Method for cell rejuvenation through initiation-phase reprogramming mediated by non-phase-separating transcription factors

Abstract

Provided in the present invention is a method for cell rejuvenation through initiation-phase reprogramming mediated by non-phase-separating transcription factors. By modifying transcription factors to not have phase-separation capacity, constructing a recombinant vector and introducing same into a target cell, and controlling the cell to be in the initiation phase of reprogramming, the technical effect of rejuvenation is achieved.

Description

A method for rejuvenating cells by initiation-stage reprogramming mediated by transcription factors without phase separation Technical Field

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

[0002] A key approach to induced pluripotent stem cell (iPSC) reprogramming is to induce differentiated cells into pluripotent stem cells through the induction of transcription factors (such as OCT4, SOX2, KLF4, and MYC, referred to as the "four factors"). This entire process, known as complete reprogramming, can be divided into the initiation, maturation, and stabilization phases (Koji et al., 2013, Nelly et al., 2019). During the reprogramming process, cells undergo dramatic changes in transcriptional levels and epigenetic modifications, resulting in significant changes in cell identity and state. In the initiation 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 maturity 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 somatic cell identity characteristic genes whose expression levels do not change much in the initiation stage decrease rapidly (Koji et al., 2013, Nelly et al., 2019), suggesting that at this stage, cells have completely established the 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 is stopped halfway. Since 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, 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 (Ocampo et al. 2016). Currently reported methods for reversing aging using transcription factors are mainly as follows: 1. Partial reprogramming with time-limited in vitro induction, also known as transient reprogramming, such as limiting the overexpression time of four factors through an inducible expression system, so that human fibroblast reprogramming is limited to the initiation stage, which can reverse the age of cells and maintain the characteristics of fibroblasts (Nelly et al., 2019, Diljeet et al., 2022); 2. Transient pulse reprogramming in vivo, such as transient pulse overexpression of four factors in mice through an inducible system, which can rejuvenate the organs of premature aging mice and also rejuvenate the organs of wild-type mice to a certain extent (Alejandro et al., 2016, Kristen et al, 2022); 3. Partial reprogramming with reduced continuous induction of transcription factors (hereinafter referred to as three-factor reprogramming), such as delivering AAV viruses loaded with OSK transcription factor sequences into the mouse eyeball to treat glaucoma (Lu et al. 2020). The above are all existing studies on the use of overexpressed transcription factors to reverse aging. However, there is still much room for improvement in the actual application of avoiding the generation of induced pluripotent stem cells and effectively reversing aging. For example, it is impossible to limit the expression time of the four factors in the human body through a small molecule induction expression system. Therefore, it is necessary to use methods such as engineering transcription factors to limit the risk of generating iPSCs at the source without affecting the effect of cell rejuvenation.

[0004] Phase separation refers to the phenomenon in which certain proteins or nucleic acid molecules within a cell, through multivalent interactions, generate a phase with distinct physicochemical properties within the surrounding homogeneous environment. This phase typically exhibits droplet-like characteristics, resulting in the formation of membraneless organelles or cellular structures. This phenomenon or structure is known as liquid-liquid phase separation (LLPS), or simply phase separation (Clifford et al. 2009, Li et al. 2012). Phase separation describes specific micron-scale structures that create relatively isolated spatial regions that selectively enrich molecules. Compared to the surrounding environment, cellular structures formed by phase separation typically have a higher protein density and reduced molecular microscopic motion, which can promote certain biochemical reactions. The protein structural features that trigger phase separation are still under investigation.

[0005] The present invention utilizes engineered transcription factors that have been deprived of phase separation (hereinafter referred to as phase-independent transcription factors) to participate in multifactorial partial reprogramming. The inventors discovered that phase-independent transcription factors cause partial reprogramming to remain in the initiation phase, rejuvenating cells while maintaining their identity. Therefore, the inventors call this initiation-phase reprogramming technology. This technology improves the efficiency of cell rejuvenation while greatly reducing the risk of iPSC generation, and has great potential for clinical application.

[0006] Summary of the Invention

[0007] Definition of general terms

[0008] 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".

[0009] In this disclosure, "comprise", "include", "contain", "have" and the like 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" and the like, "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.

[0010] 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 reasonably be expected within the recited range, and particularly, with respect to a given quantity, is intended to include a deviation of plus or minus 5 percent.

[0011] 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 as mutually exclusive between alternatives.

[0012] 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.

[0013] The terms "identity" and "homology" as used in the present invention refer to the use of amino acid sequences or nucleotide sequences. Those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, %, 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%, 99.9% similarity, and still have the same function as the original amino acid sequence or nucleotide sequence.

[0014] 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 that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are generally considered in the art to involve minor changes. As understood in the art, these minor changes can be identified, in part, by considering the hydropathic index of the amino acids. (Kyte et al., 1982, J. Mol. Biol. 157: 105-132). The hydropathic index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids having a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that result in a protein retaining biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows calculation of the maximum local average hydrophilicity of the peptide, a useful measurement that is reported to be closely related to antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substituting 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 those of their side chains, such as hydrophobicity, hydrophilicity, charge, size and other properties.

[0015] 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 under stringent conditions to a reference nucleic acid, its complementary sequence, or a sequence substantially identical thereto. A variant can be a nucleic acid sequence that is substantially identical over the entire length of a gene sequence or a fragment thereof. A nucleic acid or protein sequence can 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.

[0016] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or 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 Foundation, Washington, DC). 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. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .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.

[0017] "Hybridization" refers to the formation of double-stranded molecules between complementary polynucleotide sequences (e.g., the genes listed in Tables 1 and 2) or portions thereof under conditions of varying stringency. (See, e.g., Wahl, GM. and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507.) Definitions of general genetic engineering and cell engineering terminology

[0018] 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 comprising, for example, a single cell, a cell population, or a tissue or organ comprising a cell.

[0019] 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 can be xenogeneic, autologous, or allogeneic. The cell can be a primary cell obtained directly from a mammalian subject. The cell can also be a cell derived from the culture and expansion of cells obtained from a subject. In some embodiments, the cell has been genetically engineered to express a recombinant protein and / or nucleic acid.

[0020] Vectors and recombinant vectors are used to carry target genes. In the present invention, preferably lentiviral vectors (LV) are a type of retrovirus and are RNA viruses that can effectively integrate exogenous genes into host chromosomes to achieve persistent expression. In the present invention, preferably adeno-associated viral vectors (AAV) are non-integrated DNA viruses that are widely used in gene therapy due to their high safety, low immunogenicity, and long 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. In the present invention, preferably circular mRNA 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.

[0021] Host cells include dividing and non-dividing cells; they can effectively infect primary cells, neurons, glial cells, stem cells, mesenchymal stem cells, cardiomyocytes, hepatocytes, T cells, tumor cells, and fibroblasts, epithelial cells, blood cells, immune cells, neural cells, and embryonic cells. More specifically, they include human skin fibroblasts such as HFF-1, HSF-1, HDF, and BJ; human skin keratinocytes such as HACAT; human T cells such as Jurkat; and human lung fibroblasts such as IMR-90.

