Method for rejuvenating cells by reprogramming in non-phase separation transcription factor mediated initiation stage

Abstract

The invention provides a method for rejuvenating cells by reprogramming in a phase-free separation transcription factor mediated initiation stage. Through improvement of phase-free separation of transcription factors, a recombinant vector is constructed and introduced into a target cell, and the cell is controlled in a reprogramming starting stage, so that the technical effect of rejuvenation is achieved.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering, gene therapy and cell therapy. Background Art

[0002] An important way of induced pluripotent stem cell (iPSC) reprogramming technology is to induce differentiated cells to generate pluripotent stem cells through transcription factors (such as OCT4, SOX2, KLF4 and MYC, hereinafter referred to as 4 factors). The whole process is complete reprogramming, which can be divided into the initial stage, the mature stage, and the stable stage (Koji et al, 2013, Nelly et al., 2019). During the reprogramming process, cells undergo drastic changes in transcription levels and epigenetic modifications, which cause huge changes in cell identity and state. In the initial stage, the expression of early pluripotency-related genes such as TRA-1-60, NANOG, SSEA4, and SALL4 increases rapidly, while the expression levels of somatic cell identity characteristic genes do not change much; in the mature stage, in addition to the above-mentioned early pluripotency-related genes, maturity-specific pluripotency-related genes such as DPPA5 and LIN28 are also highly expressed, while the somatic cell identity characteristic genes whose expression levels do not change much in the initial stage decrease rapidly (Koji et al, 2013, Nelly et al., 2019), suggesting that it is difficult for cells to maintain their original somatic cell identity characteristics at this stage; in the stable stage, the cells have completely established a pluripotency gene expression network and become iPSCs. In addition to expressing the above-mentioned pluripotency-related genes, they also highly express classic core pluripotency transcription factors such as OCT4, SOX2, and KLF4, while somatic cell identity characteristic genes are basically not expressed. In addition, this process can also rejuvenate cells, which can be detected by changes in the transcription levels of specific genes, cell aging markers, and especially epigenetic age (epiAge) based on DNA methylation (Nelly et al., 2019, Antoine, et al, 2022, Diljeet et al., 2022).

[0003] Partial reprogramming is when the above-mentioned complete reprogramming stops halfway. Since cell rejuvenation occurs in the early stages of reprogramming, and the loss of somatic cell identity characteristics occurs only in the middle and late stages of reprogramming, there is a time window that allows cells to be rejuvenated while maintaining their original identity characteristics. This is the technical principle of partial reprogramming to rejuvenate cells (Ocampo et al. 2016). The methods currently reported for reversing age using transcription factors are mainly as follows: 1. Partial reprogramming with limited induction time in vitro, also known as transient reprogramming, such as limiting the overexpression time of 4 factors through an inducible expression system, so that human fibroblast reprogramming is limited to the initial stage, which can reverse the age of cells and maintain the characteristics of fibroblasts (Nelly et al., 2019, Diljeet et al., 2022); 2. Reprogramming with transient pulses in vivo, such as transient pulse overexpression of 4 factors in mice through an inducible system, which can make the organs of premature mice younger, and can also make the organs of wild-type mice younger to a certain extent (Alejandro et al., 2016, Kristen et al, 2022); 3. Partial reprogramming that reduces the continuous induction of transcription factors (hereinafter referred to as 3-factor reprogramming), such as delivering AAV viruses loaded with OSK transcription factor sequences into the mouse eyeball, which can treat glaucoma (Lu et al. 2020). The above mentioned are all existing studies on reversing age by overexpressing transcription factors. However, there is still much room for improvement in the actual application of avoiding the generation of induced pluripotent stem cells and effectively reversing age. For example, it is impossible to limit the expression time of the four factors in the human body through a small molecule induced expression system. Therefore, it is necessary to limit the risk of generating iPSCs at the source through methods such as engineering transcription factors, while not affecting the effect of cell rejuvenation.

[0004] Phase separation refers to the phenomenon that some proteins or nucleic acid molecules in cells can produce another phase with different physical and chemical properties in the surrounding originally uniform environment through multivalent interactions, usually showing droplet-like characteristics, resulting in the formation of membrane-less organelles or cell structures. This phenomenon or structure is called liquid-liquid phase separation (LLPS), or phase separation for short (Clifford, et al. 2009, Li, et al. 2012). Phase separation describes a specific structure at the micron level, which creates a relatively independent spatial region that selectively enriches molecules; compared with the surrounding environment, the cell structure formed by phase separation usually has a higher protein density and weakened molecular microscopic motion, which can promote certain biochemical reactions. The protein structural characteristics that can form phase separation are still under study.

[0005] The present invention utilizes engineered transcription factors that remove the phase separation ability (hereinafter referred to as phase-free transcription factors) to participate in multi-factor partial reprogramming. The inventors found that the phase-free transcription factors caused partial reprogramming to remain in the initial stage, making the cells younger while maintaining the cell identity characteristics, so the inventors called this the initial stage reprogramming technology. This technology greatly reduces the risk of iPSC generation while improving the efficiency of cell rejuvenation, and is more likely to be applied in clinical practice. Summary of the invention

[0006] Definition of General Terms

[0007] In the claims and / or description, when used in conjunction with the term "comprising", the word "a" or "an" can mean "one", but can also mean "one or more", "at least one" and "one or more than one".

[0008] In this disclosure, "comprise", "include", "contain", "have", etc. have the meanings ascribed to them in the patent laws of China, the United States, Europe and other countries, and may refer to "encompass", "cover", etc. "essentially composed of...", "essentially composed of", etc. have the meanings ascribed to them in the patent laws of China, the United States, Europe and other countries. The term is open-ended and allows the existence of more than the cited matters as long as the basic or novel features of the cited matters are not changed by the existence of more than the cited matters, but excludes implementation schemes of the prior art.

[0009] Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value, indicates that alternative characteristics can be reasonably expected within the recited range, and particularly refers to a given quantity, for example, is intended to include a deviation of plus or minus 5 percent.

[0010] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or the alternatives are mutually exclusive.

[0011] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0012] The terms "identity" and "homology" as used in the present invention refer to the fact that when using 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.

[0013] 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.

[0014] 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.

[0015] 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 Molecular 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 Altschul 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 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul 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.

[0016] "Hybridization" refers to the formation of double-stranded molecules between complementary polynucleotide sequences (e.g., genes listed in Tables 1 and 2) or parts thereof under different stringency conditions. (See, for example, Wahl, GM. and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507). General genetic engineering, cell engineering professional terminology definition

[0017] The term "cell" refers to a naturally occurring or modified intact living cell. A cell can be isolated from other cells, mixed with other cells in culture, or mixed in a tissue (partial or complete) or organism. The methods described herein can be performed on a sample, for example, comprising a single cell, a cell population, or a tissue or organ comprising a cell.

[0018] As used herein, the term "mammalian cell" refers to any cell derived from a mammalian subject that is suitable for transplantation into the same or a different subject. The cell may be xenogeneic, autologous, or allogeneic. The cell may be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a subject. In some embodiments, the cell has been genetically engineered to express a recombinant protein and / or nucleic acid.

[0019] Vectors and recombinant vectors are used to carry target genes. Lentiviral vectors (LV) are preferred in the present invention. They are a type of retrovirus and RNA viruses that can effectively integrate exogenous genes into host chromosomes to achieve persistent expression. Adeno-associated viral vectors (AAV) are preferred in the present invention. They are non-integrated DNA viruses that are widely used in gene therapy because they have higher safety, lower immunogenicity, and longer in vivo expression time. Engineered mRNA, including linear mRNA and circular mRNA, is a novel gene vector suitable for non-viral gene delivery systems and can be used for in vivo gene therapy. Circular mRNA is preferred in the present invention, has lower immunogenicity and higher stability than linear mRNA, is suitable for long-term expression of therapeutic proteins in vivo, and is therefore increasingly used in gene therapy. However, as long as the carrier that can be used for carrying is within the scope of the present invention.

[0020] Host cells include dividing cells and non-dividing cells; they can effectively infect primary cells, neuronal cells, glial cells, stem cells, mesenchymal stem cells, cardiomyocytes, hepatocytes, T cells, tumor cells, etc., and also fibroblasts, epithelial cells, blood cells, immune cells, neural cells, embryonic cells. More specifically, human skin fibroblasts, such as HFF-1, HSF-1, HDF, BJ, etc.; human skin keratinocytes, such as HACAT, etc.; human T cells, such as Jurkat, etc.; human lung fibroblasts, such as IMR-90, etc.

[0021] Definition of professional terms in the field of reprogramming

[0022] The term "regenerative cell" refers to an aged cell that has been treated with one or more cell reprogramming factors or transiently reprogrammed such that the cell has the transcriptomic characteristics of a young cell while still retaining one or more cell identity markers.

[0023] The term "stem cell" refers to a cell that retains the ability to renew itself through mitotic cell division and can differentiate into a variety of specialized cell types. Totipotent stem cells are produced by the fusion of egg and sperm cells. The cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Embryonic stem cells are the descendants of totipotent cells, have pluripotency, and can differentiate into cell types from any of the three germ layers. Induced pluripotent stem cells (iPSC) are derived from adult cells, which are induced by cell reprogramming factors to become an embryonic stem cell-like pluripotent state. Induced pluripotent stem cells can be derived from adult somatic cells such as skin or blood cells, and also have the potential to differentiate into any cell type in the three germ layers.

[0024] As used herein, the term "reprogramming" refers to the use of cell reprogramming factors to induce the conversion of somatic cells into iPSCs. The reprogramming process can be divided into three stages: the initiation stage, the maturity stage, and the stability stage. In the initiation stage, the cells still maintain their original somatic cell identity, but early pluripotency-related genes such as NANOG and SALL4 are rapidly expressed; in the maturity stage, somatic cell identity characteristics are rapidly lost, and late pluripotency-related genes such as DPPA5 and LIN28 are expressed; in the stable stage, cells completely lose their cell identity and become iPSCs. In addition, this process can also rejuvenate old cells (i.e., eliminate all or some signs of aging).

[0025] As used herein, the term "loss of cell identity" refers to the fact that after reprogramming, somatic cells lose their original somatic cell-specific morphology, function, epigenetic pattern, gene expression profile and other characteristics (i.e., cell identity), acquire various characteristics of pluripotent stem cells, and become iPSCs. It should be noted that the cell identity of somatic cells is gradually lost during the reprogramming process. In the initial stage, the cells still maintain their original cell identity; in the mature stage, the cells have lost most of their cell identity characteristics and have acquired a higher degree of pluripotent stem cell characteristics. At this time, even if reprogramming is terminated, there is still a great risk that the cells will not be able to restore their original cell identity.

[0026] The term "partial reprogramming" refers to the use of one or more cell reprogramming factors to treat or transiently reprogram aged cells, allowing the cells to restore a certain degree of vitality, but not enough to dedifferentiate into iPSCs, retaining their original cell identity. "Transient reprogramming" is a way to achieve partial reprogramming, which means that the cells are exposed to cell reprogramming factors for a time sufficient to restore the cells, but not enough to cause dedifferentiation into iPSCs. Because the exposure time is very short, this transient reprogramming has a lower degree of rejuvenation.

