Method for promoting rejuvenation of vascular endothelial cells and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a method for promoting rejuvenation of vascular endothelial cells and application of the method. The invention provides a method for promoting rejuvenation of vascular endothelial cells. Specific circular RNA molecules with the purity exceeding 85% can be obtained. Through effective expression of the three key reprogramming factors, while the identity characteristics of the cells are maintained, accurate induction of rejuvenation of the vascular endothelial cells is successfully realized, and after transfection of the human vascular endothelial cells, the senescence phenotype of the vascular endothelial cells can be improved, specifically, SA-beta-Gal staining is reduced, and expression of p16 and gammaH2A.X is reduced.

Description

Technical Field

[0001] The present application belongs to the field of bioengineering technology, and specifically relates to a method for promoting rejuvenation of vascular endothelial cells and its application. Background Art

[0002] Vascular aging is one of the key manifestations of physiological system degeneration and is a complex biological process involving multi-level structural and functional changes in the vascular system. At the molecular and cellular levels, vascular aging is mainly manifested as a progressive imbalance and dysfunction of endothelial cell homeostasis. This change is not only a passive result of aging, but also an active factor that induces chronic vascular diseases.

[0003] Epigenetic changes associated with aging gradually destroy the normal gene regulatory network of vascular endothelial cells, leading to the gradual degeneration of cell function. This degeneration process is specifically manifested in decreased cell proliferation, increased oxidative stress, enhanced inflammatory response, and weakened vascular repair ability. These changes jointly promote the continuous deterioration of vascular endothelial function and eventually become an independent risk factor for cardiovascular disease.

[0004] Atherosclerosis, as a chronic cardiovascular disease (CVD), poses a major risk to human health and is the root cause of peripheral vascular disease, coronary heart disease and stroke. The pathogenesis of atherosclerosis is complex and is a complex pathological process involving multiple cell types. Endothelial dysfunction is considered to be the first step of atherosclerosis, covering a series of non-adaptive changes in function and phenotype, and has an important impact on regulating hemostasis and thrombosis, balancing local vascular tension, coordinating redox balance, and acute and chronic inflammatory responses in the arterial wall.

[0005] Vascular endothelial cells (VECs) are single-layer flat epithelial cells lining the inner surface of heart, blood vessels and lymphatic vessels. They constitute the inner wall of blood vessels and serve as the interface between blood in the vascular cavity and other parts of the vascular wall. Vascular endothelial cells are widely distributed in arteries, veins, microvessels and lymphatic vessels in various tissues and organs of the human body. They play a key role in maintaining the metabolic exchange of blood and tissue fluid, regulating vascular tension, promoting angiogenesis and hemostasis. In addition, endothelial cells also provide an anti-oxidant, anti-inflammatory and anti-thrombotic interface, which is essential for maintaining the normal physiological functions of the body.

[0006] As a key risk factor for endothelial dysfunction and CVD, the senescence of VECs marks the beginning of a series of diseases that lead to CVD and other endothelial dysfunction-related diseases. The characteristics of VECs cell senescence include reduced nitric oxide production, damage and abnormality of barrier function, and reduced endothelial coverage caused by the disintegration of intercellular adhesion junctions and tight junctions. In addition to oxidative stress and chronic inflammation in the vascular endothelium, which are involved in the process of endothelial cell senescence, excessive stressors may trigger VECs cell senescence, including inflammatory molecules and chemotherapeutic drugs. These related factors not only affect the biological functions of endothelial cells, but also promote the development of CVD. Senescent VECs cells also enhance the ability to attract leukocytes by upregulating the expression of adhesion molecules such as VCAM-1 and ICAM-1, thereby exacerbating the chronic inflammatory environment of the atherosclerotic wall.

[0007] At present, vascular aging has been recognized as a key driving mechanism of cardiovascular disease and organ decline, and its importance is no less than traditional risk factors such as blood lipids and hypertension. By effectively intervening in the aging process of vascular endothelial cells, it is expected to fundamentally delay and improve the aging process of the cardiovascular system.

[0008] Currently, there are no reports on epigenetic reprogramming-based methods for promoting the rejuvenation of vascular endothelial cells. Summary of the invention

[0009] Based on this, an embodiment of the present application provides a method for promoting rejuvenation of vascular endothelial cells and its application.

[0010] On the one hand, the present application provides a method for promoting rejuvenation of vascular endothelial cells, which adopts a phase-separation-free engineered reprogramming factor approach to prepare a phase-separation-free transcription factor.

[0011] The phase-separation-free transcription factor is introduced into the target cells to perform initial stage reprogramming on the target cells.

[0012] The method of engineering reprogramming factors without phase separation includes mutating the regions related to the phase separation function of the transcription factors so that the transcription factors lack the function of inducing phase separation, and the target cells include vascular endothelial cells.

[0013] In some of these embodiments, the region associated with the phase separation function of the transcription factor includes the IDR region.

[0014] In some embodiments, the mutation treatment includes mutating the polar charged amino acids or acidic amino acids in the IDR region.

[0015] In some of these embodiments, the polar charged amino acid or the acidic amino acid is replaced with a neutral amino acid.

[0016] In some of these embodiments, the acidic amino acids are mutated to neutral amino acids.

[0017] In some of the embodiments, the amino acid sequence shown in SEQ ID NO.1 is mutated at positions 1 to 140 and / or positions 288 to 360.

[0018] In some of the embodiments, positions 1 to 140 and / or positions 288 to 360 of the amino acid sequence further include 10 to 20 amino acids before and after.

[0019] In one embodiment, the method comprises: mutating at least one of positions 8, 20, 31, 108, 138, 291, 297, 26, 56, 68, 91, 96, 98, 104, 113, 125, 127, 130, 134, 135, 296, 299, 341, 343, 145, 147, 166, 188, 209, 210, 215, 224, 238, 246, 270 and 272 of the amino acid sequence shown in SEQ ID NO.1.

[0020] In some embodiments, the amino acid sequence of the phase-separation-free transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.2.

[0021] In some embodiments, before the phase-separation-free transcription factor is introduced into cells, the step of preparing the phase-separation-free transcription factor into an element comprising the phase-separation-free transcription factor is further included.

[0022] In some embodiments, the elements include: one or more of DNA, RNA, a recombinant vector, a recombinant protein, and a recombinant cell.

