Methods and compositions for cell and tissue rejuvenation

Abstract

To provide a composition for rejuvenation of target cells.SOLUTION: A) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of a telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).SELECTED DRAWING: None

Description

[Background technology]

[0001] Related Patents This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 899,861, filed September 13, 2019, the contents of which are incorporated herein by reference in their entirety. Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on August 31, 2020, is named "UNCO-028_001WO_SeqList.txt" and is approximately 241KB in size.

[0002] The shortening of telomeres, which are repetitive DNA sequences located at the ends of chromosome chains, can lead to cellular senescence, apoptosis, or malignant tumors. Telomere shortening, especially in cells cultured in vitro, not only limits the further proliferation of therapeutic cell populations but can also reduce the biological activity of the cells, thereby reducing clinical efficacy, making it an obstacle to the generation of therapeutic cell populations. Increasing the length of telomeres in cells can lead to cell rejuvenation, but can also cause adverse side effects, such as the immortalization of oncogenic cells. Therefore, there is a need in the art for compositions, kits, and methods that can efficiently and safely increase the length of telomeres in cells in a controllable manner, thereby rejuvenating cells. Summary of the Invention

[0003] The present disclosure provides compositions comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of a telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of a telomerase RNA component (TERC).

[0004] The present disclosure provides compositions comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of a telomerase reverse transcriptase (TERT); and b) at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of a telomerase RNA component (TERC).

[0005] The first polynucleotide molecule may comprise an mRNA molecule encoding at least a portion of TERT. The first polynucleotide molecule may comprise a plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of at least a portion of TERT.

[0006] The second polynucleotide molecule may comprise an mRNA molecule encoding at least a portion of at least one DNA target polypeptide. The second polynucleotide molecule may comprise a plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide operably linked to at least one promoter sufficient to drive expression of at least a portion of the at least one DNA target polypeptide.

[0007] The DNA targeting polypeptide may comprise at least one Cas9 molecule, at least one Cas9 mutant molecule, at least one Cas9 homologous molecule, or any combination thereof. The Cas9 molecule, Cas9 mutant molecule, or Cas9 homologous molecule may be nuclease-deficient or nuclease-inactive. The Cas9 mutant molecule may comprise eSpCas9(K855A), eSpCas9(1.0), eSpCas9(1.1), SpCas9-HF1(VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9(VQR), Cas9(EQR), Cas9(VRER), Cas9(D1135E), Cas9(QQR1), SaCas9(KKH), Nme1Cas9, Nme2Cas9, Nme3Cas9, or any combination thereof. Cas9 homologues include Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), and Acidaminococcus sp.BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (SCCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casφ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str. DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str.Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. The vector may include Streptomyces cerevisiae Cas9, ...

[0008] The DNA targeting polypeptide may comprise at least one TALE molecule, at least one zinc finger molecule, at least one meganuclease molecule, or any combination thereof.

[0009] The DNA-targeting polypeptide may comprise at least one transactivating molecule. The transactivating molecule may comprise at least one P65 molecule, at least one Rta molecule, at least one VP16 molecule, at least one VP64 molecule, at least one VP160 molecule, at least one VP64-P65-Rta (VPR) molecule, at least one SunTag peptide, at least one single-guide RNA-MS2 (sgRNA-MS2) molecule, or any combination thereof. In some aspects, the DNA-targeting polypeptide may be a DNA-targeting ribonucleoprotein (RNP) complex. The DNA-targeting ribonucleoprotein complex may comprise both at least one protein component and at least one nucleic acid component. The DNA-targeting polypeptide may comprise at least one guide RNA. The transactivating molecule may comprise at least one single-guide RNA-MS2 (sgRNA-MS2) molecule. The sgRNA-MS2 molecule may comprise a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene, and at least about 1, or at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10 MS2 RNA aptamers.

[0010] The DNA targeting polypeptide may comprise a dCas9 molecule and a VPR molecule.

[0011] The DNA target polypeptide may bind upstream, 5', within, downstream, or 3' of the endogenous TERC gene.

[0012] The mRNA molecule may be a modified mRNA molecule. The modified mRNA molecule may contain at least one modified ribonucleoside base. The modified ribonucleoside base may be a pseudouridine (Ψ) residue, a 5-methylcytidine (m 5The modified mRNA molecule may contain at least one modified nucleoside. The modified nucleoside may be a 5-methylcytidine (mC) residue, a 5-methylcytidine (mC) residue, or any combination thereof. 5 C), 5-methyluridine (m 5 U), N6-methyladenosine (m 6 A), inosine 2'-O-methylated nucleosides, or any combination thereof.

[0013] Any composition of the present disclosure may further comprise a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA of the plurality of guide RNAs is complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene. The plurality of gRNA molecules may comprise at least about 1, or at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10 different species of gRNA molecules, each species having a different nucleic acid. Any composition of the present disclosure may further comprise at least one plasmid comprising at least one nucleic acid sequence encoding at least one gRNA operably linked to at least one promoter sufficient to drive expression of at least one gRNA. The plurality of gRNA molecules may comprise a plurality of single guide RNA (sgRNA) molecules, crRNA:tracrRNA molecules, truncated sgRNA molecules, high-fidelity scaffold gRNA molecules, or any combination thereof. The guide RNA molecule may be a modified guide RNA (mod gRNA) molecule. The guide RNA molecule may comprise any sequence listed in Table 1 or Table 2.

[0014] The present disclosure provides compositions comprising: a) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of human telomerase reverse transcriptase (hTERT); b) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide; and c) a plurality of guide RNA (gRNA) molecules, wherein the at least one DNA target polypeptide comprises a dCas9 and a VP64-P65-Rta (VPR) molecule, and at least one gRNA of the plurality of gRNAs is complementary to a nucleic acid sequence located upstream of an endogenous hTERC gene.

[0015] Any composition of the present disclosure may comprise at least one mRNA and / or polynucleotide encoding at least one rejuvenating factor. The rejuvenation factor may comprise telomerase RNA component (TERC), telomerase-associated reverse transcriptase (TERT), protection of telomeres 1 (POT1), insulin-like growth factor 1 (IGF1), WD repeat-containing antisense to TP53 (WRAP53), nuclear protein family A, member 3 (NOP3), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), shelterin complex subunit and telomerase recruitment factor (ACD / TPP1), TRF-1-interacting ankyrin-related ADP-ribose polymerase (TNKS), telomeric repeat-binding factor 1 (TRF-1), telomeric repeat-binding factor 2 (TRF-2), TERF1-interacting nuclear factor 2 (TIN2), telomeric repeat-binding factor 2 (Rap1), dyskerin pseudouridine synthase 1 (DKC1), ribonucleoprotein NHP2, or any combination thereof.

[0016] TERT may be human TERT (hTERT). TERC may be human TERC (hTERC).

[0017] The present disclosure provides a composition comprising at least one virus particle, including any composition of the present disclosure.The virus particle can be adeno-associated virus (AAV) particle, adenovirus particle, lentivirus particle, foamy virus particle, herpes simplex virus (HSV) particle, retrovirus particle, alphavirus particle, flavivirus particle, rhabdovirus particle, measles virus particle, Newcastle disease virus particle, poxvirus particle, picornavirus particle, or any combination thereof.The AAV particle can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2 / 1, AAV2 / 2, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV-DJ or AAV-DJ8 particle. The retroviral particle may be an MMSV or MSCV particle. The lentiviral particle may be an HIV-1 or HIV-2 particle. The alphavirus particle may be an SFV, SIN, VEE, or M1 particle. The flavivirus particle may be a Kunjin virus, West Nile virus, or Dengue virus particle.

[0018] The present disclosure provides a composition comprising at least one exosome, microvesicle, or liposome, wherein the at least one exosome, microvesicle, or liposome comprises any of the compositions of the present disclosure. The present disclosure provides a composition comprising at least one nanoparticle, wherein the at least one nanoparticle comprises any of the compositions of the present disclosure. The nanoparticle may comprise a liposome, a micelle, a polymer-based nanoparticle, a lipid-polymer-based nanoparticle, a metal-based nanoparticle, a nanocrystal, a carbon nanotube-based nanoparticle, or a polymeric micelle.

[0019] The present disclosure provides kits comprising any of the compositions of the present disclosure.

[0020] The present disclosure provides a method for rejuvenating at least one cell, the method comprising contacting the at least one cell with any of the compositions or kits of the present disclosure. The aforementioned method may further comprise expanding the at least one cell contacted with any of the compositions or kits of the present disclosure to generate a plurality of rejuvenated cells.

[0021] The present disclosure provides a method for treating and / or preventing a disease in a subject, the method comprising the steps of: a) contacting at least one cell with any of the compositions or kits of the present disclosure; b) proliferating at least one cell contacted with any of the compositions or kits of the present disclosure to generate a plurality of rejuvenated cells; and c) administering the plurality of rejuvenated cells to the subject.

[0022] The present disclosure provides methods for treating and / or preventing a disease in a subject, the methods comprising the steps of: a) contacting at least one cell with any of the compositions or kits of the present disclosure; b) proliferating the at least one cell contacted with any of the compositions or kits of the present disclosure to generate a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform them into at least one tissue or organ; and d) administering the at least one tissue or organ to a subject.

[0023] The present disclosure provides a method for generating a tissue or organ in vitro, the method comprising the steps of: a) contacting at least one cell with any of the compositions or kits of the present disclosure; b) proliferating the at least one cell contacted with any of the compositions or kits of the present disclosure to generate a plurality of rejuvenated cells; and c) culturing the plurality of rejuvenated cells under conditions sufficient to convert the plurality of rejuvenated cells into at least one tissue or organ.

[0024] The present disclosure provides a method for generating a plurality of rejuvenated edited cells, the method comprising: a) contacting a plurality of cells with a gene editing system to edit at least one gene in the genome of at least one cell of the plurality of cells, thereby generating at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with any of the compositions or kits of the present disclosure; and d) propagating the at least one cell contacted with any of the compositions or kits of the present disclosure to generate a plurality of rejuvenated edited cells.

[0025] The present disclosure provides methods for treating and / or preventing a disease in a subject, the method comprising the steps of: a) contacting a plurality of cells with a gene editing system to edit at least one gene in the genome of at least one cell of the plurality of cells, thereby generating at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with any of the compositions or kits of the disclosure; d) expanding the at least one cell contacted with any of the compositions or kits of the disclosure to generate a plurality of rejuvenated, edited cells; and e) administering the plurality of rejuvenated, edited cells to the subject.

[0026] The present disclosure provides a method of treating epidermolysis bullosa (EB) in a subject, the method comprising: a) contacting a plurality of cells, including keratinocytes, dermal fibroblasts, mesenchymal stem / stromal cells, or any combination thereof, with a gene editing system to edit at least one gene in the genome of at least one cell of the plurality of cells, thereby generating at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with any of the compositions or kits of the present disclosure; d) expanding the at least one cell contacted with any of the compositions or kits of the present disclosure to generate a plurality of rejuvenated edited cells; and e) administering the plurality of rejuvenated edited cells to the subject.

[0027] Expanding the at least one cell may include culturing the at least one cell with conditioned Opti-MEM, unconditioned Opti-MEM, human serum, fetal bovine serum (FBS), or any combination thereof.

[0028] Rejuvenating at least one cell may include increasing expression of TERC in the at least one cell, increasing expression of TERT in the at least one cell, increasing the total number of population doublings exhibited by the at least one cell, increasing telomere length in the at least one cell, increasing mitochondrial DNA copy number in the at least one cell, increasing the amount of mitochondrial DNA in the at least one cell, increasing the number of mitochondria in the at least one cell, increasing migration activity of the at least one cell, restoring thiol group oxidation levels in proteins in the at least one cell to a youthful state, reducing aging-associated DNA methylation in the at least one cell, or any combination thereof.

[0029] The at least one cell may be a fibroblast, a keratinocyte, a mesenchymal stem cell / stromal cell, a peripheral blood mononuclear cell, a chimeric antigen receptor T cell (CAR-T cell), an endothelial cell, a chondrocyte, a muscle stem cell, a neural stem cell, a hepatocyte, a limbic stem cell, a retinal pigment epithelial cell, a hematopoietic stem cell, a macrophage, a cardiomyocyte, a pancreatic cell, a beta cell, or a combination thereof.

[0030] The disease may include graft-versus-host disease (GvHD), autoimmune diseases, epidermolysis bullosa (EB), recessive dystrophic EB (RDEB), zygotic EB (JEB), simplex EB (EBS), congenital ichthyosis, dyskeratosis congenita, macular degeneration, Parkinson's disease, Alzheimer's disease, aging, type I and type II diabetes, burns, chronic skin wounds, diabetes-related ulcers / wounds, heart disease, osteoporosis, cancer, connective tissue diseases such as Ehlers-Danlos syndrome (EDS) or Marfan syndrome, liver disease, lung disease, and any combination thereof.

[0031] The step of contacting at least one cell may comprise gene transfer, transduction, electroporation, nucleic acid injection, at least one cell-penetrating peptide, or any combination thereof.

[0032] The present disclosure provides a method of rejuvenating at least one cell in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one of any of the compositions or kits of the present disclosure.

[0033] The present disclosure provides methods of rejuvenating at least one subject, the methods comprising administering to the subject a therapeutically effective amount of at least one of any of the compositions or kits of the present disclosure.

[0034] The subject may be a mammal. The subject may be a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, a pig, or any other mammal. The subject may be a bird.

[0035] Any of the above aspects may be combined with other aspects.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the singular includes the plural unless the context clearly dictates otherwise; for example, the terms "a," "an," and "the" are understood to be singular or plural, and the term "or" is understood to be inclusive. For example, the term "an element" means one or more elements. Throughout this specification, the term "comprise" or its variations "comprises" or "comprising" will mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of other elements, integers, or steps, or group of elements, integers, or steps. The term "about" may mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term "about."

[0037] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the following detailed description and claims.

