Recombinant reprogramming factors and their use

Abstract

This application provides a method for rejuvenating a population of cells, for example, skin cells, comprising introducing an effective amount of a recombinant reprogramming factor without liquid-liquid phase separation (LLPS) into the population of cells, for example, skin cells.

Description

Technical Field

[0001]

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority of Chinese Patent Application No. CN202310793813.X, filed on June 30, 2023, the content of which is incorporated herein by reference in its entirety.

[0002] [Reference to Electronic Sequence List] The content of the electronic sequence list (313682000140 SEQLIST.xml; size: 365104 bytes; and creation date: June 30, 2024) is incorporated herein by reference in its entirety.

[0003] [Technical Field to Which the Invention Belongs] This application relates to a method for rejuvenating a population of cells (e.g., skin cells), which includes introducing a recombinant reprogramming factor without effective liquid - liquid phase separation (LLPS) into the population of cells (e.g., skin cells). Also provided are methods for delaying or preventing the progression of aging of tissues (e.g., skin) of an individual (e.g., a human) using either the skin rejuvenation method described herein and / or an LLPS - deficient recombinant reprogramming factor, and methods for treating and / or preventing aging, damage, or disease or condition of tissues (e.g., skin tissue) of an individual.

Background Art

[0002]

[0004] A key approach to induced pluripotent stem cell (iPSC) reprogramming is to reprogram somatic cells into a pluripotent state using transcription factors such as OCT4, SOX2, KLF4, and MYC ("OSKM" or "4 factors"). This process is called complete reprogramming and can be divided into initiation, maturation, and stabilization phases (Non-Patent Literature 1 and 2). During the reprogramming process, cells undergo dramatic changes in transcriptional levels and epigenetic modifications, dramatically altering their identity and state. In the initial stage, the expression levels of early pluripotency genes such as TRA-1-60, NANOG, SSEA4, and SALL4 rapidly increase, while the expression levels of somatic identity genes do not change significantly. In the maturation stage, the above early pluripotency genes and late pluripotency genes such as DPPA5 and LIN28 are highly expressed, while the expression levels of somatic identity genes rapidly decrease (Non-Patent Literature 1 and 2). Finally, during the stabilization phase, the cell fully establishes its pluripotency gene expression network and becomes an iPSC, which expresses the aforementioned pluripotency genes and other classical core pluripotency transcription factors, such as OCT4, SOX2, and KLF4, at high levels, but does not express somatic identity marker genes. This reprogramming process rejuvenates the cell, which can be detected by changes in transcription levels of specific marker genes, cellular senescence markers, and especially epigenetic age (EpiAge) based on DNA methylation (Non-patent Literature 2-4).

[0003]

[0005] Partial reprogramming refers to the cessation of complete reprogramming midway through the process described above. Ocampo rejuvenation occurs in the early stages of the reprogramming process, while loss of somatic identity occurs in the middle and later stages of the reprogramming process, thus there is a time frame for rejuvenating cells without altering their original identity (Non-Patent Literature 2 and 5). Methods for rejuvenating cells through partial reprogramming using transcription factors have been reported mainly as follows: 1) In vitro induction of time-limited partial reprogramming, also known as transient reprogramming. For example, transient induction of four-factor expression using an inducible transgene expression system in human fibroblasts has been found to reverse the cell's EpiAge while maintaining fibroblast identity (Non-Patent Literature 2 and 4). 2) In vivo induction of transient pulsed partial reprogramming. For example, transient pulse induction of four-factor expression in transgenic mice with an inducible expression system has been found to rejuvenate organs in mice with progeria (e.g., skin, spleen, liver, kidney, stomach, and artery), and to some extent, rejuvenate organs in wild-type aged mice (e.g., pancreas and muscle) (Non-Patent Literature 5 and 6). 3) Partial reprogramming with a reduced number of transcription factors (called three-factor reprogramming). For example, when an AAV vector expressing three transcription factors (OSK) was delivered to the eye of a mouse glaucoma model, remarkable therapeutic efficacy was shown (Non-Patent Literature 7).

[0006] Certain proteins and / or nucleic acid molecules within a cell can interact with each other to create phases with different physical and chemical properties in the surrounding homogeneous environment, typically exhibiting droplet-like characteristics, resulting in the formation of membraneless organelles or intracellular structures, i.e., biomolecular condensates. This process is called phase separation or liquid-liquid phase separation (LLPS) (Non-Patent Documents 8 and 9). Biomolecular condensates are micron-sized and form relatively isolated spatial regions that selectively concentrate molecules. Biomolecular condensates formed by phase separation typically have a higher protein density and lower molecular micromobility compared to their surroundings, allowing them to more efficiently facilitate certain biochemical reactions. The structural characteristics of proteins capable of forming condensates are still being studied.

[0007] In 2021, Non-Patent Document 10 found that OCT4 undergoes phase separation to regulate topology-associated domain (TAD) rearrangement, and that TAD rearrangement contributes to cell fate transition.

[0008] All publications, patents, patent applications and published patent applications referred to herein are incorporated herein by reference in their entirety. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] K. Tanabe et al., Proc Natl Acad Sci US A. 2013;110(30):12172-12179 [Non-Patent Document 2] N. Olova et al., Aging Cell. 2019;18(1):e12877 [Non-Patent Document 3] A. Daunay, et al., Aging (Albany NY). 2022;14(19):7718-7733 [Non-Patent Document 4] D. Gill et al., eLife, 2022; 11: e71624 [Non-Patent Document 5] A. Ocampo et al., Cell. 2016;167(7):1719-1733.e12 [Non-Patent Document 6] K. Browder et al., Nat Aging. 2022;2(3):243-253 [Non-Patent Document 7] Lu et al.2020;588(7836):124-129 [Non-Patent Document 8] C. Brangwynne et al., Science. 2009;324(5935):1729-1732 [Non-Patent Document 9] P. Li et al. Nature. 2012;483(7389):336-340 [Non-Patent Document 10] Wang et al. Cell Stem Cell. 2021;28(10):1868-1883.e11 [Overview of the Initiative]

[0005]

[0009] One aspect of this application provides a method for rejuvenating a population of cells (e.g., skin cells), comprising introducing an effective amount of LLPS-free recombinant reprogramming factor into the population of cells (e.g., skin cells).

[0010] In one embodiment, the method is for rejuvenating the skin or skin cells. In one embodiment of any of the above skin rejuvenation methods, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof.

[0011] In one embodiment of any of the above skin rejuvenation methods, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after cosmetic, dermatological or surgical treatment. In one embodiment, the skin tissue is photoaged skin tissue.

[0012] In one embodiment of any of the above skin rejuvenation methods, the method reprograms a population of skin cells but does not reprogram them into stem cells.

[0006]

[0013] In one embodiment of any of the above skin rejuvenation methods, compared to a reference skin in which recombinant reprogramming factors have not been introduced, the rejuvenated population of skin cells exhibits: (i) a lower proportion of cells positive for cellular senescence biomarkers; (ii) reduced expression and / or function of biomarkers for cellular senescence; (iii) a reduced epigenetic age (EpiAge); (iv) increased expression and / or function of biomarkers associated with healthy skin cell function; (v) increased expression and / or function of biomarkers for skin cell identity; (vi) decreased expression and / or function of biomarkers for apoptosis; (vii) decreased expression and / or function of biomarkers for ferroptosis; (viii) decreased expression and / or function of biomarkers for inflammation; (ix) decreased expression and / or function of biomarkers for endoplasmic reticulum stress; and / or (x) decreased expression and / or function of biomarkers associated with unhealthy skin cell function.In one embodiment, (i) EpiAge biomarkers are selected from the group consisting of senescence-related β-galactosidase (SA-β-gal), p16 and p21 or a combination thereof; (ii) cellular senescence is measured using Horvat's clock, Levine's clock, Hanham's clock and / or AltumAge; (iii) biomarkers related to the function of healthy skin cells are selected from the group consisting of COL1A1, COL3A1, COL4A1, ELN, FN1 and LAMA5 or a combination thereof; (iv) biomarkers for skin cell identity are selected from the group consisting of THY1, P4HB, S100A4 and SERPINH1 or a combination thereof; (v) biomarkers for apoptosis are loss of membrane asymmetry, cleavage of Bcl-2 family proteins, caspase activation, caspase substrate cleavage, and non-caspase proteases. (vi) a biomarker for ferroptosis is HMOX1, SAT1, or a combination thereof; (vii) a biomarker for inflammation is IL6, CCL3, CXCL2, CXCL8, CSF2, or a combination thereof; (viii) a biomarker for endoplasmic reticulum stress is Hsp70 heat shock protein, Hsp40 heat shock protein, DDIT3, and HERPUD1, or a combination thereof; and / or (ix) a biomarker for unhealthy skin cell function is selected from the group consisting of IL6, CXCL8, IL1A, IL1B, IGFBP3, CDKN1A, TGFB3, MMP1, MMP2, and SOD2, or a combination thereof.

[0007]

[0014] In one embodiment of any of the above skin rejuvenation methods, compared to reference skin cells into which an effective amount of LLPS-containing (LLPS-free) reference reprogramming factor has been introduced, the rejuvenated skin cell population exhibits (i) increased expression and / or function of initiation phase reprogramming (IPR) biomarkers, (ii) decreased expression and / or function of maturation phase reprogramming biomarkers, and / or (iii) decreased expression and / or function of stabilization phase reprogramming biomarkers. In one embodiment, (i) the IPR biomarker is NANOG and / or SALL4, (ii) the maturation phase reprogramming biomarker is DPPA5 and / or LIN28A, and / or (iii) the stabilization phase reprogramming biomarker is OCT4 and / or XACT.

[0015] In one embodiment of any of the above skin rejuvenation methods, skin tissue containing a population of skin cells into which an effective amount of recombinant reprogramming factor has been introduced shows higher expression of one or more substances selected from the group consisting of elastin, collagen I, collagen III, and collagen IV, compared to reference skin tissue that does not contain skin cells into which an effective amount of recombinant reprogramming factor has been introduced. In one embodiment, skin tissue containing a population of skin cells into which an effective amount of recombinant reprogramming factor has been introduced shows higher expression of one or more substances selected from the group consisting of elastin, collagen I, collagen III, and collagen IV, compared to reference skin tissue containing skin cells into which an effective amount of LLPS-containing reference reprogramming factor has been introduced. In one embodiment, the reference skin tissue and the skin tissue originate from the same individual. In one embodiment, the reference skin tissue and the skin tissue originate from different individuals.

[0016] In one embodiment of any of the above skin rejuvenation methods, the recombinant reprogramming factor contains a mutation in the LLPS-related domain compared to the corresponding wild-type reprogramming factor. In one embodiment, the LLPS-related domain is selected from the group consisting of an endogenous irregular region (IDR), a region outside the DNA-binding domain (DBD), a ubiquitination site, or any combination thereof. In one embodiment, the mutations include: (i) substituting an acidic amino acid in the IDR with a neutral amino acid; (ii) substituting a ubiquitizable amino acid with an ubiquitizable amino acid; (iii) deleting a ubiquitizable amino acid; (iv) deleting an amino acid in the IDR; and / or (v) deleting an amino acid outside the DBD region. In one embodiment, (i) the ubiquitizable amino acid is K; and / or (ii) the amino acid to be deleted is an acidic amino acid. In one embodiment, the wild-type reprogramming factor is derived from a family selected from the group consisting of the OCT family, SOX family, KLF family, MYC family, LIN28 family, NANOG family, and GLIS family. In another embodiment, the wild-type reprogramming factor is selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG, and GLIS1.

[0017] In one embodiment of any of the above skin rejuvenation methods, the recombinant reprogramming factor is recombinant OCT4. In one embodiment, recombinant OCT4 contains mutations at amino acid positions 1-140 and / or 288-360 with reference to SEQ ID NO: 2. In one embodiment, recombinant OCT4 contains substitutions at amino acid positions selected from the group consisting of 8, 20, 26, 31, 56, 68, 91, 96, 98, 104, 108, 113, 125, 127, 130, 134, 135, 138, 291, 296, 297, 299, 341, and 343 with reference to SEQ ID NO: 2. In some embodiments, recombinant OCT4 includes substitutions at amino acid positions selected from the group consisting of D8, D20, E26, D31, E56, E68, E91, E96, E98, E104, D108, E113, E125, E127, E130, E134, E135, D138, D291, E296, D297, E299, E341, and E343. In some embodiments, the substitution includes substituting an acidic amino acid with a neutral amino acid, such as substituting D or E with A. In some embodiments, recombinant OCT4 includes substitutions selected from the group consisting of D8A, D20A, D31A, D108A, E134A, D138A, D291A, or any combination thereof.In one embodiment, recombinant OCT4 is: (i) D8A, D20A, E26A, D31A, E56A, E68A, E91A, E96A, E98A, E104A, D108A, E113A, E125A, E127A, E130A, E134A, E135A, D138A, D291A, E296A, D297A, E299A, E341A and E34 3A; (ii) D8A, D20A, E26A, D31A, E56A, E68A, E91A, E96A, E98A, E104A, D108A, E113A, E125A, E127A, E130A, E134A, E135A and D138A; (iii) D291A, E296A, D297A, E299A, E341A and E343A; (iv) D8A, D20A, E26A, D31A, E56A, E68A, E91A, E96A, E98A, E104A, D108A, E113A, E125A, E127A, E130A, E134A, D138A, D291A, E296A, E299A, E341A and E343A; (v) E134A, E135A, E296A and D297A; (vi) D8A, D The recombinant OCT4 includes substitutions selected from the group consisting of 20A, D31A, D108A, D138A, D291A and D297A; and (vii) E26A, E56A, E68A, E91A, E96A, E98A, E104A, E113A, E125A, E127A, E130A, E134A, E135A, E296A, E299A, E341A and E343A. In some embodiments, recombinant OCT4 includes a mutant having at least about 85% sequence identity with any of the amino acid sequences of SEQ ID NOs: 4-10 or any of SEQ ID NOs: 4-10. In some embodiments, recombinant OCT4 includes deletions at amino acid positions 2-140 and / or 288-360 with reference to SEQ ID NO: 2. In one embodiment, recombinant OCT4 includes a deletion selected from the group consisting of (i) deletions at amino acid positions 2-140 and 301-360; (ii) deletions at amino acid positions 301-360; and (iii) deletions at amino acid positions 2-140. In another embodiment, it includes a mutant having an amino acid sequence of any of SEQ ID NOs. 44-46 or a sequence identity of at least approximately 85% with any of SEQ ID NOs. 44-46.

[0008]

[0018] In one embodiment of any of the above skin rejuvenation methods, the recombinant reprogramming factor is recombinant c-MYC. In one embodiment, recombinant c-MYC includes a substitution of a ubiquitizable amino acid with a non-ubiquitizable amino acid, where the amino acid position refers to SEQ ID NO: 12 or 85. In one embodiment, the substitution includes substituting K with R. In one embodiment, recombinant c-MYC includes a substitution at an amino acid position selected from the group consisting of 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, with reference to SEQ ID NO: 12 or 85. In one embodiment, recombinant c-MYC includes substitutions selected from the group consisting of K51R, K52R, K148R, K275R, K289R, and K389R or any combination thereof. In one embodiment, recombinant c-MYC is one of the following: (i) K51R, K52R, K126R, K143R, K148R, K157R, K206R, K269R, K275R, K289R, K298R, K317R, K323R, K326R, K341R, K355R, K371R, K389R, K392R, K397R, K3 The substitutions include (ii) K148R, K412R, K422R, K428R and K430R; (ii) K148R; (iii) K389R; (iv) K148R and K389R; (v) K51R and K52R; (vi) K275R and K289R; and (vii) substitutions selected from the group consisting of K51R, K52R, K275R and K289R. In one embodiment, the recombinant c-MYC includes a mutant having at least about 85% sequence identity with any of the amino acid sequences of SEQ ID NOs. 14-20 or any of SEQ ID NOs. 14-20. In one embodiment, the recombinant c-MYC includes a deletion at amino acid positions 2-360 (e.g., a deletion at amino acid positions 2-140) with reference to SEQ ID NOs. 12 or 85. In one embodiment, recombinant c-MYC includes a mutant having at least approximately 85% sequence identity with the amino acid sequence of SEQ ID NO: 47 or 48, or either SEQ ID NO: 47 or 48.

[0009]

[0019] In one embodiment of any of the above skin rejuvenation methods, the recombinant reprogramming factor is recombinant KLF4. In one embodiment, recombinant KLF4 contains deletions at amino acid positions 2-374 with reference to SEQ ID NO: 22. In one embodiment, recombinant KLF4 contains the amino acid sequence of SEQ ID NO: 40 or a variant having at least about 85% sequence identity with SEQ ID NO: 40.

[0010]

[0020] In one embodiment of any of the above skin rejuvenation methods, the recombinant reprogramming factor is recombinant SOX2. In one embodiment, recombinant SOX2 contains deletions at amino acid positions 2-40 and / or 201-317 with reference to SEQ ID NO: 24. In one embodiment, recombinant SOX2 contains the amino acid sequence of any of SEQ ID NOs: 41-43 or a variant having at least about 85% sequence identity with any of SEQ ID NOs: 41-43.

[0011]

[0021] In one embodiment of any of the above skin rejuvenation methods, the recombinant reprogramming factor is encoded by a nucleic acid. In one embodiment, the nucleic acid includes the nucleic acid sequence of SEQ ID NO: 3 or 13. In one embodiment, the nucleic acid is RNA, such as circular RNA (circRNA), autoamplified mRNA, or linear mRNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is contained within a vector, such as a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector. In one embodiment, the nucleic acid is delivered by an exosome, liposome, lipid nanoparticle (LNP), or virus.

[0012]

[0022] In one embodiment of any of the above skin rejuvenation methods, the population of skin cells is present in an individual in need of treatment, and the method includes intra vivo administration of an effective amount of recombinant reprogramming factor to the individual. In one embodiment, the method includes administration of RNA encoding the recombinant reprogramming factor (e.g., circRNA or linear mRNA), the RNA being encapsulated in exosomes or LNPs. In one embodiment, the exosomes or LNPs are delivered by microneedles. In one embodiment, the population of skin cells is obtained from a donor individual and cultured ex vivo. In one embodiment, the method includes transplanting the rejuvenated population of skin cells into an individual in need of treatment. In one embodiment, the donor individual and the individual in need of treatment are the same. In one embodiment, the donor individual and the individual in need of treatment are different. In one embodiment, the method includes ex vivo administration of RNA encoding the recombinant reprogramming factor to a population of skin cells, the RNA being encapsulated in exosomes or LNPs.

[0013]

[0023] In one embodiment of any of the above skin rejuvenation methods, the method comprises introducing a plurality of reprogramming factors into a population of skin cells, where at least one of the plurality of reprogramming factors is a recombinant reprogramming factor without LLPS. In one embodiment, the plurality of reprogramming factors comprises two or more reprogramming factors derived from two or more families selected from the group consisting of the OCT family, SOX family, KLF family, MYC family, LIN28 family, NANOG family, and GLIS family. In one embodiment, the plurality of reprogramming factors comprises two or more reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG, GLIS1, and their variants. In one embodiment, the plurality of reprogramming factors comprises OCT4 and KLF4, where at least one of OCT4 and KLF4 is a recombinant reprogramming factor without LLPS. In one embodiment, the plurality of reprogramming factors comprises three or all of OCT4, KLF4, c-MYC, and SOX2, where at least one of the plurality of reprogramming factors is a recombinant reprogramming factor without LLPS. In some embodiments, OCT4 is recombinant OCT4 without LLPS. In some embodiments, KLF4 is recombinant KLF4 without LLPS. In some embodiments, the multiple reprogramming factors include wild-type reprogramming factors. In some embodiments, the multiple reprogramming factors include (i) recombinant OCT4 without (deleted) LLPS, SOX2, and KLF4; (ii) recombinant OCT4 without LLPS, KLF4, and recombinant c-MYC without LLPS; or (iii) recombinant OCT4 without LLPS, KLF4, recombinant c-MYC, and SOX2 without LLPS. In some embodiments, the multiple reprogramming factors are encoded by multiple nucleic acids. In some embodiments, the multiple nucleic acids reside on separate nucleic acid constructs. In some embodiments, at least two of the multiple nucleic acids reside on a single nucleic acid construct. In some embodiments, at least two of the multiple nucleic acids are under the control of different promoters. In some embodiments, at least two of the multiple nucleic acids are under the control of the same promoter.In certain embodiments, at least two of the plurality of nucleic acids are connected by one or more linker nucleic acids. In certain embodiments, the one or more linker nucleic acids include a linker sequence encoding an IRES or a self-cleaving peptide. In certain embodiments, the one or more linker nucleic acids include a linker sequence encoding a self-cleaving peptide, and the self-cleaving peptide is P2A, T2A, or E2A. In certain embodiments, the nucleic acid construct, in the 5' to 3' direction, comprises the following: (i) a first nucleic acid encoding a recombinant OCT4 without LLPS - a first linker sequence encoding P2A - a second nucleic acid encoding SOX2 - a second linker sequence encoding T2A - a third nucleic acid encoding KLF4; (ii) a first nucleic acid encoding a recombinant OCT4 without LLPS - a first linker sequence encoding P2A - a second nucleic acid encoding KLF4 - a second linker sequence encoding T2A - a third nucleic acid encoding a recombinant c-MYC without LLPS; (iii) a first nucleic acid encoding a recombinant OCT4 without LLPS - a first linker sequence encoding P2A - a second nucleic acid encoding KLF4 - a second linker sequence encoding T2A - a third nucleic acid encoding a recombinant c-MYC without LLPS - a third linker sequence encoding E2A - a fourth nucleic acid encoding SOX2; or (iv) a first nucleic acid encoding a recombinant OCT4 lacking LLPS - a first linker sequence encoding P2A - a second nucleic acid encoding KLF4 - a second linker sequence encoding T2A - a third nucleic acid encoding SOX2. In certain embodiments, (i) the recombinant OCT4 without LLPS comprises the amino acid sequence of SEQ ID NO: 4, and / or (ii) the recombinant c-MYC without LLPS comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the nucleic acid construct comprises a nucleic acid sequence of any one of SEQ ID NOs: 28, 30, 32, 35, 37, 39, 57, 59, 61, 94, 98, and 102.

[0024] In certain embodiments according to any of the above skin rejuvenation methods, the method further comprises introducing an effective amount of a therapeutic agent, such as B18R, vitamin C, vitamin E, or any combination thereof, into the population of skin cells.

[0014]

[0025] In certain embodiments according to any of the above skin rejuvenation methods, the individual is a human (e.g., of any age), such as a human 10 years or older (e.g., 22 years or older, 30 years or older).

[0026] Another aspect of the present application is a method of delaying or preventing the progression of aging of skin tissue of an individual (e.g., a human, such as a human 22 years or older), the method comprising introducing an effective amount of a recombinant reprogramming factor without LLPS (e.g., any of the recombinant reprogramming factors described herein) into a population of skin cells of the skin tissue. In certain embodiments, the method of delaying or preventing the progression of aging of the skin tissue of an individual comprises rejuvenating a population of skin cells of the skin tissue using any of the above skin rejuvenation methods.

[0027] Another aspect of the present application is a method of treating aging, damage, disease or condition of skin tissue of an individual (e.g., a human), the method comprising introducing an effective amount of a recombinant reprogramming factor without LLPS (e.g., any of the recombinant reprogramming factors described herein) into a population of skin cells of the skin tissue. In certain embodiments, the method of treating aging, damage, disease or condition of the skin tissue of an individual comprises rejuvenating a population of skin cells of the skin tissue using any of the above skin rejuvenation methods.

[0028] Another aspect of the present application is a method of preserving or improving the condition and / or appearance of skin tissue of an individual (e.g., a human, such as a human 22 years or older), the method comprising introducing an effective amount of a recombinant reprogramming factor without LLPS (e.g., any of the recombinant reprogramming factors described herein) into a population of skin cells of the skin tissue. In certain embodiments, the method of preserving or improving the condition and / or appearance of the skin tissue of an individual comprises rejuvenating a population of skin cells of the skin tissue using any of the above skin rejuvenation methods.

[0029] Other aspects of this application provide a method for enhancing the recovery of skin tissue in an individual (e.g., a human, e.g., a human aged 10 years or older or 22 years or older) after injury or cosmetic, dermatological or surgical treatment, comprising introducing an effective amount of an LLPS-free recombinant reprogramming factor (e.g., any of the recombinant reprogramming factors described herein) into a population of skin cells in the skin tissue. In one embodiment, the method for enhancing the recovery of skin tissue in an individual after injury or cosmetic, dermatological or surgical treatment comprises rejuvenating a population of skin cells in the skin tissue using any of the above-described skin rejuvenation methods.

[0030] Other aspects of this application provide a method for preventing aging, damage or disease or condition of skin tissue of an individual (e.g., human), comprising introducing an effective amount of LLPS-free recombinant reprogramming factor (e.g., any of the recombinant reprogramming factors described herein) into a population of skin cells in the skin tissue. In one embodiment, the method for preventing aging, damage or disease or condition of skin tissue of an individual comprises rejuvenating a population of skin cells in the skin tissue using any of the above skin rejuvenation methods. In one embodiment, the skin tissue is (i) susceptible to aging or prone to aging; (ii) susceptible to damage or prone to skin damage; (iii) susceptible to developing or prone to developing skin disease or condition; and / or (iv) subjected to cosmetic, dermatological or surgical treatment. [Brief explanation of the drawing]

[0015] [Figure 1]

[0031] This figure shows the expression and distribution patterns of GFP-OCT4 and the GFP-OCT4 mutant (GFP-OCT4') in DAPI-stained 293T cells. Figure 1A shows the distribution patterns of GFP-OCT4 and GFP-OCT4' in 293T cells, including after treatment with 1,6-hexanediol. Figure 1B shows the fluorescence recovery (FRAP) after photobleaching of GFP-Oct4 and GFP-Oct4' droplets. [Figure 2]

[0032] This figure shows schematic diagrams of AnaPIE circular RNA (circRNA) vector structures containing one or two nucleic acids encoding different reprogramming factors. Figure 2A shows the vector structure of AnaPIE-OS (encoding OCT4 and SOX2). Figure 2B shows the vector structure of AnaPIE-M (encoding c-MYC). Figure 2C shows the vector structure of AnaPIE-K (encoding KLF4). Figure 2D shows the vector structure of AnaPIE-LN (encoding LIN28 and NANOG). [Figure 3]

[0033] This figure shows the results of 1% agarose gel electrophoresis after digestion with RNase R enzyme to verify the formation of AnaPIE-OS, AnaPIE-M, AnaPIE-K, and AnaPIE-LN circRNAs. [Figure 4]

[0034] Figure 4 shows the results of 2% agarose gel electrophoresis of test circRNAs AaPiE-OS, AaPiE-M, AaPiE-K, and AaPiE-LN compared to the control circRNA AaPiE-GFP. [Figure 5]

[0035] This figure shows the Western blot analysis of protein expression from AnaPIE-OS, AnaPIE-M, AnaPIE-K, and AnaPIE-LN circRNAs after transfection of HEK 293T cells. [Figure 6]

[0036] This figure shows schematic diagrams of AzoPIE circRNA plasmid structures containing three or four nucleic acids encoding different reprogramming factors. Figure 6A shows the vector structure of AzoPIE-OSK (encoding OCT4, SOX2, and KLF4). Figure 6B shows the vector structure of AzoPIE-O'SK (encoding OCT4', SOX2, and KLF4). Figure 6C shows the vector structure of AzoPIE-OKM (encoding OCT4, KLF4, and c-MYC). Figure 6D shows the vector structure of AzoPIE-O'KM' (encoding OCT4', KLF4, and c-MYC'). Figure 6E shows the vector structure of AzoPIE-OKMS (encoding OCT4, KLF4, c-MYC, and SOX2). Figure 6F shows the vector structures of AzoPIE-O'KM's (encoding OCT4', KLF4', c-MYC', and SOX2). [Figure 7]

[0037] This figure shows the results of electrophoresis of circRNAs of AzoPIE-OSK, AzoPIE-O'SK, AzoPIE-O'KM', and AzoPIE-OKM on a 1% agarose gel. [Figure 8]

[0038] This figure shows the electrophoresis results of 1% agarose gels containing AzoPIE-OKMS and AzoPIE-O'KM'S circRNA. [Figure 9]

[0039] Figure 9 shows the results of Western blot analysis of protein expression from AzoPIE-OKM, AzoPIE-O'KM', and AzoPIE-OKMS circRNA after transfection of HEK 293T cells. [Figure 10]

[0040] This is a schematic diagram of the AzoPIE circRNA vector structure containing nucleic acids encoding mouse OCT4 (wild-type (MsO) or mutant (MsOmut)), mouse KLF4 (MsKLF4), and mouse SOX2 (MsSOX2). Figure 10A shows the vector structure of AzoPIE-MsOKS. Figures 10B-10C show the vector structures of AzoPIE MsOmutKS, which encode two different mouse OCT4 mutants. Figure 10B shows the vector structure of AzoPIE-MsOmut1KS. Figure 10C shows the vector structure of AzoPIE-MsOmut2KS. [Figure 11]

[0041] This figure shows the results of a Western blot analysis of protein expression from AzoPIE MsOmut 1 KS circRNA after transfection of HEK 293T cells. [Figure 12]

[0042] This figure shows the characterization of HEK cell exosomes. Figure 12A shows a transmission electron microscope (TEM) image of an exosome sample exhibiting a typical cup-shaped morphology. Figure 12B shows nanoflow cytometry results indicating that the size of exosomes is generally 60–80 nm. [Figure 13]

[0043] This figure shows the results of nanoflow cytometry measuring the concentration and size of HEK cell exosomes (HEK-exo) and human MSC exosomes (huMSC-exo) with and without circular RNA loading. [Figure 14]

[0044] This figure shows the results of RT-qPCR, indicating the copy number of circRNA in HEK-exo, huMSC-exo, and control samples. [Figure 15]

[0045] This figure shows the verification of circRNA delivery by exosomes using the fluorescent reporter eGFP. Figure 15A shows a microscopic image of eGFP expression 24 hours after incubation of HEK-exo with circRNA-eGFP. Figure 15B shows a microscopic image of eGFP expression 24 hours after incubation of huMSC-exo with circRNA-eGFP. [Figure 16]

[0046] This figure shows nanoflow cytometry results indicating no significant difference in loading efficiency between HEK-exo OSK-circRNA and O'SK circRNA. [Figure 17]

[0047] This image shows immunofluorescence validation of human KLF4, OCT4, and SOX4 protein expression from HEK-exosomes loaded with OSK-circRNA after transfection of human dermal fibroblasts. DAPI staining is shown in the nuclei. [Figure 18]