[0023] In some embodiments, the recombinant vector comprises one or more of an adeno-associated virus vector, a poxvirus vector, and a lentivirus vector.

[0024] In some embodiments, the RNA comprises one or more of linear mRNA, siRNA, circular RNA, circular mRNA and tRNA.

[0025] In some embodiments, the circular RNA comprises a plurality of transcription factors connected in series.

[0026] In some embodiments, the transcription factors are linked via a linker peptide.

[0027] In some of the embodiments, the nucleotide sequence of the connecting peptide is shown as SEQ ID NO.3 to SEQ ID NO.4.

[0028] In some embodiments, the element comprises one or more of Oct4, Sox2, Klf4, c-Myc, L-Myc, Lin28, Nanog and Glis1.

[0029] In some embodiments, the nucleotide sequence of the element is as shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.8.

[0030] In some embodiments, introducing the phase separation-free transcription factor into the target cell comprises using a delivery system;

[0031] In some embodiments, the delivery system includes one or more of liposome LNP, exosome delivery, GalNAc system, VLP delivery, protamine, high molecular polymer, inorganic nanoparticles, exosomes, polymer matrix and viral transfection.

[0032] On the other hand, the present application provides the use of a phase-separation-free transcription factor or an element comprising a phase-separation-free transcription factor in the preparation of a drug that promotes rejuvenation of vascular endothelial cells;

[0033] The amino acid sequence of the phase-separation-free transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.2.

[0034] In some embodiments, the nucleotide sequence of the element containing the phase-separation-free transcription factor is as shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.8.

[0035] In some embodiments, the drug includes a drug for cell therapy, gene therapy, immune cell proliferation, cancer treatment, atherosclerosis or aging-related diseases.

[0036] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0038] Figure 1 is the order of the components of O'SK circular RNA (FlexCirc system);

[0039] Figure 2 This is the result of in vitro transcription and circularization verification;

[0040] Figure 3 The results of HPLC purification and testing;

[0041] Figure 4 Validate cell-level results for circular RNA protein expression;

[0042] Figure 5 Characterization of the phase separation of Oct4 transcription factor Static phase separation observation results;

[0043] Figure 6 Characterization of the phase separation of Oct4 transcription factor Dynamic phase separation observation results;

[0044] Figure 7 To verify the expression level of SA-β-Gal in HUVEC cells after transfection by β-galactosidase staining;

[0045] Figure 8 To verify the expression levels of p16 and γ-H2AX in HUVEC cells after transfection by Western Blot;

[0046] Fig. 9 Immunofluorescence was used to detect the expression level of γ-H2AX;

[0047] Fig.10 ELISA was used to detect the expression level of IL-6 in transfected HUVEC cells. DETAILED DESCRIPTION

[0048] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0050] the term

[0051] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0052] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0053] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0054] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0055] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0056] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0057] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0058] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0059] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0060] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0061] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0062] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0063] The terms "identity" and "homology" as used in this application refer to the use of amino acid sequences or nucleotide sequences. Those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9% similarity and still have the same function as the original amino acid sequence or nucleotide sequence.

[0064] The term "variant" used in connection with a peptide or polypeptide refers to a peptide or polypeptide whose amino acid sequence differs due to insertion, deletion or conservative substitution of amino acids but retains at least one biological activity. A variant may also refer to a protein having an amino acid sequence substantially identical to a reference protein, wherein the reference protein has an amino acid sequence retaining at least one biological activity. Conservative substitution of amino acids, i.e., replacing amino acids with different amino acids having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered to involve minor changes in the art. As understood in the art, these minor changes can be identified in part by considering the hydropathic index of amino acids. (Kyte et al., 1982, J. Mol. Biology. 157: 105-132). The hydropathic index of amino acids is based on considerations of their hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. On the one hand, amino acids have a hydropathic index of ±2 and are replaced. The hydrophilicity of amino acids can also be used to reveal substitutions that result in proteins retaining biological function. Considering the hydrophilicity of amino acids in the context of peptides allows calculation of the maximum local average hydrophilicity of the peptide, which is a useful measurement reported to be closely related to antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Amino acids with hydrophilicity values ​​that differ from each other by ±2 can be substituted. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of the amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, especially the side chains of those amino acids, such as hydrophobicity, hydrophilicity, charge, size and other properties.

[0065] As used herein, the term "variant" with respect to nucleic acids refers to (i) a portion or fragment of a reference nucleotide sequence; (ii) a complementary sequence of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complementary sequence; (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complementary sequence, or a sequence substantially identical thereto under stringent conditions. A variant may be a nucleic acid sequence that is substantially identical over the entire length of a gene sequence or a fragment thereof. A nucleic acid, protein sequence may be 66%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the entire length of a gene, protein sequence, or a fragment thereof.

[0066] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in MolecμLar Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altsch μL et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altsch μL et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altsch μL et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0067] The term "epigenetic reprogramming" refers to the process of changing cell fate through epigenetic modifications such as DNA methylation without changing the gene sequence. Epigenetic reprogramming is a key biological process that involves the erasure and re-establishment of epigenetic marks across the genome. The term "reprogramming factor" has the ability to regulate the epigenetic properties of cells, restore cells to early differentiation or even undifferentiated states, and can convert adult or differentiated cells into pluripotent stem cells. Specifically, reprogramming factors are factors from the Oct family, Sox family, Klf family, Myc family, Nanog family, Glis family or Lin family. The "Yamanaka factors" include but are not limited to a combination of four transcription factor genes, namely Oct-4, Sox4, Klf4 and c-Myc (collectively referred to as OSKM genes). In some aspects, they may also be Oct4, Sox-2 and Klf4 genes (OSK genes). In some aspects, they may also be Oct4, Klf4 and c-Myc genes (OKM genes). In some aspects, they may also be Sox-2, Klf4 and c-Myc genes (OKMS genes).

[0068] Initiation phase reprogramming refers to the process of engineering cell reprogramming factors to keep the reprogramming of old cells in the initiation phase, thereby providing a longer safe time window for rejuvenation and greatly reducing the risk of becoming iPSCs. The main difference between initiation phase reprogramming and transient reprogramming is the engineering of cell reprogramming factors, which allows the reprogramming phase to be artificially controlled at the molecular level.

