A method for rejuvenating cells by reprogramming them to the transcription factor-mediated initiation stage, which lacks phase separation ability.

Abstract

This invention provides a method for rejuvenating cells through initial reprogramming via a transcription factor that lacks phase separation ability. By modifying the transcription factor to lack phase separation ability, constructing a recombinant carrier, introducing target cells, and controlling the reprogramming of the cells in the initial stage, the technical effect of rejuvenation is achieved.

Description

Technical Field

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

Background Art

[0002] The reprogramming technology of induced pluripotent stem cells (iPSCs) has an important application in inducing differentiated cells into induced pluripotent stem cells by transcription factors (such as OCT4, SOX2, KLF4, and MYC, hereinafter simply referred to as the four factors). The whole process is completely reprogrammed and can be divided into an initiation stage, a maturation stage, and a stabilization stage (Koji et al, 2013, Nelly et al., 2019). In the reprogramming process, cells undergo huge changes in cell identity and state via drastic changes in transcriptional levels and epigenetic gene modifications. In the initiation stage, early pluripotency-related genes, such as TRA-1-60, NANOG, SSEA4, SALL4, etc., rapidly increase and are expressed. However, the change in the expression level of somatic cell identity characteristic genes is not significant. In the maturation stage, in addition to the above-mentioned early pluripotency-related genes, maturation-stage-specific pluripotency-related genes such as DPPA5 and LIN28 are also highly expressed, but the somatic cell identity characteristic genes with insignificant changes in expression levels in the initiation stage rapidly decrease (Koji et al, 2013, Nelly et al., 2019), suggesting that it is difficult for cells to maintain their original somatic cell identity characteristics at this stage. In the stabilization stage, cells have already completely constructed a pluripotent gene expression network and become iPSCs. In addition to expressing the above-mentioned pluripotency-related genes, typical key pluripotency transcription factors such as OCT4, SOX2, and KLF4 are also highly expressed, and at the same time, somatic cell identity characteristic genes are hardly expressed. Also, this process can rejuvenate cells, and rejuvenation can be measured by changes in specific gene transcription levels, cell aging markers, especially epigenetic age (epiAge) based on DNA methylation (Nelly et al., 2019, Antoine, et al, 2022, Diljeet et al., 2022).

[0003] Partial reprogramming refers to the complete reprogramming process being halted midway. Cell rejuvenation occurs at the initiation stage of reprogramming, while the loss of somatic cell characteristics occurs in the mid-to-late stages. Therefore, a time interval exists between these stages, allowing for cell rejuvenation while simultaneously maintaining the cell's original characteristics. This is the principle behind cell rejuvenation through partial reprogramming (Ocampo et al. 2016). Currently reported methods for age reversal using transcription factors are mainly as follows: 1. Partial reprogramming with limited in vitro induction time, also called transient reprogramming. For example, by limiting the overexpression time of four factors using an inducible expression system, the reprogramming of human fibroblasts is restricted to the initiation stage, thereby reversing cell age and maintaining fibroblast characteristics (Nelly et al., 2019, Diljeet et al., 2022). 2. Reprogramming of intracellular instantaneous pulses, for example, using a system induced in the mouse body, can overexpress four factors via instantaneous pulses, thereby rejuvenating organs in progeria mice and consistently rejuvenating each organ in wild-type aged mice (Alejandro et al., 2016, Kristen et al., 2022). 3. Partial reprogramming that reduces the sustained induction of transcription factors (hereinafter simply referred to as three-factor reprogramming) can, for example, deliver an AAV virus carrying the OSK transcription factor sequence into the eyeball of a mouse to treat glaucoma (Lu et al. 2020). All of the above are age reversal studies using overexpressed transcription factors conducted to date. However, there is a significant need for improvement in practical use to effectively reverse age while avoiding the induction of pluripotent stem cell generation. For example, in the human body, it is not possible to limit the expression time of the four factors by a small molecule-induced expression system. Therefore, methods such as engineering and modification of transcription factors limit the risk of iPSC generation without affecting the cell rejuvenation effect.

[0004] Phase separation refers to the process in which certain proteins or nucleic acid molecules within a cell, through multivalent interactions, generate another phase with different physicochemical properties in the otherwise homogeneous surrounding environment. This phase typically exhibits droplet-like characteristics, resulting in the formation of membrane-free cell 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 structures on a micron scale, creating relatively independent spatial regions and selectively concentrating molecules. Cellular structures formed by phase separation typically have higher protein density and weaker molecular micromotion compared to their surrounding environment, which can facilitate certain biochemical reactions. The characteristics of protein structures capable of forming phase separation are still being studied.

[0005] This invention utilizes a transcription factor that removes engineered phase separation ability (hereinafter simply referred to as a transcription factor lacking phase separation ability) to engage in multifactorial reprogramming. The inventors discovered that the transcription factor lacking phase separation ability stalls partial reprogramming at the initial stage, rejuvenating cells while maintaining their characteristic features. Therefore, the inventors refer to this technique as the initial stage reprogramming technique. This technique improves the efficiency of cell rejuvenation while significantly reducing the risk of iPSC generation, making its clinical use highly desirable. [Overview of the project]

[0006] General term definitions When used in the claims and / or specification with the term “including,” “a” or “an” may mean “one,” but may also mean “one or more,” “at least one,” and “one or more.”

[0007] In this disclosure, "includes," "equipped with," "contains," "possesses," etc., have the meanings given to them in the patent laws of China, the United States, Europe, etc., and can refer to "comprehensible," "covers," etc., and "basically consists of," "basic configuration," etc., have the meanings given to them in the patent laws of China, the United States, Europe, etc., and such terms are open and allow for more than the cited material to exist, except in cases where the basic or novel features of the cited material are not altered by more than the cited material, however, embodiments of the prior art are excluded.

[0008] In this disclosure, the term “approximately” means that the value includes the standard deviation of the error of the apparatus or method used to measure the value, and that any substitutable features reasonably expected within the range listed, and in particular include a predetermined number, e.g., ±5% deviation.

[0009] In this disclosure, the definition of the term "or" means only substitutes and only "and / or", however, unless otherwise expressly indicated, the term "or" in the claims means "and / or".

[0010] In this disclosure, the terms “optional,” “optional,” or “optional” generally mean that the events or conditions described below do not necessarily occur, and such descriptions include cases in which the events or conditions occur and cases in which they do not.

[0011] In this disclosure, "identity" and "homology" refer to amino acid sequences or nucleotide sequences in which a person skilled in the art can adjust the sequence according to the actual work requirements and compare the sequence used with a sequence obtained by the prior art, without limiting the following: 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%, They have similarity densities of 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9%, and still have the same function as the original amino acid sequence or nucleotide sequence.

[0012] The term "mutant" in relation to peptides or polypeptides refers to a peptide or polypeptide whose amino acid sequence differs due to an insertion, deletion, or conserved substitution of amino acids, but which retains at least one biological activity. A mutant refers to a protein that has substantially the same amino acid sequence as a reference protein that has an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., substitution of amino acids with different amino acids that have similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are generally considered to be minute changes in this field. As understood in this field, such minute changes can be partially identified by considering the hydrophilicity index of amino acids (Kyte et al., 1982, J. Mol. Biology. 157:105-132). The hydrophilicity index of amino acids is considered based on their hydrophobicity and charge. It is known in this field that amino acids with similar hydrophilicity indices can be substituted and still retain protein function. On the other hand, amino acids with a hydrophilicity index of ±2 have been substituted. The hydrophilicity of amino acids can further be used to identify protein substitutions that lead to the retention of biological function. In the context of peptides, considering the hydrophilicity of amino acids is a useful measurement that allows for the calculation of the maximum local mean hydrophilicity of the peptide, which has been reported to be closely related to antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference. As understood in the art, substitution of amino acids with similar hydrophilicity values ​​may result in peptides that retain biological activity, such as immunogenicity. Substitutions can be made with amino acids having a difference in hydrophilicity of ±2 or less from each other. Both the hydrophobicity index and hydrophilicity of amino acids are influenced by the specific side chains of those amino acids. Consistent with these observations, it is understood that amino acid substitutions suitable for biological function depend on the relative similarities of the amino acids, particularly their side chains, such as hydrophobicity, hydrophilicity, charge, size, and other properties.

[0013] As used in this disclosure, the term "mutant" in relation to nucleic acids means (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, or (iv) a nucleic acid obtained by strictly crossing a reference nucleic acid, its complementary sequence, or a sequence substantially identical thereto. A mutant may be a nucleic acid sequence substantially identical over the entire length of the entire gene sequence or fragment thereof. Nucleic acid and protein sequences are identical over the entire length of the gene, protein sequence or fragment thereof in 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%.

[0014] In this disclosure, the terms “identity,” “homology,” or “similarity” are all used in the usual manner when describing amino acid sequences or nucleic acid sequences relative to a reference sequence to determine the percentage of identical amino acid sequences or nucleic acid sequences between two sequences. For example, Ausubel, et al. (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). See Foundation, Washington, DC. Many algorithms are used for sequence comparison and sequence identity determination, including the identity comparison algorithm by Needleman et al. (1970) J.Mol.Biol.48:443. Other algorithms include the local identity algorithm by Smith et al. (1981) Adv.Appl.Math.2:482, the similarity search method by Pearson et al. (1988) Proc.Natl.Acad.Sci.85:2444, and the Smith-Waterman algorithm (Meth.Mol.Biol.7 These include 0:173-187 (1997), and the BLASTP, BLASTN, and BLASTX algorithms (Altschul et al. (1990) J.Mol.Biol.215:403-410). Computer programs utilizing these algorithms are also available, including, but are not limited to, ALIGN or Megal (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)). Alternatively, GAP, BESTFIT, BLAST Altschul, etc., as well as the above FASTA and TFASTA, are available from the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA.This includes CLUSTAL, a PC / Gene program provided by Intelligenetics, Mountain View, California.

[0015] "Crossing" refers to the formation of double-stranded molecule pairs between complementary polynucleotide sequences (e.g., the genes listed in Tables 1 and 2) or portions thereof under different strict conditions. (See, for example, Wahl, GM. and SLBerger (1987) Methods Enzymol. 152:399, and Kimmel, AR (1987) Methods Enzymol. 152:507).

[0016] Definitions of technical terms in general genetic engineering and cell engineering. The term "cell" means a complete, living cell, whether naturally occurring or modified. Cells can be isolated from other cells, mixed with other cells in culture, or mixed in tissue (partial or complete) or in a living organism. The methods relating to this disclosure are performed, for example, on a sample containing a single cell, a group of cells, or a tissue or organ containing cells.

[0017] As used in this disclosure, the term “mammalian cells” means any cells derived from a mammalian subject suitable for transplantation into the same or different subjects. The cells may be heterogeneous, autologous, or homogeneous. The cells may be primary cells obtained directly from a mammalian subject. The cells may further be cells derived from cultures and amplifications of cells obtained from a subject. In some embodiments, the cells are genetically engineered to express recombinant proteins and / or nucleic acids.

[0018] Carriers and recombinant carriers are used to support target genes. In the present invention, lentiviral vectors (LVs) are preferred, which are a type of reverse transcription virus, an RNA virus, and can effectively integrate exogenous genes into host chromosomes, thereby achieving sustained expression. In the present invention, adeno-associated viral vectors (AAVs) are preferred, which are non-incompatible DNA viruses and have relatively high safety, relatively low immunogenicity, and relatively long in vivo expression times, and are therefore widely used in gene therapy. Engineered mRNAs, including linear mRNA and circular mRNA, are novel gene carriers applied to non-viral gene delivery systems and are used in in vivo gene therapy. In the present invention, circular mRNA is preferred, which has even lower immunogenicity and higher stability compared to linear mRNA, and is suitable for long-term expression of therapeutic proteins in the body, and is therefore increasingly used in gene therapy. However, all carriers used for support fall within the scope of protection of the present invention.

[0019] Host cells include dividing and non-dividing cells and can effectively infect primary cells, neurons, glial cells, stem cells, mesenchymal stem cells, cardiomyocytes, hepatocytes, T cells, tumor cells, etc., as well as fibroblasts, epithelial cells, blood cells, immune cells, nerve cells, and germ cells. More specifically, for example, human skin fibroblasts such as HFF-1, HSF-1, HDF, BJ, etc. For example, human skin keratinocytes such as HACAT, for example. For example, human T cells such as Jurkat, for example. For example, human lung fibroblasts such as IMR-90, for example.