[0027] 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.

[0028] The term "liquid-liquid phase separation" (LLPS), also known as phase separation, refers to the fact that some 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. It usually appears as 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 type 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. Another type depends on the ubiquitination of lysine, which can also form phase separation through the interaction of polyubiquitination chains. "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.

[0029] As used herein, the term "cell reprogramming factor" refers to a group of transcription factors and combinations thereof that can convert adult or differentiated cells into pluripotent stem cells. Specifically, the reprogramming factor is a factor from the Oct family, Sox family, Klf family, Myc family, Nanog family, Glis family or Lin family. Exemplary reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28, NANOG and / or GLIS1. Other exemplary reprogramming factors include CMYC, DPPA2, DPPA4, ESRRB, GDF3, GLIS1, KLF2, KLF4, KLF5, LIN28, LMYC, NANOG, NMYC, NR5A1, NR5A2, OCT-4, RCOR2, SALL1, SALL4, SOX1, SOX2, SOX3, TDRD12, TET1, TH2A, TH2B, UTF1, ZFP42, MDM2, CyclinD1, SV40 large T antigen, SIRT6, TCL1A, and RARy. 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 (KMS genes).

[0030] Oct reprogramming factor refers to any naturally occurring member of the transcription factor octamer family, or a variant thereof that retains transcription factor activity, a naturally occurring family member similar to the closest related factor (within at least 50%, 80% or 90% of the activity range), or a polypeptide comprising at least the DNA binding domain of a naturally occurring family member, and may further comprise a transcriptional activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11, such as Oct3 / 4 (referred to herein as "Oct4") comprising a POU domain. In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity in its entire sequence compared to a naturally occurring Oct polypeptide family member. In some embodiments, the OCT4 reprogramming factor protein / polypeptide provided herein is encoded by an optimized polynucleotide sequence SEQ ID NO: 1. Therefore, compared to wild-type OCT4, SEQ ID NO: 1 constitutes a changed polynucleotide sequence. In some embodiments, the amino acid sequence encodes a more robust OCT4 reprogramming factor compared to the amino acid sequence SEQ ID NO: 2. In some embodiments, the OCT4 reprogramming factor protein / polypeptide is encoded by a polynucleotide or amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%.

[0031] "Sox family" refers to the gene encoding SRY (sex determining region Y)-box 2, also known as SOX2, which is associated with maintaining pluripotency. Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15 or Sox18) can be from humans, mice, rats, cattle, pigs or other animals. Therefore, the "protein", "SOX2 protein" and the like referred to herein include any natural form of Sox2 transcription factor or its Sox2 transcription factor activity (e.g., at least 50%, 80%, 90% or 100% activity compared to wild-type Sox2 as measured by methods known in the art). In some embodiments, the variant has at least 90% amino acid sequence identity throughout its entire sequence compared to the naturally occurring Sox2 polypeptide. Specifically, sequence improvements with identity control are performed on the basis of SEQ ID NO: 23-24.

[0032] "Klf family" refers to Kruppel-like factor 4 or "Klf' gene encoding Klf4 protein, which is a protein encoded by mouse klf4 gene and human klf4 gene. Exemplary Klf family members include Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16 and Klf17. Therefore, the terms "KLF4", "KLF4 protein" and the like referred to herein include any naturally occurring form of KFF4 transcription factor or its KLF4 transcription factor activity (e.g., at least 50%, 80%, 90% or 100% activity compared to wild-type KFF4, as measured by methods known in the art). In some embodiments, the variant has at least 85% or 90% or 95% amino acid sequence identity throughout its entire sequence compared to the naturally occurring KFF4 polypeptide. For example, SEQ ID The variant structures shown in NO:21-22.

[0033] The factors of the Myc family refer to factors encoded by the myc proto-oncogene associated with cancer. N-Myc or L-myc are also used as possible reprogramming factors to replace c-Myc. Therefore, the terms "c-Myc", "C-MYC", "c-Myc protein", "C-MYC protein" and the like referred to herein include any naturally occurring form or variant of the cMyc transcription factor that maintains the activity of the cMyc transcription factor (e.g., compared to the wild-type cMyc, the activity is at least within 50%, 80%, 90% or 100%, as measured by methods known in the art). Specifically, variant improvements can be made based on the wild-type sequences of SEQ ID NO: 11-12.

[0034] In one embodiment of this aspect and all other aspects described herein, the reprogramming includes one of the following: introducing nucleic acid sequences encoding transcription factors Oct4, Sox2, c-Myc and Klf4, etc. into the differentiated somatic cells, wherein the sequences are operably linked to regulatory elements for expressing the factors; introducing one or more protein factors that reprogram the differentiation state of the cells; and contacting the cells with small molecules that induce reprogramming of the differentiation state of the cells.

[0035] Variants

[0036] The present invention also includes fragments, derivatives and analogs of the transcription factor mutant. The terms "fragment", "derivative" and "analog" refer to proteins that substantially maintain the same biological function or activity as the transcription factor mutant of the present invention. The protein fragment, derivative or analog of the present invention may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein having a substitution group in one or more amino acid residues, or (iii) a protein formed by the fusion of an additional amino acid sequence to this protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify this protein, or a fusion protein). According to the definition herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art. However, in the amino acid sequence of the transcription factor mutant and its fragments, derivatives and analogs, corresponding to the wild-type sequence, there must be a mutation at any site described in the above paragraph of the present invention.

[0037] Definitions of Terms in the Therapeutic Area of ​​Drugs

[0038] As used herein, the term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases of mammals, particularly cats, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0039] The terms "subject", "individual", "patient" or "subject" include (selectively added if there is a therapeutic use) mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0040] "Prevention" as used in the present invention refers to all actions to avoid symptoms or delay the onset of specific symptoms by administering the product described in the present invention before or after the onset of the disease.

[0041] The "effective amount" of the present invention refers to the amount or dosage of the product of the present invention that provides the desired treatment or prevention after being administered to a patient or an organ in a single or multiple doses.

[0042] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0043] The term "gene therapy" refers to the transient or permanent genetic modification (e.g., expression, insertion, alteration, or removal) of individual cells and / or biological tissues to treat diseases, such as tumors, genetic diseases, aging-related diseases, and autoimmune diseases. The most common forms of gene therapy include the introduction of functional genes into cells for expression to treat diseases, the insertion of functional genes into specific or non-specific genomic locations to replace mutant genes, while other forms include directly correcting mutations or modifying normal genes susceptible to pathogen infection, or transferring genes or gene fragments into cells for their transcription.

[0044] The term "cell therapy" refers to pharmaceutical products that achieve therapeutic, diagnostic and preventive purposes through a series of actions (such as proliferating and screening living autologous, allogenic, or xenogenic cells in vitro to restore cell and tissue function, or to change cell biological characteristics in other ways).

[0045] The first aspect of the present invention relates to a method for cell rejuvenation

[0046] The inventor studied the mechanism of three stages of cell reprogramming, the initiation phase: the cell identity continues to be maintained, early pluripotency genes are upregulated, epigenetic age (epiAge) decreases, and the cells begin to become younger; the maturation phase: the cell identity characteristics are greatly lost, and the late pluripotency genes are upregulated; the stabilization phase: the cell identity is completely lost and becomes iPSC; therefore, the time window before the maturity phase is a partial reprogramming (PR) window. The inventor has discovered a new method to prevent cell reprogramming from entering the maturity phase, extend the PR time window, and make the cells younger without losing their cell identity. The method is to use an engineered phase-separation-free reprogramming factor, based on mutations in the phase separation function-related regions of the transcription factor, so that the transcription factor loses the function of inducing phase separation, resulting in its inability to drive cell reprogramming into the maturity phase, thereby causing reprogramming to remain in the initiation phase, continuously rejuvenating the cells, but greatly reducing the risk of losing cell identity when reprogramming enters the late stage. This method is called "initiation phase reprogramming".

[0047] Furthermore, the phase-separation-free engineered transcription factor may be factors such as OCT4, SOX2, KLF4, c-MYC, L-myc, LIN28, NANOG and GLIS1.

[0048] The present invention relates to engineering the transcription factor to make it lose its phase separation ability. The inventors have found that there is no need to limit the method of modifying and improving the three-dimensional structure of the transcription factor. As long as it can make it lose its phase separation ability and make cell reprogramming remain in the initial stage, it is within the protection scope of the present invention.

[0049] Therefore, the present invention relates to a method for cell initiation phase reprogramming mediated by phase-separation-free engineered transcription factors, characterized in that the method is achieved by introducing phase-separation-free engineered transcription factors into cells.

[0050] A cell rejuvenation method, characterized in that phase-separation-free engineered transcription factors are used to mediate cell initiation stage reprogramming.

[0051] A cell reprogramming method, characterized in that it is achieved by introducing a vector containing a phase-separation-free engineered transcription factor into a cell.

[0052] Specifically, the criterion for determining cell rejuvenation is that, compared with a reference value, the regenerative cells show a change in gene expression of one or more epigenetic markers, wherein the change is an increase or a decrease.

[0053] Specifically, the criterion for determining cell rejuvenation is that, compared with a reference value, regenerative cells exhibit a change in the cell DNA methylation epigenetic age epiAge, wherein the change is a decrease.

[0054] Specifically, the criterion for determining cell rejuvenation is, for example, that the regenerative cells show a change in the proportion of senescence-associated β-galactosidase (SA-β-Gal)-positive cells compared to a reference value, wherein the change is a decrease.

[0055] Specifically, examples of criteria for determining cell rejuvenation: Compared with reference values, the genes related to rejuvenation function: COL1A1, COL3A1, COL4A1, FN1, LAMA5, are detected, and the gene expression levels are increased; the characteristic genes of the initial stage of reprogramming: NANOG, SALL4, are detected, and the gene expression levels are increased; the characteristic genes of the mature stage of reprogramming: DPPA5, LIN28A, are detected, and the gene expression levels are decreased. Cell identity genes can be used for cell verification, such as the detection of fibroblast identity characteristic genes: THY1, P4HB, S100A4, SERPINH1; different identity genes are selected according to different cells; after detection, the expression of identity genes can be increased relative to the reference value.

[0056] The reference value can be a blank cell control of the same cell type, or a wild-type transcription factor recombinant vector-introduced cell as a control; in short, the reference value is not an absolute standard, but a relative comparison benchmark for comparison after the modified transcription factor recombinant vector is introduced into the cell. Therefore, the above "criteria for determining rejuvenation" are only examples, not absolute standards. As long as it can be proved that the cell reprogramming is stuck in the initial stage, it is within the range of the cell rejuvenation judgment standard.

[0057] The protection scope of the present invention is not limited to the above exemplary functional genes. As long as they can be used to detect and determine that partial cell reprogramming is in the initial stage, they should be within the protection scope of the present invention.

[0058] It is worth noting that the method of cell rejuvenation or the method of cell reprogramming may or may not be used for treatment or diagnosis, and may be used to treat cells in vivo or in vitro, but does not necessarily directly treat the subject in need of treatment; in a specific embodiment, the method of cell rejuvenation or the method of cell reprogramming is not used for the diagnosis, prevention or treatment of disease.