[0069] The term "liquid-liquid phase separation" (LLPS), also known as phase separation, refers to the fact that certain proteins or nucleic acid molecules in cells can produce another phase with different physical and chemical properties in the surrounding originally uniform liquid environment through multivalent interactions, usually in the form of micron-scale droplets (similar to oil droplets in water). These droplets form a relatively independent space, selectively enriching certain protein molecules, making them have higher protein density and weakened molecular microscopic movement, which can promote certain biochemical reaction results, such as gene transcription regulation, changes in chromosome spatial structure, etc. There are many mechanisms for protein phase separation, one of which depends on the intrinsically disordered domain (IDR) of the protein. The acidic polar amino acids in this domain carry negative charges and can interact to form phase separation. "No phase separation" refers to the mutation of the IRD sequence or key amino acids that form phase separation, resulting in the inability of the variant protein to form LLPS, thereby affecting (reducing) its biological function in the cell.

[0070] The term "O'SK" refers to the Oct4mut-Sox2-Klf4 tandem sequence, and OSK refers to the Oct4-Sox2-Klf4 tandem sequence.

[0071] The term "LNP" lipid nanoparticles are nanoparticles composed of ionizable lipids, cholesterol, phospholipids and PEG lipids in a certain proportion. They are often used to encapsulate nucleic acids. Their components are similar to cell membranes and can achieve delivery to specific cells.

[0072] The term "HUVEC (human umbilical vein endothelial cell)" refers to human umbilical vein endothelial cells.

[0073] The term "SA-β-Gal (senescence-associated β-galatosidase)" refers to senescence-associated β-galactosidase.

[0074] The term "SASP (senescence-associated secretory phenotype)" refers to the senescence-associated secretory phenotype.

[0075] Methods for rejuvenating vascular endothelial cells: Induced pluripotent stem cell (iPSC) reprogramming technology is a key method for converting differentiated cells into pluripotent stem cells through transcription factors (such as OCT4, SOX2, KLF4 and MYC, hereinafter referred to as "4 factors"). The entire reprogramming process can be divided into the initiation stage, the maturation stage and the stabilization stage. During this process, cells undergo significant changes in transcription levels and epigenetic modifications, which fundamentally change the cell identity and state.

[0076] In the initial stage, early pluripotency-related genes (such as TRA-1-60, NANOG, SSEA4, and SALL4) are rapidly upregulated, while the expression of somatic cell identity characteristic genes changes little; in the mature stage, in addition to early pluripotency genes, maturity-specific pluripotency genes (such as DPPA5 and LIN28) also begin to be highly expressed, while the expression of somatic cell characteristic genes decreases significantly, indicating that the cells no longer maintain the original somatic cell identity characteristics; in the stable stage, the cells have completely established a pluripotency gene expression network and converted into iPSCs, and in addition to the above-mentioned pluripotency genes, they also highly express core transcription factors such as OCT4, SOX2, and KLF4, while somatic cell identity characteristic genes are basically no longer expressed. In addition, transcriptional changes during the reprogramming process can also promote cell rejuvenation, and the rejuvenation process can be monitored by changes in gene transcription levels, aging markers, and especially epigenetic age (epiAge) based on DNA methylation.

[0077] Partial reprogramming technology refers to the process in which cells undergo rejuvenation changes in the early stages of the complete reprogramming process, while the loss of somatic cell identity characteristics occurs in the middle and late stages. Therefore, there is a time window in this process, during which cells can achieve rejuvenation while maintaining their original somatic cell identity characteristics. This feature provides a technical basis for partial reprogramming to achieve cell rejuvenation. Compared with traditional complete reprogramming, partial reprogramming can effectively restore the youthful functions of cells by briefly activating reprogramming factors without completely changing the identity characteristics of cells. Therefore, partial reprogramming not only has the potential to solve aging-related diseases, but also can promote the repair and regeneration of aging cells without losing the original functions of cells.

[0078] The present application provides a method for producing circular RNA integrated with reprogramming factors. Through the combination of different reprogramming factors, after transfecting human vascular endothelial cells, the cells are still in a differentiated state, which promotes their rejuvenation and restores their functionality, while maintaining the identity characteristics of the cells, providing a safer treatment strategy for the regeneration of aged vascular endothelial cells.

[0079] This application uses engineered transcription factors that remove the ability to phase separate (hereinafter referred to as phase-free transcription factors) to participate in multi-factor partial reprogramming. This application found that phase-free transcription factors cause partial reprogramming to remain in the initial stage, while maintaining cell identity characteristics while rejuvenating cells, so this application refers to this as the initial stage reprogramming technology. This technology greatly reduces the risk of iPSC generation while improving the efficiency of vascular endothelial cell rejuvenation, and is more likely to be used in clinical practice.

[0080] On the one hand, a method for promoting rejuvenation of vascular endothelial cells is provided, in which phase-separation-free engineering reprogramming factors are used to prepare phase-separation-free transcription factors.

[0081] The non-phase separation transcription factors are introduced into the target cells to perform initial stage reprogramming of the target cells.

[0082] The method of engineering reprogramming factors without phase separation includes mutating the relevant regions of the phase separation function of the transcription factor so that the transcription factor lacks the function of inducing phase separation, wherein the target cells include vascular endothelial cells.

[0083] In some of these embodiments, the region associated with the phase separation function of the transcription factor includes the IDR region.

[0084] In some of these embodiments, the region associated with the phase separation function of the transcription factor includes the IDR region.

[0085] In some embodiments, the mutation treatment includes mutating the polar charged amino acid or acidic amino acid in the IDR region;

[0086] In some of these embodiments, polar charged amino acids or acidic amino acids are replaced with neutral amino acids;

[0087] In some of these embodiments, the acidic amino acids are mutated to neutral amino acids.

[0088] In some embodiments, the method comprises mutating positions 1 to 140 and / or positions 288 to 360 of the amino acid sequence shown in SEQ ID NO.1.

[0089] In some of these embodiments, positions 1 to 140 and / or positions 288 to 360 of the amino acid sequence further include 10 to 20 amino acids before and after.

[0090] In some embodiments, the invention comprises: mutating at least one of positions 8, 20, 31, 108, 138, 291, 297, 26, 56, 68, 91, 96, 98, 104, 113, 125, 127, 130, 134, 135, 296, 299, 341, 343, 145, 147, 166, 188, 209, 210, 215, 224, 238, 246, 270 and 272 of the amino acid sequence shown in SEQ ID NO.1.