[0020] Definitions of technical terms in the field of reprogramming The term "regenerated cells" refers to senescent cells that have been treated or transiently reprogrammed with one or more cell transcription factors to possess the transcriptomics characteristics of young cells while retaining one or more cell-identifying markers.

[0021] The term "stem cell" refers to a cell that has the ability to renew itself through the division of mitotic cells and can differentiate into multiple specialized cell types. Pluripotent stem cells are produced by the fusion of an egg and a sperm cell. Cells produced by the first few divisions of a fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Embryonic stem cells are pluripotent offspring of totipotent cells that can differentiate into cell types from any of the three germ layers. Induced pluripotent stem cells (iPSCs) are derived from adult cells and are induced into an embryonic stem cell-like pluripotency state by cell transcription factors. Induced pluripotent stem cells can be derived from adult cells such as skin or blood cells and may also differentiate into cell types from any of the three germ layers.

[0022] As used in this disclosure, the term “reprogramming” refers to the use of cell reprogramming factors to induce the conversion of somatic cells into iPSCs. The reprogramming process is divided into three stages: the initiation stage, the maturation stage, and the stabilization stage. In the initiation stage, the cell maintains its original somatic cell identity, but the expression of early pluripotency-related genes such as NANOG and SALL4 rapidly increases. In the maturation stage, the characteristics of somatic cell identity are rapidly lost, and the expression of late pluripotency-related genes such as DPPA5 and LIN28 increases. In the stabilization stage, the cell completely loses its somatic identity and becomes an iPSC. The process can also rejuvenate senescent cells (remove all or some of the signs of aging).

[0023] As used in this disclosure, the term "loss of cell identity" refers to the process by which somatic cells, through reprogramming, lose their original somatic cell-specific characteristics (i.e., cellular characteristics), such as morphology, function, epigenetic patterns, and gene expression profiles, and acquire various characteristics of pluripotent stem cells to become iPSCs. It should be particularly noted that the cellular characteristics of somatic cells are gradually lost during the reprogramming process, and in the initial stages, the cells retain their original cellular characteristics. By the time of maturation, the cells have already lost most of their cellular characteristics and have acquired a relatively high degree of pluripotent stem cell characteristics, and at this point, even if reprogramming is stopped, there is a high risk that the cells will not be able to recover their original cellular characteristics.

[0024] The term "partial reprogramming" refers to treating or temporarily reprogramming senescent cells with one or more cell reprogramming factors to restore some degree of vitality to the cells, but without sufficient differentiation into iPSCs, and retaining the original cellular characteristics. "Transient reprogramming" is a form of partial reprogramming in which the cells are exposed to cell reprogramming factors for a sufficient time to restore cellular vitality, but do not differentiate into iPSCs. Because the exposure time is very short, the degree to which transient reprogramming restores vitality is relatively low.

[0025] Initiation phase reprogramming involves engineering and modifying cellular reprogramming factors to stall the reprogramming of senescent cells in the initiation phase, thereby allowing for a longer, safer time interval for vitality recovery and significantly reducing the risk of developing into iPSCs. The main difference from temporary reprogramming is the engineering and modification of cellular reprogramming factors, thus allowing for artificial control of the reprogramming stage at the molecular level.

[0026] The term "liquid-liquid phase separation" (LLPS), simply referred to as phase separation, means that certain protein or nucleic acid molecules inside cells generate another phase with different physicochemical properties in the surrounding homogeneous liquid environment through multivalent interactions. Usually, it becomes micron-level droplets (similar to oil in water). These droplets form relatively independent spaces, selectively concentrate certain protein molecules, have a higher protein density, reduce the microscopic movement of molecules, and can promote the results of biochemical reactions such as gene transcriptional regulation and changes in the spatial structure of chromosomes. Protein phase separation has several mechanisms. One of them depends on the intrinsically disordered domain (IDR) of the protein. Since the acidic polar amino acids in this domain are negatively charged, phase separation occurs through interactions. Another depends on the ubiquitination of lysine, and phase separation occurs through the interaction of polyubiquitin chains. "Having no phase separation ability" refers to mutating the IRD sequence or key amino acids that form phase separation, resulting in the mutant protein being unable to form LLPS and affecting (reducing) the biological functions within the cell.

[0027] As used in this disclosure, the term “cell reprogramming factors” refers to transcription factors and combinations thereof that convert living or differentiated cells into pluripotent stem cells. Specifically, reprogramming factors are factors derived 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 transcription factors include CMYC, DPPA2, DPPA4, ESRRB, GDF3, GLIS1, KLF2, KLF4, KLF5, LIN28, LMYC, NANOG, NMYC, NR5A1, NR5A2, OCT-4, RCOR2, SALL1, SALL4, SOX1, SOX2, SOX3, TDRD12, TET1, TH2A, TH2B, UTF1, ZFP42, MDM2, CyclinD1, SV40 large T antigen, SIRT6, TCL1A, and RARy. The "Yamanaka factor" among these includes a combination of four genes: Oct-4, Sox4, Klf4, and c-Myc (referred to as the OSKM gene combination). In some respects, it includes the Oct4, Sox-2, and Klf4 genes (OSK gene), in other respects, the Oct4, Klf4, and c-Myc genes (OKM gene), and in other respects, the Sox-2, Klf4, and c-Myc genes (KMS gene), but is not limited to these.

[0028] Among these, the Oct reprogramming factors are any natural member of the transcription factor octamer family that maintains transcription factor activity compared to the most similar related factor (within the activity range of 50%, 80%, or 90% or more), or a variant thereof, or a polypeptide containing at least the DNA-binding structural domain of a natural family member, and further containing a transcription-activating structural domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11, where, for example, Oct3 / 4 (referred to as "Oct4" in this disclosure) contains the POU structural domain. In some embodiments, the variants have at least 85%, 90%, or 95% amino acid sequence identity in their entire sequence compared to a naturally occurring member of the Oct polypeptide family. In some embodiments, the OCT4 reprogramming factor protein / polypeptide provided in this disclosure is encoded by an optimized polynucleotide sequence, SEQ ID NO: 1. Therefore, Sequence ID: 1 constitutes a modified polynucleotide sequence compared to wild-type OCT4. In some embodiments, a more stable amino acid sequence of the OCT4 reprogramming factor is encoded compared to amino acid sequence Sequence ID: 2. In some embodiments, the protein / polypeptide of the OCT4 reprogramming factor 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%, and about 99%.

[0029] The "Sox family" refers to the gene encoding SRY (sex-determining region Y)-box 2, also known as SOX2, which is related to the maintenance of pluripotency. Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15 or Sox18) can be derived from humans, mice, rats, cows, pigs or other animals. Thus, the "protein", "SOX2 protein", etc. referred to in the present disclosure include any native Sox2 transcription factor having at least 50%, 80% activity compared to wild-type Sox2, e.g., 90% or 100% activity compared to wild-type Sox2 measured by methods known in the art. In some embodiments, the variant has at least 90% amino acid sequence identity in its entire sequence compared to the naturally occurring Sox2 polypeptide. Specifically, sequence refinement for identity control is performed based on SEQ ID NOs: 23-24.

[0030] The "Klf family" means that the "Klf" gene encoding Kruppel-like factor 4 or Klf-four protein is the protein encoded by the mouse klf4 gene and the human klf4 gene. Exemplary members of the Klf family include Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16 and Klf17. Thus, terms such as "KLF4", "KLF4 protein", etc. referred to in the present disclosure include any of the KFF4 transcription factors in their native form or the retained KLF4 transcription factor activity (e.g., at least 50% activity, 80%, 90% or 100% activity compared to wild-type KFF4 measured by methods known in the art). In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity in its entire sequence compared to the native KFF4 polypeptide. For example, the structure of the variant shown in SEQ ID NOs: 21-22.

[0031] 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 transcription factors that may replace c-Myc. Accordingly, "c-Myc," "C-MYC," "c-Myc protein," "C-MYC protein," etc., as referred to in this disclosure include both the natural form of cMyc transcription factor activity and variants that maintain the activity of cMyc transcription factor (for example, activity of at least 50%, 80%, 90%, or 100% or less compared to wild-type cMyc as measured by methods known in the art). In particular, variants can be improved based on the wild-type sequences of SEQ ID NOs: 11-12.

[0032] In one embodiment of the embodiments described herein and all other embodiments, the reprogramming includes introducing nucleic acid sequences encoding transcription factors such as Oct4, Sox2, c-Myc, and Klf4 into differentiated cells operably connected to a regulatory element for expressing the factors; introducing one or more protein factors that reprogram the cell differentiation state; and contacting the cells with a small molecule that induces the reprogramming of the cell differentiation state.

[0033] mutant The present invention further includes fragments, derivatives, and analogues of the above-mentioned transcription factor variants. The terms “fragment,” “derivative,” and “analog” essentially refer to proteins that retain the same biological function or activity as the transcription factor variants according to the present invention. The protein fragments, derivatives, or analogues according to the present invention may be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be proteins encoded by the genetic code; or (ii) proteins having substituents on one or more amino acid residues; or (iii) proteins formed by the fusion of an additional amino acid sequence to the protein sequence (e.g., a read sequence, a secretion sequence, a sequence used for protein purification, a prothrombin sequence, or a fusion protein). According to the definitions of this disclosure, these fragments, derivatives, and analogues are well known to those skilled in the art. However, the amino acid sequences of the above-mentioned transcription factor variants and their fragments, derivatives, and analogues always contain a mutation in one of the sites described above in the present invention, corresponding to the wild-type sequence.

[0034] Definitions of terms in the field of pharmaceuticals and therapeutics In this disclosure, the term “treatment” means being used to obtain a desired pharmacological and / or physiological effect. Such effect is prophylactic in the sense of complete or partial prevention of a disease or its symptoms, and / or therapeutic in the sense of partial or complete cure of a disease and / or adverse effects resulting from the disease. “Treatment” as used in this disclosure refers to a disease of a mammal, including cats, and includes (a) preventing the onset of a disease or illness in a susceptible and undiagnosed individual, (b) suppressing a disease in such a way that it stops the progression of the disease, or (c) reducing a disease in such a way that it alleviates symptoms associated with the disease. “Treatment” as used in this disclosure includes, but is not limited to, any administration of a pharmaceutical or compound to an individual to treat, cure, alleviate, improve, reduce or suppress a disease in that individual, and includes administering a pharmaceutical containing a compound relating to this disclosure to an individual in need.

[0035] The terms “object,” “individual,” “patient,” or “subject” include mammals to which the drug is administered, whether or not it is for therapeutic purposes. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0036] In this invention, "prevention" refers to all actions that avoid symptoms or alleviate the tension of specific symptoms by administering the product according to this invention before or after the progression of a disease.

[0037] The “effective dose” according to the present invention refers to the amount or dose of the product according to the present invention that, after being administered to a patient or organ in one or more doses, provides the desired treatment or prevention.

[0038] In this disclosure, the term “pharmaceutical composition” usually refers to a unit dose and may be prepared by any method well known in the pharmaceutical field. All methods include the step of binding an active ingredient to a carrier constituting one or more accessory components. Typically, compositions are prepared by uniformly and sufficiently binding an active compound to a liquid carrier, a finely ground solid carrier, or both.

[0039] The term "gene therapy" refers to the transient or permanent genetic modification (e.g., gene expression, insertion, alteration, or removal) of an individual's cells and / or tissues to treat diseases such as tumors, genetic disorders, age-related diseases, and autoimmune diseases. The most common forms of gene therapy are the introduction of functional genes into cells to be expressed in order to treat a disease, or the insertion of functional genes into specific or non-specific genomic locations to replace mutated genes. Other forms include directly correcting mutations, modifying normal genes that are susceptible to pathogens, or introducing genes or gene fragments into cells for transcription.

[0040] The term "cell therapy" refers to a series of actions (for example, growing, screening, restoring the function of cells and tissues, or otherwise altering the biological properties of cells) that are performed outside the body to achieve therapeutic, diagnostic, or preventive purposes.

[0041] The first aspect of the present invention relates to a method for rejuvenating cells.