[0022] Definition of professional terms in the field of reprogramming

[0023] The term "regenerative cells" refers to aged cells that have been treated with one or more cell reprogramming factors or transiently reprogrammed such that the cells have transcriptomic characteristics of young cells while still retaining one or more markers of cell identity.

[0024] 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 and have pluripotency and can differentiate into cell types from any of the three germ layers. Induced pluripotent stem cells (iPSCs) 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.

[0025] 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 stabilization stage. In the initiation stage, the cells still maintain their original somatic cell identity, but the expression of early pluripotency-related genes such as NANOG and SALL4 increases rapidly; in the maturity stage, the somatic cell identity characteristics are rapidly lost, and the expression of late pluripotency-related genes such as DPPA5 and LIN28 increases; in the stabilization stage, the 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).

[0026] As used herein, the term "loss of cell identity" refers to the process by which somatic cells, after reprogramming, lose their original somatic cell-specific morphology, function, epigenetic pattern, gene expression profile, and other characteristics (i.e., cell identity) and acquire various characteristics of pluripotent stem cells, thus becoming 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 acquired a higher degree of pluripotent stem cell characteristics. At this point, even if reprogramming is terminated, there is still a high risk that the cells will not be able to recover their original cell identity.

[0027] The term "partial reprogramming" refers to the treatment or transient reprogramming of aged cells with one or more cell reprogramming factors, restoring a certain degree of vitality to the cells, but not enough to dedifferentiate into iPSCs, retaining their original cellular identity. "Transient reprogramming," a form of partial reprogramming, involves exposing cells to cell reprogramming factors for a period of time sufficient to restore vitality but insufficient to induce dedifferentiation into iPSCs. Because the exposure time is very brief, this transient reprogramming results in a lower degree of rejuvenation.

[0028] Initiation phase reprogramming involves engineering reprogramming factors to retain the reprogramming of aged cells at the initiation phase, allowing for a longer window of safe recovery and significantly reducing the risk of developing iPSCs. This differs from transient reprogramming primarily in the engineering of reprogramming factors, allowing for molecular control of the reprogramming phases.

[0029] The term "liquid-liquid phase separation" (LLPS), also known as phase separation, refers to the ability of certain intracellular proteins or nucleic acid molecules to generate a second phase with distinct physicochemical properties within the surrounding homogeneous liquid environment through multivalent interactions. These phases typically manifest as micron-sized droplets (similar to oil droplets in water). These droplets form relatively isolated compartments that selectively enrich certain proteins, resulting in higher protein density and reduced molecular microscopic motion. This can promote biochemical reactions such as gene transcriptional regulation and alterations in chromatin spatial organization. There are various mechanisms for protein phase separation. One relies on the internally disordered domains (IDRs) of proteins. The acidic polar amino acids in these domains carry a negative charge and can interact to form phase separation. Another relies on the ubiquitination of lysines, which can also form phase separation through the interaction of polyubiquitinated chains. "Phase-free" refers to mutant proteins that are unable to form LLPS by mutating the IRD sequence or key amino acids that promote phase separation, thereby impairing (or reducing) their biological function within the cell.

[0030] 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. Among them, 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 can also be Oct4, Sox-2 and Klf4 genes (OSK genes). In some aspects, they can also be Oct4, Klf4 and c-Myc genes (OKM genes). In some aspects, they can also be Sox-2, Klf4 and c-Myc genes (KMS genes).

[0031] 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 that is similar (within the range of at least 50%, 80% or 90% activity) to the closest related factor, 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 throughout 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 an altered polynucleotide sequence. In some embodiments, the amino acid sequence encodes a more robust OCT4 reprogramming factor compared to the amino acid sequence of 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%, or about 99%.

[0032] The "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, cows, pigs, or other animals. Therefore, the "protein," "SOX2 protein," and the like referred to herein include any naturally occurring form of the Sox2 transcription factor or a variant thereof that retains 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 NOs: 23-24.

[0033] "Klf family" refers to Kruppel-like factor 4 or the "Klf" gene encoding the Klf4 protein, which is a protein encoded by the mouse klf4 gene and the human klf4 gene. Exemplary Klf family members include Klf1, Klf2, Klf3, Klf4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16, and Klf17. Thus, the terms "KLF4," "KLF4 protein," and the like as referred to herein include any naturally occurring form of the KFF4 transcription factor or a form thereof that retains KLF4 transcription factor activity (e.g., at least 50%, 80%, 90%, or 100% of the activity compared to wild-type KFF4, as measured by methods known in the art). In some embodiments, the variant has at least 85%, 90%, or 95% amino acid sequence identity throughout its sequence compared to the naturally occurring KFF4 polypeptide. For example, SEQ ID The variant structures shown in NO:21-22.

[0034] The Myc family of factors refers 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," etc. referred to herein include any naturally occurring form or variant of the cMyc transcription factor that maintains cMyc transcription factor activity (e.g., at least 50%, 80%, 90%, or 100% activity compared to wild-type cMyc, as measured by methods known in the art). Specifically, variant improvements can be made based on the wild-type sequences of SEQ ID NOs: 11-12.

[0035] 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.

[0036] Variants

[0037] The present invention also includes fragments, derivatives and analogs of the transcription factor mutants. The terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity as the transcription factor mutants of the present invention. The protein fragments, derivatives or analogs of the present invention may be (i) proteins 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) proteins with a substitution group in one or more amino acid residues, or (iii) proteins 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 the protein, or a fusion protein). According to the definition herein, these fragments, derivatives and analogs fall within the scope known to those skilled in the art. However, in the amino acid sequence of the transcription factor mutants and their fragments, derivatives and analogs, corresponding to the wild-type sequence, there must be a mutation at any of the sites described in the previous paragraph of the present invention.

[0038] Definitions of Terms in the Field of Drug Therapy

[0039] 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 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 arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the 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, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0040] The terms "subject," "individual," "patient," or "subject" include (optionally added if therapeutic use is desired) 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).

[0041] "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 of the present invention before or after the onset of the disease.

[0042] 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 administration to a patient or organ in single or multiple doses.

[0043] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0044] 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 introducing functional genes into cells for expression to treat diseases, inserting functional genes into specific or non-specific genomic locations to replace mutant genes, but other forms include directly correcting mutations or modifying normal genes that are susceptible to pathogen infection, or transferring genes or gene fragments into cells for their transcription.

[0045] The term "cell therapy" refers to pharmaceuticals that achieve therapeutic, diagnostic, and preventive purposes through a series of actions (such as the proliferation and screening of living autologous, allogenic, or xenogenic cells in vitro to restore cell and tissue function, or to change cell biological characteristics by other methods).

