Polypeptide and composition for use in cell rejuvenation, and use thereof

Abstract

Provided in the present invention are a polypeptide and composition for use in cell rejuvenation, and the use thereof. Provided are a novel truncated c-Myc factor, a novel truncated OCT4 factor, a novel truncated SOX2 factor, and a novel truncated KLF4 factor. Further provided is a truncated-OSKM composition for use in the rejuvenation of cells, tissues, or organs in a subject. The composition comprises the truncated c-Myc factor and one or more factors selected from the OCT4 factor, the SOX2 factor, and the KLF4 factor. The truncated-OSKM composition provided in the present invention not only retains the aging reprogramming ability of original OSKM, but also significantly reduces the gene length thereof and the risk of iPSC formation.

Description

Polypeptides, compositions and uses thereof for cell rejuvenation TECHNICAL FIELD

[0001] The present application belongs to the field of cell rejuvenation, tissue engineering and regenerative medicine, and in particular relates to OSKM truncations for cell, tissue or organ rejuvenation in a subject, compositions comprising the same and uses thereof. BACKGROUND

[0002] Induced pluripotent stem cell (iPSC) reprogramming refers to the reprogramming of differentiated cells into pluripotent stem cells by specific transcription factors, and the most frequently used factor combination is the OSKM four-factor combination (OCT4, SOX2, KLF4, c-Myc).

[0003] Due to the characteristics of iPSC reprogramming, there have been individual documents in recent years reporting that OSKM can reverse the aging symptoms of normal aging and progeria mice, which researchers call aging reprogramming, indicating that it has great application value in the field of anti-aging. However, due to the risk of potential teratoma formation in vivo and the difficulty in delivering the gene length due to the formation of iPSC by OSKM, its clinical application is limited. Therefore, how to achieve anti-aging while reducing the ability to form iPSC and improve its safety, and optimizing the gene length has great practical significance for clinical application. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides an OSKM truncation composition for cell, tissue or organ rejuvenation in a subject, which not only retains the aging reprogramming ability of the original OSKM (OCT4, SOX2, KLF4, c-Myc four-factor combination), but also significantly reduces the gene length and the risk of iPSC formation.

[0005] The first aspect of the present application provides a polypeptide selected from a c-Myc factor truncation, an OCT4 factor truncation, a SOX2 factor truncation and a KLF4 factor truncation, or any combination thereof.

[0006] In some embodiments, the c-Myc factor truncation lacks a CP domain.

[0007] In some embodiments, the OCT4 factor truncation lacks a CTD domain, lacks a NTD domain, or lacks both a NTD domain and a CTD domain.

[0008] In some embodiments, the TAD domain of the SOX2 factor truncation is replaced by amino acids 92-100 of the TAD domain.

[0009] In some embodiments, the KLF4 factor truncation lacks a ZnF domain or lacks an ID domain.

[0010] In some embodiments, the polypeptide is a c-Myc factor truncation that lacks a CP domain.

[0011] In some embodiments, the c-Myc factor truncation comprises an MTAD domain and a bHLHLZ domain of c-Myc. In some embodiments, the c-Myc factor truncation lacks a CP domain and comprises an MTAD domain and a bHLHLZ domain of c-Myc.

[0012] In some embodiments, the MTAD domain and the bHLHLZ domain are directly connected or connected via a linker.

[0013] In some embodiments, the c-Myc factor truncation comprises a fusion protein formed by the MTAD domain and the bHLHLZ domain.

[0014] In some embodiments, the c-Myc factor truncation has an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0015] In some embodiments, a nucleic acid encoding the c-Myc factor truncation has a nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

[0016] In some embodiments, the c-Myc factor truncation is capable of being used in combination with full OSK, has comparable rejuvenation marker ability as OSKM, while its iPSC formation efficiency is reduced to 4.3% of OSKM and total length of fragments is reduced to 86.2% of OSKM.

[0017] In some embodiments, the c-Myc factor truncation is capable of being used in combination with full OSK, has comparable rejuvenation marker ability as OSKM-W, while its iPSC formation efficiency is reduced to 3.3% of OSKM-W and total length of fragments is reduced to 86.3% of OSKM-W.

[0018] In other embodiments, the polypeptide is an OCT4 factor truncation that lacks a CTD domain.

[0019] In some embodiments, the OCT4 factor truncation has an amino acid sequence as set forth in SEQ ID NO: 56 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 56.

[0020] In some embodiments, the nucleic acid encoding the OCT4 factor truncation has a nucleotide sequence as set forth in SEQ ID NO: 55 or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 55.

[0021] In other embodiments, the polypeptide is an OCT4 factor truncation that lacks the NTD domain.

[0022] In other embodiments, the polypeptide is an OCT4 factor truncation that lacks the NTD domain and the CTD domain.

[0023] In some embodiments, the polypeptide is a combination of an OCT4 factor truncation and a c-Myc factor truncation, which is denoted as an OCT4-Myc factor truncation.

[0024] In some embodiments, the OCT4-Myc factor truncation comprises a c-Myc factor MTAD domain, an OCT4 factor POU domain, and an OCT4 factor CTD domain.

[0025] In some embodiments, the OCT4-Myc factor truncation comprises an OCT4 factor NTD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain.

[0026] In some embodiments, the OCT4-Myc factor truncation comprises an OCT4 factor NTD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain and does not comprise other domains of the c-Myc factor and other domains of the OCT4 factor.

[0027] In some embodiments, the OCT4 factor NTD domain, the OCT4 factor POU domain, and the c-Myc factor bHLHLZ domain are directly linked or linked via a linker.

[0028] In some embodiments, the OCT4-Myc factor truncation comprises a fusion protein formed by an NTD domain of an OCT4 factor, a POU domain of an OCT4 factor, and a bHLHLZ domain of a c-Myc factor.

[0029] In some embodiments, the OCT4-Myc factor truncation has an amino acid sequence set forth in SEQ ID NO: 58 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58.

[0030] In some embodiments, a nucleic acid encoding the OCT4-Myc factor truncation has a nucleotide sequence set forth in SEQ ID NO: 57 or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 57.

[0031] In some embodiments, the OCT4-Myc factor truncation comprises a c-Myc factor MTAD domain, an OCT4 factor POU domain, and an OCT4 factor CTD domain and does not comprise other domains of the c-Myc factor and other domains of the OCT4 factor.

[0032] In some embodiments, the c-Myc factor MTAD domain, the OCT4 factor POU domain, and the OCT4 factor CTD domain are directly linked or linked by a linker.

[0033] In some embodiments, the OCT4-Myc factor truncation comprises a fusion protein formed by a c-Myc factor MTAD domain, an OCT4 factor POU domain, and an OCT4 factor CTD domain.

[0034] In some embodiments, the OCT4-Myc factor truncation has an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8.

[0035] In some embodiments, the nucleic acid encoding the OCT4-Myc factor truncation has a nucleotide sequence set forth in SEQ ID NO: 2 or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

[0036] In some embodiments, the OCT4-Myc factor truncation comprising a fusion protein formed by a c-Myc factor MTAD domain, an OCT4 factor POU domain, and an OCT4 factor CTD domain can be used in combination with the full SK, has comparable ability to improve aging indicators as OSKM, while its iPSC formation efficiency is reduced to 2.1% of OSKM, and the total length of the fragment is reduced to 71.9% of OSKM.

[0037] In some embodiments, the OCT4-Myc factor truncation comprising a fusion protein formed by a c-Myc factor MTAD domain, an OCT4 factor POU domain, and an OCT4 factor CTD domain can be used in combination with the full SK, has comparable ability to improve aging indicators as OSKM-W, while its iPSC formation efficiency is reduced to 0.6% of OSKM-W, and the total length of the fragment is reduced to 72.1% of OSKM-W.

[0038] In some embodiments, the OCT4-Myc factor truncation comprises a c-Myc factor MTAD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain.

[0039] In some embodiments, the OCT4-Myc factor truncation comprises a c-Myc factor MTAD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain and does not comprise other domains of the c-Myc factor and other domains of the OCT4 factor.

[0040] In some embodiments, the c-Myc factor MTAD domain, the OCT4 factor POU domain, and the c-Myc factor bHLHLZ domain are directly linked or linked by a linker.

[0041] In some embodiments, the OCT4-Myc factor truncation comprises a fusion protein formed by a c-Myc factor MTAD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain.

[0042] In some embodiments, the OCT4-Myc factor truncation has an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0043] In some embodiments, the nucleic acid encoding the OCT4-Myc factor truncation has a nucleotide sequence as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3.

[0044] In some embodiments, the OCT4-Myc factor truncation comprising a fusion protein formed by a c-Myc factor MTAD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain is capable of having comparable rejuvenation index ability as OSKM when used in combination with intact SK, while its iPSC formation efficiency is reduced to 0% of OSKM, and the total length of the fragment is reduced to 73.0% of OSKM.

[0045] In some embodiments, the OCT4-Myc factor truncation comprising a fusion protein formed by a c-Myc factor MTAD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain is capable of having comparable rejuvenation index ability as OSKM-W when used in combination with intact SK, while its iPSC formation efficiency is reduced to 0% of OSKM-W, and the total length of the fragment is reduced to 73.2% of OSKM-W.

[0046] In other embodiments, the polypeptide is a SOX2 factor truncation, wherein the TAD domain is replaced with amino acids 92-100 of the TAD domain.

[0047] In some embodiments, the SOX2 factor truncation comprises an HMG domain, a DIM domain, and amino acids 92-100 of the TAD domain.

[0048] In some embodiments, the HMG domain, the DIM domain, and amino acids 92-100 of the TAD domain are directly connected or connected via a linker.

[0049] In some embodiments, the SOX2 factor truncation comprises a fusion protein formed by an HMG domain, a DIM domain, and amino acids 92-100 of the TAD domain.

[0050] In some embodiments, the SOX2 factor truncation has an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10.

[0051] In some embodiments, the nucleic acid encoding the SOX2 factor truncation has a nucleotide sequence as set forth in SEQ ID NO: 4 or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4.

[0052] In some embodiments, the SOX2 factor truncation is capable of being used in combination with the complete OKM, has comparable ability to improve aging indicators as OSKM, while its iPSC formation efficiency is reduced to 0% of OSKM, and the total length of fragments is reduced to 90.2% of OSKM.

[0053] In some embodiments, the SOX2 factor truncation is capable of being used in combination with the complete OKM, has comparable ability to improve aging indicators as OSKM-W, while its iPSC formation efficiency is reduced to 0% of OSKM-W, and the total length of fragments is reduced to 90.1% of OSKM-W.

[0054] In other embodiments, the polypeptide is a KLF4 factor truncation that lacks a ZnF domain.

[0055] In some embodiments, the KLF4 factor truncation lacks a ZnF domain and comprises an AD domain and an ID domain.