[0042] By studying the three-stage mechanism of cell reprogramming, the inventors discovered that in the Initiation Phase, the cell's identity is maintained, early pluripotency genes are increased, epigenetic age (epiAge) decreases, and the cell begins to rejuvenate; in the Maturation Phase, the cell's identity is largely lost, late pluripotency genes are increased; and in the Stabilization Phase, the cell's identity is completely lost, resulting in the formation of iPSCs. Therefore, the time interval before the Maturation Phase is a period of partial reprogramming (PR). The inventors discovered a new method to prevent cell reprogramming from entering the Maturation Phase, extend the PR time interval, and rejuvenate the cell while preserving its identity. The above method uses a reprogramming factor that lacks phase separation ability, and based on mutations in the phase separation function-related region of the transcription factor, the transcription factor loses its ability to induce phase separation. This eliminates the power to drive cell reprogramming into the maturation stage, thereby stalling reprogramming in the initial stage and continuously rejuvenating the cell. However, the risk of reprogramming entering the terminal stage and losing the cell's identity is significantly reduced, and this method is referred to as "initial-stage reprogramming."

[0043] Furthermore, the engineered transcription factors that do not possess the above-mentioned phase separation ability may be factors such as OCT4, SOX2, KLF4, c-MYC, L-myc, LIN28, NANOG, and GLIS1.

[0044] The present invention relates to the engineering modification of transcription factors to cause them to lose their phase separation ability. The inventors have discovered that the manner in which the three-dimensional structure of transcription factors is modified and improved is not limited, and that as long as it causes them to lose their phase separation ability and stalls the cell's reprogramming at the initial stage, it falls within the scope of the present invention.

[0045] Accordingly, the present invention relates to a method for reprogramming cells in the initial stages involving an engineered transcription factor that does not have phase separation ability, and is characterized by being implemented by introducing the engineered transcription factor that does not have phase separation ability into cells.

[0046] A method for rejuvenating cells, characterized by using an engineered transcription factor that lacks phase separation ability to reprogram the cells at the initial stage.

[0047] A method for reprogramming cells, characterized by introducing them into cells using a carrier containing an engineered transcription factor that does not possess phase separation ability.

[0048] Specifically, as a criterion for determining the youthfulness of cells, regenerated cells show changes such as an increase or decrease in the expression of one or more epigenetic marker genes compared to a reference value.

[0049] Specifically, as a criterion for determining the youthfulness of cells, regenerated cells show changes such as an increase or decrease in cellular DNA methylation epigenetic age (epiAge) compared to a reference value.

[0050] Specifically, as a criterion for determining the youthfulness of cells, regenerated cells show changes such as a decrease in the proportion of senescence-related galactosidase (SA-β-Gal) positive cells compared to a reference value.

[0051] Specifically, as an example of a criterion for determining cell youthfulness, the expression levels of youthfulness-related genes such as COL1A1, COL3A1, COL4A1, FN1, and LAMA5 can be increased compared to a reference value. Similarly, the characteristic genes for reprogramming at the initiation stage of NANOG and SALL4 can be increased, and the expression levels of characteristic genes for reprogramming at the maturation stage of DPPA5 and LIN28A can be decreased. For example, the characteristic genes of fibroblasts such as THY1, P4HB, S100A4, and SERPINH1 can be examined to verify the identity genes of the cells. Different identity genes can be selected for different cells, and their detection can improve the expression of these identity genes compared to a reference value.

[0052] The above reference values ​​can be selected using either blank cell controls of the same cell type or cells introduced with wild-type transcription factor recombinant carriers as controls. In short, the reference value is not an absolute standard, but rather a relative comparison standard for comparing cells after they have been introduced with modified transcription factor recombinant carriers. Therefore, the above "criteria for determining rejuvenation" are merely examples and not absolute standards; if it can be demonstrated that cell reprogramming remains in the initial stage, it falls within the scope of criteria for determining cell rejuvenation.

[0053] The scope of protection of the present invention is not limited to the exemplary functional genes described above, but includes any genes that can be used to detect and confirm that partial cell reprogramming is in its initiation stage.

[0054] It should be noted that the cell rejuvenation method, or cell reprogramming method, may or may not be used for treatment or diagnosis, and can be used to process cells in vivo or in vivo, and does not necessarily have to directly treat the target of treatment. In a specific embodiment, the above cell rejuvenation method, or cell reprogramming method, is not used for the diagnosis, prevention, or treatment of disease.

[0055] The specific modifications, mutations, and alterations of the engineered transcription factor element that lacks the phase separation ability described above are as follows; the following modifications are examples only.

[0056] (1) By mutating charged amino acids, mutating acidic charged amino acids, and / or mutating alkaline charged amino acids, the ability to form aggregates is adjusted by changing the charged amino acids in the disordered region (IDR region) of the protein.

[0057] The inventors discovered that one of the principles is that "the negative charge of the IDR is a phase separation of the transcription factor," and therefore, by predicting the IDR region in a transcription factor, for example, by performing a Disorder Score using the VSL2 algorithm, the IDR of OCT4 can be predicted. For example, a continuous region with a score greater than a certain value is relatively likely to be the IDR, and the above value is not particularly limited. However, it may be in the range of 0.4 to 0.6, and preferably 0.5. From the results of exemplary prediction for OCT4, it was found that the IDR region of OCT4 is distributed in the activation domains (ADs) at both ends, more specifically, amino acids 1 to 140 and 288 to 360. The intermediate DNA binding domain (DBD) does not belong to the IDR. It is also possible to use alternative evaluation criteria such as the IUPred2 and ANCHOR2 criteria, but as long as they can be used for predictive evaluation of the IDR region, they are within the scope of the present invention. The inventors found that the conclusions obtained by multiple evaluation criteria are the same (within the error range), so the present invention can protect the IDR region structure predicted by multiple evaluation criteria.

[0058] Specifically, it is possible to perform amino acid modifications on the disordered region (IDR region) of transcription factor proteins, or modifications on the activated structural region, and further modifications on charged amino acids. This removes the phase separation ability from transcription factors.

[0059] Specifically, polar charged amino acids can be replaced with neutral amino acids, and the polar charged amino acids may be acidic amino acids.

[0060] Preferably, the polar charged amino acid in the IDR region can be replaced with a neutral amino acid, and the polar charged amino acid may be an acidic amino acid.

[0061] Specifically, the acidic amino acids aspartic acid (Asp) and glutamic acid (Glu) can be mutated into the neutral amino acid alanine.

[0062] Specifically, the acidic amino acids in the IDR regions of aspartic acid (Asp) and glutamic acid (Glu) can be mutated to the neutral amino acids of alanine.

[0063] In a specific embodiment, this involves performing mutational improvements on the wild-type amino acid sequence of OCT4.

[0064] In the IDR region of the OCT4 wild-type gene protein sequence described above, the N-terminus accounts for 38-40% of the total sequence, while the C-terminus accounts for 19-24%.

[0065] Preferably, the above mutation improvement is performed on positions 1 to 140 of SEQ ID NO: 2, and / or on positions 288 to 360. In particular, the above locations can have an error range of approximately 10 to 20 amino acid positions before and after.

[0066] Preferably, the mutation point 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, and 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 may be 1, 2, 3, ..., 36.

[0067] For example, the wild-type OCT4 gene is mutated to change the acidic polar amino acid (from D / E to A) in the IDR region of the protein, and this is hereinafter referred to as OCT4'. For example, this may be the sequence shown in sequence numbers 4-10, or a sequence that can achieve at least 85%, 90%, or 95% or more of the same function.

[0068] The present invention further protects the nucleotide sequence expressing the amino acid sequence of the above protein, which may be, for example, the sequence shown in SEQ ID NO: 3, but it will be clear to those skilled in the art that all different nucleotide sequences obtained by a reasonable change in codon preference are within the protected range, as long as the target protein is obtained by codon expression.

[0069] Of course, the present invention is not limited to the above-mentioned transcription factors, but encompasses all modifications of transcription factors that can achieve a function without phase separation ability by modifying charged amino acids in the IDR region.

[0070] (2) Ubiquitination modification The inventors discovered that after an amino acid is modified with a polyubiquitin chain, a polyubiquitin-mediated phase separation occurs. Therefore, by, for example, replacing lysine K with arginine R, polyubiquitin chain modification does not occur, and thus the occurrence of phase separation can be avoided. However, it should be noted that the scope of protection of the present invention is to provide a method for performing ubiquitination mutations on transcription factors to avoid phase separation, and is not limited to specific mutation patterns.

[0071] For example, a ubiquitinated lysine mutation (from K to R) may be introduced into the C-MYC transcription factor, and ubiquitination mutations may also be introduced into other transcription factors.

[0072] Specifically, mutations can be performed on sequence numbers 11-12, and based on sequence number 12, mutations can be performed on at least one lysine (K) from among 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.

[0073] Preferably, mutations are performed on 25 of the above-mentioned sites, numbered 1, 2, 3, ... Preferably, mutations are performed on one or both of the amino acids at positions 148 and 389. Preferably, mutations are performed on 20 to 25 sites. Preferably, mutations are performed on all 25 sites. However, the number of specific mutation sites is not particularly limited as long as the removal of the ubiquitin chain is achieved and the target effect, i.e., the avoidance of phase separation, is achieved.

[0074] Preferably, the modification involves replacing lysine K with arginine. However, the specific modification is not limited; any modification that achieves ubiquitination removal and avoids phase separation is within the protected range.

[0075] For example, examples include the variants shown in SEQ ID NOs: 14-20, or sequences that can achieve at least 85%, 90%, or 95% or more identity of the same function.

[0076] The present invention further protects the nucleotide sequence expressing the amino acid sequence of the above protein, exemplified by sequences shown in SEQ ID NO: 13, or sequences that can achieve at least 85%, 90%, or 95% or more identity of the same function. However, those skilled in the art will see that if the target protein is obtained by codon expression, then all different nucleotide sequences can be obtained by reasonably changing the codon preference according to the needs of the cell to be expressed, and all of these fall within the protected range.

[0077] Of course, the present invention is not limited to the above-mentioned transcription factors, and any modification of a transcription factor that can achieve a function without phase separation ability by removing ubiquitination treatment is all covered by the present invention.

[0078] (3) Partial deletion treatment, preferably terminal deletion treatment, can be performed on the N-terminus or C-terminus, or on the intermediate part of the gene. Alternatively, this is called partial deletion treatment of the IDR region of a protein, i.e., partial deletion treatment of charged amino acids in the IDR region. Alternatively, this is called deletion treatment of the key regulatory region of a protein, i.e., key lysine deletion treatment with ubiquitination chain modification, or amino acid deletion treatment that has a key role in phase separation generation.

[0079] For example, deletion can be performed on the N-terminus or C-terminus of a transcription factor, or on non-key regions other than its DBD region. For instance, deletion can be performed on positions 1-20, and further deletion can be performed on positions 2-20, 1-19, 2-29, 2-18, 2-17, 2, and 16. Such a treatment does not affect the DBD region, i.e., it does not affect the transcription factor's original DNA site recognition function, but it can remove its IDR region, lysine of ubiquitinated chain modifications, or other key amino acids, thus avoiding the occurrence of phase separation.

[0080] Specifically, by partially deleting the sequence of KLF transcription factor 4 (KLF4) shown in SEQ ID NOs: 21-22, for example, since its important DBD region is located at positions 375-470 of the C-terminus, by deleting all or part of the IDR region at positions 2-374 of the N-terminus, it is possible to obtain the amino acid sequence shown in SEQ ID NO: 40, or a sequence that achieves at least 85%, 90%, or 95% or more of the same functional identity.

[0081] Specifically, by partially deleting the sequence of sex-determining Y-box transcription factor 2 (SOX2) shown in SEQ ID NOs: 23-24, for example, by deleting all or part of the IDR region from positions 2-40 at the N-terminus and / or positions 201-317 at the C-terminus, since its important DBD region and transcriptional activation structural region are located at positions 41-200 in the center, it is possible to obtain the amino acid sequence shown in SEQ ID NOs: 41-43, or a sequence that achieves at least 85%, 90%, or 95% or more identity with the same function.

[0082] Specifically, by partially deleting the OCT4 sequence shown in sequence numbers 1-2, for example, by deleting all or part of the IDR region at positions 2-140 of the N-terminus and / or positions 288-360 of the C-terminus, since its important DBD region is located at positions 141-287 in the center, it is possible to obtain the amino acid sequence shown in sequence numbers 44-46, or a sequence that achieves at least 85%, 90%, or 95% or more of the same functional identity.