[0046] The first aspect of the present invention relates to a method for cell rejuvenation

[0047] The inventors studied the mechanisms of the three stages of cell reprogramming: the initiation phase: cell identity continues to be maintained, early pluripotency genes are upregulated, epigenetic age (epiAge) decreases, and cells begin to rejuvenate; the maturation phase: cell identity characteristics are significantly lost, and late pluripotency genes are upregulated; the stabilization phase: cell identity is completely lost and becomes iPSC; therefore, the time window before the maturation phase is a time window for partial reprogramming (PR). The inventors discovered a new method to prevent cell reprogramming from entering the maturation phase, extend the PR time window, and rejuvenate cells without losing their cell identity. The method uses engineered phase-separation-free reprogramming factors, based on mutations in the phase separation function-related regions of the transcription factors, so that the transcription factors lose their function of initiating phase separation, resulting in their inability to drive cell reprogramming into the maturation phase, thereby causing reprogramming to remain in the initiation phase and continuously rejuvenate the cells, but greatly reducing the risk of reprogramming entering the late phase and losing cell identity. This method is called "initiation phase reprogramming."

[0048] Furthermore, the phase-separation-free engineered transcription factors may be factors such as OCT4, SOX2, KLF4, c-MYC, L-myc, LIN28, NANOG and GLIS1.

[0049] The present invention relates to engineering a transcription factor to cause it to lose its phase separation ability. The inventors have found that there is no need to limit the method for modifying and improving the three-dimensional structure of the transcription factor. As long as it can cause it to lose its phase separation ability and cause cell reprogramming to remain in the initial stage, it is within the scope of protection of the present invention.

[0050] Therefore, the present invention relates to a method for cell initiation stage reprogramming mediated by phase-separation-free engineered transcription factors, which is characterized in that the method is achieved by introducing phase-separation-free engineered transcription factors into cells.

[0051] A cell rejuvenation method characterized by using phase-separation-free engineered transcription factors to mediate cell initiation stage reprogramming.

[0052] A cell reprogramming method is characterized by using a vector containing a phase-separation-free engineered transcription factor to introduce it into a cell.

[0053] Specifically, the criterion for determining cell rejuvenation is that, compared with a reference value, the regenerative cells exhibit a change in gene expression of one or more epigenetic markers, wherein the change is an increase or decrease.

[0054] Specifically, the criterion for determining cell rejuvenation is that, compared with a reference value, regenerative cells exhibit a change in the epigenetic age of cell DNA methylation, epiAge, wherein the change is a decrease.

[0055] Specifically, the criterion for determining cell rejuvenation is, for example, a change in the proportion of senescence-associated β-galactosidase (SA-β-Gal)-positive cells in regenerative cells compared to a reference value, wherein the change is a decrease.

[0056] Specific examples of criteria for determining cell rejuvenation include: Compared to reference values, testing for increased expression of genes associated with rejuvenation: COL1A1, COL3A1, COL4A1, FN1, and LAMA5; testing for increased expression of genes characteristic of the initial reprogramming phase: NANOG and SALL4; and testing for decreased expression of genes characteristic of the mature reprogramming phase: DPPA5 and LIN28A. Cell identity verification can be performed by testing cell identity genes, for example, testing for fibroblast identity genes: THY1, P4HB, S100A4, and SERPINH1. Different identity genes are selected for different cell types; after testing, identity gene expression can be increased relative to the reference value.

[0057] The reference value can be a blank cell control of the same cell type, or a control cell introduced with a wild-type transcription factor recombinant vector. In short, the reference value is not an absolute standard, but rather a relative benchmark for comparison after the modified transcription factor recombinant vector is introduced into the cell. Therefore, the above "criteria for determining rejuvenation" are merely examples, not absolute standards. As long as it can be demonstrated that cell reprogramming is stuck in the initial stage, it is considered within the range of the cell rejuvenation criteria.

[0058] 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.

[0059] It is worth noting that the cell rejuvenation method or cell reprogramming method 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 cell rejuvenation method or cell reprogramming method is not used for the diagnosis, prevention and treatment of disease.

[0060] The modification of the phase-separation-free engineered transcription factor element may be carried out in a specific transformation / mutation / modification manner. The following improvements are merely examples:

[0061] (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.

[0062] The inventors discovered that "negative charge of IDR indicates phase separation of transcription factors" is one of the principles. Therefore, by predicting the internal IDR region of the transcription factor, for example, the Disorder Score of OCT4 is predicted by the VSL2 algorithm. 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. The exemplary prediction results for OCT4 show 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 (DBD) does not belong to the 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 scope of protection of the present invention. After prediction, the inventors found that the conclusions obtained by multiple scoring standards are the same (within the error range), so the present invention can protect the IDR region structure predicted by multiple scoring standards.

[0063] Specifically, amino acid modification can be performed on the internal disordered region (IDR region) of the transcription factor protein, or the activation domain can be modified. Furthermore, charged amino acids can be modified to remove the phase separation ability of the transcription factor.

[0064] Specifically, polar charged amino acids may be replaced with neutral amino acids, and the polar charged amino acids may be acidic amino acids;

[0065] Preferably, the polar charged amino acids in the IDR region may be replaced with neutral amino acids, and the polar charged amino acids may be acidic amino acids;

[0066] More specifically, acidic amino acids such as aspartic acid (Asp) and glutamic acid (Glu) can be mutated into neutral amino acids such as alanine;

[0067] 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.

[0068] In a specific embodiment, the wild-type amino acid sequence of OCT4 may be mutated and improved;

[0069] 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.

[0070] Preferably, the above mutation improvements can 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 can have an error range of about 10-20 amino acid positions before and after.

[0071] 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.

[0072] Exemplarily, the wild-type OCT4 gene can be mutated so that the acidic polar amino acid in the IDR region of its 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.

[0073] The present invention further protects the nucleotide sequence expressing the amino acid sequence of the above-mentioned protein. For example, it can be shown in SEQ ID NO: 3. However, those skilled in the art will know that as long as the target protein can be obtained through codon expression, reasonable changes in codon preference to obtain different nucleotide sequences are all within the scope of protection.

[0074] 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 function of phase-free transcription factor transformation, it is within the protection scope of the present invention.

[0075] (2) Ubiquitination

[0076] The inventors discovered that amino acid modification with polyubiquitin chains can lead to polyubiquitin-mediated phase separation. Therefore, replacing lysine K with arginine R, for example, eliminates polyubiquitin chain modification and can prevent phase separation. However, it is worth noting that the scope of the present invention is to provide a method for ubiquitinating transcription factors to prevent phase separation, and is not limited to a specific mutation method.

[0077] For example, for the C-MYC transcription factor, its ubiquitinated lysine is mutated (K→R), and other transcription factors can also be subjected to ubiquitination mutations.