[0048] This figure shows the construction of lentiviral vectors. Figure 18A shows schematic diagrams of four lentiviral vector structures: LV-TRE3G-OKM, LV-TRE3G-O'KM', LV-TRE3G-OKMS, and LV-TRE3G-O'KM'S. Figure 18B shows the results of enzymatic digestion of the four lentiviral vector plasmids. [Figure 19]

[0049] This figure shows the lentiviral vector quality control assays. Figure 19A shows the lentiviral titer and sterility test results for LV-TRE3G-OKM, LV-TRE3G-O'KM', LV-TRE3G-OKMS, and LV-TRE3G-O'KM'S. Figure 19B shows the detection of KLF4 RNA expression levels of the four lentiviral vector plasmids after transduction into HEK 293T cells. [Figure 20]

[0050] This is a schematic diagram of the AAV-TRE3G-OKS vector structure. [Figure 21]

[0051] This figure shows the results of enzymatic digestion of the AAV-TRE3G-OKS vector plasmid. [Figure 22]

[0052] This is a schematic diagram of the AAV-TRE3G-O'KS vector structure. [Figure 23]

[0053] This figure shows the results of enzymatic digestion of the AAV-TRE3G-O'KS vector plasmid. [Figure 24]

[0054] This figure shows the results of RT-qPCR for the AAV-TRE3G vector. Figure 24A shows the original amplification curve set. Figure 24B shows the standard curve. Figure 24C shows the titer measurements of AAV-TRE3G-OKS and AAV-TRE3G-O'KS using RT-qPCR. [Figure 25]

[0055] This figure shows immunohistochemical staining of KLF4 in mouse retinal ganglion cells two weeks after intravitreous injection of AAV-TRE3G-eGFP, AAV-TRE3G-OKS, or AAV-TRE3G-O'KS together with AAV-CMV-TET3G, followed by doxycycline induction. [Figure 26]

[0056] This figure shows the Western blot analysis of KLF4 expression from the AAV-TRE3G-OKS and AAV-TRE3G-O'KS treatment groups. [Figure 27]

[0057] This is a schematic diagram of the mouse AAV-CMV-OKS (AAV-CMV-MsOKS) vector structure. [Figure 28]

[0058] This figure shows the results of enzymatic degradation of the AAV-CMV-MsOKS vector plasmid. [Figure 29]

[0059] This is a schematic diagram of the AAV-CMV-MsOmut1KS vector encoding mouse Oct4 mutant 1. [Figure 30]

[0060] This figure shows the results of enzymatic degradation of the AAV-CMV-MsOmut1KS vector plasmid. [Figure 31]

[0061] This is a schematic diagram of the AAV-CMV-MsOmut2KS vector encoding OCT4 mutant 2. [Figure 32]

[0062] This figure shows the results of enzymatic degradation of the AAV-CMV-MsOmut2KS vector plasmid. [Figure 33]

[0063] This figure shows the results of RT-qPCR for the AAV2-CMV vector. Figure 33A shows the original amplification curve set. Figure 33B shows the standard curve. Figure 33C shows the viral particle titer measurements of AAV2-CMV-MsOKS, AAV2-CMV-MsOmut1KS, and AAV2-CMV-MsOmut2KS using RT-qPCR. [Figure 34]

[0064] This figure shows the results of RT-qPCR for the AAV9-CMV vector. Figure 34A shows the original amplification curve set. Figure 34B shows the standard curve. Figure 34C shows the viral particle titer measurement of AAV9-CMV-MsOKS and AAV9-CMV-MsOmut1KS using RT-qPCR. [Figure 35]

[0065] These are schematic diagrams of the structures of the Tet-On lentiviral vectors LV-TRE3G-O'KM'S, LV-TRE3G-OKMS, and LV-EFS-Tet3G. [Figure 36]

[0066] This figure shows the results of senescence-associated galactosidase (SA-β-Gal) staining experiments on skin cells FY-009 transfected with lentiviral particles LV-TRE3G-O'KM'S or LV-TRE3G-OKMS and TET3G (Tet-On 3G)-expressing lentiviral particles LV-EFS-TET3G. Figure 36A shows a representative image of SA-β-Gal staining results for the blank control group (no virus added). Figure 36B shows a representative image of SA-β-Gal staining results for the OKMS reprogrammed group. Figure 36C shows a representative image of SA-β-Gal staining results for the O'KM reprogrammed group. Figure 36D shows statistics of the percentage of SA-β-Gal positive cells in each group. [Figure 37]

[0067] This figure shows the detection of gene expression levels related to dermal fibroblast rejuvenation function or healthy skin cell function. Significance was determined by t-tests. **: P<0.01, ***: P<0.001. [Figure 38]

[0068] This figure shows the detection of gene expression levels for dermal fibroblast identification markers. Significance was determined by t-tests. *: P<0.05, ***: P<0.001. [Figure 39]

[0069] This figure shows the detection of marker gene expression levels during initiation phase reprogramming (IPR). Significance was determined by t-tests. **: P<0.01. [Figure 40]

[0070] This shows the detection of gene expression levels during mature phase reprogramming. Significance was determined by t-tests. **: P<0.01, ***: P<0.001. [Figure 41]

[0071] This figure shows the epigenetic age (EpiAge) analysis of cellular DNA methylation. [Figure 42]

[0072] These are schematic diagrams of the structures of the Tet-On lentiviral vectors LV-TRE3G-O'KM', LV-TRE3G-OKM, and LV-EFS-Tet3G. [Figure 43]

[0073] This figure shows the results of SA-β-GAL staining of skin cells FY-009 transfected with lentiviral particles LV-TRE3G-OKM or LV-TRE3G-O'KM' and TET3G (Tet-On 3G) expressing lentiviral particles LV-EFS-TET3G. Figure 43A shows a representative image of SA-β-Gal staining results for the blank control group (no virus added). Figure 43B shows a representative image of SA-β-Gal staining results for the OKM reprogrammed group. Figure 43C shows a representative image of SA-β-Gal staining results for the O'KM' reprogrammed group. Figure 43D shows statistics of the percentage of SA-β-GAL stain-positive cells in each group. [Figure 44]

[0074] This figure shows the detection of gene expression levels related to skin cell rejuvenation function or healthy skin cell function. Significance was determined by t-tests. **: P<0.01, ***: P<0.001. [Figure 45]

[0075] This figure shows the detection of gene expression levels for dermal fibroblast identification markers. Significance was determined by a t-test. ***: P<0.001. [Figure 46]

[0076] This figure shows the detection of gene expression levels during initiation phase reprogramming (IPR). Significance was determined by a t-test. ***: P<0.001. [Figure 47]

[0077] This figure shows the detection of gene expression levels during mature phase reprogramming. Significance was determined by a t-test. ***: P<0.001. [Figure 48]

[0078] This figure shows the results of SA-β-GAL staining of SBK-M57 skin cells treated with OSK-LNP or O'SK-LNP. Untreated cells were used as a blank control. Figure 48A shows representative SA-β-GAL stained images from the OSK-LNP-reprogrammed group, O'SK LNP-reprogrammed group, and blank control at four time points. Figure 48B shows statistics of the percentage of SA-β-GAL stain-positive cells in each group at different time points. [Figure 49]

[0079] This figure shows the epigenetic age analysis of cellular DNA methylation in SBK-M57 dermal fibroblasts treated with OSK-LNP and O'SK-LNP. Untreated cells were used as a control. [Figure 50]

[0080] This figure shows the single-cell RNA sequencing (scRNA-seq) results of upregulated genes in the OSK-LNP reprogrammed group and the O'SK-LNP reprogrammed group. [Figure 51]

[0081] This figure shows the results of scRNA-seq analysis confirming the decrease in gene expression in the OSK-LNP reprogramming group and the O'SK-LNP reprogramming group. [Figure 52]

[0082] This figure shows the scRNA-seq cell cluster analysis for the OSK-LNP reprogrammed group, the O'SK-LNP reprogrammed group, and the blank control. Figure 52A shows the cluster pattern for each group. Figure 52B shows the statistics of proportional changes in cell number for each cluster across the samples. Figure 52C shows the top gene ontology-biological process (GO-BP) and KEGG pathway analysis for cluster 1. Figure 52D shows the top GO-BP and KEGG pathway analysis for cluster 6. [Figure 53]

[0083] This figure shows the immunohistochemical analysis of elastin content in isolated skin tissue treated with OSK-LNP or O'SK-LNP compared to a control. Figure 53A shows representative images of staining results for the OSK-LNP-reprogrammed group, the O'SK-LNP-reprogrammed group, and the three control groups. Figures 53B to 53C show summary statistics of the relative integrated optical density (IOD) mean for each group. ##: P<0.01 compared to blank control (BC), *: P<0.05 compared to negative control (NC), **: P<0.01 compared to negative control. [Figure 54]

[0084] This figure shows the immunohistochemical analysis of collagen I content in isolated skin tissue treated with OSK-LNP or O'SK-LNP compared to a control. Figure 54A shows representative images of the staining results for the OSK-LNP reprogrammed group, the O'SK-LNP reprogrammed group, and the three control groups. Figures 54B to 54C show summary statistics of the relative IOD mean for each group. ##: P<0.01 compared to blank control, **: P<0.01 compared to negative control. [Figure 55]

[0085] This figure shows the immunohistochemical analysis of collagen III content in isolated skin tissue treated with OSK-LNP or O'SK-LNP compared to a control. Figure 55A shows representative images of the staining results for the OSK-LNP-reprogrammed group, the O'SK-LNP-reprogrammed group, and the three control groups. Figures 55B to 55C show summary statistics of the relative IOD mean for each group. ##: P<0.01 compared to blank control, **: P<0.01 compared to negative control. [Figure 56]

[0086] This figure shows the immunohistochemical analysis of collagen IV content in isolated skin tissue treated with OSK-LNP or O'SK-LNP compared to a control. Figure 56A shows representative images of the staining results for the OSK-LNP-reprogrammed group, the O'SK-LNP-reprogrammed group, and the three control groups. Figures 56B to 56C show summary statistics of the relative IOD mean for each group. ##: P<0.01 compared to blank control, *: P<0.05 compared to negative control, **: P<0.01 compared to negative control. [Figure 57]

[0087] This figure shows representative images of cutaneous fibroblasts transfected with circRNA-OSKMLN LNP at 1 and 7 days post-transfection, compared to negative controls. Transfected cutaneous fibroblasts eliminate iPSC-like clones at 7 days. [Figure 58]

[0088] This protocol evaluates the effects of AAV-TRE3G-OKS and AAV-TRE3G-O'KS on optic nerve regeneration and retinal ganglion cell (RGC) function after laser coagulation-induced retinal damage. Figure 58A shows the experimental timeline for evaluating the regenerative effect after laser coagulation-induced retinal damage. Figure 58B shows an overview of the experimental group. AAV-luciferase was used as a negative control. [Figure 59]

[0089] This figure shows the effects of AAV-TRE3G-OKS and AAV-TRE3G-O'KS on laser-induced retinal lesions. Figure 59A shows representative optical coherence tomography (OCT) images. Figure 59B shows a statistical analysis of the laser-induced retinal lesion area in each experimental group. AAV-luciferase was used as a negative control. [Figure 60]

[0090] This figure shows the effects of AAV-TRE3G-OKS and AAV-TRE3G-O'KS on ERG after laser coagulation. Figure 60A shows a representative full-field ERG image. Figure 60B shows the statistical analysis of photopic negative response (PhNR) amplitude by experimental group. PhNR is a measure of the electrophysiological function of RGC. AAV-luciferase was used as a negative control. [Figure 61]

[0091] This figure shows the effects of AAV-TRE3G-OKS and AAV-TRE3G-O'KS on optic nerve regeneration. Figure 61A shows a representative immunofluorescence image of βIII-tubulin, a neuronal marker for the optic nerve. Figure 61B shows a statistical analysis of βIII-tubulin density. AAV-luciferase was used as a negative control. [Figure 62]

[0092] This figure shows the effects of AAV-TRE3G-OKS and AAV-TRE3G-O'KS on RGC survival. Figure 62A shows a representative immunofluorescence image of the retinal RGC marker BRN3A. Figure 62B shows the quantification of the number of RGCs per 1 mm² area. AAV-luciferase was used as a negative control. [Figure 63]

[0093] This protocol evaluates the effects of AAV9-CMV-MsOKS and AAV9-CMV-MsOmut1KS on optic nerve regeneration and RGC function after optic nerve contusion injury. Figure 63A shows the experimental timeline for evaluating the regeneration effect after optic nerve contusion injury. Figure 63B shows an overview of the experimental group. [Figure 64]

[0094] This figure shows the effects of AAV9-CMV-MsOKS and AAV9-CMV-MsOmut1KS on RGC electrophysiological function in response to optic nerve crush. Figure 64A shows a representative waveform graph of the pERG results. Figure 64B shows a statistical analysis of the P50-N95 amplitude of the pERG. AAV-EGFP (undisrupted) and AAV-EGFR (disrupted) were used as negative controls. [Figure 65]

[0095] This figure shows the P1-N2 waveform graphs of fVEP for the groups treated with AAV9-CMV-MsOKS and AAV9-CMV-MsOmut1KS. AAV-EGFR (crush) served as a negative control. [Figure 66]

[0096] This protocol evaluates the effects of AAV2-CMV-MsOKS, AAV2-CMV-MsOmut1KS, and AAV2-CMV-MsOmut2KS on optic nerve regeneration and RGC survival after optic nerve contusion injury. Figure 66A shows the experimental timeline for evaluating the regeneration effect after optic nerve contusion injury. Figure 66B shows an overview of the experimental group. [Figure 67]

[0097] This figure shows the effects of AAV2-CMV-MsOKS, AAV2-CMV-MsOmut1KS, and AAV2-CMV-MsOmut2KS on optic nerve regeneration. Figure 67A shows representative images of regenerated CTB 555-labeled axons in the optic nerve after crushing. Figure 67B shows the quantification of CTB intensity at different distances from the crush site. [Figure 68]

[0098] This figure shows the effects of AAV2-CMV-MsOKS, AAV2-CMV-MsOmut1KS, and AAV2-CMV-MsOmut2KS on RGC survival. Figure 68A shows a representative whole-mount retinal image showing RGC marker RBPMS staining. Figure 68B shows the quantification of the number of RGCs per 0.04 mm2 area. [Figure 69]

[0099] This figure shows the evaluation of the anti-aging effects of OSK-LNP and O'SK-LNP administration to HepG2 cells, as measured by senescence-related β-galactosidase (SA-β-gal) staining. [Figure 70]

[0100] This figure shows the evaluation of the anti-aging effects of O'SK-LNP on three different lots of primary human liver (PHH). Figure 70A shows SA-β-gal staining. Figure 70B shows the quantification of the aging marker p16 by Western blotting. [Figure 71]

[0101] This figure shows the evaluation of the antifibrotic effects of OSK-LNP and O'SK-LNP administration to TGFβ-induced LX-2. Figure 71A shows cell morphology evaluation. Figure 71B shows the results of immunostaining for collagen I. Figure 71C shows the quantification of collagen I by Western blotting. [Figure 72]

[0102] This figure shows the effect of O'SK-LNP on liver regeneration in C57 / B6J mice that underwent 70% partial hepatectomy (PHx). Figure 72A shows a schematic diagram of the 5-day experimental protocol. Figure 72B shows the change in body weight observed on day 5. Figure 72C shows the change in the anatomical structure of the liver observed on day 5. Figure 72D shows the change in liver weight on day 5. Figure 72E shows the change in the liver-to-body weight ratio on day 5. [Figure 73]

[0103] This figure shows the effect of O'SK-LNP on serum markers compared to PBS controls in C57 / B6J mice subjected to 70% PHx. Figure 73A shows ALT levels. Figure 73B shows AST levels. Figure 73C shows total bilirubin levels. Figure 73D shows albumin levels. [Figure 74]

[0104] This figure shows the expression of exogenous human OCT4mut in mouse liver sections. Figure 74A shows the expression of OCT4mut on day 2. Figure 74B shows the expression of OCT4mut on day 5. [Figure 75]

[0105] This figure shows the histological results of mouse liver tissue comparing O'SK-LNP to PBS control for sections obtained on days 2 and 5. [Figure 76]

[0106] This figure shows the effect of O'SK-LNP on Ki67+ hepatocyte count levels. Figure 76A shows the liver histological results of O'SK-LNP versus PBS control for sections obtained on days 2 and 5. Figure 76B shows the Ki67+ liver levels on day 2. Figure 76C shows the Ki67+ liver levels on day 5. [Figure 77]

[0107] This figure shows immunohistochemical staining to evaluate the effects of OSK-LNP and O'SK-LNP treatment on TGFβ-induced epithelial-mesenchymal transition. Figure 77A shows a representative immunohistochemical staining image for N-cadherin. Figure 77B shows the quantification of immunofluorescence for N-cadherin. Figure 77C shows a representative immunohistochemical staining image for E-cadherin. Figure 77D shows the quantification of immunofluorescence for E-cadherin. [Figure 78]

[0108] This figure shows Western blot analysis of N-cadherin and E-cadherin levels to evaluate the effects of OSK-LNP and O'SK-LNP treatment on TGFβ-induced epithelial-mesenchymal transition. [Figure 79]

[0109] This figure shows the results of a wound healing assay to measure cell proliferation after treatment with OSK-LNP, O'SK-LNP, and pirfenidone. Figure 79A shows the quantification of cell-free areas at 0, 48, and 72 hours after wound introduction. Figure 79B shows the normalization of wound healing data for the cell-free area at 0 hours and the effect compared to the untreated group. *: p<0.05, **: p<0.01 indicates a statistical difference compared to the untreated group at 48 hours. #: p<0.05, ##: p<0.01 indicates a statistical difference at 72 hours compared to the untreated group. [Figure 80]

[0110] This figure shows collagen I and / or α-SMA immunostaining and Western blotting. OSK-LNP and O'SK-LNP improved TGFβ-induced fibrillation in HFL-1 cells. [Figure 81]

[0111] This figure shows a schematic diagram of treatment with AAV-OSK and AAV-O'SK in mouse models of bleomycin-induced idiopathic pulmonary fibrosis (IPF). [Figure 82]

[0112] This figure shows immunohistochemical staining that reveals the co-localization of AAV-derived GFP and AAV-specific antibodies in the alveoli. [Figure 83]

[0113] The results of αSMA immunohistochemistry of lung tissue are shown. Figure 83A shows a representative immunohistochemistry image. Figure 83B shows the quantification of αSMA staining. [Modes for carrying out the invention]

[0016]

[0114] The inventors have found that recombinant reprogramming factors lacking (or deficient in) liquid-liquid phase separation (LLPS) (hereinafter also referred to as "LLPS-deficient reprogramming factors") can mediate initiation phase reprogramming (IPR). Because LLPS (or the condensates it forms) is absent, cell reprogramming is maintained in the initiation phase. Advantages of the methods described herein include, but are not limited to, i) the ability to effectively rejuvenate cells and tissues (e.g., skin cells / tissues), including effective restoration of EpiAge, reduction of biomarkers for cellular senescence, apoptosis, ferroptosis, inflammatory responses, endoplasmic reticulum stress, and unhealthy cell function, increase of biomarkers for healthy cell function, and effective repair of damaged or injured cells and tissues (e.g., photoaged skin cells / tissues); ii) the rejuvenated cells retain their cellular identity (e.g., dermal fibroblasts) without becoming iPSCs; and iii) conventional Compared to what is achieved by the four-factor (unmanipulated OSKM) or three-factor reprogramming methods, the risk of iPSC induction is significantly reduced while the efficacy of cell rejuvenation is significantly improved; iv) Various delivery methods, such as DNA or RNA (e.g., circRNA) delivery, viral vector delivery, or delivery by exosomes or lipid nanoparticles (LNPs), are compatible with the LLPS-deficient reprogramming factors and rejuvenation methods described herein, meaning they can be reliably applied to various organs and tissue types (e.g., skin using microneedles). These findings can be reliably applied to various clinical settings, such as skin rejuvenation, prevention or delay of skin aging progression, and treatment of aged or damaged skin tissue.

[0017]

[0115] Accordingly, in one embodiment, the present application provides a method for rejuvenating a population of skin cells (hereinafter also referred to herein as the "skin rejuvenation method"), comprising introducing an effective amount of LLPS-deficient recombinant reprogramming factor into a population of skin cells. The present application also provides a method for slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human being 22 years of age or older) and a method for treating aging, damage, disease or condition of skin tissue in an individual, comprising (i) rejuvenating a population of skin cells in skin tissue by any of the skin rejuvenation methods described herein and / or (ii) introducing an effective amount of any of the LLPS-deficient recombinant reprogramming factors described herein into a population of skin cells in skin tissue.

[0116] In other embodiments, the application provides LLPS-free recombinant reprogramming factors for use in any of the skin rejuvenation methods or treatment or preventive methods described herein. A method for preventing aging, damage or disease or condition of an individual's skin tissue, comprising a recombinant reprogramming factor (e.g., any of the recombinant reprogramming factors described herein) that is lacking an effective amount of LLPS in a population of skin cells of the skin tissue, for example, before the individual reaches a certain age (e.g., before the age of 22 or older) or before the skin tissue is exposed to a damaging factor (e.g., UV irradiation or cosmetic, dermatological or surgical procedure).

[0018]

[0117] In a further embodiment, the present application provides a method for rejuvenating a cell population (e.g., cells of skin, eyes, liver, lungs, heart, muscle, brain, spleen, stomach, bone, blood, pancreas, bladder, intestines, kidneys, gallbladder, etc.) (hereinafter also generally referred to as the "rejuvenation method" herein) comprising introducing an effective amount of LLPS-deficient recombinant reprogramming factor (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein) into the cell population. Also provided are methods for delaying or preventing the progression of aging of cells (or related tissues or organs) of an individual (e.g., a human being 22 years of age or older), methods for treating aging, injury, disease, or condition of tissues or organs of an individual, methods for preserving or improving the viability, tissue structure, and / or function of cells (or related tissues or organs) of an individual, methods for enhancing the recovery of cells (or related tissues or organs) of an individual after injury or treatment (e.g., cosmetic or surgical), methods for promoting the regeneration of cells (or related tissues or organs) of an individual, and methods for preventing aging, injury, disease, or condition of tissues or organs of an individual, comprising (i) rejuvenating a population of cells (e.g., of tissue or organs) by any of the rejuvenation methods described herein and / or (ii) introducing an effective amount of LLPS-deficient recombinant reprogramming factor described herein into a population of cells (e.g., of tissue or organs).

[0019] I. Definition

[0118] As used herein, the term "condensate" means a non-membrane-encapsulated compartment formed by the phase separation (including all stages of phase separation) of one or more proteins and / or other macromolecules such as nucleic acids.

[0119] The term "liquid-liquid phase separation" (LLPS) or "phase separation" refers to the process by which macromolecules (e.g., proteins, nucleic acids) condense into a liquid-like, dense phase and separate from their diluted surrounding environment. LLPS forms the basis for the formation of biomolecular condensates. It concentrates specific factors while excluding other factors from the condensate, creating a unique environment that either supports or restricts specific biochemical reactions.

[0020]

[0120] As used herein, the term "stem cell" refers to a cell that retains the ability to regenerate through mitotic cell division while being able to differentiate into a wide range of specialized cell types. This includes both embryonic stem cells (ESCs) found in blastocysts and adult stem cells found in adult tissues. "Pluripotent cell" refers to a cell that has the ability to develop into cells derived from all three embryonic germ layers (mesoderm, endoderm, and ectoderm) and from the whole organism (for example, in humans, when located in the female uterus, from the human body). Pluripotent stem cells are produced by the fusion of an egg and a sperm. Cells produced in the first few divisions of a fertilized egg are also totipotent. Pluripotent cells can become the embryo, the extraembryonic membrane, and all metaembryonic tissues and organs. ESCs are the offspring of totipotent cells. ESCs are pluripotent and can differentiate into cell types from any of the three germ layers.

[0021]

[0121] As used herein, the term "pluripotency" refers to cells that, under appropriate conditions, have the ability to produce offspring that can differentiate into a cell type exhibiting collectively characteristics associated with cell lineages derived from the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells (PSCs) can contribute to the tissues of prenatal, postnatal, or adult organisms. Pluripotency of cell populations can be established using standard, industry-accepted tests for teratoma formation and other functions in 8-12 week-old SCID mice. However, pluripotent cells can also be identified by identifying various pluripotent stem cell characteristics. Human pluripotent stem cells may express at least some, and possibly all, of the following non-limiting list of markers: SSEA-3, SSEA-4, TRA-I-60, TRA-I-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-I, Oct4, Lin28, REX-1, and Nanog-derived markers.

[0122] As used herein, the term "induced pluripotent stem cell" (iPSC) refers to a pluripotent stem cell artificially induced from non-pluripotent cells. Non-pluripotent cells may be cells with lower self-renewal and differentiation capabilities than pluripotent stem cells. Cells with lower potency may be, but are not limited to, somatic stem cells, tissue-specific progenitor cells, primary cells, or secondary cells. iPSCs may originate from adult cells induced to ESC-like pluripotency by cell reprogramming factors. iPSCs may originate from adult somatic cells such as skin or blood cells. iPSCs may differentiate into one of three germ layer cell types.

[0022]

[0123] The term "cellular reprogramming" refers to the process of altering the epigenome of a cell using reprogramming factors (e.g., reversing or preventing epigenetic changes in cells that cause dysfunction, deterioration, cell death, senescence, or aging). Cellular reprogramming may be complete, such as when differentiated cells (e.g., somatic cells) are reprogrammed into PSCs. Cellular reprogramming may be incomplete, resulting in differentiated cells (e.g., somatic cells) retaining their cellular identity (e.g., lineage-specific stem cells). Cellular reprogramming may be incomplete, for example, if stem cells are not produced, resulting in rejuvenation or younger attributes (e.g., increased survival, inflammation, or decreased mitotic function). Cellular reprogramming may provide further cellular function or prevent cellular senescence (e.g., transition to differentiation or cellular senescence). Cellular reprogramming may induce temporary or permanent changes in gene expression. In some embodiments, cellular reprogramming prevents the onset of senescence.

[0023]

[0124] As used herein, the term "complete reprogramming" refers to the use of cell reprogramming factors to induce the conversion of somatic cells into iPSCs. The complete reprogramming process can be divided into three phases: the initiation phase, the maturation phase, and the stabilization phase. In the initiation phase, the cell still maintains its original somatic identity, but the expression of early pluripotency-related genes such as NANOG and SALL4 increases rapidly. In the maturation phase, the characteristics of somatic identity are rapidly lost, and the expression of late pluripotency-related genes such as DPPA5 and LIN28 increases. In the stabilization phase, the cell completely loses its cellular identity and becomes an iPSC.

[0024]

[0125] As used herein, “loss of cellular identity” refers to the reprogramming of a somatic cell into an iPSC, in which it loses its morphology, function, epigenetic pattern, gene expression profile, and other characteristics of the original somatic cell (i.e., “cellular identity”) and acquires various PSC characteristics. Somatic cells gradually lose their cellular identity during the reprogramming process. In the initial stage, the cell still retains its original somatic cell identity, but in the maturation stage, most of the cell's cellular identity characteristics are lost, and the acquired PSC characteristics become more pronounced.

[0025]

[0126] As used herein, the term "partial reprogramming" refers to the treatment or reprogramming of senescent or damaged cells using one or more cell reprogramming factors, which restores some degree of cell viability but is insufficient to dedifferentiate the cells into iPSCs, while retaining their original somatic identity. Transient reprogramming is the implementation of partial reprogramming, in which cells are exposed to cell reprogramming factors for a period sufficient to rejuvenate the cells but insufficient to dedifferentiate them into iPSCs. Due to the extremely short exposure time, the degree of recovery from this instantaneous reprogramming is low.

[0127] "Initiation-phase reprogramming," or "IPR," refers to the reprogramming of aging or damaged cells during the initiation phase to extend the safe time frame for cell rejuvenation while significantly reducing the risk of developing iPSCs.

[0026]

[0128] A “reprogramming factor” is a factor that can be used to reprogram target cells, and may be, for example, a transcription factor. The term “reprogramming factor” further includes any analog molecules that mimic the function of the factor with respect to its reprogramming function.

[0129] As used herein, the terms “rejuvenation” or “rejuvenation” include preventing or reversing the cellular causes of aging or damage without inducing a pluripotent state.

[0130] As used herein, the term “rejuvenated cells” means senescent or damaged cells that have been treated or reprogrammed with one or more cell reprogramming factors so that they have a transcriptome profile of younger or healthier cells, while still retaining one or more cell identity markers.

[0027]

[0131] "Regeneration" refers to a function that contributes to the repair or new construction of cells, tissues, or organs. In some embodiments, cell rejuvenation includes cell regeneration.

[0132] The term "tissue regeneration" refers to the restoration of existing tissue or cells (e.g., aged, damaged, or diseased) and / or the production of new tissue or cells within tissue that are of the same type as the target tissue (e.g., the same type as the damaged tissue or cells).

[0133] In the context of damaged tissue, the term "tissue repair" refers to the restoration of tissue structure, function after tissue damage, or a combination thereof. Examples of tissue repair include tissue regeneration, cell proliferation, tissue replacement, and / or rewiring (reprogramming) of existing tissue.

[0028]

[0134] As used herein, the terms “treatment,” “to treat,” and “to treat” mean reversing, alleviating, delaying, or inhibiting the progression of a disease or disorder or one or more of its symptoms as described herein. In some embodiments, treatment may be administered after the onset of one or more symptoms. In other embodiments, treatment may be administered when there are no symptoms. For example, treatment may be administered before the onset of symptoms to a susceptible individual who has not yet been diagnosed with the disease or condition, or may be treated with other damaging agents (for example, in light of the history of the symptoms, genetic or other susceptibility factors, disease therapy or any combination thereof). Treatment may also be continued after the disappearance of symptoms, for example, to prevent or delay relapse. Desired effects of treatment include a reduction in the rate of progression of the disease or condition, improvement or alleviation of the disease state or symptoms, achievement of desired pharmacological and / or physiological effects, and remission or improved prognosis. The effect may be preventive in terms of the complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of the partial or complete cure of the disease and / or side effects arising from the disease. For example, an individual is successfully “treated” if one or more symptoms associated with the disease or condition are alleviated or eliminated, which includes, but is not limited to, delaying or preventing the progression of the disease or condition, reducing the symptoms arising from the disease or condition, improving the quality of life of the person suffering from the disease or condition, reducing the dose of other drugs necessary to treat the disease or condition, and / or extending the individual’s survival.

[0135] As used herein, the term "prevention" refers to all actions taken to avoid symptoms or delay the onset of specific symptoms by administering a drug before or after the onset of a disease or condition.