[0059] The modification of the phase-free engineered transcription factor element may be carried out in a specific transformation / mutation / modification manner. The following improvements are only examples:

[0060] (1) Mutating charged amino acids, including mutating acidic charged amino acids and / or mutating basic charged amino acids, thereby changing the charged amino acids in the internal disordered region (IDR region) of the protein to regulate the ability to form condensed states.

[0061] The inventors found that "IDR with negative charge is the phase separation of transcription factors" is one of the principles. Therefore, by predicting the internal IDR region of the transcription factor, for example, the IDR of OCT4 is predicted by using the VSL2 algorithm to perform Disorder Score. For example, a continuous region with a score greater than a certain value has a higher probability of being an IDR. The value is not particularly limited, but can be in the range of 0.4-0.6, preferably 0.5. From the exemplary prediction results of OCT4, it can be seen that the IDR of OCT4 is distributed in the activation domain (AD) at both ends, more specifically, the 1st to 140th and 288th to 360th amino acids; the middle DNA binding domain-(DNA binding domain, DBD) does not belong to IDR. It is worth noting that alternative scoring standards can also be used, such as the scoring standards of IUPred2 and ANCHOR2, but as long as they can be used for the prediction and scoring of the IDR region, they are within the protection scope of the present invention. After prediction, the inventors found that the conclusions obtained by various scoring standards are the same (within the error range), so the present invention can protect the IDR region structure predicted by various scoring standards.

[0062] Specifically, amino acid modification can be performed on the disordered region (IDR region) inside the transcription factor protein, or the activation domain can be modified. Furthermore, charged amino acids can be modified, so that the transcription factor loses its phase separation ability.

[0063] Specifically, the polar charged amino acid may be replaced with a neutral amino acid, and the polar charged amino acid may be an acidic amino acid;

[0064] Preferably, the polar charged amino acid in the IDR region may be replaced with a neutral amino acid, and the polar charged amino acid may be an acidic amino acid;

[0065] More specifically, acidic amino acids such as aspartic acid (Asp) and glutamic acid (Glu) can be mutated into neutral amino acids such as alanine;

[0066] More specifically, the acidic amino acids in the IDR region, such as aspartic acid (Asp) and glutamic acid (Glu), can be mutated to neutral amino acids, such as alanine.

[0067] In a specific embodiment, the wild-type amino acid sequence of OCT4 may be mutated and improved;

[0068] The IDR region of the protein sequence of the wild-type OCT4 gene may account for 38-40% of the entire sequence at the N-terminus and 19-24% at the C-terminus.

[0069] Preferably, the above mutation improvement may be performed on positions 1-140 of SEQ ID NO: 2, and / or on positions 288-360; it is worth noting that the above positions may have an error range of about 10-20 amino acid positions before and after.

[0070] Preferably, the mutation site is selected from at least one of 8, 20, 31, 108, 138, 291, 297; 26, 56, 68, 91, 96, 98, 104, 113, 125, 127, 130, 134, 135, 296, 299, 341, 343 of SEQ ID NO: 2; preferably, the mutation point is selected from at least one of 145, 147, 166, 188, 209, 210, 215, 224, 238, 246, 270, 272 of SEQ ID NO: 2. Specifically, the number of mutations can be 1, 2, 3 ... 36.

[0071] Exemplarily, the gene of wild-type OCT4 may be mutated so that the acidic polar amino acid in the IDR region of the protein is mutated (D / E→A), hereinafter referred to as OCT4'; for example, any one of SEQ ID NOs: 4-10, or a sequence with at least 85% or 90% or more identity that can achieve the same function.

[0072] The present invention further protects the nucleotide sequence expressing the amino acid sequence of the above-mentioned protein, illustratively, which may be shown in SEQ ID NO: 3. However, those skilled in the art know that as long as the target protein can be obtained through codon expression, different nucleotide sequences obtained by reasonable codon preference changes are all within the scope of protection.

[0073] Of course, the present invention is not limited to the above-mentioned transcription factors. As long as the modification of charged amino acids in the IDR region can achieve the transcription factor transformation without phase separation function, it is within the protection scope of the present invention.

[0074] (2) Ubiquitination

[0075] The inventors found that after amino acids are modified by polyubiquitin chains, polyubiquitin chains mediate phase separation. Therefore, replacing lysine K with arginine R, for example, does not produce polyubiquitin chain modification and can avoid phase separation. However, it is worth noting that the scope of protection of the present invention is to provide a method for ubiquitination mutation of transcription factors to avoid phase separation, and is not limited to a specific mutation method.

[0076] For example, for the C-MYC transcription factor, its ubiquitinated lysine is mutated (K→R), and other transcription factors can also be mutated for ubiquitination.

[0077] Specifically, SEQ ID NOs: 11-12 may be mutated, and based on SEQ ID NO: 12, at least one lysine (K) in positions 51, 52, 126, 143, 148, 157, 206, 269, 275, 289, 298, 317, 323, 326, 341, 355, 371, 389, 392, 397, 398, 412, 422, 428, 430 may be mutated;

[0078] Preferably, mutations are performed on 1, 2, 3 ... 25 of the above positions; preferably, mutations are performed on one of the amino acids at positions 148 and 389, or both; preferably, mutations are performed on 20-25 positions; more preferably, mutations are performed on all 25 positions; however, as long as the removal of ubiquitinated chains can be achieved and the intended effect, i.e., phase separation can be avoided, the number of specific mutation positions does not need to be particularly limited.

[0079] Preferably, the modification method is to mutate lysine K to arginine; however, the specific method is not limited, as long as it can remove ubiquitination and avoid phase separation, it is within the scope of protection.

[0080] For example, variants shown in SEQ ID NO: 14-20 are used as examples, or sequences with at least 85%, 90% or more identity that can achieve the same function.

[0081] The present invention further protects the nucleotide sequence expressing the amino acid sequence of the above-mentioned protein, which can be exemplified as shown in SEQ ID NO: 13, or a sequence with at least 85% or 90% or more identity that can achieve the same function; however, those skilled in the art know that as long as the target protein can be obtained through codon expression, different nucleotide sequences obtained by reasonable codon preference changes according to the needs of the expression cell are all within the scope of protection.

[0082] Of course, the present invention is not limited to the above-mentioned transcription factors. As long as the ubiquitination treatment is removed and the transcription factor modification can achieve the function of phase separation, it is within the protection scope of the present invention.

[0083] (3) Partial deletion treatment, preferably terminal deletion treatment, which can be partial deletion treatment of the N-terminus or C-terminus, or partial deletion treatment of the middle part of the gene; or partial deletion treatment of the IDR region of the protein, that is, partial deletion treatment of charged amino acids in the IDR region; or deletion treatment of the key regulatory region of phase separation of the protein, that is, deletion treatment of key lysine modified with ubiquitination chain, or deletion treatment of amino acids that play a key role in producing phase separation.

[0084] Exemplarily, the non-critical regions other than the N-terminus or C-terminus of the transcription factor and its DBD can be deleted, for example, positions 1-20 can be deleted, and further, positions 2-20, 1-19, 2-29, 2-18, 2-17, 2, and 16 can be deleted. This treatment does not affect the DBD region, that is, it does not affect the original DNA site recognition function of the transcription factor, but it is possible to remove its IDR region, ubiquitinated chain-modified lysine or other key amino acids, thereby avoiding phase separation;

[0085] Specifically, the sequence of KLF transcription factor 4 (KLF4) shown in SEQ ID NO: 21-22 can be partially deleted, for example, its important DBD region is located at 375-470 of the C-terminus, so the IDR region at 2-374 of the N-terminus can be completely or partially deleted to obtain, for example, the amino acid sequence shown in SEQ ID NO: 40, or a sequence with at least 85% or 90% or more identity that can achieve the same function;

[0086] Specifically, the sequence of the sex determining Y-box transcription factor 2 (SOX2) shown in SEQ ID NOs: 23-24 can be partially deleted. For example, its important DBD region and transcription activation domain are located in the middle 41-200 positions. Therefore, the IDR region at positions 2-40 at the N-terminus and / or positions 201-317 at the C-terminus can be completely or partially deleted to obtain, for example, the amino acid sequence shown in SEQ ID NOs: 41-43, or a sequence with at least 85% or 90% or more identity that can achieve the same function.

[0087] Specifically, the OCT4 sequence shown in SEQ ID NO: 1-2 can be partially deleted. For example, its important DBD region is located in the middle 141-287. Therefore, the IDR region at 2-140 at the N-terminus and / or 288-360 at the C-terminus can be completely or partially deleted to obtain, for example, the amino acid sequence shown in SEQ ID NO: 44-46, or a sequence with at least 85% or 90% or more identity that can achieve the same function.

[0088] Specifically, the sequence of c-MCY shown in SEQ ID NO: 11-12 can be partially deleted, for example, its most important DBD is the bHLH-LZ domain at 361-439 of the C-terminus, so the N-terminal 2-360 can be completely or partially deleted to obtain, for example, the amino acid sequence shown in SEQ ID NO: 47-48, or a sequence that can achieve at least 85% or 90% or 95% identity or more of the same function. The present invention further protects the nucleotide sequence expressing the amino acid sequence of the above protein, which can be shown in SEQ ID NO: 13, or a sequence that can achieve at least 85% or 90% or 95% identity or more of the same function; however, those skilled in the art know that as long as the target protein can be obtained through codon expression, reasonable codon preference changes to obtain different nucleotide sequences are within the scope of protection.

[0089] Of course, the present invention is not limited to the above-mentioned transcription factors. As long as partial deletion treatment is performed to achieve the transcription factor modification without phase separation function, it is within the protection scope of the present invention.

[0090] A second aspect of the invention provides an element comprising an engineered transcription factor

[0091] Nucleic acid elements

[0092] The present invention further includes the nucleic acid elements of the aforementioned engineered transcription factors, which may be DNA elements, RNA elements, and further may be mRNA elements, siRNA elements, circular RNA elements, circular mRNA elements, and tRNA elements.

[0093] The element further comprises a Kozak sequence, a promoter sequence, at least one connecting peptide sequence, a UTR region, a terminator sequence, a 5' cap, a polyA tail, etc.; the nucleic acid element can be a DNA, RNA sequence, or a further expressed protein element. The promoter can be selected from inducible promoters such as TRE, TRE3G, TRE3GS, TRE3GV, etc., constitutive promoters CMV, EFS, CAG, PGK, etc., post-transcriptional regulatory sequences such as WPRE, etc., poly (A) tails such as SV40 poly (A), pGH poly (A), etc.

[0094] The mRNA elements include, but are not limited to, linear mRNA and circular mRNA. The circular mRNA may contain IRES and self-cleaving polypeptide 2A elements. The RNA may contain appropriate modifications, including but not limited to pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), 5-methoxy-uridine (5moU), N6-methyl-adenosine (m6A), 5-methyl-cytidine (5m5C), etc. The RNA may contain sequences that bind to L7Ae, TAT, etc., which are required for enriching the RNA.

[0095] Recombinant vector elements

[0096] The elements of the present invention can be further constructed into vectors, which can be monocistronic, bicistronic or polycistronic. In a specific embodiment of the present invention, the coding regions of the bicistronic or polycistronic genes can be linked by at least one connecting peptide, and the connecting peptide can be an internal ribosome entry site (IRES) or a self-cleaving 2A peptide (2A).