[0038] The above-mentioned and further features will be more clearly understood from the following detailed description when taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0039] [Figure 1] 1 is a schematic diagram of a DNA targeting molecule of the present disclosure that binds upstream of the endogenous hTERC locus. In this non-limiting example, the DNA targeting molecule comprises dCas9 and a transactivation molecule, which is a VP64-P65-Rta (VPR) molecule. [Figure 2] FIG. 2 is a schematic diagram of the disclosed method of treatment and / or method of generating tissue ex vivo. [Figure 3]FIG. 3 is a schematic diagram of a method for generating multiple rejuvenated edited cells of the present disclosure. [Figure 4] FIG. 4 is a diagram showing hTERC transcription levels in various cell types. [Figure 5] FIG. 5 is a chart showing human TERC RNA levels in F50 cells transfected with compositions of the present disclosure. [Figure 6] FIG. 6 is a series of charts showing levels of human TERC RNA in HEKn cells (left) and human mesenchymal stem / stromal cells (hMSCs) (right) transfected with a composition of the disclosure (+dCas9-VPR+gmix) compared to non-transfected HEKn cells, non-transfected hMSC cells, and F50-derived induced pluripotent stem cells. [Figure 7] FIG. 7 is a series of charts showing human TERC RNA levels in F50 cells (left) and hMSCs (right) transfected with a composition of the disclosure that does not include a guide RNA (+dCas9-VPR(no guide)). [Figure 8] FIG. 8 is a schematic diagram of one gene transfer regimen of the present disclosure. [Figure 9] FIG. 9 is a chart showing total population doublings of senescent F50S cells transfected with compositions of the present disclosure. [Figure 10] FIG. 10 is a schematic diagram of another gene transfer treatment scheme of the present disclosure. [Figure 11] FIG. 11 is a schematic diagram of another gene transfer treatment scheme of the present disclosure. [Figure 12] FIG. 12 is a chart showing relative telomere length in senescent F50S cells transfected with a composition of the disclosure (+TERT+dCas9-VPR / gmix) compared to non-transfected F50S cells and F50-derived induced pluripotent stem cells. [Figure 13] FIG. 13 is a chart showing relative telomere length in F50 cells transfected with a composition of the disclosure (+hTERT+dCas9-VPR / gmix) compared to non-transfected F50 cells and F50-derived induced pluripotent stem cells. [Figure 14]FIG. 14 is a series of charts showing relative telomere length in HEKn cells (left) and hMSCs (right) transfected with a composition of the disclosure (+TERT+dCas9-VPR / gmix) compared to non-transfected HEKn cells, non-transfected hMSCs, and F50-derived induced pluripotent stem cells. [Figure 15] FIG. 15 is a series of charts showing the relative amounts of mitochondrial DNA in F50 cells (left), HEKn cells (center), and hMSCs (right) transfected with a composition of the disclosure (+TERT+dCas9-VPR / gmix) compared to non-transfected F50 cells, non-transfected HEKn cells, and non-transfected hMSCs. [Figure 16] FIG. 16 is a gel image showing the results of telomerase activity measurements in F50 cells transfected with various compositions of the present disclosure (F50+TERT and F50+TERT+dCas9-VPR / gmix), as well as non-transfected F50 cells and F50-derived induced pluripotent stem cells. [Figure 17] FIG. 17 is a series of representative microscopy images of adult human primary fibroblasts grown from single cells that were not transfected (top two rows) or transfected with a composition of the disclosure (+hTERT / dCas9-VPR+gRNA) (bottom two rows). [Figure 18] FIG. 18 is a schematic diagram of the transendothelial migration (TEM) assay. [Figure 19] FIG. 19 is a chart showing the migration activity of hMSCs transfected with compositions of the present disclosure as measured using TEM assays. [Figure 20] FIG. 20 is a series of charts showing the oxidation levels of thiol groups detected within selected proteins in aged hMSCs transfected with compositions of the present disclosure compared to non-transfected young, low-passage hMSCs and aged, high-passage hMSCs. [Figure 21] FIG. 21 is a series of charts showing the levels of methylation at nine senescence-associated DNA methylation sites in various types of aged cells transfected with compositions of the present disclosure compared to non-transfected young, low-passage cells and aged, high-passage cells. DETAILED DESCRIPTION OF THE INVENTION

[0040] Telomeres comprise repeated DNA sequences located at the ends of chained chromosomes, where, when long enough, they can form loops that protect the ends of each chromosome from acting as double- or single-stranded DNA breaks. Telomeres shorten over time, in part due to oxidative damage and incomplete DNA replication, ultimately leading to extremely short telomeres that are unable to form protective loops, exposed chromosome ends, chromosome-chromosome fusions, the DNA damage response, and cellular senescence, apoptosis, or malignant transformation.

[0041] The enzyme complex telomerase elongates telomeres and contains two essential components: telomerase reverse transcriptase (TERT) and an RNA component known as the telomerase RNA component (TERC). Other components of the telomerase complex include the proteins TCAB1, dyskerin, Garl, Nhp2, Nop10, and RHAU.

[0042] Because maintaining telomere length is crucial for preventing cellular senescence, apoptosis, and the resulting cellular dysfunction, mutations in the TERT and TERC genes are associated with fatal genetic disorders resulting from telomere failure, such as idiopathic pulmonary fibrosis, dyskeratosis congenita, and aplastic anemia. In these diseases, the effects of premature cellular senescence and apoptosis due to telomere shortening can be devastating in themselves and exacerbated by an increased risk of cancer. Furthermore, telomere shortening in ex vivo cultured cells poses significant challenges for generating therapeutic cell populations, forming synthetic tissues and tumors in vitro for research and drug testing, and generating noncancerous somatic cell lines in vitro. Repeated ex vivo passaging can lead to senescence and a loss of further proliferative potential, potentially resulting in a loss of clinically relevant bioactivity in therapeutic cell populations.

[0043] Therefore, there is a clear need in the art for compositions, kits, and methods for elongating telomeres to rejuvenate cells.Existing methods for increasing the expression of TERT and / or TERC in target cells rely on the use of integrating viruses to achieve the desired increase in the expression of TERT and / or TERC.However, these methods are plagued by safety concerns, as integrating viruses can potentially cause dangerous and permanent genome modifications.In addition, persistent overexpression of TERT and / or TERC and the associated increase in telomere length are associated with the immortalization of cancer cells, making integrating viruses a dangerous method in clinical settings.

[0044] Without wishing to be bound by theory, the compositions, kits, and methods of the present disclosure allow for transiently increasing expression of TERT and / or TERC for a time period long enough to rejuvenate target cells, but short enough to avoid harmful and dangerous off-target effects. The use of non-integrating RNA molecules in the present disclosure allows for fine-tuning of the expression levels and stoichiometry of rejuvenation factors in a clinically safe manner.

[0045] The compositions, kits, and methods of the present disclosure may be used in a variety of research and clinical applications, including, but not limited to, the generation of therapeutic cell populations (e.g., CAR-T cell populations, mesenchymal stem / stromal cell populations), the generation of tissues and organs in vitro for subsequent transplantation, research, or drug testing, the generation of genome-edited cell populations for therapeutic and research uses, and the rejuvenation of cell lines associated with senescence, aging, and disease.

[0046] The various compositions, kits, and methods of the present disclosure are all described in detail herein.

[0047] Rejuvenating compositions

[0048] In some aspects, the present disclosure provides compositions comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of a telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of a telomerase RNA component (TERC).

[0049] In some aspects, the present disclosure provides compositions comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of a telomerase reverse transcriptase (TERT); and b) at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of a telomerase RNA component (TERC).

[0050] In some aspects, the at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT) can be an mRNA molecule encoding at least a portion of TERT. In some aspects, the at least one first polynucleotide molecule can be a plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of at least a portion of TERT.

[0051] In some aspects, the at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide can be an mRNA molecule encoding at least a portion of at least one DNA target polypeptide. In some aspects, the at least one second polynucleotide molecule can be a plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide operably linked to at least one promoter sufficient to drive expression of at least a portion of the at least one DNA target polypeptide.

[0052] Accordingly, the present disclosure provides compositions comprising: a) at least one first mRNA molecule encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of the telomerase RNA component (TERC).

[0053] The present disclosure also provides compositions comprising: a) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of at least a portion of TERT; and b) at least one second mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of a telomerase RNA component (TERC).

[0054] The present disclosure also provides compositions comprising: a) at least one first mRNA molecule encoding at least a portion of a telomerase reverse transcriptase (TERT); and b) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide operably linked to at least one promoter sufficient to drive expression of at least a portion of at least one DNA target polypeptide, wherein the DNA target polypeptide increases transcription of a telomerase RNA component (TERC).

[0055] The present disclosure also provides compositions comprising: a) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of at least a portion of TERT; and b) at least one plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of a telomerase RNA component (TERC).

[0056] In some aspects, the DNA targeting polypeptide may comprise at least one Cas9 molecule, at least one Cas9 mutant molecule, at least one Cas9 homologous molecule, or any combination thereof.

[0057] In some aspects, Cas9 molecules, Cas9 mutant molecules, or Cas9 homologous molecules may be nuclease-deficient or nuclease-inactive. As used herein, the term "dCas9" is used in its broadest sense to refer to Cas9 molecules, homologs, and / or mutants that are nuclease-deficient or nuclease-inactive. In a non-limiting example, a Cas9 molecule, Cas9 mutant molecule, or Cas9 homologous molecule may contain at least one mutation, deletion, or insertion that renders the Cas9 molecule, Cas9 mutant molecule, or Cas9 homologous molecule nucleic acid-deficient or nucleic acid-inactive.

[0058] In some aspects, the Cas9 mutant molecule can include eSpCas9(K855A), eSpCas9(1.0), eSpCas9(1.1), SpCas9-HF1(VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9(VQR), Cas9(EQR), Cas9(VRER), Cas9(D1135E), Cas9(QQR1), SaCas9(KKH), Nme1Cas9, Nme2Cas9, Nme3Cas9, or any combination thereof.

[0059] In some aspects, Cas9 homologues include Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria osteomyelitis Cas9 (NmCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), and Lachnospiraceae bacterium ND2006. Cpf1 (LbCpf1), Streptococcus canis Cas9 (SCCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casφ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum strain F Cas9, Nitratifructah sarsuginis strain DSM 16511 Cas9, Parvibaculum labamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azosopyrilum B510 The causative agent may include, for example, Sphaerocaeta globus strain buddy Cas9, Flavobacterium columnarum Cas9, Fluviicola taphenensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudomeriae Cas9, Filifactor allocis Cas9, Legionella pneumophila strain paris Cas9, Saterella wadswarsensis Cas9, Corynebacter diphtheriae Cas9, or any combination thereof.

[0060] In some aspects, Cas9 homologues include Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria osteomyelitis Cas9 (NmCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), and Lachnospiraceae bacterium ND2006. Cpf1 (LbCpf1), Streptococcus canis Cas9 (SCCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casφ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum strain F Cas9, Nitratifructah sarsuginis strain DSM 16511 Cas9, Parvibaculum labamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azosopyrilum B510 The Cas9 may include chimeric mutants of Sphaerocaeta globus strain buddy Cas9, Flavobacterium columnarum Cas9, Fluviicola taphenensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudomeriae Cas9, Filifactor allocis Cas9, Legionella pneumophila strain paris Cas9, Saterella wadswarsensis Cas9, Corynebacter diphtheriae Cas9, or any combination thereof.

[0061] In some aspects, the DNA targeting polypeptide may comprise at least one TALE molecule, at least one zinc finger molecule, at least one meganuclease molecule, or any combination thereof.

[0062] In some aspects, the DNA target polypeptide may include at least one transactivating molecule. In some aspects, the transactivating molecule is a molecule that binds to a transcription factor and / or a transcriptional coregulator that can drive transcription of the target gene.

[0063] In some aspects, the transactivation molecule can include at least one P65 molecule, at least one Rta molecule, at least one VP16 molecule, at least one VP64 molecule, at least one VP160 molecule, at least one VP64-P65-Rta (VPR) molecule, at least one SunTag peptide, at least one single guide RNA-MS2 (sgRNA-MS2) molecule, or any combination thereof.

[0064] In some aspects, the DNA-targeting polypeptide may be a DNA-targeting ribonucleoprotein (RNP) complex. The DNA-targeting ribonucleoprotein complex may include both at least one protein component and at least one nucleic acid component. The at least one protein component may include any of the protein components described herein, such as, but not limited to, a transactivation molecule, a Cas9 molecule, a Cas9 mutant molecule or a Cas9 homologous molecule, a TALE molecule, a zinc finger molecule, a meganuclease molecule, or any combination thereof. The at least one nucleic acid component may be a ribonucleic acid component. The at least one nucleic acid component may include any of the nucleic acid components described herein, such as, but not limited to, a guide RNA molecule, a single guide RNA molecule, a single guide RNA-MS2 (sgRNA-MS2) molecule, or any combination thereof.

[0065] In some aspects, the DNA targeting polypeptide can further comprise at least one cell-penetrating peptide, which can include at least a portion of the TAT protein from HIV, polyarginine, any other cell-penetrating peptide known in the art, or any combination thereof.

[0066] In some aspects, the DNA target polypeptide can include at least one guide RNA. In some aspects, the transactivation molecule can include at least one single-guide RNA-MS2 (sgRNA-MS2) molecule. In some aspects, the sgRNA-MS2 molecule can include a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of an endogenous TERC gene, and at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten MS2 RNA aptamers.

[0067] In some aspects, the DNA targeting polypeptide can include a dCas9 molecule and a VPR molecule.

[0068] In some aspects, the DNA target polypeptide may be bound upstream, 5', within, downstream, or 3' of an endogenous TERC gene, eg, an endogenous human TERC gene.

[0069] In some aspects, the at least one DNA target polypeptide is at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about It may be linked upstream of an endogenous TERC gene, for example, an endogenous human TERC gene, by 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1,000 kb, or at least 5,000 kb, or at least about 10,000 kb.

[0070] In some aspects, the at least one DNA target polypeptide is at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about It may be linked 3' to an endogenous TERC gene, e.g., an endogenous human TERC gene, for 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1,000 kb, or at least 5,000 kb, or at least about 10,000 kb.

[0071] In some aspects, the at least one DNA target polypeptide is at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about It may be linked downstream of an endogenous TERC gene, for example, an endogenous human TERC gene, by 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1,000 kb, or at least 5,000 kb, or at least about 10,000 kb.