[0056] In some embodiments, the AD domain and the ID domain are directly linked or linked via a linker.

[0057] In some embodiments, the KLF4 factor truncation comprises a fusion protein formed by the AD domain and the ID domain.

[0058] In some embodiments, the KLF4 factor truncation has an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0059] In some embodiments, the nucleic acid encoding the KLF4 factor truncation has a nucleotide sequence set forth in SEQ ID NO: 5 or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0060] In some embodiments, the KLF4 factor truncation comprising a fusion protein formed by an AD domain and an ID domain can be used in combination with the full OSM, has an ability to improve aging indicators comparable to OSKM, while its iPSC formation efficiency is reduced to 0% of OSKM, and the total length of the fragment is reduced to 89.9% of OSKM.

[0061] In some embodiments, the KLF4 factor truncation lacks the ID domain and comprises an AD domain and a ZnF domain.

[0062] In some embodiments, the AD domain and the ZnF domain are directly connected or connected through a linker.

[0063] In some embodiments, the KLF4 factor truncation comprises a fusion protein formed by an AD domain and a ZnF domain.

[0064] In some embodiments, the KLF4 factor truncation has an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12.

[0065] In some embodiments, the nucleic acid encoding the KLF4 factor truncation has a nucleotide sequence set forth in SEQ ID NO: 6 or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6.

[0066] In some embodiments, the KLF4 factor truncation comprising a fusion protein formed by an AD domain and a ZnF domain can be used in combination with the full OSM, has an ability to improve aging indicators comparable to OSKM, while its iPSC formation efficiency is reduced to 0% of OSKM, and the total length of the fragment is reduced to 79.9% of OSKM.

[0067] The second aspect of the present application provides a fusion protein comprising the polypeptide of the first aspect or any combination thereof.

[0068] In some embodiments, the fusion protein comprises any one selected from the group consisting of:

[0069] (1) a c-Myc factor truncation, an OCT4 factor, a SOX2 factor, and a KLF4 factor;

[0070] (2) an OCT4-Myc factor truncation, a SOX2 factor, and a KLF4 factor;

[0071] (3) a c-Myc factor, an OCT4 factor, a SOX2 factor truncation, and a KLF4 factor; or

[0072] (4) a c-Myc factor, an OCT4 factor, a SOX2 factor, and a KLF4 factor truncation.

[0073] In some embodiments, the fusion protein comprises, in order from N-terminus to C- terminus: a c-Myc factor MTAD domain, a c-Myc factor bHLHLZ domain, an OCT4 factor, a SOX2 factor, and a KLF4 factor. In other embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: a c-Myc factor MTAD domain, a c-Myc factor bHLHLZ domain, an OCT4 factor, a SOX2 factor, and a KLF4 factor, arranged in any other order.

[0074] In some embodiments, the fusion protein comprises, in order from N-terminus to C- terminus: a c-Myc factor MTAD domain, an OCT4 factor POU domain, an OCT4 factor CTD domain, a SOX2 factor, and a KLF4 factor. In other embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: a c-Myc factor MTAD domain, an OCT4 factor POU domain, an OCT4 factor CTD domain, a SOX2 factor, and a KLF4 factor, arranged in any other order.

[0075] In some embodiments, the fusion protein comprises, in order from N-terminus to C- terminus: a c-Myc factor MTAD domain, an OCT4 factor POU domain, a c-Myc factor bHLHLZ domain, a SOX2 factor, and a KLF4 factor. In other embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: a c-Myc factor MTAD domain, an OCT4 factor POU domain, a c-Myc factor bHLHLZ domain, a SOX2 factor, and a KLF4 factor, arranged in any other order.

[0076] In some embodiments, the fusion protein comprises, in order from N-terminus to C- terminus: an OCT4 factor NTD domain, an OCT4 factor POU domain, a c-Myc factor bHLHLZ domain, a SOX2 factor, and a KLF4 factor. In other embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: an OCT4 factor NTD domain, an OCT4 factor POU domain, a c-Myc factor bHLHLZ domain, a SOX2 factor, and a KLF4 factor, in any other order.

[0077] In some embodiments, the fusion protein comprises, in order from N-terminus to C- terminus: a c-Myc factor, an OCT4 factor, a SOX2 factor truncation, and a KLF4 factor. In other embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: a c-Myc factor, an OCT4 factor, a SOX2 factor truncation, and a KLF4 factor, in any other order.

[0078] In some embodiments, the fusion protein comprises, in order from N-terminus to C- terminus: a c-Myc factor, an OCT4 factor, a SOX2 factor, a KLF4 factor AD domain, and a KLF4 factor ID domain. In other embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: a c-Myc factor, an OCT4 factor, a SOX2 factor, a KLF4 factor AD domain, and a KLF4 factor ID domain, in any other order.

[0079] In some embodiments, the fusion protein comprises, in order from N-terminus to C- terminus: a c-Myc factor, an OCT4 factor, a SOX2 factor, a KLF4 factor AD domain, and a KLF4 factor ZnF domain. In other embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: a c-Myc factor, an OCT4 factor, a SOX2 factor, a KLF4 factor AD domain, and a KLF4 factor ZnF domain, in any other order.

[0080] In some embodiments, each factor, truncation thereof, or domain thereof comprised in the fusion protein is directly connected to each other or connected via a linker.

[0081] In some embodiments, the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NOs: 21-26, 50-54, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 21-26, 50-54.

[0082] A third aspect of the present application provides an isolated nucleic acid molecule encoding the polypeptide of the first aspect or the fusion protein of the second aspect.

[0083] In some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the RNA comprises one or more of mRNA, srRNA (self-replicating RNA), siRNA (Small interfering RNA), circRNA (circular RNA).

[0084] The fourth aspect of the present application provides a vector comprising the nucleic acid molecule of the third aspect.

[0085] In some embodiments, the vector is an expression vector.

[0086] In some embodiments, the vector is a delivery vector.

[0087] In some embodiments, the c-Myc factor truncation, the OCT4 factor truncation, the SOX2 factor truncation, and / or the KLF4 factor truncation are present on the same expression vector or delivery vector, or are present on multiple expression vectors or delivery vectors.

[0088] In some embodiments, the delivery vector is an LNP, an LPP, a virus-like particle (VLP), an exosome, an extracellular vesicle, a micelle, a metallic nanoparticle, a nanoscale microsphere, a nanoemulsion, a dendrimer, or a hydrogel.

[0089] In some embodiments, the expression vector is a plasmid vector, an episomal vector, a transposon, or a viral vector.

[0090] In some embodiments, the viral vector is an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, a herpes viral vector, a retroviral vector, a Sendai viral vector, a cytomegalovirus vector, or a vaccinia viral vector.

[0091] In some embodiments, the viral vector can serve as a delivery vector.

[0092] The fifth aspect of the present application provides a composition for rejuvenation of a cell, tissue, or organ of a subject, the composition comprising the polypeptide, the fusion protein, the nucleic acid molecule, the vector, or a combination thereof of the present application.

[0093] In some embodiments, the composition comprises one or more of a c-Myc factor truncation, an OCT4 factor truncation, a SOX2 factor truncation, or a KLF4 factor truncation.

[0094] In some embodiments, the composition further comprises one or more of a c-Myc factor, an OCT4 factor, a SOX2 factor, and a KLF4 factor.

[0095] In some embodiments, the composition comprises the c-Myc factor truncation and one or more selected from the group consisting of an OCT4 factor, a SOX2 factor, and a KLF4 factor. In some embodiments, the composition comprises the c-Myc factor truncation and an OCT4 factor, a SOX2 factor, and a KLF4 factor. In some embodiments, the composition comprises a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 21 or 51 or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity thereto. In some embodiments, the composition comprises a polypeptide encoded by a nucleotide having a nucleotide sequence as set forth in SEQ ID NO: 14 or 46 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0096] In some embodiments, the composition comprises the OCT4-Myc factor truncation and one or more selected from the group consisting of a SOX2 factor and a KLF4 factor. In some embodiments, the composition comprises the OCT4-Myc factor truncation and a SOX2 factor and a KLF4 factor. In some embodiments, the composition comprises a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 22-23 or 52-53 or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity thereto. In some embodiments, the composition comprises a polypeptide encoded by a nucleotide having a nucleotide sequence as set forth in SEQ ID NO: 15-16 or 47-48 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0097] In some embodiments, the composition comprises the SOX2 factor truncation and one or more selected from the group consisting of an OCT4 factor, a KLF4 factor, and a c-Myc factor. In some embodiments, the composition comprises the SOX2 factor truncation and an OCT4 factor, a KLF4 factor, and a c-Myc factor. In some embodiments, the composition comprises a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 24 or 54, or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity thereto. In some embodiments, the composition comprises a polypeptide encoded by a nucleotide having a nucleotide sequence as set forth in SEQ ID NO: 17 or 49, or having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0098] In some embodiments, the composition comprises the KLF4 factor truncation and one or more selected from the group consisting of an OCT4 factor, a SOX2 factor, and a c-Myc factor. In some embodiments, the composition comprises the KLF4 factor truncation and an OCT4 factor, a SOX2 factor, and a c-Myc factor. In some embodiments, the composition comprises a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 25 or 26, or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity thereto. In some embodiments, the composition comprises a polypeptide encoded by a nucleotide having a nucleotide sequence as set forth in SEQ ID NO: 18 or 19, or having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0099] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor is a protein, a nucleic acid encoding the same, or a mixture thereof.

[0100] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA comprises one or more of mRNA, srRNA (self-replicating RNA), siRNA (Small interfering RNA), circRNA (circular RNA).

[0101] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor is a human protein, or a non-human protein, or a protein having at least 70%, at least 75%, at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the human protein or non-human protein and retains activity.

[0102] In some embodiments, the non-human protein is a corresponding protein of mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, or bird.

[0103] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor is a nucleic acid encoding a human protein, or a nucleic acid encoding a non-human protein, or a nucleic acid having at least 80% identity to the corresponding nucleic acid encoding a human protein, or a nucleic acid encoding a non-human protein and retains activity.

[0104] In some embodiments, the nucleic acid is a nucleic acid encoding a corresponding protein of mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, or bird.

[0105] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor is in the form of DNA or RNA.

[0106] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor is in the form of mRNA.

[0107] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor are in a complex form of different mRNA molecules.

[0108] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor are synthetic mRNA selected from the group consisting of wild-type form, mutant, or genetically engineered form.

[0109] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor are in the form of an expression vector or delivered by a delivery vector.

[0110] In some embodiments, the expression vector is a plasmid vector, episomal vector, transposon, or viral vector.

[0111] In some embodiments, the expression vector is an episomal vector, i.e., a non-integrated extrachromosomal plasmid that is able to replicate autonomously. In some embodiments, the episomal vector comprises an autonomous DNA replication sequence, i.e., a sequence that is able to cause replication of the vector.