[0136] As used herein, the term “effective dose” refers to the amount of drug that is effective in providing the desired treatment or prevention after administration in single or multiple doses. As understood in clinical contexts, the effective dose of a drug or pharmaceutical composition may or may not be achieved in combination with other drugs or pharmaceutical compositions. Therefore, “effective dose” may be considered in the context of administering one or more drugs, and an effective dose may be given if a single drug can or does achieve the desired result when used in combination with one or more other drugs.

[0029]

[0137] As used herein, the terms “individual” or “subject” refer to mammals including, but not limited to, humans, cattle, horses, sheep, goats, pigs, deer, donkeys, cats, dogs, rodents (e.g., rats, mice, rabbits, hamsters), or non-human primates (e.g., monkeys, chimpanzees). In some embodiments, the individual is a human. In some embodiments, the individual is a pet, e.g., a horse, dog, cat, rabbit, guinea pig, hamster, rat, mouse, ferret, chinchilla, etc. In some embodiments, the individual is livestock, e.g., a horse, cattle, cow, goat, sheep, pig, donkey, etc.

[0138] "Tissue" refers to a collection of cells (identical or non-identical) and extracellular matrix (ECM) that perform a specific function or set of functions together.

[0030]

[0139] As used herein, the term "mutant" refers to a sequence that includes modifications to the wild-type sequence. Non-restrictive modifications to amino acid sequences include insertions, deletions, and point mutations. Non-restrictive recombinations to nucleic acid sequences (e.g., recombinant nucleic acids) include frameshift mutations, nucleotide insertions, and nucleotide deletions.

[0140] As used herein, the terms “percentage (%) amino acid sequence identity” and “homology” relating to peptide or polypeptide sequences are defined as the ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after the sequences have been aligned and gaps introduced as necessary to achieve maximum percentage sequence identity, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity ratio can be achieved in various ways within the skill of a person skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0141] Amino acid substitutions include, but are not limited to, the substitution of one amino acid in a polypeptide with another amino acid. Examples of substitutions are shown in Table 1.

[0031] [Table 1]

[0142] Amino acids can be classified by common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: Leu, Ile; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. In non-conservative substitutions, one member of a class is exchanged for one of another.

[0032]

[0143] As used herein, the term "transfection" refers to the uptake of exogenous DNA or RNA by a cell. A cell is "transfected" when exogenous DNA or RNA is introduced into the cell membrane. Numerous transfection techniques are generally known in the industry. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13: 197. These techniques can be used to introduce one or more exogenous DNA or RNA molecules into a cell. The term refers to the stable and transient uptake of DNA or RNA molecules. For example, transfection can be used to transiently introduce mRNA encoding cell reprogramming factors into cells that require rejuvenation.

[0033]

[0144] The terms “host cell,” “host cell line,” and “host cell culture” are interchangeable and refer to cells into which exogenous nucleic acids have been introduced, including their offspring. Examples of host cells include “transformed organisms” and “transformed cells,” and include primary transformed cells and their offspring regardless of passage number. The offspring do not need to have exactly the same nucleic acid content as the parent cells and may contain mutations. Mutant offspring exhibiting the same function or biological activity as those screened or selected from the initially transformed cells are included herein.

[0145] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient is effective, and which does not contain any further ingredients that are unacceptably toxic to the subject to which the preparation is administered. Such a preparation is sterile. A "sterile" preparation is sterile or completely free of viable microorganisms and their spores.

[0146] Those skilled in the art will understand that uracil and thymine can both be represented by "t" instead of uracil "u" ​​and thymine "t"; in the context of ribonucleic acid, it will be understood that "t" is used to represent uracil unless otherwise specified.

[0147] It is understood that embodiments of the present invention described herein include embodiments consisting of and / or essentially consisting of.

[0034]

[0148] The term "about" used herein refers to (and describes) a value or parameter that includes (and describes) a variation in the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X".

[0149] In this specification, references to the term "not" a value or parameter generally mean and describe something "other than" a value or parameter. For example, a method not used to treat type X cancer means a method used to treat cancers other than type X.

[0150] As used herein, the term "approximately X to Y" is synonymous with "approximately X to approximately Y".

[0151] As used herein and in the appended claims, the singular forms "a," "or," and "the" refer to multiple subjects unless the context clearly indicates otherwise.

[0035] II. Methods to rejuvenate skin cell populations

[0152] In one embodiment, a method is provided for rejuvenating a population of cells (e.g., skin cells), comprising introducing into the population of cells (e.g., skin cells) an effective amount of a recombinant reprogramming factor lacking LLPS (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein) or a nucleic acid (DNA or RNA), nucleic acid construct (e.g., circRNA or plasmid), or virus, or a composition containing the same (e.g., a pharmaceutical composition), which encodes the same.In one embodiment, a method for (i) slowing or preventing the progression of aging in the tissue (e.g., skin tissue) of an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of the tissue (e.g., skin tissue) of an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of the tissue (e.g., skin tissue) of an individual (e.g., a human, e.g., 22 years of age or older) (e.g., improving by at least about 10%, 30%, 50%, 70%, 90%, 1x, 2x, 5x, or more), (iv) enhancing the recovery of the tissue (e.g., skin tissue) of an individual (e.g., a human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) preventing aging, damage, disease, or condition of the tissue (e.g., skin tissue) of an individual (e.g., a human), wherein (a) an effective amount L A method is provided comprising (b) introducing an LPS-deficient recombinant reprogramming factor (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein) or a nucleic acid (DNA or RNA), nucleic acid construct (e.g., circRNA or plasmid), or virus, or a composition containing the same (e.g., a pharmaceutical composition) that encodes the same; or (b) introducing an effective amount of an LLPS-deficient recombinant reprogramming factor (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein) or a nucleic acid (DNA or RNA), nucleic acid construct (e.g., circRNA or plasmid), or virus, or a composition containing the same (e.g., a pharmaceutical composition) into a population of cells (e.g., skin cells) (e.g., from the same or different individuals) and transplanting the rejuvenated population of cells (e.g., skin cells) into an individual (e.g., the skin tissue of an individual).In one embodiment, (i) slows or prevents the progression of aging in the tissue (e.g., skin tissue) of an individual (e.g., a human, e.g., 22 years of age or older), (ii) treats aging, damage, disease, or condition of the tissue (e.g., skin tissue) of an individual (e.g., a human), (iii) preserves or improves the condition and / or appearance of the tissue (e.g., skin tissue) of an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhances the recovery of the tissue (e.g., skin tissue) of an individual (e.g., a human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) an individual (e.g., A method is provided for preventing aging, damage, disease, or condition of a tissue (e.g., skin tissue) of a person (e.g., human), comprising (a) rejuvenating a population of cells in the tissue (e.g., skin cells in skin tissue) using any of the rejuvenation methods described herein (e.g., skin rejuvenation methods), or (b) rejuvenating a population of cells (e.g., skin cells) (e.g., from the same or different individuals) and transplanting the rejuvenated population of cells (e.g., skin cells) into an individual (e.g., the skin tissue of an individual) using any of the rejuvenation methods described herein (e.g., skin rejuvenation methods). In some embodiments, the cell population is a population of skin cells. In some embodiments, the tissue is skin tissue. In some embodiments, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In some embodiments, a population of cells (e.g., skin cells) is derived from tissue (e.g., skin tissue) that is (i) aged tissue (e.g., skin tissue); (ii) damaged tissue (e.g., skin tissue); (iii) tissue with a disease or condition (e.g., skin disease or condition); and / or (iv) tissue after cosmetic, dermatological or surgical treatment (e.g., skin tissue). In some embodiments, the tissue (e.g., skin tissue) is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to injury (e.g., skin injury); (iii) susceptible to or predisposed to developing a disease or condition (e.g., skin disease or condition); and / or (iv) subjected to cosmetic, dermatological or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue.In some embodiments, the method does not reprogram a population of cells (e.g., skin cells) into reprogrammed stem cells. In some embodiments, the recombinant reprogramming factor includes a mutation in the LLPS-related domain compared to the corresponding wild-type reprogramming factor. In some embodiments, the LLPS-related domain is selected from the group consisting of an endogenous irregular region (IDR), a region outside the DNA-binding domain (DBD), a ubiquitination site, or any combination thereof. In some embodiments, the mutation includes (i) substituting an acidic amino acid (e.g., D or E) in the IDR with a neutral amino acid (e.g., A); (ii) substituting a ubiquitizable amino acid (e.g., K) with an unubiquitizable amino acid (e.g., R); (iii) deleting a ubiquitizable amino acid (e.g., K); (iv) deleting an amino acid (e.g., an acidic amino acid) in the IDR; and / or (v) deleting an amino acid (e.g., an acidic amino acid) in a region outside the DBD. In one embodiment, the wild-type reprogramming factor is derived from a family selected from the group consisting of the OCT family, SOX family, KLF family, MYC family, LIN28 family, NANOG family, and GLIS family. In one embodiment, the wild-type reprogramming factor is selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG, and GLIS1. In one embodiment, the recombinant reprogramming factor is recombinant OCT4 containing any of the amino acid sequences of SEQ ID NOs. 4-10 and 44-46, such as SEQ ID NO 4. In one embodiment, the recombinant reprogramming factor is recombinant c-MYC containing any of the amino acid sequences of SEQ ID NOs. 14-20, 47, and 48, such as SEQ ID NO 14. In one embodiment, the recombinant reprogramming factor is recombinant SOX2 containing any of the amino acid sequences of SEQ ID NOs. 41-43. In one embodiment, the recombinant reprogramming factor is recombinant KLF4 containing the amino acid sequence of SEQ ID NO 40, for example. In one embodiment, the recombinant reprogramming factor is encoded by nucleic acids, such as RNA (e.g., circRNA) or DNA.In some embodiments, the nucleic acid is contained within a vector, such as a viral vector, such as a lentiviral vector or an AAV vector. In some embodiments, the nucleic acid is delivered by exosomes, liposomes, LNPs, or viruses. In some embodiments, a population of cells (e.g., skin cells) is present in an individual in need of treatment or prevention, and the method comprises intra vivo administration of an effective amount of recombinant reprogramming factor to the individual (e.g., into skin tissue). In some embodiments, the method comprises administration of RNA (e.g., circRNA) encoding the recombinant reprogramming factor, the RNA being encapsulated within an exosome or LNP. In some embodiments, the exosome or LNP is delivered by a microneedle. In some embodiments, a population of cells (e.g., skin cells) is obtained from a donor individual and cultured ex vivo. In some embodiments, the method comprises transplanting a rejuvenated population of cells (e.g., skin cells) into an individual in need of treatment or prevention (e.g., into skin tissue). The donor individual and the individual in need of treatment or prevention may be the same or different. In one embodiment, the method comprises ex vivo administration of RNA encoding a recombinant reprogramming factor (e.g., circRNA) to a population of cells (e.g., skin cells), wherein the RNA is encapsulated within an exosome or LNP. In another embodiment, the method further comprises introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, to a population of skin cells. In another embodiment, the individual is a human (e.g., of any age) aged 10 years or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years or older). In yet another embodiment, the method is used to prevent aging, damage, or disease or condition of tissue / cells (e.g., skin tissue / cells) that are aging, exposed to damaging factors (e.g., UV irradiation or cosmetic, dermatological or surgical procedures), or have a disease or condition (e.g., have been diagnosed with one).In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours or more) before the tissue / cells (e.g., skin tissue / cells) are exposed to a damaging factor (e.g., UV irradiation).

[0036]

[0153] Individuals that can be treated preventively or therapeutically may include, but are not limited to, any mammals including, humans, cattle, horses, sheep, goats, pigs, deer, donkeys, cats, dogs, rodents (e.g., rats, mice, rabbits, hamsters) or non-human primates (e.g., monkeys, chimpanzees). In some embodiments, the individual is human. In some embodiments, the individual is a non-human mammal (e.g., a pet or livestock). The human being treated may be 10, 15, 20, 22, 30, 40, 50, 60, 70, 80, 90 years of age or older. In some embodiments, the human may be 22 years of age or older, for example, 30 years of age or older, 50 years of age or older, or 60 years of age or older. If the individual is a non-human mammal, the human ages described herein may be converted to the equivalent non-human age of the corresponding non-human mammal.

[0037]

[0154] Recombinant reprogramming factors can be introduced into cell populations (e.g., skin cells) as proteins, nucleic acids (DNA or RNA such as circRNA), nucleic acid constructs (e.g., plasmids), viruses (e.g., lentiviruses or AAVs), or compositions containing them (e.g., pharmaceutical compositions). Therefore, when referring to the introduction of a recombinant reprogramming factor without an effective amount of LLPS into a population of cells (e.g., skin cells), the introduction is not limited to proteins. The methods described herein are intended to be any methods of introduction or administration in which recombinant reprogramming factors can be introduced into cells (e.g., skin cells) or expressed in cells (e.g., skin cells).

[0038]

[0155] In one embodiment, the method comprises introducing a plurality of reprogramming factors into a population of cells (e.g., skin cells), wherein at least one of the plurality of reprogramming factors is a recombinant reprogramming factor without LLPS (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein). Thus, in one embodiment, a method is provided for rejuvenating a population of cells (e.g., skin cells), comprising introducing an effective amount of a plurality of reprogramming factors (or a pharmaceutical composition containing them) into a population of cells (e.g., skin cells), wherein at least one of the plurality of reprogramming factors is a recombinant reprogramming factor without LLPS (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein).In one embodiment, (i) slowing or preventing the progression of aging in the tissue (e.g., skin tissue) of an individual (e.g., human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of the tissue (e.g., skin tissue) of an individual (e.g., human), (iii) preserving or improving the condition and / or appearance of the tissue (e.g., skin tissue) of an individual (e.g., human, e.g., 22 years of age or older), (iv) enhancing the recovery of the tissue (e.g., skin tissue) of an individual (e.g., human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) the tissue (e.g., skin tissue) of an individual (e.g., human) A method is provided for preventing aging, damage, disease, or condition, comprising: (a) introducing an effective amount of a plurality of reprogramming factors (or a pharmaceutical composition containing them) into a population of tissue cells (e.g., skin cells of skin tissue), wherein at least one of the plurality of reprogramming factors is an LLPS-free recombinant reprogramming factor (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein); and (b) introducing an effective amount of a plurality of reprogramming factors (or a pharmaceutical composition containing them) into a population of cells (e.g., skin cells) (e.g., from the same or different individuals), wherein the population of cells (e.g., skin cells) is an LLPS-free recombinant reprogramming factor (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein); and transplanting the rejuvenated population of cells (e.g., skin cells) into an individual (e.g., the skin tissue of an individual).In one embodiment, (i) slowing or preventing the progression of aging in the tissue (e.g., skin tissue) of an individual (e.g., human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of the tissue (e.g., skin tissue) of an individual (e.g., human), (iii) preserving or improving the condition and / or appearance of the tissue (e.g., skin tissue) of an individual (e.g., human, e.g., 22 years of age or older), (iv) enhancing the recovery of the tissue (e.g., skin tissue) of an individual (e.g., human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) the tissue (e.g., skin tissue) of an individual (e.g., human) A method is provided for preventing aging, damage, disease, or condition of a person, comprising (a) rejuvenating a population of tissue cells (e.g., skin cells of skin tissue) using any of the rejuvenation methods described herein (e.g., skin rejuvenation methods), or (b) rejuvenating a population of cells (e.g., skin cells) (e.g., from the same or different individuals) and transplanting the rejuvenated population of cells (e.g., skin cells) into an individual (e.g., the skin tissue of an individual) using any of the rejuvenation methods described herein (e.g., skin rejuvenation methods). In one embodiment, the plurality of reprogramming factors comprises two or more reprogramming factors from two or more families selected from the group consisting of the OCT family, SOX family, KLF family, MYC family, LIN28 family, NANOG family, and GLIS family. In one embodiment, the plurality of reprogramming factors comprises two or more reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG, GLIS1, and their variants. In one embodiment, the multiple reprogramming factors include three or all of the reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, and c-MYC, and at least one of the multiple reprogramming factors is a recombinant reprogramming factor without LLPS. In another embodiment, the multiple reprogramming factors include OCT4 and KLF4, and at least one of OCT4 and KLF4 is a recombinant reprogramming factor without LLPS.In one embodiment, all of the multiple reprogramming factors are recombinant reprogramming factors without LLPS. In one embodiment, the multiple reprogramming factors include wild-type reprogramming factors. In one embodiment, OCT4 is recombinant OCT4 without LLPS, containing any of the amino acid sequences of SEQ ID NOs: 4-10 and 44-46, for example, SEQ ID NO: 4. In one embodiment, c-MYC is recombinant c-MYC without LLPS, containing any of the amino acid sequences of SEQ ID NOs: 14-20, 47 and 48, for example, SEQ ID NO: 14. In one embodiment, the cell population is a population of skin cells. In one embodiment, the tissue is skin tissue. In one embodiment, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In some embodiments, a population of cells (e.g., skin cells) is derived from tissue (e.g., skin tissue) that is (i) aged tissue (e.g., skin tissue); (ii) damaged tissue (e.g., skin tissue); (iii) tissue with a disease or condition (e.g., skin disease or condition); and / or (iv) tissue after cosmetic, dermatological or surgical treatment (e.g., skin tissue). In some embodiments, the tissue (e.g., skin tissue) is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to injury (e.g., skin injury); (iii) susceptible to developing a disease or condition (e.g., skin disease or condition); and / or (iv) subjected to cosmetic, dermatological or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In some embodiments, the method does not reprogram the population of cells (e.g., skin cells) into reprogrammed stem cells. In one embodiment, a population of cells (e.g., skin cells) is present in an individual in need of treatment or prevention, and the method involves intra vivo administration of an effective amount of multiple reprogramming factors to the individual (e.g., into skin tissue). In one embodiment, the multiple reprogramming factors are encapsulated within exosomes, liposomes, or LNPs. In one embodiment, the exosomes, liposomes, or LNPs are delivered by microneedles.In one embodiment, a population of cells (e.g., skin cells) is obtained from a donor individual and cultured ex vivo. In one embodiment, the method includes transplanting a rejuvenated population of cells (e.g., skin cells) into an individual (e.g., skin tissue) that is in need of treatment or prevention. The donor individual and the individual in need of treatment or prevention may be the same or different. In one embodiment, the method includes ex vivo administration of an effective amount of several reprogramming factors to the population of cells (e.g., skin cells). In one embodiment, the method further includes introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, into the population of skin cells. In one embodiment, the individual is a human (e.g., of any age), such as a human aged 10 years or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years or older). In one embodiment, to prevent aging, damage, or disease or condition of tissue / cells (e.g., skin tissue / cells), the method is performed before the tissue / cells (e.g., skin tissue / cells) age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before the tissue / cells (e.g., skin tissue / cells) are exposed to damaging factors (e.g., UV irradiation).

[0039]

[0156] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into the population of skin cells, wherein the multiple reprogramming factors include LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), SOX2 (e.g., SEQ ID NO. 24), and recombinant OCT4 without KLF4 (e.g., SEQ ID NO. 22). In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) preventing aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), comprising (a) introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into a population of skin cells in skin tissue, wherein the multiple reprogramming factors are LLPS (e.g., any of SEQ ID NOs: 4-10 and 44-46, e.g., SEQ ID NO: 4) The present invention provides a method comprising (b) introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into a population of skin cells (e.g., from the same or different individuals) and transplanting the rejuvenated population of skin cells into an individual (e.g., the skin tissue of the individual), wherein the multiple reprogramming factors include LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), SOX2 (e.g., SEQ ID NO. 24), and KLF4 (e.g., SEQ ID NO. 22), and introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into a population of skin cells (e.g., from the same or different individuals) and transplanting the rejuvenated population of skin cells into an individual (e.g., the skin tissue of the individual).In some embodiments, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In some embodiments, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to skin injury; (iii) prone to developing or predisposed to developing a skin disease or condition; and / or (iv) has undergone cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In some embodiments, the method does not reprogram the population of skin cells into reprogramming stem cells. In some embodiments, the population of skin cells is present in an individual in need of treatment or prevention, and the method includes intra vivo administration of effective amounts of multiple reprogramming factors to the individual (e.g., into skin tissue). In one embodiment, multiple reprogramming factors are encapsulated within exosomes, liposomes, or LNPs. In one embodiment, the exosomes, liposomes, or LNPs are delivered by microneedles. In one embodiment, a population of skin cells is obtained from a donor individual and cultured ex vivo. In one embodiment, the method includes transplanting the rejuvenated population of skin cells into an individual (e.g., skin tissue) that requires treatment or prevention. The donor individual and the individual requiring treatment or prevention may be the same or different. In one embodiment, the method includes ex vivo administration of an effective amount of multiple reprogramming factors to the population of skin cells. The method further includes introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, to the population of skin cells. In one embodiment, the individual is a human (e.g., of any age), such as a human aged 10 years or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years or older).In one embodiment, the method is performed to prevent aging, damage, or disease or condition of tissue / cells (e.g., skin tissue / cells) before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0040]

[0157] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into a population of skin cells in skin tissue, wherein the multiple reprogramming factors include recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 22), and recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48, e.g., SEQ ID NO. 14). In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological or surgical treatment, and / or (v) preventing aging, damage, disease or condition of skin tissue in an individual (e.g., a human), comprising (a) introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into a population of skin cells in skin tissue, wherein the multiple reprogramming factors are recombinant OCT4 without LLPS (e.g., The present invention provides a method comprising (b) introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into a population of skin cells, wherein the multiple reprogramming factors include recombinant OCT4 without LLPS (e.g., any of sequence numbers 4-10 and 44-46, e.g., sequence number 4), KLF4 (e.g., sequence number 22), and recombinant c-MYC without LLPS (e.g., any of sequence numbers 14-20, 47, and 48, e.g., sequence number 14), and transplanting the rejuvenated population of skin cells into an individual (e.g., the skin tissue of an individual).In some embodiments, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In some embodiments, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to skin injury; (iii) prone to developing or predisposed to developing a skin disease or condition; and / or (iv) has undergone cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In some embodiments, the method does not reprogram the population of skin cells into reprogramming stem cells. In some embodiments, the population of skin cells is present in an individual in need of treatment or prevention, and the method includes intra vivo administration of effective amounts of multiple reprogramming factors to the individual (e.g., into skin tissue). In some embodiments, multiple reprogramming factors are encapsulated within exosomes, liposomes, or LNPs. In some embodiments, the exosomes, liposomes, or LNPs are delivered by microneedles. In some embodiments, a population of skin cells is obtained from a donor individual and cultured ex vivo. In some embodiments, the method includes transplanting a rejuvenated population of skin cells (e.g., into skin tissue) into an individual in need of treatment or prevention. The donor individual and the individual in need of treatment or prevention may be the same or different. In some embodiments, the method includes ex vivo administration of an effective amount of multiple reprogramming factors to the population of skin cells. The method further includes introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, into the population of skin cells. In some embodiments, the individual is a human (e.g., of any age), such as a human aged 10 years or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years or older).In one embodiment, the method is performed to prevent aging, damage, or disease or condition of skin tissue / cells before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0041]

[0158] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into the population of skin cells, wherein the multiple reprogramming factors include recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 22), recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48, e.g., SEQ ID NO. 14), and SOX2 (e.g., SEQ ID NO. 24).In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) preventing aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), comprising (a) introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into a population of skin cells in skin tissue, wherein the multiple reprogramming factors are recombinant OCT4 without LLPS (e.g., SEQ ID NOs: 4-10 and 44-46) The present invention provides a method comprising (b) introducing an effective amount of multiple reprogramming factors (or a pharmaceutical composition containing them) into a population of skin cells, wherein the multiple reprogramming factors include recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 22), recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47 and 48, e.g., SEQ ID NO. 14), and SOX2 (e.g., SEQ ID NO. 24), and transplanting the rejuvenated population of skin cells into an individual (e.g., the skin tissue of an individual). In one embodiment, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In one embodiment, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after a cosmetic, dermatological or surgical procedure.In some embodiments, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to skin injury; (iii) susceptible to developing or predisposed to developing skin diseases or conditions; and / or (iv) subjected to cosmetic, dermatological or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In some embodiments, the method does not reprogram a population of skin cells into reprogrammed stem cells. In some embodiments, the population of skin cells is present in an individual in need of treatment or prevention, and the method comprises intra vivo administration of an effective amount of multiple reprogramming factors to the individual (e.g., into skin tissue). In some embodiments, the multiple reprogramming factors are encapsulated in exosomes, liposomes, or LNPs. In some embodiments, the exosomes, liposomes, or LNPs are delivered by microneedles. In some embodiments, the population of skin cells is obtained from a donor individual and cultured ex vivo. In some embodiments, the method comprises transplanting a rejuvenated population of skin cells into an individual in need of treatment or prevention (e.g., into skin tissue). The donor individual and the individual requiring treatment or prevention may be the same or different. In one embodiment, the method comprises ex vivo administration of an effective amount of several reprogramming factors to a population of skin cells. The method further comprises introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, to the population of skin cells. In one embodiment, the individual is a human (e.g., of any age) who is 10 years of age or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years or older). In one embodiment, the method is performed to prevent aging, damage, or disease or condition of skin tissue / cells before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological or surgical procedures), or before disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0042]

[0159] In one embodiment, the method comprises introducing a plurality of nucleic acids (e.g., RNA (e.g., circRNA) or DNA) encoding a plurality of reprogramming factors into a population of cells (e.g., skin cells), wherein at least one of the plurality of reprogramming factors is a recombinant reprogramming factor without LLPS (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein). Thus, in one embodiment, a method is provided for rejuvenating a population of cells (e.g., skin cells), comprising introducing an effective amount of a plurality of nucleic acids (RNA (e.g., circRNA) or DNA, e.g., any of the nucleic acids described herein) (or a pharmaceutical composition containing the same) encoding a plurality of reprogramming factors, wherein at least one of the plurality of reprogramming factors is a recombinant reprogramming factor without LLPS (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein).In one embodiment, (i) slowing or preventing the progression of aging in the tissue (e.g., skin tissue) of an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of the tissue (e.g., skin tissue) of an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of the tissue (e.g., skin tissue) of an individual (e.g., a human, e.g., 22 years of age or older), (iv) treating the tissue (e.g., skin tissue) of an individual (e.g., a human) after damage or cosmetic, dermatological, or surgical treatment. A method is provided for enhancing the recovery of and / or (v) preventing aging, damage or disease or condition of tissue (e.g., skin tissue) of an individual (e.g., human), comprising (a) introducing a population of tissue cells (e.g., skin cells of skin tissue) a plurality of nucleic acids (RNA (e.g., circRNA) or DNA, e.g., any of the nucleic acids described herein) encoding a plurality of reprogramming factors, wherein at least one of the plurality of reprogramming factors is an LLPS-free recombinant reprogramming factor (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein), or (b) introducing a population of cells (e.g., skin cells) (e.g., from the same or different individuals) a plurality of nucleic acids (RNA (e.g., circRNA) or DNA, e.g., any of the nucleic acids described herein) encoding a plurality of reprogramming factors, wherein at least one of the plurality of reprogramming factors is an LLPS-free recombinant reprogramming factor (e.g., any of the LLPS-deficient recombinant reprogramming factors described herein), and transplanting the rejuvenated population of cells (e.g., skin cells) into an individual (e.g., skin tissue of an individual).In one embodiment, (i) slowing or preventing the progression of aging in the tissue (e.g., skin tissue) of an individual (e.g., human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of the tissue (e.g., skin tissue) of an individual (e.g., human), (iii) preserving or improving the condition and / or appearance of the tissue (e.g., skin tissue) of an individual (e.g., human, e.g., 22 years of age or older), (iv) enhancing the recovery of the tissue (e.g., skin tissue) of an individual (e.g., human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) the tissue (e.g., skin tissue) of an individual (e.g., human) A method is provided for preventing aging, damage, disease, or condition of an organism, comprising (a) rejuvenating a population of tissue cells (e.g., skin cells of skin tissue) using any of the rejuvenation methods described herein (e.g., skin rejuvenation methods), or (b) rejuvenating a population of skin cells (e.g., from the same or different organisms) and transplanting the rejuvenated population of cells (e.g., skin cells) into an organism (e.g., the skin tissue of an organism) using any of the rejuvenation methods described herein (e.g., skin rejuvenation methods). In one embodiment, the plurality of reprogramming factors comprises two or more reprogramming factors from two or more families selected from the group consisting of the OCT family, SOX family, KLF family, MYC family, LIN28 family, NANOG family, and GLIS family. In one embodiment, the plurality of reprogramming factors comprises two or more reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG, GLIS1, and their variants. In one embodiment, the multiple reprogramming factors include three or all of the reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, and c-MYC, and at least one of the multiple reprogramming factors is a recombinant reprogramming factor without LLPS. In another embodiment, the multiple reprogramming factors include OCT4 and KLF4, and at least one of OCT4 and KLF4 is a recombinant reprogramming factor without LLPS.In one embodiment, all of the multiple reprogramming factors are recombinant reprogramming factors without LLPS. In one embodiment, the multiple reprogramming factors include wild-type reprogramming factors. In one embodiment, OCT4 is recombinant OCT4 without LLPS, containing any of the amino acid sequences of SEQ ID NOs: 4-10 and 44-46, for example, SEQ ID NO: 4. In one embodiment, c-MYC is recombinant c-MYC without LLPS, containing any of the amino acid sequences of SEQ ID NOs: 14-20, 47 and 48, for example, SEQ ID NO: 14. In one embodiment, the cell population is a population of skin cells. In one embodiment, the tissue is skin tissue. In one embodiment, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In some embodiments, a population of cells (e.g., skin cells) is derived from tissue (e.g., skin tissue) that is (i) aged tissue (e.g., skin tissue); (ii) damaged tissue (e.g., skin tissue); (iii) tissue with a disease or condition (e.g., skin disease or condition); and / or (iv) tissue after cosmetic, dermatological or surgical treatment (e.g., skin tissue). In some embodiments, the tissue (e.g., skin tissue) is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to injury (e.g., skin injury); (iii) susceptible to developing a disease or condition (e.g., skin disease or condition); and / or (iv) subjected to cosmetic, dermatological or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In some embodiments, the method does not reprogram the population of cells (e.g., skin cells) into reprogrammed stem cells. Multiple nucleic acids may reside on separate nucleic acid constructs (e.g., circRNA, plasmid, or recombinant viral genome) or on a single nucleic acid construct. Multiple nucleic acids can be delivered using exosomes, liposomes, LNPs, or viruses (e.g., LV or AAV). In some embodiments, at least two (e.g., all) of the multiple nucleic acids reside on a single nucleic acid construct.In some embodiments, at least two (e.g., all) of a plurality of nucleic acids are under the control of different promoters. In some embodiments, at least two (e.g., all) of a plurality of nucleic acids are under the control of the same promoter. In some embodiments, at least two (e.g., all) of a plurality of nucleic acids are linked by one or more linked nucleic acids. In some embodiments, one or more linked nucleic acids include linked sequences encoding IRES or self-cleaving peptides, e.g., P2A, T2A, or E2A. In some embodiments, the nucleic acid construct includes any of the nucleic acid sequences SEQ ID NOs: 28, 30, 32, 35, 37, 39, 57, 59, 61, 94, 98, and 102. In some embodiments, a population of cells (e.g., skin cells) is present in an individual in need of treatment or prevention, and the method includes intra vivo administration of an effective amount of a plurality of nucleic acids (or a nucleic acid construct(s) or a virus containing them) to the individual (e.g., skin tissue). In some embodiments, the plurality of nucleic acids are one or more RNAs (e.g., one circRNA). In one embodiment, one or more RNAs (e.g., one circRNA) are encapsulated within an exosome, liposome, or LNP. In one embodiment, the exosome, liposome, or LNP is delivered by a microneedle. In one embodiment, a population of cells (e.g., skin cells) is obtained from a donor individual and cultured ex vivo. In one embodiment, the method includes transplanting a rejuvenated population of cells (e.g., skin cells) into an individual in need of treatment or prevention (e.g., skin tissue). The donor individual and the individual in need of treatment or prevention may be the same or different. In one embodiment, the method includes ex vivo administration of an effective amount of multiple nucleic acids (or nucleic acid constructs or viruses containing them) to a population of cells (e.g., skin cells). In one embodiment, the multiple nucleic acids are one or more RNAs (e.g., one circRNA). In one embodiment, one or more RNAs (e.g., one circRNA) are encapsulated within an exosome, liposome, or LNP. The method further includes introducing an effective amount of a therapeutic agent, such as B18R, vitamin C, vitamin E, or any combination thereof, into a population of skin cells.In one embodiment, the individual is a human being of any age, such as a human being 10 years of age or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years of age or older). In one embodiment, the method is performed to prevent aging, damage, or disease or condition of tissue / cells (e.g., skin tissue / cells) before the tissue / cells (e.g., skin tissue / cells) age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before the tissue / cells (e.g., skin tissue / cells) are exposed to damaging factors (e.g., UV irradiation). In one embodiment, multiple nucleic acids encoding multiple reprogramming factors reside on a single nucleic acid construct (e.g., RNA (e.g., circRNA) or DNA). In one embodiment, the multiple nucleic acids are under the control of the same promoter. In one embodiment, multiple nucleic acids are linked by one or more linking nucleic acids. In one embodiment, one or more linking nucleic acids include linking sequences encoding IRES or self-cleaving peptides, such as P2A, T2A, or E2A.