[0083] Specifically, by partially deleting the c-MCY sequence shown in SEQ ID NOs: 11-12, for example, since its most important DBD is the bHLH-LZ structural region at positions 361-439 of the C-terminus, by deleting all or part of positions 2-360 of the N-terminus, it is possible to obtain the amino acid sequence shown in SEQ ID NOs: 47-48, or a sequence that can achieve at least 85%, 90%, or 95% or more identity of the same function. The present invention further protects the nucleotide sequence expressing the above protein amino acid sequence, and may, exemplary, be the sequence shown in SEQ ID NO: 13, or a sequence that can achieve at least 85%, 90%, or 95% or more identity of the same function. However, those skilled in the art will understand that if the target protein can be obtained by codon expression, all different nucleotide sequences obtained by a reasonable change in codon preference are within the protected range.

[0084] Of course, the present invention is not limited to the above-mentioned transcription factors, and any modification of a transcription factor that can achieve a function without phase separation by partial deletion treatment falls within the scope of the present invention.

[0085] A second aspect of the present invention is to provide an element containing an engineered transcription factor.

[0086] nucleic acid elements The present invention further relates to a nucleic acid element comprising the above-mentioned engineered transcription factor, wherein the element may be a DNA element, an RNA element, or more precisely, an mRNA element, an siRNA element, a circular RNA element, a circular mRNA element, or a tRNA element.

[0087] The above element further includes a Kozak sequence, a promoter sequence, at least one linked peptide sequence, a UTR region, a terminator sequence, a 5' cap, a polyA tail, etc. The above nucleic acid element may be a DNA or RNA sequence, or it may be an expressed protein element. The promoter can be selected from inducible promoters such as TRE, TRE3G, TRE3GS, and TRE3GV, compositional 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).

[0088] The above mRNA elements include, but are not limited to, linear mRNA and circular mRNA. The above circular mRNA has an IRES and a self-cleaving polypeptide 2A element. The above RNA is appropriately modified and includes, but is not limited to, pseudouridine (Ψ), N1-methyl-psoidouridine (m1Ψ), 5-methoxyuridine (5moU), N6-methyladenosine (m6A), 5-methylcytidine (5m5C), etc. The above RNA has a sequence that binds to L7Ae, TAT, etc., which are necessary for enriching the above RNA.

[0089] Recombinant carrier element The element according to the present invention can be further constructed with a support, which may be a monocistrone, a doublecistrone, or a multicistrone. In one specific embodiment of the present invention, the coding region of the doublecistrone or multicistrone gene can be linked by at least one linking peptide, which may be an internal ribosome entry site (IRES) or a self-cleaving polypeptide 2A (2A).

[0090] The IRES sequences that can be used include, but are not limited to, small RNA viruses (e.g., FMDV), plague viruses (e.g., CFFV), poliovirus (e.g., PV), encephalomyocarditis virus (e.g., ECMV), foot-and-mouth disease virus (e.g., FMDV), hepatitis C virus (e.g., HCV), classical swine fever virus (e.g., CSFV), mouse corneal leukovirus (e.g., MLV), ape immunodeficiency virus (e.g., SIV), or cricket paralysis virus (e.g., CrPV). The coding region can also be divided by "2A connectors." These "2A connectors" are short peptides (18-25 amino acids) derived from viruses, also called "self-cleaving" peptides, and can produce multiple proteins from a single transcript, including, but are not limited to, P2A / T2A / E2A / F2A.

[0091] When multiple transcription factors are arranged in series, this can be specifically shown in sequence numbers 25-27. Of course, the present invention also provides carriers for transcription factors and allows for mixing in certain proportions, for example, 1:1:1:1, 1:2:1:2, 1:2:2:1, and 2:1:1:1. Mixtures are also within the scope of protection of the present invention.

[0092] The present invention further relates to recombinant carriers of the above-mentioned engineered transcription factors, and includes, but is not limited to, clonal carriers, expression carriers, shuttle carriers, and viral carriers (e.g., lentiviral carriers, adeno-associated virus carriers, poxvirus carriers, etc.).

[0093] Recombinant protein The present invention further includes recombinant protein elements expressed by translating the above nucleic acid elements, and all nucleotide sequences obtained by expressing the above protein elements. The protein elements maintain some degree of function and may include certain variants.

[0094] Recombinant cells The present invention further relates to recombinant cells into which the above-mentioned carrier is introduced.

[0095] Specifically, the methods according to the present invention can be applied to any type of cell requiring regeneration. Cells may be isolated from other cells, mixed with other cells in culture, or mixed in tissue (partial or complete) or in vivo. The methods according to the present invention are performed, for example, on a sample containing a single cell, a group of cells, or a tissue or organ containing cells. The selection of cells for rejuvenation depends on the therapeutic effect required to treat age-related diseases or symptoms. The cells are mammalian cells, including, but not limited to, prokaryotic and eukaryotic cells, and further including cells from mammalian animals such as humans, monkeys, mice (rats, rats), horses, cattle, sheep, pigs, dogs, and cats. Examples include CRFK feline kidney cells, PG-4(S+L-) feline brain cells, feline kidney cells, FCA-S1 feline skin cells, CATK1 feline kidney cells, MDCK(NBL-2) canine kidney cells, SCBN canine duodenal cells, DH82 canine renal malignant tissue proliferative disease cells, HEK293T, HEK293, BHK, CHO, 3T3, NS0, Hela, HT-1080, PERC6, CAP, HKB-11, and Huh-7. In one embodiment, the cells are human cells. In one embodiment, the cells are obtained from an elderly subject.

[0096] The methods according to the present invention can be performed on cells, tissues, or organs of the nervous system, muscular system, respiratory system, cardiovascular system, skeletal system, reproductive system, endothelial system, lymphatic system, excretory system, endocrine system (e.g., nervous system), or digestive system. As described herein, all types of cells are regenerative and include, but are not limited to, epithelial cells (e.g., squamous epithelium, cuboidal epithelium, columnar epithelium, and pseudostratified epithelium), endothelial cells (e.g., venous, arterial, and lymphatic endothelial cells) and connective tissue, muscle, and nervous system cells. These cells 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, satellite cells), columnar cells, adipocytes, pericytes, astrocytes, lung cells, blood and immune system cells (e.g., erythrocytes, monocytes, dendritic cells, macrophages, neutrophils, eosinophils, mast cells, T cells, B cells, natural killer cells), hormone-secreting cells, germ cells, stromal cells, lens cells, photoreceptor cells, taste cells, and olfactory cells. These cells originate from the kidneys, liver, pancreas, stomach, spleen, gallbladder, intestines, bladder, lungs, prostate, breast, genitourinary tract, pituitary cells, oral cavity, esophagus, skin, hair, nails, thyroid gland, parathyroid gland, adrenal gland, eye, nose, or brain.

[0097] 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.

[0098] Introduction and transportation The above transfection methods include, but are not limited to, liposome transfection, microvesicle transfection, exosome transfection, electroporation, nanocarrier transfection, and chemical transfection using transfection reagents. The nanocarriers include lipids, polymers, or lipid-polymer hybrids.

[0099] The transport system is used to transport nucleic acid molecules, recombinant carriers containing nucleic acid molecules, recombinant DNA molecules, mRNA, recombinant proteins, etc. In some embodiments, lipid nanoparticles or "LNPs" are used to transport nucleic acids to cells. As described above, LNPs may include natural or synthetic lipids, including conjugated lipids or polymers (e.g., pegylated lipids). LNPs may include one or more neutral lipids, amphoteric lipids, ionizable lipids, cationic lipids, and anionic lipids. In some embodiments, LNPs may include natural or synthetic monoacyl or diacyl phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA), or monoacyl, diacyl, triacyl, or tetraacyl cardiolipin. In some embodiments, LNPs are micelles or reverse micelles. In other embodiments, LNPs are monolayer liposomes or multilayer liposomes.

[0100] The present invention further relates to other transport systems, such as GalNAc-conjugated transport systems, liposome LNP nanotransport systems (for small molecule ligands, antibodies, and other molecules), virus-like particle (VLP) transport, protamine, high molecular weight polymers, inorganic nanoparticles, exosomes, polymer matrices, and viral transfection.

[0101] A third aspect of the present invention relates to preventive and / or therapeutic use.

[0102] The nucleic acid elements (DNA, RNA elements), recombinant carriers, recombinant proteins, and recombinant cells according to the present invention can be used for prevention and / or therapeutic purposes in various fields and are also used in the manufacture of pharmaceuticals used to treat diseases. Specific therapeutic diseases include, but are not limited to, cell therapy, gene therapy, immune cell proliferation, cancer treatment, and age-related disease treatment.

[0103] The method according to the present invention is used to rejuvenate cultured cells (e.g., ex vivo or in vitro) to improve their function and efficacy for use in cell therapy. The cells used to treat a patient may be autologous or allogeneic. Preferably, the cells are derived from the patient or a suitable donor. For example, in ex vivo therapy, cells are obtained directly from the patient being treated, transfected with mRNA encoding a cell reprogramming factor as in the present invention, and then re-implanted in the patient's body. The cells may be obtained, for example, from a biopsy or surgery performed on the patient. Alternatively, cells requiring regeneration may be directly transfected in vivo using mRNA encoding a cell reprogramming factor. In some embodiments, the immune cell process is performed ex vivo, for example, in the manufacture of a cell therapy product, such as autologous or allogeneic chimeric antigen receptor (CAR)-T, CAR-NK, CAR-M, or CAR-NKT cells.

[0104] The methods according to the present invention are used for gene therapy. "Gene therapy" is the treatment and / or prevention of diseases in mammalian subjects (e.g., humans) using genes (i.e., genetic material). In some embodiments, the genetic material according to the present invention (nucleic acid elements (DNA, RNA elements), recombinant carriers, etc.) is directly delivered to at least some of the cells of a mammalian subject. The present invention is used in any specific method or composition of gene therapy. The goals of gene therapy can be achieved by introducing the above-mentioned nucleic acid elements, recombinant carriers, etc., into mammalian cells. Specifically, this includes autologous or ex vivo gene therapy. Gene therapy is further divided into "germ cell gene therapy" and "somatic cell gene therapy." In "germ cell gene therapy," germ cells, i.e., sperm or egg cells, are genetically modified. Genetic modifications are usually incorporated into their genomes. Therefore, therapeutic modifications can be inherited and passed on to offspring. The methods are used to treat genetic disorders and genetic diseases. In "somatic cell gene therapy," therapeutic genes are delivered to the somatic cells of an individual. Any modifications and effects are limited to the individual concerned and are not inherited by its offspring or descendants. Specific strategies include, but are not limited to, forms of DNA modification, DNA substitution, DNA supplementation, and DNA inactivation.

[0105] In another aspect, the present invention provides a method for inducing the proliferation of cells such as immune cells. In some embodiments, the method involves exposing cells to mRNA encoding one or more reprogramming factors, so that the exposure results in the expression of one or more transcription factors in the cells, thereby enhancing cell proliferation while simultaneously preserving its properties. In some embodiments, the method for inducing proliferation does not induce failure. In some embodiments, proliferation is induced by the prevention or reversal of failure.

[0106] A specific embodiment of the cell rejuvenation therapy according to the present invention is used for "age-related diseases or illnesses," specifically referring to any disease, illness, or condition related to aging, such as neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, dementia, stroke), cardiovascular and peripheral vascular diseases (e.g., arteriosclerosis, peripheral artery disease (PAD), hematoma, calcification, thrombosis, embolism, arterial (e.g., tumors), eye diseases (e.g., age-related macular degeneration, glaucoma, cataracts, dry eye, diabetic retinopathy, visual impairment), skin diseases (atrophic and thinned dermis, elastic hyperplasia and wrinkles, sebaceous gland hyperplasia or hypoplasia, pigmentation abnormalities such as senile freckles, gray hair, hair loss or thinning hair, chronic skin ulcers), autoimmune diseases (e.g., polymyalgia rheumatica (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), crystalline arthropathy and spinal cord disease) Spondyloarthritis (SPA), internal medicine metabolic disorders (e.g., adult hypopituitarism, hypothyroidism, slender hyperthyroidism, osteoporosis, diabetes mellitus, adrenal insufficiency, various forms of hypogonadism, endocrine tumors), musculoskeletal disorders (e.g., arthritis, osteoporosis, myeloma, gout, Paget's disease, bone fractures, bone marrow failure syndrome, ankylosing spondylitis, diffuse idiopathic osteoporosis, hematogenous osteomyelitis, muscle atrophy, peripheral neuropathy, multiple sclerosis) This includes, but is not limited to, diseases such as amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis, myasthenia gravis, gastrointestinal diseases (e.g., cirrhosis, hepatic fibrosis, Barrett's esophagus), respiratory diseases (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism (PE), lung cancer, infections), diseases associated with cell proliferation, and any other diseases and conditions associated with aging.