[0112] In some embodiments, the viral vector is an adenoviral vector, adeno-associated viral vector (AAV), lentiviral vector, herpes viral vector, retroviral vector, Sendai viral vector, cytomegalovirus vector, or vaccinia viral vector.

[0113] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor are present on the same expression vector or delivery vector, or are present on multiple expression vectors or delivery vectors.

[0114] In some embodiments, the delivery vehicle is an LNP, LPP, virus-like particle (VLP), exosome, extracellular vesicle, micelle, metallic nanoparticle, nanoscale microsphere, nanoemulsion, dendrimer, or hydrogel.

[0115] A sixth aspect of the present application provides a method of rejuvenating a subject cell, tissue, or organ, comprising administering to the subject a polypeptide, fusion protein, nucleic acid molecule, vector, or composition described herein.

[0116] A seventh aspect of the present application provides use of a polypeptide, fusion protein, nucleic acid molecule, vector, or composition described herein in the manufacture of a medicament for rejuvenating a subject cell, tissue, or organ.

[0117] In some embodiments, the rejuvenated subject cell, tissue, or organ exhibits increased or decreased gene expression of one or more markers of senescence.

[0118] In some embodiments, the marker of senescence is selected from one or more of HP1y, H3K9me3, pl6, p21, SA-b-gal, and Lamin A / C protein.

[0119] In some embodiments, the rejuvenated subject cell, tissue, or organ exhibits decreased expression of one or more inflammatory factors.

[0120] In some embodiments, the inflammatory factor comprises one or more of IL-1b, IL-6, TNFa, IL22, and IL9.

[0121] In some embodiments, the rejuvenated subject cell, tissue, or organ exhibits increased or decreased gene expression of one or more markers of senescence and / or decreased expression of one or more inflammatory factors compared to a cell induced to age.

[0122] In some embodiments, the cell is a human cell or a non-human mammalian cell.

[0123] In some embodiments, the cell is selected from a skin-derived cell, a bone joint-derived cell, a muscle-derived cell, an eye-derived cell, a blood vessel-derived cell, a lung-derived cell, or a neural cell.

[0124] In some embodiments, the cell is selected from a fibroblast, an endothelial cell, a lung epithelial cell, a chondrocyte, a synoviocyte, a keratinocyte, an optic nerve cell, a corneal cell, a muscle cell, and other cells.

[0125] In some embodiments, the aged chondrocytes exhibit aging indicators (increased positive rate of β-gal staining, increased expression of inflammatory factor IL-6, increased p21, p16 mRNA levels, decreased epigenetic modification H3K9me3 levels, and decreased cell proliferation rate).

[0126] In some embodiments, the composition is administered intravenously, intraperitoneally, nasally, orally, intramuscularly, subcutaneously, intraocularly, parenterally, intratumorally, or topically. In some embodiments, the composition is directly injected into the target cells.

[0127] In some embodiments, the target cells are selected from the group consisting of fibroblast cells, endothelial cells, lung epithelial cells, chondrocytes, synoviocytes, keratinocytes, neural cells, and corneal cells, and other cells.

[0128] In some embodiments, rejuvenating the subject’s cells, tissues, or organs comprises improving, reversing, or inhibiting aging of the cells, tissues, or organs.

[0129] In some embodiments, rejuvenating the subject’s cells, tissues, or organs comprises restoring at least one of the transcriptional profile or epigenetic information of the cells, tissues, or organs that is lost due to aging, injury, disease, or any combination thereof.

[0130] In some embodiments, rejuvenating the subject’s cells, tissues, or organs comprises restoring the function of the cells, increasing the potency of the cells, enhancing the survival capacity of the cells, or increasing the replicative capacity or lifespan of the cells, or a combination thereof.

[0131] In some embodiments, rejuvenating the subject’s cells, tissues, or organs comprises promoting axonal regeneration of neuronal cells in the subject; promoting Retinal ganglion cell (RGC) cell regeneration in the subject; promoting fibroblast cell rejuvenation in the skin of the subject; promoting chondrocyte rejuvenation in the subject; promoting muscle stem cell proliferation in the subject; promoting vascular endothelial cell rejuvenation; promoting lung epithelial cell rejuvenation.

[0132] In some embodiments, the subject has, is suspected of having, or is at risk of having a disease or condition associated with aging, an ocular disease, a pulmonary disease, a skin disease, a bone and joint disease, a vascular disease.

[0133] In some embodiments, the subject has, is suspected of having, or is at risk of having a degenerative disease, a neurodegenerative disease, a cardiovascular disease, a peripheral vascular disease, a skin disease, an autoimmune disease, an endocrine entity disorder, a metabolic dysfunction, a musculoskeletal disease, a digestive system disease, a respiratory system disease, an ocular disease related to optic nerve damage, or a cartilage deformative condition.

[0134] In some embodiments, the subject is a mammal, including but not limited to a human, a mouse, a rat, a cow, a sheep, a horse, a dog, a cat, a pig, or a monkey.

[0135] In some embodiments, the subject cell, tissue, or organ is from, but not limited to, an eye, an ear, a nose, a mouth, including a gum and a tooth root; a bone, a lung, a breast, a pancreas, a stomach, an esophagus; a muscle, including a cardiac muscle; a liver, a blood vessel; a skin, including a hair; a heart, a brain, a neural tissue, a kidney, a testis, a prostate, a penis, a cloaca, a fin, an ovary, or an intestine.

[0136] The OSKM truncated body composition of the present application retains the ability of OSKM to reverse cell aging, while greatly reducing the risk of inducing pluripotent stem cells (iPSC) and gene length and greatly improving the safety and feasibility of clinical application of OSKM in restoring functionality while rejuvenating aging cells. BRIEF DESCRIPTION OF DRAWINGS

[0137] Figure 1 shows the modification strategy of OSKM factor protein truncated body.

[0138] Figure 2 shows the evaluation results of the anti-aging effect of the truncated body combination, wherein A and E are the SA-β-gal positive rates of different truncated body combinations, B and F are the H3K9me3 expression levels of different truncated body combinations, C and G are the p16 and p21 mRNA levels of different truncated body combinations, and D and H are the expression levels of inflammatory factor IL6 of different truncated body combinations.

[0139] Figure 3 shows the fluorescence staining results of different truncated body combinations.

[0140] Figure 4 shows the safety evaluation results of the truncated body combination CSK, wherein A is a transcriptome clustering heat map analysis chart of the truncated body combination CSK and OSKM, B is a signal pathway analysis chart of the truncated body combination CSK and OSKM, C is a clustering analysis chart of stem cell pluripotency related genes of the truncated body combination CSK and OSKM, and D is a clustering analysis chart of reprogramming process markers.

[0141] Figure 5 shows the long-term expression safety evaluation results of the truncated body combination CSK, wherein A is the ips reprogramming efficiency of the truncated body combination CSK and OSKM, B is a signal pathway analysis chart of the truncated body combination CSK and OSKM, C is a clustering analysis chart of stem cell pluripotency related genes of the truncated body combination CSK and OSKM, and D is a clustering analysis chart of reprogramming process markers.

[0142] Figure 6 shows the results of the pharmacodynamic evaluation of the truncated combination CSK and CSK-W in the optic nerve injury mouse ophthalmic model, wherein A is a schematic diagram of the in vivo pharmacodynamic experiment, B is a schematic diagram of the protein expression principle of AAV virus, C is a graph of RGC cell survival analysis of the truncated combination CSK and CSK+dox groups, D is a graph of axon regeneration analysis of the truncated combination CSK and CSK+dox groups, D is a graph of RGC cell survival analysis of the truncated combination CSK-W and CSK-W+dox groups, F is a graph of axon regeneration analysis of the truncated combination CSK-W and CSK-W+dox groups.

[0143] Figure 7 shows the results of the pharmacodynamic evaluation of the truncated combination CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, DOKM-W on the improvement of respiratory system lung cells.

[0144] Figure 8 shows the results of the pharmacodynamic evaluation of the truncated combination CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, DOKM-W on the improvement of skin cell aging.

[0145] Figure 9 shows the results of the pharmacodynamic evaluation of the truncated combination CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, DOKM-W on the improvement of vascular cell aging.

[0146] Figure 10 shows the results of the pharmacodynamic evaluation of the truncated combination CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, DOKM-W on the promotion of myoblast cells. DETAILED DESCRIPTION

[0147] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description of the present application is made below in combination with the drawings and examples. The specific examples described herein are only used to explain the present application and do not constitute any limitation to the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0148] Unless the context clearly indicates otherwise, the indication of a quantity in the present application without a specific number includes its plural form. For example, the mention of "cells" includes one or more such cells and equivalents known to those skilled in the art, etc.

[0149] The term "about" as used herein denotes a range of ±20% of the numerical value that follows. In some embodiments, the term "about" denotes a range of ±10% of the numerical value that follows. In some embodiments, the term "about" denotes a range of ±5% of the numerical value that follows.

[0150] The term "c-Myc" as used herein refers to a transcription factor encoded by the gene c-myc that controls cell proliferation. c-Myc is capable of forming a heterodimer with the transcription factor MAX, and the heterodimer is capable of binding to an E box sequence (E Box, with the sequence 5'-CACGTG-3') on a nucleic acid (e.g., an engineered nucleic acid) to regulate transcription of a target gene. In some embodiments, a c-Myc factor includes, from N-terminus to C-terminus, a transactivation domain (MTAD domain, SEQ ID NO: 33), a central domain (CP domain, SEQ ID NO: 34), and a basic region alpha helix 1-loop-alpha helix 2 leucine zipper domain (bHLHLZ domain, SEQ ID NO: 35). In some embodiments, a c-Myc factor truncation lacks any one or both of the MTAD domain, the CP domain, the bHLHLZ domain relative to a c-Myc factor.

[0151] The term "OCT4" as used herein refers to a member of the family of octamer transcription factors that plays a key role in maintaining the pluripotency of a cell. "OCT4" can also be referred to as octamer-binding transcription factor, OCT3, OCT3 / 4, POU5F1, or POU class 5 homeobox 1, and is a transcription factor involved in embryonic development and determining cell fate. Like other OCT transcription factors, OCT4 is characterized by a bipartite DNA-binding domain known as the POU domain. In some embodiments, an OCT4 factor includes, from N-terminus to C-terminus, an N-terminal transactivation domain (NTD domain, SEQ ID NO: 36), a Pit-Oct-Unc domain (POU domain, SEQ ID NO: 37), and a C-terminal transactivation domain (CTD domain, SEQ ID NO: 38). In some embodiments, an OCT4 factor truncation lacks any one or both of the NTD domain, the POU domain, the CTD domain relative to an OCT4 factor. In some embodiments, the OCT4 factor truncation lacks the NTD domain. In some embodiments, the DNA sequence of the OCT4 factor truncation is set forth in SEQ ID NO: 55, and the amino acid sequence is set forth in SEQ ID NO: 57. In some embodiments, the OCT4 factor truncation includes only the POU domain.