[0043]

[0160] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of multiple nucleic acids (RNA (e.g., circRNA) or DNA or nucleic acid constructs or viruses containing the same) (or a pharmaceutical composition containing the same) encoding multiple reprogramming factors into a population of skin cells, wherein the multiple reprogramming factors include LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), SOX2 (e.g., SEQ ID NO. 24), and recombinant OCT4 without KLF4 (e.g., SEQ ID NO. 22). In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) preventing aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), (a) A method is provided comprising introducing a plurality of nucleic acids (RNA (e.g., circRNA) or DNA; or nucleic acid constructs (or viruses) containing the same) (or pharmaceutical compositions containing the same) encoding a plurality of reprogramming factors into a population of skin cells and transplanting the rejuvenated population of skin cells into an individual (e.g., the skin tissue of an individual), wherein the plurality of reprogramming factors include LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), SOX2 (e.g., SEQ ID NO. 24), and recombinant OCT4 without KLF4 (e.g., SEQ ID NO. 22).In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after injury or cosmetic, dermatological or surgical treatment, and / or (v) preventing aging, injury, disease or condition of skin tissue in an individual (e.g., a human), wherein (a) A method is provided comprising introducing a population of skin cells into a population of nucleic acids (RNA (e.g., circRNA) or DNA; or nucleic acid constructs (or viruses) containing the same) (or pharmaceutical compositions containing the same) encoding multiple reprogramming factors, wherein the multiple reprogramming factors include LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO 4), SOX2 (e.g., SEQ ID NO 24), and recombinant OCT4 without KLF4 (e.g., SEQ ID NO 22). In one embodiment, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In one embodiment, the population of skin cells is derived from skin tissue which is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after a cosmetic, dermatological or surgical procedure. In some embodiments, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to skin injury; (iii) susceptible to or predisposed to developing skin diseases or conditions; and / or (iv) subjected to cosmetic, dermatological or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In some embodiments, the method does not reprogram a population of skin cells into reprogrammed stem cells. Multiple nucleic acids may be on separate nucleic acid constructs (e.g., circRNA, plasmid, or recombinant viral genome) or on a single nucleic acid construct.Multiple nucleic acids can be delivered using exosomes, liposomes, LNPs, or viruses (e.g., LV or AAV). In some embodiments, at least two (e.g., all) of the multiple nucleic acids reside on a single nucleic acid construct. In some embodiments, at least two (e.g., all) of the multiple nucleic acids are under the control of different promoters. In some embodiments, at least two (e.g., all) of the multiple nucleic acids are under the control of the same promoter. In some embodiments, at least two (e.g., all) of the multiple nucleic acids are linked by one or more linked nucleic acids. In some embodiments, one or more linked nucleic acids include IRES or self-cleaving peptides, e.g., linked sequences encoding P2A, T2A, or E2A. In some embodiments, a population of skin cells is present in an individual in need of treatment or prevention, and the method involves intra vivo administration of an effective amount of multiple nucleic acids (or nucleic acid constructs or viruses containing them) to the individual (e.g., skin tissue). In some embodiments, the multiple nucleic acids are one or more RNAs (e.g., one circRNA). In one embodiment, one or more RNAs (e.g., one circRNA) are encapsulated within an exosome, liposome, or LNP. In one embodiment, the exosome, liposome, or LNP is delivered by a microneedle. In one embodiment, a population of skin cells is obtained from a donor individual and cultured ex vivo. In one embodiment, the method includes transplanting a rejuvenated population of skin cells (e.g., into skin tissue) into an individual in need of treatment or prevention. The donor individual and the individual in need of treatment or prevention may be the same or different. In one embodiment, the method includes ex vivo administration of an effective amount of multiple nucleic acids (or nucleic acid constructs or viruses containing them) to a population of skin cells. In one embodiment, the multiple nucleic acids are one or more RNAs (e.g., one circRNA). In one embodiment, one or more RNAs (e.g., one circRNA) are encapsulated within an exosome, liposome, or LNP. The method further includes introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, to a population of skin cells.In one embodiment, the individual is a human being of any age, such as a human being 10 years of age or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years of age or older). In one embodiment, the method is performed to prevent aging, damage, or disease or condition of skin tissue / cells, before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0044]

[0161] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of a nucleic acid construct (RNA (e.g., circRNA) or DNA; or a virus containing the same) (or a pharmaceutical composition containing the same) into the population of skin cells, wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding SOX2 (e.g., SEQ ID NO. 24) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22). In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological or surgical treatment, and / or (v) preventing aging, damage, disease or condition of skin tissue in an individual (e.g., a human), wherein (a) A method is provided comprising (b) introducing an effective amount of a nucleic acid construct (RNA (e.g., circRNA) or DNA; or a virus containing the same) (or a pharmaceutical composition containing the same) into a population of skin cells, and (b) transplanting the rejuvenated population of skin cells into an individual (e.g., the skin tissue of an individual), wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding SOX2 (e.g., SEQ ID NO. 24) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22).In one embodiment, a method is provided which includes (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), and (iv) transplanting a rejuvenated population of skin cells into an individual (e.g., skin tissue of an individual), wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding SOX2 (e.g., SEQ ID NO. 24) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22). In one embodiment, the nucleic acid construct encodes the protein sequence of SEQ ID NO. 93. In one embodiment, the nucleic acid construct further comprises an IRES at 5' of the first nucleic acid. In one embodiment, the nucleic acid construct is circRNA. In some embodiments, the nucleic acid construct is a DNA vector (e.g., a lentibal vector, an AAV vector, or a plasmid). In some embodiments, the nucleic acid construct includes any of the nucleic acid sequences SEQ ID NOs: 39, 57, and 94. In some embodiments, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In some embodiments, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to damage or predisposed to skin damage; (iii) prone to developing or predisposed to developing a skin disease or condition; and / or (iv) has undergone cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In one embodiment, the method does not reprogram a population of skin cells into reprogrammed stem cells. The nucleic acid construct can be delivered using exosomes, liposomes, LNPs, or viruses (e.g., LV or AAV).In some embodiments, exosomes, liposomes, or LNPs are delivered by microneedles. In some embodiments, a population of skin cells is present in an individual in need of treatment or prevention, and the method comprises intra vivo administration of an effective amount of nucleic acid construct (or a virus containing it) to the individual (e.g., into skin tissue). In some embodiments, the population of skin cells is obtained from a donor individual and cultured ex vivo. In some embodiments, the method comprises transplanting a rejuvenated population of skin cells into an individual in need of treatment or prevention (e.g., into skin tissue). The donor individual and the individual in need of treatment or prevention may be the same or different. In some embodiments, the method comprises ex vivo administration of an effective amount of nucleic acid construct (or a virus containing it) to a population of skin cells. The method further comprises introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, into the population of skin cells. In one embodiment, the individual is a human being of any age, such as a human being 10 years of age or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years of age or older). In one embodiment, the method is performed to prevent aging, damage, or disease or condition of skin tissue / cells, before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0045]

[0162] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of a nucleic acid construct (RNA (e.g., circRNA) or DNA; or a virus containing the same) (or a pharmaceutical composition containing the same) into the population of skin cells, wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding SOX2 (e.g., SEQ ID NO. 24). In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological or surgical treatment, and / or (v) preventing aging, damage, disease or condition of skin tissue in an individual (e.g., a human), wherein (a) A method is provided comprising (b) introducing an effective amount of a nucleic acid construct (RNA (e.g., circRNA) or DNA; or a virus containing the same) (or a pharmaceutical composition containing the same) into a population of skin cells, and (b) transplanting the rejuvenated population of skin cells into an individual (e.g., the skin tissue of an individual), wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding SOX2 (e.g., SEQ ID NO. 24).In one embodiment, a method is provided which includes (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), and (iv) transplanting a rejuvenated population of skin cells into an individual (e.g., skin tissue of an individual), wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding SOX2 (e.g., SEQ ID NO. 24). In one embodiment, the nucleic acid construct further comprises IRES at 5' of the first nucleic acid. In one embodiment, the nucleic acid construct is circRNA. In some embodiments, the nucleic acid construct is a DNA vector (e.g., a lentibal vector, an AAV vector, or a plasmid). In some embodiments, the nucleic acid construct includes the nucleic acid sequence of SEQ ID NO: 32 or 39. In some embodiments, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In some embodiments, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to skin injury; (iii) prone to developing a skin disease or condition; and / or (iv) has undergone cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In some embodiments, the method does not reprogram the population of skin cells into reprogrammed stem cells. Nucleic acid constructs can be delivered using exosomes, liposomes, LNPs, or viruses (e.g., LV or AAV). In some embodiments, exosomes, liposomes, or LNPs are delivered by microneedles.In one embodiment, the population of skin cells is present in an individual in need of treatment or prevention, and the method comprises intra vivo administration of an effective amount of nucleic acid construct (or a virus containing it) to the individual (e.g., into skin tissue). In one embodiment, the population of skin cells is obtained from a donor individual and cultured ex vivo. In one embodiment, the method comprises transplanting a rejuvenated population of skin cells into an individual in need of treatment or prevention (e.g., into skin tissue). The donor individual and the individual in need of treatment or prevention may be the same or different. In one embodiment, the method comprises ex vivo administration of an effective amount of nucleic acid construct (or a virus containing it) to the population of skin cells. The method further comprises introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, into the population of skin cells. In one embodiment, the individual is a human (e.g., of any age), such as a human aged 10 years or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years or older). In one embodiment, the method is performed to prevent aging, damage, or disease or condition of skin tissue / cells before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0046]

[0163] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of multiple nucleic acids (RNA (e.g., circRNA) or DNA or nucleic acid constructs or viruses containing the same) (or a pharmaceutical composition containing the same) encoding multiple reprogramming factors into a population of skin cells, wherein the multiple reprogramming factors include recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 22), and recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48, e.g., SEQ ID NO. 14). In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological or surgical treatment, and / or (v) preventing aging, damage, disease or condition of skin tissue in an individual (e.g., a human), comprising (a) an effective amount of multiple reprogramming to a population of skin cells The present invention provides a method comprising introducing multiple nucleic acids (RNA (e.g., circRNA) or DNA; or nucleic acid constructs or viruses containing the same) (or pharmaceutical compositions containing the same) that encode reprogramming factors, and transplanting a rejuvenated population of skin cells (e.g., from the same or different individuals) into an individual (e.g., the skin tissue of the individual), wherein the multiple reprogramming factors include recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 22), and recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48, e.g., SEQ ID NO. 14).In one embodiment, the method provides a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease or condition of skin tissue in an individual (e.g., a human), or (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), comprising introducing (or a pharmaceutical composition containing) (or containing) into a population of skin cells and transplanting a rejuvenated population of skin cells (e.g., from the same or different individuals) into an individual (e.g., skin tissue of an individual), wherein the multiple reprogramming factors include recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 22), and recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48, e.g., SEQ ID NO. 14). In some embodiments, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In some embodiments, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to skin injury; (iii) prone to developing or predisposed to developing a skin disease or condition; and / or (iv) has undergone cosmetic, dermatological, or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In some embodiments, the method does not reprogram the population of skin cells into reprogrammed stem cells. Multiple nucleic acids may be on separate nucleic acid constructs (e.g., circRNA, plasmid, or recombinant viral genome) or on a single nucleic acid construct. Multiple nucleic acids can be delivered using exosomes, liposomes, LNPs, or viruses (e.g., LV or AAV). In one embodiment, at least two (e.g., all) of the multiple nucleic acids reside on a single nucleic acid construct.In some embodiments, at least two (e.g., all) of a plurality of nucleic acids are under the control of different promoters. In some embodiments, at least two (e.g., all) of a plurality of nucleic acids are under the control of the same promoter. In some embodiments, at least two (e.g., all) of a plurality of nucleic acids are linked by one or more linked nucleic acids. In some embodiments, one or more linked nucleic acids include IRES or self-cleaving peptides, e.g., linked sequences encoding P2A, T2A, or E2A. In some embodiments, a population of skin cells is present in an individual in need of treatment or prevention, and the method includes intra vivo administration of an effective amount of a plurality of nucleic acids (or nucleic acid constructs or viruses containing them) to the individual (e.g., skin tissue). In some embodiments, the plurality of nucleic acids are one or more RNAs (e.g., one circRNA). In some embodiments, one or more RNAs (e.g., one circRNA) are encapsulated in exosomes, liposomes, or LNPs. In some embodiments, exosomes, liposomes, or LNPs are delivered by microneedles. In some embodiments, a population of skin cells is obtained from a donor individual and cultured ex vivo. In one embodiment, the method includes transplanting a rejuvenated population of skin cells (e.g., into skin tissue) into an individual in need of treatment or prevention. The donor individual and the individual in need of treatment or prevention may be the same or different. In one embodiment, the method includes ex vivo administration of an effective amount of multiple nucleic acids (or nucleic acid constructs(s) or viruses containing them) to the population of skin cells. In one embodiment, the multiple nucleic acids are one or more RNAs (e.g., one circRNA). In one embodiment, one or more RNAs (e.g., one circRNA) are encapsulated in exosomes, liposomes or LNPs. The method further includes introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, into the population of skin cells. In one embodiment, the individual is a human (e.g., of any age), such as a human aged 10 years or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years or older).In one embodiment, the method is performed to prevent aging, damage, or disease or condition of skin tissue / cells before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0047]

[0164] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of a nucleic acid construct (RNA (e.g., circRNA) or DNA; or a virus containing the same) (or a pharmaceutical composition containing the same) into the population of skin cells, wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47 and 48, e.g., SEQ ID NO. 14). In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) preventing aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), comprising: (a) an effective amount of nucleic acid construct (RNA (e.g., circ A method is provided which includes (b) introducing RNA) or DNA; or a virus containing the same) (or a pharmaceutical composition containing the same) and (b) implanting a nucleic acid construct into a population of skin cells (e.g., from the same or different individuals) comprising a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47 and 48, e.g., SEQ ID NO. 14).In one embodiment, a method is provided which includes (i) introducing (or a pharmaceutical composition containing) a first nucleic acid encoding recombinant OCT4 without an effective amount of LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first linkage sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22) - a second linkage sequence (e.g., encoding T2A) - a third nucleic acid encoding recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47 and 48, e.g., SEQ ID NO. 14) - a first linkage sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22) - a second linkage sequence (e.g., encoding T2A) - a third nucleic acid encoding recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47 and 48, e.g., SEQ ID NO. 14). In one embodiment, the nucleic acid construct encodes the protein sequence of SEQ ID NO: 97. In one embodiment, the nucleic acid construct further comprises an IRES at the 5' of the first nucleic acid. In one embodiment, the nucleic acid construct is circRNA. In one embodiment, the nucleic acid construct is a DNA vector (e.g., a lentibal vector, an AAV vector, or a plasmid). In one embodiment, the nucleic acid construct comprises any of the nucleic acid sequences of SEQ ID NOs: 30, 35, 59, and 98. In one embodiment, the population of skin cells comprises fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In one embodiment, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after a cosmetic, dermatological, or surgical procedure. In some embodiments, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or predisposed to skin injury; (iii) prone to developing or predisposed to developing skin diseases or conditions; and / or (iv) subjected to cosmetic, dermatological or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue.In some embodiments, the method does not reprogram a population of skin cells into reprogrammed stem cells. The nucleic acid construct can be delivered using exosomes, liposomes, LNPs, or viruses (e.g., LV or AAV). In some embodiments, exosomes, liposomes, or LNPs are delivered by microneedles. In some embodiments, the population of skin cells is present in an individual in need of treatment or prevention, and the method includes intra vivo administration of an effective amount of the nucleic acid construct (or a virus containing it) to the individual (e.g., into skin tissue). In some embodiments, the population of skin cells is obtained from a donor individual and cultured ex vivo. In some embodiments, the method includes transplanting a rejuvenated population of skin cells into an individual in need of treatment or prevention (e.g., into skin tissue). The donor individual and the individual in need of treatment or prevention may be the same or different. In some embodiments, the method includes ex vivo administration of an effective amount of the nucleic acid construct (or a virus containing it) to a population of skin cells. The method further includes introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, to the population of skin cells. In one embodiment, the individual is a human being of any age, such as a human being 10 years of age or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years of age or older). In one embodiment, the method is performed to prevent aging, damage, or disease or condition of skin tissue / cells, before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed). In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0048]

[0165] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of multiple nucleic acids (RNA (e.g., circRNA) or DNA or nucleic acid constructs or viruses containing the same) (or a pharmaceutical composition containing the same) encoding multiple reprogramming factors into a population of skin cells, wherein the multiple reprogramming factors include recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 22), recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48, e.g., SEQ ID NO. 14), and SOX2 (e.g., SEQ ID NO. 24). In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological or surgical treatment, and / or (v) preventing aging, damage, disease or condition of skin tissue in an individual (e.g., a human), comprising (a) a population of skin cells containing a plurality of nucleic acids (RN) encoding a plurality of reprogramming factors in an effective amount. A method is provided comprising (b) introducing A (e.g., circRNA) or DNA; or nucleic acid constructs or viruses containing the same) (or pharmaceutical compositions containing the same), and (c) introducing a population of skin cells (e.g., from the same or different individuals) a plurality of reprogramming factors comprising recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 22), and recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48, e.g., SEQ ID NO. 14), and SOX2 (e.g., SEQ ID NO. 24), and transplanting the rejuvenated population of skin cells into an individual (e.g., the skin tissue of an individual).In one embodiment, (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (b) introducing a population of skin cells into a population of nucleic acids (RNA (e.g., circRNA) or DNA; or nucleic acid constructs or viruses containing the same) (or pharmaceutical compositions containing the same) that encode multiple reprogramming factors, and (b) the multiple reprogramming factors in a population of skin cells (e.g., from the same or different individuals) that are recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 2) (2) Introducing recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48, e.g., SEQ ID NO. 14) and SOX2 (e.g., SEQ ID NO. 24) and (b) Introducing a population of skin cells (e.g., from the same or different individuals) into which multiple reprogramming factors are introduced, including recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4), KLF4 (e.g., SEQ ID NO. 22) and recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48, e.g., SEQ ID NO. 14) and SOX2 (e.g., SEQ ID NO. 24) and transplanting the rejuvenated population of skin cells into an individual (e.g., the skin tissue of an individual). In one embodiment, the population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In one embodiment, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after cosmetic, dermatological, or surgical treatment. In one embodiment, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to damage or predisposed to damage; (iii) prone to developing a skin disease or condition; and / or (iv) has undergone cosmetic, dermatological, or surgical treatment. In one embodiment, the skin tissue is photoaged skin tissue.In some embodiments, the method does not reprogram a population of skin cells into reprogrammed stem cells. Multiple nucleic acids may be on separate nucleic acid constructs (e.g., circRNA, plasmid, or recombinant viral genome) or on a single nucleic acid construct. Multiple nucleic acids can be delivered using exosomes, liposomes, LNPs, or viruses (e.g., LV or AAV). In some embodiments, at least two (e.g., all) of the multiple nucleic acids are on a single nucleic acid construct. In some embodiments, at least two (e.g., all) of the multiple nucleic acids are under the control of different promoters. In some embodiments, at least two (e.g., all) of the multiple nucleic acids are under the control of the same promoter. In some embodiments, at least two (e.g., all) of the multiple nucleic acids are linked by one or more linked nucleic acids. In some embodiments, one or more linked nucleic acids include IRES or self-cleaving peptides, e.g., linked sequences encoding P2A, T2A, or E2A. In one embodiment, the population of skin cells is present in an individual in need of treatment or prevention, and the method comprises intra vivo administration of an effective amount of multiple nucleic acids (or nucleic acid constructs or viruses containing them) to the individual (e.g., into skin tissue). In one embodiment, the multiple nucleic acids are one or more RNAs (e.g., one circRNA). In one embodiment, one or more RNAs (e.g., one circRNA) are encapsulated in exosomes, liposomes, or LNPs. In one embodiment, the exosomes, liposomes, or LNPs are delivered by microneedles. In one embodiment, the population of skin cells is obtained from a donor individual and cultured ex vivo. In one embodiment, the method comprises transplanting a rejuvenated population of skin cells into an individual in need of treatment or prevention (e.g., into skin tissue). The donor individual and the individual in need of treatment or prevention may be the same or different. In one embodiment, the method comprises ex vivo administration of an effective amount of multiple nucleic acids (or nucleic acid constructs or viruses containing them) to a population of skin cells. In one embodiment, the plurality of nucleic acids are one or more RNAs (e.g., one circRNA).In one embodiment, one or more RNAs (e.g., one circRNA) are encapsulated within an exosome, liposome, or LNP. The method further comprises introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, into a population of skin cells. In one embodiment, the individual is a human (e.g., of any age) who is 10 years of age or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years of age or older). In one embodiment, the method is performed before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed) in order to prevent aging, damage, or disease or condition of skin tissue / cells. In one embodiment, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0049]

[0166] In one embodiment, a method is provided for rejuvenating a population of skin cells, comprising introducing an effective amount of a nucleic acid construct (RNA (e.g., circRNA) or DNA; or a virus containing the same) (or a pharmaceutical composition containing the same) into the population of skin cells, wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding recombinant c-MYC without LLPS (e.g., any of SEQ ID NOs. 14-20, 47 and 48, e.g., SEQ ID NO. 14) - a third ligation sequence (e.g., encoding E2A) - a fourth nucleic acid encoding SOX2 (e.g., SEQ ID NO. 24). In one embodiment, a method for (i) slowing or preventing the progression of aging of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (ii) treating aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), (iii) preserving or improving the condition and / or appearance of skin tissue in an individual (e.g., a human, e.g., 22 years of age or older), (iv) enhancing the recovery of skin tissue in an individual (e.g., a human) after damage or cosmetic, dermatological, or surgical treatment, and / or (v) preventing aging, damage, disease, or condition of skin tissue in an individual (e.g., a human), comprising (a) an effective amount of nucleic acid construct (RNA (e.g., circRNA) or DNA) to a population of skin cells in skin tissue;(b) a virus containing the same (or a pharmaceutical composition containing the same), wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first nucleic acid (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO. 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO. 22) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding recombinant c-MYC without LLPS (e.g., SEQ ID NOs. 14-20, 47 and 48, e.g., SEQ ID NO. 14) - a third ligation sequence (e.g., encoding E2A) - a fourth nucleic acid encoding SOX2 (e.g., SEQ ID NO. 24); or (b) a population of skin cells (e.g., from the same or different individuals), containing an effective amount of the nucleic acid construct (RNA (e.g., circRN) A) the introduction of DNA or a virus containing it (or a pharmaceutical composition containing it) and the transplantation of a rejuvenated population of skin cells into an individual (e.g., the skin tissue of an individual), wherein the nucleic acid construct comprises, from 5' to 3', a first nucleic acid encoding recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO 4) - a first nucleic acid (e.g., any of SEQ ID NOs. 4-10 and 44-46, e.g., SEQ ID NO 4) - a first ligation sequence (e.g., encoding P2A) - a second nucleic acid encoding KLF4 (e.g., SEQ ID NO 22) - a second ligation sequence (e.g., encoding T2A) - a third nucleic acid encoding recombinant c-MYC without LLPS (e.g., SEQ ID NOs. 14-20, 47 and 48, e.g., SEQ ID NO 14) - a third ligation sequence (e.g., encoding E2A) - a fourth nucleic acid encoding SOX2 (e.g., SEQ ID NO 24). In one embodiment, the nucleic acid construct encodes the protein sequence of SEQ ID NO: 101. In one embodiment, the nucleic acid construct further comprises an IRES at the 5' of the first nucleic acid. In one embodiment, the nucleic acid construct is circRNA. In one embodiment, the nucleic acid construct is a DNA vector (e.g., a lentibal vector, an AAV vector, or a plasmid). In one embodiment, the nucleic acid construct comprises any of the nucleic acid sequences of SEQ ID NOs: 28, 37, 61, and 102. In one embodiment, the population of skin cells comprises fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In one embodiment, the population of skin cells is derived from skin tissue that is (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after a cosmetic, dermatological, or surgical procedure. In some embodiments, the skin tissue is (i) prone to aging or predisposed to aging; (ii) susceptible to injury or prone to skin injury; (iii) susceptible to or predisposed to developing skin diseases or conditions; and / or (iv) subjected to cosmetic, dermatological or surgical treatment. In some embodiments, the skin tissue is photoaged skin tissue. In some embodiments, the method does not reprogram the population of skin cells into reprogrammed stem cells. The nucleic acid construct can be delivered using exosomes, liposomes, LNPs, or viruses (e.g., LV or AAV). In some embodiments, the exosomes, liposomes, or LNPs are delivered by microneedles. In some embodiments, the population of skin cells is present in an individual in need of treatment or prevention, and the method comprises intra vivo administration of an effective amount of the nucleic acid construct (or a virus containing it) to the individual (e.g., skin tissue). In some embodiments, the population of skin cells is obtained from a donor individual and cultured ex vivo. In one embodiment, the method includes transplanting a rejuvenated population of skin cells (for example, into skin tissue) into an individual in need of treatment or prevention. The donor individual and the individual in need of treatment or prevention may be the same or different. In one embodiment, the method includes ex vivo administration of an effective amount of nucleic acid construct (or a virus containing it) to the population of skin cells.The method further comprises introducing an effective amount of a therapeutic agent, e.g., B18R, vitamin C, vitamin E, or any combination thereof, into a population of skin cells. In some embodiments, the individual is a human being of any age, such as a human being 10 years of age or older (e.g., at least about 15, 22, 30, 40, 50, 60, 70 years of age or older). In some embodiments, the method is performed before skin tissue / cells age, before exposure to damaging factors (e.g., UV irradiation or cosmetic, dermatological, or surgical procedures), or before a disease or condition exists (e.g., is diagnosed) in order to prevent aging, damage, or disease or condition of skin tissue / cells. In some embodiments, the method is performed at least about 12 hours (e.g., at least about 24 hours, 48 ​​hours, or more) before skin tissue / cells are exposed to damaging factors (e.g., UV irradiation).

[0050] Skin cells and tissues and related conditions

[0167] The skin is typically composed of two main tissue layers. The outermost layer is the epidermis, which is made up of several layers. The dermis consists of the upper papillary layer and the lower reticular layer.

[0168] The human epidermis is primarily composed of keratinocytes. It also contains other types of cells, including melanocytes and Langerhans cells.

[0169] The dermis provides a solid support structure for the epidermis and also serves as its feeder layer. The dermis is mainly composed of fibroblasts. Leukocytes, mast cells, or tissue macrophages are also present. The dermis further contains blood vessels and nerve fibers. The cell-free portion of the dermis (i.e., the intercellular region) is called the extracellular matrix (ECM). The cutaneous ECM is composed of various extracellular components, including proteins, particularly collagen fibers and elastin. Other dermal extracellular membrane (ECM) components include glycosaminoglycans (e.g., hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, etc.), proteoglycans (e.g., fibromodulin, decorin, biglycan, perlecan, heparan sulfate proteoglycan 2, agrin, versican, aggrecan, lumican, type IX collagen, type XII collagen, type XIV collagen, testis 1, testis 2, etc.) and various glycoproteins (e.g., fibrillin 1, thrombospondin-1 and -2, tenascin-C and -X, osteopontin, fibronectin, laminin-5 and -6, vitronectin, etc.). These extracellular components are synthesized by dermal fibroblasts, making dermal fibroblasts the main components of the dermal structure.

[0051]

[0170] ECM is a highly heterogeneous amalgam of morphologically diverse structural entities. In addition to regulating cellular behavior by interacting with cell surface receptors and soluble growth factors, it organizes individual tissues and confers structural integrity to them. Dysfunction and alterations of ECM components can impair both tissue integrity and cellular performance. Dysfunction and alterations of ECM components in human skin and mucous membranes can result in skin aging, skin atrophy, damaged skin, wounded skin, vulvar and vaginal atrophy (vulvovaginal atrophy), or any other conditions, disorders, and diseases of the skin and mucous membranes associated with alterations of ECM components.

[0052]

[0171] The five most common types of collagen in the human body are collagen I, II, III, IV, and V. However, over 90% of the collagen in the body is type I.