[0078] Specifically, SEQ ID NOs: 11-12 can be mutated. Based on SEQ ID NO: 12, at least one lysine (K) at 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 can be mutated.

[0079] Preferably, one, two, three, ... 25 of the above positions are mutated; preferably, one of the amino acids 148 and 389, or both, are mutated; preferably, 20-25 positions are mutated; more preferably, all 25 positions are mutated; however, as long as the removal of ubiquitinated chains can be achieved and the desired effect, i.e., phase separation can be avoided, the specific number of mutation positions does not need to be particularly limited.

[0080] 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.

[0081] For example, variants shown in SEQ ID NO: 14-20 are used as examples, or sequences with at least 85%, 90%, or 95% or more identity that can achieve the same function.

[0082] The present invention further protects the nucleotide sequence expressing the amino acid sequence of the above-mentioned protein, which can be exemplified by SEQ ID NO: 13, or a sequence with at least 85%, 90%, or 95% or more identity that can achieve the same function; however, those skilled in the art will appreciate that as long as the target protein can be expressed through codons, different nucleotide sequences obtained by reasonable changes in codon preference according to the needs of the expression cell are all within the scope of protection.

[0083] 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.

[0084] (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 the charged amino acids in the IDR region; or deletion treatment of the key regulatory region of the protein for phase separation, that is, deletion treatment of the key lysine with ubiquitination chain modification, or deletion treatment of amino acids that play a key role in producing phase separation.

[0085] For example, deletions can be performed on the N-terminus or C-terminus of the transcription factor, or on non-critical regions outside its DBD, such as positions 1-20. Furthermore, deletions can be performed on positions 2-20, 1-19, 2-29, 2-18, 2-17, 2, and 16. This approach does not affect the DBD region, i.e., does not affect the original DNA site recognition function of the transcription factor, but may remove its IDR region, ubiquitinated chain-modified lysine, or other key amino acids, thereby avoiding phase separation.

[0086] Specifically, the sequence of KLF transcription factor 4 (KLF4) shown in SEQ ID NOs: 21-22 can be partially deleted. For example, the important DBD region is located at positions 375-470 of the C-terminus. Therefore, the IDR region at positions 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 that is at least 85%, 90%, or more identical to the amino acid sequence that can achieve the same function.

[0087] 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.

[0088] Specifically, the OCT4 sequence shown in SEQ ID NOs: 1-2 can be partially deleted. For example, the important DBD region is located in the middle at positions 141-287. Therefore, the IDR region at positions 2-140 at the N-terminus and / or positions 288-360 at the C-terminus can be completely or partially deleted to obtain, for example, the amino acid sequence shown in SEQ ID NOs: 44-46, or a sequence with at least 85%, 90%, or more than 95% identity that can achieve the same function.

[0089] Specifically, the c-MCY sequence shown in SEQ ID NOs: 11-12 can be partially deleted. For example, its most important DBD is the bHLH-LZ domain at positions 361-439 at the C-terminus. Therefore, positions 2-360 at the N-terminus can be completely or partially deleted to obtain, for example, the amino acid sequence shown in SEQ ID NOs: 47-48, or a sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence. The present invention further protects the nucleotide sequence that expresses the amino acid sequence of the above-mentioned protein. For example, it can be shown in SEQ ID NO: 13, or a sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence. However, those skilled in the art will appreciate that as long as the target protein can be expressed through codons, reasonable changes in codon preference to obtain different nucleotide sequences are within the scope of protection.

[0090] Of course, the present invention is not limited to the above-mentioned transcription factors. As long as a partial deletion process is performed to achieve the function of phase-free transcription factor modification, it is within the scope of protection of the present invention.

[0091] A second aspect of the present invention provides an element comprising an engineered transcription factor

[0092] Nucleic acid elements

[0093] 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, or tRNA elements.

[0094] The elements further include a Kozak sequence, a promoter sequence, at least one connecting peptide sequence, a UTR region, a terminator sequence, a 5' cap, a poly(A) tail, and the like. The nucleic acid element can be a DNA or RNA sequence, or a protein element that is further expressed. The promoter can be selected from inducible promoters such as TRE, TRE3G, TRE3GS, and TRE3GV; constitutive promoters such as CMV, EFS, CAG, and PGK; post-transcriptional regulatory sequences such as WPRE; and poly(A) tails such as SV40 poly(A) and pGH poly(A).

[0095] The mRNA elements include, but are not limited to, linear mRNA and circular mRNA. The circular mRNA may contain an IRES and a self-cleaving polypeptide 2A element. 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 enrichment of the RNA.

[0096] Recombinant vector elements

[0097] The elements of the present invention can be further constructed into vectors, which can be monocistronic, bicistronic, or polycistronic. In one embodiment of the present invention, the coding regions of the bicistronic or polycistronic genes can be linked by at least one connecting peptide, which can be an internal ribosome entry site (IRES) or a self-cleaving 2A peptide (2A).

[0098] IRES sequences that can be used include, but are not limited to, picornaviruses (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 leukoma viruses (e.g., MLV), simian immunodeficiency viruses (e.g., SIV), or cricket paralysis viruses (e.g., CrPV). 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, that enables a single transcript to produce multiple proteins, including, but not limited to, P2A / T2A / E2A / F2A.

[0099] When multiple transcription factors are connected in series, they may be specifically represented by SEQ ID NOs: 25-27. Of course, the present invention also provides vectors for individual transcription factors, which can be mixed in certain ratios, such as a 1:1:1:1 mixture, a 1:2:1:2 mixture, a 1:2:2:1 mixture, or a 2:1:1:1 mixture. Such mixtures are also within the scope of protection of the present invention.

[0100] The present invention further includes recombinant vectors of the aforementioned engineered transcription factors, including but not limited to cloning vectors, expression vectors, shuttle vectors, and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, poxvirus-associated vectors, etc.).

[0101] recombinant protein

[0102] The present invention further includes recombinant protein elements obtained by translation and expression of the above nucleic acid elements, as well as all nucleotide sequences capable of expressing the above protein elements. The protein elements maintain their functions to a certain extent and can also contain certain variants.

[0103] recombinant cells

[0104] The present invention further includes recombinant cells introduced with the aforementioned vector.

[0105] Specifically, the methods provided by the present invention can be applied to any type of cell that needs to be regenerated. The cells can be isolated 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 containing, for example, single cells, cell populations, or tissues or organs containing cells. The cells selected for rejuvenation will depend on the therapeutic effect desired for treating age-related diseases or conditions. The cells are mammalian cells. Including but not limited to prokaryotic cells and eukaryotic cells, further, can be mammalian cells, such as human, monkey, mouse (rats, mice), horse, cow, sheep, pig, dog, cat, 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.

[0106] 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 cell type can 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., red blood cells, 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, gallbladder, intestine, bladder, lung, prostate, breast, genitourinary tract, pituitary gland, mouth, esophagus, skin, hair, nails, thyroid, parathyroid glands, adrenal glands, eyes, nose, or brain.