[0152] The term "SOX2", also known as "SRY-2" and "Sex-determining Region Y", as used herein, is a transcription factor involved in self-renewal of undifferentiated embryonic stem cells, maintenance of embryonic stem cells and neural stem cells, and malignant phenotype of certain cancers. SOX2 is one of the members of the Sox family of transcription factors, which share a high-mobility group (HMG) box domain of about 80 amino acids. In some embodiments, the SOX2 factor includes, from N- to C-terminus, a high-mobility group domain (HMG domain, SEQ ID NO: 39), a dimerization domain (DIM domain, SEQ ID NO: 40), and a transactivation domain (TAD domain, SEQ ID NO: 41). In some embodiments, the SOX2 factor truncates missing any one or both of the HMG domain, the DIM domain, the TAD domain, relative to the SOX2 factor. In some embodiments, the SOX2 factor truncates replacing the TAD domain with amino acids 92-100 of the TAD domain, relative to the SOX2 factor.

[0153] The term "KLF4", as used herein, denotes a member of the Krüppel-like family of transcription factors, which can also be referred to as Kruppel-like factor 4, EZF, or GKLF, and is a zinc-finger transcription factor. KLF4 is involved in the regulation of differentiation and proliferation, and is capable of interacting with coactivators including members of the p300-CBP coactivator family. In some embodiments, the KLF4 factor includes, from N- to C-terminus, a transcription activation domain (AD domain, SEQ ID NO: 42), a transcription inhibition domain (ID domain, SEQ ID NO: 43), and a zinc-finger domain (ZnF domain, SEQ ID NO: 44). In some embodiments, the KLF4 factor truncates missing any one or both of the AD domain, the TD domain, the ZnF domain, relative to the KLF4 factor.

[0154] The OSKM four-factor combination (OCT4, SOX2, KLF4, c-Myc) can reprogram cells, and the four transcription factor combinations are transferred into differentiated somatic cells by using a viral vector, so that the somatic cells are reprogrammed to obtain a cell type similar to embryonic stem cells, thereby de-differentiating the somatic cells to a state similar to embryonic stem cells. This cell is similar to human embryonic stem cells and has super strong differentiation ability, and can differentiate into human blood cells, bone cells, nerve cells, etc., and further cultivate human organs, bones, cornea, pancreas, etc. The genes contained in the reprogramming factors can be listed as OCT3 / 4, SOX2, SOX1, SOX3, SOX15, SOX17, KLF4, KLF2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc.

[0155] The term "induced pluripotent stem cell (iPSC)" as used herein refers to a stem cell produced from a differentiated adult cell that has been induced or altered (i.e., reprogrammed) to be able to differentiate into cells having all three germ layers or dermal layers of tissue: mesoderm, endoderm, and ectoderm.

[0156] The term "truncation" or specifically "truncation of c-Myc factor, OCT4 factor, SOX2 factor or KLF4 factor" as used herein refers to a gene sequence of each factor of OSKM or any combination thereof being modified by genetic engineering technology, and a part of the sequence being deleted, so as to express a factor with shorter length. Such truncation usually has similar biological properties as the factor or any combination thereof, but may have better performance in certain specific aspects or be suitable for specific application scenarios.

[0157] In some embodiments, the c-Myc factor truncation and the OCT4 factor truncation are combined to obtain an OCT4-Myc truncation. In some embodiments, the OCT4-Myc truncation comprises an OCT4 factor NTD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain. In some embodiments, the OCT4-Myc truncation comprises a c-Myc factor MTAD domain, and an OCT4 factor POU domain and a CTD domain. In some embodiments, the OCT4-Myc truncation comprises a c-Myc factor MTAD domain and a bHLHLZ domain, and an OCT4 factor POU domain. In some embodiments, the OCT4-Myc truncation comprises, in order from N-terminus to C-terminus, an OCT4 factor NTD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain. In some embodiments, the OCT4-Myc truncation comprises, in order from N-terminus to C-terminus, a c-Myc factor MTAD domain, an OCT4 factor POU domain, and an OCT4 factor CTD domain. In some embodiments, the OCT4-Myc truncation comprises, in order from N-terminus to C-terminus, a c-Myc factor MTAD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain. The domains are connected directly or through a linker. In some embodiments, the OCT4-Myc truncation has an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the OCT4-Myc truncation has an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the OCT4-Myc truncation has an amino acid sequence set forth in SEQ ID NO: 58 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58.

[0158] In some embodiments, the c-Myc factor truncation, the c-Myc factor, the OCT4 factor truncation, the OCT4 factor, the OCT4-Myc truncation, the SOX2 factor truncation, the SOX2 factor, the KLF4 factor truncation, and / or the KLF4 factor are present on multiple expression vectors.

[0159] In some embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor are each present on separate expression vectors, which are combined by mixing the respective expression vectors together for use in combination, as shown in Table 1 below. There are two variants of the KLF4 factor, one of which (referred to herein as KLF4-W) has nine additional amino acids after the start codon than the other, both of which were tested by the inventors.

[0160] Table 1

[0161] In other embodiments, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation, and / or KLF4 factor are present on the same expression vector. In combination, the respective genes are synthesized according to the different truncation combinations shown in Table 2 below, and then incorporated into an expression vector for expression.

[0162] Table 2

[0163] As used herein, the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some embodiments, two or more linkers can be connected in series. When multiple linkers are present, each linker can be the same or different. Generally, linkers provide flexibility or prevent / improve steric hindrance. Linkers are generally not cleaved; however, in certain aspects, such cleavage can be desirable. Thus, in some embodiments, a linker can include one or more protease-cleavable sites, which can be within the linker sequence or flanking the linker sequence on either end of the linker.

[0164] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker can include at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids. In some embodiments, the linker includes one or more amino acids. In some embodiments, the linker includes a Gly-Ser (GS) linker. In some embodiments, the GS linker includes (G4S)n, where n is an integer between 1 and 10. In some embodiments, the GS linker includes (G3S)n, where n is an integer between 1 and 10.

[0165] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a linked sequence of amino acids, and also to refer to gene products, homologs, orthologs, paralogs, fragments, any protease-derived peptides (fragments), and other equivalents, variants, and analogs of polymers of amino acids. The term can include not only polypeptides in isolated form, but also active fragments and derivatives thereof. Polypeptides can be naturally occurring polypeptides or recombinantly, engineered, or synthetically produced polypeptides. Particular polypeptides can be derived or excised from natural proteins, for example, by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques, or can be strategically truncated or fragmented to yield active fragments that maintain activity against the same or similar activities.

[0166] "Domain" as used herein is a contiguous or non-contiguous sequence of amino acid residues that refers to a set of amino acids that are conserved at a particular position along a sequence alignment of evolutionarily related proteins. While amino acids at other positions can vary between homologs, highly conserved amino acids at a particular position indicate amino acids that can be essential for structure, stability, or function of the protein. Domains are identified by high degree of conservation in aligned sequences of a family of protein homologs, and they can be used as identifiers to determine whether an arbitrary polypeptide in question belongs to a previously identified family of polypeptides.

[0167] As used herein, "fusion protein" refers to a polypeptide composed of at least two polypeptides or domains and optionally a linker, resulting in a fusion protein that generally has two or more domains or segments with different properties or functions. In some embodiments, the fusion protein also refers to a polypeptide comprising two or more polypeptides or peptides covalently linked, directly or via an amino acid or linker. The polypeptides forming the fusion protein are generally linked C-terminus to N-terminus, they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The linker operably links the two polypeptides into one continuous polypeptide, e.g., produced by recombinant methods. The two polypeptides can be operably attached directly or indirectly.

[0168] "Isolated" when used to describe various polypeptides or polynucleotides herein refers to a polypeptide or polynucleotide that has been identified and separated and / or recovered from a component of its natural environment. Recombinant polynucleotides and polypeptides, as well as chemically synthesized polynucleotides and polypeptides, are also encompassed.

[0169] As used herein, "polynucleotide" is used interchangeably with "nucleic acid" and refers to a biological molecule comprising two or more nucleotides. A polynucleotide can be single-stranded or double-stranded nucleic acid. Polynucleotides include RNA genomic sequences, cDNA sequences, RNA sequences transcribed from cDNA, and analogs of natural polynucleotides. Generally, the polynucleotide consists of nucleotides naturally occurring in DNA or RNA, such as adenine, thymine, guanine, cytosine, uracil, deoxyadenine, deoxythymine, deoxyguanine, and deoxycytosine, joined by phosphodiester bonds. In some embodiments, a polynucleotide comprises molecules of nucleotides or nucleotide analogs that contain chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules can be preferred for certain applications. When the present application refers to polynucleotides, it is understood that both DNA, RNA are provided and in each case single-stranded and double-stranded forms are provided (as well as the complement of each single-stranded molecule). As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or to sequence information that biochemically characterizes a particular nucleic acid (i.e., the sequence of letters used as base abbreviations).

[0170] In this text, amino acids can be represented by the commonly known three-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission or by the one-letter symbols well known in the art. Nucleotides can be represented by the commonly known one-letter symbols well known in the art.

[0171] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matches between the sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A match is any position in which the same nucleotide or amino acid is found in both the target and reference sequences. Gaps are not nucleotides or amino acids, so a gap in the target sequence is not counted. Likewise, a gap in the reference sequence is not counted because it is not a nucleotide or amino acid from the reference sequence that is being counted. Percent sequence identity can be calculated by determining the number of positions in which the same amino acid residue or nucleic acid base occurs in both sequences, resulting in a number of matches, dividing the number of matches by the total number of positions in the comparison window, and multiplying the result by 100 to yield percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software readily available for online use and download.

[0172] As used herein, "vector" refers to a macromolecule or association of macromolecules that comprises or is associated with a polynucleotide and that can be used to mediate delivery of the polynucleotide to a cell. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.

[0173] As used herein, "expression vector" refers to any type of genetic construct comprising a nucleic acid of the application, wherein some or all of the nucleic acid coding sequence is capable of being transcribed for the purpose of effecting expression of a polypeptide or protein in an intended target cell. The expression vector can be a vector known in the art for expressing foreign proteins in plants, animals, or microorganisms, such as plasmids, minicircles, viral vectors, liposomes, and the like. It can be formed using various methods known in the art. The expression vector further comprises control elements operably linked to the coding region to facilitate expression of the polypeptide or protein in the target cell. Control elements such as promoters, enhancers, UTRs, miRNA targeting sequences, and the like. Many such control elements are known in the art or can be readily constructed from components available in the art.