[0091] In some embodiments, the amino acid sequence of the non-phase separation transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.2.

[0092] The present application performs amino acid modification on a specific site of Oct4 protein to weaken its phase separation ability, including selecting the most important transcription factor, performing amino acid modification on a specific site to weaken its phase separation ability, and converting the amino acid sequence into a DNA sequence that can express circular RNA through codon optimization, adding necessary auxiliary sequences, and preparing circular RNA.

[0093] The amino acid sequence of the Oct4 protein before modification is as shown in SEQ ID NO.1:

[0094] MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEVWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASPEPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN

[0095] The amino acid sequence of the modified Oct4mut protein is as shown in SEQ ID NO.2:

[0096] MAGHLAS A FAFSPPPGGGG A GPGGP A PGWV A PRTWLSFQGPPGGPGIGPGVGPGS A VWGIPPCPPPY A FCGGMAYCGPQVGVGLVPQGGL A TSQP A G A AGVGV A SNS A GASP A PCTVTPGAVKL A K A KL A QNP AA SQ AIKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLF LQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN

[0097] In some of the embodiments, before the phase-separation-free transcription factor is introduced into the cell, the method further comprises the step of preparing the phase-separation-free transcription factor into an element comprising the phase-separation-free transcription factor.

[0098] In some embodiments, the element comprises one or more of DNA, RNA, a recombinant vector, a recombinant protein, and a recombinant cell.

[0099] In some embodiments, the recombinant vector comprises one or more of an adeno-associated virus vector, a poxvirus vector, and a lentivirus vector.

[0100] In some of these embodiments, the RNA includes one or more of linear mRNA, siRNA, circular RNA, circular mRNA, and tRNA.

[0101] In one embodiment, the circular RNA comprises multiple transcription factors in series.

[0102] In some of these embodiments, the transcription factor is linked via a linker peptide;

[0103] In some of the embodiments, the nucleotide sequence of the connecting peptide is shown as SEQ ID NO.3 to SEQ ID NO.4.

[0104] In one embodiment, the element comprises one or more of Oct4, Sox2, Klf4, c-Myc, L-Myc, Lin28, Nanog and Glis1.

[0105] In one embodiment, the nucleotide sequence of the element is as shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.8.

[0106] In one embodiment, a linker sequence is designed, such as P2A and T2A sequences, as shown in SEQ ID NO.3 to SEQ ID NO.4.

[0107]

[0108] Oct4 or Oct4mut is connected to Sox2 and Klf4 through the above elements to form a tandem sequence, which is only an example and can be shown in SEQ ID NO.5 to SEQ ID NO.6, and the names are Oct4-P2A-Sox2-T2A-Klf4 tandem sequence (abbreviated as OSK, corresponding to SEQ ID NO.5) and Oct4mut-P2A-Sox2-T2A-Klf4 tandem sequence (abbreviated as O'SK, corresponding to SEQ ID NO.6).

[0109]

[0110]

[0111] In some of these embodiments, the Oct4mut-P2A-Sox2-T2A-Klf4 tandem sequence further comprises one or more of an internal ribosome entry site of the target fragment, a 5' homology arm, a ribozyme, a spacer sequence, a 3' homology arm, and a regulatory element.

[0112] In some of the embodiments, the nucleotide sequence of the internal ribosome entry site of the target fragment is shown as SEQ ID NO.7, and the internal ribosome entry site can allow the ribosome to directly bind and start translation in the absence of a 5' cap.

[0113] Wherein, the nucleotide sequence of SEQ ID NO.7 is:

[0114] TTTGCTCAGCGTAACTTCTCCGGGTTACGTGGAGACCAAAAGGCTACGGAGACTCGGGCTACGGCCCTGGAGCACCTAGGTGCTCCTAAAGACGTTAGAAGTTGTACAAACTCGCCCAATAGGGCCCCCCAACCAGGGGGGTAGCGGGCAAGCACTTCTGTTTCCCCG GTATGATCTCATAGGCTGTACCCACGGCTGAAAGAGAGATTATCGTTACCCGCCTCACTACTTCGAGAAGCCCAGTAATGGTTCATGAAGTTGATCTCGTTGACCCGGTGTTCCCCCACCAGAAACCTGTGATGGGGGTGGTCATCCCGGTCATGGCGACATGACG GACCTCCCCGCGCCGGCACAGGGCCTCTCGGAGGACGAGTGACATGGATTCAACCGTGAAGAGCCTATTGAGCTAGTGTTGATTCCTCCGCCCCCGTGAATGCGGCTAATCCCAACTCCGGAGCAGGCGGGCCCAAACCAGGGTCTGGCCTGTCGTAACGCGAAAGTC TGGAGCGGAACCGACTACTTTCGGGAAGGCGTGTTTCCTTTTGTTCCTTTTATCAAGTTTTATGGTGACAACTCCTGGTAGACGTTTTATTGCGTTTATTGAGAGATTTCCAACAATTGAACAGACTAGAACCACTTGTTTTTATCAAACCCTCACAGAATAAGATAACA

[0115] In some embodiments, the auxiliary looping fragment includes one or more of: a 5' homology arm, a ribozyme, a spacer sequence, a 3' homology arm, and a regulatory element.

[0116] In some of these embodiments, the spacer sequence includes a 5' spacer sequence and a 3' spacer sequence.

[0117] In some embodiments, the regulatory element includes one or more of substrate E1, IG3, IG5 and a promoter.

[0118] In some embodiments, the Oct4mut-P2A-Sox2-T2A-Klf4 tandem sequence can be a circular sequence, for example, the nucleotide sequence of the circular RNA is shown in SEQ ID NO.8.

[0119]

[0120] On the other hand, the present application provides the use of the circular RNA shown in SEQ ID NO.8 in the preparation of a product that promotes the rejuvenation of vascular endothelial cells.

[0121] On the one hand, the present application provides a nucleic acid molecule, which includes a fragment encoding a circular RNA.

[0122] The nucleic acid molecules of the present application mainly refer to isolated nucleic acid molecules. "Isolated" refers to molecules that are substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of these materials or the absence of water, buffer or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compounds as described herein.

[0123] Another aspect of the present application provides a vector, which includes a nucleic acid molecule.