[0107] Specifically, the cells are selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells. Specifically, the cells are fibroblasts. Specifically, the cells are endothelial cells. Specifically, the cells are chondrocytes. Specifically, the cells are skeletal muscle stem cells. Specifically, the cells are keratinocytes. Specifically, the cells are mesenchymal stem cells. Specifically, the cells are corneal epithelial cells.

[0108] Import and delivery

[0109] 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 lipids, polymers, or lipid-polymer hybrids.

[0110] 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 can comprise natural lipids or synthetic lipids, including conjugated lipids or polymers (e.g., PEGylated lipids). LNPs can comprise any one or more of neutral lipids, zwitterionic lipids, ionizable lipids, cationic lipids, and anionic lipids. In some embodiments, LNPs comprise natural or synthetic monoacyl or diacyl forms of phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA), or monoacyl, diacyl, triacyl, or tetraacyl forms of cardiolipin. In some embodiments, LNPs are micelles or reverse micelles (reverse micelles). In other embodiments, LNPs are unilamellar liposomes or multilamellar liposomes.

[0111] 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.

[0112] A third aspect of the present invention relates to prophylactic and / or therapeutic uses

[0113] The nucleic acid elements (DNA, RNA elements), recombinant vectors, recombinant proteins, and recombinant cells described herein can be used for preventive and / or therapeutic purposes in a variety of fields, and can also be used in the preparation of medicaments for treating diseases. Specific diseases treated include, but are not limited to, cell therapy, gene therapy, immune cell proliferation, cancer treatment, and treatment of aging-related diseases.

[0114] 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 the patient to be treated, transfected with mRNA encoding cell reprogramming factors as described herein, and re-implanted into the patient's body. This type of cell can be obtained, for example, from a biopsy or surgery performed on the 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.

[0115] 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 a mammalian subject. 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, it can include autologous or ex vivo gene therapy. 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. This method can be used to treat genetic disorders and genetic diseases. In the case of "somatic cell gene therapy", therapeutic genes are transferred into the somatic cells of an individual. Any modification and effect will be limited only to that individual and will not be inherited by the individual's offspring or descendants. Specific strategies include, but are not limited to, DNA correction, DNA replacement, DNA supplementation, and DNA inactivation.

[0116] In another aspect, methods for inducing proliferation of cells, such as immune cells, are provided. In some embodiments, the methods comprise exposing the cells to mRNA encoding one or more reprogramming factors, whereby the exposure results in expression of the one or more reprogramming factors in the cells to enhance cell proliferation while preserving their properties. In some embodiments, the methods for inducing proliferation do not induce exhaustion. In some embodiments, proliferation is induced by preventing or reversing exhaustion.

[0117] Specifically, the cell rejuvenation treatment method of the present invention can be used for "age-related diseases or conditions", which specifically refers to 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 eye, diabetic retinopathy, vision loss), skin diseases (dermal atrophy and thinning, elastic hyperplasia and skin wrinkles, sebaceous gland hyperplasia or hypoplasia, pigmentation abnormalities such as senile lentigo, graying, 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 mellitus, 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, ankylosis, diffuse idiopathic skeletal hyperostosis, hematogenous osteomyelitis, muscle 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.

[0118] 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,

[0119] 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, kidney failure, eating disorders, malnutrition, starvation, infection, or treatment with glucocorticoids.

[0120] 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.

[0121] "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 gland cancer; anal cancer; angiosarcomas (e.g., lymphangiosarcoma, lymphangioendothelial sarcoma, 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 cancer); brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchogenic carcinoma; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, 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, Rett's adenocarcinoma); Ewing's sarcoma; eye cancers (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gallbladder cancer; stomach cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumors (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 cancer, oropharyngeal cancer)); hematopoietic cancers (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 lymphomas (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 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, Sézary 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 (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; Neurofibromas (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancers (e.g., gastroenteropancreatic neuroendocrine tumors (GEP-NETs), carcinoid tumors); osteosarcomas (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, embryonal carcinoma, adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic cancer, intraductal papillary mucinous neoplasms (IPMNs), islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pineal gland; primitive neuroectodermal tumors (PNTs); plasma cell tumors; paraneoplastic syndromes; intraepithelial neoplasia; prostate cancer (e.g., prostate cancer); rectal 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., appendiceal 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).

[0122] The fourth aspect of the present invention provides a pharmaceutical composition

[0123] A pharmaceutical composition is generally 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).

[0124] The main ingredients are mainly the nucleic acid elements, recombinant vectors, and recombinant cells mentioned in the first and second aspects of the present invention. The recombinant vector can be formed in series, or by constructing vectors separately to form multiple separate transcription factors and mixing them to form a mixture. The mixing ratio is preferably equal, but different ratios are also allowed on the basis of maintaining the same active function.

[0125] The present invention further provides the use of the aforementioned nucleic acid elements, recombinant vectors, recombinant cells, etc., in the preparation of medicaments for treating diseases that can be prevented or treated by mediating cellular rejuvenation. Furthermore, pharmaceutical compositions for treating diseases that can be prevented or treated by mediating cellular rejuvenation are provided. Furthermore, pharmaceutical compositions for treating diseases that can be prevented or treated by mediating cellular rejuvenation are provided.

[0126] 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.

[0127] Examples of growth factors include, but are not limited to, fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor beta (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.

[0128] In addition to the main ingredients, the pharmaceutical composition may optionally further comprise one or more pharmaceutically acceptable excipients, carriers, adjuvants, and vehicles. Examples of excipients, carriers, adjuvants, and vehicles include, but are not limited to, anti-adherents, adhesives, coatings, compression aids, disintegrants, dyes, lubricants, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, adsorbents, suspending or dispersing agents, or sweeteners. Exemplary excipients, carriers, adjuvants, and vehicles include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (monohydrogen), calcium stearate, croscarmellose, 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.

[0129] The pharmaceutical composition can be prepared in the form of an injectable preparation, an oral preparation, an inhalation preparation, or a microneedle preparation. Injectable preparations are categorized by physical state into liquid injections, powders for injection, and tablets for injection; and by the site of injection into the body into intradermal, subcutaneous, intramuscular, intravenous, intraperitoneal, and spinal injections. Preferred solvents for the injectable preparations include water for injection or physiological saline. Intravenous, intramuscular, and microneedle injections are preferred.

[0130] Formulations for oral use include tablets containing the active ingredient mixed with nontoxic pharmaceutically acceptable excipients. These excipients may 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 and disintegrants (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, croscarmellose 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 carboxymethylcellulose, 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). Formulations 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.

[0131] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavorings, plasticizers, humectants, and buffers. 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 in a conventional manner using, for example, a mixer, fluidized bed apparatus, or spray drying apparatus using the ingredients mentioned above under tablets or capsules.