[0174] As used herein, “viral vector” refers to a vector created from at least a portion of a viral genome that can be used to carry or deliver one or more polynucleotide regions encoding or comprising molecules of interest (e.g., proteins, polypeptides, and oligonucleotides, or any combination thereof). Viral vectors can be used to deliver genetic material into a cell. Viral vectors can be modified for specific applications. In some embodiments, the expression vector is a viral vector selected from an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector. In some embodiments, the viral vector refers to a virion or viral particle that functions as a nucleic acid delivery vehicle, comprising a vector genome packaged within the virion or viral particle. Viral vectors include adeno-associated viral (AAV), retroviral vectors, bovine papilloma virus, adenoviral vectors, vaccinia virus, or polyoma virus.

[0175] In some embodiments, the viral vector is an AAV vector. As used herein, an AAV vector refers to any vector comprising or derived from components of an adeno-associated vector and suitable for infecting mammalian cells, preferably human cells. An AAV vector can be derived from various serotypes, including combinations of serotypes (i.e., “pseudotyped” AAV) or from various genomes (e.g., single-stranded or self-complementary). Further, an AAV vector can be replication-defective and / or targeted. Illustratively, the AAV vector includes, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutant forms thereof, as well as any other AAV vector now known or later discovered. An AAV vector can have all or a portion of one or more AAV wild-type genes deleted, preferably the rep and / or cap genes, but retaining functional flanking ITR sequences.

[0176] In some embodiments, the viral vector is a retroviral vector. As used herein, the term "retroviral vector" refers to a vector containing structural and functional genetic elements derived primarily from a retrovirus. In some embodiments, the retroviral vector is derived from an alpha retrovirus (e.g., avian leukosis virus), a beta retrovirus (e.g., mouse mammary tumor virus), a gamma retrovirus (e.g., murine leukemia virus), a delta retrovirus (e.g., bovine leukemia virus), an epsilon retrovirus (e.g., Walley dermatosarcoma virus), a lentivirus (e.g., HIV-1, HIV-2), and a spumavirus (e.g., human spumavirus). In some embodiments, the viral vector is a lentivirus vector. Lentivirus vectors are viral vectors developed based on HIV-1 (human immunodeficiency virus type 1). Lentivirus vectors have a broader host range than retroviral vectors, and lentiviruses can efficiently infect cells that are not in the cell cycle and post-mitotic cells. Lentivirus expression vectors contain the genetic information required for packaging, transfection, and stable integration. Lentivirus packaging plasmids provide all the packaging RNAs and accessory proteins required for recombination of the pseudoviral vector after transcription and translation. The vector can effectively integrate exogenous genes into the target cell chromosome, thereby achieving persistent expression. For some cells that are difficult to transfect, such as neuronal cells, liver cells, myocardial cells, tumor cells, endothelial cells, stem cells, undifferentiated cells, etc., the transduction efficiency of the target gene can be greatly improved. Moreover, the probability of the target gene integrating into the target cell genome is greatly increased, thereby achieving good prevention or treatment effect. In some embodiments, the lentivirus vector includes, but is not limited to, HIV-1, HIV-2, SIV, FIV, EIAV, BIV, VISNA, and CAEV vectors. In some embodiments, the lentivirus vector includes a tetracycline-on (Tet-On) expression regulation system. The Tet-On system generally uses a reverse tetracycline transactivator (rtTA) to induce gene expression. The reverse tetracycline transactivator (rtTA) contains a mutant tetracycline repressor DNA binding protein (TetR) and a transactivation domain. These transactivators can be activated in the presence of tetracycline and then bind to the promoter containing the tetracycline response element (TRE) to induce gene expression.

[0177] Nanoparticles that can be used as delivery vehicles include LNPs, PNPs, LPPs, INPs, and the like. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP). As used herein, the term "lipid nanoparticle (LNP)" refers to any small vesicle formed from one or more lipid components that can partition hydrophobic molecules within a lipid membrane or encapsulate water-soluble particles or molecules in an aqueous core. These LNPs include four lipids 1) an ionizable lipid, (2) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), (3) cholesterol, and (4) a polyethylene glycol-lipid conjugate (PEG-DMG). These components help to form uniform nanoparticles, enhance the stability of the nanoparticles, facilitate efficient encapsulation of nucleic acids, assist in cellular uptake, and promote endosomal escape of the lipid nanoparticle. In some embodiments, the delivery vehicle is a lipopolyplex (LPP), a new delivery system that combines a negatively charged nucleic acid molecule and a positively charged polymer to form a dense core, which is then encapsulated in a lipid bilayer to form a core-shell structure similar to a viral particle.

[0178] As used herein, "extracellular vesicle (EV)" refers to a cell-derived vesicle comprising a membrane that encloses an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) that are smaller in diameter than the cell from which they are derived. Typically, extracellular vesicles range in diameter from 20 nm to 1000 nm, and can include various macromolecular payloads or cargos within the internal space, displayed on the outer surface of the extracellular vesicle, and / or across the membrane. The payloads can include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. Various types of extracellular vesicles exist, which are named according to their site of origin in the cell, size, and structural and / or functional properties. Illustratively, extracellular vesicles include, but are not limited to, exosomes (e.g., small and large exosomes), ectosomes, macropinocytotic vesicles, microparticles, apoptotic bodies, vesicular organelles, oncosomes, exosome granules, cell-derived nanovesicles (e.g., produced by grinding or shearing cells), liposomes, or analogs known to one of ordinary skill in the art. Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some embodiments, the extracellular vesicles are produced by cells comprising the expression vector.

[0179] Extracellular vesicles can be produced naturally through known or unknown biosynthetic pathways. Production of extracellular vesicles can be facilitated by using mechanical methods such as cell homogenization or cell shearing, in which cells are homogenized or sheared, causing partial or fractional cell membranes to form vesicles. For example, CDNs can be formed by using mechanical methods such as cell homogenization or cell shearing, in which cells are homogenized or sheared, causing partial or fractional cell membranes to form vesicles. Further non-limiting examples of mechanical methods that can be used to form cell-derived nanovesicles are described in further detail in, e.g., Goh, W.J. et al. Bioinspired Cell-Derived Nanovesicles versus Exosomes as Drug Delivery Systems: A Cost-Effective Alternative. Sci Rep. (2017) 7: 14322 https: / / doi.org / 10.1038 / s41598-017-14725-x, the contents of which are incorporated herein by reference in their entirety.

[0180] As used herein, “Exosome” or “Exo” refers to an extracellular vesicle with a diameter between 20-300 nm (e.g., between 40-200 nm). Exosomes comprise a membrane enclosing an internal space (lumen), and in some embodiments, can be produced from a cell (e.g., a producer cell) by direct plasma membrane budding or by fusion of a late endosome with the plasma membrane.

[0181] As used herein, the term “virus-like particle” or “VLP” refers to a structure that resembles a viral particle but does not contain a viral genome, is not capable of replication, and is not pathogenic. The particle typically comprises at least one structural protein from a virus. Preferably, only one structural protein is present. Most preferably, no other non-structural components of the virus are present. Thus, a virus-like particle can spontaneously self-assemble from viral structural proteins in vitro under appropriate conditions, while excluding genetic material and potential replication possibilities. Virus-like particles of about 20 to 150 nm in diameter also have the characteristics of nanomaterials, such as large surface area, surface accessible amino acids with reactive moieties (e.g., lysine and glutamic acid residues), inerratic spatial structure, and good biocompatibility. Thus, assembled virus-like particles have great potential as specialized delivery systems for carrying various forms of compositions described herein in combination.

[0182] As used herein, "hydrogel" refers to a three-dimensional, hydrophilic or amphiphilic polymer network capable of absorbing large amounts of water. The network is composed of homopolymers or copolymers and is insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks. The crosslinks provide the network structure and physical integrity. The hydrogel exhibits thermodynamic compatibility with water, swelling in aqueous media. The network's chains are connected in a manner that allows diffusion of water-soluble solutes within and outside the network, with the presence of pores and dimensions smaller than the pores.

[0183] Any of the polypeptides and / or fusion proteins described herein can be used in gene therapy via an appropriate polynucleotide (e.g., DNA or RNA) encoding the desired polypeptide and / or fusion protein. The "gene therapy" refers to a method of treatment provided by using a composition comprising a delivery system and a polynucleotide of the present application. Exemplary gene therapy delivery systems include, but are not limited to, viral vectors, liposomes, lipid-nucleic acid nanoparticles, exosomes, and gene editing systems. For example, a gene editing system such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein 9 (CRISPR-Cas-9), Transcription Activator-like Effector Nuclease (TALEN), or ZNF (zinc finger protein) can be used to insert the polynucleotide into the DNA of a target cell.

[0184] As used herein, "aging" refers to a process of loss and deterioration of an organism over time in terms of constituent materials, tissue structure, physiological function, and the like. In the present application, aging refers to biological aging. As used herein, "anti-aging" refers to the delay, retardation, reduction, cessation, and / or reversal of the effects or progression of aging. As used herein, "aging cell" refers to the state of aging of all kinds of cells (e.g., muscle cells, connective cells, lung cells, vascular cells, skin cells, etc.) known to those skilled in the art. In the present application, the feature can be the restoration of the function or morphology of the aging cell to that of a normal cell by the change in the above-mentioned cell aging phenotype. In the present application, the aging cell includes, but is not limited to, an aging fibroblast, myoblast, lung epithelial cell, retinal pigment epithelial cell, vascular cell, and skin cell.

[0185] The term "H3K9me3" as used herein refers to histone H3 lysine 9 trimethylation (H3 lysine 9 trimethylation, H3K9me3). The term "SA-β-gal" as used herein refers to senescence-associated β-galactosidase. The term "P16 protein" as used herein refers to the protein expression product of the CDKN2A gene, which is a cell senescence marker. The term "Lamin A / C" as used herein refers to a nuclear membrane protein encoded by the LMNA gene, which plays a key role in maintaining the stability of the nuclear structure and regulating gene expression. The term "HP1 γ" as used herein refers to senescence-associated heterochromatin protein 1 gamma.

[0186] The term "administering" as used herein refers to introducing a polypeptide, fusion protein, nucleic acid, virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV), or any combination or composition thereof of the present application. The polypeptide, fusion protein, nucleic acid, virus (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV), or any combination or composition thereof of the present application can be administered intravenously, intradermally, intra-arterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostatically, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesically, mucosally, intrapericardially, intranipple, intraocularly, orally, topically, systemically, by injection, by infusion, by continuous infusion, by direct local perfusion into an

[0187] Examples and drawings are provided below to assist in the understanding of the present application. It is understood that the examples and drawings are merely used to illustrate the present application and do not constitute any limitation. The actual protection scope of the present application is set forth in the claims. It is understood that any modification and change can be made without departing from the spirit of the present application.