[0097] IRES sequences that can be used include, but are not limited to, small RNA viruses (e.g., FMDV), pestiviruses (e.g., CFFV), polioviruses (e.g., PV), encephalomyocarditis viruses (e.g., ECMV), foot-and-mouth disease viruses (e.g., FMDV), hepatitis C viruses (e.g., HCV), classical swine fever viruses (e.g., CSFV), mouse corneal white eye viruses (e.g., MLV), simian immunodeficiency viruses (e.g., SIV), or cricket paralysis viruses (e.g., CrPV), etc. The coding region can also be divided by a "2A linker", which is a short peptide (18-25 amino acids) derived from a virus, also known as a "self-cleaving" peptide, which can produce multiple proteins from one transcription product, including, but not limited to, P2A / T2A / E2A / F2A, etc.

[0098] When multiple transcription factors are connected in series, the specific ones may be as shown in SEQ ID NO: 25-27. Of course, the present invention also provides vectors of individual transcription factors, and mixes them in a certain ratio, such as 1:1:1:1, 1:2:1:2, 1:2:2:1, 2:1:1:1. The mixture is also within the protection scope of the present invention.

[0099] The present invention further includes recombinant vectors of the aforementioned engineered transcription factors, including but not limited to cloning vectors, expression vectors, shuttle vectors, viral vectors (eg, lentiviral vectors, adeno-associated viral vectors, poxvirus-associated vectors, etc.).

[0100] Recombinant protein

[0101] The present invention further includes the recombinant protein element obtained by translating and expressing the above nucleic acid element, and all nucleotide sequences that can express the above protein element. The protein element maintains its function to a certain extent and can also contain certain variants.

[0102] Recombinant cells

[0103] The present invention further includes recombinant cells introduced with the aforementioned vector.

[0104] Specifically, the methods provided by the present invention can be applied to any type of cells that need to be regenerated. Cells can be separated from other cells, mixed with other cells in culture, or mixed in tissues (partial or complete) or living organisms. The methods described herein can be performed on samples, such as single cells, cell groups, or tissues or organs containing cells. The cells selected for rejuvenation will depend on the therapeutic effect required for treating age-related diseases or disorders. The cells are mammalian cells. Including but not limited to prokaryotic cells, eukaryotic cells, further, can be mammalian cells, such as humans, monkeys, mice (rats, mice), horses, cattle, sheep, pigs, dogs, cats, etc. Such as CRFK cat kidney cells, PG-4 (S + L-) cat brain cells, F81 cat kidney cells, FCA-S1 cat skin cells, CATK1 cat kidney cells, MDCK (NBL-2) canine kidney cells, SCBN canine duodenal cells, DH82 canine renal malignant histiocytosis cells, HEK293T, HEK293, BHK, CHO, 3T3, NS0, Hela, HT-1080, PERC6, CAP, HKB-11, Huh-7, etc.; in an embodiment, the cell is a human cell. In an embodiment, the cell is from an elderly subject.

[0105] The methods provided herein can be performed on cells, tissues or organs of the nervous system, muscular system, respiratory system, cardiovascular system, skeletal system, reproductive system, integumentary system, lymphatic system, excretory system, endocrine system (e.g., nervous system), (endocrine and exocrine) or digestive system. As described herein, any type of cell may be regenerated, including but not limited to epithelial cells (e.g., squamous, cuboidal, columnar and pseudostratified epithelial cells), endothelial cells (e.g., venous, arterial and lymphatic endothelial cells) and cells of the connective tissue, muscle and nervous system. Such cells may include, but are not limited to, epidermal cells, fibroblasts, chondrocytes, skeletal muscle cells, satellite cells, cardiomyocytes, smooth muscle cells, keratinocytes, basal cells, ameloblasts, exocrine cells, myoepithelial cells, osteoblasts, osteoclasts, neurons (e.g., sensory neurons, motor neurons, and interneurons), glial cells (e.g., oligodendrocytes, astrocytes, ependymal cells, microglia, Schwann cells, and satellite cells), columnar cells, adipocytes, pericytes, stellate cells, pneumocytes, Blood and immune system cells (e.g., erythrocytes, monocytes, dendritic cells, macrophages, neutrophils, eosinophils, mast cells, T cells, B cells, natural killer cells), hormone-secreting cells, germ cells, interstitial cells, lens cells, photoreceptor cells, taste cells, and olfactory cells; and cells from the kidney, liver, pancreas, stomach, spleen, gall bladder, intestine, bladder, lung, prostate, breast, genitourinary tract, pituitary gland, mouth, esophagus, skin, hair, nails, thyroid, parathyroid glands, adrenal glands, eyes, nose, or brain.

[0106] Specifically, the cell is selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells and corneal epithelial cells. Specifically, the cell is a fibroblast. Specifically, the cell is an endothelial cell. Specifically, the cell is a chondrocyte. Specifically, the cell is a skeletal muscle stem cell. Specifically, the cell is a keratinocyte. Specifically, the cell is a mesenchymal stem cell. Specifically, the cell is a corneal epithelial cell.

[0107] Import and delivery

[0108] The introduction method includes but is not limited to liposome transfection, microvesicle transfection, exosome transfection, electroporation, nanocarrier transfection, and chemical transfection using a transfection reagent; the nanocarrier includes a lipid, a polymer, or a lipid-polymer hybrid.

[0109] A delivery system 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 cardiolipin in the form of monoacyl, diacyl, triacyl, or tetraacyl groups. In some embodiments, LNPs are micelles or reverse micelles (reverse micelles). In other embodiments, LNPs are unilamellar liposomes or multilamellar liposomes.

[0110] The present invention 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.

[0111] A third aspect of the invention relates to preventive and / or therapeutic uses

[0112] The nucleic acid elements (DNA, RNA elements), recombinant vectors, recombinant proteins, and recombinant cells involved in the present invention can be used for prevention and / or treatment in many fields, and can also be used in the preparation of drugs for treating diseases. Specific diseases to be treated include, but are not limited to, cell therapy, gene therapy, immune cell proliferation, cancer treatment, and treatment of aging-related diseases.

[0113] The method to which the present invention relates can be used to rejuvenate cultured cells (e.g., ex vivo or in vitro) to improve the function and efficacy of cell therapy. The cells used to treat patients can be autologous or allogeneic. Preferably, the cells are derived from patients or matching donors. For example, in ex vivo treatment, cells are obtained directly from patients to be treated, transfected with mRNA encoding cell reprogramming factors as described in the present invention, and reimplanted into the patient's body. This type of cell can be obtained, for example, from a biopsy or surgical operation performed on a patient. Alternatively, cells that need to be regenerated can be directly transfected in vivo with mRNA encoding cell reprogramming factors. In some embodiments, this immune cell engineering is performed ex vivo, for example, in the manufacture of cell therapy products, such as autologous or allogeneic chimeric antigen receptor (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells.

[0114] The method of the present invention can be used for gene therapy. "Gene therapy" is used to refer to the use of genes (i.e., genetic material) to treat and / or prevent diseases in mammalian subjects (e.g., humans). In some embodiments, the genetic material involved in the present invention (nucleic acid elements (DNA, RNA elements), recombinant vectors, etc.) is directly delivered to at least some cells of mammalian subjects. The present invention can be used for any specific method or composition of gene therapy. The aforementioned nucleic acid elements, recombinant vectors, etc. can be used to introduce into mammalian cells to achieve the purpose of gene therapy. Specifically, autologous or ex vivo gene therapy can be included. Gene therapy can also be divided into "germline cell gene therapy" and "somatic cell gene therapy". In the case of "germ cell gene therapy", germ cells, i.e., sperm or egg cells, are genetically modified. Genetic changes are usually integrated into their genome. Therefore, the changes caused by the treatment can be inherited and will be passed on to future generations. The method can be used to treat genetic disorders and genetic diseases. In the case of "somatic cell gene therapy", therapeutic genes are transferred into individual somatic cells. Any modification and effect will be limited only to the individual and will not be inherited by the descendants or offspring of the individual. Specific strategies include, but are not limited to, DNA correction, DNA replacement, DNA supplementation, and DNA inactivation.

[0115] In another aspect, a method for inducing proliferation of cells, such as immune cells, is provided. In some embodiments, the method comprises exposing the cell to mRNA encoding one or more reprogramming factors, whereby the exposure achieves expression of the one or more reprogramming factors in the cell to enhance cell proliferation while retaining its properties. In some embodiments, the method of inducing proliferation does not induce exhaustion. In some embodiments, the proliferation is caused by the prevention or reversal of exhaustion.

[0116] Specifically, the cell rejuvenation treatment method of the present invention can be used for "age-related diseases or conditions", specifically any condition, disease or condition related to aging, such as but not limited to neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, dementia, and stroke), cardiovascular and peripheral vascular diseases (such as atherosclerosis, peripheral arterial disease (PAD), hematoma, calcification, thrombosis, embolism and aneurysm), eye diseases (such as age-related macular degeneration, glaucoma, cataracts, dry eyes, diabetic retinopathy, vision loss), skin diseases (dermal atrophy and thinning, elastic hyperplasia and skin wrinkles, sebaceous gland hyperplasia or hypoplasia, senile freckles and other pigmentation abnormalities, graying hair, hair loss or thinning, chronic skin ulcers), autoimmune diseases (such as polymyalgia rheumatica (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), crystalline arthropathy and spondyloarthropathies (SPAs), medical metabolic disorders (e.g., adult hypopituitarism, hypothyroidism, apathetic thyrotoxicosis, osteoporosis, diabetes, adrenal insufficiency, various forms of hypogonadism, and endocrine malignancies), musculoskeletal disorders (e.g., arthritis, osteoporosis, myeloma, gout, Paget's disease, bone fractures, bone marrow failure syndrome, ankylosing spondylosis, diffuse idiopathic skeletal hyperostosis, hematogenous osteomyelitis, muscular atrophy, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis, and myasthenia gravis), diseases of the digestive system (e.g., cirrhosis, liver fibrosis, Barrett's esophagus), respiratory disorders (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism (PE), lung cancer, and infections), conditions related to cell proliferation, and any other diseases and conditions associated with aging.

[0117] As used herein, the term "disease or condition involving cartilage degradation" is any disease or condition involving cartilage and / or joint degradation. The term "disease or condition involving cartilage degradation" includes conditions, disorders, syndromes, diseases and injuries affecting the intervertebral discs or joints (e.g., joints) of animals (including humans), and includes, but is not limited to, arthritis, cartilage deformities, spondyloarthropathies, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjögren's syndrome. The term "muscle degenerative disease or condition" is any disease or condition involving muscle degeneration. The term includes conditions, disorders, syndromes,

[0118] Diseases and injuries affecting muscle tissue, such as, but not limited to, muscle wasting, muscle disuse, muscle tears, burns, surgery, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis, myasthenia gravis, cancer, AIDS, congestive heart failure, chronic obstructive pulmonary disease (COPD), liver disease, renal failure, eating disorders, malnutrition, starvation, infection, or glucocorticoid treatment.