[0132] The injectable preparations include injectable solutions, injectable emulsions, lyophilized preparations, and suspension injections. Excipients for the injectable preparations 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. Injectable preparations also include, but are not limited to, protein nanoparticles, cyclodextrin inclusions, liposomes, microspheres, reservoir-type controlled-release injections, or needle-free drug delivery systems.

[0133] Combination therapy

[0134] The nucleic acid elements, recombinant vectors, recombinant cells, and pharmaceutical compositions comprising the same can be used in combination with other drugs for treatment. For example, in the context of anti-aging therapy, initial reprogramming can also be combined with protein therapy, such as B18R protein, for anti-aging treatment. 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, resulting in cells being 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, it reduced the risk of generating iPSCs and did not completely reprogram, so it was called "initiation stage reprogramming". The phenotype of the functional genes was used to judge and verify the cell reprogramming stage, proving that the cells achieved the technical effect of rejuvenation.

[0136] Clinically, as cells age, their functions gradually decline, leading to aging and disorders at the tissue, organ, and system levels. Numerous age-related diseases, including but not limited to neurodegenerative diseases, cardiovascular and peripheral vascular diseases, eye diseases, skin diseases, autoimmune diseases, metabolic dysfunction, musculoskeletal disorders, digestive system diseases, cell proliferation-related disorders, and tumors, are rooted in the aging and dysfunction of related tissues and organs. Cell rejuvenation therapies targeting specific tissues and organs can effectively alleviate and treat these diseases, benefiting the general public. Furthermore, extending healthy lifespan will significantly improve social productivity, alleviate medical needs and pressures, and create 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. Lentiviral quality testing. (A) Lentiviral titer and sterility test results; (B) Expression level of transgenic KLF4 after lentiviral transduction of 293T cells.

[0139] FIG3 is a schematic diagram of the structure of the lentiviral vector used in Example 2.

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

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

[0142] Figure 6 Detection of skin fibroblast identity marker gene expression levels. 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 marker gene expression levels 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 significance of the 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] Figure 9 shows the results of cell DNA methylation epigenetic age (epiAge) analysis.

[0146] Figure 10 Structural diagram of the lentiviral vector used in Example 3

[0147] Figure 11: Senescence-associated β-galactosidase (SA-β-gal) staining. A) Representative images of the blank control group. B) Representative images of the OKM reprogramming group. C) Representative images of the O'KM' reprogramming group. D) Statistical analysis of the percentage of cells positive for staining in each group.

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

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

[0150] Figure 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] Figure 15 Detection of expression levels of marker genes in the mature 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. ***: P < 0.001.

[0152] Figure 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] Figure 17 qPCR determination of AAV vector titer. (A) Original amplification curve set; (B) Standard curve; (C) AAV vector titer determination results.

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

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

[0156] Figure 20. WB verification results of protein expression in HEK293T cells transfected with circular RNA of some reprogramming factors

[0157] FIG21 Fibroblasts were transfected with wild-type OKSMLN factor circular RNA to induce iPSCs, and clones appeared after 7 days. DETAILED DESCRIPTION

[0158] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning Laboratory Manual, 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 element sequence design

[0161] For designing the sequences of OCT4' and variants OCT4'1-6, refer to SEQ ID NOs: 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 NOs: 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, such as that shown in SEQ ID NO: 63; design SV40 poly (A) sequence, such as that shown in SEQ ID NO: 64

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

[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) Enzyme digestion of vector backbone

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

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

[0172] 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.

[0173] 3) Connection conversion

[0174] The synthetic gene and other related elements were sequentially ligated to the linearized vector backbone using the C115 Infusion Ligase System as follows:

[0175] After the ligation reaction is completed, the corresponding resistance plate is taken to a 37°C incubator in advance and inverted to dry, and 500 μL of resistance-free culture medium is preheated at the same time; take out the competent engineered bacteria from the -80°C refrigerator, place it on ice for 15-20 minutes until it melts, draw half of the ligation liquid into the competent state, flick it 5 times with your hand, and bathe it on ice for 30 minutes; quickly transfer the competent state to a preheated 42°C water bath for heat shock for 1 minute (heat shock time is 45-90s, time control is strict); after the heat shock time is over, immediately transfer it to ice and place it for 2 minutes, then transfer the competent state to 500 μl of preheated culture medium and place it on a 37°C shaker for 1 hour; take the bacterial solution and centrifuge it at 4000rpm / 2min to remove the supernatant (discard 400 μl 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.

[0176] After overnight incubation, perform a bacterial test. Turn on the ultraviolet light on the clean bench. After 20 minutes, turn off the ultraviolet light and turn on the third setting of the fan. Wipe the clean bench with 75% ethanol and begin the bacterial test. On the clean bench, add 25μl of sterile water to the bottom of the 96-well PCR plate and check for air at the bottom by holding it up to the light. Use a sterilized 10μl white pipette tip to gently dip the monoclonal bacterial plaque on the overnight culture plate and transfer it to the corresponding 96-well reaction plate. Use a 10μl pipette to mix 2-3 times. Pipette 4μl per row and transfer it to a new 96-well reaction plate as a template for the PCR reaction. Add 50μl of antibiotic-free broth culture medium to the original 96-well plate template, cover with a sterilized 96-well PCR plate lid, and place in a 30°C incubator.

[0177] 4) Bacterial examination

[0178] The bacterial test uses the P222-AA Taq enzyme reaction system as follows

[0179] P222-AA Taq enzyme PCR reaction procedure

[0180] 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 3.5 mL of Amp resistance broth culture medium to a 48-deep-well plate and place it in a 37-degree shaker for overnight culture.

[0181] 5) Plasmid extraction

[0182] Plasmids were extracted using Qiagen's QIAprep Spin Miniprep Kit (27106). 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 allowed for enzyme digestion identification.

[0183] 6) Enzyme digestion identification

[0184] Use NTI / SnapGene software to find available enzyme digestion schemes based on existing enzymes to identify whether the plasmid is correct. The enzyme digestion system is as follows

[0185] After the system is prepared, it is placed at the adaptation temperature of the corresponding enzyme for 45 minutes. The enzyme-digested system is taken out, 5 μl of bromophenol blue / methyl orange is added to the TAE detection gel, and the band size is checked by taking a photo with a gel imager (see Figure 1 for the results).

[0186] If the bands are correct, pipette 30 μL of plasmid for testing on the same day. Send the plasmid to GENEWIZ for sequencing to determine the sequence of the junction region.

[0187] 7) Plasmid sent for sequencing

[0188] The constructed plasmid was sent to GeneWeiZ for sequencing, and sequencing proved that the vector was successfully constructed.

[0189] Material situation

[0190] The successfully constructed lentiviral vectors include LV-TRE3G-OKM, LV-TRE3G-O'KM', LV-TRE3G-OKMS, and LV-TRE3G-O'KM'S. The plasmid sequences thereof are shown in SEQ ID NOs: 34-37.