[0124] The term "vector", which may also be referred to as "nucleic acid construct", refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which another DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can direct the expression of the genes to which they are effectively connected. This vector is referred to herein as a "recombinant expression vector" (or simply "expression vector"). Generally, expression vectors useful in recombinant DNA technology are usually present in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), which play equivalent functions. Engineered mRNA, including linear mRNA and circular mRNA, is a new type of gene vector suitable for non-viral gene delivery systems and can be used for in vivo gene therapy. Circular mRNA is preferred in this application, which has lower immunogenicity and higher stability than linear mRNA, and is suitable for long-term expression of therapeutic proteins in vivo. Therefore, it is increasingly used in gene therapy. As long as the vector can be used for carrying, it is within the scope of protection of this application.

[0125] In some embodiments, the vector comprises an adeno-associated virus or a plasmid.

[0126] On the other hand, the present application provides a cell, which includes the above-mentioned circular RNA, the above-mentioned nucleic acid molecule or the above-mentioned vector. The term "cell" or "host cell" refers to a cell into which an expression vector has been introduced. The host cell may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells.

[0127] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of passages. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, this is clear from the context.

[0128] In some embodiments, introducing the phase separation-free transcription factor into the target cell comprises using a delivery system;

[0129] In some of these embodiments, the delivery system includes one or more of liposome LNP, exosome delivery, GalNAc system, VLP delivery, protamine, high molecular polymer, inorganic nanoparticles, exosomes, polymer matrix and viral transfection.

[0130] On the other hand, the present application provides the use of a phase-separation-free transcription factor or an element comprising a phase-separation-free transcription factor in the preparation of a drug that promotes rejuvenation of vascular endothelial cells.

[0131] The amino acid sequence of the non-phase separation transcription factor is shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.2.

[0132] In some embodiments, the nucleotide sequence comprising the element of the phase separation-free transcription factor is as shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.8.

[0133] In some of these embodiments, the drug includes a drug for cell therapy, gene therapy, immune cell proliferation, cancer treatment, or treatment of aging-related diseases.

[0134] On the other hand, the present application provides a method for preparing cells, which comprises the step of introducing a nucleic acid molecule or a vector into a target cell.

[0135] In one example, the introduction was carried out by lipofectamine transfection.

[0136] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MolecμLar Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methodsin MolecμLar Biology, Elsevier; Chu et al, 1981, Gene 13:197. The introduction method includes, but is not limited to, liposome transfection, microvesicle transfection, exosome transfection, electroporation, nanocarrier transfection, chemical transfection using a transfection reagent; the nanocarrier includes a lipid, a polymer, or a lipid-polymer hybrid.

[0137] The present application provides a delivery system on the other hand for delivering the above-mentioned nucleic acid molecules, recombinant vectors comprising nucleic acid molecules, recombinant DNA molecules, mRNA, recombinant proteins, etc. In some embodiments, lipid nanoparticles or "LNPs" are used to deliver nucleic acids to cells. As described above, LNPs may include natural lipids or synthetic lipids, including conjugated lipids or polymers (e.g., pegylated lipids). LNPs may include any one or more of neutral lipids, zwitterionic lipids, ionizable lipids, cationic lipids, and anionic lipids. In some embodiments, LNPs include phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA) in the form of natural or synthetic monoacyl or diacyl groups, or monoacyl, diacyl, triacyl, or tetraacyl forms of cardiolipin. In some embodiments, LNPs are micelles or reverse micelles (reverse micelles). In other embodiments, LNPs are unilamellar liposomes or multilamellar liposomes.

[0138] It is understandable that the present application also relates to other delivery systems, such as GalNAc conjugated delivery system, liposome LNP nano delivery system (small molecule ligands, antibodies and other molecules) and virus-like particle VLP delivery, protamine, high molecular polymers, inorganic nanoparticles, exosomes, polymer matrices, viral transfection, etc.

[0139] On the other hand, the present application provides the use of the above-mentioned circular RNA, the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned cell in the preparation of a product for promoting the rejuvenation of vascular endothelial cells.

[0140] On the other hand, the present application provides a method for constructing a circular RNA, comprising:

[0141] A tandem sequence of Oct4mut-P2A-Sox2-T2A-Klf4 was constructed to prepare a vector. The template of the vector was linearized, and then an in vitro transcription reaction and a cyclization catalytic reaction were performed. An exoribonuclease was used for digestion reaction to construct a circular RNA.

[0142] This application cleverly integrates three key transcription factors into circular RNA molecules, which significantly reduces the risk of forming induced pluripotent stem cells during cell reprogramming compared to the six-transcription factor combination scheme commonly used in the prior art, effectively avoids the complete dedifferentiation of cell identity, and thus improves the safety and controllability of the reprogramming process at the molecular level.

[0143] Furthermore, this application makes full use of the unique circular structure characteristics of circular RNA, substantially prolongs the molecular stability of transcription factors, and effectively overcomes the inherent limitations of the short biological half-life (about 24 hours) of traditional linear mRNA. By enhancing the sustained expression ability of transcription factors, this technology significantly improves the efficiency and reliability of partial cell reprogramming, bringing an important breakthrough to partial cell reprogramming technology.

[0144] In some embodiments, the method comprises linearizing the template of the vector using restriction endonuclease EcoR I for linearization.

[0145] In some embodiments, the exonuclease described in the above method includes RNase R enzyme. RNase R (Ribonuclease R) is a 3'-5' exoribonuclease derived from the Escherichia coli RNR superfamily. RNase R can cut and degrade RNA from the 3'-5' direction, digesting almost all linear RNA molecules, but it is not easy to digest circular RNA, lasso structure or double-stranded RNA molecules with less than 7 nucleotides at the 3' end protruding end. RNase R is mainly used for the identification and enrichment experiments of circRNA (circular RNA). It enriches circRNA by digesting linear RNA, thereby playing a role in gene expression and variable splicing research.

[0146] On the other hand, the present application provides a method for treating or a method for promoting rejuvenation of vascular endothelial cells, which comprises administering an effective dose of the above-mentioned circular RNA to a subject.

[0147] "Administering," "giving," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0148] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments disclosed herein, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., treatment methods or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0149] "Effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For preventive uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity or delaying the onset of a condition, including the biochemical, histological and / or behavioral symptoms of the condition, its complications and the intermediate pathological phenotypes presented during the development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related conditions of the present application or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of the present disclosure in the patient. Diseases treated include diseases related to aging of vascular endothelial cells, including atherosclerosis.