[0119] A disorder associated with cell proliferation refers to a disease that occurs due to abnormal growth or extension caused by cell proliferation (Walker, Cambridge Dictionary of Biology, Cambridge University Press: Cambridge, UK, 1990). Proliferative diseases may be associated with: 1) pathological proliferation of normally quiescent cells; 2) pathological migration of cells from their normal location (e.g., metastasis of tumor cells); 3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) pathological angiogenesis, such as proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancer (i.e., "malignant tumors"), benign tumors, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0120] "Cancer" refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelioma, angiosarcoma); appendix cancer; benign monoclonal gammopathy; bile duct cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, breast cancer, medullary breast carcinoma); brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchial carcinoma; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer); carcinoma, colorectal adenocarcinoma); connective tissue cancer; epithelial cancer; ependymoma; endothelial sarcoma (e.g., Kaposi sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing's sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familiar eosinophilia; gallbladder cancer; gastric cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), pharyngeal cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal carcinoma, oropharyngeal cancer)); hematopoietic system systemic cancer (e.g., leukemias, such as acute lymphoblastic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphomas, such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL, such as diffuse large cell lymphoma ( DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma;and T-cell NHL, such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); mixtures of one or more leukemias / lymphomas as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharyngeal cancer; inflammatory myofibroblastic tumor; immune cell amyloidosis; renal cancer (e.g., Wilms' tumor also known as Wilms tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorders (MPD) (e.g., polycythemia vera (PV), essential thrombocythemia (ET), idiopathic myeloid metaplasia (AMM) also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia disease (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic cancer, intraductal papillary mucinous neoplasm (IPMN), islet cell tumor); penile cancer (e.g., Paget's disease of the penis and scrotum); pineal gland; primitive neuroectodermal tumor (PNT); plasma cell tumor; paraneoplastic syndrome; intraepithelial neoplasm; prostate cancer (e.g., e.g., prostate cancer); colorectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small intestinal cancer (e.g., appendix cancer); soft tissue sarcomas (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland cancer; small intestinal cancer; sweat gland cancer; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva). ;

[0121] A fourth aspect of the present invention provides a pharmaceutical composition

[0122] The pharmaceutical composition is usually composed of a main ingredient and an auxiliary ingredient. The pharmaceutical composition can be used for prevention or treatment, and can be used for standardized treatment or personalized treatment (cell therapy, gene therapy).

[0123] The selection of the main ingredients is mainly the nucleic acid elements, recombinant vectors, recombinant cells, etc. mentioned in the first and second aspects of the present invention as the main components, which can be formed by a series connection to form a recombinant vector, or by constructing a vector separately to form a mixture of multiple separate transcription factors. The mixing ratio is preferably equal, of course, different ratios are also allowed on the basis of maintaining the same active function.

[0124] The present invention further provides the use of the above-mentioned nucleic acid elements, recombinant vectors, recombinant cells, etc. in the preparation of drugs for treating diseases that can be prevented and treated by mediating cell rejuvenation. And provides a pharmaceutical composition for treating diseases that can be prevented and treated by mediating cell rejuvenation. And provides a pharmaceutical composition for treating diseases that can be prevented and treated by mediating cell rejuvenation.

[0125] When the pharmaceutical composition is a cellular composition, especially a regenerative cell composition, it may also contain one or more additional factors, such as nutrients, cytokines, growth factors, extracellular matrix (ECM) components, antibiotics, antioxidants or immunosuppressants to improve cell function or viability.

[0126] Examples of growth factors include, but are not limited to, fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor β (TGF-β), epiregulin, epidermal growth factor ("EGF"), endothelial cell growth factor ("ECGF"), nerve growth factor ("NGF"), leukemia inhibitory factor ("LIF"), bone morphogenetic protein 4 ("BMP-4"), hepatocyte growth factor ("HGF"), vascular endothelial growth factor-A ("VEGF-A"), and cholecystokinin octapeptide.

[0127] On the basis of the main ingredients, the pharmaceutical composition may optionally further comprise one or more pharmaceutically acceptable excipients, carriers, auxiliary materials, and vehicles. Examples of excipients, carriers, auxiliary materials, and vehicles include, but are not limited to, anti-adherents, adhesives, coatings, compression aids, disintegrants, dyes, lubricants, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, aromatics, glidants (flow enhancers), lubricants, adsorbents, suspending or dispersing agents, or sweeteners. Exemplary excipients, carriers, adjuvants, vehicles include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (monohydrogen), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked povidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0128] The pharmaceutical composition can be prepared in the form of an injection preparation, an oral preparation, an inhalation preparation or a microneedle preparation. The injection preparation is classified according to the physical state into liquid injection, injection powder, and injection tablet; according to the injection part, it is classified into intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, and spinal cavity injection; preferably, the solvent of the injection preparation includes injection water or physiological saline. Intravenous, intramuscular, and microneedle injection are preferred.

[0129] Formulations for oral use include tablets containing the active ingredient mixed with nontoxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); granulating agents and disintegrants (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, cross-linked carboxymethyl cellulose sodium, alginates or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinyl pyrrolidone or polyethylene glycol); and lubricants, glidants and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil or talc). Preparations for oral use may also be in the form of chewable tablets, or in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil). Powders, granules and pills can be prepared using the ingredients mentioned above under tablets or capsules in a conventional manner using, for example, a mixer, fluidized bed equipment or spray drying equipment.

[0130] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavoring agents, plasticizers, humectants, and buffers. Formulations for oral use may also be in the form of chewable tablets, or in the form of hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or in the form of soft gelatin capsules, wherein the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil). Powders, granules, and pills may be prepared in a conventional manner using, for example, a mixer, a fluidized bed apparatus, or a spray drying apparatus using the ingredients mentioned above under tablets or capsules.

[0131] The injection preparation includes injection solution, injection emulsion, lyophilized agent, suspension injection, etc. The excipients of the injection preparation include water for injection and at least one solubilizer. The solubilizer includes at least one of Tween 80, ethanol, propylene glycol, and polyethylene glycol 400. Preferably, the solubilizer is Tween 80 or polyethylene glycol 400. The injection preparation also includes but is not limited to protein nanoparticles, cyclodextrin inclusions, liposomes, microspheres, reservoir-type controlled release injections or needle-free injection drug delivery systems.

[0132] Combination therapy

[0133] The nucleic acid elements, recombinant vectors, recombinant cells and pharmaceutical compositions comprising the same involved in the present invention can be combined with other drugs for treatment. Taking anti-aging treatment as an example, the initial stage reprogramming can also be combined with protein therapy, such as B18R protein, for anti-aging treatment.

[0134] Beneficial Effects

[0135] Cell rejuvenation occurs in the early stages of reprogramming, while the loss of somatic cell identity characteristics occurs in the middle and late stages of reprogramming. Therefore, there is a time window here that allows cells to be rejuvenated while maintaining their original identity characteristics. This is the technical principle of partial reprogramming to rejuvenate cells. The inventors found that after the transcription factors lost their phase separation ability, it was difficult for reprogramming to enter the mature stage, so that the cells were stuck in the initial stage, so that the cells did not lose their cell identity (Loss of cell identity). Therefore, while increasing the degree of cell rejuvenation, there was a risk of reducing the generation of iPSCs, and it would not be completely reprogrammed, so it was called "initial stage reprogramming". It was judged by the phenotype of the functional gene to ensure the verification of the cell reprogramming stage, proving that the cells achieved the technical effect of rejuvenation.

[0136] Clinically, as cells age, the function of the cells themselves gradually declines, leading to aging and disorder at the tissue, organ and system levels. A large number of age-related diseases, including but not limited to neurodegenerative diseases, cardiovascular and peripheral vascular diseases, eye diseases, skin diseases, autoimmune diseases, metabolic dysfunction, musculoskeletal diseases, digestive system diseases, cell proliferation-related diseases and tumors, are rooted in the aging and disorder of related tissues and organs. Through cell rejuvenation treatment for specific tissues and organs, related diseases can be effectively alleviated and treated, benefiting the general public. At the same time, the extension of healthy life expectancy is conducive to greatly improving social productivity, alleviating medical needs and pressure, and creating new wealth for an aging society. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1 Lentiviral vector construction. (A) Schematic diagram of the lentiviral vector structure; (B) Enzyme digestion and identification of the lentiviral vector plasmid.

[0138] Figure 2 Lentivirus quality detection. (A) Lentivirus titer and sterility test results; (B) Expression level of transgenic KLF4 after lentivirus transduction of 293T cells.

[0139] Figure 3 This is a schematic diagram of the structure of the lentiviral vector used in Example 2.

[0140] Figure 4Senescence-associated β-galactosidase (SA-β-Gal) staining experiment. A) Representative images of the blank control group staining results. B) Representative images of the OKMS reprogramming group staining results. C) Representative images of the O'KM'S reprogramming group staining results. D) Statistics of the proportion of SA-β-Gal staining positive cells in each group.

[0141] Figure 5 Detection of expression levels of genes related to skin fibroblast rejuvenation function. The significant difference between the OKMS reprogramming group and the O'KM'S reprogramming group was confirmed by t-test. **: P < 0.01, ***: P < 0.001.

[0142] Figure 6 Detection of the expression level of skin fibroblast identity marker genes. The significant difference between the OKMS reprogramming group and the O'KM'S reprogramming group was confirmed by t-test. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0143] Figure 7 . Detection of the expression level of marker genes at the initial stage of cell reprogramming. The significant difference between the OKMS reprogramming group and the O'KM'S reprogramming group was confirmed by t-test. **: P<0.01.

[0144] Figure 8 Detection of expression levels of marker genes in the mature stage of cell reprogramming. The significant difference between the OKMS reprogramming group and the O'KM'S reprogramming group was confirmed by t-test. **: P<0.01, ***: P<0.001.

[0145] Fig. 9 Results of cell DNA methylation epigenetic age (epiAge) analysis.

[0146] Fig.10 Structural map of the lentiviral vector used in Example 3

[0147] Fig.11 Senescence-associated β-galactosidase (SA-β-gal) staining experiment. A) Representative picture of the blank control group staining results. B) Representative picture of the OKM reprogramming group staining results. C) Representative picture of the O'KM' reprogramming group staining results. D) Statistics of the proportion of positive cells in each group.

[0148] Fig.12 Detection of expression levels of genes related to skin fibroblast rejuvenation function. The significant difference between the OKM reprogramming group and the O'KM' reprogramming group was confirmed by t-test. **: P<0.01, ***: P<0.001.

[0149] Fig.13Detection of the expression level of skin fibroblast identity marker genes. The significance of the difference between the OKM reprogramming group and the O'KM' reprogramming group was confirmed by t-test. ***:

[0150] Fig.14 Detection of marker gene expression levels at the initial stage of cell reprogramming. The significance of the difference between the OKM reprogramming group and the O'KM' reprogramming group was confirmed by t-test. ns: not significant, ***: P<0.001.

[0151] Fig.15 Detection of expression levels of marker genes in the mature stage of cell reprogramming. The significant difference between the OKM reprogramming group and the O'KM' reprogramming group was confirmed by t-test. ***: P<0.001.