[0188] Example 1

[0189] By splitting the OSKM factor functional domain and recombining, six truncated bodies were constructed (see Figure 1), wherein,

[0190] The DNA sequence of c-Myc Truncation A is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 7;

[0191] The DNA sequence of OCT4-Myc Truncation B is shown as SEQ ID NO: 2, and the amino acid sequence is shown as SEQ ID NO: 8;

[0192] The DNA sequence of OCT4-Myc Truncation C is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 9;

[0193] The DNA sequence of OCT4-Myc Truncation G is shown as SEQ ID NO: 57, and the amino acid sequence is shown as SEQ ID NO: 58;

[0194] The DNA sequence of SOX2 Truncation D is shown as SEQ ID NO: 4, and the amino acid sequence is shown as SEQ ID NO: 10;

[0195] The DNA sequence of KLF4 Truncation E is shown as SEQ ID NO: 5, and the amino acid sequence is shown as SEQ ID NO: 11;

[0196] The DNA sequence of KLF4 Truncation F is shown as SEQ ID NO: 6, and the amino acid sequence is shown as SEQ ID NO: 12.

[0197] As shown in FIG. 1, the constructed truncations are respectively recombined to construct corresponding six kinds of truncation compositions:

[0198] c-Myc Truncation A is combined with complete OSK (OCT4, SOX2 and KLF4 / KLF4-W) to form AOSK / AOSK-W, and the total length of the DNA sequence fragments of AOSK is reduced to 86.2% of OSKM, and the total length of the DNA sequence fragments of AOSK-W is reduced to 86.3% of OSKM-W;

[0199] OCT4-Myc Truncation B is combined with complete SK (SOX2 and KLF4 / KLF4-W) to form BSK / BSK-W, and the total length of the DNA sequence fragments of BSK is reduced to 71.9% of OSKM, and the total length of the DNA sequence fragments of BSK-W is reduced to 72.1% of OSKM-W;

[0200] OCT4-Myc Truncation C is combined with complete SK (SOX2 and KLF4 / KLF4-W) to form CSK / CSK-W, and the total length of the DNA sequence fragments of CSK is reduced to 73.0% of OSKM, and the total length of the DNA sequence fragments of CSK-W is reduced to 73.2% of OSKM-W;

[0201] SOX2 truncation D is combined with complete OKM (OCT4, KLF4 / KLF4-W and c-Myc) to form DOKM / DOKM-W, the total length of DNA sequence fragments of DOKM is reduced to 90.2% of OSKM, and the total length of DNA sequence fragments of DOKM-W is reduced to 90.1% of OSKM-W;

[0202] KLF4 truncation E is combined with complete OSM (OCT4, SOX2 and c-Myc) to form EOSM, the total length of DNA sequence fragments is reduced to 89.9% of OSKM;

[0203] KLF4 truncation F is combined with complete OSM (OCT4, SOX2 and c-Myc) to form FOSM, the total length of DNA sequence fragments is reduced to 79.9% of OSKM.

[0204] After the above-mentioned truncated body and the coding nucleic acid sequence of the four factors of OCT4 (Gene ID: 5450), SOX2 (Gene ID: 6657), KLF4 (Gene ID: 9314) and c-Myc (Gene ID: 4609) are synthesized by Nanjing Kingsriver Biotechnology Co., Ltd., the synthesized genes are respectively transferred to FUW-TetON lentivirus vector by EcoRI single enzyme digestion and connection, and the packaged lentivirus is transduced into 293T cells. After 72 hours, the virus supernatant is collected, filtered through a 0.22 μm membrane and stored at -80°C. By mixing the above-mentioned virus supernatant, the above-mentioned different truncated body compositions are obtained, wherein OSKM and OSKM-W are control compositions.

[0205] Example 2

[0206] In order to explore the influence of truncation modification on the reprogramming ability of aging, the chondrocyte cell line C28 / I2 (Qingqi (Shanghai) Biotechnology Development Co., Ltd., #BFN60803901) is used for effect evaluation. The specific method is as follows:

[0207] 1. On day 0, C28 / I2 is inoculated in a 24-well plate containing complete culture medium (DMEM, 10% fetal bovine serum, 1% PS) at 4×10 4 cells / well;

[0208] 2. On day 1, the fresh virus supernatant obtained in Example 1 is added to the above-mentioned culture medium together with polybrene;

[0209] 3. On day 2, the culture medium is replaced with aging induction culture medium (DMEM, 10% fetal bovine serum, 20 μM etoposide, 1% PS);

[0210] 4. On day 3, the aging induction medium was replaced with complete medium (DMEM medium containing 10% fetal bovine serum (FBS)) containing 1 pg / ml doxycycline;

[0211] 5. On day 9, the supernatant was collected for IL6 content detection using a human IL-6 ELISA kit (Duo, #1110602), the cells were collected, and RNA extraction was performed using an RNA extraction kit (Genorbid, #AE311-02) for subsequent RT-qPCR experiments to detect p16 / p21 expression (primer sequences are shown in Table 3). The collected cells were stained using a β-gal staining kit (Bi Yun Tian, #C0602) for immunofluorescence experiments (abcam, #ab176916), and then the SA-β-gal positive rate, H3K9me3 expression, and p16 / p21 mRNA level were detected.

[0212] Table 3

[0213] The results show that each combination of truncations can significantly inhibit the aging of chondrocytes (see Figure 2). When chondrocytes are affected by etoposide, they exhibit symptoms of aging. Specifically, the SA-β-gal positive rate increases, the expression level of the epigenetic modifier H3K9me3 decreases, the mRNA level of the aging-related genes p16 / p21 increases, and the expression level of the inflammatory factor IL6 increases. When the cells are transfected with OSKM or the combination of truncations, the symptoms of aging are significantly improved, the SA-β-gal positive rate decreases (A, E in Figure 2), the expression level of the epigenetic modifier H3K9me3 increases (B, F in Figure 2), the mRNA level of the aging-related genes p16 and p21 decreases (C, G in Figure 2), and the expression level of the inflammatory factor IL6 decreases (D, H in Figure 2). No significant difference was found between OSKM / OSKM-W and the combination of truncations, indicating that each combination of truncations retains the anti-aging ability of OSKM / OSKM-W.

[0214] Example 3

[0215] To further detect whether the truncation modification can significantly inhibit the iPSC induction ability or even make it lose, an ips reprogramming experiment was performed using mouse embryonic fibroblasts (MEF cells). The specific method is as follows:

[0216] 1. On day 0, P1 generation MEF cells were inoculated in DMEM with 10% fetal bovine serum (FBS) in a 12-well plate;

[0217] 2. On day 1, the fresh virus supernatant obtained in Example 1 was added to the culture medium together with polybrene;

[0218] 3. On day 2, the medium was replaced with iPSC induction medium (DMEM, 10% fetal bovine serum, 10% serum replacement (Gibco, #10828010), 1x non-essential amino acid solution (Gibco, #11140050), 1 mM glutamine, 1% PS, 55 mM beta-mercaptoethanol, 1 pg / ml doxycycline) and cultured for 7 days;

[0219] 4. On day 9, replaced with iPSC maintenance medium (Neurolbasal / B27 mix: DMEM / F12 / N2 mix = 1:1, 1x non-essential amino acid solution, 1 mM glutamine, 1% PS, 55 mM beta-mercaptoethanol, 10 3 units of LIF, 1 mM Mirdametinib, 3 mM Laduviglusib HC1) and cultured for 7 days;

[0220] 5. On day 16, immunofluorescence staining (CST, #8822S) was performed to detect the number of Nanog-positive clones. In addition, MEF cells on day 0, OSKM cells on day 6, truncated combination cells on day 12, and mouse ESC cells were collected for transcriptome sequencing (the sequencing work was completed by Lianchuan Biology).

[0221] The results showed that OSKM and OSKM-W could reprogram MEF into iPSC, with efficiencies of 0.3525% and 0.3775%, respectively, while the iPSC formation ability of truncated combination was almost lost (truncated combination DOKM, EOSM, FOSM, CSK, and DOKM-W, CSK-W) or greatly weakened (truncated combination AOSK, BSK, and AOSK-W, BSK-W) (see Figure 3 and Tables 4-1 and 4-2).

[0222] Table 4-1

[0223] Table 4-2

[0224] Example 4

[0225] To further explore the differences between truncated combination and OSKM in reprogramming, transcriptome sequencing was performed in this example, and the results of OSKM and CSK are exemplarily shown (see Figure 4). Cluster heat map analysis showed that the expression profile of OSKM was closer to embryonic stem cells (ESC), while CSK was more similar to mouse embryonic fibroblasts (MEF cells) (Figure 4A), indicating that after reprogramming, MEF induced by OSKM was significantly dedifferentiated, and gradually transformed into ESC, while CSK did not significantly dedifferentiate MEF cells. The signal pathway analysis was consistent with the cluster heat map analysis (Figure 4B).

[0226] Next, the present embodiment selects stem cell pluripotency-related genes for cluster analysis again, and the results again prove that OSKM induces MEF cells to reprogram, causing pluripotency-related genes to be expressed, while CSK hardly does (Fig. 4C). The reprogramming process is a gradual process, during which the cells will go through the initial, mature and stable stages, each stage having its own unique markers, so that the progress of reprogramming can be reflected by detecting the expression of the markers. The results show that OSKM is mainly in the stable and part of the mature stage, while CSK is in the initial safe stage (Fig. 4D).

[0227] Example 5

[0228] In order to further explore whether the long-term expression of the truncated body combination will cause the cell to dedifferentiate into iPSC, the present embodiment prolongs the action time of the ips induction medium in Example 3. The specific method is as follows:

[0229] 1. P1 generation MEF cells were inoculated in DMEM with 10% fetal bovine serum (FBS) in a 12-well plate on day 0;

[0230] 2. Fresh virus supernatant obtained in Example 1 was added to the culture medium together with polybrene on day 1;

[0231] 3. The culture medium was replaced with iPSC induction medium (DMEM, 10% fetal bovine serum, 10% serum replacement, 1x non-essential amino acid solution, 1 mM glutamine, 1% PS, 55 μM β-mercaptoethanol, 1 μg / ml doxycycline) on day 2, and the culture was continued for 12 days;

[0232] 4. The iPSC maintenance medium (Neurolbasal / B27 mixture: DMEM / F12 / N2 mixture = 1:1, 1x non-essential amino acid solution, 1 mM glutamine, 1% PS, 55 μM β-mercaptoethanol, 10^3 units of LIF, 1 μM Mirdametinib, 3 μM Laduviglusib HCl) was replaced on day 14, and the culture was continued for 7 days;

[0233] 5. The cells were immunofluorescently stained (CST, #8822S) on day 21, and the fluorescence staining method was the same as that in Example 3. In addition, the CSK combination cells collected on day 19 were subjected to transcriptome sequencing (the sequencing work was completed by Lianchuan Biology).