[0150] This application proposes a method for promoting the rejuvenation of vascular endothelial cells, which can obtain specific circular RNA molecules with a purity of more than 85%. Through the effective expression of three key reprogramming factors, while maintaining the cell identity characteristics, the precise induction of vascular endothelial cell rejuvenation was successfully achieved. For example, after transfection of human vascular endothelial cells, the aging phenotype of vascular endothelial cells can be improved, which is specifically manifested by reduced SA-β-Gal staining, reduced γH2A.X and IL-6 expression.

[0151] Furthermore, this application has significantly reduced the risk of formation of induced pluripotent stem cells during cell reprogramming, and effectively avoided the complete dedifferentiation of cell identity, thereby improving the safety and controllability of the reprogramming process at the molecular level by cleverly integrating three key transcription factors into circular RNA molecules, compared to the six transcription factor combination schemes commonly used in the prior art. And the application makes full use of the unique annular structure characteristics of circular RNA, substantially prolongs the molecular stability of transcription factors, and effectively overcomes the inherent limitations of the short biological half-life (about 24 hours) of traditional linear mRNA. By enhancing the sustained expression ability of transcription factors, this technology significantly improves the efficiency and reliability of partial cell reprogramming, bringing important breakthroughs to partial cell reprogramming technology.

[0152] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0153] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0154] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0155] Example 1

[0156] 1. Design, production and validation of O'SK circular RNA

[0157] 1. Vector construction

[0158] The order of each element of O'SK circular RNA (FlexCirc system) is as follows Figure 1As shown: 5' homology arm, IG5, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, IG3, 3' homology arm, the above-mentioned FlexCirc system ring system sequence, including the 5' end T7 promoter sequence, and the DNA template linearization restriction site EcoR I sequence (see SEQ ID NO.8 for details) were recombined into the pBlue Script plasmid, and the required plasmid was obtained by direct synthesis by a third-party sequence synthesis company, and the DNA sequence was confirmed to be correct by sequencing.

[0159] 2. In vitro transcription and circularization verification

[0160] (1) Preparation of DNA linearized template: The mutation vector plasmid synthesized by a third party is linearized using restriction endonuclease EcoRI to obtain a transcription template.

[0161] Specifically, EcoR I restriction endonuclease was used to linearize and digest the plasmid qualified by PCR to prepare the IVT transcription template. The enzyme digestion system is shown in Table 1.

[0162] The purity of the linearized template was tested by HPLC to ensure that the prepared linearized template met the requirements of subsequent IVT transcription.

[0163] Table 1: EcoR I enzyme 500μL digestion system formula

[0164] Components Volume (μL) Plasmids 50μg EcoRI (10U / μL) 50 Tingo Buffer 50 Enzyme-free water Up to 500

[0165] (2) In vitro transcription and circularization verification

[0166] Prepare 20 μL of reaction system according to Table 2, place the transcription system in the PCR instrument, and react at 37°C for 2 hours. After the reaction is completed, add 1 μL of DNase I and 2 μL of 10×DNase I buffer to each reaction system, and react at 37°C for 20 minutes. Transfer each transcription system to a 1.5 mL centrifuge tube, add RNase R enzyme and incubate at 37°C for 30 minutes, then add 0.5 times the volume of 7.5M lithium chloride and 50mM EDTA RNA precipitation solution, mix well and precipitate in a -20°C refrigerator for 1 hour. After precipitation is completed, centrifuge at 18360×g for 15 minutes at 4°C, remove the supernatant as much as possible, add 500 μL of 70% ethanol solution, blow and wash the precipitate, centrifuge again under the same conditions, remove the supernatant as much as possible, evaporate the ethanol, and resuspend each experimental group with 100 μL of enzyme-free water. Samples were taken to detect RNA concentration and purity, and 2% E-Gel electrophoresis was used to determine the circRNA synthesis effect.

[0167] The results are as follows Figure 2As shown, in 2% E-gel electrophoresis, lanes 5 (OSK circular RNA) and 6 (O'SK circular RNA) can form a single band with a slower migration rate, and its size is higher than 6000 nt, which is consistent with the theory.

[0168] Table 2 In vitro transcription system

[0169]

[0170] (3) HPLC purification and testing

[0171] This part of the work was completed by Shenzhen XingGene Technology Co., Ltd. using its internal methods, such as Figure 3 As shown, the main peak of O'SK circular RNA is obvious, and the impurity peak is relatively low. The main peak is integrated to obtain its purity. The concentration and purity of the prepared O'SK circular RNA are shown in Table 3 below:

[0172] Table 3: In vitro transcription system

[0173] Sample Concentration Purity O'SK circular RNA 0.096 mg / mL 86.2％

[0174] Verification results:

[0175] 1. Circular RNA protein expression verification

[0176] 1. Cell culture: HEK293T cells were cultured in a 37°C 5% CO2 biological incubator using DMEM (Gibco) complete medium containing 10% fetal bovine serum and 1% double antibody. When the cells grew to 90%, 0.05% Trypsin-EDTA was used to digest the cells and count them. The cells were counted according to 1.0×10 5 The cells were seeded at a density of 1.54 × 10 / well in a 24-well plate and mRNA transfection was performed the next day.

[0177] 2. Cell transfection: According to the instructions, use lipofectamine 3000 (Thermo Fisher) to transfect O'SK circular RNA in HEK293T cells. Add 1.0 μg of circular RNA to 50 μL Opti-MEM and mix thoroughly; take another tube, add 50 μL Opti-MEM and 1.0 μL lipofectamine 3000, mix the two tubes thoroughly, incubate at room temperature for 15 minutes, and add to the corresponding cells. After 48 hours of transfection, add 50 μL RIPA lysis buffer (Biyuntian) to each well, place on ice for 30 minutes, fully lyse, and collect protein lysate for WB (protein blotting) experiments. Among them, Oct4mut antibody (Wuhan Proleger) and Sox2 antibody (Jingjie Bio, PTM-6930) were used for WB experiments.

[0178] The results are as follows Figure 4As shown, the internal reference gene ( Figure 4 Group A in), Oct4mut( Figure 4 in Group B) and Sox2( Figure 4 Group C in the sample was only detected in sample 1.