[0152] Fig.16 AAV vector construction. (A) Schematic diagram of the AAV-TRE3G-OKS vector structure; (B) Enzyme digestion and identification of the AAV-TRE3G-OKS vector plasmid; (C) Schematic diagram of the AAV-TRE3G-O'KS vector structure; (D) Enzyme digestion and identification of the AAV-TRE3G-O'KS vector plasmid.

[0153] Fig.17 qPCR method was used to determine the titer of AAV vector. (A) Original amplification curve group; (B) Standard curve; (C) AAV vector titer determination results.

[0154] Fig.18 Construction of circular RNA vectors containing different combinations of some reprogramming factors.

[0155] Fig.19 Validation of circular RNA formation by different combinations of some reprogramming factors. (A) 2% E-gel electrophoresis results show that MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, Lin28A-P2A-Nanog-circRNA have circular RNA formation as shown by arrows. (B and C) 0.6% agarose gel electrophoresis for 30 minutes (upper) and 90 minutes (lower) results show that OSK-circRNA, O'SK-circRNA and O'KM'-circRNA, OKM-circRNA, OKMS-circRNA have circular RNA formation as shown by arrows.

[0156] Fig. 20 HEK293T cells were transfected with circular RNA of some reprogramming factors, and the protein expression was verified by WB

[0157] Fig.21 Wild-type OKSMLN factor circular RNA was transfected into fibroblasts to induce iPSCs, and clones appeared after 7 days. DETAILED DESCRIPTION

[0158] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0159] Example 1 Gene synthesis and lentiviral (LV) vector construction

[0160] 1 Transcription factor single component sequence design

[0161] For designing the sequences of OCT4' and variants OCT4'1-6, refer to SEQ ID NO:4-10, and designing the corresponding nucleotide sequences, for example, as shown in SEQ ID NO:3; for designing the sequences of C-MYC and variants C-MYC, refer to SEQ ID NO:14-20, and designing the corresponding nucleotide sequences, for example, as shown in SEQ ID NO:13; for designing the KLF4 sequence, refer to SEQ ID NO:22, and designing the corresponding nucleotide sequences, for example, as shown in SEQ ID NO:21; for designing the SOX2 sequence, refer to SEQ ID NO:24, and designing the corresponding nucleotide sequences, for example, as shown in SEQ ID NO:23.

[0162] Design linker sequences, P2A, T2A, E2A sequences, such as those shown in SEQ ID NOs: 25-27; design Kozak sequence, GCCGCCACC; design TRE3G promoter sequence as shown in SEQ ID NO: 63; design SV40 poly (A) sequence as shown in SEQ ID NO: 64

[0163] 2. Construction of tandem sequences of transcription factor elements

[0164] The sequences formed by connecting the above elements in series are only examples, and can be shown in SEQ ID NOs: 28-33, and their names are OCT4'-P2A-KLF4s-T2A-MYC'-E2A-SOX2 tandem sequence (abbreviated as O'KM'S, corresponding to SEQ ID NO: 28), OCT4-P2A-KLF4s-T2A-MYC-E2A-SOX2 tandem sequence (abbreviated as OKMS, corresponding to SEQ ID NO: 29), OCT4'-P2A-KLF4s-T2A-MYC' tandem sequence (abbreviated as O'KM', corresponding to SEQ ID NO: 30), OCT4-P2A-KLF4s-T2A-MYC tandem sequence (abbreviated as OKM, corresponding to SEQ ID NO: 31), OCT4'-P2A-KLF4s-T2A-SOX2 tandem sequence (abbreviated as O'KS, corresponding to SEQ ID NO: 32). NO:32), OCT4-P2A-KLF4s-T2A-SOX2 tandem sequence (abbreviated as OKS, corresponding to SEQ ID NO:33).

[0165] 3. Lentiviral vector construction and packaging (performed by Saiye Biotechnology Co., Ltd.)

[0166] 3.1 Lentiviral vector construction:

[0167] 1) Gene synthesis

[0168] O'KM'S, OKMS, O'KM' and OKM fragments were gene synthesized.

[0169] 2) Vector backbone digestion

[0170] Select appropriate restriction sites on the vector backbone for enzyme cutting.

[0171] Enzyme digestion using NEB's CutSmart system is as follows

[0172]

[0173]

[0174] After the enzyme digestion is completed, the gel is recovered and the bands are recovered using a gel recovery kit (NEB Monarch DNA). The required backbone fragments and target fragments are prepared for subsequent ligation experiments.

[0175] 3) Connection conversion

[0176] The synthetic gene and other related elements were connected to the linearized vector backbone in sequence. The C115 Infusion ligase system was used for the connection as follows:

[0177] Adding materials volume The processed skeleton 100ng Connect fragments 50ng 2× Enzyme Mix 5μL Sterile water Up to 10μL

[0178]

[0179] After the ligation reaction is completed, the corresponding resistance plate is taken to a 37°C incubator in advance and inverted to dry, and 500μL of non-resistance culture medium is preheated at the same time; take out the competent engineering bacteria from the -80°C refrigerator, place it on ice for 15-20min until it melts, draw half of the ligation solution into the competent state, flick it 5 times with your hand, and ice bath it for 30min; quickly transfer the competent state to a preheated 42°C water bath for heat shock for 1min (the heat shock time is 45-90s, and the time is strictly controlled); after the heat shock time is over, immediately transfer it to ice and place it for 2min, then transfer the competent state to 500μl of preheated culture medium, and place it on a 37°C shaker for 1h; take the bacterial solution and centrifuge it at 4000rpm / 2min to remove the supernatant (discard 400μl of supernatant), pour about 20 glass beads on the plate, pipette the bacterial solution to mix it evenly and apply it to the corresponding resistance plate, and place it in the corresponding temperature incubator for overnight culture.

[0180] After overnight culture, turn on the ultraviolet light of the clean bench, turn off the ultraviolet light and turn on the third gear wind after 20 minutes, take 75% ethanol to wipe the clean bench and start the bacterial test; on the clean bench, add 25μl sterilized water to the bottom of the 96-well PCR plate, and check whether there is air at the bottom against the light; use the sterilized 10μl white gun tip to gently dip the monoclonal plaque on the overnight culture plate, pick it into the corresponding 96-well reaction plate, use a 10μl discharge gun to blow and mix 2-3 times, and transfer 4μl row by row to the new 96-well reaction plate as a template for PCR reaction. Add 50μl of antibiotic-free broth culture medium to the original 96-well plate template, cover the sterilized 96-well PCR plate cover and place it in a 30℃ incubator.

[0181] 4) Bacteria inspection

[0182] The bacterial test uses the P222-AA Taq enzyme reaction system as follows

[0183] Adding materials system 2× Taq Enzyme Mix 12.5μL Single colony water suspension 4μL 2× Enzyme Mix 2×0.75μL Sterile water Up to 25μL

[0184] P222-AA Taq PCR Reaction Procedure

[0185]

[0186] After the bacterial test reaction is completed, add TBE test gel to check whether the band size is consistent with the theoretical band size, then add 3.5mL Amp resistance broth culture base to a 48-deep-well plate and place it in a 37-degree shaking incubator for overnight culture.

[0187] 5) Plasmid extraction

[0188] Qiagen's QIAprep Spin Miniprep Kit (27106) was used for plasmid extraction. The concentration of the extracted plasmid was measured and marked on the wall of a 1.5 mL EP tube. A concentration greater than 50 ng / μL indicated successful extraction and could be identified by enzyme digestion.

[0189] 6) Enzyme digestion identification

[0190] Use NTI / SnapGene software to find available restriction enzyme solutions based on the existing restriction enzymes to identify whether the plasmid is correct. The restriction enzyme system is as follows

[0191] Adding materials system Buffer 2μL Single enzyme / double enzyme 0.7μL / 0.5μL Plasmids 600~800ng Sterile water Up to 20μL

[0192] After the system is prepared, place it at the adaptation temperature of the corresponding enzyme for 45 minutes, take out the enzyme-cut system, add 5ul bromophenol blue / methyl orange to TAE detection gel, and then use a gel imager to take pictures to check the band size (see the results in the attached Figure 1 ).

[0193] If the bands are correct, take 30 μL of plasmid and prepare it for testing on the same day. Send it to GENEWISE for sequencing of the sequence of the junction region.

[0194] 7) Plasmid sent for sequencing

[0195] The constructed plasmid was sent to GeneWeiZi for sequencing, and sequencing proved that the vector was successfully constructed.

[0196] Material situation

[0197] materials source PCR enzymes Novozyme P515 PCR gel recovery kit QIA Gel Extraction Kit(28706) Ligase Novozyme C115 Bacteria detection Taq enzyme Novozyme P222 Competent cells DH5α Plasmid extraction QIAprep Spin Miniprep Kit (27106) PCR, bacterial picking and sequencing primer synthesis and gene synthesis Jin Weizhi

[0198] The successfully constructed lentiviral vectors include LV-TRE3G-OKM, LV-TRE3G-O'KM', LV-TRE3G-OKMS, and LV-TRE3G-O'KM'S, and their plasmid sequences are shown in SEQ ID NOs: 34-37.

[0199] 3.2 Lentivirus packaging:

[0200] 1) Lentiviral plasmids and helper plasmids (SL3, SL4 and SL5) were co-transfected into 293T cells.

[0201] 2) Virus harvesting: 48 hours after transfection, the cell supernatant was collected by centrifugation.

[0202] 3) Conversion and concentration: The cell supernatant was concentrated with PEG8000 and subjected to sucrose density gradient ultracentrifugation.

[0203] 4) Quality testing: physical state testing, sterility testing and titer testing, etc. The titer testing uses the qPCR method (see the attached results). Figure 2A).

[0204] 5) Transgenic expression detection: The above lentiviral vectors were co-transduced with the lentiviral vector expressing TET3G into 293T cells, and dox was used to induce expression. RNA samples were extracted 48 hours later, and the expression level of transgenic KLF4 was detected using transgenic KLF4-specific primers (see the attached results). Figure 2 B).

[0205] Example 2: Four-factor initiation phase reprogramming rejuvenates human skin fibroblasts

[0206] Experimental protocol:

[0207] 1) Select a primary human skin fibroblast cell line FY-009 from a female donor over 60 years old, culture it in a dedicated epidermal cell culture medium, pass it through generations, and select a cell line in good condition for subsequent viral infection experiments;

[0208] 2) After the 4th generation of in vitro culture of fibroblasts, they were seeded in 12-well plates and divided into 3 groups when the confluence reached about 40%: the first group was a blank control group without virus addition; the second group was an OKMS reprogramming group, in which lentivirus LV-TRE3G-OKMS and LV-EFS-Tet3G were added; the third group was an O'KM'S reprogramming group, in which lentivirus LV-TRE3G-O'KM'S and LV-EFS-Tet3G were added. (The lentiviral vector was prepared by Saiye Biotechnology Co., Ltd., and the structural diagram is attached. Figure 3 ). At the same time, polybrene is added to promote the efficiency of viral infection.

[0209] 3) After 24 hours, discard the supernatant and replace with high-glucose DMEM culture medium containing 20% ​​fetal bovine serum

[0210] 4) Continue to change the medium, add doxycycline (dox) to induce transcription factor expression, and change the medium every two days thereafter

[0211] 5) After 14 days of dox induction, cells from each group were collected for detection.