[0172] In human skin, collagen types I and III are the major types of collagen. They exist as fibrils and are involved in the stiffness and strength of the dermis. Type I collagen is the major collagen in adult skin, accounting for about 80% of the total collagen volume, and therefore plays a major role in providing tensile strength to the skin. However, type III collagen, which makes up about 10% of total dermal collagen, is also clearly important in providing further tensile properties to the skin and other tissues. See Journal of Dermatological Science, 24, Suppl. 1, 2000, S60-S69.

[0173] Skin that ages over time shows a decrease in the synthesis of both type I and type III collagen. Regarding photoaging, Schwarz et al. (Photochem Photobiol 1993, 58, 841-844) demonstrated that collagen loss in sun-damaged human skin is due to increased degradation of both type I and type III collagen. Furthermore, it was shown that fibroblasts derived from sun-exposed skin synthesize a lower percentage of collagen III than cells derived from sun-protected skin. See J Photochem Photobiol B. 1995, 27:33-38.

[0174] Collagen production in the body requires activation of the collagen biosynthesis pathway, in which transcription in the cell nucleus promotes polypeptide synthesis via translation from mRNA. In the cytoplasm, the polypeptides are organized into a procollagen triple helix structure, and procollagen is secreted from the cell. Subsequently, cleavage reactions, fibril assembly, and extracellular cross-linking occur. Unlike many proteins that are stored in secretory granules and then secreted from the cell on demand, collagen is secreted continuously.

[0175] Changes in the content and structure of collagen, as well as other components of the ECM, including but not limited to elastin and hyaluronic acid, are characteristic of aged human skin.

[0053]

[0176] Elastin is a vital component of the extracellular matrix (ECM) in vertebrates, providing many tissues and organs, including skin, with excellent properties, including elasticity and tensile strength. Mature elastin is an insoluble and extremely durable protein with little turnover; however, sustained exposure to proteases can lead to irreversible and severe damage, and thus loss of function of the elastic fiber network. Generally, elastin content decreased with age in skin not exposed to sunlight (i.e., buttocks) (i.e., elastin content decreased by approximately 44% between ages 50 and 70). A similar decrease was observed in heavily sun-exposed skin (i.e., face) (i.e., elastin content decreased by approximately 31% between ages 50 and 70). Interestingly, elastin content in moderately sun-exposed areas (i.e., forearms) did not change significantly during aging. This phenomenon may be explained by a combination of age-induced decrease and sun-dependent increase in elastin, which appears to be at least partially regulated by UV-induced deposition of lysozyme in elastin fibers. See JEADV 2006, 20, 980-987.

[0054]

[0177] Besides skin aging, collagen I and III are also major ECM components involved in scar formation. Scars occur after trauma, injury, or surgery to any tissue or organ of the body. Such scars are the result of a repair mechanism that replaces the missing normal tissue with the ECM, which consists mainly of collagen types I and III, as well as fibronectin and several other ECM components. Scars represent incomplete tissue regeneration. Skin wounds in early mammalian embryos (e.g., up to about 24 weeks of gestation in humans) heal completely without signs of scarring and fully restore normal skin structure, while postnatal wounds heal with scarring. See Dang C et al., Clin Plast Surg 2003: 30, 13-23. Of the many different types of collagen identified, fetal skin is known to contain a larger proportion of type III collagen, while adult skin consists mainly of type I collagen.

[0055]

[0178] Inflammation plays a crucial role in scar formation. As the immune system develops and the resulting inflammatory response increases, scar formation occurs at the repair site. The synthesis and remodeling of the extracellular matrix (ECM) by wound fibroblasts are likely major determinants of post-repair dermal structure. The difference between scarred and non-scarred collagen structures can be partially explained by phenotypic differences between adult and fetal fibroblasts.

[0056]

[0179] Fetal and adult fibroblasts exhibit differences in the synthesis rates of collagen, hyaluronic acid (HA), and other ECM components. In vitro, fetal fibroblasts synthesize more type III and type IV collagen than their adult counterparts. Fetal fibroblasts can proliferate and synthesize collagen simultaneously. Fetal fibroblasts have a greater ability to migrate into collagen gel than adult fibroblasts. Increased cell density reduces adult HA production, but does not affect fetal fibroblast HA synthesis.

[0057]

[0180] Skin aging is classified into intrinsic and extrinsic aging depending on the cause. Intrinsic aging is the process by which skin structure and physiological function deteriorate as a person ages, regardless of environmental changes. Extrinsic aging is caused by continuous exposure to external environmental factors such as sunlight and air pollutants. In particular, skin aging caused by sunlight is called photoaging. Ultraviolet (UV) light from the sun is the main cause of physiological and morphological changes in aged skin.

[0181] As intrinsic skin aging progresses, the skin dries out, and fine lines and wrinkles form, which become more visible and deeper with age. Furthermore, due to structural and functional changes in the epidermis and dermis, the skin loses its elasticity and appears to sag. The dermis thins with age, and visible skin folds (e.g., nasolabial folds) are formed. The total amount of collagen loss per year in adults is estimated to be about 1%. In addition, remaining collagen fibers gradually become thicker, and the cross-linking of collagen fibers increases, reducing their solubility, elasticity, etc. Furthermore, elastin fibers become thicker, and their cross-linking also increases. Moreover, the proliferative activity of dermal fibroblasts decreases over time, and the ability of aging fibroblasts to form (i.e., synthesize) new collagen, elastin, hyaluronic acid, and other components of the ECM also decreases.

[0058]

[0182] Continuous exposure to sunlight is a major cause of extrinsic aging of the skin. The UV components of sunlight, particularly UVA and UVB, are generally considered to be the main causative factors of this process known as photoaging. While the amount of UV exposure required to cause "photoaging" is not currently known, the amount sufficient to cause erythema (redness, commonly described as sunburn) in human skin is quantified as the "minimum erythemal dose" (MED) from a given UV light source. Nevertheless, repeated exposure to sunlight UV at levels that cause erythema and sunburn is generally associated with photoaging.

[0059]

[0183] In comparison to photoaging of the skin, there are differences in the physiological functions of intrinsic aging (i.e., aging over time). Skin that has aged over time usually maintains a smooth, blemish-free appearance compared to photoaged skin, which is leathery, blemish-like, and often deeply wrinkled. Photoaging is clinically characterized by roughness, wrinkles, mottled pigmentation, yellowing, flaccidity, telangiectasia, lentigo, purpura and bruising, atrophy, depigmented areas, and ultimately pre-malignant and ultimately malignant neoplasms (i.e., abnormal masses of tissue as a result of neoplasms, which are abnormal proliferations of cells). Photoaging generally occurs in skin that is commonly exposed to sunlight, such as the face, ears, bald areas of the scalp, neck, décolleté, forearms and hands.

[0184] Nevertheless, it has been found that skin that has aged over time and skin exposed to UV radiation share important molecular characteristics, including alterations in signaling pathways that promote the expression of matrix metalloproteinases (e.g., collagenase, gelatinase) that cause ECM degradation, decreased collagen formation, and alterations or damage to the skin's ECM, such as the accumulation of amorphous elastin-containing material beneath the epidermal-dermal junction.

[0060]

[0185] Wrinkles are generally the result of the natural aging process of the skin and exposure to ultraviolet (UV) radiation from the sun. Wrinkles are morphological changes on the surface of the skin that do not involve specific structural changes at the histological level. Generally, wrinkles are described by Ligman et al. (1985) Br. J. Derm. 113:37-42. Ligman classifies wrinkles into three classes: linear wrinkles, glyphic wrinkles, and creases. Linear wrinkles are straight lines, commonly found on the skin of the face, and are caused by natural aging or exposure to UV radiation. Glyphic wrinkles form as distinct triangular or rectangular creases and are found on the face, hands, and neck exposed to sunlight, and are aggravated by UV radiation exposure or cutaneous heliosis. Wrinkles are thin, wrinkled lines on loose skin that can be found anywhere on the skin, but are usually seen on the back of the hands and around the eyelids.

[0061]

[0186] The skin tissue described herein may be abnormal, damaged, or unhealthy skin tissue that may require treatment. The skin tissue may also be normal or healthy skin tissue that is at a higher-than-normal risk of becoming abnormal or unhealthy (e.g., undergoing cosmetic, dermatological, or surgical procedures or being exposed to UV radiation) that may require preventative measures. In some embodiments, the skin tissue is healthy but is considered suboptimal to the capacity or survival of its current or future condition.

[0187] The skin tissue may originate from any of the following areas of the human body: face, neck, décobe, scalp, hands, palms, arms, legs, feet, soles, chest, back, abdomen, buttocks, vulva, penis, scrotum, anus, and / or any other area of ​​skin of the human body. In one embodiment, the skin tissue is facial skin.

[0062]

[0188] In some embodiments, the population of skin cells administered with the LLPS-deficient recombinant reprogramming factor described herein includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof. In some embodiments, the population of skin cells includes (or is essentially thereof or consists of) fibroblasts. In some embodiments, the population of skin cells is derived from skin tissue, which is (i) aged skin tissue or skin tissue that is prone to aging or predisposed to aging; (ii) damaged skin tissue or skin tissue that is susceptible to damage or prone to skin damage; (iii) skin tissue that has a skin disease or condition or is prone to developing a skin disease or condition or is predisposed to developing one; and / or (iv) skin tissue that is administered or skin tissue that has undergone cosmetic, dermatological or surgical treatment.

[0189] In some embodiments, the population of skin cells is derived from aged skin tissue or skin tissue that is prone to aging or predisposed to aging. Skin aging may be intrinsic aging (i.e., aging over time) or exogenous aging (e.g., due to exogenous damaging factors such as UV irradiation or air pollutants). In some embodiments, the aged skin tissue is derived from a human being 22 years of age or older (e.g., at least about 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 years or older), for example, a human being 24 years of age or older, 30 years of age or older, 50 years of age or older, 55 years of age or older, or 60 years of age or older. In some embodiments, the skin tissue is photoaged skin tissue or skin tissue that is prone to or predisposed to photodamage. The photoaged skin tissue or skin tissue that is prone to aging or predisposed to aging may be derived from an individual (e.g., a human) of any age.

[0190] Exogenous skin aging can be caused by skin damaging factors including, but are not limited to, air pollution, solar radiation (e.g., UV), tobacco, alcohol, climate (e.g., high temperatures, high altitude, or desert environments), nutrition (e.g., fats, refined flour, highly processed foods), stress, lack of sleep, lack of exercise, or a combination thereof.

[0063]

[0191] In some embodiments, the population of skin cells originates from damaged skin tissue or skin tissue that is susceptible to damage or predisposed to skin damage. In some embodiments, the damage is ultraviolet (e.g., UVA or UVB) induced and, for example, causes wrinkles. In some embodiments, the damage is due to invasive cutting, abrasion, or wound, such as cutting with a knife or physical stretching. In some embodiments, the damage is due to fire or chemical burns. In some embodiments, the damage is due to cosmetic, dermatological, or surgical procedures. Skin damage or injury includes, but is not limited to, cuts, lacerations, deep wounds, lacerations, abrasions, scratches, bed burns, and contusions. Damaged skin is usually characterized by one or more of the following: inflammation, epidermal hyperplasia, dermal elastic fibrosis and matrix proteolysis, and the presence of perivenule lymphohistiocytic dermal infiltrations.

[0064]

[0192] In some embodiments, the population of skin cells is derived from skin tissue that has a skin disease or condition, or is prone to or predisposed to developing a skin disease or condition. Any skin disease or condition that is related to skin aging or damage or to changes in ECM components (e.g., not related to aging) can be treated or prevented herein and includes, but is not limited to, inflammatory skin conditions, neurogenic or neuroinflammatory skin conditions, acute or chronic wounds, acute or chronic ulcers or burns. In some embodiments, the skin disease or condition requires scar-free healing and / or a balanced response to skin inflammation. In some embodiments, the skin tissue is scarred or prone to or predisposed to scarring. In some embodiments, the skin disease or condition is hereditary. In some embodiments, the skin disease or condition is a skin aging-related disease or condition, e.g., dry skin, itchy skin, blemishes, skin tags, chronic wounds, etc. In some embodiments, the skin disease or condition is cutaneous porosis. See also, for skin aging-related diseases or conditions, see, for example, Blume-Peytavi et al. 2016:56 Suppl 2:S230-42.

[0193] Inflammatory skin conditions may include vulvovaginal vestibular syndrome, dysvaginal vulva, vulva pain, psoriasis, exfoliative dermatitis, atopic dermatitis, eczema, contact dermatitis, allergic contact dermatitis, herpetiform dermatitis, systemic dermatitis, vulvovaginal lichen, sebaceous cyst, seborrheic dermatitis, rosacea, acne, keloids, itching, keratosis pilaris, dandruff, diaper rash, actinitis, periulcer, scarring, or xerosis.

[0194] Wounds may occur during and / or after skin allografts, autologous skin grafts, or three-dimensional skin structures. Postoperative wounds may also be treated, including, but not limited to, non-cosmetic, cosmetic, reconstructive, and / or reconstructive surgical procedures. Acute and chronic wounds may include minor burns, minor wounds, lacerations, fissures, scratches, accidental wounds, ulcers, periulcerated skin, wound closure, and / or post-healing skin.

[0195] Cosmetic, plastic, and reconstructive surgical procedures include, but are not limited to, breast lifts, breast augmentation or reduction, facelifts, forehead lifts, nose surgery, ear surgery, eyelid surgery, abdominal surgery, fat sculpting, liposuction, scar revision, fat grafting, soft tissue augmentation, cryosurgery or cryotherapy, hair transplants, nail surgery, sclerotherapy, laser surgery, tattoo removal and / or venous surgery.

[0065]

[0196] In one embodiment, the population of skin cells is derived from the administered skin tissue or from skin tissue after cosmetic, dermatological, or surgical procedures.

[0197] Cosmetic and / or dermatological treatments include, but are not limited to, mild chemical peels, moderate chemical peels, deep chemical peels, physical peels, waxing, microdermabrasion, dermabrasion, phototherapy (intense pulsed light, photomodulation, visible light, invisible light, infrared light and / or ultraviolet light), laser treatment, radiofrequency treatment, thermal treatment (heat, cold, heat and cold cycles), electrical treatment, ultrasonic treatment, mechanical treatment (massage, pressure, suction, vibration, friction, abrasion), oxygen and / or ozone treatment, injections and / or any combination thereof.

[0198] Surgical procedures on the skin include, but are not limited to, skin biopsies (e.g., punch biopsy, shave biopsy, incision biopsy, and excision biopsy), electrosurgery, curettage and cauterization, wound closure, skin flaps, skin grafts, Mohs excision, cryotherapy, subcision, and excision of skin lesions.

[0199] The skin rejuvenation methods, treatments, or preventive measures described herein, or the LLPS-deficient recombinant reprogramming factors, may be used in combination with the aforementioned cosmetic and / or dermatological procedures or the aforementioned cosmetic, plastic, and / or reconstructive surgical procedures, either before, after, and / or during (in parallel with) the procedures.

[0066] Therapeutic and / or preventive skin rejuvenation effects

[0200] The skin rejuvenation methods described herein may be therapeutic or prophylactic (i.e., preventive), and are also referred to herein as “therapeutic skin rejuvenation method” or “treatment method” and “prophylactic skin rejuvenation method” or “preventive method,” respectively. The methods or LLPS-deficient recombinant reprogramming factors (or compositions thereof) described herein may be used prophylactically for the purpose of reducing, improving, mitigating, modifying, treating, improving or influencing the appearance of skin damage, or may be used to reduce, treat and / or prevent further skin aging or damage (e.g., wrinkles) or reduce the appearance of skin aging or damage in individuals who are predisposed to or likely to cause skin damage (e.g., UV radiation such as UVB radiation). Therapeutic and prophylactic methods described herein that involve introducing LLPS-deficient recombinant reprogramming factors (or compositions thereof) may function by any mechanism, including but not limited to skin rejuvenation.

[0201] For therapeutic skin rejuvenation, the skin tissue to be treated may be (i) aged skin tissue; (ii) damaged skin tissue; (iii) skin tissue with a skin disease or condition; and / or (iv) skin tissue after a cosmetic, dermatological, or surgical procedure (or the population of skin cells to be treated is derived from there).

[0202] In the case of preventive skin rejuvenation, the skin tissue (or population of skin cells) treated may be (i) skin tissue that is prone to aging or is prone to aging (e.g., before the onset of endogenous aging of the skin); (ii) skin tissue that is prone to injury or is susceptible to skin damage (e.g., before exposure to skin damaging factors such as UV irradiation or exposure to drugs that are likely to cause skin damage); (iii) skin tissue that is prone to developing or is susceptible to developing a skin disease or condition (e.g., before a skin disease or condition is diagnosed); and / or (iv) skin tissue that is to undergo cosmetic, dermatological or surgical treatment (e.g., before laser treatment or skin grafting).

[0067]

[0203] The therapeutic skin rejuvenation methods described herein include: i) improving the condition of aged skin and / or visible signs of skin aging (including, for example, fine lines, wrinkles, skin folds, enlarged pores, roughness, dryness, loss of elasticity, and loss of volume); ii) improving other defects in skin texture, such as striae (caused by pregnancy, trauma, or other effects); iii) reducing under-eye bags (or "eye puffiness"); and iv) dark circles under the eyes. a) reducing (dark circles under the eyes) (both caused by thinning of the skin, poor blood circulation and sagging of tissue); v) reducing the severity of atrophic skin (e.g., atrophic vulva, atrophic vagina); vi) reducing scarring of damaged skin; vii) restoring damaged skin; viiii) enhancing the recovery of skin after injury(s) or cosmetic, dermatological and / or surgical(s), e.g., enhancing scar or wound healing, enhancing skin regeneration or reducing inflammation; ix) skin One or more of the following can be achieved: (x) preventing or slowing the progression of aging; (x) reducing the severity of a skin disease or condition; (xi) preventing or slowing the progression of a skin disease or condition (e.g., inflammation); (xii) treating, alleviating or improving symptoms associated with a skin disease or condition; (xiii) improving the individual's quality of life (e.g., self-confidence or satisfaction); and / or (xiv) preserving or improving the condition (e.g., texture, moisture, elasticity, volume or biomarkers) and / or appearance of skin tissue (including the extracutaneous tissue). In some embodiments, compared to reference, the aging, damage or disease or condition (or associated symptoms) of the skin treated by the skin rejuvenation method described herein is reduced by at least about 5%, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In one embodiment, compared to the reference, the progression of skin aging, damage, or disease or condition (or related symptoms) treated by the skin rejuvenation method described herein is delayed by at least about one week, for example, at least about two weeks, one month, three months, six months, one year, two years, five years, or any of these.In some embodiments, compared to a reference, the appearance, condition, state and / or function of the skin (e.g., protection against microorganisms, dehydration, ultraviolet and mechanical damage, pain, temperature, contact and deep pressure, etc.), and / or the quality of life of the individual are improved by at least about 5%, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2 times, 5 times, 10 times or more. In some embodiments, the appearance, condition, state and / or function of the skin are restored to at least about 20% of healthy skin and / or young skin (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The reference may be i) reference skin cells or tissue that have not been introduced with LLPS-deficient recombinant reprogramming factor; and / or ii) reference skin cells or tissue that have been introduced with LLPS-containing reference reprogramming factor. Reference skin tissue / cells and skin tissue / cells may originate from the same or different individuals.

[0068]

[0204] The preventive skin rejuvenation methods described herein can achieve one or more of the following: i) maintaining healthy skin and / or ii) preventing or delaying the onset of skin aging, damage (e.g., scarring, sunburn) or disease or condition (or related symptoms). In some embodiments, compared to a reference, the onset of skin aging, damage or disease or condition (or related symptoms) treated by the skin rejuvenation method (or LLPS-deficient recombinant reprogramming factor) described herein is delayed by at least about one week, for example, at least about two weeks, one month, three months, six months, one year, two years, five years or more. In some embodiments, compared to a reference, healthy skin is maintained for at least about one week longer, such as at least about two weeks, one month, three months, six months, one year, two years, five years or more. The reference may be i) reference skin cells or tissue in which the LLPS-deficient recombinant reprogramming factor has not been introduced; and / or ii) reference skin cells or tissue in which the LLPS-containing reference reprogramming factor has been introduced. Reference skin tissue / cells and skin tissue / cells may originate from the same or different individuals.

[0069]

[0205] In some embodiments, the skin rejuvenation method (or therapeutic or preventive method) or the therapeutic or preventive effect of LLPS-deficient recombinant reprogramming factor described herein can be determined or quantified using one or more biomarkers.

[0070]

[0206] In one embodiment, the skin rejuvenation method (or treatment or prevention method) does not reprogram a population of skin cells into reprogrammed stem cells (i.e., iPSCs). The reprogrammed stem cells or iPSCs may express at least some and optionally all of the following non-limiting list of markers: SSEA-3, SSEA-4, TRA-I-60, TRA-I-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Lin28, Klf4, REX-1, and Nanog. OCT4, LIN28, and / or KLF4 may be expressed at high levels in iPSCs, but skin identity marker genes are not expressed or are barely expressed. In one embodiment, compared to reference skin cells into which an effective amount of LLPS-containing reference reprogramming factor has been introduced, the rejuvenated population of skin cells is (i) (ii) Increase the expression and / or function of biomarkers in initiation phase reprogramming (IPR) (e.g., increase by at least about 5%, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2x, 3x, 5x, 10x or more); (ii) Decrease the expression and / or function of biomarkers in maturation phase reprogramming (e.g., at least about 5%, e.g. (iii) reducing by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%; and / or (iii) reducing the expression and / or function of the biomarkers of the stabilization phase reprogramming (e.g., at least about 5%, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). In one embodiment, compared to reference skin cells that have not been introduced recombinant reprogramming factors, the rejuvenated population of skin cells shows increased expression and / or function of IPR biomarkers (e.g., at least about 5%, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2x, 3x, 5x, 10x or more).In one embodiment, (i) the biomarker for IPR is NANOG and / or SALL4, (ii) the biomarker for maturation reprogramming is DPPA5 and / or LIN28A, and / or (iii) the biomarker for stabilization reprogramming is OCT4 and / or XACT. The reference skin tissue / cells and skin tissue / cells may originate from the same or different individuals.

[0071]

[0207] In one embodiment, compared to reference skin cells without recombinant reprogramming factor introduced and / or reference skin cells with an effective amount of LLPS-containing reference reprogramming factor introduced, the rejuvenated skin cell population exhibited: (i) a reduced proportion of cells positive for cellular senescence biomarkers; (ii) reduced expression and / or function of biomarkers for cellular senescence; (iii) reduced epigenetic age (EpiAge); (iv) increased expression and / or function of biomarkers associated with healthy skin cell function; (v) increased expression and / or function of biomarkers for skin cell identity; (vi) decreased expression and / or function of biomarkers for apoptosis; (vii) decreased expression and / or function of biomarkers for ferroptosis; (viii) decreased expression and / or function of biomarkers for inflammation; (ix) decreased expression and / or function of biomarkers for endoplasmic reticulum (ER) stress; and / or (x) decreased expression and / or function of biomarkers associated with unhealthy skin cell function. A reduction in biomarker expression and / or function may be a reduction of at least about 5% of the biomarker expression and / or function of reference skin cells (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). An increase in biomarker expression and / or function may be an increase of at least about 5% of the biomarker expression and / or function of reference skin cells (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2x, 3x, 5x, 10x, or more).In one embodiment, (i) EpiAge biomarkers are selected from the group consisting of senescence-related β-galactosidase (SA-β-gal), p16 and p21 or a combination thereof; (ii) cellular senescence is measured using Horvat's clock, Levine's clock, Hanham's clock and / or AltumAge; (iii) biomarkers related to the function of healthy skin cells are selected from the group consisting of COL1A1, COL3A1, COL4A1, ELN, FN1 and LAMA5 or a combination thereof; (iv) biomarkers for skin cell identity are selected from the group consisting of THY1, P4HB, S100A4 and SERPINH1 or a combination thereof; (v) biomarkers for apoptosis are loss of membrane asymmetry, cleavage of Bcl-2 family proteins, caspase activation, caspase substrate cleavage, and non-caspase proteases. (i) Biomarkers for ferroptosis are selected from the group consisting of activation, decreased mitochondrial membrane potential (ΔΨm), cytochrome C release, increased sub-G1 population, nuclear condensation, DNA fragmentation, and membrane vesicle formation, or combinations thereof; (vi) Biomarkers for inflammation are HMOX1, SAT1, or combinations thereof; (vii) Biomarkers for inflammation are IL6, CCL3, CXCL2, CXCL8, CSF2, or combinations thereof; (viii) Biomarkers for endoplasmic reticulum stress include Hsp70 heat shock protein, Hsp40 heat shock protein, DDIT3, and HERPUD1, or combinations thereof; and / or (ix) Biomarkers associated with unhealthy skin cell function are selected from the group consisting of IL6, CXCL8, IL1A, IL1B, IGFBP3, CDKN1A, TGFB3, MMP1, MMP2, and SOD2, or combinations thereof. Reference skin tissue / cells and skin tissue / cells may originate from the same or different individuals.

[0072]

[0208] In one embodiment, skin tissue containing a population of skin cells introduced with an effective amount of recombinant reprogramming factor shows increased expression of one or more selected from the group consisting of elastin, collagen I, collagen III, and collagen IV, compared to reference skin tissue not containing skin cells introduced with an effective amount of recombinant reprogramming factor and / or reference skin cells introduced with an effective amount of LLPS-containing reference reprogramming factor. The reference skin tissue and the skin tissue may originate from the same or different individuals.

[0073]

[0209] Methods for measuring elastin and / or collagen are well known. For example, elastin can be measured by desmosine immunoassay. Collagen can be measured by hydroxyproline assay (hydroxyproline is a characteristic amino acid of fibrillary collagen and constitutes approximately 13.5% of the protein). Total protein is a useful denominator for normalizing elastin or collagen levels in tissues or cells. Protein levels can be measured rapidly and at high throughput from amino acid hydrolysates using a simple colorimetric assay in a convenient 96-well plate format (Martener, 2001). For example, see Stoilov et al., "Chapter 7 - Measurement of elastin, collagen, and total protein levels in tissues," Methods in Cell Biology, Volume 143, 2018, Pages 133-146.

[0210] The biomarkers described herein can be detected and / or measured using any method known in the art, such as RNA and / or protein expression tests, DNA methylation assays, and functional assays. Such methods include, but are not limited to, Northern blotting, RNA sequencing (RNA-Seq), RT-qPCR, RNA-fluorescence in situ hybridization (RNA-FISH), Western blotting, mass spectrometry (MS), immunofluorescence (IF), reporter assays (in vitro or intracellular), and signal transduction assays.

[0211] Any cellular senescence assay, such as a colorimetric assay (e.g., SA-β-gal assay), a fluorescence assay (e.g., C12FDG or CellEvent Senescence Green Reagent functioning as a fluorescent β-gal substrate), or a flow cytometry assay (e.g., using a combination of antibody markers such as p16ARF and p21), may be used herein.

[0212] Any apoptosis assay may be used herein, including but not limited to annexin V assays, DNA condensation and fragmentation (TUNEL) assays, caspase activation and detection assays, mitochondrial voltage-gated dye assays, cytochrome C release assays, and glutathione assays. For example, see the “Apoptosis assays and markers guide,” abcam, at abcam.com / kits / apoptosis-assays, the entire content of which is incorporated herein by reference.

[0074]

[0213] This specification may use, but is not limited to, any ferroptosis assay, including, measuring ferroptosis responses related to iron and / or lipid metabolism (e.g., fatty ROS), such as Prussian blue staining, Mito-FerroGreen, fluorescent probes Ferrum 430, Ferrum 560 and Ursa 520-R, green fluorescent heavy metal indicator Phen Green SK, FIP-1 (endoperoxide reactivity-based fluorescence resonance energy transfer probe), staining of TFRCs with antibodies (e.g., 3F3-FMA), fluorescent probes C11-BODIPY, Click-It LAA and LiperFluo, fluorescent probes MitoPerOx, MitoPeDPP and MitoCLox, and monoclonal antibodies specific to HMOX1, SAT1, MDA or 4 HNE. For example, see Chen et al., Front Cell Dev Biol. 2021:9:637162; Tang et al., Cell Res. 2021;31(2):107-125; the contents of each of these are incorporated herein by reference in their entirety.

[0214] Any ER stress assay may be used herein, including but not limited to electron microscopy (e.g., detecting ER lumen dilation), real-time redox measurements during ER stress (e.g., using eroGFP (ER-targeted redox-sensitive GFP)), and detection of misfolded and unfolded protein accumulation in the ER. ER stress can be detected using thioflavin T (ThT), a small molecule that exhibits enhanced fluorescence upon binding to protein aggregates. Enhanced ThT fluorescence directly correlates with proven indicators of UPR (endoplasmic reticulum stress response) activation. ER stress can also be detected indirectly by measuring the levels of specific UPR factors. Commonly measurable indicators of UPR activation include: phosphorylated PERK, phosphorylated eIF2α, Gadd153 / CHOP, ATF4, Grp78 / BiP, Grp94, calreticulin, and protein disulfide isomerase (PDI). These are typically detected by immunoblotting or immunohistochemistry. For example, see Oslowski (Barton) and Urano, Methods Enzymol. 2011;490:71-92, the entirety of which is incorporated herein by reference.

[0075]

[0215] EpiAge is measured by age-related changes in a set of DNA methylation sites (e.g., clock CpG or age-related CpG sites), and the methylation state of this set (e.g., weighted methylation mean) measures biological age. Different EpiAge models use different sets of methylation sites and different weighting algorithms. Horvath's clock is the most common model, but any EpiAge model such as Levins' clock, Hanham's clock, or AltumAge may be used herein (see, for example, Lucas Paulo de Lima Carnillo et al. 2022;8(1):4; Horvath and Raj, Nat Rev Genet. 2018;19(6):371-384; the contents of each of these are incorporated herein by reference in their entirety). Methods for detecting and / or measuring DNA methylation are well known, and should be seen in relation to methods described in methylation microarrays, Reduced Representation Bisulfite Sequencing (RRBS), Tagmentation-based Indexing for Methylation Sequencing (TIME-Seq; Griffin et al., bioRxiv. 2021), bisulfite barcoded amplicon sequencing (BBA-seq), pyrosequencing, droplet digital PCR (ddPCR), etc. Han et al., BMC Biol. 2020;18:71; Kurdyukov and Bullock, Biology (Basel). 2016;5(1):3. The contents of each of these are incorporated herein by reference in their entirety.

[0076]

[0216] In some embodiments, morphological features and / or histology of skin cells and / or skin tissues are used to evaluate the therapeutic or preventive effects of the skin rejuvenation method (or treatment or preventive method) or LLPS-deficient recombinant reprogramming factor described herein. Such methods include, but are not limited to, tissue biopsy, immunohistochemical staining, immunofluorescence, light microscopy, and transmission electron microscopy. For example, fibroblasts are elongated spindle-shaped or star-shaped with numerous cytoplasmic processes. Skin fibroblasts have a very different phenotype compared to iPSCs (see Figure 57). See Agarwal and Krishnamurthy, "Histology, Skin," StatPearls. May 2023; Dick et al., "Histology, Fibroblasts," StatPearls. May 2023; and Moroki, J Toxicol Pathol. 2023 Apr.; 36(2):85-94.