[0234] Figure 5 shows the results of CSK, which shows that prolonging the induction time of CSK does not cause MEF cells to transform into iPSCs, and the iPSC reprogramming efficiency is still 0 (Figure 5A), the stem cell pluripotency regulation signal pathway is not activated (Figure 5B), and the stem cell pluripotency related gene expression profile is highly similar to that of MEF cells on day 12 (Figure 5C), and the reprogramming progress is still in the initial safe stage (Figure 5D). In summary, CSK does not significantly cause cells to change their identity like OSKM, triggering iPSCs and creating safety risks, but rather preserves the original identity of the target cells as much as possible, and does not change with the extension of the action time.

[0235] Example 6

[0236] In order to further detect whether each truncated body combination can significantly promote the optic nerve regeneration of mice in vivo and play an aging reprogramming effect, an in vivo pharmacodynamic test was performed using a mouse optic nerve injury model. Figure 6A shows a schematic diagram of the in vivo pharmacodynamic test, and the specific method is as follows:

[0237] 1. Preparation of AAV-TRE-CSK, AAV-TRE-CSK-W, and AAV-CAG-tTA viruses

[0238] The CSK gene fragment (SEQ ID NO: 16) or CSK-W gene fragment (SEQ ID NO: 48) was synthesized into an AAV2 vector containing a tetracycline response element (TRE3G promoter) and an SV40 element to form an AAV-TRE-CSK or AAV-TRE-CSK-W vector (Figure 6B), and AAV-CAG-tTA was purchased from Wuhan Privy Brain Science and Technology Co., Ltd.; AAV was injected into the vitreous body.

[0239] 2. Construction of optic nerve clip injury model

[0240] After about 4-week-old mice were anesthetized, 1.5 μl of virus solution was injected into the vitreous body, of which 1 μl was AAV-TRE-CSK or AAV-TRE-CSK-W, and 0.5 μl was AAV-CAG-rTA. After completing the virus injection, the mice were divided into a virus group and a virus+dox group, each containing 5 mice, wherein the virus+dox group was added with 2 mg / ml of doxycycline hydrochloride (Dalian Meilun Bio, #MB1088-2) in the drinking water of the mice to inhibit the expression of CSK or CSK-W in the virus, and the virus group was normal drinking water.

[0241] 2 weeks after AAV injection, mice were deeply anesthetized, and the optic nerve was clamped for about 5 seconds with scissors about 2 mm from the eyeball under a dissecting microscope. After the clamp was removed, the mice were euthanized 2 days later for eye collection.

[0242] 3. Eye collection:

[0243] After the mice were euthanized with CO2, the eyeball and optic nerve were removed.

[0244] 4. Retinal flat mount:

[0245] After the eyeball was removed, it was rinsed with PBS and then placed in a 4% paraformaldehyde solution for 1.5-2 hours of room temperature fixation. After fixation, the mouse eyeball was removed from the PBS and the anterior cornea, lens, and vitreous body were removed. The retina was then divided into four equal parts. Triton X-100 was added at a concentration of 0.5% and the membrane was permeabilized on a horizontal shaker for 30 minutes. The 0.5% Triton X-100 was removed and blocking solution (5% BSA solution containing 0.1% Triton X-100 prepared with PBS) was added and incubated overnight at 4°C on a horizontal shaker. RBPMS antibody working solution (RBPMS, Abeam, #AB152101, dilution ratio 1:300) was added and incubated for 2 days at 4°C in a refrigerator. PBS was added and washed 3 times on a shaker. Alexa Fluor 488 donkey anti-rabbit IgG antibody working solution (Thermo Fisher, #A-21206, dilution ratio 1:1000) was added and incubated for 2.5 hours at room temperature on a horizontal shaker. The retina was then removed and mounted on a coverslip. A confocal microscope was used to take pictures at 20x magnification. Image software was used to count the number of RBPMS positive cells in each picture, which were RGC cells. TM

[0246] 5. Optic nerve frozen section:

[0247] After the eyeball was removed, it was rinsed with PBS and then placed in a 4% paraformaldehyde solution for 1.5-2 hours of room temperature fixation. After fixation, the mouse eyeball was removed from the PBS and the anterior cornea, lens, and vitreous body were removed. The retina was then divided into four equal parts. Triton X-100 was added at a concentration of 0.5% and the membrane was permeabilized on a horizontal shaker for 30 minutes. The 0.5% Triton X-100 was removed and blocking solution (5% BSA solution containing 0.1% Triton X-100 prepared with PBS) was added and incubated overnight at 4°C on a horizontal shaker. RBPMS antibody working solution (RBPMS, Abeam, #AB152101, dilution ratio 1:300) was added and incubated for 2 days at 4°C in a refrigerator. PBS was added and washed 3 times on a shaker. Alexa Fluor 488 donkey anti-rabbit IgG antibody working solution (Thermo Fisher, #A-21206, dilution ratio 1:1000) was added and incubated for 2.5 hours at room temperature on a horizontal shaker. The retina was then removed and mounted on a coverslip. A confocal microscope was used to take pictures at 20x magnification. Image software was used to count the number of RBPMS positive cells in each picture, which were RGC cells. TM

[0248] Results show that, compared with the CSK+dox group, the CSK group can significantly inhibit RGC cell apoptosis caused by crush injury (Fig. 6C), and significantly promote axon regeneration of the optic nerve (Fig. 6D), and compared with the CSK-W+dox group, the CSK-W group can significantly inhibit RGC cell apoptosis caused by crush injury (Fig. 6E), and significantly promote axon regeneration of the optic nerve (Fig. 6F), indicating that the CSK or CSK-W obtained after truncation still has the effect of aging reprogramming.

[0249] Example 7

[0250] In order to explore the effects of the truncated combinations CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, and DOKM-W on aging reprogramming, human lung epithelial cell-related A549 cells (Chinese Academy of Sciences Cell Bank, SCSP-503) were used for effect evaluation. The specific method is as follows:

[0251] 1. On day 0, A549 cells were inoculated in a 24-well plate containing complete culture medium (F12k, 10% FBS fetal bovine serum, 1% PS);

[0252] 2. On day 1, the fresh virus supernatant obtained in Example 1 was added to the above-mentioned culture medium together with polybrene;

[0253] 3. On day 2, the culture medium was replaced with an aging induction culture medium (F12k, 10% fetal bovine serum, 5 μM bleomycin sulfate, 1% PS);

[0254] 4. On day 3, the aging culture medium was replaced with 1 μg / ml doxycycline and 5 μM bleomycin sulfate in complete culture medium (F12k culture medium containing 10% fetal bovine serum (FBS)), and then a cell aging SA-β-gal staining kit (Bi Yun Tian, #C0602) was used for staining, and the SA-β-gal positive level was observed.

[0255] Results: An inverted phase contrast microscope (Leica, DMi8) was used to take pictures at 100 times magnification. As shown in Fig. 7, the SA-β-gal positive rate decreased in the groups added with CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, and DOKM-W, and the aging symptoms of lung epithelial cell-related A549 cells were significantly improved, indicating that CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, and DOKM-W have anti-aging ability on respiratory lung cells.

[0256] Example 8

[0257] To explore the effect of CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, DOKM-W on the aging reprogramming ability, mouse skin cells MEF (mouse embryonic fibroblast) were used for effect evaluation. The specific method is as follows:

[0258] 1. On day 0, MEF cells were inoculated in a well plate containing complete culture medium (DMEM, 10% FBS fetal bovine serum, 1% PS);

[0259] 2. On day 1, the fresh virus supernatant obtained in Example 1 was added to the above-mentioned culture medium together with polybrene;

[0260] 3. On day 3, the culture medium was replaced with 1 μg / ml doxycycline complete culture medium (DMEM medium containing 10% fetal bovine serum (FBS));

[0261] 4. On day 6, the culture medium was replaced with aging induction medium (DMEM, 10% fetal bovine serum, 20 μM etoposide, 1% PS);

[0262] 5. On day 7, the aging medium was replaced with 1 μg / ml doxycycline complete culture medium (DMEM medium containing 10% fetal bovine serum (FBS)) for continuous culture, and then stained using a cell aging β-gal staining kit (Bi Yun Tian, #C0602), and the SA-β-gal positive level was observed.

[0263] Results: The picture was taken using an inverted phase contrast microscope (Leica, DMi8) with 100 times magnification. As shown in Figure 8, the SA-β-gal positive rate decreased in the groups after adding CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, DOKM-W, and the aging symptoms of mouse skin cells were significantly improved, indicating that CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, DOKM-W had anti-aging ability for aging skin

[0264] Example 9

[0265] To explore the effect of CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, DOKM-W on the aging reprogramming ability, human endothelial cells HUVEC cells (Human umbilical vein endothelial cell) were used for effect evaluation. The specific method is as follows:

[0266] 1. On day 0, HUVEC cells were inoculated in a 6-well plate containing complete culture medium (ECM medium, 10% FBS fetal bovine serum, 1% PS, 1% ECGS);

[0267] 2. On day 1, each fresh virus supernatant obtained from Example 1 was added to the above culture medium together with polybrene;

[0268] 3. On day 2, the culture medium was replaced with complete medium containing 1 μg / ml doxycycline (ECM medium, 10% FBS fetal bovine serum, 1% PS, 1% ECGS) and continued to culture, and then stained using a cell aging β-gal staining kit (Bi Yun Tian, #C0602), and the SA-β-gal positive level was observed.

[0269] Results: The pictures were taken using an inverted phase contrast microscope (Leica, DMi8) at 100 times magnification. As shown in Figure 9, the SA-β-gal positive rate decreased in the groups after adding CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, and DOKM-W, and the aging symptoms of human endothelial cells were significantly improved, indicating that CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, and DOKM-W have anti-aging ability for aged blood vessels.

[0270] Example 10

[0271] The main purpose of this example is to investigate the promoting effect of the related gene combination on myoblasts.

[0272] Method: C2C12 cells (Punuo Sai, CL-0044) were selected for plating, and after being stable for 48 h, the culture medium containing 2% HBS (i.e. 2% HBS + 2% PS + 96% DMEM) was replaced for culture to make the C2C12 cells enter the differentiation state, and the CSK virus liquid in Example 1 was added at the same time, and after continuous culture for 7 days, immunohistochemical staining was performed using MYH1 antibody (Sigma, ZRB1214), the cell nucleus was stained with DAPI, and the degree of cell differentiation was analyzed by taking pictures using a fluorescence inverted microscope (Leica, DMIL LED FLUO) at 100 times magnification.

[0273] Results: As shown in Figure 10, the MYH1 expression amount in the groups after adding CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, and DOKM-W was significantly higher than that in the control group, and MYH1 indicated newly born muscle cells, indicating that the CSK, BSK, DOKM, EOSM, CSK-W, BSK-W, and DOKM-W gene combination can promote myoblasts.