[0072] In some aspects, the at least one DNA target polypeptide is at least about 0.1 kilobases (kb), or at least about 0.5 kb, or at least about 1.0 kb, or at least about 1.5 kb, or at least about 2.0 kb, or at least about 2.5 kb, or at least about 3.0 kb, or at least about 3.5 kb, or at least about 4.0 kb, or at least about 4.5 kb, or at least about 5.0 kb, or at least about 5.5 kb, or at least about 6.0 kb, or at least about 6.5 kb, or at least about 7.0 kb, or at least about 7.5 kb, or at least about 8.5 kb, or at least about 9.0 kb, or at least about It may be linked 9.5 kb, or at least about 10.0 kb, or at least about 15 kb, or at least about 20 kb, or at least about 30 kb, or at least about 40 kb, or at least about 50 kb, or at least about 60 kb, or at least about 15 kb, or at least about 70 kb, or at least about 80 kb, or at least about 90 kb, or at least about 100 kb, or at least about 250 kb, or at least about 500 kb, or at least about 750 kb, or at least about 1,000 kb, or at least 5,000 kb, or at least about 10,000 kb 5' of an endogenous TERC gene, e.g., an endogenous human TERC gene.

[0073] In some aspects, the mRNA molecules of any of the compositions of the present disclosure may be modified mRNA molecules.

[0074] In some aspects, the modified mRNA molecule may include at least one modified ribonucleoside base. The modified ribonucleoside base may be a pseudouridine (Ψ) residue, a 5-methylcytidine (m 5 C) residues, or any combination thereof.

[0075] In some aspects, the modified mRNA molecule may include at least one modified nucleoside. Modified nucleosides include N7-methylguanosine (m 7 G), 2-thiouridine (s2 U), pseudouridine (Ψ), 2'-O-methyl-U, m 1 In addition to A (1-methyladenosine), 5-methylcytidine (m 5 C), 5-methyluridine (m 5 U), N6-methyladenosine (m 6 A), inosine, and 2'-O-methylated nucleosides; m 2 A(2-methyladenosine); Am(2'-O-methyladenosine); ms 2 m 6 A(2-methylthio-N 6 -methyladenosine);i 6 A(N 6 -Isopentenyladenosine);ms 2 i 6 A(2-methylthio-N 6 Isopentenyladenosine);io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine);ms 2 i 6 A(2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine);g 6 A(N 6 -glycinylcarbamoyl adenosine);t 6 A(N 6 -threonylcarbamoyl adenosine);ms 2 t 6 A(2-methylthio-N 6 -threonylcarbamoyl adenosine); m 6 t 6 A(N 6 -methyl-N 6 -threonylcarbamoyl adenosine);hn 6 A(N 6 -hydroxynorvalylcarbamoyl adenosine);ms 2 hn 6 A(2-methylthio-N 6 -Hydroxynorvalylcarbamoyl adenosine; Ar(p)(2'-O-ribosyladenosine(phosphate)); I(inosine); m 1 I(1-methylinosine);m 1Im(l,2'-O-dimethylinosine);m 3 C(3-methylcytidine); Cm(2'-O-methylcytidine); S 2 C(2-thiocytidine);ac 4 C(N 4 -acetylcytidine);f 5 C(5-formylcytidine);m 5 Cm(5,2'-O-dimethylcytidine);ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine);k 2 C(lycidin);m 1 G(1-methylguanosine);m 2 G(N 2 -methylguanosine);m 7 G(7-methylguanosine); Gm(2'-O-methylguanosine); m 2 2G(N 2 ,N 2 -dimethylguanosine);m 2 Gm(N 2 ,2'-O-dimethylguanosine);m 2 2G m(N 2 ,N 2 ,2'-O-trimethylguanosine;Gr(p)(2'-O-ribosylguanosine (phosphate));yW(wybutosine);o2yW(peroxywybutosine);OHyW(hydroxywybutosine);OHyW*(undermodified hydroxywybutosine);imG(wybutosine);mimG(methylwybutosine);Q(queuosine);oQ(epoxyqueuosine);galQ(galactosylqueuosine);manQ(mannosylqueuosine);preQ0(7-cyano-7-deazaguanosine);preQ1(7-aminomethyl-7-deazaguanosine);G + (Archeosin); D(Dihydrouridine); m 5 Um(5,2'-O-dimethyluridine);s 4 U(4-thiouridine);m 5 s 2 U(5-methyl-2-thiouridine);s 2 Um(2-thio-2'-O-methyluridine); acp 3U(3-(3-amino-3-carboxypropyl)uridine);ho 5 U(5-hydroxyuridine);mo 5 U(5-methoxyuridine);cmo 5 U(uridine 5-oxyacetic acid);mcmo 5 U(uridine 5-hydroxyacetic acid methyl ester);chm 5 U(5-(carboxyhydroxymethyl)uridine);mchm 5 U(5-(carboxyhydroxymethyl)uridine methyl ester);mcm 5 U(5-methoxycarbonylmethyluridine); mcm 5 Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine);nm 5 s 2 U(5-aminomethyl-2-thiouridine);mnm 5 U(5-methylaminomethyluridine); mnm 5 s 2 U(5-methylaminomethyl-2-thiouridine);mnm 5 se 2 U(5-methylaminomethyl-2-selenouridine);ncm 5 U(5-carbamoylmethyluridine);ncm 5 Um(5-carbamoylmethyl-2'-O-methyluridine);cmnm 5 U(5-carboxymethylaminomethyluridine);cmnm 5 Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm 5 s 2 U(5-carboxymethylaminomethyl-2-thiouridine);m 6 2A(N 6 ,N 6 -dimethyladenosine; Im (2'-O-methylinosine); m 4 C(N 4 -methylcytidine);m 4 Cm(N 4 ,2'-O-dimethylcytidine);hm 5C(5-hydroxymethylcytidine);m 3 U(3-methyluridine); cm 5 U(5-carboxymethyluridine);m 6 Am(N 6 ,2'-O-dimethyladenosine);m 6 2Am(N 6 ,N 6 ,0-2'- 2 7 2 2 2 7 2 2 trimethyladenosine);m 2,7 G(N 2 ,7-dimethylguanosine);m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine);m 3 Um(3,2'-O-dimethyluridine);m 5 D(5-methyldihydrouridine);f 5 Cm(5-formyl-2'-O-methylcytidine);m 1 Gm(1,2'-O-dimethylguanosine);m 1 Am(1,2'-O-dimethyladenosine);τm 5 U(5-taurinomethyluridine);τm 5 s 2 U(5-taurinomethyl-2-thiouridine);imG-14(4-demethylwyosine);imG2(isowyosine);ac 6 A(N 6 -acetyladenosine), or any combination thereof.

[0076] In some aspects, mRNA molecules may be chemically synthesized using methods standard in the art. In some aspects, mRNA molecules may be chemically synthesized such that the mRNA molecule contains at least one chemical modification. In some aspects, mRNA molecules may be produced by in vitro transcription methods standard in the art, including, but not limited to, in vitro transcription using a plasmid template, in vitro transcription using a PCR-based template. In some aspects, in vitro transcription methods may be performed such that the produced mRNA molecule contains at least one chemical modification.

[0077] In some aspects, purified DNA target polypeptides may be produced using standard methods in the art, including, but not limited to, recombinant protein expression and purification in bacterial, fungal, insect, and / or mammalian systems, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, and / or other standard protein production / purification methods known in the art.

[0078] In some aspects, purified DNA-targeted ribonucleoprotein (RNP) complexes may be produced using standard methods in the art, including, but not limited to, recombinant protein expression and purification in bacterial, fungal, insect, and / or mammalian systems, in vitro RNA transcription, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, other standard protein production / purification methods known in the art, and / or other standard nucleic acid production / purification methods known in the art. In some aspects, pre-assembled RNP complexes comprising both at least one protein component and at least one nucleic acid may be assembled and co-purified in vivo (i.e., in bacterial, fungal, insect, and / or mammalian recombinant expression systems). In some aspects, RNP complexes may be assembled in vitro after the at least one protein component and at least one nucleic acid component are purified separately.

[0079] In some aspects, any of the compositions of the present disclosure may further comprise a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA of the plurality of gRNAs is complementary to a nucleic acid sequence located upstream, within, or downstream of an endogenous TERC gene. In some aspects, the plurality of gRNA molecules can comprise at least about 1, or at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, at least about 16, or at least about 17, or at least about 18, or at least about 19, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 500, or at least about 1,000 distinct species of gRNA molecules, each having a different nucleic acid sequence.

[0080] In some aspects, any of the compositions of the present disclosure may further comprise at least one plasmid comprising at least one nucleic acid sequence encoding at least one gRNA operably linked to at least one promoter sufficient to drive expression of the at least one gRNA. In some aspects, any of the compositions of the present disclosure may comprise at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, at least about 16, or at least about 17, or at least about 29, or at least about 30, or at least about 31, or at least about 32, or at least about 33, or at least about 34, or at least about 35, or at least about 36, or at least about 37, or at least about 38, or at least about 39, or at least about 40, or at least about 41, or at least about 42, or at least about 43, or at least about 44, or at least about 45, or at least about 46, or at least about 47, or at least about 48, or at least about 49, or at least about 50, or at least about 51, or at least about 52, or at least about 53, or at least about 54, or at least about 55, or at least about 56, or at least about 57, or at least about 58, or at least about 59, or at least about 60, or at least about 61, or at least about 62, or at least about 63, or at least about 64, or at least about 65, or at least about 66, or at least about 67, or at least about 68, or at least about 6 The vector may further include at least one plasmid comprising at least one nucleic acid sequence encoding about 17, or at least about 18, or at least about 19, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 500, or at least about 1,000 distinct species of gRNA molecules, each species having a different nucleic acid sequence.

[0081] In some aspects, the multiple gRNA molecules may comprise multiple single guide RNA (sgRNA) molecules, crRNA:tracrRNA molecules, truncated sgRNA molecules, high-fidelity scaffold gRNA molecules, or any combination thereof.

[0082] In some aspects, the plurality of gRNA molecules can comprise a plurality of single guide RNA (sgRNA) molecules. In some aspects, the sgRNA molecules can comprise a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of an endogenous TERC gene and at least one MS2 RNA aptamer. In some aspects, the sgRNA molecules can comprise at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about ten MS2 RNA aptamers.

[0083] In some aspects, the guide RNA molecule of any of the compositions of the present disclosure may be a modified guide RNA (mod gRNA) molecule.

[0084] In some aspects, the modified guide RNA may include at least one modified ribonucleoside base. The modified ribonucleoside base may be a pseudouridine (Ψ) residue, a 5-methylcytidine (m 5 C) residues, or any combination thereof.

[0085] In some aspects, the modified guide RNA may include at least one modified nucleoside. The modified nucleoside may be N7-methylguanosine (m 7 G), 2-thiouridine (s 2 U), pseudouridine (Ψ), 2'-O-methyl-U, m 1 In addition to A (1-methyladenosine), 5-methylcytidine (m 5 C), 5-methyluridine (m 5 U), N6-methyladenosine (m 6 A), inosine, and 2'-O-methylated nucleosides; m 2 A(2-methyladenosine); Am(2'-O-methyladenosine); ms 2 m 6 A(2-methylthio-N 6 -methyladenosine);i 6 A(N 6-Isopentenyladenosine);ms 2 i 6 A(2-methylthio-N 6 Isopentenyladenosine);io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine);ms 2 i 6 A(2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine);g 6 A(N 6 -glycinylcarbamoyl adenosine);t 6 A(N 6 -threonylcarbamoyl adenosine);ms 2 t 6 A(2-methylthio-N 6 -threonylcarbamoyl adenosine); m 6 t 6 A(N 6 -methyl-N 6 -threonylcarbamoyl adenosine);hn 6 A(N 6 -hydroxynorvalylcarbamoyl adenosine);ms 2 hn 6 A(2-methylthio-N 6 -Hydroxynorvalylcarbamoyl adenosine; Ar(p)(2'-O-ribosyladenosine(phosphate)); I(inosine); m 1 I(1-methylinosine);m 1 Im(l,2'-O-dimethylinosine);m 3 C(3-methylcytidine); Cm(2'-O-methylcytidine); S 2 C(2-thiocytidine);ac 4 C(N 4 -acetylcytidine);f 5 C(5-formylcytidine);m 5 Cm(5,2'-O-dimethylcytidine);ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine);k 2 C(lycidin);m 1 G(1-methylguanosine);m 2 G(N2 -methylguanosine);m 7 G(7-methylguanosine); Gm(2'-O-methylguanosine); m 2 2G(N 2 ,N 2 -dimethylguanosine);m 2 Gm(N 2 ,2'-O-dimethylguanosine);m 2 2G m(N 2 ,N 2 ,2'-O-trimethylguanosine;Gr(p)(2'-O-ribosylguanosine (phosphate));yW(wybutosine);o2yW(peroxywybutosine);OHyW(hydroxywybutosine);OHyW*(undermodified hydroxywybutosine);imG(wybutosine);mimG(methylwybutosine);Q(queuosine);oQ(epoxyqueuosine);galQ(galactosylqueuosine);manQ(mannosylqueuosine);preQ0(7-cyano-7-deazaguanosine);preQ1(7-aminomethyl-7-deazaguanosine);G + (Archeosin); D(Dihydrouridine); m 5 Um(5,2'-O-dimethyluridine);s 4 U(4-thiouridine);m 5 s 2 U(5-methyl-2-thiouridine);s 2 Um(2-thio-2'-O-methyluridine); acp 3 U(3-(3-amino-3-carboxypropyl)uridine);ho 5 U(5-hydroxyuridine);mo 5 U(5-methoxyuridine);cmo 5 U(uridine 5-oxyacetic acid);mcmo 5 U(uridine 5-hydroxyacetic acid methyl ester);chm 5 U(5-(carboxyhydroxymethyl)uridine);mchm 5 U(5-(carboxyhydroxymethyl)uridine methyl ester);mcm 5 U(5-methoxycarbonylmethyluridine); mcm 5Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine);nm 5 s 2 U(5-aminomethyl-2-thiouridine);mnm 5 U(5-methylaminomethyluridine); mnm 5 s 2 U(5-methylaminomethyl-2-thiouridine);mnm 5 se 2 U(5-methylaminomethyl-2-selenouridine);ncm 5 U(5-carbamoylmethyluridine);ncm 5 Um(5-carbamoylmethyl-2'-O-methyluridine);cmnm 5 U(5-carboxymethylaminomethyluridine);cmnm 5 Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm 5 s 2 U(5-carboxymethylaminomethyl-2-thiouridine);m 6 2A(N 6 ,N 6 -dimethyladenosine; Im (2'-O-methylinosine); m 4 C(N 4 -methylcytidine);m 4 Cm(N 4 ,2'-O-dimethylcytidine);hm 5 C(5-hydroxymethylcytidine);m 3 U(3-methyluridine); cm 5 U(5-carboxymethyluridine);m 6 Am(N 6 ,2'-O-dimethyladenosine);m 6 2Am(N 6 ,N 6 ,0-2'- 2 7 2 2 2 7 2 2 trimethyladenosine);m 2,7 G(N 2 ,7-dimethylguanosine);m 2,2,7 G(N 2 ,N 2,7-trimethylguanosine);m 3 Um(3,2'-O-dimethyluridine);m 5 D(5-methyldihydrouridine);f 5 Cm(5-formyl-2'-O-methylcytidine);m 1 Gm(1,2'-O-dimethylguanosine);m 1 Am(1,2'-O-dimethyladenosine);τm 5 U(5-taurinomethyluridine);τm 5 s 2 U(5-taurinomethyl-2-thiouridine);imG-14(4-demethylwyosine);imG2(isowyosine);ac 6 A(N 6 -acetyladenosine), or any combination thereof.