[0179] 2. Phase separation characterization of Oct4 transcription factor

[0180] 1. Plasmid synthesis: GFP-Oct4 plasmid and GFP-Oct4mut (O'SK) plasmid were synthesized at GENEWISE (Suzhou GENEWISE Biotechnology Co., Ltd.) and cloned into pCDNA3.4 vector. Subsequent experiments were performed after the DNA sequence was confirmed to be correct.

[0181] The nucleotide sequence of GFP-Oct4 plasmid is shown in SEQ ID NO.9:

[0182]

[0183] The nucleotide sequence of GFP-O'SK plasmid is shown in SEQ ID NO.10:

[0184]

[0185] 2. Cell culture: HEK293T cells were cultured in a 37°C 5% CO2 biological incubator using DMEM (Gibco) complete medium containing 10% fetal bovine serum and 1% double antibody. When the cells grew to 90%, 0.05% Trypsin-EDTA was used to digest the cells and count them according to 1.0×10 5 The cells were seeded at a density of 1.54 × 10 / well in a 24-well plate and mRNA transfection was performed the next day.

[0186] 3. Cell transfection: According to the instructions, lipofectamine 3000 (Thermo Fisher) was used to transfect GFP-Oct4 plasmid and GFP-Oct4mut plasmid in HEK293T cells. 1.0 μg of plasmid was added to 50 μL Opti-MEM and mixed thoroughly; in another tube, 50 μL Opti-MEM and 1.0 μL lipofectamine 3000 were added, the two tubes were mixed thoroughly, incubated at room temperature for 15 minutes, and added to the corresponding cells.

[0187] 4. Static phase separation observation: 48 hours after transfection, cells were fixed with 4% paraformaldehyde (Biyuntian, P0099). After being fully washed with PBS, DAPI (Biyuntian, P0131) was used for nucleus staining and sealing. For cells in the 1,6-hexanediol treatment group, 48 hours after transfection, 1.5% 1,6-hexanediol (Sangong, A601513) was added and incubated for 5 minutes. After washing with PBS, cells were fixed and stained. The morphology and aggregation degree of GFP in the cell nucleus were observed under a 100x oil lens using a Nikon A1 confocal fluorescence microscope. GFP-Oct4 exists in the cell nucleus in the form of highly condensed droplets. After treatment with 1,6-hexanediol, it becomes a uniformly distributed form, indicating that Oct4 has the characteristics of classic phase separation; while GFP-Oct4mut has always been a diffusely distributed form, indicating that its phase separation characteristics can be destroyed by amino acid modification. The results are as follows: Figure 5 shown.

[0188] 5. Dynamic phase separation observation (fluorescence decolorization and fluorescence recovery)

[0189] The cells were transfected in the same way, and the GFP signal in the nucleus of living cells was dynamically observed under a Nikon A1 confocal fluorescence microscope. The GFP fluorescence spots were first quenched with a 2-second laser (excitation light 488nm, fluorescence intensity 21%), and then recorded every 2 seconds within a 10-minute observation window. It can be found that GFP-Oct4 has the ability to recover quickly, while the fluorescence spots of GFP-Oct4mut do not have the ability to recover after quenching. The results are shown in Figure 2. Figure 6 shown.

[0190] 3. Evaluation of endothelial cell rejuvenation after transfection

[0191] 1. Cell culture: HUVEC cells were cultured in a 37°C 5% CO2 biological incubator using Endothelial Cell Medium (ScienCell) complete culture medium containing 10% fetal bovine serum and 1% double antibody. When the cells grew to 90%, 0.05% Trypsin-EDTA was used to digest the cells and count them. The cells were counted according to 5.0×10 5 The cells were seeded at a density of 1.54 × 10 / well in a 24-well plate and mRNA transfection was performed the next day.

[0192] 2. Cell transfection: According to the instructions, lipofectamine 3000 (Thermo Fisher) was used to transfect O'SK circular RNA in HUVEC cells. 1.0 μg circular RNA was added to 50 μL Opti-MEM and mixed thoroughly; in another tube, 500 μL Opti-MEM and 1.0 μL lipofectamine 3000 were added, the two tubes were mixed thoroughly, incubated at room temperature for 15 minutes, and added to the corresponding cells. After 48 hours of transfection, HUVEC cell senescence markers were detected.

[0193] 3. β-Galactosidase staining to detect the expression level of SA-β-Gal in HUVEC cells after transfection

[0194] After 48 hours of transfection, the morphology of HUVEC cells was observed and recorded. According to the instructions, the cells in each group were stained with senescence-associated β-galactosidase (SA-β-gal) to detect their senescence degree: β-galactosidase fixative was used for 15 minutes, and then washed three times with PBS. Subsequently, the staining working solution was prepared for staining and incubated in a 37°C carbon dioxide-free incubator overnight. During this period, the 24-well plate was sealed with plastic wrap to prevent evaporation. The results are shown in Figure 2. Figure 7 shown.

[0195] The results showed that in HUVECs of three passages, the expression level of SA-β-Gal in the O'SK group was lower than that in the control group.

[0196] 4. Western Blot detection of p16 expression level in HUVEC cells after transfection: 48 hours after transfection, add 50 μL of RIPA lysis solution (Biyuntian) to each well, place on ice for 30 minutes, fully lyse, and collect the protein lysate for WB experiment. Use the BCA method to quantify protein concentration. Load equal amounts of protein samples (about 30 μg) into SDS-PAGE gel, separate them by electrophoresis, and transfer them to PVDF membrane. After the membrane is blocked, incubate with specific primary antibodies against p16INK4a (abcam) and GAPDH (abcam) at 4°C overnight. Subsequently, incubate with HRP-labeled secondary antibody for 1 hour and detect using chemiluminescence, such as Figure 8 shown.

[0197] The results showed that the expression level of p16 in HUVEC cells of O'SK group was significantly lower than that of control group, and the degree of cell senescence was milder than that of control group.

[0198] 5. Immunofluorescence detection of γH2A.X

[0199] First, wash the cells with PBS, then fix with fixative for 15 minutes, and then wash with washing solution for 3 times. After that, add blocking solution and block at room temperature for 10 to 20 minutes. Add γ-H2A.X rabbit monoclonal antibody and incubate at 4°C overnight. After washing, add anti-rabbit secondary antibody and incubate at room temperature for 1 hour. After washing again, add DAPI for staining for 5 minutes, and finally wash and observe under a microscope. γ-H2A.X is red and DAPI is blue. The results are as follows Fig. 9 shown.