[0274] The above only describes the preferred embodiments of the present application, and for researchers in the technical field, several improvements and modifications made without departing from the principles of the present application should also be considered as the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A polypeptide selected from a c-Myc factor truncation, an OCT4 factor truncation, a SOX2 factor truncation, a KLF4 factor truncation, or any combination thereof: the c-Myc factor truncation lacks a CP domain; the OCT4 factor truncation lacks a CTD domain, lacks a NTD domain, or lacks both a NTD domain and a CTD domain; the SOX2 factor truncation has a TAD domain replaced with amino acids 92-100 of the TAD domain; and the KLF4 factor truncation lacks a ZnF domain or lacks an ID domain. wherein the c-Myc factor truncation comprises a MTAD domain and a bHLHLZ domain of c-Myc; preferably, the MTAD domain and the bHLHLZ domain are directly linked or linked via a linker; more preferably, the c-Myc factor truncation has an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7; further preferably, a nucleic acid encoding the c-Myc factor truncation has a nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

1.

2. The polypeptide of claim 1, wherein, the polypeptide is a combination of an OCT4 factor truncation and a c-Myc factor truncation, the combination denoted as an OCT4-Myc factor truncation, the OCT4-Myc factor truncation comprising a c-Myc factor MTAD domain, an OCT4 factor POU domain, and an OCT4 factor CTD domain; or the OCT4 factor-Myc truncation comprises a c-Myc factor MTAD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain; or the OCT4-Myc factor truncation comprises an OCT4 factor NTD domain, an OCT4 factor POU domain, and a c-Myc factor bHLHLZ domain; preferably, the c-Myc factor MTAD domain, the OCT4 factor POU domain, and the OCT4 factor CTD domain are directly linked or linked via a linker; 3. The polypeptide of claim 1, wherein, preferably, the c-Myc factor MTAD domain, the OCT4 factor POU domain, and the c-Myc factor bHLHLZ domain are directly linked or linked via a linker; preferably, the OCT4 factor NTD domain, the OCT4 factor POU domain, and the c-Myc factor bHLHLZ domain are directly linked or linked via a linker; ​ ​ ​ ​ More preferably, the OCT4-Myc factor truncation has an amino acid sequence as set forth in SEQ ID NO: 8, 9, or 58, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8, 9, or 58; Further preferably, the nucleic acid encoding the OCT4-Myc factor truncation has a nucleotide sequence as set forth in SEQ ID NO: 2, 3, or 57, or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2, 3, or 57.

4. The polypeptide of claim 1, wherein, the SOX2 factor truncation comprises HMG domain, DIM domain and TAD domain 92-100th amino acids; Preferably, the HMG domain, DIM domain and TAD domain 92-100th amino acids are directly connected or connected through a linker; More preferably, the SOX2 factor truncation has an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; Further preferably, the nucleic acid encoding the SOX2 factor truncation has a nucleotide sequence as set forth in SEQ ID NO: 4, or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

4.

5. The polypeptide of claim 1, wherein the KLF4 factor truncation lacks ZnF domain and comprises AD domain and ID domain; or the KLF4 factor truncation lacks ID domain and comprises AD domain and ZnF domain; Preferably, the AD domain and ID domain are directly connected or connected through a linker; Preferably, the AD domain and ZnF domain are directly connected or connected through a linker; More preferably, the KLF4 factor truncation has an amino acid sequence as set forth in SEQ ID NO: 11 or 12, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11 or 12; Further preferably, the nucleic acid encoding the KLF4 factor truncation has a nucleotide sequence as set forth in SEQ ID NO: 5 or 6, or a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or 6.

6. A fusion protein comprising the polypeptide of any one of claims 1-5 or any combination thereof.

7. The fusion protein of claim 6, wherein, The fusion protein comprises any one selected from the following: (1) a c-Myc factor truncation, an OCT4 factor, a SOX2 factor, and a KLF4 factor; (2) an OCT4-Myc factor truncation, a SOX2 factor, and a KLF4 factor; (3) a c-Myc factor, an OCT4 factor, a SOX2 factor truncation, and a KLF4 factor; or (4) a c-Myc factor, an OCT4 factor, a SOX2 factor, and a KLF4 factor truncation, Preferably, the factors or truncations are directly connected to each other or connected via a linker; More preferably, the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NOs: 21-26, 50-54, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 21-26, 50-54.

8. An isolated nucleic acid molecule encoding the polypeptide of any one of claims 1-5 or the fusion protein of claim 6 or 7.

9. The nucleic acid molecule of claim 8, wherein, The nucleic acid molecule is DNA or RNA.

10. A vector containing the nucleic acid molecule of claim 8 or 9.

11. The carrier of claim 10, wherein, The vector is an expression vector or a delivery vector, Preferably, the c-Myc factor truncation, the OCT4 factor truncation, the SOX2 factor truncation, and / or the KLF4 factor truncation are present on the same expression vector or delivery vector, or are present on multiple expression vectors or delivery vectors, Preferably, the delivery vector is an LNP, an LPP, a virus-like particle (VLP), an exosome, an extracellular vesicle, a micelle, a metal nanoparticle, a nanoscale microsphere, a nanoemulsion, a dendrimer, or a hydrogel; Preferably, the expression vector is a plasmid vector, an episomal vector, a transposon, or a viral vector; More preferably, the viral vector is an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, a herpes viral vector, a retroviral vector, a Sendai viral vector, a cytomegalovirus vector, or a vaccinia viral vector.

12. A composition for restoring viability of a cell, tissue, or organ of a subject, the composition comprising the polypeptide of any one of claims 1-5, the fusion protein of claim 6 or 7, the nucleic acid molecule of claim 8 or 9, the vector of claim 9 or 10, or a combination thereof.

13. The composition of claim 12, wherein, The composition further comprises one or more of a c-Myc factor, an OCT4 factor, a SOX2 factor, and a KLF4 factor; The composition further comprises one or more of a c-Myc factor, an OCT4 factor, a SOX2 factor, and a KLF4 factor; Preferably, the composition comprises the c-Myc factor truncation and one or more selected from the group consisting of an OCT4 factor, a SOX2 factor and a KLF4 factor; Preferably, the composition comprises the OCT4-Myc factor truncation and one or more selected from the group consisting of a SOX2 factor and a KLF4 factor; Preferably, the composition comprises the SOX2 factor truncation and one or more selected from the group consisting of an OCT4 factor, a KLF4 factor and a c-Myc factor; Preferably, the composition comprises the KLF4 factor truncation and one or more selected from the group consisting of an OCT4 factor, a SOX2 factor and a c-Myc factor.

14. The composition of claim 13, wherein, The c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation and / or KLF4 factor is a protein, a nucleic acid encoding the same or a mixture thereof; Preferably, the nucleic acid molecule is DNA or RNA; Preferably, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation and / or KLF4 factor is in the form of an expression vector or a delivery vector, Preferably, the expression vector is a plasmid vector, an episomal vector, a transposon or a viral vector, More preferably, the viral vector is an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, a herpes viral vector, a retroviral vector, a Sendai viral vector, a cytomegalovirus vector or a vaccinia viral vector, Preferably, the c-Myc factor truncation, c-Myc factor, OCT4 factor truncation, OCT4 factor, OCT4-Myc factor truncation, SOX2 factor truncation, SOX2 factor, KLF4 factor truncation and / or KLF4 factor is present on the same expression vector or delivery vector, or is present on multiple expression vectors or delivery vectors, Preferably, the delivery vector is an LNP, an LPP, a virus-like particle (VLP), an exosome, an extracellular vesicle, a micelle, a metal nanoparticle, a nanoscale microsphere, a nanoemulsion, a dendrimer or a hydrogel.

15. A method of revitalizing a cell, tissue or organ of a subject, the method comprising administering to the subject in vivo the polypeptide of any one of claims 1-5, the fusion protein of claim 6 or 7, the nucleic acid molecule of claim 8 or 9, the vector of claim 10 or 11 or the composition of any one of claims 12-14.

16. Use of the polypeptide of any one of claims 1-5, the fusion protein of claim 6 or 7, the nucleic acid molecule of claim 8 or 9, the vector of claim 10 or 11 or the composition of any one of claims 12-14 in the manufacture of a medicament for revitalizing a cell, tissue or organ of a subject.

17. The method of claim 15 or use of claim 16, wherein, The rejuvenated subject cell, tissue or organ exhibits an increase or decrease in expression of one or more senescence-associated markers and / or a decrease in expression of one or more inflammatory factors; Preferably, the senescence-associated markers are selected from one or more of HP1y, H3K9me3, pl6, p21, SA-b-gal and Lamin A / C protein; Preferably, the inflammatory factors include one or more of IL-1 b, IL-6, TNFa, IL22 and IL9; Preferably, the cell is a human cell or a non-human mammalian cell; Preferably, the cell is selected from a skin-derived cell, a bone joint-derived cell, a muscle-derived cell, an eye-derived cell, a blood vessel-derived cell, a lung-derived cell or a neural cell; More preferably, the cell is selected from a fibroblast cell, an endothelial cell, an epithelial cell, a chondrocyte cell, a synoviocyte cell, a keratinocyte cell, a retinal cell, a corneal cell, a muscle cell and other cells.

18. The method of claim 15 or use of claim 16, wherein, the rejuvenating the subject cell, tissue or organ comprises ameliorating, reversing or inhibiting cell, tissue or organ senescence; or the rejuvenating the subject cell, tissue or organ comprises restoring at least one of the transcriptional profile or epigenetic information of the cell, tissue or organ lost due to senescence, injury, disease or any combination thereof; or the rejuvenating the subject cell, tissue or organ comprises restoring the function of the cell, increasing the potency of the cell, enhancing the survival capacity of the cell or increasing the replicative capacity or lifespan of the cell, or a combination thereof; or the rejuvenating the subject cell, tissue or organ comprises promoting axonal regeneration of a subject neuronal cell; promoting RGC cell regeneration of a subject; promoting fibroblast cell rejuvenation of a subject skin; promoting chondrocyte cell rejuvenation of a subject; promoting muscle stem cell proliferation of a subject; promoting vascular endothelial cell rejuvenation; promoting lung epithelial cell rejuvenation.

19. A composition according to any one of claims 12 to 14, a method according to claim 15, a use according to claim 16, or a method or use according to claim 17 or 18, characterised in that, the subject has, is suspected of having, or is at risk of having a disease or condition associated with aging, an ocular disease, a pulmonary disease, a skin disease, a bone joint disorder or a vascular disease; Preferably, the subject has, is suspected of having, or is at risk of having a degenerative disease, a neurodegenerative disease, a cardiovascular disease, a peripheral vascular disease, a skin disease, an autoimmune disease, an endocrine entity disorder, a metabolic dysfunction, a musculoskeletal disease, a digestive system disease, a respiratory system disease, an ocular optic nerve damage related disease or a cartilage degenerative disorder.

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