[0107] As used in this invention, the term “disease or illness relating to cartilage degeneration” is any disease or illness relating to the degeneration of cartilage and / or joints. The term “disease or illness relating to cartilage degeneration” includes, but is not limited to, diseases, illnesses, syndromes, disorders and injuries affecting the intervertebral discs or joints (e.g., joints) of animals, including humans, and includes arthritis, cartilage deformities, spondyloarthropathy, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjögren’s syndrome. The term “disease or illness relating to muscle degeneration” refers to any disease or illness relating to muscle degeneration. The term includes diseases, disorders and syndromes.

[0108] Diseases and injuries affecting muscles include, but are not limited to, muscle atrophy, disuse atrophy, muscle tears, burns, surgery, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis, myasthenia gravis, cancer, acquired immunodeficiency syndrome, congestive heart failure, chronic obstructive pulmonary disease (COPD), liver disease, renal failure, eating disorders, malnutrition, starvation, infections, and corticosteroid therapy.

[0109] Diseases related to cell proliferation refer to illnesses resulting from abnormal growth or elongation caused by cell proliferation (Walker, Cambridge Dictionary of Biology, Cambridge University Press: Cambridge, UK, 1990). Proliferative disorders are related to 1) pathological proliferation of normally quiescent cells, 2) pathological migration of cells from normal sites (e.g., metastasis of tumor cells), 3) pathological expression of matrix metalloproteinase proteolytic enzymes such as collagenase, gelatinase, and elastase, or 4) pathological angiogenesis such as proliferative retinopathy and tumor metastasis. Representative proliferative disorders include cancer (i.e., malignant tumors), benign tumors, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0110] "Cancer" refers to a disease characterized by the development of abnormal cells that have the ability to grow uncontrollably and invade and destroy normal body tissues. Representative cancers include acoustic neuroma, adenocarcinoma, adrenal cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, intralymphatic sarcoma, angiosarcoma), appendiceal cancer, benign monoclonal immunoglobulinemia, bile duct cancer (e.g., bile duct cancer), bladder cancer, breast cancer (e.g., mammary gland cancer, papillary mammary gland cancer, mammary gland cancer, medullary mammary gland cancer), brain cancer (e.g., meningioma), glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma, medulloblastoma), bronchial cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, cranial candoma, colorectal cancer. For example, colon cancer, rectal cancer, colorectal adenocarcinoma), connective tissue cancer, epithelial cancer, ependymoma, endosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), typical eosinophilia, gallbladder cancer, gastric cancer (e.g., gastric adenocarcinoma), gastrointestinal stromal tumor (GIST), germ cell carcinoma, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), oral cancer (oral squamous cell carcinoma), pharyngeal cancer (For example, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer), hematopoietic cancers (for example, leukemia, acute lymphoblastic leukemia (ALL) (for example, B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (for example, B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (for example, B-cell CML, T-cell CML), chronic lymphocytic leukemia (CLL) (for example, B-cell CLL, T-cell CLL), lymphoma, Hodgkin lymphoma (HL) (for example, B-cell HL, T-cell HL), non-Hodgkin lymphoma (NHL), for example, diffuse Large B-cell lymphoma and other diffuse large B-cell lymphomas (DLCL), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasma-cell lymphoma (i.e., Waldenström macroglobulinemia), pilar cell leukemia (HCL), immunoblastic large B-cell lymphoma,Precursor B-cell lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma, T-cell non-Hodgkin lymphoma (e.g., precursor T-cell 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, extratubular NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma), mixtures of one or more of the above leukemias or lymphomas, and multiple myeloma (MM), heavy chain disease (e.g.) (α-chain disease, γ-chain disease, μ-chain disease), hemangioblastoma, hypopharyngeal cancer, inflammatory myofibroblastoma, immune cell amyloidosis, kidney cancer (e.g., intraocular melanoma, renal cell tumor), liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatocellular carcinoma), lung cancer (e.g., bronchial cancer, 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 thrombocytosis (ET), myelofibroblastic disease) Idiopathic myeloid metaplasia (AMM), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), eosinophilic syndrome (HES), neuroblastoma, neurofibroma (e.g., neurofibromatosis type 1 or 2 (NF), schwannoma), neuroendocrine carcinoma (e.g., gastrointestinal pancreatic neuroendocrine neoplasm (GEP-NET), carcinoid tumor), osteosarcoma (e.g., bone cancer), ovarian cancer (e.g., cystadenocarcinoma, ovarian germ cell tumor, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), islet cell neoplasm) Cystoma), penile cancer (e.g., Paget's disease of the penis and scrotum), pineal gland tumor, primitive neuroectodermal tumor (PNT), plasmacytoma, paraneoplastic syndrome, neoplasm in situ, prostate cancer (e.g., prostate cancer), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratogenic echinoblastoma (KA), melanoma, basal cell carcinoma (BCC)), small intestine cancer (e.g., appendiceal cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve schwannoma (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland cancer, small intestine cancer, sweat gland cancer, synoviomas,This includes, but is not limited to, testicular cancer (e.g., seminomas, prenatal testicular cancer), thyroid cancer (e.g., papillary thyroid carcinoma (PTC), medullary thyroid carcinoma), urethral cancer, vaginal cancer, and vulvar cancer (e.g., vulvar Paget's disease).

[0111] The fourth aspect of the present invention is to provide a pharmaceutical composition.

[0112] Pharmaceutical compositions typically consist of a main material and auxiliary materials, and these pharmaceutical compositions are used for prevention or treatment, and are used in standardized treatment or personalized treatment (cell therapy, gene therapy).

[0113] The main components are primarily nucleic acid elements, recombinant carriers, recombinant cells, etc., as mentioned in the first and second aspects of this invention. Recombinant carriers can be formed by connecting them in series, and by constructing carriers individually, various single transcription factors can be formed to create a mixture. The mixing ratio is preferably equal, but of course, different ratios are acceptable as long as similar active functions are maintained.

[0114] The present invention further provides the use of the above-mentioned nucleic acid elements, recombinant carriers, recombinant cells, etc., for the manufacture of pharmaceuticals that can be prevented and treated by cell rejuvenation. It also provides pharmaceutical compositions that can be prevented and treated by cell rejuvenation. And it provides pharmaceutical compositions for treating diseases that can be prevented and treated by cell rejuvenation.

[0115] If the pharmaceutical composition is a cellular composition, and especially if it is a regenerative cell composition, it may further contain one or more additional factors, such as nutrients, cytokines, growth factors, extracellular matrix (ECM) components, antibiotics, antioxidants, or immunosuppressants, which improve cell function or viability.

[0116] Examples of growth factors include, but are not limited to, fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor β (TGF-P), epidermal regulatory proteins, epidermal growth factor ("EGF"), endothelial cell factor / cell growth factor ("ECGF"), nerve growth factor ("NGF"), leukemia suppressor factor ("LIF"), bone morphogenetic protein 4 ("BMP-4"), hepatocyte growth factor ("HGF"), vascular / endothelial growth factor-A ("VEGF-A"), and cholecystokinin octapeptide.

[0117] Based on the main material, the above pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, carriers, auxiliary materials, or mediators of selectability. Examples of the above excipients, carriers, auxiliary materials, or mediators include, but are not limited to, anti-tacks, adhesives, coatings, compression aids, disintegrants, dyes, lubricants, emulsifiers, fillers (thinners), film-forming agents or coatings, flavorings, fragrances, flow accelerators (flow enhancers), lubricants, adsorbents, suspending agents, dispersants, or sweeteners. Exemplary excipients, carriers, auxiliary materials, and media include, but are not limited to, butylhydroxytoluene (BHT), calcium carbonate, calcium phosphate (monohydrogen), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinylpyrrolidone, citric acid, cross-linked povidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltol, mannitol, methionine, methylcellulose, p-hydroxybenzoate methyl, microcrystalline cellulose, polyethylene glycol, povidone, proglued starch, p-hydroxybenzoate propyl, retinyl palmitate, shellac, silica, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0118] The above pharmaceutical compositions are manufactured as injectable preparations, oral preparations, inhalation preparations, or microneedle preparations. The above injectable preparations are classified by form and include liquid injectable preparations, injectable powders, and injectable tablets. They are also classified by injection site and include intradermal injectable preparations, subcutaneous injectable preparations, intramuscular injectable preparations, intravenous injectable preparations, intraperitoneal injectable preparations, and spinal injectable preparations. Preferably, the solvent for the above injectable preparations is sterile water for injection or physiological saline. Preferably, they are administered by intravenous, intramuscular, or microneedle injection.

[0119] Preparations for oral use comprise tablets containing an active ingredient to be mixed with a non-toxic and pharmaceutically acceptable excipient. The excipient may be, for example, an inert diluent or filler (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch such as potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, sodium phosphate). It may also be a granulator and disintegrant (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, cross-linked carboxymethylcellulose sodium, alginate, or alginic acid), adhesive (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, gelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, polyethylene glycol), and a lubricant, flow promoter and anti-adhesive (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, talc). Preparations for oral use are further available in the form of chewable tablets or 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, kaolin, etc.), 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, olive oil). Powders, granules, and pills can usually be prepared using the ingredients mentioned above for tablets or capsules, for example, by using a mixer, fluidized bed apparatus, or spray dryer.

[0120] Other pharmaceutically acceptable excipients for oral use include, but are not limited to, colorants, flavorings, plasticizers, humectants, and buffers. Oral formulations may also be used in the form of chewable tablets or 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, kaolin), or in the form of soft gelatin capsules, in which the active ingredient is mixed with water or an oil-based medium (e.g., peanut oil, liquid paraffin, olive oil). Powders, granules, and pills may be prepared using the ingredients mentioned above for tablets or capsules, typically by using, for example, a mixer, fluidized bed apparatus, or spray dryer.

[0121] The above-mentioned injectable formulations include injectable solutions, injectable emulsions, lyophilized agents, and injectable suspensions. The auxiliary materials of the injectable formulations include water for injection and at least one solubilizer. The solubilizers include at least one of Twain 80, ethyl alcohol, propylene glycol, and polyethylene glycol 400. Preferably, the solubilizer is Twain 80 or polyethylene glycol 400. The injectable formulations further include, but are not limited to, protein nanoparticles, cyclodextrin-containing materials, liposomes, microspheres, storage-type release-controlled injectable formulations, or needleless injection-release systems.

[0122] Combination therapy The nucleic acid element, recombinant carrier, recombinant cells, and pharmaceutical compositions containing the same according to the present invention are used in combination with other pharmaceuticals for therapeutic purposes. Taking anti-aging therapy as an example, both initial reprogramming and protein therapy may be combined, for example, by combining with the B18R protein to perform anti-aging therapy. [Effects of the Invention]

[0123] Cell rejuvenation occurs at the initial stage of reprogramming, while the loss of somatic cell characteristics only occurs in the middle and later stages of reprogramming. Therefore, there is a time interval between these stages, allowing for cell rejuvenation while maintaining the cell's original characteristics. This is the principle behind the technology of rejuvenating cells through partial reprogramming. The inventor discovered that after transcription factors lose their phase separation ability, reprogramming becomes less likely to enter the maturation stage, remaining in the initial stage, and the cell's identity is not lost (Loss of cell identity). Therefore, it is possible to increase the degree of cell rejuvenation while simultaneously reducing the risk of generating iPSCs. Complete reprogramming is not possible, hence it is called "initial stage reprogramming." By making judgments based on the phenotype of functional genes, verification at each stage of cell reprogramming was ensured, proving that the technology achieved the effect of cell rejuvenation.