[0077]

[0217] Depending on the therapeutic or prophylactic use, the effective dose of the LLPS-deficient recombinant reprogramming factor (or its composition) described herein is the amount that, upon administration to a population of skin cells, skin tissue, or an individual, achieves one or more of the therapeutic or prophylactic effects, biomarkers, and / or morphological features described above. For example, the effective dose can prevent or reduce one or more signs of skin aging or damage, such as inflammation, epidermal hyperplasia, dermal elastic fibrosis and matrix proteolysis, perivenule lymphohistiocytic dermal infiltration, and the formation of wrinkles (e.g., fine wrinkles). The effective dose administered to skin tissue or an individual is usually based on a variety of factors, including age, sex, surface area, body weight, and skin condition. Body surface area can be roughly determined from an individual's height and weight. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardley, NY, 1970, 537. The effective dose varies depending on the route of administration, the use of excipients, and the possibility of concomitant use with other treatments (e.g., the use of other wrinkle or scar reducing agents, wound healing agents, or cosmetic or dermatological procedures), as will be recognized by those skilled in the art. Experimental animals may be used in a method for determining the effective dose of LLPS-deficient recombinant reprogramming factor (or a composition thereof).

[0078] Recombinant reprogramming factors without LLPS

[0218] In one aspect, this application provides a recombinant reprogramming factor that lacks LLPS (also referred to herein as a "recombinant reprogramming factor" or "LLPS-deficient recombinant reprogramming factor"). Any of the recombinant reprogramming factors provided herein can be used in the rejuvenation methods (or therapeutic or preventive methods) described herein.

[0219] Any recombinant reprogramming factor that lacks LLPS (for example, reducing LLPS by at least about 50%, such as reducing LLPS by at least about 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100%) may be used herein. In some embodiments, LLPS deficiency includes condensate formation deficiency, such as deficiency of any condensate formation. In some embodiments, LLPS deficiency includes deficiency in forming condensates containing the same condensate components as those formed by the corresponding wild-type reprogramming factor. For example, wild-type OCT4 may form condensates with both wild-type SOX2 and wild-type KLF4, while LLPS-deficient OCT4 may form condensates with wild-type SOX2 only, but wild-type KLF4 is not present in the condensates. In some embodiments, the LLPS-deficient recombinant reprogramming factor does not reprogram a population of skin cells into reprogrammed stem cells (or iPSCs), such as by retaining the population in initiation phase reprogramming (IPR).

[0079]

[0220] Any method known in the art capable of detecting LLPS or condensate formation can be used herein to determine whether a recombinant reprogramming factor is LLPS deficient or whether a mutation in a reprogramming factor causes LLPS deficiency. For example, for imaging techniques such as microscopy (e.g., fluorescence microscopy), see Basturea (Barton, GN) ("Biological Condensates", MATER METHODS 2019;9:2794) and Alberti et al. (J Mol Biol. 2018;430(23):4806-4820). Condensate formation of LLPS or recombinant reprogramming factors can be detected in vitro or in cells. For example, recombinant reprogramming factors may include detectable properties such as fluorescence properties. In some embodiments, recombinant reprogramming factors include labels such as fluorescent labels. In some embodiments, the recombinant reprogramming factor is further labeled with a detection tag such as fluorescein, a fluorescent polypeptide (e.g., GFP), or a radioisotope label. In some embodiments, the label or tag does not affect the action and / or activity of the recombinant reprogramming factor in association / dissociation with one or more biomolecules (e.g., DNA, other condensed protein components, or other proteins that form condensates) or condensates. In some embodiments, the recombinant reprogramming factor or condensates containing it are detected using an antibody that specifically recognizes the recombinant reprogramming factor or a label / tag of the recombinant reprogramming factor. The antibody may be fluorescently labeled or can be detected using a fluorescently labeled secondary antibody. In some embodiments, deficiency of the recombinant reprogramming factor in LLPS with other condensed components (e.g., DNA or other proteins) is investigated by detecting whether colocalization of the recombinant reprogramming factor with other condensed components exists, for example, by using fluorescence imaging techniques. The dynamic properties of the LLPS or condensates can be investigated via FRAP.

[0080]

[0221] In some embodiments, the imaging technique includes one or more of the following: immunofluorescence (IF), in situ hybridization (ISH, e.g., FISH), gene fusion (e.g., GFP labeling), or recombinant reprogramming factor-specific dyes. In some embodiments, the imaging technique includes the use of agents to visualize cellular features such as cell membranes or organelles such as the nucleus (e.g., by DAPI staining). In some embodiments, the imaging technique further includes measuring (e.g., quantifying) the signal of a detectable reagent. The microscopy technique may be one or more of the following: confocal microscopy, fluorescence microscopy, high-resolution microscopy, stimulated emission suppression (STED) microscopy, SoRa super-resolution spinning disk microscopy, electron microscopy (such as cryo-EM or cryo-ET), and / or total internal reflection fluorescence (TIRF) microscopy.

[0081]

[0222] In one embodiment, a recombinant reprogramming factor is provided that contains a mutation (e.g., one or more mutations such as insertions, deletions, and / or substitutions) in an LLPS-related domain relative to the corresponding wild-type reprogramming factor. The mutation may be located in any LLPS-related domain, for example, a domain selected from the group consisting of an endogenously impaired region (IDR), an activation domain (AD), a region outside the DNA-binding domain (DBD), a ubiquitination site, or any combination thereof. Mutations in other domains are also considered in this application insofar as such mutations can reduce or eliminate the LLPS of the reprogramming factor (e.g., by reducing at least about 50% of LLPS, such as reducing any of the following LLPS: at least about 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more). Mutations include (i) substituting a charged amino acid in the IDR or AD (e.g., acidic amino acids such as D or E, or basic amino acids such as K, R, or H) with a neutral amino acid (e.g., Gly, Ala, Leu, Ile, Val, Phe, Pro, Met, Trp, Tyr, Ser, Thr, Cys, Gln, or Asn, e.g., A); (ii) substituting a ubiquitizable amino acid (e.g., K in S, T, or K) with an unubiquitizable amino acid (e.g., R); (iii) deleting a ubiquitizable amino acid (e.g., K in S, T, or K); (iv) deleting an amino acid in the IDR or AD (e.g., an acidic amino acid (e.g., D or E) or a basic amino acid (e.g., K, R, or H), or a charged amino acid); and / or (v) This may include one or more deletions of amino acids in regions outside the DBD region (e.g., acidic amino acids (e.g., D or E) or basic amino acids (e.g., K, R, or H) that are charged). In some embodiments, the amino acid to be deleted includes an acidic amino acid (e.g., D or E). In some embodiments, substituting a charged amino acid in the IDR or AD with a neutral amino acid includes substituting an acidic amino acid (e.g., D or E) in the IDR or AD with a neutral amino acid (e.g., A). In some embodiments, the amino acid that can be ubiquitinated is K.In one embodiment, the recombinant reprogramming factor does not contain mutations in the IDR. In another embodiment, the recombinant reprogramming factor does not contain mutations in the DBD.

[0082]

[0223] In some embodiments, mutations causing LLPS deficiency in recombinant reprogramming factor include partial or complete deletions, such as partial or complete deletions of LLPS-related domains. In some embodiments, the mutation is a partial or complete deletion of IDR and / or AD, e.g., a partial deletion of a charged amino acid in IDR. In some embodiments, the mutation is a terminal deletion, such as a partial or complete deletion of the N-terminal domain and / or C-terminal domain of the wild-type reprogramming factor. In some embodiments, the N-terminal domain and / or C-terminal domain of the wild-type reprogramming factor includes IDR and / or AD. In some embodiments, the mutation is a partial or complete deletion of the central portion of the wild-type reprogramming factor. In some embodiments, the central portion of the wild-type reprogramming factor includes IDR and / or AD. In some embodiments, the mutation is a partial or complete deletion of a regulatory region of the wild-type reprogramming factor, e.g., a partial or complete deletion of lysine that can undergo ubiquitination modification or a partial or complete deletion of another amino acid that contributes to LLPS.

[0083]

[0224] In some embodiments, the recombinant reprogramming factor contains (or further contains) mutations (e.g., insertions, deletions, and / or substitutions) in the DBD. In some embodiments, the mutations in the DBD do not affect the DNA binding activity (e.g., affinity and / or specificity) of the recombinant reprogramming factor, or affect it by up to about 10% (e.g., up to about 9%, 8%, 7%, 6%, 5%, 2%, 1%, or less).

[0225] In some embodiments, the DBD is located in the center of the wild-type reprogramming factor, and therefore, the region outside the DBD region may be at the N-terminus and / or C-terminus of the wild-type reprogramming factor. In some embodiments, the DBD is located at the N-terminus of the wild-type reprogramming factor, and therefore, the region outside the DBD region may be at the C-terminus of the DBD or at the C-terminus of the wild-type reprogramming factor. In some embodiments, the DBD is located at the C-terminus of the wild-type reprogramming factor, and therefore, the region outside the DBD region may be at the N-terminus of the DBD or at the N-terminus of the wild-type reprogramming factor. In some embodiments, the DBD is located at both the N-terminus and C-terminus of the wild-type reprogramming factor, and therefore, the region outside the DBD region may be at the center of the wild-type reprogramming factor, the C-terminal side of the N-terminal DBD, and the N-terminal side of the C-terminal DBD.

[0084]

[0226] DBDs can be identified by many methods known in the field. Experimental approaches include, but are not limited to, electrophoretic mobility shift assays (EMSA), DNase I footprinting, exonuclease III footprinting, southwestern blotting, DNA pull-down assays, reporter assays, microplate capture and detection assays, X-ray crystallography, and nuclear magnetic resonance spectroscopy (NMR). Computer or database methods such as DP-Bind, DeepDISE, DNAbinder, DNABIND, DRNApred, GraphSite, DBS-pred, DBS-PSSM, BindN, DNABindR, DISIS, BindN-RF, DBindR, DBD-Threader, ProteDNA, BindN+, NAPS, MetaDBSite, DNABR, TargetS, SNBRFinder, DisoRDPbind, DQPred-DBR, TargetDNA, PRODNA, DRNApred, PDRLGB, ENSEMBLE-CNN, NucBind, hybridNAP, DNAPred, ProNA2020, NCBRPred, MTDsite, DNAgenie, DeepDISOBind, HybridDBRpred, PDNAsite, RANSFAC, JASPA, PROSITE, Pfam, and SMART can also be used.

[0085]

[0227] The activating domain (AD) or transactivating domain (TAD) is a transcription factor scaffold domain that contains binding sites for other proteins, such as transcription coregulators. Typically, TADs belong to four classes: acidic domains, glutamine-rich domains, proline-rich domains, and isoleucine-rich domains. ADs can be identified by many methods known in the field. Experimental approaches include, but are not limited to, reporter assays, co-IP, and tAD seq (Arnold et al., EMBO J. 2018;37(16):e98896). Computational or database methods such as ADpred, PADDY, and the 9 aAtAD Prediction Tool can also be used.

[0086]

[0228] Protein regions lacking a stable three-dimensional structure are called IDRs (Intra-Defined Regions). IDRs can be inferred from sequence characteristics (see, for example, Lu et al., PLoS Comput Biol. 2022; Vol. 18 (No. 6): e1010238; Trivedi and Nagarajaram, Int J Mol Sci. 2022; Vol. 23 (No. 22): p. 14050; R. van der Lee et al., Chem Rev. 2014; Vol. 114 (No. 13): pp. 6589-6631; the contents of each of these references are incorporated herein by reference in their entirety). IDRs are usually rich in charged polar amino acids and depleted of bulky hydrophobic residues, resulting in weakened hydrophobicity, which is usually the main driving force for polypeptide folding into a compact tertiary structure (the naturally folded state). Methods for identifying IDRs are well known and can all be used herein. The computational methods for identifying IDRs can be classified into three categories: (1) physicochemistry-based methods such as FoldIndex, GlobPlot, IUPred, FoldUnfold, and IsUnstruct, which depend on the physiological and chemical properties of amino acids to identify disorders; (2) machine learning-based methods such as DISvgg, RFPR-IDP, IDP-Seq2Seq, SPOT-Disorder, SPOT-Disorder2, DISOPRED3, SPINE-D, ESpritz, BVDEA, POODLE-S, RONN, and PONDRs, which treat IDR identification as labeling each amino acid in the protein sequence or as a classification problem; and (3) meta-methods, including MFDp, MetArPDOS, and meta-disorder predictors, which fuse multiple predictors to obtain a final prediction of IDRs. Other computational techniques, such as VSL2, VSL2B, IUPred2, ANCHOR2, DisEMBL, DISOPRED, and IUPred, can also be used. Experimental methods used to detect IDR include X-ray crystallography, NMR, small-angle X-ray scattering, circular dichroism, and Förster resonance energy transfer.

[0087]

[0229] Negatively charged IDRs in reprogramming factors can contribute to LLPS. IDR regions within reprogramming factors can be predicted by algorithms that provide disability scores. Regions with disability scores greater than a certain value have a high probability of being IDRs. Using human OCT4 as an example, VSL2 was used as the algorithm for predicting IDRs. Contiguous regions with disability scores greater than a certain value have a high probability of being IDRs—this value is not particularly limited but may be in the range of 0.4 to 0.6, preferably 0.5. Exemplary prediction results for OCT4 showed that the IDRs of OCT4 are distributed at both ends of the protein, more specifically in the activation domain (AD) at amino acid residues 1-140 and 288-360, while the intermediate DBD does not belong to the IDR. Alternative scoring criteria such as IUPred2 and ANCHOR2 may also be used. All scoring algorithms and criteria may be used herein as long as they are usable for predicting IDRs. For example, as we predicted, the IDR predictions obtained by various scoring criteria were the same (within the margin of error).

[0088]

[0230] Methods for identifying ubiquitination sites are well-known in this field. Biochemical approaches such as immunoblotting using anti-Ub antibodies can be used. If immunoblotting results suggest ubiquitination on the substrate, the presumed ubiquitinated lysine can be further mutated and analyzed by immunoblotting to evaluate whether the mutant lysine is ubiquitinated. Ubiquitination sites can also be identified by MS through the detection of ubiquitin-derived peptide adducts. Other methods for profiling ubiquitination sites include, but are not limited to, approaches based on Ub tagging, approaches based on anti-diGly antibodies, approaches based on UbiSite antibodies, and antibody-free approaches (e.g., Ub-conjugated diagonal chromatography (COFRADIC), StUbEx+). Computational methods, such as ESA-UbiSite and HUbipPred, can also be used. See also, for example, M. Pourmirzaei (M. Horton) et al., BMC Bioinformatics. 2023 Nov;24(1):449; Sun and Zhang, Cell Biosci. 2022;12:126; the contents of each of these are incorporated herein by reference in their entirety.

[0089]

[0231] When amino acids are modified by polyubiquitin, the polyubiquitin chain can mediate LLPS. Substituting a ubiquitizable amino acid (e.g., K) with a non-ubiquitizable amino acid (e.g., R) prevents polyubiquitin modification and thus avoids LLPS. Any other mutations that can reduce or eliminate ubiquitination of reprogramming factors may also be used herein.

[0090]

[0232] Any transcription factor capable of reprogramming cells (partially or completely) may be used herein as a wild-type or reference reprogramming factor. The wild-type reprogramming factor may be derived from a family selected from the group consisting of the OCT family, SOX family, KLF family, MYC family, LIN28 family, NANOG family, and GLIS family. In one embodiment, the wild-type reprogramming factor is selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG, and GLIS1. Other exemplary reprogramming factors that may be used herein or used as reference reprogramming factors include DPPA2, DPPA4, ESRRB, GDF3, KLF2, KLF5, LMYC, NMYC, NR5A1, NR5A2, RCOR2, SALL1, SALL4, SOX1, SOX3, TDRD12, TET1, TH2A, TH2B, UTF1, ZFP42, MDM2, cyclin D1, SV40 large T antigen, SIRT6, TCL1A, and RARγ.

[0091]

[0233] Recombinant reprogramming factors may originate from any source, including but not limited to humans, cattle, horses, sheep, cats, dogs, rodents (e.g., rats, mice, rabbits), birds (e.g., chickens), and non-human primates (e.g., monkeys). In some embodiments, the recombinant reprogramming factor is a human recombinant reprogramming factor. In some embodiments, the recombinant reprogramming factor is a mouse recombinant reprogramming factor.

[0092] OCT

[0234] The Oct family is a family of octameric ("Oct") transcription factors that play a crucial role in maintaining pluripotency. As used herein, Oct reprogramming factors refer to functional variants of any naturally occurring member of the Oct family or the corresponding wild-type Oct reprogramming factor that maintain at least about 50% (e.g., at least about 60%, 70%, 80%, 90%, 95%, or 100%) of the reprogramming activity. In some embodiments, an Oct reprogramming factor variant may include at least the DBD of the corresponding wild-type Oct reprogramming factor, and further include the AD. Exemplary Oct reprogramming factors include Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11.

[0093]

[0235] POU5F1 (POU domain, class 5, transcription factor 1), also known as Oct3 / 4, is a representative of the Oct family. The absence of Oct3 / 4 in Oct-3 / 4+ cells, such as blastomeres and embryonic stem cells, results in spontaneous trophoblast differentiation, and therefore, the presence of Oct3 / 4 gives rise to pluripotency and differentiation ability in embryonic stem cells. Exemplary Oct3 / 4 proteins are those encoded by the mouse Oct3 / 4 gene (Genbank accession number NM_013633) and the human Oct3 / 4 gene (Genbank accession number NM_002701).

[0236] The terms "Oct3 / 4," "OCT4," "OCT4," "OCT4 protein," and "OCT4 protein" refer to any naturally occurring form of Octomer 4 transcription factor or its variant that maintains OCT4 transcription factor activity (e.g., activity of at least about 50%, 80%, 90%, or 100% compared to wild-type OCT4, as measured by methods known in the art). In some embodiments, the Oct4 variant has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) of amino acid sequence identity across the entire sequence compared to the naturally occurring Oct4 polypeptide. In some embodiments, the Oct4 protein is the protein identified by Genbank reference number ADW77327.1.

[0094]

[0237] In one embodiment, the LLPS-deficient recombinant reprogramming factor is recombinant OCT4, such as recombinant human OCT4 or recombinant mouse OCT4.

[0238] In one embodiment, recombinant OCT4 is provided, with reference to SEQ ID NO: 2, containing mutations (e.g., one or more mutations such as deletions, insertions, and / or substitutions) in the IDR(IDR). The IDR of OCT4 can be identified using any method known in the art or described herein, such as VSL2. The IDR of wild-type OCT4 may occupy about 38% to about 40% of the entire sequence of SEQ ID NO: 2 at the N-terminus and / or about 19% to about 24% of the entire sequence of SEQ ID NO: 2 at the C-terminus. In one embodiment, the IDR of OCT4 is located at amino acid positions 1-140 and 288-360 (with an error range of about 10 to about 20 amino acids), with reference to SEQ ID NO: 2. In one embodiment, the DBD of OCT4 is located at amino acid positions 141-287 (with an error range of about 10 to about 20 amino acids), with reference to SEQ ID NO: 2. In one embodiment, recombinant OCT4 is provided, which includes mutations (e.g., deletions, insertions, and / or substitutions) at amino acid positions 1-140 and / or 288-360, where the amino acid positions refer to SEQ ID NO: 2. In another embodiment, human wild-type OCT4 is GFP-tagged and encoded by the nucleic acid sequence, for example, SEQ ID NO: 109.

[0095]

[0239] In one embodiment, recombinant OCT4 is provided with respect to SEQ ID NO: 2, wherein the substitution includes substituting a polar amino acid (charged or uncharged, e.g., D, E, R, K, H, S, T, N, Q, C, Y) with a neutral amino acid (e.g., Gly, Ala, Leu, Ile, Val, Phe, Pro, Met, Trp, Tyr, Ser, Thr, Cys, Gln, or Asn, e.g., A). In one embodiment, recombinant OCT4 is provided with respect to SEQ ID NO: 2, wherein the substitution includes substituting a charged amino acid (e.g., an acidic amino acid such as D or E, or a basic amino acid such as R, K, or H) with a neutral amino acid (e.g., A). In one embodiment, the substitution includes substituting an acidic amino acid (e.g., D or E) with a neutral amino acid (e.g., A) with respect to SEQ ID NO: 2. In one embodiment, the substitution includes substituting an acidic amino acid (e.g., D or E) in the IDR with a neutral amino acid (e.g., A) with reference to SEQ ID NO: 2. In one embodiment, recombinant OCT4 includes substitutions (e.g., substitutions to neutral amino acids such as A) at amino acid positions selected from the group consisting of 8, 20, 26, 31, 56, 68, 91, 96, 98, 104, 108, 113, 125, 127, 130, 134, 135, 138, 145, 147, 166, 188, 209, 210, 215, 224, 238, 246, 270, 272, 291, 296, 297, 299, 341, and 343, where the amino acid positions refer to SEQ ID NO: 2. In one embodiment, recombinant OCT4 includes substitutions (e.g., substitutions to neutral amino acids such as A) at amino acid positions selected from the group consisting of 8, 20, 26, 31, 56, 68, 91, 96, 98, 104, 108, 113, 125, 127, 130, 134, 135, 138, 291, 296, 297, 299, 341, and 343, where the amino acid positions refer to SEQ ID NO: 2. In one embodiment, recombinant OCT4 includes substitutions (e.g., substitutions to neutral amino acids such as A) at amino acid positions selected from the group including D8, D20, E26, D31, E56, E68, E91, E96, E98, E104, D108, E113, E125, E127, E130, E134, E135, D138, D291, E296, D297, E299, E341, and E343, where the amino acid positions refer to SEQ ID NO: 2.In some embodiments, the substitution includes substituting D or E with A. In some embodiments, recombinant OCT4 includes substitutions selected from the group consisting of D8A, D20A, D31A, D108A, E134A, D138A, D291A, or any combination thereof, where the amino acid position refers to SEQ ID NO: 2. In some embodiments, recombinant OCT4 is provided that includes substitutions referring to SEQ ID NO: 2, where the substitutions are (i) D8A, D20A, E26A, D31A, E56A, E68A, E91A, E96A, E98A, E104A, D108A, E113A, E125A, E127A, E130A, E134A, E135A, D138A, D291A, E296A, D297A, E299A, E341A, and E343A; (ii) D8A, D20A, E26A, D31A, E56A, E68A, E91A, E96A, E98A, E104A, D108A, E113A, E125A, E127A, E130A, E134A, E135A, and D138A; (iii) D291A, E296A, D297A, E299A, E341A, and E343A; (iv) D8A, D20A, E26A, D31A, E56A, E68A, E91A, E96A, E98A, E104A, D108A, E113A, E125A, E127A, E130A, E134A, D138A, D291A, E296A, E299A, E341A, and E343A; (v) E134A, E135A, E296A, and D297A; (vi) D8A, D20A, D31A, D108A, D138A, D291A, and D297A; (vii) Selected from the group including E26A, E56A, E68A, E91A, E96A, E98A, E104A, E113A, E125A, E127A, E130A, E134A, E135A, E296A, E299A, E341A, and E343A. In some embodiments, recombinant OCT4 includes any amino acid sequence of SEQ ID NOs. 4-10 or a variant thereof that has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) sequence identity with any of SEQ ID NOs. 4-10. In some embodiments, recombinant OCT4 is encoded by the nucleic acid sequence of SEQ ID NO. 3.

[0096]

[0240] In one embodiment, recombinant OCT4 includes deletions at amino acid positions 2-140 and / or 288-360, where the amino acid positions refer to SEQ ID NO: 2. In another embodiment, recombinant OCT4 includes deletions selected from the group consisting of (i) deletions at amino acid positions 2-140 and 301-360; (ii) deletions at amino acid positions 301-360; and (iii) deletions at amino acid positions 2-140. In yet another embodiment, recombinant OCT4 includes a variant thereof that has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) sequence identity with any of the amino acid sequences of SEQ ID NOs: 44-46 or any of SEQ ID NOs: 44-46. In yet another embodiment, recombinant OCT4 is GFP-tagged and encoded by, for example, the nucleic acid sequence of SEQ ID NO: 110.

[0097]

[0241] In one embodiment, mouse wild-type OCT4 (MsOCT4) is encoded by the amino acids of SEQ ID NO: 66 or by the nucleic acid sequence of SEQ ID NO: 67. In another embodiment, recombinant mouse OCT4 (MsOCT4mut) is provided, which has mutations (e.g., one or more mutations such as deletions, insertions, and / or substitutions) in IDRs (may be multiple) with respect to SEQ ID NO: 66. The IDRs of MsOCT4 can be identified by any method known in the art or described herein, such as VSL2. In one embodiment, the IDRs of MsOCT4 are located at positions 1-140 and 280-352 with respect to SEQ ID NO: 66 (with an error range of about 10-20 amino acids). In another embodiment, the DBDs of MsOCT4 are located at amino acids 141-279 with respect to SEQ ID NO: 66 (with an error range of about 10-20 amino acids). In one embodiment, recombinant MsOCT4 is provided that includes mutations (e.g., deletions, insertions, and / or substitutions) at positions 1 to 140 and / or 280 to 352 relative to sequence number 66.

[0098]

[0242] In one embodiment, recombinant MsOCT4 is provided with respect to SEQ ID NO: 66, wherein the substitution includes substituting a polar amino acid (charged or uncharged, e.g., D, E, R, K, H, S, T, N, Q, C, Y) with a neutral amino acid (e.g., Gly, Ala, Leu, Ile, Val, Phe, Pro, Met, Trp, Tyr, Ser, Thr, Cys, Gln, or Asn, e.g., A). In one embodiment, recombinant MsOCT4 is provided with respect to SEQ ID NO: 66, wherein the substitution includes substituting a charged amino acid (e.g., an acidic amino acid such as D or E, or a basic amino acid such as R, K, or H) with a neutral amino acid (e.g., A). In one embodiment, the substitution includes substituting an acidic amino acid (e.g., D or E) with a neutral amino acid (e.g., A) with respect to SEQ ID NO: 66. In one embodiment, the substitution includes substituting an acidic amino acid (e.g., D or E) in the IDR with respect to SEQ ID NO: 66 with a neutral amino acid (e.g., A). In one embodiment, recombinant MsOCT4 is provided, comprising substitutions (e.g., substitutions to neutral amino acids such as A) at amino acid positions selected from the group consisting of D8, D19, E25, D30, E51, E63, E86, E91, E99, E103, E108, D112, E120, E123, E127, E128, D131, E284, E289, E290, E292, E333, and E335, where the amino acid positions are based on SEQ ID NO: 66. In one embodiment, recombinant MsOCT4 comprises the amino acids of SEQ ID NO: 68 or a variant thereof having at least about 85% (e.g., at least about 90%, 95%, 99%, or more) sequence identity to SEQ ID NO: 68. In one embodiment, recombinant MsOCT4 is encoded by the nucleotide sequence of SEQ ID NO: 69.

[0099]

[0243] In one embodiment, recombinant MsOCT4 is provided that contains a deletion (partial or complete) of IDR(or IDR) in relation to SEQ ID NO: 66. In one embodiment, recombinant MsOCT4 contains a deletion at positions 2–140 and / or 280–352 relative to SEQ ID NO: 66. In one embodiment, recombinant MsOCT4 contains a deletion at amino acids 324–326 relative to SEQ ID NO: 66. In one embodiment, recombinant MsOCT4 contains amino acids of SEQ ID NO: 70 or a variant thereof that has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) sequence identity with respect to SEQ ID NO: 70. In one embodiment, recombinant MsOCT4 is encoded by the nucleotide sequence of SEQ ID NO: 71.

[0100] MYC

[0244] Factors of the Myc family are factors encoded by the myc proto-oncogene involved in cancer. c-Myc has been shown to be a factor involved in the generation of mouse and human iPSCs. Exemplary c-Myc proteins are proteins encoded by the mouse c-Myc gene (Genbank accession number NM_010849) and the human c-Myc gene (Genbank accession number NM_002467). N-Myc or L-myc have also been used as possible reprogramming factors to replace c-Myc. N-Myc or L-myc can also be used as possible reprogramming factors to replace c-Myc. Myc reprogramming factors as used herein refer to any naturally occurring member of the Myc family or a functional variant of the corresponding wild-type Myc reprogramming factor that maintains at least about 50% (e.g., at least about 60%, 70%, 80%, 90%, 95%, or 100%) of its reprogramming activity.

[0245] Terms used herein such as “c-Myc,” “c-Myc protein,” and “c-Myc protein” include any of the naturally occurring forms of the cMyc transcription factor or recombinants thereof that maintain cMyc transcription factor activity (e.g., within at least about 50%, 80%, 90%, or 100% of the activity compared to wild-type cMyc, as measured by methods known in the art). In some embodiments, a c-Myc variant has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) of amino acid sequence identity across the entire sequence compared to a naturally occurring c-Myc polypeptide. In some embodiments, the c-Myc protein is a protein identified by NCBI reference number NP_002458.2.

[0101]

[0246] In one embodiment, the LLPS-deficient recombinant reprogramming factor is recombinant c-MYC such as recombinant human c-MYC or recombinant mouse c-MYC.

[0247] In some embodiments, human wild-type c-MYC comprises the amino acid sequence of SEQ ID NO: 12 or is encoded by the nucleic acid sequence of SEQ ID NO: 11 or 53. In some embodiments, human wild-type c-MYC comprises the amino acid sequence of SEQ ID NO: 85 or is encoded by the nucleic acid sequence of SEQ ID NO: 86. In some embodiments, recombinant c-MYC is provided that comprises a mutation (e.g., one or more mutations, e.g., deletion, insertion, and / or substitution) in the ubiquitination site (e.g., S, T, or K, e.g., K) with respect to SEQ ID NO: 12 or 85. The ubiquitination site of c-MYC can be identified by any method known in the art or described herein. In some embodiments, recombinant c-MYC is provided that comprises a mutation (e.g., one or more mutations such as deletion, insertion, and / or substitution) in the IDR with respect to SEQ ID NO: 12 or 85. The IDR of c-MYC can be identified by any method known in the art or described herein, such as VSL2. In one embodiment, the IDR of c-MYC is located at amino acid positions 1–360 (with an error margin of approximately 10–20 amino acids) with reference to SEQ ID NO: 12 or 85. In another embodiment, the DBD (bHLH-LZ domain) of c-MYC is located at amino acid positions 361–439 (with an error limit of approximately 10–20 amino acids) with reference to SEQ ID NO: 12 or 85. In yet another embodiment, recombinant c-MYC contains a deletion at amino acid positions 2–360, where the amino acid positions refer to SEQ ID NO: 12 or 85.