[0086] In some aspects, the guide RNA molecule may comprise any sequence listed in Table 1 or Table 2.

[0087] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0088] [Table 2-1] [Table 2-2]

[0089] In some aspects, a guide RNA molecule may comprise a portion of any sequence listed in Table 3 or Table 4. In some aspects, a guide RNA molecule may comprise about the first 20 nucleotides of any sequence listed in Table 3. In some aspects, a guide RNA molecule may comprise about the first 21 nucleotides of any sequence listed in Table 4.

[0090] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 4-1] [Table 4-2]

[0091] In some aspects, guide RNA molecules may be chemically synthesized using methods standard in the art. In some aspects, guide RNA molecules may be chemically synthesized such that the guide RNA molecule comprises at least one chemical modification. In some aspects, guide RNA molecules may be generated by in vitro transcription methods standard in the art, including, but not limited to, in vitro transcription using a plasmid template or in vitro transcription using a PCR-based template. In some aspects, in vitro transcription methods may be performed such that the generated guide RNA molecule comprises at least one chemical modification.

[0092] In some aspects, any of the compositions of the present disclosure may further comprise at least one mRNA and / or polynucleotide encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one transactivator molecule. In some aspects, the at least one polynucleotide may be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one transactivator molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

[0093] In some aspects, any of the compositions of the present disclosure may further comprise at least one mRNA and / or polynucleotide encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one VP64 transactivator molecule. In some aspects, the at least one polynucleotide may be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least a portion of MS2 coat protein (MCP) and at least one VP64 transactivator molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

[0094] In some aspects, any of the compositions of the present disclosure may further comprise at least one mRNA and / or polynucleotide encoding a fusion protein comprising at least a portion of an MS2 coat protein (MCP) and at least one P65-HSF transactivator molecule. In some aspects, the at least one polynucleotide may be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least a portion of an MS2 coat protein (MCP) and at least one P65-HSF transactivator molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

[0095] In some aspects, any of the compositions of the present disclosure may further comprise at least one mRNA and / or polynucleotide encoding a fusion protein comprising at least one antibody that binds the SunTag peptide and at least one transactivator molecule. In some aspects, the at least one polynucleotide may be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least one antibody that binds the SunTag peptide and at least one transactivator molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

[0096] In some aspects, any of the compositions of the present disclosure may further comprise at least one mRNA and / or polynucleotide encoding a fusion protein comprising at least one antibody that binds the SunTag peptide and at least one P65-HSF transactivator molecule. In some aspects, the at least one polynucleotide may be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least one antibody that binds the SunTag peptide and at least one P65-HSF transactivator molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

[0097] In some aspects, any of the compositions of the present disclosure may further comprise at least one mRNA and / or polynucleotide encoding a fusion protein comprising at least one antibody that binds the SunTag peptide and at least one VP64 transactivator molecule. In some aspects, the at least one polynucleotide may be a plasmid comprising a nucleic acid encoding a fusion protein comprising at least one antibody that binds the SunTag peptide and at least one VP64 transactivator molecule operably linked to at least one promoter sufficient to drive expression of the fusion protein.

[0098] In some aspects, any composition of the present disclosure may further comprise at least one mRNA and / or polynucleotide encoding at least one rejuvenation factor. In some aspects, the at least one polynucleotide may be a plasmid comprising a nucleic acid encoding at least one rejuvenation factor operably linked to at least one promoter sufficient to drive expression of the at least one rejuvenation factor. The rejuvenation factor may comprise telomerase RNA component (TERC), telomerase-associated reverse transcriptase (TERT), protection of telomeres 1 (POT1), insulin-like growth factor 1 (IGF1), WD repeat-containing antisense to TP53 (WRAP53), nuclear protein family A, member 3 (NOP3), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), shelterin complex subunit and telomerase recruitment factor (ACD / TPP1), TRF-1-interacting ankyrin-related ADP-ribose polymerase (TNKS), telomeric repeat-binding factor 1 (TRF-1), telomeric repeat-binding factor 2 (TRF-2), TERF1-interacting nuclear factor 2 (TIN2), telomeric repeat-binding factor 2 (Rap1), dyskerin pseudouridine synthase 1 (DKC1), ribonucleoprotein NHP2, or any combination thereof.

[0099] The compositions of the present disclosure may be diluted in at least one cell culture medium. In some aspects, the at least one cell culture medium may include conditioned Opti-MEM (Opti-MEM adjusted to pH 8.2 or any pH value between 7.4 and 8.6), unconditioned Opti-MEM, human serum, fetal bovine serum (FBS), 1x phosphate buffered saline (PBS) with a pH between 7.0 and 8.6, or any combination thereof.

[0100] In some aspects, any composition of the present disclosure can be packaged into any cellular delivery system known in the art.Cellular delivery systems include, but are not limited to, adeno-associated virus (AAV; all serotypes, pseudotypes, and hybrids), adenovirus, lentivirus, foamy virus, herpes simplex virus (HSV) particles, retrovirus particles, alphavirus particles, flavivirus particles, rhabdovirus particles, measles virus particles, Newcastle disease virus particles, poxvirus particles, picornavirus particles, nanoparticles, exosomes, and any combination thereof.

[0101] In some aspects, the adeno-associated virus may include, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2 / 1, AAV2 / 2, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV-DJ, AAV-DJ8, or any combination thereof.

[0102] The present disclosure provides at least one viral particle, wherein the at least one viral particle comprises any of the compositions of the present disclosure. In some aspects, the at least one viral particle may be an adeno-associated viral (AAV) particle. In some aspects, the at least one viral particle may be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2 / 1, AAV2 / 2, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV-DJ, or AAV-DJ8 particle. In some aspects, the at least one viral particle may be an adenovirus particle. In some aspects, the at least one viral particle may be a foamy virus particle. In some aspects, the at least one viral particle may be a lentivirus particle. The retroviral particle may be an MMSV particle or an MSCV particle. The lentiviral particle may be an HIV-1 particle or an HIV-2 particle. The alphaviral particle may be an SFV particle, a SIN particle, a VEE particle, or an M1 particle. The flaviviral particle may be a Kunjin virus particle, a West Nile virus particle, or a Dengue virus particle.

[0103] The present disclosure provides at least one exosome, microvesicle, or liposome, wherein the at least one exosome, microvesicle, or liposome comprises any of the compositions of the present disclosure.

[0104] The present disclosure provides at least one nanoparticle, wherein the at least one nanoparticle comprises any of the compositions of the present disclosure. In some aspects, the nanoparticle may comprise a liposome, a micelle, a polymer-based nanoparticle, a lipid-polymer-based nanoparticle, a metal-based nanoparticle, a nanocrystal, a carbon nanotube-based nanoparticle, or a polymeric micelle. In some aspects, the polymer-based nanoparticle may comprise a multiblock copolymer, a diblock copolymer, a polymeric micelle, or a hyperbranched polymer. In some aspects, the polymer-based nanoparticle may comprise a multiblock copolymer or a diblock copolymer. In some aspects, the polymer-based nanoparticle comprises a PLGA polymer, which is poly(lactic-co-glycolic acid).

[0105] In some aspects, the present disclosure provides compositions comprising: a) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of human telomerase reverse transcriptase (hTERT); b) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide; and c) a plurality of guide RNA (gRNA) molecules, wherein the at least one DNA target polypeptide comprises a dCas9 and a VP64-P65-Rta (VPR) molecule, and at least one gRNA of the plurality of gRNAs is complementary to a nucleic acid sequence located upstream of an endogenous hTERC gene.

[0106] kit

[0107] In some aspects, the present disclosure provides kits comprising any of the compositions of the present disclosure. In some aspects, the present disclosure provides kits comprising any portion of any of the compositions of the present disclosure. In some aspects, any of the kits of the present disclosure may be used in any of the methods of the present disclosure.

[0108] In a non-limiting example, the present disclosure provides a kit comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of a telomerase RNA component (TERC).

[0109] Rejuvenation Method of the Present Disclosure

[0110] The present disclosure provides a method for rejuvenating at least one cell, the method comprising contacting the at least one cell with at least one composition of the present disclosure, the method further comprising proliferating the at least one cell contacted with the at least one composition of the present disclosure to produce a plurality of rejuvenated cells.

[0111] Accordingly, the present disclosure provides a method of rejuvenating at least one cell, the method comprising contacting the at least one cell with a composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of a telomerase RNA component (TERC).

[0112] The present disclosure provides a method for treating and / or preventing a disease in a subject, the method comprising the steps of: a) contacting at least one cell with at least one composition of the present disclosure; b) proliferating at least one cell contacted with at least one composition of the present disclosure to generate a plurality of rejuvenated cells; and c) administering the plurality of rejuvenated cells to the subject.

[0113] The present disclosure provides methods for treating and / or preventing disease in a subject, the methods comprising: a) contacting at least one cell with at least one composition of the present disclosure; b) proliferating the at least one cell contacted with the at least one composition of the present disclosure to generate a plurality of rejuvenated cells; c) culturing the plurality of rejuvenated cells under conditions sufficient to transform the plurality of rejuvenated cells into at least one tissue or organ; and d) administering the at least one tissue or organ to a subject.

[0114] The present disclosure provides a method for generating a tissue or organ in vitro, the method comprising: a) contacting at least one cell with a composition of the present disclosure; b) proliferating the at least one cell contacted with at least one composition of the present disclosure to generate a plurality of rejuvenated cells; and c) culturing the plurality of rejuvenated cells under conditions sufficient to convert the plurality of rejuvenated cells into at least one tissue or organ. The at least one tissue or organ may be used for further in vitro testing, including, but not limited to, testing of drugs and / or therapeutic compounds.

[0115] The present disclosure provides a method for generating a plurality of rejuvenated cells, the method comprising: a) contacting at least one cell with at least one composition of the present disclosure; and b) proliferating the at least one cell contacted with at least one composition of the present disclosure to generate a plurality of rejuvenated cells.

[0116] The present disclosure provides a method for generating a plurality of rejuvenated edited cells, the method comprising: a) contacting a plurality of cells with a gene editing system to edit at least one gene in the genome of at least one cell of the plurality of cells, thereby generating at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with at least one composition of the disclosure; and d) expanding the at least one cell contacted with the at least one composition of the disclosure to generate a plurality of rejuvenated edited cells.

[0117] The present disclosure provides methods for treating and / or preventing a disease in a subject, the method comprising the steps of: a) contacting a plurality of cells with a gene editing system to edit at least one gene in the genome of at least one cell of the plurality of cells, thereby generating at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with at least one composition of the disclosure; d) expanding the at least one cell contacted with the at least one composition of the disclosure to generate a plurality of rejuvenated edited cells; and e) administering the plurality of rejuvenated edited cells to the subject.

[0118] In some aspects, the present disclosure provides methods of treating epidermolysis bullosa (EB) in a subject, the method comprising: a) contacting a plurality of cells, including keratinocytes, dermal fibroblasts, mesenchymal stem / stromal cells, or any combination thereof, with a gene editing system to edit at least one gene in the genome of at least one cell of the plurality of cells, thereby generating at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with at least one composition of the disclosure; d) expanding the at least one cell contacted with the at least one composition of the disclosure to generate a plurality of rejuvenated, edited cells; and e) administering the plurality of rejuvenated, edited cells to the subject.

[0119] In some aspects, the present disclosure provides methods of rejuvenating at least one cell in a subject, the methods comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure.In some aspects, the present disclosure provides methods of rejuvenating at least one cell in a subject, the methods comprising administering to the subject at least one therapeutically effective amount of at least a portion of at least one composition of the present disclosure.

[0120] In some aspects, the present disclosure provides methods of rejuvenating at least one subject, the methods comprising administering at least one therapeutically effective amount of at least one composition of the present disclosure.In some aspects, the present disclosure provides methods of rejuvenating at least one subject, the methods comprising administering at least one therapeutically effective amount of at least a portion of at least one composition of the present disclosure.

[0121] In some aspects of the disclosed methods, the step of contacting at least one cell with at least one composition of the present disclosure includes contacting the at least one cell with a first portion of the at least one composition of the present disclosure and then contacting the at least one cell with the first portion of the at least one composition of the present disclosure at least 1 hour, or at least about 2 hours, or at least about 3 hours, or at least about 4 hours, or at least about 5 hours, or at least about 6 hours, or at least about 7 hours, or at least about 8 hours, or at least about 9 hours, or at least about 10 hours, or at least about 11 hours, or at least about 12 hours, or at least about 16 hours, or at least about 20 hours, or at least The method may include contacting the at least one cell with a second portion of at least one composition of the present disclosure after at least about 24 hours, or at least about 28 hours, or at least about 32 hours, or at least about 36 hours, or at least about 40 hours, or at least about 44 hours, or at least about 48 hours, or at least about 52 hours, or at least about 56 hours, or at least about 60 hours, or at least about 64 hours, or at least about 68 hours, or at least about 72 hours, or at least about 76 hours, or at least about 80 hours, or at least about 84 hours, or at least about 88 hours, or at least about 92 hours, or at least about 96 hours.

[0122] Thus, contacting at least one cell with at least one composition of the present disclosure may include: a) contacting the at least one cell with at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) contacting the at least one cell with at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide, wherein the DNA target polypeptide increases transcription of the telomerase RNA component (TERC) at least about 24 hours after step (a). Optionally, steps (a) and (b) may be repeated about every 1 day, or about every 2 days, or about every 3 days, or about every 4 days, or about every 5 days, or about every 6 days, or about every 7 days, or about every 8 days, or about every 9 days, or about every 10 days.