[0124] Clinically, as cells age, their function gradually declines, and aging and dysfunction occur in tissues, organs, and systems. A large number of age-related diseases include, but are not limited to, neurodegenerative diseases, cardiovascular and peripheral vascular diseases, ocular diseases, skin diseases, autoimmune diseases, metabolic disorders, musculoskeletal diseases, digestive system diseases, cell proliferation-related diseases, and tumors. The underlying cause is the aging and dysfunction of the relevant tissues and organs. Cellular rejuvenation therapy targeting specific tissues and organs can effectively alleviate and treat related diseases, bringing well-being to a vast number of people. At the same time, extending healthy life expectancy greatly increases societal productivity, reduces medical demand and pressure, and creates new assets in aging societies. [Brief explanation of the drawing]

[0125] [Figure 1] The diagram shows the construction of a lentivirus carrier. (A) is a schematic diagram of the lentivirus carrier structure, and (B) is an analysis of plasmid cleavage of the lentivirus carrier. [Figure 2]This figure shows the quality control of lentiviruses. (A) shows the results of lentivirus titer testing and sterility testing, and (B) shows the expression level of transgenic KLF4 after lentiviruses were introduced into 293T cells. [Figure 3] This figure shows the structure of the lentivirus carrier used in Example 2. [Figure 4] This figure shows staining experiments for senescence-related galactosidase (SA-β-Gal). A) is a representative photograph of the staining results in the blank control group. B) is a representative photograph of the staining results in the OKMS reprogramming group. C) is a representative photograph of the staining results in the O'KM'S reprogramming group. D) is a statistical analysis of the percentage of SA-β-Gal-positive cells in each group. [Figure 5] This figure shows the measurement of gene expression levels related to rejuvenation function in dermal fibroblasts. A significant difference between the OKMS reprogramming group and the O'KM'S reprogramming group was confirmed by a t-test. **: P<0.01, ***: P<0.001. [Figure 6] This figure shows the measurement of the expression levels of characteristic genes in dermal fibroblasts. Significant differences between the OKMS reprogramming group and the O'KM'S reprogramming group were confirmed by t-test. *:P<0.05, **:P<0.01, ***:P<0.001. [Figure 7] This figure shows the measurement of indicator gene expression levels at the initiation stage of cell reprogramming. A significant difference between the OKMS reprogramming group and the O'KM'S reprogramming group was confirmed by a t-test. **: P<0.01. [Figure 8] This figure shows the measurement of indicator gene expression levels during the maturation stage of cell reprogramming. A significant difference between the OKMS reprogramming group and the O'KM'S reprogramming group was confirmed by a t-test. **: P<0.01, ***: P<0.001. [Figure 9] This is the result of an analysis of cellular DNA methylation epigenetic age (epiAge). [Figure 10] This is a structural diagram of the lentivirus carrier used in Example 3. [Figure 11] This is a staining experiment for senescence-related galactosidase (SA-β-gal). A) is a representative photograph of the staining results in the blank control group. B) is a representative photograph of the staining results in the OKM reprogramming group. C) is a representative photograph of the staining results in the O'KM' reprogramming group. D) is a statistical analysis of the percentage of stain-positive cells in each group. [Figure 12] This figure shows the measurement of gene expression levels related to rejuvenation function in skin fibroblasts. A significant difference between the OKM reprogramming group and the O'KM' reprogramming group was confirmed by a t-test. **: P<0.01, ***: P<0.001. [Figure 13] This figure shows the measurement of the expression levels of characteristic genes in skin fibroblasts. A significant difference between the OKM reprogramming group and the O'KM' reprogramming group was confirmed by a t-test. ***: [Figure 14] This figure shows the measurement of indicator gene expression levels at the initiation stage of cell reprogramming. Significant differences between the OKM reprogramming group and the O'KM' reprogramming group were confirmed by a t-test. ns: no significant difference, ***: P<0.001. [Figure 15] This figure shows the measurement of indicator gene expression levels during the maturation stage of cell reprogramming. A significant difference between the OKM reprogramming group and the O'KM' reprogramming group was confirmed by a t-test. ***: P<0.001. [Figure 16] The diagram shows the construction of the AAV carrier. (A) is a schematic diagram of the AAV-TRE3G-OKS carrier structure, (B) is a diagram showing the cleavage identification of the AAV-TRE3G-OKS carrier plasmid, (C) is a schematic diagram of the AAV-TRE3G-O'KS carrier structure, and (D) is a diagram showing the cleavage identification of the AAV-TRE3G-O'KS carrier plasmid. [Figure 17] This figure shows the measurement of AAV carrier titer by qPCR. (A) is the original amplification curve, (B) is the standard curve, and (C) is the measurement result of AAV carrier titer. [Figure 18]This figure shows the construction of circular RNA carriers with different combinations of partial reprogramming factors. [Figure 19] This figure shows the verification of ring formation of circular RNAs in different combinations using partial reprogramming factors. (A) shows the results of 2% E-gel electrophoresis, in which MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, and Lin28A-P2A-Nanog-circRNA formed circular RNAs as indicated by the arrows. (B and C) show the results of 0.6% agarose gel electrophoresis for 30 minutes (upper) and 90 minutes (lower), in which OSK-circRNA, O'SK-circRNA, O'KM'-circRNA, OKM-circRNA, and OKMS-circRNA formed circular RNAs as indicated by the arrows. [Figure 20] This figure shows the results of transfecting HEK293T cells with a circular RNA of a partial reprogramming factor and verifying protein expression in a Western blotting (WB). [Figure 21] iPSCs were induced by transfecting fibroblasts with circular RNA of the wild-type OKSMLN factor, and clones were displayed after 7 days. [Modes for carrying out the invention]

[0126] The present invention will be described in more detail below with reference to specific examples. These examples are merely illustrative and should be understood not to limit the scope of the present invention. In the following examples, experimental methods for which specific conditions are not explicitly stated are usually carried out according to the conditions described in J. Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd edition (Science Press, 2002), or the conditions recommended by the manufacturer. [Examples]

[0127] Gene synthesis and construction of lentivirus (LV) carriers 1. Design of single-element sequences of transcription factors Design the sequences for OCT4' and the mutant OCT4'1-6. Refer to sequence numbers 4-10 and design the corresponding nucleotide sequences, for example, as shown in sequence number 3. Refer to sequence numbers 14-20 and design the sequences for C-MYC and the mutant C-MYC, for example, as shown in sequence number 13. Refer to sequence number 22 and design the sequence for KLF4. For example, design the corresponding nucleotide sequence, as shown in sequence number 21. Refer to sequence number 24 and design the sequence for SOX2. For example, design the corresponding nucleotide sequence, as shown in sequence number 23.

[0128] For example, design the linker sequences P2A, T2A, and E2A as shown in sequence numbers 25-27. Design the GCCGCCACC sequence of Kozak. For example, design the promoter sequence of TRE3G as shown in sequence number 63. Design the sequence of SV40 poly(A) as shown in sequence number 64.

[0129] Construction of a sequence in which two transcription factor elements are linked in series. The arrays formed by connecting the above elements in series are only illustrative examples, and as shown in sequence numbers 28-33, they include the OCT4'-P2A-KLF4s-T2A-MYC'-E2A-SOX2 series array (simply called O'KM'S, corresponding to sequence number 28), the OCT4-P2A-KLF4s-T2A-MYC-E2A-SOX2 series array (simply called OKMS, corresponding to sequence number 29), and the OCT4'-P2A-KLF These are referred to as 4s-T2A-MYC' series sequence (simply called O'KM', corresponding to sequence number: 30), OCT4-P2A-KLF4s-T2A-MYC series sequence (simply called OKM, corresponding to sequence number: 31), OCT4'-P2A-KLF4s-T2A-SOX2 series sequence (simply called O'KS, corresponding to sequence number: 32), and OCT4-P2A-KLF4s-T2A-SOX2 series sequence (simply called OKS, corresponding to sequence number: 33).

[0130] 3. Construction and packaging of lentivirus carriers (conducted by Saiye Biotechnology Co., Ltd.) 3.1 Construction of a lentiviral carrier: 1) Gene synthesis The O'KM'S, OKMS, O'KM', and OKM fragments were synthesized as genes.

[0131] 2) Enzymatic cleavage of the carrier skeleton Enzymatic cleavage was performed by selecting an appropriate site on the carrier skeleton.

[0132] The CutSmart system using NEB for enzymatic cleavage is as follows:

[0133] [Table 1] After enzymatic cleavage, the adhesive was recovered, and the tape was retrieved using an adhesive recovery kit (NEB Monarch DNA). The necessary skeletal and target fragments were then prepared and used for the subsequent linking experiment.

[0134] 3) Conversion The above-mentioned synthetic genes and other related elements are sequentially linked to linearized carrier skeletons. The following linkage enzyme system using C115 Infusion is used for linking.

[0135] [Table 2]

[0136] [Table 3] After the coupling reaction is complete, the corresponding resistant plates are pre-dried in a 37°C incubator, inverted, and at the same time, 500 μL of antibody-free culture medium is preheated. Remove the responsive process bacteria from the -80°C refrigerator, leave them on ice for 15-20 minutes until thawed, absorb half of the connecting solution to make them responsive, lightly flick them five times by hand, leave them in an ice bath for 30 minutes, quickly transfer the responsive state to a pot of water bath preheated to 42°C, and subject them to thermal shock for 1 minute (the thermal shock time should be strictly controlled to 45-90 seconds), immediately transfer them to ice after the thermal shock is complete and leave them for 2 minutes, then transfer the responsive state to 500 μl of preheated culture medium, leave them on a 37°C shaking bed for 1 hour, take the bacterial suspension, centrifuge at 4000 rpm / 2 minutes and remove the supernatant (discard 400 μl of supernatant), place 20 glass beads on each side of a plate, aspirate the bacterial suspension and mix uniformly, spread it onto the corresponding resistant plate, and incubate overnight in an incubator at the corresponding temperature.

[0137] After overnight incubation, a bacterial test is performed, and the plates are treated with UV irradiation on a sterile workbench. After 20 minutes, the UV irradiation is complete, a third stage of airflow is applied, 75% ethyl alcohol is taken, and the sterile workbench is wiped down before the bacterial test is performed. 25 μl of sterile water is added to the bottom of a 96-well PCR plate on the sterile workbench, and the bottom is exposed to light to check for air bubbles. Using a sterilized 10 μl white pipette tip, lightly apply the monoclonal bacterial flecks from the overnight-incubated plate, transfer them to the corresponding 96-well reaction plate, and using a 10 μl pipette, blow two or three times to mix uniformly. Aspirate 4 μl from each row and transfer to a new 96-well reaction plate to use as a template for the PCR reaction. 50 μl of antibody-free broth medium is added to the original 96-well plate template, the plate is covered with a sterilized 96-well PCR plate, and the plate is left in a 30°C incubator.

[0138] 4) Bacteriological examination The bacterial testing is performed using an enzyme reaction system with P222-AA Taq, as follows:

[0139] [Table 4]

[0140] [Table 5] After the bacterial testing reaction was complete, TBE test adhesive was added to check if the band size matched the logical band size, and then the 3.5mLAmp-resistant broth culture was incubated overnight in a 48-depth plate in a vibrator at 37°C.

[0141] 5) Plasmid extraction Plasmid extraction was performed using the QIAprep Spin Miniprep Kit (27106) from Qiagen. The concentration of the extracted plasmid was measured and indicated on the wall of a 1.5 mL EP tube. If the concentration was 50 ng / μL or higher, the extraction procedure was considered appropriate, and identification was performed by enzymatic digestion.

[0142] 6) Identification by enzymatic cleavage Using NTI / SnapGene software, we searched for available enzymatic cleavage methods based on existing endonucleases, verified the plasmid's correctness, and determined the following enzymatic cleavage systems:

[0143] [Table 6] After preparing the system, it was left at the appropriate temperature for the corresponding enzyme for 45 minutes. The enzyme-cleaved system was then removed, 5 μl of bromophenol blue / methyl orange was added, and the TAE detection gel was added. A photograph was then taken using a gel scanner, and the size of the band was confirmed (see Figure 1 for results).

[0144] If the band is correct, collect 30 μL of plasmid on the same day and prepare it for analysis. It was delivered to the ligation region sequence of the GENEWIZ sequencing.

[0145] 7) Plasmid sequencing The constructed plasmid was delivered to GENEWIZ sequencing company for sequencing, which proved that the carrier structure had been successfully constructed through sequencing.

[0146] Material Information

[0147] [Table 7] A lentiviral carrier containing LV-TRE3G-OKM, LV-TRE3G-O'KM', LV-TRE3G-OKMS, and LV-TRE3G-O'KM'S was constructed, and the plasmid sequences refer to SEQ ID NOs: 34-37.

