, providing a safer and more efficient reprogramming strategy for clinical applications.

[0122] 4. Functional testing.

[0123] (1) Differentiation of the three germ layers in vitro.

[0124] To verify the pluripotency of iPSCs, embryoid bodies (EBs) can be used to induce differentiation into cells derived from the three germ layers. Under specific induction conditions, iPSCs can differentiate into ectoderm (such as neural cells), mesoderm (such as cardiomyocytes, skeletal muscle cells), and endoderm (such as hepatocytes, 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 cells and confirm their pluripotency characteristics.

[0125] Through in vitro differentiation experiments, it was verified whether the iPSCs obtained by reprogramming with a single plasmid system were pluripotent. These iPSCs can be successfully induced to differentiate into three germ layer-derived cells, including ectoderm (neurons), mesoderm (cardiomyocytes) and endoderm (hepatocytes), and their differentiation ability is comparable to that of iPSCs obtained by embryonic stem cells (ESCs) and traditional reprogramming methods. In addition, in the Teratoma Formation Assay, after iPSCs were injected into immunodeficient mice, tissue structures derived from the three germ layers could be observed, further confirming their pluripotency. These results show that the single plasmid vector system of the present invention can efficiently generate fully 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 tissues derived from the three germ layers in vivo. After the tumor is formed, different tissue types are analyzed by histopathology and specific markers are detected to confirm whether they are derived from the ectoderm, mesoderm, and endoderm, thereby proving the pluripotency of iPSCs. The results are as follows Figure 6 The results showed that the obtained tumors were derived from ectoderm, mesoderm and endoderm.

[0128] 5. Quality control and precautions.

[0129] Quality control runs through the entire process of cell reprogramming to ensure the repeatability of the experiment and compliance with GMP standards. First, the electrotransfection efficiency and cell viability should be evaluated regularly, and the electrotransfection parameters should be optimized according to the experimental requirements to improve the success rate of reprogramming. Secondly, aseptic operation should be strictly implemented 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 to record in detail the key information of each operation, cell passaging, freezing and thawing, ensure data traceability, meet GMP quality management requirements, and provide a reliable basis for subsequent experiments and clinical applications.

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

[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 the experimental group and the control group. P value < 0.05 was considered significant difference; p < 0.05; p < 0.01; p < 0.001.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A reprogramming vector for blood cells, characterized in that: The reprogramming vector comprises: (1) OCT4 gene, SOX2 gene, KLF4 gene and c-MYC gene; (2) a first expression frame, wherein the first expression frame comprises an operably linked first gene transcription regulatory element; and (3) a second expression frame, wherein the second expression frame comprises an operably linked second gene transcription regulatory element; Among them, the first gene transcription regulatory element is used to regulate the expression of the OCT4 gene, SOX2 gene and 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 includes a regulatory element that inhibits the expression of the KLF4 gene before reprogramming induction.

2. The reprogramming vector according to claim 1, characterized in that The first gene transcription regulatory element or the second gene transcription regulatory element comprises a promoter, an enhancer or a silencer.

3. The reprogramming vector according to claim 2, characterized in that The promoter is selected from SFFV, CMV, EF1α, Sox2, Nanog, RUNX1, GATA2, and PU.

1.

4. The reprogramming vector according to claim 2, characterized in that The enhancer is selected from WPRE, EBNA1, SV40, CMV, and CAG.

5. The reprogramming vector according to claim 1, characterized in that The regulatory element that inhibits the expression of the KLF4 gene before reprogramming induction is a miRNA regulatory element.

6. The reprogramming vector according to claim 5, characterized in that The miRNA regulatory element is at least one selected from the group consisting of 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, and miR-142-3pT (2c).

7. The reprogramming vector according to claim 1, characterized in that 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.

8. The reprogramming vector according to claim 7, characterized in that The anti-apoptotic gene is at least one selected from the group consisting of BCL-2 family, IAP family, FLIP, Akt / PKB, HSPs, NF-κB, ARC, DAD1, and SODD.

9. The reprogramming vector according to claim 1, 7 or 8, characterized in that The gene sequences in the first expression frame also contain sequences encoding self-cleavage peptides.

10. The reprogramming vector according to claim 9, characterized in that The self-cleaving peptide is at least one selected from the group consisting of 2A peptide family, intein, and Sortase A.

11. The reprogramming vector according to claim 1, characterized in that The reprogramming vector is SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-EF1s-KLF4-polyA-142-3pT (2c).

12. A product for blood cell reprogramming, characterized in that: The product is a plasmid or a virus, wherein the plasmid or the virus comprises an OriP / EBNA1 replication system and a reprogramming vector according to any one of claims 1 to 11.

13. The product according to claim 12, characterized in that The virus is a lentivirus, an adenovirus, an adeno-associated virus or a retrovirus, and the plasmid is 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 EO2S ET2K, epiCRISPR, pEB-TRE-Cas9, pOriP-Hygro, pEB-Multi, pEB-CAG-GOI-IRES-Hygro, HEK293-EBNA, CHO-EBNA or pCMV-EBNA1.

14. A plasmid or virus composition for blood cell reprogramming, characterized in that: The composition includes plasmid or virus A and plasmid or virus B; Wherein, the plasmid or virus A comprises the reprogramming vector according to claim 7 or 8, wherein the OCT4 gene, SOX2 gene and c-MYC gene in the reprogramming vector are located downstream of the anti-apoptotic gene, The plasmid or virus B comprises the reprogramming vector according to claim 7 or 8, wherein the OCT4 gene, SOX2 gene and c-MYC gene in the reprogramming vector are located upstream of the anti-apoptotic gene.

15. A plasmid or virus composition for blood cell reprogramming, characterized in that: The composition includes plasmid or virus D, plasmid or virus E and plasmid or virus F; Wherein, the plasmid or virus D comprises the first expression cassette as described in the reprogramming vector of claim 1, The plasmid or virus E comprises the anti-apoptotic gene and the first gene transcription regulatory element as described in the reprogramming vector of claim 7 or 8, The plasmid or virus F comprises the second expression cassette as described in the reprogramming vector of claim 1.

16. A method for reprogramming blood cells into induced pluripotent stem cells (iPSCs), characterized in that: include: Step 1) preparing the product as claimed in claims 12 and 13, or the plasmid or virus composition as claimed in claims 14 and 15; Step 2) introducing the product obtained in step 1) into the blood cells; Step 3) reprogramming and inducing the blood cells obtained in step 2) to obtain iPSC clones; Step 4) Screening the clones obtained in step 3).

17. The method according to claim 16, characterized in that The blood cells are peripheral blood mononuclear cells (PBMNCs).

18. Use of the reprogramming vector according to any one of claims 1 to 11, the product according to claims 12 and 13, or the plasmid or virus composition according to claims 14 and 15 in preparing induced pluripotent stem cells.

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