[0123] In some aspects of the disclosed methods, contacting at least one cell with at least one composition of the present disclosure may further include pretreating the at least one cell. In some aspects, pretreating the cell may include contacting the at least one cell with at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT) about once every 4 hours, or about once every 8 hours, or about once every 12 hours, or about once every 16 hours, or about once every 20 hours, or about once every 24 hours, or about once every 28 hours, or about once every 32 hours, or about once every 36 hours, or about once every 40 hours, or about once every 44 hours, or about once every 48 hours. In some aspects, the at least one cell is pretreated for at least about 2 days, or at least about 4 days. Alternatively, the pretreatment may be for at least about 6 days, or at least about 8 days, or at least about 10 days.

[0124] In some aspects, contacting at least one cell with at least one composition of the present disclosure may include: a) contacting the at least one cell with at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of a telomerase reverse transcriptase (TERT) and at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide that increases transcription of a telomerase RNA component (TERC); and b) repeating step (a) about every day, or about every 2 days, or about every 3 days, or about every 4 days, or about every 5 days, or about every 6 days, or about every 7 days, or about every 8 days, or about every 9 days, or about every 10 days.

[0125] Examples of gene transfer treatment schemes are shown in Figures 8, 10, and 11.

[0126] In some aspects of the disclosed methods, contacting at least one cell with a composition of the present disclosure may include gene transfer. In some aspects, gene transfer may include the use of lipofectamine. In some aspects, gene transfer may include any standard gene transfer method known in the art. In some aspects of the disclosed methods, contacting at least one cell with a composition of the present disclosure may include electroporation.

[0127] In some aspects of the methods of the present disclosure, the step of contacting at least one cell may comprise gene transfer, transduction, electroporation, nucleic acid injection, at least one cell-penetrating peptide, or any combination thereof.

[0128] In some aspects of the disclosed methods, contacting at least one cell with a composition of the present disclosure can include nucleic acid injection, which can include any standard nucleic acid injection method known in the art.

[0129] In some aspects of the disclosed methods, contacting at least one cell with a composition of the present disclosure may include contacting the cell with at least one cell-penetrating peptide. In some aspects, the cell-penetrating peptide may be the TAT protein from HIV. In some aspects, the cell-penetrating peptide may include polyarginine. Without wishing to be bound by theory, the at least one cell-penetrating peptide may assist in delivery of the protein or RNP complex of the present disclosure into the cytoplasm of the target cell.

[0130] In some aspects, at least one composition of the present disclosure or at least a portion of at least one composition may be administered to a subject orally, intranasally, transdermally, intrapulmonary, by inhalation, buccal, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrathoracically, intrathecally, and / or parenterally.

[0131] In some aspects of the methods of the present disclosure, the step of expanding at least one cell may include culturing the at least one cell with conditioned Opti-MEM, unconditioned Opti-MEM, human serum, fetal bovine serum (FBS), or any combination thereof.

[0132] In some aspects of the disclosed methods, rejuvenating at least one cell can include increasing expression of TERC in the at least one cell. In some aspects, rejuvenating at least one cell can include increasing expression of TERC by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or may include an increase of at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 10,000%, or at least about 100,000%, or at least about 1,000,000%, or at least about 10,000,000%, or at least about 100,000,000%.In some aspects, the step of rejuvenating the at least one cell comprises increasing the expression level of TERC after contacting the at least one cell with at least one composition of the disclosure by at least about 0.5-fold, or at least about 1.0-fold, or at least about 1.5-fold, or at least about 2.0-fold, or at least about 2.5-fold, or at least about 3.0-fold, or at least about 3.5-fold, or at least about 4.0-fold, or at least about 4.5-fold, or at least about 5.0-fold, or at least about 5.5-fold, or at least about 6.0-fold, or at least about 6.5-fold, or at least about 7.0-fold, or at least about 7.5-fold, or at least about 8.0-fold, compared to the expression level of TERC before contacting the at least one cell with at least one composition of the disclosure. fold, or at least about 8.5 fold, or at least about 9.0 fold, or at least about 9.5 fold, or at least about 10.0 fold, or at least about 25 fold, or at least about 50 fold, or at least about 75 fold, or at least about 100 fold, or at least about 1,000 fold, or at least about 10,000 fold, or at least about 20,000 fold, or at least about 30,000 fold, or at least about 40,000 fold, or at least about 50,000 fold, or at least about 60,000 fold, or at least about 70,000 fold, or at least about 80,000 fold, or at least about 90,000 fold, or at least about 100,000 fold higher.

[0133] In some aspects of the disclosed methods, rejuvenating at least one cell results in an expression level of TERC that is at least about the same as, or at least about 1.5-fold, or at least about 2.0-fold, or at least about 2.5-fold, or at least about 3.0-fold, or at least about 3.5-fold, or at least about 4.0-fold, or at least about 4.5-fold, or at least about 5.0-fold, or at least about 5.5-fold, or at least about 6.0-fold, or at least about 6.5-fold, or at least about 7.0-fold, or at least about 7.5-fold, or at least about 8.0-fold, or at least about 8.5-fold, or at least about 9.0-fold, or less, relative to expression of TERC in a control cell. In some embodiments, the method may include increasing expression of TERC in at least one cell by at least about 9.5 fold, or at least about 10.0 fold, or at least about 25 fold, or at least about 50 fold, or at least about 75 fold, or at least about 100 fold, or at least about 1,000 fold, or at least about 10,000 fold, or at least about 20,000 fold, or at least about 30,000 fold, or at least about 40,000 fold, or at least about 50,000 fold, or at least about 60,000 fold, or at least about 70,000 fold, or at least about 80,000 fold, or at least about 90,000 fold, or at least about 100,000 fold.

[0134] In some aspects of the disclosed methods, rejuvenating at least one cell can include increasing expression of TERT in the at least one cell. In some aspects, rejuvenating at least one cell can include increasing expression of TERT by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or may include an increase of at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1,000%, or at least about 10,000%, or at least about 100,000%, or at least about 1,000,000%, or at least about 10,000,000%, or at least about 100,000,000%.In some aspects, the step of rejuvenating the at least one cell comprises increasing the expression level of TERT after contacting the at least one cell with at least one composition of the disclosure by at least about 0.5-fold, or at least about 1.0-fold, or at least about 1.5-fold, or at least about 2.0-fold, or at least about 2.5-fold, or at least about 3.0-fold, or at least about 3.5-fold, or at least about 4.0-fold, or at least about 4.5-fold, or at least about 5.0-fold, or at least about 5.5-fold, or or at least about 6.0 fold, or at least about 6.5 fold, or at least about 7.0 fold, or at least about 7.5 fold, or at least about 8.0 fold, or at least about 8.5 fold, or at least about 9.0 fold, or at least about 9.5 fold, or at least about 10.0 fold, or at least about 25 fold, or at least about 50 fold, or at least about 75 fold, or at least about 100 fold, or at least about 1,000 fold, or at least about 10,000 fold, or at least about 20,000 fold, or at least about 30,000 fold, or at least about 40,000 fold, or at least about 50,000 fold, or at least about 60,000 fold, or at least about 70,000 fold, or at least about 80,000 fold, or at least about 90,000 fold, or at least about 100,000 fold higher.

[0135] In some aspects of the disclosed methods, rejuvenating at least one cell results in an expression level of TERT that is at least about the same as, or at least about 1.5-fold, or at least about 2.0-fold, or at least about 2.5-fold, or at least about 3.0-fold, or at least about 3.5-fold, or at least about 4.0-fold, or at least about 4.5-fold, or at least about 5.0-fold, or at least about 5.5-fold, or at least about 6.0-fold, or at least about 6.5-fold, or at least about 7.0-fold, or at least about 7.5-fold, or at least about 8.0-fold, or at least about 8.5-fold, or at least about 9.0-fold, or less, relative to expression of TERT in a control cell. In some embodiments, the method may include increasing expression of TERT in at least one cell by at least about 9.5 fold, or by at least about 10.0 fold, or by at least about 25 fold, or by at least about 50 fold, or by at least about 75 fold, or by at least about 100 fold, or by at least about 1,000 fold, or by at least about 10,000 fold, or by at least about 20,000 fold, or by at least about 30,000 fold, or by at least about 40,000 fold, or by at least about 50,000 fold, or by at least about 60,000 fold, or by at least about 70,000 fold, or by at least about 80,000 fold, or by at least about 90,000 fold, or by at least about 100,000 fold.

[0136] In some aspects of the disclosed methods, rejuvenating at least one cell may include increasing the total number of population doublings exhibited by the at least one cell. In some aspects, rejuvenating at least one cell increases the total number of population doublings exhibited by the at least one cell by at least 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%. , or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000% This may include increasing the concentration of phospholipids by at least about 0%, or by at least about 8,000%, or by at least about 9,000%, or by at least about 10,000%, or by at least about 20,000%, or by at least about 30,000%, or by at least about 40,000%, or by at least about 50,000%, or by at least about 60,000%, or by at least about 70,000%, or by at least about 80,000%, or by at least about 90,000%, or by at least about 100,000%.

[0137] In some aspects, rejuvenating at least one cell can be achieved by increasing the number of population doublings exhibited by the at least one cell by at least about 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8 times, or at least about 9 times, or at least about 10 times, or at least about 11 times, or at least about 12 times, or at least about 13 times, or at least about 14 times, or at least about 15 times, or at least about 16 times, or at least about 17 times, or at least about 18 times, or at least about 19 times, or at least about 20 times, or at least about 21 times, or at least about 22 times, or at least about 23 times, or at least about 24 times, or at least about 25 times, or at least about 26 times, or at least about 27 times, or at least about 28 times, or at least about 29 times, or at least about 30 times, or at least about 31 times, or at least about 32 times, or at least about 33 times, or at least about 34 times, or at least about 35 times, or at least about 36 times, or at least about 37 times, or at least about 38 times, or at least about 39 times, or at least about 40 times, or at least about 41 times, or at least about 42 times, or at least about 43 times, or at least about 44 times, or at least about 45 times, or at least about 46 times The method may include increasing the total population doublings by at least about 0.0 fold, or at least about 8.5 fold, or at least about 9.0 fold, or at least about 9.5 fold, or at least about 10.0 fold, or at least about 25 fold, or at least about 50 fold, or at least about 75 fold, or at least about 100 fold, or at least about 200 fold, or at least about 300 fold, or at least about 400 fold, or at least about 500 fold, or at least about 600 fold, or at least about 700 fold, or at least about 800 fold, or at least about 900 fold, or at least about 1,000 fold.

[0138] In some aspects of the disclosed methods, rejuvenating at least one cell may include increasing telomere length in the at least one cell. In some aspects, rejuvenating at least one cell reduces telomere length in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%. , or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000% This may include increasing the concentration of phospholipids by at least about 0%, or by at least about 8,000%, or by at least about 9,000%, or by at least about 10,000%, or by at least about 20,000%, or by at least about 30,000%, or by at least about 40,000%, or by at least about 50,000%, or by at least about 60,000%, or by at least about 70,000%, or by at least about 80,000%, or by at least about 90,000%, or by at least about 100,000%.

[0139] In some aspects, rejuvenating at least one cell can result in telomere length in the at least one cell being the same, or at least 1.5-fold, or at least about 2.0-fold, or at least about 2.5-fold, or at least about 3.0-fold, or at least about 3.5-fold, or at least about 4.0-fold, or at least about 4.5-fold, or at least about 5.0-fold, or at least about 5.5-fold, or at least about 6.0-fold, or at least about 6.5-fold, or at least about 7.0-fold, or at least about 7.5-fold, or at least about 8.0-fold, or In some embodiments, the method may include increasing the length of telomeres in at least one cell by at least about 8.5 times, or at least about 9.0 times, or at least about 9.5 times, or at least about 10.0 times, or at least about 25 times, or at least about 50 times, or at least about 75 times, or at least about 100 times, or at least about 200 times, or at least about 300 times, or at least about 400 times, or at least about 500 times, or at least about 600 times, or at least about 700 times, or at least about 800 times, or at least about 900 times, or at least about 1,000 times.

[0140] In some aspects of the disclosed methods, rejuvenating at least one cell may include increasing mitochondrial DNA copy number in the at least one cell. In some aspects, rejuvenating at least one cell reduces the mitochondrial DNA copy number in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 500%. 50%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%. 00%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

[0141] In some aspects, the step of rejuvenating at least one cell comprises increasing the mitochondrial DNA copy number in the at least one cell relative to the mitochondrial DNA copy number in at least one control cell by at least 1.5 times, or at least about 2.0 times, or at least about 2.5 times, or at least about 3.0 times, or at least about 3.5 times, or at least about 4.0 times, or at least about 4.5 times, or at least about 5.0 times, or at least about 5.5 times, or at least about 6.0 times, or at least about 6.5 times, or at least about 7.0 times, or at least about 7.5 times, or at least about 8.0 times, Alternatively, the method may include increasing the mitochondrial DNA copy number in at least one cell by at least about 8.5-fold, or at least about 9.0-fold, or at least about 9.5-fold, or at least about 10.0-fold, or at least about 25-fold, or at least about 50-fold, or at least about 75-fold, or at least about 100-fold, or at least about 200-fold, or at least about 300-fold, or at least about 400-fold, or at least about 500-fold, or at least about 600-fold, or at least about 700-fold, or at least about 800-fold, or at least about 900-fold, or at least about 1,000-fold.

[0142] In some aspects of the disclosed methods, rejuvenating at least one cell may include increasing the amount of mitochondrial DNA in the at least one cell. In some aspects, the step of rejuvenating at least one cell increases the amount of mitochondrial DNA in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 55%. 0%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%. 00%, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

[0143] In some aspects, the step of rejuvenating at least one cell results in an increase in the amount of mitochondrial DNA in the at least one cell relative to the amount of mitochondrial DNA in at least one control cell, or at least 1.5-fold, or at least about 2.0-fold, or at least about 2.5-fold, or at least about 3.0-fold, or at least about 3.5-fold, or at least about 4.0-fold, or at least about 4.5-fold, or at least about 5.0-fold, or at least about 5.5-fold, or at least about 6.0-fold, or at least about 6.5-fold, or at least about 7.0-fold, or at least about 7.5-fold, or at least about 8.0-fold, or or at least about 8.5-fold, or at least about 9.0-fold, or at least about 9.5-fold, or at least about 10.0-fold, or at least about 25-fold, or at least about 50-fold, or at least about 75-fold, or at least about 100-fold, or at least about 200-fold, or at least about 300-fold, or at least about 400-fold, or at least about 500-fold, or at least about 600-fold, or at least about 700-fold, or at least about 800-fold, or at least about 900-fold, or at least about 1,000-fold.