[0102]

[0248] In one embodiment, a recombinant c-MYC is provided, comprising the substitution of a ubiquitinated amino acid (e.g., S, T, or K, e.g., K) with a non-ubiquitinated amino acid (e.g., R), where the amino acid position refers to SEQ ID NO: 12 or 85. In one embodiment, the substitution comprises substituting K with R. In another embodiment, the recombinant c-MYC comprises (e.g., one or more) substitutions at an amino acid position selected from the group consisting of 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, where the amino acid position refers to SEQ ID NO: 12 or 85. In one embodiment, with respect to SEQ ID NO: 12 or 85, 20 to 25 amino acid positions are substituted from any of 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. In one embodiment, recombinant c-MYC includes substitutions selected from the group consisting of K51R, K52R, K148R, K275R, K289R, and K389R or any combination thereof, where the amino acid positions refer to SEQ ID NO: 12 or 85. In one embodiment, recombinant c-MYC is (i) K51R, K52R, K126R, K143R, K148R, K157R, K206R, K269R, K275R, K289R, K298R, K317R, K323R, K326R, K341R, K355R, K371R, K389R, K392R, K397R, K398R, K412R, K422R , K428R and K430R; (ii) K148R; (iii) K389R; (iv) K148R and K389R; (v) K51R and K52R; (vi) K275R and K289R; and (vii) substitutions selected from the group consisting of K51R, K52R, K275R and K289R; where the amino acid position is as shown in SEQ ID NO: 12 or 85.In one embodiment, recombinant c-MYC includes a variant thereof that has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) sequence identity to any of the amino acid sequences of SEQ ID NOs. 14-20 or any of the SEQ ID NOs. 13

[0249] In one embodiment, a recombinant c-MYC is provided that includes a (partial or complete) deletion of the IDR in reference to SEQ ID NO: 12 or 85. In one embodiment, the recombinant c-MYC includes a deletion at amino acid positions 2-360, where the amino acid positions refer to SEQ ID NO: 12 or 85. In one embodiment, the recombinant c-MYC includes a deletion at amino acid positions 2-140, referencing SEQ ID NO: 12 or 85. In one embodiment, the recombinant c-MYC includes the amino acid sequence of SEQ ID NO: 47 or 48 or a variant thereof that has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) sequence identity to SEQ ID NO: 47 or 48.

[0103]

[0250] In some embodiments, recombinant c-MYC includes substitutions in the IDR with reference to SEQ ID NO: 12 or 85, the substitutions including the substitution of a polar amino acid (charged or uncharged, e.g., D, E, R, K, H, S, T, N, Q, C, Y) with a neutral amino acid (e.g., Gly, Ala, Leu, Ile, Val, Phe, Pro, Met, Trp, Tyr, Ser, Thr, Cys, Gln, or Asn, e.g., A). In some embodiments, recombinant c-MYC includes substitutions in the IDR with reference to SEQ ID NO: 12 or 85, the substitutions including the substitution of a charged amino acid (e.g., an acidic amino acid such as D or E, or a basic amino acid such as R, K, or H) with a neutral amino acid (e.g., A). In some embodiments, the substitutions include the substitution of an acidic amino acid (e.g., D or E) in the IDR related to SEQ ID NO: 12 or 85 with a neutral amino acid (e.g., A). In some embodiments, the IDR related to SEQ ID NO: 12 or 85 is located at amino acid positions 2 to 360.

[0104]

[0251] In one embodiment, mouse wild-type c-MYC (Msc-MYC) contains the amino acid sequence of SEQ ID NO: 111. In another embodiment, mouse wild-type c-MYC (Msc-MYC) is encoded by the nucleic acid sequence of SEQ ID NO: 112. In another embodiment, recombinant mouse c-MYC (Msc-MYCmut) is provided, having a mutation (e.g., one or more mutations such as deletions, insertions, and / or substitutions) in the IDR with reference to SEQ ID NO: 111. The IDR of Msc-MYC can be identified using any method known in the art or described herein, such as VSL2. In another embodiment, the IDR of Msc-MYC is located at amino acid positions 1 to 360 (with an error limit of about 10 to about 20 amino acids) relative to SEQ ID NO: 111. In another embodiment, recombinant Msc-MYC is provided, having a mutation (e.g., deletions, insertions, and / or substitutions) at amino acid positions 2 to 360, where the amino acid positions are relative to SEQ ID NO: 111. In one embodiment, a recombinant Msc-MYC is provided that includes a mutation (e.g., one or more mutations, e.g., deletion, insertion, and / or substitution) in the ubiquitination site (e.g., S, T, or K, e.g., K) with respect to SEQ ID NO: 111. The ubiquitination site of Msc-MYC can be identified by any method known in the art or described herein. In one embodiment, the substitution includes substituting K with R.

[0105] KLF

[0252] The Klf family (or Kruppel-like family of transcription factors) refers to Klf genes that were initially identified as factors for the generation of mouse iPSCs and have also been demonstrated to be factors for the generation of human iPSCs. The exemplary klf4 protein is the protein encoded by the mouse klf4 gene (Genbank accession number NM_010637) and the human klf4 gene (Genbank accession number NM_004235). Exemplary Klf family members include Klf1, Klf2, Klf3, Klf4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16, and Klf17. As used herein, KLF reprogramming factors refer to any naturally occurring member of the Klf family or a functional variant of the corresponding wild-type Klf reprogramming factor that retains at least about 50% (e.g., at least about 60%, 70%, 80%, 90%, 95%, or 100%) of its reprogramming activity.

[0106]

[0253] Terms such as “KLF4” and “KLF4 protein” as used herein include any of the naturally occurring forms of the KLF4 transcription factor or its variants that maintain KLF4 transcription factor activity (e.g., within at least about 50%, 80%, 90%, or 100% of the activity compared to wild-type KLF4, as measured by methods known in the art). In some embodiments, the KLF4 variant has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) of amino acid sequence identity across the entire sequence compared to the naturally occurring KLF4 polypeptide. In other embodiments, the KLF4 protein is the protein identified by NCBI reference number NP_004226.3.

[0254] In one embodiment, the LLPS-deficient recombinant reprogramming factor is recombinant KLF4 such as recombinant human KLF4 or recombinant mouse KLF4.

[0107]

[0255] In some embodiments, human wild-type KLF4 is encoded by the amino acid sequence of SEQ ID NO: 22 or 87, or by the nucleic acid sequence of SEQ ID NO: 21, 54, or 88. In some embodiments, recombinant KLF4 is provided, comprising a mutation (e.g., one or more mutations such as deletions, insertions, and / or substitutions) in the IDR with respect to SEQ ID NO: 22. The IDR of KLF4 can be identified by any method known in the art or described herein, such as VSL2. In some embodiments, the IDR of KLF4 is located at amino acid positions 1 to 374 (with an error limit of about 10 to about 20 amino acids) with respect to SEQ ID NO: 22. In some embodiments, the DBD of KLF4 is located at amino acid positions 375 to 470 (with an error limit of about 10 to about 20 amino acids) with respect to SEQ ID NO: 22. In some embodiments, recombinant KLF4 is provided, comprising a mutation (e.g., deletion, insertion, and / or substitution) at amino acid positions 2 to 374, wherein the amino acid positions are relative to SEQ ID NO: 22. In one embodiment, recombinant KLF4 includes deletions at amino acid positions 2-374, with the amino acid positions relative to SEQ ID NO: 22. In another embodiment, recombinant KLF4 includes the amino acid sequence of SEQ ID NO: 40 or a variant thereof that has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) sequence identity to SEQ ID NO: 40.

[0108]

[0256] In one embodiment, recombinant KLF4 includes a substitution in the IDR with respect to SEQ ID NO: 22, the substitution comprising substituting a polar amino acid (charged or uncharged, e.g., D, E, R, K, H, S, T, N, Q, C, Y) with a neutral amino acid (e.g., Gly, Ala, Leu, Ile, Val, Phe, Pro, Met, Trp, Tyr, Ser, Thr, Cys, Gln, or Asn, e.g., A). In one embodiment, recombinant KLF4 includes a substitution in the IDR with respect to SEQ ID NO: 22, the substitution comprising substituting a charged amino acid (e.g., an acidic amino acid such as D or E, or a basic amino acid such as R, K, or H) with a neutral amino acid (e.g., A). In one embodiment, the substitution, with reference to SEQ ID NO: 22, comprises substituting an acidic amino acid (e.g., D or E) in the IDR with a neutral amino acid (e.g., A). In one embodiment, the IDR with respect to SEQ ID NO: 22 is located at amino acid positions 2 to 374.

[0109]

[0257] In one embodiment, mouse wild-type KLF4 (MsKLF4) is encoded by the amino acids of SEQ ID NO: 72 or by the nucleic acid sequence of SEQ ID NO: 73. In one embodiment, recombinant mouse KLF4 (MsKLF4mut) is provided, having a mutation (e.g., one or more mutations such as deletions, insertions, and / or substitutions) in the IDR with reference to SEQ ID NO: 73. The IDR of MsKLF4 can be identified by any method known in the art or described herein, such as VSL2. In one embodiment, the IDR of MsKLF4 is located at positions 1 to 374 (with an error range of about 10 to about 20) with respect to SEQ ID NO: 73. In one embodiment, recombinant MsKLF4 is provided, having a mutation (e.g., deletion, insertion, and / or substitution) at amino acid positions 2 to 374, such as a partial or complete deletion of amino acids at positions 2 to 374 with respect to SEQ ID NO: 73.

[0110] SOX

[0258] The Sox family refers to Sox genes that, like Oct-3 / 4, are associated with maintaining pluripotency, but in contrast to Oct-3 / 4, which is exclusively expressed in pluripotent stem cells, the Sox family is associated with both pluripotent and unipotent stem cells. SOX genes (SRY-related HMG box genes) encode a family of transcription factors that bind to the minor groove of DNA and belong to a superfamily of genes characterized by homologous sequences called HMG boxes (high mobility groups). These HMG boxes are DNA-binding domains that are highly conserved across eukaryotic species. Homologouss have been identified in insects, nematodes, amphibians, reptiles, birds, and a range of mammals. Sox proteins (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) can originate from humans, mice, rats, cattle, pigs, or other animals. As used herein, a SOX reprogramming factor refers to any naturally occurring member of the Sox family or a functional variant of the corresponding wild-type Sox reprogramming factor that retains at least about 50% (e.g., at least about 60%, 70%, 80%, 90%, 95%, or 100%) of its reprogramming activity.

[0259] Exemplary Sox2 proteins are those encoded by the mouse Sox2 gene (Genbank accession number NM_011443) and the human Sox2 gene (Genbank accession number NM_003106).

[0260] Accordingly, terms such as “SOX2,” “SOX2,” “SOX2 protein,” and “SOX2 protein” as used herein include any of the naturally occurring forms of the SOX2 transcription factor or its variants that maintain SOX2 transcription factor activity (e.g., within at least about 50%, 80%, 90%, or 100% of the activity compared to wild-type SOX2, as measured by methods known in the art). In some embodiments, the SOX2 variant has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) of amino acid sequence identity across the entire sequence compared to the naturally occurring SOX2 polypeptide. In some embodiments, the Sox2 protein is the protein identified by NCBI reference number NP_003097.1.

[0111]

[0261] In one embodiment, the LLPS-deficient recombinant reprogramming factor is recombinant SOX2 such as recombinant human SOX2 or recombinant mouse SOX2.

[0262] In one embodiment, human wild-type SOX2 is encoded by the amino acid sequence of SEQ ID NO: 24 or by the nucleic acid sequence of SEQ ID NO: 23. In one embodiment, recombinant SOX2 is provided, comprising mutations (e.g., one or more mutations such as deletions, insertions, and / or substitutions) in IDRs (may be multiple) with respect to SEQ ID NO: 24. The IDRs of SOX2 can be identified by any method known in the art or described herein, such as VSL2. In one embodiment, the IDRs of SOX2 are located at amino acid positions 1-40 and 201-317 (with an error limit of about 10-20 amino acids) with respect to SEQ ID NO: 24. In one embodiment, the DBDs of SOX2 are located at amino acid positions 41-200 (with an error limit of about 10-20 amino acids) with respect to SEQ ID NO: 24. In one embodiment, recombinant SOX2 is provided, comprising mutations (e.g., deletions, insertions, and / or substitutions) at amino acid positions 2-40 and / or 201-317, with respect to SEQ ID NO: 24. In one embodiment, recombinant SOX2 is provided that includes (i) a deletion at amino acid positions 2-40, (ii) a deletion at amino acid positions 201-317, or (iii) a deletion at positions 2-40 and 201-317, with the amino acid positions relative to SEQ ID NO: 24. In another embodiment, recombinant SOX2 includes a variant thereof that has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) sequence identity with any of the amino acid sequences of SEQ ID NOs: 41-43 or any of SEQ ID NOs: 41-43.

[0112]

[0263] In some embodiments, recombinant SOX2 includes substitutions in the IDR(s) with respect to SEQ ID NO: 24, the substitutions include substituting a polar amino acid (charged or uncharged, e.g., D, E, R, K, H, S, T, N, Q, C, Y) with a neutral amino acid (e.g., Gly, Ala, Leu, Ile, Val, Phe, Pro, Met, Trp, Tyr, Ser, Thr, Cys, Gln, or Asn, e.g., A). In some embodiments, recombinant SOX2 includes substitutions in the IDR(s) with respect to SEQ ID NO: 24, the substitutions include substituting a charged amino acid (e.g., an acidic amino acid such as D or E, or a basic amino acid such as R, K, or H) with a neutral amino acid (e.g., A). In some embodiments, the substitutions include substituting an acidic amino acid (e.g., D or E) in the IDR(s) with respect to SEQ ID NO: 24 with a neutral amino acid (e.g., A). In one embodiment, the IDR for SEQ ID NO: 24 is located at amino acid positions 1-40 and 201-317.

[0113]

[0264] In one embodiment, mouse wild-type SOX2 (MsSOX2) is encoded by the amino acids of SEQ ID NO: 74 or by the nucleic acid sequence of SEQ ID NO: 75. In one embodiment, recombinant mouse SOX2 (MsSOX2mut) is provided, which contains mutations (e.g., one or more mutations such as deletions, insertions, and / or substitutions) in IDRs (may be multiple IDRs) with respect to SEQ ID NO: 74. The IDRs of MsSOX2 can be identified using any method known in the art or described herein, such as VSL2. In one embodiment, the IDRs of MsSOX2 are located at positions 1-40 and 203-319 (with an error range of about 10-20 amino acids) with respect to SEQ ID NO: 74. In one embodiment, the DBDs of MsSOX2 are located at amino acid residues 41-202 (with an error range of about 10-20 amino acid residues) with respect to SEQ ID NO: 74. In one embodiment, recombinant MsSOX2 is provided that includes mutations (e.g., deletions, insertions, and / or substitutions) at amino acid positions 2-40 and / or 203-319 relative to SEQ ID NO: 74, such as partial or complete deletions of amino acids at positions 2-40 and / or 203-319.

[0114] NANOG

[0265] Nanog is a transcription factor critically involved in the self-renewal of undifferentiated embryonic stem cells. It helps embryonic stem cells (ESCs) maintain pluripotency by repressing cell-determining factors. In humans, this protein is encoded by the NANOG gene. Exemplary nanog proteins are those encoded by the mouse gene (Genbank accession number XM_132755) and the human Nanog gene (Genbank accession number NM_024865). The human hNanog protein encoded by the NANOG gene consists of 305 amino acids and has three functional domains: the N-terminal domain, the C-terminal domain, and a conserved homeodomain motif (for DNA binding). The homeodomain of hNANOG is in the range of residues 95-155. Further NANOG genes (NANOG2, NANOG p8) also exist. As used herein, Nanog reprogramming factors refer to any naturally occurring member of the Nanog family or a functional variant of the corresponding wild-type Nanog reprogramming factor that retains at least about 50% (e.g., at least about 60%, 70%, 80%, 90%, 95%, or 100%) of its reprogramming activity.

[0115]

[0266] Accordingly, terms such as “Nanog” or “nanog” as used herein include any of the naturally occurring forms of the Nanog transcription factor or its variants that maintain Nanog transcription factor activity (e.g., within at least about 50%, 80%, 90%, or 100% of the activity compared to wild-type Nanog as measured by methods known in the art). In some embodiments, the Nanog variant has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) amino acid sequence identity across the entire sequence compared to the naturally occurring Nanog polypeptide. In some embodiments, the Nanog protein is the protein identified by NCBI reference number NP_079141.

[0267] LLPS-deficient NANOG may include any of the mutations described herein that result in LLPS deficiency. For example, partial or complete deletion of the N-terminal and / or C-terminal domain outside the DBD region, substitution of a charged amino acid (e.g., acidic amino acid such as D or E, or basic amino acid such as R, K, or H) with a neutral amino acid (e.g., A), or a combination thereof.

[0116] LIN28

[0268] The LIN28 family of RNA-binding proteins that promote pluripotency has two members, LIN28A and LIN28B. Lin28 reprogramming factors as used herein refer to any naturally occurring member of the Lin28 family or a functional variant of the corresponding wild-type Lin28 reprogramming factor that maintains at least about 50% (e.g., at least about 60%, 70%, 80%, 90%, 95%, or 100%) of its reprogramming activity.

[0269] LIN28, or Lin-28 homolog A, is a protein encoded by the LIN28 gene in humans. It is a marker of undifferentiated human embryonic stem cells and encodes a cytoplasmic mRNA-binding protein that binds to IGF-2 (insulin-like growth factor 2) mRNA and enhances its translation. Lin28 has also been shown to bind to let-7 pre-miRNA and block the production of mature let-7 microRNA in mouse embryonic stem cells. This is a non-essential but important factor in iPSC generation. Exemplary Lin28 proteins are those encoded by the mouse gene (Genbank accession number NM_145833) and the human Lin28 gene (Genbank accession number NM_024674).

[0270] Accordingly, terms such as “Lin28” or “Lin28 homolog A” as used herein include any of the naturally occurring forms of the Lin28 transcription factor or its variants that maintain Lin28 transcription factor activity (e.g., within at least about 50%, 80%, 90%, or 100% of the activity compared to wild-type Lin28 as measured by methods known in the art). In some embodiments, the LIN28 variant has at least about 85% (e.g., at least about 90%, 95%, 99%, or more) amino acid sequence identity across the entire sequence compared to the naturally occurring LIN28 polypeptide. In other embodiments, the Lin28 protein is the protein identified by NCBI reference number NP_078950.

[0271] Human LIN28 has IDRs at amino acid positions 1-31 and 178-209. LLPS-deficient LIN28 may include any of the mutations described herein that result in LLPS deficiency. For example, partial or complete deletion of an IDR, substitution of a charged amino acid (e.g., acidic amino acid such as D or E, or basic amino acid such as R, K, or H) within an IDR with a neutral amino acid (e.g., A), or a combination thereof.

[0117] Nucleic acids, structures, and carriers

[0272] In some embodiments, the recombinant reprogramming factor is encoded by a nucleic acid (e.g., RNA or DNA) or a nucleic acid construct (e.g., an RNA construct such as circRNA or a DNA construct such as a viral or bacterial vector). Therefore, nucleic acids (e.g., codon-optimized nucleic acids) and nucleic acid constructs encoding any of the LLPS-deficient recombinant reprogramming factors described herein are also provided. In some embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 3 or 13. In some embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 69 or 71. In the rejuvenation methods (or therapeutic or preventive methods) described herein, introducing a recombinant reprogramming factor lacking an effective amount of LLPS into a population of cells (e.g., skin cells) also includes introducing a nucleic acid encoding a recombinant reprogramming factor lacking an effective amount of LLPS into a population of cells (e.g., skin cells).

[0273] Any carrier or method capable of introducing the nucleic acids described herein into skin cells may be used herein. In some embodiments, nucleic acid delivery is viral delivery (e.g., AAV or lentivirus). In some embodiments, nucleic acid delivery is non-viral delivery such as lipofection, nucleofection, microinjection, bioristic, virosome, liposome, immunoliposome, polycation or lipid:nucleic acid conjugate, electroporation, nanoparticles, exosome, microvesicle, gene gun, naked DNA, and artificial virions. Delivery using GalNAc-binding ligation systems, protamine, polymers, inorganic nanoparticles, or polymer matrices is also conceivable.

[0118]

[0274] In some embodiments, the nucleic acid is RNA (e.g., mRNA), such as circular RNA (circRNA) or linear RNA (e.g., linear mRNA). Circular mRNA has lower immunogenicity and higher stability than linear mRNA, making it suitable for long-term in vivo expression of the encoded protein. In some embodiments, the nucleic acid is contained within a vector. In some embodiments, the nucleic acid (e.g., circRNA) is delivered by exosomes, liposomes, or lipid nanoparticles (LNPs). In some embodiments, the nucleic acid is delivered by electroporation. In some embodiments, the mRNA includes IRES. In some embodiments, the RNA may include sequences conjugated to L7Ae, TAT, etc., for RNA enrichment. In some embodiments, the RNA includes modifications. In some embodiments, the RNA is self-amplified mRNA.

[0275] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is contained within a vector such as a viral vector. In some embodiments, the viral vector is selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, retrovirus vectors, herpes simplex virus (HSV) vectors, mouse stem cell virus (MSCV) vectors, Moloney's mouse leukemia virus (MoMuLV) vectors, human immunodeficiency virus (HIV) vectors, bovine papillomavirus vectors, lentivirus vectors, vaccinia virus vectors, and polyomavirus vectors. In some embodiments, the viral vector is a lentivirus vector or an AAV vector. In some embodiments, the nucleic acid is delivered by a virus, such as a lentivirus or an AAV. In some embodiments, the nucleic acid is delivered by electroporation.

[0119]

[0276] As used herein, the terms “vector” or “nucleic acid construct” refer to a DNA or RNA molecule containing a coding nucleotide sequence that can be transcribed into RNA or expressed in a protein. This application also provides vectors or nucleic acid constructs into which the nucleic acids of this application are inserted. Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe-generating vectors and sequencing vectors.

[0277] In some embodiments, the vector includes a promoter operably ligated to a coding nucleotide sequence so that the promoter controls the transcription or expression of the coding nucleotide sequence. The promoter may be located 5' (upstream) of the coding nucleotide sequence under its control. The distance between the promoter and the coding sequence may be approximately the same as the distance between the promoter and the gene controlled by the promoter in the gene from which the promoter originates. As is known in the art, this change in distance can be adapted without loss of promoter function. In some embodiments, the vector includes a 5'UTR and / or 3'UTR that regulates the transcription or expression of the coding nucleotide sequence. In some embodiments, a suitable vector includes a functional origin of replication in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers. (See, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6, 326193). Exemplary selection markers may include, but are not limited to, peptide protein tags (e.g., FLAG tags), fluorescent tags, and antibiotic resistance cassettes. Both the selection marker and reporter genes may be flanked by appropriate regulatory sequences to enable expression in target cells. In some embodiments, the vector also includes or encodes a Kozak sequence, a terminator sequence, a 5' cap, a poly(A) tail, or a WPRE.

[0278] Suitable vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing or not containing one or more free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and various other polynucleotides known in the art. One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which additional DNA segments can be inserted by standard molecular cloning techniques, for example. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomatic mammalian vectors). Other vectors (e.g., non-episomatic mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome. Furthermore, certain vectors can instruct the transcription or expression of coding nucleotide sequences into which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0120]

[0279] Recombinant expression vectors may contain nucleic acids of the present invention in a form suitable for transcription or expression of nucleic acids in host cells (e.g., skin cells). In some embodiments, a recombinant expression vector includes one or more regulatory elements, which may be selected based on the host cell used for transcription or expression, and which are operably ligated to the nucleic acid sequence to be transcribed or expressed. Within a recombinant expression vector, "operably ligated" is intended to mean that the nucleotide sequence of interest is ligated to the regulatory element in a manner that enables the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell if the vector is introduced into a host cell).

[0280] An expression vector may contain various elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection marker, and a signal sequence. These elements can be appropriately selected by those skilled in the art. For example, a promoter sequence may be selected to promote the transcription of polynucleotides in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, CAG promoter, PGK promoter, and SV40 promoter. An enhancer sequence may be selected to enhance the transcription of nucleic acids. A selection marker may be selected to allow selection of a host cell into which the vector has been inserted from a host cell into which the vector has not been inserted; for example, the selection marker may be a gene that confers antibiotic resistance. A signal sequence may be selected to allow the expressed polypeptide to be transported outside the host cell.

[0281] Expression vectors may be in the form of viral vectors. Viral vector technology is well known in the field and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as in other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In one embodiment, a mouse stem cell virus (MSCV) vector is used to express a desired nucleic acid. MSCV vectors have been demonstrated to efficiently express desired nucleic acids in cells. Other examples of viral vectors are based on Moloney's mouse leukemia virus (MoMuLV) and human immunodeficiency virus (HIV). The use of RNA or DNA virus-based systems for nucleic acid delivery is highly efficient in targeting viruses to specific cells and transporting the viral payload to the cell nucleus.

[0121]

[0282] In one embodiment, the viral vector is a recombinant AAV (rAAV) vector. In one embodiment, the rAAV vector is a vector derived from an AAV serotype, and is not limited to AAV ITRs such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV capsid serotype. In some embodiments, the nucleic acids in the AAV include ITRs such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV capsid serotypes. In some embodiments, the nucleic acids in the AAV further encode recombinant reprogramming factors as described herein. The use of any AAV serotype is considered to be within the scope of this disclosure. In some embodiments, the vector is capsid-formed in rAAV particles. In one embodiment, the AAV virus particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2 V708K, AAV2-HBKO, AAVDJ8, AAVPHP.B, AAVPHP.eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV, or raAV2 / HboV1 serotype capsids.

[0122]

[0283] In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is pseudotyped. In some embodiments, the lentiviral vector contains a G glycoprotein. In some embodiments, the G glycoprotein is selected from the group consisting of G glycoproteins of vesicular stomatitis virus (VSV), pirivirus, Chandipla virus, spring viremia virus of carp, and mocola virus. In some embodiments, the lentiviral vector contains a terminal repeat sequence (LTR) selected from the group consisting of terminal repeat sequences of HIV and equine infectious anemia.

[0123]

[0284] Viral vectors containing nucleic acids encoding reprogramming factors (e.g., transfer vectors) can be introduced into producing cells. In some embodiments, the nucleic acids encoding the reprogramming factors include terminal repeat sequences (LTRs) adjacent to the transgene to enable integration into the target cell genome. In some embodiments, the LTRs (5'LTR and / or 3'LTR) are mutated (e.g., cleaved, such as by cleaving U3) to produce a self-inactivating viral vector. One or more packaging vectors can be introduced into producer cells simultaneously or sequentially (in any order) to produce recombinant viruses. Recombinant viruses can be produced, for example, by culturing producing cells under conditions sufficient to produce recombinant viruses. In some embodiments, producing cells already contain one or more nucleic acids encoding components for producing recombinant viruses. These may be structural or non-structural viral components or proteins. The producing cell line is known in the art and can be recombinant with the molecule of interest to produce the recombinant virus of interest. Any suitable producing cell line known in the art, such as HEK293, 293T, HeLa, D-17, MDCK, BHK, and Cf2Th cells, can be used. In one embodiment, the producing cell line is 293T.

[0124]

[0285] In one embodiment, the viral vector promoter is selected from the group consisting of the Rous sarcoma virus (RSV) promoter, Simian virus 40 (SV40) promoter, cytomegalovirus pre-early gene promoter (CMV IE), mouse stem cell virus promoter (MCSV), elongation factor 1 alpha promoter (EF1-α), phosphoglycerate kinase-1 (PGK) promoter, ubiquitin-C (UBQ-C) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, polyoma enhancer / herpes simplex thymidine kinase (MC1) promoter, beta-actin (β-ACT) promoter, "myeloproliferative sarcoma virus enhancer, negative regulatory region deletion, d1587rev primer binding site substitution (MND)" promoter, NFAT promoter, TETON® promoter, and NFκB promoter. The transfer vector (e.g., one containing the transgene) may use the same or a different promoter as the envelope vector and / or packaging vector.

[0125]

[0286] The genes necessary to produce recombinant viruses may be split across two or more vectors to enhance the safety of the recombinant viruses. Recombinant viruses are typically produced using an envelope vector encoding viral glycoproteins, a packaging vector encoding viral structural proteins, and a transfer vector encoding the desired transgene. The genes necessary to produce recombinant viruses include gag, pol, rev, and optionally tat (depending on the vector used). gag encodes viral structural proteins, pol encodes enzymes involved in reverse transcription and genome integration, and rev and tat encode gene regulatory proteins. If the transfer plasmid contains a 5'LTR fused to a heterologous promoter, tat is no longer necessary, as transgene expression is tat-independent. Promoters may be selected for each vector based on the desired application, for example, depending on the cell type being transduced (e.g., primary cell-versus-cell line) or the gene being expressed (e.g., a dual promoter for the co-expression of two transgenes).

[0126]

[0287] In one embodiment, the rejuvenation method (or treatment or prevention method) described herein comprises introducing a plurality of reprogramming factors (e.g., a plurality of nucleic acids encoding the plurality of reprogramming factors) into a population of cells (e.g., skin cells), wherein at least one of the plurality of reprogramming factors is a recombinant reprogramming factor without LLPS (e.g., any of the recombinant reprogramming factors described herein). In one embodiment, the plurality of reprogramming factors comprises two or more reprogramming factors from two or more families selected from the group consisting of the OCT family, SOX family, KLF family, MYC family, LIN28 family, NANOG family, and GLIS family. In one embodiment, the plurality of reprogramming factors comprises two or more reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG, GLIS1 and their variants, wherein at least one of the plurality of reprogramming factors is a recombinant reprogramming factor without LLPS. In some embodiments, the multiple reprogramming factors include three or all of OCT4, KLF4, SOX2, and c-MYC, and at least one of the multiple reprogramming factors is a recombinant reprogramming factor without LLPS. In some embodiments, SOX2 is recombinant SOX2 without LLPS, for example, any of the recombinant SOX2s described herein, for example, any of SEQ ID NOs. 41 to 43. In some embodiments, c-MYC is recombinant c-MYC without LLPS, for example, any of the recombinant c-MYCs described herein, for example, any of SEQ ID NOs. 14 to 20, 47, and 48, for example, SEQ ID NO. 14. In some embodiments, the multiple reprogramming factors include OCT4 and KLF4, and at least one of OCT4 and KLF4 is a recombinant reprogramming factor without LLPS. In some embodiments, OCT4 is recombinant OCT4 without LLPS, for example, any of the recombinant OCT4s described herein, for example, any of SEQ ID NOs. 4 to 10 and 44 to 46, for example, SEQ ID NO. 4.In one embodiment, KLF4 is recombinant KLF4 without LLPS, for example, any of the recombinant KLF4s described herein, e.g., Sequence ID No. 40. In one embodiment, all of the multiple reprogramming factors are recombinant reprogramming factors without LLPS. In one embodiment, the multiple reprogramming factors include (e.g., one or more) wild-type reprogramming factors. In one embodiment, the multiple reprogramming factors include (i) recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46), SOX2 (e.g., SEQ ID NOs. 24), and KLF4 (e.g., SEQ ID NOs. 22); (ii) recombinant OCT4 without recombinant c-MYC (e.g., any of SEQ ID NOs. 4-10 and 44-46), KLF4 (e.g., SEQ ID NOs. 22), and LLPS (e.g., any of SEQ ID NOs. 14-20, 47, and 48); or (iv) recombinant OCT4 without LLPS (e.g., any of SEQ ID NOs. 4-10 and 44-46), KLF4 (e.g., SEQ ID NOs. 22), and SOX2 (e.g., SEQ ID NOs. 24).