[0191] 3.2 Lentiviral packaging:

[0192] 1) Lentiviral plasmids and helper plasmids (SL3, SL4, and SL5) were co-transfected into 293T cells.

[0193] 2) Virus harvest: 48 hours after transfection, the cell supernatant was collected by centrifugation.

[0194] 3) Conversion and concentration: The cell supernatant was concentrated with PEG8000 and subjected to sucrose density gradient ultracentrifugation.

[0195] 4) Quality testing: physical state testing, sterility testing and titer testing, etc. Titer testing uses qPCR method (results

[0196] See Figure 2A).

[0197] 5) Transgene Expression Detection: 293T cells were co-transduced with the aforementioned lentiviral vectors and a lentiviral vector expressing TET3G. Expression was induced with doxorubicin (dox). RNA samples were extracted 48 hours later, and transgenic KLF4 expression levels were detected using transgenic KLF4-specific primers (see Figure 2B for results).

[0198] Example 2: Four-factor priming phase reprogramming rejuvenates human skin fibroblasts

[0199] Experimental plan:

[0200] 1) Select a primary human skin fibroblast cell line, FY-009, from a female donor aged 60 or older. Culture and passage the cell line using a dedicated epidermal cell culture medium, and select cells in good condition for subsequent viral infection experiments.

[0201] 2) After the fourth passage of in vitro culture, fibroblasts were seeded into 12-well plates and, when confluency reached approximately 40%, divided into three groups: a blank control group (no virus added); an OKMS reprogramming group (additional lentiviruses, LV-TRE3G-OKMS and LV-EFS-Tet3G); and an O'KM'S reprogramming group (additional lentiviruses, LV-TRE3G-O'KM'S and LV-EFS-Tet3G). (The lentiviral vectors were produced by Saiye Biotechnology Co., Ltd.; see Figure 3 for a schematic diagram of their structure.) Polybrene was also added to enhance viral infection efficiency.

[0202] 3) After 24 hours, discard the supernatant and replace with high-glucose DMEM culture medium containing 20% ​​fetal bovine serum

[0203] 4) Continue to change the medium and add doxycycline (dox) to induce transcription factor expression. Change the medium every two days thereafter.

[0204] 5) After 14 days of dox induction, cells from each group were collected for detection.

[0205] 6) Cells in each group were stained for senescence-associated β-galactosidase (SA-β-gal) to assess their senescence (see Figure 4 for experimental results). The results showed that both reprogramming groups rejuvenated cells, with the O'KM'S reprogramming group exhibiting a higher degree of rejuvenation.

[0206] 7) RNA samples were extracted from each cell group and subjected to real-time quantitative polymerase chain reaction (q-PCR) to detect genes associated with rejuvenation: COL1A1, COL3A1, COL4A1, FN1, and LAMA5 (see Figure 5 for experimental results); fibroblast identity signature genes: THY1, P4HB, S100A4, and SERPINH1 (see Figure 6 for experimental results); genes characteristic of the initiation phase of reprogramming: NANOG and SALL4 (see Figure 7 for experimental results); and genes characteristic of the mature phase of reprogramming: DPPA5 and LIN28A (see Figure 8 for experimental results). Results: The O'KM'S reprogramming group expressed significantly higher levels of genes associated with rejuvenation and identity signature genes than the OKMS reprogramming group. Furthermore, judging by the reprogramming signature genes, the former was still in the initiation phase, while the latter had entered the mature phase.

[0207] 8) Cells from each group were collected for methylation microarray analysis, and the results were analyzed for epiage (see Figure 9 ). The results showed that the epiage of the blank control group was over 60 years old, close to the chronological age of the donor. The OKMS partial reprogramming group reduced the epiage to 42 years old, while the O'KM'S partial reprogramming group reduced the epiage even further, to 39 years old.

[0208] Example 3: Three-factor initiation phase reprogramming rejuvenates human skin fibroblasts

[0209] Experimental plan:

[0210] 1) Human skin fibroblasts were cultured in a dedicated epidermal cell culture medium, passaged, and cell lines in good condition were selected for subsequent viral infection experiments;

[0211] 2) After the fourth passage of in vitro culture, fibroblasts were seeded into 12-well plates and, when confluency reached approximately 40%, divided into three groups: a blank control group (no virus added); an OKM reprogramming group (additional lentiviruses LV-TRE3G-OKM and LV-EFS-Tet3G); and an O'KM' reprogramming group (additional lentiviruses LV-TRE3G-O'KM' and LV-EFS-Tet3G). (The lentiviral vector was prepared by Saiye Biotechnology Co., Ltd.; see Figure 10 for a schematic diagram of its structure.) Polybrene was also added to enhance viral infection efficiency.

[0212] 3) After 24 hours, discard the supernatant and replace with high-glucose DMEM culture medium containing 20% ​​fetal bovine serum

[0213] 4) Continue to change the medium and add doxycycline (dox) to induce transcription factor expression. Change the medium every two days thereafter.

[0214] 5) After 14 days of dox induction, cells from each group were collected for detection.

[0215] 6) Cells in each group were stained for senescence-associated β-galactosidase (SA-β-gal) to assess their senescence (see Figure 11 for experimental results). The results showed that both reprogramming groups rejuvenated cells, but the degree of rejuvenation was lower in the OKM reprogramming group, while higher in the O'KM' reprogramming group.

[0216] 7) RNA samples were extracted from each cell group and subjected to real-time quantitative polymerase chain reaction (q-PCR) to detect genes associated with rejuvenation: COL1A1, COL3A1, COL4A1, FN1, and LAMA5 (see Figure 12 for experimental results); fibroblast identity signature genes: THY1, P4HB, S100A4, and SERPINH1 (see Figure 13 for experimental results); genes characteristic of the initiation phase of reprogramming: NANOG and SALL4 (see Figure 14 for experimental results); and genes characteristic of the mature phase of reprogramming: DPPA5 and LIN28A (see Figure 15 for experimental results). Results: The O'KM' reprogramming group expressed significantly higher levels of genes associated with rejuvenation and identity signature genes than the OKM reprogramming group. Furthermore, judging by the reprogramming signature genes, the former was still in the initiation phase, while the latter had entered the mature phase.

[0217] Example 4 Gene Synthesis and Adeno-Associated Virus (AAV) Vector Construction

[0218] 1. Transcription factor single element sequence design

[0219] For designing the sequences of OCT4' and variants OCT4'1-6, refer to SEQ ID NOs: 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 of C-MYC, refer to SEQ ID NOs: 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.

[0220] Design linker sequences, P2A, T2A, and E2A sequences, such as those shown in SEQ ID NOs: 25-27, design Kozak sequence, gccgccaccatgg, design TRE3G promoter sequence, such as that shown in SEQ ID NO: 63, and design hGH poly(A) sequence, such as that shown in SEQ ID NO: 65.