[0212] 6) The cells in each group were stained with senescence-associated β-galactosidase (SA-β-gal) to detect their senescence degree (the experimental results are shown in the attached Figure 4 ). The results showed that both reprogramming groups could rejuvenate cells, but the O'KM'S reprogramming group was more rejuvenated.

[0213] 7) Extract RNA samples from each group of cells and perform real-time fluorescence quantitative polymerase chain reaction (q-PCR) to detect genes related to rejuvenation function: COL1A1, COL3A1, COL4A1, FN1, LAMA5 (see the attached experimental results) Figure 5); Detection of fibroblast identity characteristic genes: THY1, P4HB, S100A4, SERPINH1 (experimental results are attached Figure 6 ); Detection of characteristic genes at the initial stage of reprogramming: NANOG, SALL4 (experimental results are attached Figure 7 ); Detection of reprogramming mature stage characteristic genes: DPPA5, LIN28A (experimental results are attached Figure 8 ). Results The O'KM'S reprogramming group expressed significantly higher levels of genes related to youth function and identity characteristic genes than the OKMS reprogramming group. In addition, from the perspective of characteristic genes in the reprogramming stage, the former was still in the initial stage, while the latter had entered the mature stage.

[0214] 8) Collect cells from each group for methylation chip detection and perform epiAge analysis on the detection results (see attached for analysis results). Fig. 9 ). The results showed that the epiAge of the blank control group was over 60 years old, close to the actual age of the donor. The OKMS partial reprogramming group reduced the epiAge of the cells to 42 years old, while the O'KM'S partial reprogramming group reduced the epiAge of the cells even lower to 39 years old.

[0215] Example 3: Three-factor initiation phase reprogramming rejuvenates human skin fibroblasts

[0216] Experimental protocol:

[0217] 1) Human skin fibroblasts were cultured with a dedicated epidermal cell culture medium, passaged, and cell lines in good condition were selected for subsequent virus infection experiments;

[0218] 2) After the 4th generation of in vitro culture of fibroblasts, they were seeded in 12-well plates and divided into 3 groups when the confluence reached about 40%: the first group was a blank control group without virus addition; the second group was an OKM reprogramming group, in which lentivirus LV-TRE3G-OKM and LV-EFS-Tet3G were added; the third group was an O'KM' reprogramming group, in which lentivirus LV-TRE3G-O'KM' and LV-EFS-Tet3G were added. (The lentiviral vector was prepared by Saiye Biotechnology Co., Ltd., and the structural diagram is attached. Fig.10 ). At the same time, polybrene is added to promote the efficiency of viral infection.

[0219] 3) After 24 hours, discard the supernatant and replace with high-glucose DMEM culture medium containing 20% ​​fetal bovine serum.

[0220] 4) Continue to change the medium, add doxycycline (dox) to induce transcription factor expression, and change the medium every two days thereafter

[0221] 5) After 14 days of dox induction, cells from each group were collected for detection.

[0222] 6) The cells in each group were stained with senescence-associated β-galactosidase (SA-β-gal) to detect their senescence degree (the experimental results are shown in the attached Fig.11 ). The results showed that both reprogramming groups could rejuvenate cells, but the degree of rejuvenation in the OKM reprogramming group was lower, while the degree of rejuvenation in the O'KM' reprogramming group was higher.

[0223] 7) Extract RNA samples from each group of cells and perform real-time fluorescence quantitative polymerase chain reaction (q-PCR) to detect genes related to rejuvenation function: COL1A1, COL3A1, COL4A1, FN1, LAMA5 (see the attached experimental results) Fig.12 ); Detection of fibroblast identity characteristic genes: THY1, P4HB, S100A4, SERPINH1 (experimental results are attached Fig.13 ); Detection of characteristic genes at the initial stage of reprogramming: NANOG, SALL4 (experimental results are attached Fig.14 ); Detection of reprogramming mature stage characteristic genes: DPPA5, LIN28A (experimental results are attached Fig.15 ). Results The O'KM' reprogramming group expressed significantly higher levels of genes related to youth function and identity characteristic genes than the OKM reprogramming group. In addition, from the perspective of characteristic genes in the reprogramming stage, the former was still in the initial stage, while the latter had entered the mature stage.

[0224] Example 4 Gene Synthesis and Adeno-Associated Virus (AAV) Vector Construction

[0225] 1 Transcription factor single component sequence design

[0226] For designing the sequences of OCT4' and variants OCT4'1-6, refer to SEQ ID NO:4-10, and designing the corresponding nucleotide sequences, for example, as shown in SEQ ID NO:3; for designing the sequences of C-MYC and variants C-MYC, refer to SEQ ID NO:14-20, and designing the corresponding nucleotide sequences, for example, as shown in SEQ ID NO:13; for designing the KLF4 sequence, refer to SEQ ID NO:22, and designing the corresponding nucleotide sequences, for example, as shown in SEQ ID NO:21; for designing the SOX2 sequence, refer to SEQ ID NO:24, and designing the corresponding nucleotide sequences, for example, as shown in SEQ ID NO:23.

[0227] Design linker sequence, P2A, T2A, E2A sequence, such as shown in SEQ ID NO: 25-27. Design Kozak sequence, gccgccaccatgg; Design TRE3G promoter sequence, such as shown in SEQ ID NO: 63, and design hGH poly (A) sequence as shown in SEQ ID NO: 65.

[0228] 2. Construction of tandem sequences of transcription factor elements

[0229] The sequences formed by connecting the above elements in series are only examples, and can be shown in SEQ ID NOs: 32-33, and are named OCT4'-P2A-KLF4s-T2A-SOX2 series sequence (abbreviated as O'KS, corresponding to SEQ ID NO: 32), OCT4-P2A-KLF4s-T2A-SOX2 series sequence (abbreviated as OKS, corresponding to SEQ ID NO: 33) respectively.

[0230] 3AAV vector construction and packaging (performed by Saiye Biotechnology Co., Ltd.)

[0231] 3.1 AAV vector construction:

[0232] 1) Select a suitable restriction site combination on the AAV backbone plasmid, incubate the AAV backbone plasmid and the enzyme at 37°C for 1 hour, and then recover the corresponding linear backbone plasmid after restriction digestion by agarose gel electrophoresis.

[0233] 2) Gene synthesis of OKS and O'KS fragments, cloning of gene fragments and other related elements into linear backbone plasmids by homologous recombination kit (Novozyme, C115), transformation of the reaction system into competent bacteria, plating, picking, shaking, extraction, enzyme digestion and sequencing identification.

[0234] 3) After the enzyme digestion and sequencing results are confirmed to be correct (see the attached Fig.16 ), select the plasmid of the correct bacterial solution and transform the competent bacteria again, plate, pick, amplify and extract the plasmid required for AAV packaging, including AAV-TRE3G-OKS and AAV-TRE3G-O'KS, and the plasmid sequence thereof is shown in SEQ ID NO:38-39.

[0235] 3.2AAV packaging:

[0236] 1) AAV viral plasmid, helper plasmid and packaging plasmid were co-infected into 293T cells.

[0237] 2) Virus harvesting: 48 hours after transfection, the cell supernatant was collected by centrifugation.

[0238] 3) Conversion and concentration: The cell supernatant was concentrated with PEG8000 and subjected to sucrose density gradient ultracentrifugation.

[0239] 4) Quality testing: physical state testing, sterility testing and titer testing, etc. The titer testing uses the qPCR method (see the attached titer test results). Fig.17 ).

[0240] Example 5 Gene synthesis and circular mRNA vector construction

[0241] 1. Construction of circular RNA vectors containing different tandem combinations of some reprogramming factors

[0242] a) Design of circular RNAs of different tandem combinations of some reprogramming factors: design single gene, double gene, triple gene and quadruple gene tandems respectively, and connect different genes with P2A, T2A and E2A tandem peptides. See the attached diagram for the vector structure of different gene tandem combinations Fig.18 And, see Table 1.

[0243]

[0244] b) Selection of looping elements: Using the Anabaena group I intron or the Azoarcus sp. BH7 group I intron rearrangement to form a looping framework PIE (permuted intron-exon) structure, different partial reprogramming factors combined with the target gene sequence are recombined within the PIE framework and recombined into the pBlueScript IISK(+) plasmid

[0245] AnaPIE components:

[0246] The 5' element sequence is shown in SEQ ID NO: 49; the 3' element sequence is shown in SEQ ID NO: 50. AzoPIE element:

[0247] 5' element sequence: as shown in SEQ ID NO: 51; 3' element sequence: as shown in SEQ ID NO: 52

[0248] c) Plasmid synthesis: The recombinant plasmid is commissioned to a third-party sequence synthesis company for synthesis. The synthesized plasmid is linearized using EcoR I to obtain a pre-transcription template.

[0249] 2. Preparation and Circularization Verification of Different Tandem Combinations of Some Reprogramming Factors

[0250] a) Circular RNA in vitro transcription and circularization:

[0251] The in vitro transcription system is as follows: Table 2

[0252]

[0253] Incubate at 37°C for 3 h in a PCR instrument, then digest the linear DNA template with DNase I (5 U / μL), digestion concentration: 5 U / μg DNA template, digestion conditions: 37°C digestion for 20 min

[0254] Cyclization reaction:

[0255] Prepare the cyclization system, the cyclization system is as follows: Table 3

[0256]

[0257] Place the prepared cyclization system in a PCR instrument and incubate at 60°C for 20 minutes.

[0258] b) Circular RNA Purification and Identification:

[0259] Transfer the cyclization system to a 1.5mL centrifuge tube, add 0.5 times the volume of 7.5M lithium chloride and 50mM EDTA RNA precipitation solution, mix well and precipitate in a -20℃ refrigerator for 1h. After precipitation is completed, centrifuge at 18360×g for 15min at 4℃ and remove the supernatant as much as possible. Add 800μL of 70% ethanol solution, blow and wash the precipitate, centrifuge again under the same conditions, remove the supernatant as much as possible and evaporate the ethanol, and resuspend each experimental group with 100μL enzyme-free water. Determine the content concentration value and identify the size of circRNA by 1% agarose gel electrophoresis or 2% E-Gel electrophoresis. The electrophoresis results show the formation of circular RNA, the results are attached Fig.19 . 3. Verification of protein expression of some reprogramming factors in eukaryotic cells

[0260] a) Cell preparation and mRNA transfection:

[0261] Take a bottle of HEK293T / 17 cells cultured in T75 and plate them when the cell confluence is 80%-90%. Discard the culture medium, add PBS to wash, discard PBS, add 0.05% trypsin to digest the cells, wait until the cells become round, discard the trypsin, and use 10mL of 10% FBS cell culture medium without antibiotics to disperse the cells. Count the cells and adjust the cell concentration to 1×10 6 cells / mL±10%, take 10 48-well plates, inoculate 0.2 mL of cell suspension respectively, and place the cell culture plates at 37°C and 5% CO 2 Incubator for 24 hours ± 4 hours

[0262] Dissolve 0.4ug circRNA of MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, and Lin28A-P2A-Nanog-circRNA in 25μL serum-free DMEM medium, mix gently, and let stand at room temperature for 5 minutes. Dissolve 1μL Lipohigh transfection reagent in 25μL serum-free DMEM medium, mix gently, and let stand at room temperature for 5 minutes. Add the medium containing LipoHigh in the previous step to the medium containing circRNA, mix gently, and let stand at room temperature for 20 minutes. Remove the cell culture plate from the incubator, drip into the seeded cells drop by drop, mix gently, and place at 37°C, 5% CO 2 After 4 to 8 hours, discard the solution and add 0.5 mL of culture medium containing 2% FBS to continue culturing.