[0148] 3.2 Packaging of lentiviruses 1) Lentiviral plasmids and auxiliary plasmids (SL3, SL4, SL5) were co-infected with 293T cells.

[0149] 2) Virus collection: After 48 hours of transfection, the cell supernatant was collected by centrifugation.

[0150] 3) Conversion and Concentration: The cell supernatant was concentrated with PEG8000 and then processed by high-speed centrifugation with a sucrose density gradient.

[0151] 4) Quality inspection: This included physical condition inspection, sterility inspection, and titer testing. Titer testing was performed using the qPCR method (results are shown in Figure 2A).

[0152] 5) Transgenic expression testing: The above-mentioned lentiviral carrier and the lentiviral carrier expressing TET3G were introduced into 293T cells, and expression was induced using doxycycline (dox). RNA samples were extracted after 48 hours, and the expression level of transgenic KLF4 (results shown in Figure 2B) was measured using a specific primer for transgenic KLF4. [Examples]

[0153] Reprogramming of four factors that rejuvenate human dermal fibroblasts at the initial stage Experimental plan 1) Primary human dermal fibroblast cell line FY-009 was selected from a female donor over 60 years of age, cultured in a dedicated epidermal cell medium, and subcultured. Cell lines in good condition were then selected for the subsequent viral infection experiment.

[0154] 2) Fibroblasts were cultured in vitro and inoculated into 12-well plates from the 4th generation onward. Once a 40% degree of left-right fusion was achieved, the cells were divided into three groups: Group 1 was a blank control group, without the addition of any virus; Group 2 was the OKMS reprogramming group, to which lentiviruses LV-TRE3G-OKMS and LV-EFS-Tet3G were added; and Group 3 was the O'KM'S reprogramming group, to which lentiviruses LV-TRE3G-O'KM'S and LV-EFS-Tet3G were added (lentivirus carriers were manufactured by Caiye Biotechnology Co., Ltd., see Figure 3 for structural diagrams). Polyglutamine (polybrene) was added simultaneously to promote viral infection efficiency.

[0155] 3) After 24 hours, the supernatant was discarded and replaced with a high-sugar DMEM culture medium containing 20% ​​fetal bovine serum.

[0156] 4) The solution was then changed, doxycycline (dox) was added to induce the expression of transcription factors, and the solution was changed every two days thereafter.

[0157] 5) After 14 days of induction with dox, cells were collected from each group and used for measurement.

[0158] 6) Cells in each group were stained with senescence-related β-galactosidase (SA-β-gal), and the degree of senescence was measured (see Figure 4 for experimental results). The results showed that both reprogramming groups rejuvenated the cells, but the O'KM'S reprogramming group showed a greater degree of rejuvenation.

[0159] 7) Cell RNA samples were extracted from each group, and real-time fluorescence quantitative polymerase chain reaction (q-PCR) was performed to measure COL1A1, COL3A1, COL4A1, FN1, and LAMA5 as genes associated with rejuvenation function (see Figure 5 for experimental results). THY1, P4HB, S100A4, and SERPINH1 were measured as characteristic genes of fibroblasts (see Figure 6 for experimental results). NANOG and SALL4 were measured as characteristic genes at the initiation stage of reprogramming (see Figure 7 for experimental results). DPPA5 and LIN28A were measured as characteristic genes at the maturation stage of reprogramming (see Figure 8 for experimental results). As a result, the O'KM'S reprogramming group showed significantly higher levels of genes associated with cell rejuvenation function and characteristic genes compared to the OKMS reprogramming group. Therefore, in terms of characteristic genes at the reprogramming stage, the former was still in the initiation stage, while the latter had already entered the maturation stage.

[0160] 8) Cells were collected from each group, methylation analysis was performed, and epiAge analysis was performed on the test results (see Figure 9 for analysis results). As a result, the epiAge of the blank control group was 60 years or older, which was close to the actual age of the donor. The OKMS partial reprogramming group reduced the epiAge of the cells to 42 years, and the O'KM'S partial reprogramming group further reduced the epiAge of the cells to 39 years. [Examples]

[0161] Reprogramming of the initial stages of three factors that rejuvenate human dermal fibroblasts Experimental plan 1) Human dermal fibroblasts were cultured in a specialized epidermal cell culture medium, passed through cell lines, and a cell line in good condition was selected for the next viral infection experiment.

[0162] 2) Fibroblasts were cultured in vitro and inoculated into 12-well plates from the 4th generation onward. Once a 40% degree of left-right fusion was achieved, the cells were divided into three groups: Group 1 was a blank control group, without the addition of virus; Group 2 was the OKM reprogramming group, to which lentiviruses LV-TRE3G-OKM and LV-EFS-Tet3G were added; and Group 3 was the O'KM' reprogramming group, to which lentiviruses LV-TRE3G-O'KM' and LV-EFS-Tet3G were added. (The lentivirus carriers were manufactured by Caiye Biotechnology Co., Ltd., and their structures are shown in Figure 10). Polydextrin (polybrene) was added simultaneously to promote viral infection efficiency.

[0163] 3) After 24 hours, the supernatant was discarded and replaced with a high-sugar DMEM culture medium containing 20% ​​fetal bovine serum.

[0164] 4) The solution was then changed, and doxycycline (DOX) was added to induce the expression of transcription factors. After that, the solution was changed every two days.

[0165] 5) After 14 days of induction with dox, cells were collected from each group and used for measurement.

[0166] 6) Cells in each group were stained with senescence-related β-galactosidase (SA-β-gal), and the degree of senescence was measured (see Figure 11 for experimental results). The results showed that both reprogramming groups rejuvenated the cells, but the OKM reprogramming group showed a lower degree of rejuvenation, while the O'KM' reprogramming group showed a higher degree of rejuvenation.

[0167] 7) Cell RNA samples were extracted from each group, and real-time fluorescence quantitative polymerase chain reaction (q-PCR) was performed to measure COL1A1, COL3A1, COL4A1, FN1, and LAMA5 as genes associated with rejuvenation function (see Figure 12 for experimental results). THY1, P4HB, S100A4, and SERPINH1 were measured as characteristic genes of fibroblasts (see Figure 13 for experimental results). Characteristic genes at the initiation stage of reprogramming: NANOG and SALL4 were measured (see Figure 14 for experimental results). Characteristic genes at the maturation stage of reprogramming: DPPA5 and LIN28A were measured (see Figure 15 for experimental results). As a result, the O'KM' reprogramming group showed significantly higher levels of genes associated with cell rejuvenation function and characteristic genes compared to the OKM reprogramming group. Therefore, looking at the characteristic genes at the reprogramming stage, the former is still in the initiation stage, while the latter has already entered the maturation stage. [Examples]

[0168] Gene synthesis and construction of adenovirus (AAV) carriers 1. Design of single-element sequences of transcription factors Refer to SEQ ID NOs: 4-10 to design the sequences of OCT4' and mutant OCT4'1-6, and design the corresponding nucleotide sequences, for example, as shown in SEQ ID NO: 3. Refer to SEQ ID NOs: 14-20 to design the sequences of C-MYC and mutant C-MYC, and design the corresponding nucleotide sequences, for example, as shown in SEQ ID NO: 13. Refer to SEQ ID NO: 22 to design the sequence of KLF4, and design the corresponding nucleotide sequence, for example, as shown in SEQ ID NO: 21. Refer to SEQ ID NO: 24, for example, to design the sequence of SOX2, and design the corresponding nucleotide sequence, for example, as shown in SEQ ID NO: 23.

[0169] For example, design the linker sequences P2A, T2A, and E2A as shown in sequence numbers 25-27. Design the Kozak sequence gccgccaccatgg. For example, design the promoter sequence for TRE3G as shown in sequence number 63. Design the hGH poly(A) sequence as shown in sequence number 65.

[0170] 2. Construction of a sequence in which transcription factor elements are linked in series. The arrays formed by connecting the above elements in series are illustrative examples only, and are referred to as the OCT4'-P2A-KLF4s-T2A-SOX2 series array (simply called O'KS, corresponding to array number 32) and the OCT4-P2A-KLF4s-T2A-SOX2 series array (simply called OKS, corresponding to array number 33), respectively, as shown by array numbers 32-33.

[0171] 3. Construction and packaging of AAV carriers (implemented by Saiye Biotechnology Co., Ltd.) 3.1 Construction of AAV carriers 1) A suitable combination of enzyme cleavage sites was selected from the AAV backbone plasmid, and the AAV backbone plasmid and enzyme were incubated at 37°C for 1 hour. Subsequently, the corresponding linear backbone plasmid after enzyme cleavage was recovered by agarose gel electrophoresis.

[0172] 2) OKS and O'KS fragments were synthesized, and the gene fragments and other related elements were cloned into linear skeleton plasmids using a recombinant kit (Vazyme#C115) that maintained identity. The reaction system was converted to susceptible bacteria, plated, picked, shaken, and extracted. Enzyme cleavage and sequencing were analyzed.

[0173] 3) After confirming the results of enzyme digestion and sequencing, if there are no problems (see Figure 16 for the results of enzyme digestion), the plasmid from the correct bacterial suspension is selected, converted back into susceptible bacteria, spread onto a plate, picked, amplified, and the plasmids necessary for AAV packaging are extracted, including AAV-TRE3G-OKS and AAV-TRE3G-O'KS, the plasmid sequences of which are referred to SEQ ID NOs: 38-39.

[0174] 3.2 AAV Packaging 1) AAV virus plasmid, auxiliary plasmid, and packaging plasmid were co-infected with 293T cells.

[0175] 2) Virus collection: 48 hours after transfection, the supernatant of the cells was collected by centrifugation.

[0176] 3) Conversion and Concentration: The cell supernatant was concentrated with PEG8000 and then processed by high-speed centrifugation with a sucrose density gradient.

[0177] 4) Quality inspection: This included physical condition inspection, sterility inspection, and titer testing. Titer testing was performed using the qPCR method (see Figure 17 for titer measurement results). [Examples]

[0178] Gene synthesis and construction of circular mRNA carriers 1. Construction of different series-linked circular RNA carriers of partial reprogramming factors. a) In the design of circular RNAs linked in series with different partial reprogramming factors, single-gene, two-gene, three-gene, and four-gene series were designed, and the different genes were linked together with P2A, T2A, and E2A series peptides. See Figure 18 and Table 1 for schematic diagrams of the carrier structures of the different gene series links.

[0179] [Table 8] b) For the selection of ring-forming elements, we performed group I intron rearrangement of either the Anabaena group I intron from the genus Vibrio or the group I intron of Azoarcus sp. BH7 from the genus Vibrio to form a ring-forming frame PIE (permuted intron-exon) structure. We then combined different partial reprogramming factors to reconstruct the target gene sequence into the PIE frame and reassembled it into a pBlueScript II SK(+) plasmid.

[0180] AnaPIE Element The 5' element array is indicated by array index: 49, and the 3' element array is indicated by array index: 50.

[0181] AzoPIE element The 5' element array is indicated by array number 51, and the 3' element array is indicated by array number 52.

[0182] c) In plasmid synthesis, the recombinant plasmid was synthesized by a third-party sequencing company. The resulting plasmid was then subjected to template linearization using EcoR I to obtain a pre-transcription template.

[0183] 2. Production of serially linked circular RNAs with different partial reprogramming factors and verification of ring formation. a) In vitro transcription and cyclization of cyclic RNA The in vitro transcription system is as follows:

[0184] Table 2

[0185] [Table 9] The DNA was incubated in a PCR machine at 37°C for 3 hours, and then digested using DNase I (5U / μL). The digestion concentration was 5U / μg of DNA template, and the digestion conditions were 37°C for 20 minutes.

[0186] Cyclization reaction A cyclic cyclization system was created, and the cyclic cyclization system is as follows:

[0187] Table 3

[0188] [Table 10] The prepared cyclization system was placed in a PCR meter and incubated at 60°C for 20 minutes.