[0144] In some aspects of the disclosed methods, rejuvenating at least one cell may include increasing the number of mitochondria in the at least one cell. In some aspects, rejuvenating at least one cell increases the number of mitochondria in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%. %, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000% This may include increasing the concentration of phospholipids by at least about 0%, or by at least about 8,000%, or by at least about 9,000%, or by at least about 10,000%, or by at least about 20,000%, or by at least about 30,000%, or by at least about 40,000%, or by at least about 50,000%, or by at least about 60,000%, or by at least about 70,000%, or by at least about 80,000%, or by at least about 90,000%, or by at least about 100,000%.

[0145] In some aspects, rejuvenating at least one cell can result in the number of mitochondria in the at least one cell being the same as, or at least 1.5-fold, or at least about 2.0-fold, or at least about 2.5-fold, or at least about 3.5-fold, or at least about 4.0-fold, or at least about 4.5-fold, or at least about 5.0-fold, or at least about 5.5-fold, or at least about 6.0-fold, or at least about 6.5-fold, or at least about 7.0-fold, or at least about 7.5-fold, or at least about 8.0-fold, or at least about In some embodiments, the method may include increasing the number of mitochondria in at least one cell by 8.5 fold, or at least about 9.0 fold, or at least about 9.5 fold, or at least about 10.0 fold, or at least about 25 fold, or at least about 50 fold, or at least about 75 fold, or at least about 100 fold, or at least about 200 fold, or at least about 300 fold, or at least about 400 fold, or at least about 500 fold, or at least about 600 fold, or at least about 700 fold, or at least about 800 fold, or at least about 900 fold, or at least about 1,000 fold.

[0146] In some aspects of the disclosed methods, rejuvenating at least one cell may include increasing the migratory activity of the at least one cell. In some aspects, rejuvenating the at least one cell increases the migratory activity of the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7,000%. %, or at least about 8,000%, or at least about 9,000%, or at least about 10,000%, or at least about 20,000%, or at least about 30,000%, or at least about 40,000%, or at least about 50,000%, or at least about 60,000%, or at least about 70,000%, or at least about 80,000%, or at least about 90,000%, or at least about 100,000%.

[0147] In some aspects, rejuvenating at least one cell results in a migratory activity of the at least one cell that is the same as, or at least 1.5-fold, or at least about 2.0-fold, or at least about 2.5-fold, or at least about 3.5-fold, or at least about 4.0-fold, or at least about 4.5-fold, or at least about 5.0-fold, or at least about 5.5-fold, or at least about 6.0-fold, or at least about 6.5-fold, or at least about 7.0-fold, or at least about 7.5-fold, or at least about 8.0-fold, or at least about 8.5-fold, or at least about 9. fold, or at least about 9.0 fold, or at least about 9.5 fold, or at least about 10.0 fold, or at least about 25 fold, or at least about 50 fold, or at least about 75 fold, or at least about 100 fold, or at least about 200 fold, or at least about 300 fold, or at least about 400 fold, or at least about 500 fold, or at least about 600 fold, or at least about 700 fold, or at least about 800 fold, or at least about 900 fold, or at least about 1,000 fold.

[0148] In some aspects, rejuvenating at least one cell can include restoring oxidation of at least one protein surface thiol group in the at least one cell to a youthful state. In a non-limiting example, the rejuvenating can include increasing oxidation of at least one protein surface thiol group in the at least one cell such that the oxidation of at least one protein surface thiol group in the at least one cell is equivalent to the oxidation of the same protein surface thiol group in a younger cell. In a non-limiting example, the rejuvenating can include decreasing oxidation of at least one protein surface thiol group in the at least one cell such that the oxidation of at least one protein surface thiol group in the at least one cell is equivalent to the oxidation of the same protein surface thiol group in a younger cell.

[0149] In some aspects, rejuvenating at least one cell can include reducing oxidation of thiol groups of at least one protein in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%. In some aspects, the at least one protein can be EIF2S1, TM9F3, or USP14.

[0150] In some aspects, the step of rejuvenating at least one cell reduces oxidation of thiol groups of at least one protein in the at least one cell by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or less. or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least 950%, or at least about 1,000%, or at least about 2,000%, or at least about 3,000%, or at least about 4,000%, or at least about 5,000%, or at least about 6,000%, or at least about 7 In some aspects, the at least one protein may be IGFB5.

[0151] In some aspects of the disclosed methods, rejuvenating at least one cell can include reducing senescence-associated DNA methylation in the at least one cell. In some aspects, reducing senescence-associated DNA methylation in the at least one cell can include reducing DNA methylation at at least one genomic location correlated with senescence-associated methylation. In some aspects, the at least one genomic location can be cg09780241, cg05099537, cg24541426, cg04316624, cg13180312, cg13316854, cg15726154, cg21507095, cg01697719, or any combination thereof.

[0152] In some aspects, rejuvenating at least one cell may include reducing DNA methylation at at least one genomic location by at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%.

[0153] In some aspects, the at least one control cell may comprise a cell not contacted with a composition of the present disclosure. In some aspects, the at least one control cell may comprise a cell not contacted with a composition of the present disclosure but grown under otherwise identical conditions to the at least one cell contacted with a composition of the present disclosure. In some aspects, the at least one control cell may be a skin fibroblast isolated from a 50-year-old human subject. In some aspects, the at least one control cell may be a neonatal human epidermal keratinocyte (HEKn). In some aspects, the at least one control cell may be an induced pluripotent stem cell (iPSC).

[0154] In some aspects, editing at least one cell may include correcting at least one gene in the at least one cell, knocking out at least one gene in the at least one cell, inserting at least one DNA sequence into the genome of the at least one cell, deleting at least one DNA sequence in the genome of the at least one cell, or any combination thereof. In some aspects, the gene editing system may include any system known in the art for modifying the genome of a target cell, including, but not limited to, CRISPR technology, viral technology, etc.

[0155] The at least one cell may be obtained and / or isolated from a subject. In some aspects, the at least one cell may be any somatic cell. In some aspects, the at least one cell may be a fibroblast, a keratinocyte, a mesenchymal stem cell / stromal cell, a peripheral blood mononuclear cell, a chimeric antigen receptor T cell (CAR-T cell), an endothelial cell, a chondrocyte, a muscle stem cell, a neural stem cell, a hepatocyte, a limbic stem cell, a retinal pigment epithelial cell, a hematopoietic stem cell, a macrophage, a cardiomyocyte, a pancreatic cell, a beta cell, or any combination thereof.

[0156] Depending on the aspect, at least one cell may be an exocrine epithelial cell, a Brunner's gland cell, a sequestering goblet cell of the respiratory and digestive tract, a gastric pit cell, a chief cell, a parietal cell, a pancreatic acinar cell, a small intestinal Paneth cell, a type II pneumocyte, a pulmonary club cell, a barrier cell, a type I pneumocyte, a gallbladder epithelial cell, a cardiac atrial cell, an intercalated duct cell, an intestinal brush border cell, a hormone-secreting cell, an enteroendocrine cell, a K cell, an L cell, an I cell, a G cell, an enterochromaffin cell, an enterochromaffin-like cell, an N cell, an S cell, a D cell, an Mo cell (or an M cell), or a thyroid cell. Thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxic cells, pancreatic islets (islets of Langerhans), alpha cells, beta cells, delta cells, epsilon cells, PP cells (gamma cells), exocrine epithelial cells, salivary gland mucous cells, salivary gland serous cells, von Ebner's gland cells, mammary gland cells, lacrimal gland cells, auditory canal gland cells, eccrine sweat gland dark cells, eccrine sweat gland clear cells, apocrine sweat gland cells, Mollus' gland cells, sebaceous gland cells, Bowman's gland cells, pituitary anterior / intermediate cells, corticotropic cells, gonadotropic cells, mammotropic cells, melanocyte-trophic cells, growth hormone-trophic cells, thyrotropic cells, magnocellular neurosecretory cells, paracellular neurosecretory cells, chromaffin cells, epithelial cells, keratinocytes, epidermal basal cells, melanocytes, follicle cells, hair shaft cells, cortical hair shaft cells, and cornified hair shaft cells.Huxley layer root sheath cells, Henle layer root sheath cells, outer root sheath hair cells, surface epithelial cells, basal cells (stem cells), intercalated duct cells, striated duct cells, mammary gland duct cells, ameloblasts, oral cells, odontoblasts, chalky blasts, nerve cells, inner ear auditory hair cells, outer ear auditory hair cells, olfactory epithelial basal cells, cold-sensing primary sensory neurons, heat-sensing primary sensory neurons, Merkel cells, olfactory receptor neurons, pain receptor neurons, photoreceptor cells, photoreceptor rod cells, photoreceptor blue-sensitive cone cells, photoreceptor green-sensitive cone cells, Photoreceptor red-sensitive cone cells, proprioceptive primary sensory neurons, tactile primary sensory neurons, chemoreceptor carotid body cells, outer hair cells, inner hair cells, taste receptor cells, autonomic neurons, cholinergic neurons, adrenergic neurons, peptidergic neurons, inner column cells, outer column cells, inner supporting cells, outer supporting cells, border cells, Hensen cells, vestibular organ supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells, satellite glial cells, enteric glial cells, glial cells, interneurons, basket cells, wheel cells. Astrocytes, Golgi cells, granule cells, Lugaro cells, unipolar brush cells, Martinotti cells, chandelier cells, Cajal-Retzius cells, double bouquet cells, glial morphocytes, retinal horizontal cells, axonal cells, spinal interneurons, Renshaw cells, principal cells, spindle neurons, tuning fork neurons, pyramidal cells, place cells, grid cells, speed cells, head direction cells, Betz cells, stellate cells, border cells, tufted cells, Purkinje cells, medium spinous cells, (various) astrocytes, oligodendrocytes, ependymal cells, elongated ependymal cells, pituitary cells, Lens cells, anterior lens epithelial cells, crystal-containing lens fiber cells, adipocytes such as white adipocytes and brown adipocytes, hepatic adipocytes, theca interna cells, luteal cells, granulosa luteal cells, theca luteal cells, Leydig cells of the testis that secrete testosterone, seminal vesicle cells, prostate cells, bulbar urethral gland cells, Bartholin's gland cells, Littlé gland cells, endometrial cells, juxtaglomerular cells, renal macular cells, renal peripolar cells, renal mesangial cells, barrier cells, parietal epithelial cells, podocytes, proximal tubule brush border cells, Henle's loop slit cells, and renal distal tubule cells.Renal collecting duct chief cells, interstitial cells, transitional epithelial cells, duct cells, testicular efferent duct cells, epididymal chief cells, epididymal basal cells, endothelial cells, meniscal epithelial cells, interdental epithelial cells, corneal fibroblasts, tendon fibroblasts, bone marrow reticular fibroblasts, other non-epithelial fibroblasts, hepatic stellate cells (Ito cells), intervertebral disc nucleus pulposus cells, hyaluronan chondrocytes, fibrochondrocytes, elastic chondrocytes, osteoblasts / osteocytes, osteoblast precursor cells, vitreous cells, auricular lymph node peri-stellate cells, pancreatic stellate cells, skeletal muscle cells, red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, muscle spindle nucleus sac cells, muscle spindle nucleus chain cells, muscle satellite cells, cardiac myocytes, SA nodule cells, pull The cell may be a kinesthetic fibrocyte, a smooth muscle cell, a myoepithelial cell, an erythrocyte, a megakaryocyte, a platelet, a monocyte, a connective tissue macrophage, an epidermal Langerhans cell, an osteoclast, a dendritic cell, a microglial cell, a neutrophil granulocyte, a myeloblast, a promyelocyte, a myelocyte, a metamyelocyte, an eosinophil, a basophil, a mast cell, a helper T cell, a suppressor T cell, a cytotoxic T cell, a natural killer T cell, a B cell, a plasma cell, a natural killer cell, a hematopoietic stem cell, a germ cell, an oogonia / oocyte, a spermatid, a spermatogonia, a spermatogonium, a trophoblast, a granulosa cell, a Sertoli cell, an epithelial reticular cell, an interstitial cell, an interstitial kidney cell, or a combination thereof.

[0157] In some aspects, the disease may include inflammatory diseases, autoimmune diseases, degenerative diseases, cardiovascular diseases, ischemic diseases, cancer, genetic diseases, metabolic diseases, idiopathic diseases, or any combination thereof. In some aspects, the disease may include, but is not limited to, direct tissue injury (burns, trauma, pressure ulcers, etc.); ischemic / vascular events (myocardial infarction, stroke, shock, hemorrhage, coagulation disorders, etc.); infections (cellulitis, pneumonia, meningitis, SIRS, etc.); tumors (e.g., breast cancer, lung cancer, lymphoma, etc.); immune / autoimmune diseases (graft-versus-host disease, multiple sclerosis, diabetes, inflammatory bowel disease, lupus erythematosus, rheumatoid arthritis, psoriasis, etc.); degenerative diseases (osteoporosis, osteoarthritis, Alzheimer's disease, etc.); congenital / genetic diseases (epidermolysis bullosa, osteogenesis imperfecta, muscular dystrophy, etc.). rheumatoid arthritis, lysosomal storage diseases, Huntington's disease, etc.); adverse drug reactions (drug-induced hepatitis, drug-induced myocardial injury, etc.); toxic injury (radiation exposure, chemical exposure, alcoholic hepatitis, alcoholic pancreatitis, alcoholic cardiomyopathy, cocaine cardiomyopathy, etc.); metabolic disorders (uremic pericarditis, metabolic acidosis, etc.); iatrogenic conditions (radiation-induced tissue damage, surgery-related complications, etc.); and / or progressive conditions of unknown cause (amyotrophic lateral sclerosis, Parsonage-Turner syndrome, etc.), or any combination thereof. In some aspects, the disease may include graft-versus-host disease (GvHD), epidermolysis bullosa (EB), zygotic EB (JEB), simplex EB (EBS), congenital ichthyosis, dyskeratosis congenita, recessive dystrophic EB (RDEB), macular degeneration, Alzheimer's disease, aging syndrome, type II diabetes, heart disease, osteoporosis, chronic skin wounds, diabetes-related ulcers / wounds, connective tissue diseases such as Ehlers-Danlos syndrome (EDS) and Marfan syndrome, cancer, or any combination thereof. In some aspects, the disease may also include injury. Injury may include burns, fractures, concussions, contusions, fractured bones, torn tendons, torn ligaments, punctured skin, scarring, and / or amputation, or any other injury known in the art. In some aspects, the disease may be Ehlers-Danlos syndrome.