[0127]

[0288] In some embodiments, the multiple reprogramming factors include three or all of mouse OCT4 (MsOCT4), mouse KLF4 (MsKLF4), mouse SOX2 (MsSOX2), and mouse c-MYC (Msc-MYC), and at least one of the multiple reprogramming factors is a recombinant reprogramming factor without LLPS. In some embodiments, MsSOX2 is a recombinant MsSOX2 without LLPS, such as any of the recombinant MsSOX2s described herein. In some embodiments, Msc-MYC is a recombinant Msc-MYC without LLPS, for example, any of the recombinant Msc-MYCs described herein. In some embodiments, the multiple reprogramming factors include MsOCT4 and MsKLF4, and at least one of MsOCT4 and MsKLF4 is a recombinant reprogramming factor without LLPS. In some embodiments, MsOCT4 is a recombinant MsOCT4 without LLPS, for example, any of the recombinant MsOCT4s described herein, for example, SEQ ID NO: 68 or 70. In some embodiments, MsKLF4 is a recombinant MsKLF4 without LLPS, such as any of the recombinant MsKLF4s described herein. In some embodiments, the reprogramming factors include (e.g., one or more) wild-type reprogramming factors. In some embodiments, the reprogramming factors include recombinant OCT4 without LLPS (e.g., SEQ ID NO: 68 or 70), MsKLF4 (e.g., SEQ ID NO: 72), and MsSOX2 (e.g., SEQ ID NO: 74).

[0128]

[0289] In one embodiment, the multiple reprogramming factors are encoded by multiple nucleic acids, such as RNA (e.g., linear mRNA or circRNA) or DNA. Nucleic acid expression may be constitutive or induced (e.g., using a Tet-On system).

[0290] In one embodiment, multiple nucleic acids reside on separate nucleic acid constructs. The nucleic acid constructs may be RNA constructs (e.g., linear RNA or circRNA) or DNA constructs (e.g., lentiviral vectors, AAV vectors, or plasmids).

[0291] In some embodiments, at least two of the multiple nucleic acids reside on a single nucleic acid construct. The nucleic acid construct may be an RNA construct (e.g., linear RNA or circRNA) or a DNA construct (e.g., a lentiviral vector, AAV vector, or plasmid). In some embodiments, at least two of the multiple nucleic acids reside on a single DNA construct (e.g., a viral vector or bacterial vector). In some embodiments, at least two of the multiple nucleic acids are under the control of different promoters. In some embodiments, at least two of the multiple nucleic acids are under the control of the same promoter. The promoter may be a constitutive promoter (e.g., CMV) or an inductive promoter (e.g., a Tet-On promoter, e.g., TRE3G (e.g., SEQ ID NO: 63), TRE, TRE3GS, TRE3GV, etc.). In some embodiments, at least two of the multiple nucleic acids are linked by a linking sequence encoding one or more linked nucleic acids or self-cleaving peptides (e.g., 2A peptides such as P2A, T2A, E2A, F2A, etc.), such as IRES. In one embodiment, one or more linked nucleic acids include linked sequences encoding self-cleaving peptides, where the self-cleaving peptides are P2A, T2A, or E2A. In one embodiment, the nucleic acid encoding P2A includes the nucleic acid sequence of SEQ ID NO: 25. In one embodiment, the nucleic acid encoding T2A includes the nucleic acid sequence of SEQ ID NO: 26. In one embodiment, the nucleic acid encoding E2A includes the nucleic acid sequence of SEQ ID NO: 27.

[0129] Circular RNA (circRNA)

[0292] In some embodiments, the nucleic acid or nucleic acid construct is circRNA. CircRNA refers to polyribonucleotides that form a circular structure via covalent bonds. Heterodouble-stranded RNA can be produced by any method known in the art, including, but not limited to, back-splicing, in vitro synthesis of linear RNA precursors, subsequent covalently closed loop formation by ligation of their ends (which may be a chemical or enzymatic approach), or production of living cells by overexpression of plasmids containing minigene sequences that spontaneously become circular after transcription. In vitro or in-cell circRNA synthesis can be performed using permuted intron-exon (PIE) systems, such as the Anabaena substitution group I intron-exon system (Ana PIE) or the Azoarcus species BH substitution group I intron circular system (AzoPIE), or Twister-optimized RNA (Tornado system) for durable overexpression systems. For example, nucleic acid sequences encoding one or more reprogramming factors can be recombined into the PIE system and constructed within a plasmid (e.g., pBluescript II SK(+)), and after in vitro transcription, linear RNA can be circularized to obtain circRNA. This is described in Obi and Chen, Methods. 2021:196:85-103, WO2023115732, WO2023024500, US20230346921, US11679120, US11981909, US20240078661 and US20240042015. For example, nucleic acid sequences encoding one or more reprogramming factors can be recombined into the Tornado system and constructed within a plasmid (e.g., pcDNA3.1(+), Piggybac), and after transfecting the plasmid into cells such as 293T, circRNA can be produced, and exosomes containing circRNA can also be collected.See U.S. Patent No. 20210340542 and U.S. Patent No. 011756183 for illustrative purposes, the contents of which are incorporated herein by reference in their entirety.

[0130]

[0293] In the PIE method, circularly rearranged group I intron precursor RNA (e.g., from Tetrahymena or Anabaena) has exons where the ends are fused, interrupting half of the intron sequence. The circularly rearranged group I intron precursor RNA can self-splice in vitro to produce circular RNA exons. Foreign sequences (e.g., encoding reprogramming factors) can be placed in the exons of the substituted group I intron self-splicing system and made circular because the exon sequence does not participate in the self-splicing reaction.

[0294] A method for producing any of the circRNAs described herein is provided. In one embodiment, the method comprises synthesizing a precursor RNA (linear RNA) by transcription (e.g., run-off transcription) using a DNA vector (e.g., a DNA vector comprising a 5' homology arm, a 3' group I intron fragment, a 5' spacer, an IRES, an expression sequence (e.g., encoding one of the reprogramming factors described herein), a 3' spacer, a 5' group I intron fragment, and a 3' homology arm in this order) as a template, and circulating the precursor RNA to form a circRNA (e.g., incubating the precursor RNA under conditions suitable for circulation, such as in the presence of a divalent cation (e.g., magnesium ion) and GTP). In one embodiment, the method further comprises constructing a DNA vector for transcription (e.g., in vitro) of the precursor RNA. DNA vector construction can be carried out using any molecular cloning method. Exemplary DNA vectors are shown in Figures 2 A-2 D, 6 A-6 F, and 10 A-10 B. In one embodiment, the DNA vector is linearized (e.g., via enzymatic digestion) to provide a transcription template. In some embodiments, the transcription template is digested (e.g., using DNase I) after the transcription of the precursor RNA is complete. In some embodiments, the method further comprises purifying the circRNA (e.g., via RNA precipitation and ethanol washing). In some embodiments, the method further comprises validating the circRNA (e.g., RNase R digestion analysis or E-Gel electrophoresis). Since circRNA lacks free ends, successfully produced circRNA remains intact and is not digested by exonuclease-like RNase R. See also the exemplary circRNA production methods in Examples 2-4.

[0295] Therefore, this application also provides DNA vectors and linear precursor RNAs for producing any of the circRNAs described herein.

[0131]

[0296] In one embodiment, the DNA vector comprises, in the following order: any 5' homology arm, a 3' group I intron fragment, any 5' internal spacer, an IRES (e.g., CVB3 IRES), an expression sequence (e.g., encoding multiple reprogramming factors), any 3' internal spacer, a 5' group I intron fragment, and any 3' homology arm. In one embodiment, the 5' and 3' homology arms are reverse complementary sequences that can bring the two ends of the linear precursor RNA closer together, potentially improving the circularization efficiency. In one embodiment, the 5' and 3' internal spacers are non-structural sequences (e.g., CAAA or CAAAAA) for separating the ribozyme element (group I intron RNA) from the IRES element or coding element. In one embodiment, the DNA vector comprises, in the following order: a 5' homology arm, a 3' group I intron fragment, a 5' internal spacer, an IRES (e.g., a CVB3 IRES), an expression sequence (e.g., encoding a recombinant reprogramming factor or a plurality of reprogramming factors as described herein), a 3' internal spacer, a 5' group I intron fragment, and a 3' homology arm. In one embodiment, at least one of the 3' or 5' internal spacers is about 3 to about 60 nucleotides long. In one embodiment, the 3' and / or 5' homology arm is about 10 to about 100 nucleotides long, e.g., about 10 to about 80 nt, about 10 to about 60 nt, about 10 to about 50 nt, about 10 to about 30 nt, about 20 to about 80 nt, about 40 to about 60 nt, or about 10 to about 20 nt. In one embodiment, the 3' and 5' internal spacers each include a double-stranding region capable of forming a double helix. In one embodiment, the double-stranding regions of the 3' and 5' spacers are each about 5 to about 100 nucleotides long, for example, about 10 to about 50 nt, about 20 to about 40 nt, or about 8 to about 20 nt. The homology arms or double-stranding regions can form a complete or incomplete double helix. For example, at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the homology arms or double-stranding regions can base pair with each other.In some embodiments, the DNA vector further comprises a transcriptional regulatory sequence. In some embodiments, the transcriptional regulatory sequence comprises a promoter and / or an enhancer. In some embodiments, the promoter comprises a T7 promoter. The DNA vector may be circular DNA or linear DNA. In some embodiments, the T7 promoter is located at the 5' end of a 5' homology arm.

[0132]

[0297] In one embodiment, the 5' element of the DNA vector upstream of the IRES (containing a 5' homology arm, a 3' group I intron fragment, and a 5' internal spacer) contains the sequence of SEQ ID NO: 49. In another embodiment, the 3' element of the DNA vector downstream of the protein coding sequence (containing a 3' internal spacer, a 5' group I intron fragment, and a 3' homology arm) contains the sequence of SEQ ID NO: 50.

[0298] In one embodiment, the upstream 5' element of the IRES (containing a 5' homology arm, a 3' group I intron fragment, and a 5' internal spacer) contains the sequence of SEQ ID NO: 51. In another embodiment, the downstream 3' element of the protein-coding sequence (containing a 3' internal spacer, a 5' group I intron fragment, and a 3' homology arm) contains the sequence of SEQ ID NO: 52.

[0133]

[0299] The term “3' Group I intron fragment” refers to a sequence that is 75% or more similar to the 3' proximal end of a native Group I intron, including a splice site dinucleotide and optionally a stretch of the native exon sequence. In some embodiments, the term “5' Group I intron fragment” refers to a sequence that is 75% or more similar to the 5' proximal end of a native Group I intron, including a splice site dinucleotide and optionally a stretch of the native exon sequence. The term “substitution site” refers to a site in a Group I intron where a cleavage occurs prior to the substitution of the intron. This cleavage produces 3' and 5' Group I intron fragments that are rearranged to be on either side of the stretch of the precursor RNA being circularized. The term “splice site” refers to a dinucleotide that is partially or completely contained within a Group I intron, between which a phosphodiester bond is cleaved during RNA circularization. The two “double-strand forming regions,” “homologous arms,” or “homologous regions” may be any two regions that are thermodynamically favorable for cross-pairing in sequence-specific interactions. In some embodiments, two double-stranding regions, homology arms, or homology regions share a sufficient level of sequence identity with respect to each other's reverse complementary strands in order to act as substrates for a hybridization reaction. As used herein, polynucleotide sequences are "homologous" if they are identical to or share sequence identity with a reverse complementary or "complementary" sequence. The sequence identity ratio between a homologous region and the reverse complementary sequence of a corresponding homologous region may be any sequence identity ratio that allows hybridization to occur. In some embodiments, an internal double-stranding region of the polynucleotide of the present invention may double-strand with another internal double-stranding region but not with an external double-stranding region.

[0134]

[0300] Two types of spacers can improve the circularization of precursor RNA and / or gene expression from circRNA. The first type of spacer is an external spacer, i.e., present in the precursor RNA but removed during circularization. While we do not wish to be constrained by theory, it is intended that external spacers can improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing other adjacent sequence elements from interfering with its folding and function. The second type of spacer is an internal spacer, i.e., present in the precursor RNA and retained in the resulting circular RNA. While we do not wish to be constrained by theory, it is thought that internal spacers can improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing other adjacent sequence elements, particularly adjacent IRESs and coding regions, from interfering with their folding and function. It is also intended that internal spacers can improve protein expression from IRESs by preventing adjacent sequence elements, particularly intron elements, from hybridizing with sequences within IRESs and inhibiting their ability to fold into their most preferred and active conformation.

[0135]

[0301] Any IRES may be used herein. In one embodiment, the IRES is a CVB3 IRES. Modification of the IRES and accessory sequences can increase or decrease IRES activity, for example, by cleaving the 5' and / or 3' ends of the IRES, adding a spacer to the 5' end of the IRES, modifying the 6 nucleotides at the 5' end of the translation initiation site (Kozak sequence), modifying an alternative translation initiation site, and creating a chimeric / hybrid IRES sequence.

[0136]

[0302] In some embodiments, the vectors, precursor RNAs, and circular RNAs provided herein include a first (5') and / or second (3') spacer. In some embodiments, including a spacer between the 3' group I intron fragment and the IRES can preserve the secondary structure of those regions by preventing them from interacting, and thus increase splicing efficiency. In some embodiments, the first (between the 3' group I intron fragment and the IRES) spacer and the second (between the expression sequence and the 5' group I intron fragment) spacer include further base-pairing regions that are expected to base-pair with each other but do not base-pair with the first and second double-strand formation regions. In some embodiments, such spacer base-pairing brings the group I intron fragments closer together, further increasing splicing efficiency. Furthermore, in some embodiments, the combination of base-pairing between the first and second double-strand formation regions, and separately, base-pairing between the first and second spacers, promotes the formation of a splicing bubble containing the group I intron fragment adjacent to the base-pairing adjacent region. A typical spacer is a continuous sequence having one or more of the following properties: 1) expected to avoid interference with proximal structures, e.g., IRES, expression sequences, or introns; 2) at least 7 nt in length and no more than 100 nt; 3) located after and adjacent to a 3' intron fragment, and / or before and adjacent to a 5' intron fragment; and 4) including one or more of the following: a) an unstructured region of at least 5 nt in length; b) a base-pairing region of at least 5 nt in length relative to a distal sequence, including other spacers; and c) a structured region of at least 7 nt in length, limited to the sequence of the spacer. A spacer can have several regions, including an unstructured region, a base-pairing region, a hairpin / structured region, and combinations thereof. In one embodiment, the spacer has a structured region with a high GC content. In one embodiment, a region within a spacer base-pairs with other regions within the same spacer. In one embodiment, a region within a spacer base-pairs with regions within other spacers. In one embodiment, the spacer includes one or more hairpin structures.In one embodiment, the spacer comprises one or more hairpin structures having a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In one embodiment, an additional spacer (internal spacer) is present between the 3' group I intron fragment and the IRES. In one embodiment, this additional spacer prevents or reduces to the extent that the structuring region of the IRES interferes with the folding of the 3' group I intron fragment. In one embodiment, the 5' spacer sequence is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides long. In one embodiment, the 5' spacer sequence is 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides long or less. In one embodiment, the 5' spacer sequence is 3-50, 5-10, 10-50, 20-50, 20-40, and / or 25-35 nucleotides long. In another embodiment, the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In one embodiment, the 5' spacer sequence is a polyA sequence. In another embodiment, the 5' spacer sequence is a polyAC sequence. In one embodiment, the spacer contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC. In another embodiment, the spacer contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U).

[0303] In one embodiment, the method includes incubating a DNA vector under conditions suitable for transcription. In one embodiment, the DNA vector is transcribed in vitro. In one embodiment, suitable conditions include ATP, GTP, CTP, UTP, and RNA polymerase (e.g., T7 RNA polymerase). In one embodiment, suitable conditions further include an RNase inhibitor.

[0137]

[0304] Circular RNAs have been found to exhibit reduced immunogenicity compared to their corresponding mRNAs, at least partially, because the mRNA contains an immunogenic 5' cap. When transcribing a DNA vector from a specific promoter (e.g., the T7 promoter) to produce precursor RNA, it is understood that the 5' end of the precursor RNA is G. To reduce the immunogenicity of circular RNA compositions containing low levels of contaminated linear mRNA, excess GMP relative to GTP can be provided during transcription so that most transcripts contain 5'GMP that cannot be capped.

[0138]

[0305] In one embodiment, linear precursor RNA can be cyclically formed by the autocatalytic action of a group I intron. Any group I intron known in the art can be used to generate circRNA via self-splicing. Examples of group I introns useful in the method of this application are described in Puttaraju, M. & Been, M., Nucleic Acids Res. 20, 5357-5364 (1992); Ford, E. & Ares, M., Proc. Natl Acad Sci. 91, 3117-3121 (1994); Vicens, Q., Paukstelis, PJ, Westof, E., Lambowitz, AM; and RNA 14, 2013-2029 (2008). Group I introns can be derived from bacterial phage group I introns or bacterial group I introns. In one embodiment, the group I intron is derived from a cyanobacterial group I intron. In some embodiments, the group I intron is derived from an anabaena group I intron. In some embodiments, the linear RNA polynucleotide includes a 3' anabaena group I intron fragment and a 5' anabaena group I intron fragment. In some embodiments, the reference 3' anabaena group I intron fragment and the reference 5' anabaena group I intron fragment are generated using an L6-5 substitution site. In some embodiments, the linear RNA polynucleotide includes a 3' azoarcus sp. BH group I intron fragment and a 5' azoarcus sp. BH group I intron fragment.

[0306] In certain embodiments, the circRNA comprises an IRES followed by an expression sequence (e.g., encoding a recombinant reprogramming factor or a plurality of reprogramming factors). In certain embodiments, the circRNA comprises, in the following order: a) the post-splicing intron fragment of the 3' group I intron fragment, b) the IRES, c) the expression sequence, and d) the post-splicing intron fragment of the 5' group I intron fragment. In certain embodiments, the circRNA does not include 5' homology arms and 3' homology arms. In certain embodiments, the circRNA does not include group I intron fragments. In certain embodiments, the circRNA retains 5' internal spacers and 3' internal spacers.

[0139]

[0307] In certain embodiments, the circular RNA polynucleotide is produced via circularization of an RNA polynucleotide comprising, in the following order, a 3' group I intron fragment, an IRES, an expression sequence (e.g., encoding a recombinant reprogramming factor or a plurality of reprogramming factors described herein), and a 5' group I intron fragment.

[0308] In certain embodiments, the circular RNA polynucleotide is produced via circularization of an RNA polynucleotide comprising, in the following order, a 5' external double-stranded forming region (or homology arm), a 3' group I intron fragment, a 5' internal spacer optionally including a 5' internal double-stranded forming region, an IRES, an expression sequence (e.g., encoding a recombinant reprogramming factor or a plurality of reprogramming factors described herein), a 3' internal spacer optionally including a 3' internal double-stranded forming region, a 5' group I intron fragment, and a 3' external double-stranded forming region (or homology arm).

[0309] In certain embodiments, the circular RNA polynucleotide is produced via circularization of an RNA polynucleotide comprising, in the following order, a 5' - external double-stranded forming region (or homology arm), a 5' - external spacer, a 3' Group I intron fragment, a 5' - internal spacer optionally containing a 5' - internal double-stranded forming region, an IRES, an expression sequence (such as encoding a recombinant reprogramming factor or multiple reprogramming factors described herein), a 3' - internal spacer optionally containing a 3' - internal double-stranded forming region, a 5' Group I intron fragment, a 3'- external spacer, and a 3' - external double-stranded forming region (or homology arm).

[0310] In certain embodiments, the circular RNA polynucleotide is produced via circularization of an RNA polynucleotide comprising, in the following order, a 3' Group I intron fragment, a 5' - internal spacer containing a 5' - internal double-stranded forming region, an IRES, an expression sequence (such as encoding a recombinant reprogramming factor or multiple reprogramming factors described herein), a 3' - internal spacer containing a 3' - internal double-stranded forming region, and a 5' Group I intron fragment.

[0311] In certain embodiments, the circular RNA polynucleotide is produced via circularization of an RNA polynucleotide comprising, in the following order, a 5' - external double-stranded forming region (or homology arm), a 5' - external spacer, a 3' Group I intron fragment, a 5' - internal spacer containing a 5' - internal double-stranded forming region, an IRES, an expression sequence (such as encoding a recombinant reprogramming factor or multiple reprogramming factors described herein), a 3' - internal spacer containing a 3' - internal double-stranded forming region, a 5' Group I intron fragment, a 3'- external spacer, and a 3' - external double-stranded forming region (or homology arm).

[0140]

[0312] In one embodiment, a circular RNA polynucleotide is prepared by circularizing an RNA polynucleotide comprising, in the following order: a first polyA sequence, a 5' outer double-strand forming region (or homology arm), a 5' outer spacer, a 3' group I intron fragment, a 5' inner spacer including a 5' inner double-strand formin...

Claims

1. A method for rejuvenating a population of skin cells, comprising introducing an effective amount of one or more recombinant reprogramming factors into the population of skin cells, wherein at least one of the reprogramming factors is devoid of liquid-liquid phase separation (LLPS) and is a recombinant reprogramming factor containing a mutation in the LLPS-related domain relative to the corresponding wild-type reprogramming factor.

2. A population of skin cells includes fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or any combination thereof, and / or the following (i) Aged skin tissue; (ii) Damaged skin tissue; (iii) Skin tissue with a skin disease or disorder; and / or (iv) Skin tissue after cosmetic, dermatological, or surgical procedures The method according to claim 1.

3. The method according to claim 1, wherein the LLPS-related domain is selected from the group consisting of an endogenous irregular region (IDR), a region outside the DNA-binding domain (DBD), a ubiquitination site, or any combination thereof.

4. The following mutations: (i) Substituting acidic amino acids in IDR with neutral amino acids; (ii) Substituting ubiquitizable amino acids with non-ubiquitizable amino acids; (iii) Deleting ubiquitinated amino acids; (iv) Deleting an amino acid in the IDR; and / or (v) Deleting amino acids outside the DBD region This includes, preferably, the following: (i) The amino acid that can be ubiquitinated is K; and / or (ii) The amino acid to be deleted is an acidic amino acid. The method according to claim 3.

5. The method according to any one of claims 1 to 4, wherein the recombinant reprogramming factor is selected from recombinant OCT4, recombinant c-MYC, recombinant KLF4, and recombinant SOX2.

6. Recombinant OCT4 contains mutations at amino acid positions 1-140 and / or 288-360 with reference to SEQ ID NO:

2. Recombinant c-MYC includes a substitution of a ubiquitinated amino acid with a non-ubiquitinated amino acid, wherein the amino acid position refers to SEQ ID NO: 12 or 85, and preferably the substitution includes a substitution of K with R. Recombinant KLF4 contains a deletion at amino acid positions 2–374 with reference to SEQ ID NO: 22, and / or Recombinant SOX2 contains deletions at amino acid positions 2-40 and / or 201-317 with reference to SEQ ID NO:

24. The method according to claim 5.

7. Recombinant OCT4 includes substitutions at amino acid positions selected from the group consisting of 8, 20, 26, 31, 56, 68, 91, 96, 98, 104, 108, 113, 125, 127, 130, 134, 135, 138, 291, 296, 297, 299, 341, and 343 with reference to SEQ ID NO: 2, or Recombinant OCT4 contains deletions at amino acid positions 2-140 and / or 288-360 with reference to SEQ ID NO: 2, and / or Recombinant c-MYC includes substitutions at amino acid positions selected from the group consisting of 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, with reference to SEQ ID NO: 12 or 85, or The method according to claim 6, wherein recombinant c-MYC includes a deletion at amino acid positions 2 to 360 with reference to SEQ ID NO: 12 or 85.

8. Recombinant OCT4 is as follows: (i) D8A, D20A, E26A, D31A, E56A, E68A, E91A, E96A, E98A, E104A, D108A, E113A, E125A, E127A, E130A, E134A, E135A, D138A, D291A, E296A, D297A, E299A, E341A and E343A; (ii) D8A, D20A, E26A, D31A, E56A, E68A, E91A, E96A, E98A, E104A, D108A, E113A, E125A, E127A, E130A, E134A, E135A and D138A; (iii) D291A, E296A, D297A, E299A, E341A and E343A; (iv) D8A, D20A, E26A, D31A, E56A, E68A, E91A, E96A, E98A, E104A, D108A, E113A, E125A, E127A, E130A, E134A, D138A, D291A, E296A, E299A, E341A and E343A; (v) E134A, E135A, E296A and D297A; (vi) D8A, D20A, D31A, D108A, D138A, D291A and D297A; and (vii) E26A, E56A, E68A, E91A, E96A, E98A, E104A, E113A, E125A, E127A, E130A, E134A, E135A, E296A, E299A, E341A and E343A, Includes a substitution selected from the group consisting of and / or Recombinant c-MYC is as follows: (i) K51R, K52R, K126R, K143R, K148R, K157R, K206R, K269R, K275R, K289R, K298R, K317R, K32 3R, K326R, K341R, K355R, K371R, K389R, K392R, K397R, K398R, K412R, K422R, K428R and K430R; (ii) K148R; (iii) K389R; (iv) K148R and K389R; (v) K51R and K52R; (vi) K275R and K289R; and (vii) K51R, K52R, K275R and K289R Includes a substitution selected from the group consisting of, The method according to claim 7.

9. Recombinant OCT4 contains an amino acid sequence of any of SEQ ID NOs: 4-10 or a mutant that has at least approximately 85% sequence identity with any of SEQ ID NOs: 4-10, or contains an amino acid sequence of any of SEQ ID NOs: 44-46 or a mutant that has at least approximately 85% sequence identity with any of SEQ ID NOs: 44-46. Recombinant c-MYC contains a mutant that has at least approximately 85% sequence identity with any of the amino acid sequences of SEQ ID NOs. 14-20 or any of SEQ ID NOs. 14-20, or contains a mutant that has at least approximately 85% sequence identity with the amino acid sequence of SEQ ID NOs. 47 or 48 or any of SEQ ID NOs. 47 or 48. Recombinant KLF4 includes the amino acid sequence of SEQ ID NO: 40 or a mutant having at least approximately 85% sequence identity with SEQ ID NO: 40; and / or Recombinant SOX2 contains an amino acid sequence of any of SEQ ID NOs. 41-43 or a mutant that has at least approximately 85% sequence identity with any of SEQ ID NOs. 41-43. The method according to claim 8.

10. Recombinant reprogramming factors are encoded by nucleic acids selected from the group consisting of RNA, circular RNA (circRNA), and DNA, and / or The nucleic acids are delivered by exosomes, lipid nanoparticles (LNPs), or viruses. The method according to any one of claims 1 to 4.

11. A population of skin cells is present in the individual requiring treatment or obtained from a donor individual, and is cultured ex vivo, preferably here, RNA is encapsulated within an exosome or LNP, and more preferably, the exosome or LNP is delivered by a microneedle. The method according to any one of claims 1 to 4.

12. The plurality of reprogramming factors include two or more reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, L-MYC, LIN28, NANOG, GLIS1 and their variants, and / or The plurality of reprogramming factors include wild-type reprogramming factors, and here preferably, the plurality of reprogramming factors (i) Recombinant OCT4 without LLPS, SOX2, and KLF4; (ii) LLPS, KLF4 and recombinant OCT4 without LLPS and recombinant c-MYC; or (iii) comprising LLPS, KLF4, recombinant c-MYC without LLPS and recombinant OCT4 without SOX2, The method according to claim 1.

13. The aforementioned multiple reprogramming factors are encoded by multiple nucleic acids, and / or, here, At least two of the aforementioned plurality of nucleic acids are located on a single nucleic acid construct, preferably, here, At least two of the plurality of nucleic acids are under the control of the same promoter, preferably, where at least two of the plurality of nucleic acids are linked by one or more linked nucleic acids, preferably, where the one or more linked nucleic acids include linked sequences encoding IRES or self-cleaving peptides, preferably, where the nucleic acid construct is in the 5' to 3' direction as follows: (i) A first nucleic acid encoding recombinant OCT4 without LLPS – a first ligation sequence encoding P2A – a second nucleic acid encoding SOX2 – a second ligation sequence encoding T2A – a third nucleic acid encoding KLF4; (ii) A first nucleic acid encoding recombinant OCT4 without LLPS – a first ligation sequence encoding P2A – a second nucleic acid encoding KLF4 – a second ligation sequence encoding T2A – a third nucleic acid encoding recombinant c-MYC without LLPS; (iii) A first nucleic acid encoding recombinant OCT4 without LLPS – a first ligature sequence encoding P2A – a second nucleic acid encoding KLF4 – a second ligature sequence encoding T2A – a third nucleic acid encoding recombinant c-MYC without LLPS – a third ligature sequence encoding E2A – a fourth nucleic acid encoding SOX2; or (iv) comprising a first nucleic acid encoding recombinant OCT4 without LLPS, a first ligation sequence encoding P2A, a second nucleic acid encoding KLF4, a second ligation sequence encoding T2A, and a third nucleic acid encoding SOX2, preferably the nucleic acid construct comprising any of the nucleic acid sequences of SEQ ID NOs: 28, 30, 32, 35, 37, 39, 57, 59, 61, 94, 98, and 102. The method according to claim 12.

14. One or more recombinant reprogramming factors for use in a method to slow or prevent the progression of aging of an individual's skin tissue, wherein the method comprises introducing an effective amount of the LLPS-free recombinant reprogramming factor into a population of skin cells of skin tissue.

15. One or more recombinant reprogramming factors for use in a method for treating aging, damage, or disease or condition of an individual's skin tissue, wherein the method comprises introducing an effective amount of the LLPS-free recombinant reprogramming factor into a population of skin cells of skin tissue.

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