[0221] 2. Construction of tandem transcription factor element sequences

[0222] The sequences formed by connecting the above elements in series are merely examples and may be those shown in SEQ ID NOs: 32-33, and are respectively named OCT4'-P2A-KLF4s-T2A-SOX2 tandem sequence (abbreviated as O'KS, corresponding to SEQ ID NO: 32) and OCT4-P2A-KLF4s-T2A-SOX2 tandem sequence (abbreviated as OKS, corresponding to SEQ ID NO: 33).

[0223] 3AAV vector construction and packaging (performed by Saiye Biotechnology Co., Ltd.)

[0224] 3.1 AAV vector construction:

[0225] 1) Select an appropriate restriction enzyme cleavage site combination on the AAV backbone plasmid, incubate the AAV backbone plasmid and enzyme at 37°C for 1 hour, and then recover the corresponding linear backbone plasmid after enzyme cleavage by agarose gel electrophoresis.

[0226] 2) Gene synthesis of OKS and O'KS fragments. The gene fragments and other related elements were cloned into linear backbone plasmids using a homologous recombination kit (Novozyme, C115). The reaction system was transformed into competent bacteria, plated, picked, shaken, and extracted. Enzyme digestion and sequencing were performed for identification.

[0227] 3) After enzyme digestion and sequencing results were confirmed to be correct (see Figure 16 for enzyme digestion results), the plasmid from the correct bacterial solution was selected for another transformation into competent bacteria. Plating, picking, amplification, and extraction yielded the plasmids required for AAV packaging, including AAV-TRE3G-OKS and AAV-TRE3G-O'KS. See SEQ ID NOs: 38-39 for their plasmid sequences.

[0228] 3.2 AAV packaging:

[0229] 1) AAV viral plasmid, helper plasmid and packaging plasmid were co-infected into 293T cells.

[0230] 2) Virus harvest: 48 hours after transfection, the cell supernatant was collected by centrifugation.

[0231] 3) Conversion and concentration: The cell supernatant was concentrated with PEG8000 and subjected to sucrose density gradient ultracentrifugation.

[0232] 4) Quality testing: physical status testing, sterility testing, and titer testing, etc. The titer testing uses the qPCR method (see Figure 17 for titer test results).

[0233] Example 5 Gene Synthesis and Circular mRNA Vector Construction

[0234] 1. Construction of circular RNA vectors containing different tandem combinations of some reprogramming factors

[0235] a) Design of circular RNAs for various tandem combinations of some reprogramming factors: Single, dual, triple, and quadruple gene concatemers were designed, with P2A, T2A, and E2A tandem peptides used to connect the different genes. Schematic diagrams of the vector structures for various tandem gene combinations are shown in Figure 18 and Table 1.

[0236] b) Selection of loop elements: Using the Anabaena group I intron or the Azoarcus sp. BH7 group I intron rearranged to form a loop framework PIE (permuted intron-exon) structure, different partial reprogramming factors combined with the target gene sequence were recombined within the PIE framework and recombined into the pBlueScript II SK(+) plasmid.

[0237] AnaPIE components:

[0238] The 5' element sequence is shown in SEQ ID NO: 49; the 3' element sequence is shown in SEQ ID NO: 50

[0239] AzoPIE elements:

[0240] 5' element sequence: as shown in SEQ ID NO: 51; 3' element sequence: as shown in SEQ ID NO: 52

[0241] c) Plasmid synthesis: The recombinant plasmid was commissioned to a third-party sequence synthesis company for synthesis. The synthesized plasmid was linearized using EcoR I to obtain the template before transcription.

[0242] 2. Preparation and Circularization Verification of Different Tandem Combinations of Some Reprogramming Factors

[0243] a) In vitro transcription and circularization of circular RNA:

[0244] The in vitro transcription system is as follows: Table 2

[0245] 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

[0246] Cyclization reaction:

[0247] Prepare the cyclization system as follows: Table 3

[0248] Place the prepared cyclization system in a PCR instrument and incubate at 60°C for 20 minutes.

[0249] b) Circular RNA Purification and Identification:

[0250] Transfer the cyclized system to a 1.5 mL centrifuge tube, add 0.5 volumes of RNA precipitation solution containing 7.5 M lithium chloride and 50 mM EDTA, mix well, and precipitate in a -20°C refrigerator for 1 hour. After precipitation, centrifuge at 18,360 × g for 15 minutes at 4°C, and remove as much of the supernatant as possible. Add 800 μL of 70% ethanol solution to rinse the precipitate. Centrifuge again under the same conditions, remove as much of the supernatant as possible, and evaporate the ethanol. Resuspend each experimental group in 100 μL of enzyme-free water. Determine the concentration and size of the circRNA using 1% agarose gel electrophoresis or 2% E-Gel electrophoresis. Electrophoresis results indicate the formation of circular RNA (see Figure 19).

[0251] 3. Verification of protein expression of some reprogramming factors in eukaryotic cells

[0252] a) Cell preparation and mRNA transfection:

[0253] Take a bottle of HEK293T / 17 cells cultured in a T75 microplate and plate them when the confluence is 80%-90%. Discard the culture medium, add PBS to wash, discard the PBS, add 0.05% trypsin to digest the cells. When the cells become round, discard the trypsin and disperse the cells by pipetting with 10 mL of 10% FBS cell culture medium without antibiotics. 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 in a 37°C, 5% CO2 incubator for 24 hours±4 hours

[0254] Dissolve 0.4 μg of each MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, and Lin28A-P2A-Nanog-circRNA in 25 μL of serum-free DMEM medium, mix gently, and incubate at room temperature for 5 minutes. Dissolve 1 μL of LipoHigh transfection reagent in 25 μL of serum-free DMEM medium, mix gently, and incubate at room temperature for 5 minutes. Add the medium containing LipoHigh from the previous step to the medium containing the circRNA, mix gently, and incubate at room temperature for 20 minutes. Remove the cell culture plate from the incubator and drip dropwise onto the seeded cells. Mix gently and incubate at 37°C, 5% CO2. After 4-8 hours, discard the solution and add 0.5 mL of medium containing 2% FBS to continue incubation.

[0255] b) Western blot analysis (WB) results are shown in Figure 20 , which showed that circular RNAs: MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, and Lin28A-P2A-Nanog-circRNA were all able to express corresponding proteins.

[0256] 4. Wild-type OKSMLN circular RNA transfected fibroblasts to induce iPSCs, functional verification experiment: Human skin fibroblasts were seeded 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 2 μg of total mRNA / well was co-incubated with fibroblasts for transfection. After 24 hours, the transfection was terminated and the cell growth was observed. On the 7th day after transfection, the cell growth was continued to observe whether clones appeared. The results are shown in Figure 21. The results showed that wild-type OSKMLN circular RNA can successfully induce fibroblasts to produce pluripotent stem cell-like clones.

[0257] circular RNA sequences

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 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 may be 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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