[0158] The terms "treating" or "treat," as used herein, refer to the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph, or solvate thereof, to ameliorate the symptoms or complications of the disease or eliminate the disease, condition, or disorder. The term "treat" may also include treatment of a cell or animal model in vitro.

[0159] As used herein, the terms "preventing," "prevent," or "protecting against" refer to reducing or eliminating the onset of symptoms or complications of such a disease, condition, or disorder.

[0160] As used herein, the terms "ameliorate," "ameliorating," and grammatical variations thereof, mean to reduce the severity of disease symptoms in a subject.

[0161] The terms "effective amount" and "therapeutically effective amount" of an agent or compound are used in the broadest sense to refer to a non-toxic but sufficient amount of an active agent or compound to provide a desired effect or benefit.

[0162] The term "benefit" is used in the broadest sense to refer to any desired effect, and particularly includes clinical benefit as defined herein. Clinical benefit may be determined by assessing various endpoints, such as some degree of inhibition of disease progression, including delay and complete halt; a reduction in the number of disease episodes and / or symptoms; a reduction in lesion size; inhibition (i.e., reduction, delay, or complete halt) of disease cell invasion into adjacent peripheral organs and / or tissues; inhibition (i.e., reduction, delay, or complete halt) of disease spread; a reduction in autoimmune responses (which may, but need not, result in regression or ablation of disease lesions); some degree of alleviation of one or more symptoms associated with the disease; the length of disease-free interval after treatment, for example, an increase in progression-free survival; an increase in overall survival; a higher response rate; and / or a decrease in mortality at a given time point after treatment.

[0163] In any of the methods, compositions, or kits of the disclosure, the TERT may be human TERT (hTERT).

[0164] In any of the methods, compositions, or kits of the disclosure, the TERC may be human TERC (hTERC).

[0165] As used herein, "subject" includes mammals. The mammal may be any mammal, such as a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, a pig, or any other mammal. In some aspects, the mammal may be a human. The subject may be male or female. [Example]

[0166] Example 1: Levels of hTERC transcription are higher in induced pluripotent stem cells compared to fibroblasts The transcription levels of human telomerase RNA component (hTERC) in fibroblasts (FB) and induced pluripotent stem cells (iPSC) were measured using NanoString's nCounter Gene Expression assay. As shown in Figure 4, hTERC expression was elevated by approximately 2.4 to 4.6 times in iPSC compared to the parental cell lines F50 (human dermal fibroblasts derived from a 50-year-old individual) and FN2 (neonatal fibroblasts).

[0167] Example 2: Contacting somatic cells with compositions of the present disclosure increases the level of hTERC in somatic cells In this example, various cell lines were transfected with various compositions of the present disclosure.

[0168] Human dermal fibroblasts (F50), neonatal human epidermal keratinocytes (HEKn), and GFP-expressing human mesenchymal stem / stromal cells (hMSC-GFP) from a 50-year-old individual were transfected with 500 ng of modified mRNA (mod-mRNA) encoding dCas9-VPR and 500 ng of hTERC guide RNA (gRNA) (individually or as a mixture of four guides at a 1:1:1:1 ratio) using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). Cells were harvested 24 hours after transfection. Real-time quantitative PCR reactions for human TERC RNA expression were then performed using a Bio-Rad CFX Connect System. Data were analyzed using the ΔΔCt method.

[0169] To test activation of endogenous hTERC transcripts, F50 cells were transfected with one individual gRNA (g1, g2, g3, and g4) and mod-mRNA encoding dCas9-VPR, or a mixture of all four guides (gmix) in a 1:1:1:1 ratio and mod-mRNA encoding dCas9-VPR. The level of hTERC transcription was quantified using quantitative reverse transcription PCR. As shown in Figure 5, the results indicate that even a single guide (g2 or g4) is sufficient to activate endogenous hTERC to a level comparable to that achieved by the mixture of four gRNAs. Transfection with the mixture of four gRNAs and mod-mRNA encoding dCas9-VPR showed the greatest increase in hTERC expression, as shown in Figure 5.

[0170] To observe the activation of endogenous hTERC by dCas9-VPR across other cell lines, HEKn and hMSC-GFP cell lines were transfected with a mixture of all four gRNAs and mod-mRNA encoding dCas9-VPR. hTERC expression was then measured using quantitative reverse transcription PCR. The results are shown in Figure 6. As shown in Figure 6, the level of hTERC activation observed in all cell lines tested was comparable to that observed in iPSCs.

[0171] Method in Example 2 Cell lines: 50-year-old human dermal fibroblast (F50, passage 5) and neonatal human epidermal keratinocyte (HEKn, passage 7) lines were obtained from ATCC. GFP-fluorescent human mesenchymal stem / stromal cells (hMSC-GFP, passage 15) were obtained from Cyagen. F50 lines were cultured in fibroblast growth medium (FEM) consisting of DMEM / F12 supplemented with 5% human serum, 1x MEM non-essential amino acid solution, 55 μM 2-mercaptoethanol (β-ME), 1x GlutaMAX™ supplement, and antibiotics (all from Thermo Fisher Scientific), along with 50 μg / mL ascorbic acid, 1 ng / mL hydrocortisone (both from Sigma), 12 ng / mL basal FGF (from Gibco), and 5 ng / mL human EGF (from Invitrogen). HEKn cells were cultured in EpiLife medium supplemented with EDGS and antibiotics (all from Thermo Fisher Scientific). hMSCs were cultured in mesenchymal stem cell growth medium (MSCGM) (available as a kit from Cyagen).

[0172] Gene transfection: All transfections of fibroblasts and keratinocytes were performed using Opti-MEM® I Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific) as the complexing buffer. Transfections of human mesenchymal stem / stromal cells (hMSCs) were performed using Opti-MEM adjusted to pH 8.2 (Opti-MEM-pH8.2) as described by Kogut et al. (Nature Communications, 2018). One transfection with 500 ng of mod-mRNA encoding dCas9-VPR and 500 ng of hTERC guide RNA (gRNA) (individually (g1, g2, g3, g4) or a 1:1:1:1 ratio of the four guides (gmix)) or 500 ng of mod mRNA encoding dCas9-VPR alone was performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in either Opti-MEM for transfection of keratinocytes and fibroblasts or Opti-MEM-pH 8.2 for transfection of hMSCs. For transfection of mod-mRNA and / or gRNA, 100ng / μL of RNA was diluted 5-fold, and 5μL of RNAiMAX per mg of mod-mRNA and / or gRNA was diluted 10-fold with Opti-MEM (for keratinocytes and fibroblasts) or Opti-MEM-pH 8.2 (for hMSCs). After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, the mod-RNA mix and / or gRNA and RNAiMAX transfection mixture was administered to cell cultures in their respective medium supplemented with 200ng / mL B18R (eBioscience).

[0173] PCR: F50, HEKn, and hMSC-GFP cells were harvested 24 hours after transfection. RNA was extracted using the RNeasy Plus Minikit (Qiagen). cDNA was synthesized using the iScript™ cDNA Synthesis Kit (BioRad). Quantitative PCR (QPCR) reactions for human TERC RNA were performed using SsoAdvanced™ Universal SYBR® Green Supermix. Data were analyzed using the ΔΔCt method.

[0174] Summary of Example 2: When the compositions of the present disclosure, more specifically, mod-mRNA encoding dCas9-VPR, are combined with multiple gRNAs, including one or more different gRNA species, and transfected into somatic cells, hTERC expression can be increased in the transfected cells to levels equivalent to those of induced pluripotent stem cells.

[0175] Example 3: Transfection of mod-RNA encoding dCas9-VPR alone does not induce hTERC expression in target cells In this example, various cell lines were transfected with various compositions of the present disclosure, specifically compositions containing only mod-RNA encoding dCas9-VPR.

[0176] Fifty-year-old human dermal fibroblasts (F50) and GFP-expressing human mesenchymal stem / stromal cells (hMSC-GFP) were transfected with 500 ng of mod-mRNA encoding dCas9-VPR using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). Cells (F50, hMSC-GFP) were harvested 24 hours after transfection. Quantitative reverse transcription PCR of human TERC RNA expression was performed using Bio-Rad's CFX Connect System, and the data were analyzed using the ΔΔCt method. The results are shown in Figure 7. As shown in Figure 7, transfection of only mod-RNA encoding dCas9-VPR into target cells in the absence of any guide RNA did not increase hTERC expression levels.

[0177] Method in Example 3: Cell lines: Fifty-year-old human dermal fibroblasts (F50, passage 5) were obtained from ATCC. GFP-fluorescent human mesenchymal stem / stromal cells (hMSC-GFP, passage 11) were obtained from Cyagen. The F50 line was cultured in fibroblast growth medium (FEM) consisting of DMEM / F12 supplemented with 5% human serum, 1x MEM non-essential amino acid solution, 55 μM 2-mercaptoethanol (β-ME), 1x GlutaMAX™ supplement, and antibiotics (all from Thermo Fisher Scientific), along with 50 μg / mL ascorbic acid, 1 ng / mL hydrocortisone (both from Sigma), 12 ng / mL basal FGF (Gibco), and 5 ng / mL human EGF (Invitrogen). hMSCs were cultured in mesenchymal stem cell growth medium (MSCGM) (obtained as a kit from Cyagen).

[0178] Gene transfection: All fibroblast transfections were performed using Opti-MEM as the complex buffer, while human mesenchymal stem / stromal cell (hMSC) transfections were performed using Opti-MEM pH 8.2. A single transfection with 500 ng of mod-mRNA encoding dCas9-VPR alone was performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in the appropriate Opti-MEM. For mod-mRNA transfections, 100 ng / μL of RNA was diluted 5-fold, and 5 μL of RNAiMAX per μg of mod-mRNA was diluted 10-fold with the appropriate Opti-MEM. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, the mod-RNA and RNAiMAX transfection mixtures were administered to these cell cultures in their respective media supplemented with 200 ng / mL B18R (eBioscience).

[0179] PCR: F50 cells and hMSC-GFP cells were harvested 24 hours after transfection. RNA was extracted using the RNeasy Plus Minikit (Qiagen). cDNA was synthesized using the iScript™ cDNA Synthesis Kit (BioRad). QPCR reactions for human TERC RNA were performed using SsoAdvanced™ Universal SYBR® Green Supermix. Data were analyzed using the ΔΔCt method. P values ​​were calculated using a paired two-tailed Student's t-test. **P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

[0180] Summary of Example 3: Increasing hTERC expression using the methods and compositions of the present disclosure relies on the specific targeting of a DNA targeting molecule containing a transactivation domain, for example, by co-administration of at least one guide RNA.

[0181] Example 4: Methods and compositions of the present disclosure cause an increase in population doublings (PD) of aged fibroblasts In this example, aged fibroblasts were contacted with the compositions of the present disclosure using the methods of the present disclosure.

[0182] A 50-year-old human dermal fibroblast (F50) cell line was obtained from ATCC and subsequently cultured until 90% of the cells exhibited a senescent phenotype, as previously described by Kogut et al. (Nature Communications, 2018). Briefly, this senescent phenotype may include expanded cell morphology and increased positivity for senescence-associated β-galactosidase to approximately 90% or greater. F50 line (F50S, 15th passage: p15, 32.5PD) was thawed and cultured in FEM:DMEM / F12 supplemented with 5% human serum, 1x MEM non-essential amino acid solution, 55µM 2-mercaptoethanol (β-ME), 1x GlutaMAX™ supplement, and antibiotics (all from Thermo Fisher Scientific) with 50µg / mL ascorbic acid, 1ng / mL hydrocortisone (both from Sigma), 12ng / mL basal FGF (from Gibco), and 5ng / mL human EGF (from Invitrogen). 10k fibroblasts (F50S, p15, 32.5PD) were initially seeded per well.

[0183] Figure 8 is a schematic diagram showing the transfection protocol for fibroblasts using the rejuvenation composition of the present disclosure. Initially, 10k aged fibroblasts (F50S, p15, 32.5PD) were seeded per well. The cells were first pretreated with 500ng of mod-mRNA encoding hTERT in three sequential rounds of transfection. After pretreatment, four sequential rounds of transfection were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific) with 500ng of mod-mRNA encoding hTERT, followed the next day by 500ng of mod-mRNA encoding dCas9-VPR and 500ng of human TERC guide RNA (gRNA) (four guides in a 1:1:1:1 ratio). RNA and RNAiMAX were first diluted in Opti-MEM® I reduced serum medium (Opti-MEM). For mod-mRNA and / or gRNA transfection, 100ng / µL of RNA was diluted 5-fold, and 5µL of RNAiMAX per mg of mod-mRNA and / or gRNA was diluted 10-fold in Opti-MEM. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, the mod-RNA mix and / or gRNA and RNAiMAX transfection mixture was applied to cell cultures in FEM supplemented with 200ng / mL B18R (eBioscience). This medium was changed after overnight incubation following each transfection.

[0184] The cumulative population doublings of untreated aged fibroblasts (F50S, p15, 32.5 PD) and the same cells treated with the rejuvenation composition were measured (using the scheme shown in Figure 8). When the cells reached 70-80% confluency, they were trypsinized, counted using a hemocytometer, and passaged. PD was calculated as the logarithm of the ratio of the final count (N) to the starting (standard) count (X0), divided by the logarithm of 2; i.e., PD = [log(N÷X0)]÷log2. P values ​​were calculated using a paired, two-tailed Student's t-test. *P≦0.05, **P≦0.01, ***P≦0.001. The results are shown in Figure 9. As shown in Figure 9, treatment with the rejuvenation composition of the present disclosure results in an increase in population doublings.

[0185] Summary of Example 4: Compositions and methods of the present disclosure can be used to rejuvenate aged cells, including aged fibroblasts, resulting in an increase in the total number of population doublings the treated cells undergo.

[0186] Example 5: Methods and compositions of the present disclosure increase telomere length and mitochondrial DNA content in transfected target cells In this example, various cell lines (low passage and aged 50-year-old human dermal fibroblasts (F50 and F50S, respectively), human mesenchymal stem / stromal cells (hMSCs), and human keratinocytes) were transfected with various compositions of the present disclosure using various methods of the present disclosure. The change in telomere length in each cell line was then measured.