[0189] b) Purification and identification of circular RNA The cyclization system was transferred to a 1.5 mL centrifuge tube, and RNA precipitation solutions of lithium chloride at a concentration of 7.5 M (0.5x by volume) and 50 mM EDTA were added. The mixture was uniformly mixed and allowed to precipitate for 1 hour in a refrigerator at -20°C. After precipitation was complete, the mixture was centrifuged at 18360 × g for 15 minutes at 4°C, and the supernatant was removed as much as possible. 800 μL of 70% ethyl alcohol solution was added, the precipitate was blown off, and the mixture was centrifuged again under the same conditions. After removing the supernatant as much as possible, ethyl alcohol was added, and each experimental group was resuspended in 100 μL of enzyme-free water. The concentration of the circRNA was measured, and the size of the circRNA was determined by 1% agarose gel electrophoresis or 2% E-Gel electrophoresis. Circular RNA was formed as a result of electrophoresis, and the results are shown in Figure 19.

[0190] 3. Verification of eukaryotic cell transfection protein expression of partial reprogramming factors. a) Cell preparation and mRNA transcription HEK293T / 17 cells were taken from one T75 culture and plated when the cell convergence rate was 80%-90%. The medium was discarded, washed with PBS, discarded the PBS, 0.05% pancreatic enzyme digested cells were added, and after waiting until the cells rounded, the pancreatic enzyme was discarded, and the cells were dispersed by spraying with 10 mL of antibiotic-free 10% FBS cell medium. The cells were counted and the cell concentration was set to 1 × 10⁻⁶. 6 The cell suspension was adjusted to cells / mL ± 10%, and 10 48-well plates were prepared. Each plate was inoculated with 0.2 mL of cell suspension, and the cell culture plates were incubated in a 37°C, 5% CO2 incubator for 24 ± 4 hours.

[0191] 0.4g each of MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, and Lin28A-P2A-Nanog-circRNA were dissolved in 25 μL of serum-free DMEM medium, gently mixed, and allowed to stand at room temperature for 5 minutes. 1 μL of LipoHigh transfection reagent was dissolved in 25 μL of serum-free DMEM medium, gently mixed, and allowed to stand at room temperature for 5 minutes. The above LipoHigh-containing medium was added to the circRNA-containing medium, gently mixed, and allowed to stand at room temperature for 20 minutes. The cell culture plate was removed from the incubator, the inoculated cells were added dropwise, gently mixed, and then cultured in a 37°C, 5% CO2 incubator. After 4-8 hours, the liquid was discarded, 0.5 mL of 2% FBS-containing medium was added, and the culture was continued.

[0192] b) Refer to Figure 20 for the results of Western blot analysis (WB). The results showed that all circular RNAs—MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, and Lin28A-P2A-Nanog-circRNA—can express their corresponding proteins.

[0193] 4. Transfect fibroblasts with wild-type OKSMLN circular RNA to induce iPSCs and conduct functional verification experiments. Human skin fibroblasts were placed in a 12-well plate and grown to a convergence degree of 80%-90%. Circular RNAs (MYC-circRNA, KLF4-circRNA, OCT4-P2A-SOX2-circRNA, and Lin28A-P2A-Nanog-circRNA) were encapsulated in the transfection reagent Lipofectamine MessengerMAX (Cimar Fisher). A total mRNA / pore volume of 2 μg was co-cultured with the fibroblasts, and transfection was performed. After 24 hours, transfection was stopped, and cell growth was observed. On day 7 after transfection, cell growth was again observed to check for the appearance of clones. The results are shown in Figure 21. The results demonstrated that wild-type OSKMLN circular RNA can induce fibroblasts and generate pluripotent stem cell-like clones.

[0194] Circular RNA sequence

[0195] [Table 11]

Claims

1. A method for rejuvenating cells, characterized by rejuvenating cells through initial reprogramming using an engineered transcription factor that lacks phase separation ability.

2. The method for determining rejuvenation described above is characterized in that the expression level of an epigenetic indicator gene is increased or decreased compared to a reference value.

3. The expression "rejuvenating the cells" refers to a change in the proportion of regenerated cells that are positive for senescence-related galactosidase (SA-β-Gal), and the method according to claim 1 is characterized in that the change is lower than the reference value.

4. The method according to claim 2, wherein the expression of rejuvenating cells is characterized by a decrease in the cellular DNA methylation epigenetic age epiAge level and / or an increase in the expression level of rejuvenation function-related genes and / or an increase in the expression level of characteristic genes at the initiation stage of reprogramming and / or a decrease in the expression level of characteristic genes at the maturation stage of reprogramming and / or an increase in the expression level of cellular identity genes, compared to a reference value.

5. The method according to claim 4, characterized in that the above-mentioned rejuvenation function-related genes include COL1A1, COL3A1, COL4A1, FN1, and LAMA5; the characteristic genes detected at the initiation stage of the above-mentioned reprogramming include NANOG and SALL4; the characteristic genes detected at the maturation stage of the above-mentioned reprogramming include DPPA5 and LIN28A; and the detected cell identity genes include THY1, P4HB, S100A4, and SERPINH1.

6. A cell initiation-stage reprogramming method characterized by being implemented using gene-transformed cells containing engineered reprogramming factors that do not possess phase separation ability.

7. An engineered transcription factor that lacks phase separation ability and is characterized by its ability to reprogram cells at the initiation stage.

8. The above-mentioned engineered reprogramming factor that lacks phase separation ability is characterized by causing the transcription factor to lose its phase separation-inducing function based on modification of the phase separation function-related region of the transcription factor, as described in any one of claims 1 to 7.

9. The above modifications include amino acid modifications, partial deletions, and ubiquitination mutations in the IDR region of the transcription factor protein, as described in claim 8.

10. Furthermore, the engineered transcription factor according to claim 9 is characterized in that it can mutate polar charged amino acids or acidic amino acids in the IDR region of a protein, can further substitute polar charged amino acids or acidic amino acids with neutral amino acids, and can further mutate the acidic amino acids aspartic acid (Asp) and glutamic acid (Glu) with the neutral amino acid alanine.

11. The engineered transcription factor according to claim 10, characterized in that it can mutate positions 1 to 140 and / or 288 to 360 of the sequence indicated by Sequence ID No. 2, including an error range of 10 to 20 amino acids before and after it.

12. The engineered transcription factor according to claim 10, characterized in that at least one of the positions 8, 20, 31, 108, 138, 291, 297, 26, 56, 68, 91, 96, 98, 104, 113, 125, 127, 130, 134, 135, 296, 299, 341, 343, 145, 147, 166, 188, 209, 210, 215, 224, 238, 246, 270, and 272 of the sequence shown by sequence number 2 can be mutated.

13. The engineered transcription factor according to claim 11 or 12, characterized in that the mutated sequence may be the sequence indicated by any of sequence numbers 4-10, or a sequence that can achieve at least 85%, 90%, or 95% or more of the same function.

14. The above modification may be a mutation that does not cause polyubiquitin chain modification, and may further be a substitution of lysine K with an amino acid that does not cause ubiquitin chain modification, and may further be a substitution with arginine R, as described in claim 9.

15. The engineered transcription factor according to claim 14, characterized in that at least one of the 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 the sequence indicated by sequence number 12 can be mutated.

16. More preferably, the engineered transcription factor according to claim 15, characterized in that at least one of the 148th and 389th amino acids is mutated, preferably the 20th to 25th amino acids are mutated, and preferably all of the sites described in claim 15 are mutated.

17. The engineered transcription factor according to claim 16, characterized in that the mutated sequence may be, for example, the variant shown in SEQ ID NOs: 14-20, or a sequence that can achieve at least 85%, 90%, or 95% or more of the same function.

18. The engineered transcription factor according to claim 9, characterized in that the above partial deletion treatment is a deletion treatment of a key regulatory region of protein phase separation, or a deletion treatment of an amino acid that plays a key role in the generation of phase separation.

19. The engineering transcription factor according to claim 18, characterized in that the above partial deletion treatment is preferably terminal deletion treatment, IDR region deletion treatment, or non-key region deletion treatment other than DBD, and more preferably charged amino acid portion deletion treatment in IDR region, or key amino acid deletion treatment of ubiquitin chain modification.

20. The engineered transcription factor according to claim 19, characterized in that the key amino acid for the ubiquitin chain modification described above is lysine.

21. The engineered transcription factor according to claim 19, characterized in that a portion of the SOX2 sequence shown in SEQ ID NOs: 21-22 can be deleted, preferably all of positions 2-40 and / or 201-317 can be deleted to obtain, for example, the amino acid sequence shown in SEQ ID NOs: 41-43, or a sequence that can achieve at least 85%, 90%, or 95% or more identity of the same function.

22. The engineered transcription factor according to claim 19, characterized in that a portion of the sequence of OCT4 shown in SEQ ID NOs: 1-2 can be deleted, preferably all of positions 2-140 and / or C-terminus positions 288-360 can be deleted to obtain, for example, the amino acid sequence shown in SEQ ID NOs: 44-46, or a sequence that can achieve at least 85%, 90%, or 95% or more identity of the same function.

23. The engineered transcription factor according to claim 19, characterized in that a portion of the c-MYC sequence shown in SEQ ID NOs: 11-12 can be deleted, preferably by performing deletion treatment on all or part of positions 2 to 360, to obtain, for example, the amino acid sequence shown in SEQ ID NOs: 47-48, or a sequence that can achieve at least 85%, 90%, or 95% or more identity of the same function.

24. A nucleic acid capable of expressing an engineered transcription factor according to any one of claims 7 to 23.

25. The nucleic acid according to claim 24, characterized by comprising a sequence indicated by sequence numbers 3, 13, and 21, or a sequence capable of achieving at least 85%, 90%, or 95% or more identity of the same function.

26. An element comprising an engineered transcription factor according to any one of claims 7 to 25, comprising DNA, RNA, a recombinant carrier, a recombinant protein, and a recombinant cell, wherein preferably the recombinant carrier is an adenovirus carrier, a poxvirus carrier, or a lentivirus carrier.

27. The element according to claim 25, characterized in that the RNA element is further a linear mRNA element, a siRNA element, a circular RNA element, a circular mRNA element, or a tRNA element.

28. The element according to claim 25, characterized in that the recombinant carrier is a monocistrone, a doublecistrone, or a multicistrone linked by at least one linker peptide.

29. The recombinant carrier is characterized in that a plurality of transcription factors are linked in series, connected by at least one linker peptide, or directly connected, and the linker peptide is selected from the sequences shown in SEQ ID NOs: 25-27.

30. An engineered transcription factor according to any one of claims 7 to 25, characterized in that it is at least one selected from OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG, and GLIS1, or an element according to any one of claims 26 to 29.

31. The above-mentioned engineered transcription factors may be connected in series, and the connection procedure may be OCT4-SOX2, LIN28-NANOG, OCT4-SOX2-KLF4, OCT4-KLF4-c-MYC, or OCT4-KLF4-c-MYC-SOX2, characterized in that the engineered transcription factor or element according to claim 30.

32. The above series-connected sequences may be those indicated by sequence numbers 28-33, and the sequence of recombinant carriers may be those indicated by sequence numbers 34-39, characterized in that the engineered transcription factor or element according to claim 31.

33. An engineered transcription factor characterized in that at least one of the above transcription factors is modified by a transcription factor according to any one of claims 8 to 23.

34. A transport system for transporting elements according to any one of claims 26 to 32.

35. Furthermore, the transport system according to claim 34 may also include liposome LNPs, exosome transport, GalNAc system, VLP transport, protamine, polymer, inorganic nanoparticles, exosomes, polymer matrix, viral transfection, etc.

36. The use of an engineered transcription factor according to any one of claims 7 to 25, characterized in that it is used for cell therapy, gene therapy, immune cell proliferation, cancer treatment, or treatment of aging-related diseases, or for the production of pharmaceuticals for cell therapy, gene therapy, immune cell proliferation, cancer treatment, or treatment of aging-related diseases, an element containing an engineered transcription factor according to any one of claims 26 to 32, or a transport system according to any one of claims 34 to 35.

37. A pharmaceutical composition characterized by comprising at least one of the following: an engineered transcription factor according to any one of claims 7 to 25, an element containing the engineered transcription factor according to any one of claims 26 to 32, or a transport system according to any one of claims 34 to 35.

38. The pharmaceutical composition according to claim 37, characterized in that the above pharmaceutical composition may be a mixture.

39. A combination therapy characterized by using the pharmaceutical composition described in claim 37 or 38 in combination with other related pharmaceuticals.

40. A combination therapy method characterized by using a combination of the pharmaceutical composition described in claim 37 or 38 and the B18R protein to perform treatment.

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