[0200] The results showed that the expression level of γH2A.X in HUVEC cells in the O'SK group was significantly lower than that in the control group, and the degree of cell senescence was milder than that in the control group.

[0201] 6. ELISA detection of IL-6 expression level in HUVEC cells after transfection

[0202] ELISA was performed on HUVECs in the control group and the O'SK treatment group. According to the instructions of the kit (Elabscience), standard wells, blank wells and sample wells were first set. 100 μL of diluted standard was added to the standard well, 100 μL of diluent was added to the blank well, and 100 μL of the sample to be tested was added to the sample well. Avoid touching the well wall when adding the sample and mix gently. The whole process was controlled within 10 minutes. After adding the sample, the membrane was covered and incubated at 37°C for 90 minutes. Then, the liquid in the well was shaken off, 100 μL of biotinylated antibody working solution was added to each well, the membrane was covered, and the well was incubated at 37°C for 60 minutes, and then washed three times. After that, 100 μL of HRP enzyme conjugate working solution was added to each well, the membrane was covered, and the well was incubated at 37°C for 30 minutes, and then washed five times. Finally, 90 μL of substrate solution (TMB) was added to each well, the membrane was covered, and the well was incubated at 37°C in the dark for 15 minutes, and the microplate reader was preheated at the same time. After the incubation, 50 μL of stop solution was added to each well to terminate the reaction, and the OD value of each well was measured with an enzyme-labeled instrument at a wavelength of 450 nm. The test results were as follows: Fig.10 shown.

[0203] The results showed that compared with the control group, the expression level of IL-6 in the O'SK group was lower, and O'SK treatment could alleviate the SASP phenotype of HUVEC.

[0204] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for promoting rejuvenation of vascular endothelial cells, characterized in that: Using a phase-separation-free engineered reprogramming factor approach to prepare a phase-separation-free transcription factor; Introducing the phase-separation-free transcription factor into target cells to perform initial phase reprogramming on the target cells; The method of engineering reprogramming factors without phase separation includes mutating the relevant regions of the phase separation function of the transcription factor so that the transcription factor lacks the function of inducing phase separation; The target cells include vascular endothelial cells.

2. The method for promoting rejuvenation of vascular endothelial cells according to claim 1, characterized in that: The region related to the phase separation function of the transcription factor includes the IDR region.

3. The method for promoting rejuvenation of vascular endothelial cells according to claim 2, characterized in that: The mutation treatment includes mutating the polar charged amino acid or acidic amino acid in the IDR region; Optionally, the polar charged amino acid or the acidic amino acid is replaced with a neutral amino acid; Optionally, the acidic amino acids are mutated to neutral amino acids.

4. The method for promoting rejuvenation of vascular endothelial cells according to claim 3, characterized in that: The method comprises mutating positions 1 to 140 and / or positions 288 to 360 of the amino acid sequence shown in SEQ ID NO.1, Optionally, positions 1 to 140 and / or positions 288 to 360 of the amino acid sequence further include 10 to 20 amino acids before and after.

5. The method for promoting rejuvenation of vascular endothelial cells according to claim 4, characterized in that: include: The invention relates to a method for mutating at least one of positions 8, 20, 31, 108, 138, 291, 297, 26, 56, 68, 91, 96, 98, 104, 113, 125, 127, 130, 134, 135, 296, 299, 341, 343, 145, 147, 166, 188, 209, 210, 215, 224, 238, 246, 270 and 272 of the amino acid sequence shown in SEQ ID NO.

1.

6. The method for promoting rejuvenation of vascular endothelial cells according to any one of claims 1 to 5, characterized in that: The amino acid sequence of the phase-separation-free transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.

2.

7. The method for promoting rejuvenation of vascular endothelial cells according to claim 6, characterized in that: Before the phase-separation-free transcription factor is introduced into cells, the method further comprises the step of preparing the phase-separation-free transcription factor into an element containing the phase-separation-free transcription factor.

8. The method for promoting rejuvenation of vascular endothelial cells according to claim 7, characterized in that: The elements include: one or more of DNA, RNA, recombinant vector, recombinant protein and recombinant cell; Optionally, the recombinant vector comprises one or more of an adeno-associated virus vector, a poxvirus vector and a lentivirus vector; Optionally, the RNA comprises one or more of linear mRNA, siRNA, circular RNA, circular mRNA and tRNA.

9. The method for promoting rejuvenation of vascular endothelial cells according to claim 8, characterized in that: The circular RNA comprises a plurality of transcription factors connected in series; Optionally, the transcription factor is linked via a linker peptide; Optionally, the nucleotide sequence of the connecting peptide is shown as SEQ ID NO.3 to SEQ ID NO.

4.

10. The method for promoting rejuvenation of vascular endothelial cells according to claim 9, characterized in that: The elements include one or more of Oct4, Sox2, Klf4, c-Myc, L-Myc, Lin28, Nanog and Glis1.

11. The method for promoting rejuvenation of vascular endothelial cells according to claim 10, characterized in that: The nucleotide sequence of the element is shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.

8.

12. The method for promoting rejuvenation of vascular endothelial cells according to any one of claims 1 to 5 or 7 to 11, characterized in that: Introducing the phase separation-free transcription factor into target cells comprises using a delivery system; Optionally, the delivery system comprises one or more of liposome LNP, exosome delivery, GalNAc system, VLP delivery, protamine, high molecular polymer, inorganic nanoparticles, exosomes, polymer matrix and viral transfection.

13. Use of a phase-separation-free transcription factor or an element containing a phase-separation-free transcription factor in the preparation of a drug that promotes rejuvenation of vascular endothelial cells; The amino acid sequence of the phase-separation-free transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.

2.

14. The use according to claim 13, characterized in that The nucleotide sequence of the element containing the phase-separation-free transcription factor is shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.

8.

15. The use according to claim 14, characterized in that: The drugs include drugs for cell therapy, gene therapy, immune cell proliferation, cancer treatment, atherosclerosis, coronary heart disease, stroke, hypertension or aging-related diseases.

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