[0263] b) Western blot analysis (WB) results are shown in the attached Fig. 20 The results showed that circular RNA: MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, Lin28A-P2A-Nanog-circRNA can all express corresponding proteins.

[0264] 4. Wild-type OKSMLN circular RNA transfected fibroblasts to induce iPSCs, functional verification experiment: Human skin fibroblasts were planted in 12-well plates and grown to a confluence of 80%-90%. The transfection reagent Lipofectamine MessengerMAX (Thermo Fisher Scientific) was used to encapsulate circular RNA: MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, Lin28A-P2A-Nanog-circRNA, and co-incubated with fibroblasts at a total mRNA volume of 2μg / well for transfection. After 24h, the transfection was terminated and the cell growth was observed. On the 7th day after transfection, the cell growth was continued to be observed to see if any clones appeared. The results are attached. Fig.21 The results showed that wild-type OSKMLN circular RNA could successfully induce fibroblasts to generate pluripotent stem cell-like clones.

[0265] Circular RNA sequences

[0266] MYC circular RNA MYC-circRNA SEQ ID NO: 53 KLF4 circular RNA KLF4-circRNA SEQ ID NO: 54 Lin28A-P2A-Nanog circular RNA LN-circRNA SEQ ID NO: 55 OCT4-P2A-SOX2 circular RNA OS-circRNA SEQ ID NO: 56 OCT4'-P2A-SOX2-T2A-KLF4s circular RNA O'SK-circRNA SEQ ID NO: 57 OCT4-P2A-SOX2-T2A-KLF4s circular RNA OSK-circRNA SEQ ID NO: 58 OCT4'-P2A-KLF4s-T2A-MYC' circular RNA O'KM'-circRNA SEQ ID NO: 59 OCT4-P2A-KLF4s-T2A-MYC circular RNA OKM-circRNA SEQ ID NO: 60 OCT4'-P2A-KLF4s-T2A-MYC'-E2A-SOX2 circular RNA O'KM'S-circRNA SEQ ID NO: 61 OCT4-P2A-KLF4s-T2A-MYC-E2A-SOX2 circular RNA OKMS-circRNA SEQ ID NO: 62

Claims

1. A cell rejuvenation method, characterized in that: Using engineered reprogramming factors without phase separation to mediate cell initiation reprogramming.

2. According to the method of claim 1, the rejuvenation is judged in that, compared with the reference value, the expression level of the epigenetic marker gene is increased or decreased relative to the reference value.

3. The method according to claim 1, wherein the cell rejuvenation is manifested in that the regenerative cells show a change in the proportion of senescence-associated β-galactosidase (SA-β-Gal)-positive cells, and the change is a decrease relative to a reference value.

4. The method described in claim 2, wherein the cell rejuvenation is characterized by a decrease in the epigenetic age epiAge level of cell DNA methylation and / or an increase in the expression level of genes related to rejuvenation functions, relative to a reference value; and / or an increase in the expression level of characteristic genes in the initial stage of reprogramming; and / or a decrease in the expression level of characteristic genes in the mature stage of reprogramming; and / or an increase in the expression level of cell identity genes.

5. The method according to claim 4, wherein the genes related to rejuvenation function include COL1A1, COL3A1, COL4A1, FN1, and LAMA5; the characteristic genes for detecting the initial stage of reprogramming include NANOG and SALL4; the characteristic genes for detecting the mature stage of reprogramming include DPPA5 and LIN28A; and the identity genes of the detected cells include THY1, P4HB, S100A4, and SERPINH1.

6. A method for reprogramming cells at the initial stage, characterized in that: This is achieved by introducing into cells components containing engineered reprogramming factors without phase separation.

7. A phase-separation-free engineered transcription factor, characterized in that: Capable of mediating cell reprogramming at the initial stage.

8. The phase-separation-free engineered transcription factor according to claims 1-7, characterized in that: The engineered phase-separation-free reprogramming factor is based on the modification of the phase-separation function-related region of the transcription factor, so that the transcription factor loses the function of inducing phase separation.

9. The engineered transcription factor of claim 8, wherein the modification comprises amino acid modification, partial deletion, or ubiquitination mutation of the IDR region of the transcription factor protein.

10. According to the engineered transcription factor of claim 9, further, the polar charged amino acids or acidic amino acids in the IDR region of the protein can be mutated; further, the polar charged amino acids or acidic amino acids can be replaced with neutral amino acids; further, the acidic amino acids such as aspartic acid (Asp) and glutamic acid (Glu) can be mutated into neutral amino acids, such as alanine.

11. The engineered transcription factor according to claim 10, wherein positions 1-140 and / or 288-360 of the sequence shown in SEQ ID NO: 2 can be mutated, and the positions include an error range of 10-20 amino acids before and after.

12. The engineered transcription factor according to claim 10, wherein 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, 272 of the sequence shown in SEQ ID NO: 2 is mutated.

13. The engineered transcription factor according to claim 11 or 12, wherein the sequence obtained after mutation can be any one of SEQ ID NOs: 4-10, or a sequence with at least 85%, 90%, or more than 95% identity that can achieve the same function.

14. The engineered transcription factor of claim 9, wherein the modification may be a mutation that does not produce polyubiquitin chain modification; further, lysine K may be replaced by an amino acid that does not produce ubiquitin chain modification; further, lysine K may be replaced by arginine R.

15. The engineered transcription factor according to claim 14, wherein at least one of positions 51, 52, 126, 143, 148, 157, 206, 269, 275, 289, 298, 317, 323, 326, 341, 355, 371, 389, 392, 397, 398, 412, 422, 428, and 430 of SEQ ID NO: 12 is mutated.

16. The engineered transcription factor according to claim 15, further preferably at least one of the amino acids 148 and 389 is mutated; preferably the amino acids at positions 20-25 are mutated; preferably all positions described in claim 15 are mutated.

17. The engineered transcription factor according to claim 16, wherein the sequence obtained after mutation can be a variant shown in SEQ ID NO: 14-20 as an example, or a sequence with at least 85% or 90% or more identity that can achieve the same function.

18. The engineered transcription factor of claim 9, wherein the partial deletion treatment is to delete the key regulatory region of the phase separation of the protein, or to delete the amino acids that play a key role in producing phase separation.

19. The engineered transcription factor according to claim 18, wherein the partial deletion treatment is preferably a terminal deletion treatment, or a deletion treatment is performed on the IDR region, or a deletion treatment is performed on the non-critical region outside the DBD; preferably, a partial deletion treatment is performed on the charged amino acids in the IDR region, or a deletion treatment is performed on the key amino acids modified by the ubiquitination chain.

20. The engineered transcription factor according to claim 19, wherein the key amino acid for modification of the ubiquitination chain is lysine.

21. According to the engineered transcription factor of claim 19, the SOX2 sequence shown in SEQ ID NO: 21-22 can be partially deleted; preferably, positions 2-40 and / or 201-317 are completely or completely deleted to obtain, for example, the amino acid sequence shown in SEQ ID NO: 41-43, or a sequence with at least 85% or 90% or more identity that can achieve the same function.

22. According to the engineered transcription factor of claim 19, the sequence of OCT4 shown in SEQ ID NO: 1-2 can be partially deleted; preferably, positions 2-140 and / or positions 288-360 at the C-terminus are completely or completely deleted to obtain, for example, the amino acid sequence shown in SEQ ID NO: 44-46, or a sequence with at least 85% or 90% or more identity that can achieve the same function.

23. According to the engineered transcription factor of claim 19, the sequence of c-MYC shown in SEQ ID NO: 11-12 can be partially deleted; preferably, positions 2-360 are completely or partially deleted to obtain, for example, the amino acid sequence shown in SEQ ID NO: 47-48, or a sequence with at least 85% or 90% or more identity that can achieve the same function.

24. A nucleic acid capable of expressing the engineered transcription factor of claims 7-23.

25. The nucleic acid of claim 24, comprising SEQ ID NO: 3, 13, 21, or a sequence having at least 85%, 90% or more identity that can achieve the same function.

26. An element comprising the engineered transcription factor described in claims 7-25, including DNA, RNA, a recombinant vector, a recombinant protein, and a recombinant cell; preferably, the recombinant vector can be an adeno-associated virus vector, a poxvirus vector, a lentivirus vector, etc.

27. The element according to claim 25, wherein the RNA element may further be a linear mRNA element, a siRNA element, a circular RNA element, a circular mRNA element, a tRNA element, etc.

28. The element of claim 25, wherein the recombinant vector can be monocistronic, bicistronic or polycistronic, connected by at least one connecting peptide.

29. The element of claim 26, wherein the recombinant vector can be composed of a plurality of transcription factors connected in series, which may be connected by or without at least one connecting peptide, and further, the connecting peptide can be selected from SEQ ID NOs: 25-27.

30. The engineered transcription factor according to claims 7-25 or the element according to claims 26-29, characterized in that At least one selected from OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG and GLIS1.

31. According to the engineered transcription factor or element of claim 30, the engineered transcription factors can be connected in series in sequence, and the connection order can be OCT4-SOX2, LIN28-NANOG, OCT4-SOX2-KLF4, OCT4-KLF4-c-MYC, OCT4-KLF4-c-MYC-SOX2.

32. The engineered transcription factor or element according to claim 31, wherein the tandem sequence may be as shown in SEQ ID NOs: 28-33, and the sequence of the recombinant vector may be as shown in SEQ ID NOs: 34-39.

33. An engineered transcription factor, at least one of which has been modified by the transcription factor of claims 8-23.

34. A delivery system for delivering the element of claims 26-32.

35. The delivery system according to claim 34 can further be liposome LNP, exosome delivery, GalNAc system, VLP delivery, protamine, high molecular polymer, inorganic nanoparticles, exosomes, polymer matrix, viral transfection, etc.

36. Use of the engineered transcription factor described in claims 7-25 or the element comprising the engineered transcription factor described in claims 26-32 or the delivery system described in claims 34-35 in cell therapy, gene therapy, immune cell proliferation, cancer therapy, and treatment of aging-related diseases, or in the preparation of a medicament for cell therapy, gene therapy, immune cell proliferation, cancer therapy, and treatment of aging-related diseases.

37. A pharmaceutical composition comprising at least one engineered transcription factor as described in claims 7-25 or an element comprising an engineered transcription factor as described in claims 26-32 or a delivery system as described in claims 34-35.

38. The pharmaceutical composition according to claim 37, which is a mixture.

39. A method of combined therapy, comprising using the pharmaceutical composition of claims 37-38 in combination with other related drugs.

40. A method of combined therapy, comprising using the pharmaceutical composition of claims 37-38 and B18R protein for combined therapy.

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