[0187] Low-passage and aged 50-year-old human dermal fibroblasts: The 50-year-old human dermal fibroblast (F50) line was obtained from ATCC and subsequently cultured until 90% of the cells exhibited a senescent phenotype as previously described in Kogut et al. (Nature Communications, 2018). Briefly, the senescent phenotype may include expanded cell morphology and increased positivity for senescence-associated β-galactosidase to approximately 90% or greater. F50 cells (F50S, passage 15, 32.5 PD) were thawed and cultured in FEM:DMEM / F12 supplemented with 5% human serum, 1x MEM non-essential amino acid solution, 55 μM 2-mercaptoethanol (β-ME), 1x GlutaMAX™ supplement, and antibiotics (all from Thermo Fisher Scientific) with 50 μg / mL ascorbic acid, 1 ng / mL hydrocortisone (both from Sigma), 12 ng / mL basal FGF (Gibco), and 5 ng / mL human EGF (Invitrogen). 10k fibroblasts (F50, p3-4 or F50S, p15, 32.5 PD) were initially seeded per well.

[0188] Figure 8 is a schematic diagram illustrating the transfection protocol for fibroblasts using the rejuvenation composition of the present disclosure. First, 10k aged fibroblasts (F50S, p15, 32.5 PD) were seeded per well. The cells were first pretreated with 500ng of mod-mRNA encoding hTERT in three sequential rounds of transfection. After pretreatment, four sequential rounds of transfection were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific) with 500ng of mod-mRNA encoding hTERT, followed the next day by 500ng of mod-mRNA encoding dCas9-VPR and 500ng of human TERC guide RNA (gRNA) (four guides in a 1:1:1:1 ratio). RNA and RNAiMAX were first diluted in Opti-MEM® I reduced serum medium (Opti-MEM). For mod-mRNA and / or gRNA transfection, 100ng / µL of RNA was diluted 5-fold, and 5µL of RNAiMAX per mg of mod-mRNA and / or gRNA was diluted 10-fold in Opti-MEM. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, the mod-RNA mix and / or gRNA and RNAiMAX transfection mixture was applied to cell cultures in FEM supplemented with 200ng / mL B18R (eBioscience). This medium was changed after overnight incubation following each transfection.

[0189] Figure 10 is a schematic diagram showing another fibroblast transfection protocol using the rejuvenation composition of the present disclosure. F50 fibroblasts were seeded in FEM at 15K cells per well in a 6-well dish. Four rounds of transfections were performed every 4 days using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific) with 500 ng of mod-mRNA encoding hTERT, along with 200 ng of mod-mRNA encoding dCas9-VPR and 500 ng of hTERC guide RNA (gRNA) (one selection guide). RNA and RNAiMAX were first diluted in Opti-MEM® I reduced serum medium (Opti-MEM). For mod-mRNA and / or gRNA transfections, 100 ng / μL of RNA was diluted 5-fold, and 5 μL of RNAiMAX per mg of mod-mRNA and / or gRNA was diluted 10-fold using Opti-MEM. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, cell cultures were treated with the mod-RNA mix and / or gRNA and RNAiMAX transfection mixture in FEM supplemented with 200ng / mL B18R (eBioscience). This medium was changed after overnight incubation following each transfection.

[0190] Human mesenchymal stem / stromal cells: Human mesenchymal stem / stromal cells (hMSCs) were cultured under low oxygen (5%) conditions using Mesenchymal Stem Cell Growth Medium (MSCGM) (Cyagen, Inc.). All transfections of hMSCs were performed using Opti-MEM pH 8.2 as described by Kogut et al. (Nature Communications, 2018).

[0191] Figure 11 is a schematic diagram showing the transfection strategy for hMSCs using the rejuvenation composition of the present disclosure. Three rounds of transfection were performed using Lipofectamine (RNAiMAX) (Thermo Fisher Scientific) with 500 ng of mod-mRNA encoding human TERT. Following pretreatment with hTERT mod-mRNA transfection, four sequential rounds of transfection were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific) with 500 ng of mod-mRNA encoding hTERT, followed the next day by 500 ng of mod-mRNA encoding dCas9-VPR and 500 ng of human TERC guide RNA (gRNA) (four guides in a 1:1:1:1 ratio). RNA and RNAiMAX were first diluted in Opti-MEM pH 8.2. For mod-mRNA transfection, 100ng / μL of RNA was diluted 5-fold, and 5μL of RNAiMAX per mg of mod-mRNA was diluted 10-fold using Opti-MEM pH 8.2. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, the mod-RNA mix and RNAiMAX transfection mixture were applied to cell cultures in their respective media supplemented with 200ng / mL B18R. The media was changed after overnight incubation following each transfection.

[0192] Human keratinocytes: Human neonatal epidermal keratinocytes (HEKn) were cultured in EpiLife medium (all ThermoFisher) supplemented with EDGS and antibiotics. All HEK transfections were performed using Opti-MEM (pH unadjusted).

[0193] Figure 11 is a schematic diagram showing the transfection strategy for HEKn cells using the rejuvenation composition of the present disclosure. Three rounds of transfection were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific) with 100 ng of mod-mRNA encoding human TERT. RNA and RNAiMAX were first diluted in Opti-MEM® I Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific). For mod-mRNA transfection, 100 ng / μL of RNA was diluted 5-fold, and 5 μL of RNAiMAX was diluted 10-fold in Opti-MEM per μg of mod-mRNA. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, the mod-RNA mix and RNAiMAX transfection mixture was applied to cell cultures in EpiLife medium supplemented with 200 ng / mL B18R (eBioscience). After pretreatment of HEKn cell lines, four sequential transfections were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific) with 100 ng of mod-mRNA encoding hTERT, followed the next day by 100 ng of mod-mRNA encoding dCas9-VPR and 100 ng of gRNA mix. RNA and RNAiMAX were first diluted in Opti-MEM (Thermo Fisher Scientific). For mod-mRNA and / or gRNA transfections, 100 ng / μL of RNA was diluted 5-fold, and 5 μL of RNAiMAX per μg of mod-mRNA and / or gRNA was diluted 10-fold in Opti-MEM. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, cell cultures were fed with mod-RNA mix and / or gRNA and RNAiMAX transfection mixtures in EpiLife supplemented with 200 ng / mL B18R (eBioscience), which was changed after each transfection overnight incubation.

[0194] F50S cells were harvested 3 days after the final transfection. Genomic DNA (gDNA) was extracted using the DNeasy® Blood and Tissue Kit (Qiagen). Quantitative PCR reactions for relative telomere length in treated and untreated cells were performed using SsoAdvanced™ Universal SYBR® Green Supermix. The results are shown in Figure 12.

[0195] F50 cells were harvested 3 days after the final transfection. Genomic DNA (gDNA) was extracted using the Quick-DNA™ Miniprep Kit (Zymo Research). Changes in average telomere length between treated and untreated cells were measured using quantitative PCR based on the ScienCell Absolute Human Telomere Length Quantification and Mitochondrial DNA Copy Number qPCR Assay Kit (#8958). The telomere and primer set recognized and amplified telomere length by comparing the samples with control genomic DNA (provided in the kit) containing a 100-base pair (bp) telomere sequence located on human chromosome 17. Primer-probe real-time PCR was performed using a BioRad CFX96 Real-Time System (BioRad, Hercules, CA). The results are shown in Figure 13.

[0196] Three days after the final transfection, hMSCs and HEKn cells were harvested. Genomic DNA (gDNA) was extracted using the DNeasy® Blood and Tissue Kit (Qiagen). Changes in average telomere length between treated and untreated cells were measured using quantitative PCR based on the ScienCell Absolute Human Telomere Length Quantification and Mitochondrial DNA Copy Number qPCR Assay Kit (#8958). The telomere and primer set recognized and amplified telomere length by comparing the samples with control genomic DNA (provided in the kit) containing a 100-base pair (bp) telomere sequence located on human chromosome 17. Primer-probe real-time PCR was performed using a BioRad CFX96 Real-Time System (BioRad, Hercules, CA). The results are shown in Figure 14.

[0197] As shown in Figures 12, 13, and 14, F50S, F50, hMSC, and HEKn cells treated with the rejuvenating compositions of the present disclosure had increased telomere length compared to untreated control cells. Furthermore, in the case of treated F50S and HEKn cells, telomere length exceeded that measured in F50-derived induced pluripotent stem cells (F50-iPSCs).

[0198] Changes in mitochondrial DNA (mtDNA) copy number were measured by quantitative PCR using ScienCell's Absolute Human Telomere Length Quantification and Mitochondrial DNA Copy Number Dual Quantification qPCR Assay Kit (#8958). The mtDNA and primer set recognize and amplify one of the most conserved regions in human mtDNA and do not amplify non-target sequences in nuclear genomic DNA. The single copy control (SCR) and primer set recognize and amplify a 100-bp region on human chromosome 17 and serve as a control for data normalization. Primer-probe real-time PCR was performed using a BioRad CFX96 Real-Time System (BioRad, Hercules, CA). The results are shown in Figure 15. As shown in Figure 15, F50S, hMSC, and HEKn cells treated with the rejuvenating composition of the present disclosure had increased mitochondrial DNA copy numbers compared to untreated cells.

[0199] Summary of Example 5: The compositions and methods of the present disclosure can be used to rejuvenate a variety of cell types, including low-passage and aged fibroblasts, human mesenchymal stem / stromal cells, and human epidermal keratinocytes, resulting in increased telomere length and mitochondrial DNA content in the treated cells.

[0200] Example 6: Methods and compositions of the present disclosure reactivate telomerase activity in fibroblasts In this example, 50-year-old human fibroblasts (F50) were transfected with various compositions of the present disclosure. Telomerase activity was analyzed in transfected target cells as well as control cells.

[0201] Fifty-year-old human dermal fibroblasts (F50, passage 6) were cultured in fibroblast growth medium (FEM) consisting of DMEM / F12 supplemented with 5% human serum, 1x MEM non-essential amino acid solution, 55 μM 2-mercaptoethanol (β-ME), 1x GlutaMAX™ supplement, and antibiotics (all from Thermo Fisher Scientific), along with 50 μg / mL ascorbic acid, 1 ng / mL hydrocortisone (both from Sigma), 12 ng / mL basal FGF (Gibco), and 5 ng / mL human EGF (Invitrogen). As an untreated control, the F50-iPSC line was grown in mTeSR™ supplement (StemCell Technologies) supplemented with 1x mTeSR™ supplement and antibiotics (Thermo Fisher Scientific) on plates coated with Matrigel base matrix (Corning). TM Cultured in medium 1.

[0202] Two sequential transfections were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific) with either 3 μg of mod-mRNA encoding hTERT or 3 μg of mod-mRNA encoding hTERT with 3 μg of mod-mRNA encoding dCas9-VPR and 500 ng of gRNA mix. RNA and RNAiMAX were first diluted in Opti-MEM® I Reduced Serum Medium (Opti-MEM) (Thermo Fisher Scientific). For mod-mRNA transfections, 100 ng / μL of RNA was diluted 5-fold, and 5 μL of RNAiMAX per μg of mod-mRNA was diluted 10-fold with Opti-MEM. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, cell cultures were treated with the RNAiMAX transfection mixture containing mod-RNA and / or gRNA in FEM supplemented with 200 ng / mL B18R (eBioscience). The medium was changed after each transfection overnight incubation.

[0203] Telomerase activity was measured using the TRAPeze® Telomerase Detection Kit (Millipore) according to the manufacturer's instructions. Extracts were obtained using CHAPS (1x) lysis buffer from positive control cells (provided with the kit), an F50-derived iPSC line (F50-iPSC), fibroblasts (F50), and fibroblasts (F50) treated with two sequential transfections of 3 μg hTERT alone or 3 μg hTERT with 3 μg dCas9-VPR and 3 μg gRNA mix. Approximately 10,000 cells were assayed for each telomeric repeat amplification procedure, and the equivalent of 1,500 cells was loaded into each well of a 15% non-denaturing TBE (Tris-borate, EDTA)-urea polyacrylamide gel. Each sample was heat-inactivated at 85°C for 10 minutes to assess assay background.

[0204] The results of the telomerase activity assay are shown in Figure 16. Brighter products indicate higher activity. As shown in Figure 16, combined treatment with mod-mRNA encoding hTERT and mod-mRNA encoding dCas9-VPR and a gRNA specific for hTERC resulted in higher levels of telomerase activity compared to untreated iPSCs or F50 cells transfected with mod-mRNA encoding hTERT alone.

[0205] Summary of Example 6: The compositions and methods of the present disclosure can reactivate and increase telomerase activity in target cells, thereby rejuvenating the target cells.

[0206] Example 7: Compositions of the present disclosure promote the proliferation of single cells In this example, primary human adult fibroblasts were transfected with the compositions of the present disclosure to determine whether the methods and compositions of the present disclosure can support proliferation from a single cell.

[0207] Primary human adult fibroblasts were obtained from skin biopsies. Adult fibroblasts were cultured in fibroblast growth medium (FEM) consisting of DMEM / F12 supplemented with 5% human serum, 1x MEM non-essential amino acid solution, 55 μM 2-mercaptoethanol (β-ME), 1x GlutaMAX™ supplement, and antibiotics (all from Thermo Fisher Scientific), along with 50 μg / mL ascorbic acid, 1 ng / mL hydrocortisone (both from Sigma), 12 ng / mL basal FGF (Gibco), and 5 ng / mL human EGF (Invitrogen). Fibroblasts from individual patients were seeded and single cells were selected using (10 x 10 mm) PYREX® cloning cylinders.

[0208] Figure 11 is a schematic diagram of the transfection protocol for selected single fibroblasts using the rejuvenation composition of the present disclosure, with adjustments made for limited tissue culture surface area. These cells were first pretreated with three sequential transfections of 50 ng of mod-mRNA encoding hTERT. Following pretreatment, four sequential transfections were performed using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific) with 50 ng of mod-mRNA encoding hTERT, followed the next day by 50 ng of mod-mRNA encoding dCas9-VPR and 50 ng of hTERC guide RNA (gRNA) (four guides in a 1:1:1:1 ratio). RNA and RNAiMAX were first diluted in Opti-MEM® I reduced serum medium (Opti-MEM). For mod-mRNA and / or gRNA transfection, 100ng / μL of RNA was diluted 5-fold, and 5μL of RNAiMAX per 1μg of mod-mRNA and / or gRNA was diluted 10-fold in Opti-MEM. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, the mod-RNA mix and / or gRNA and RNAiMAX transfection mixture was applied to cell cultures in FEM supplemented with 200ng / mL B18R (eBioscience). This medium was changed after overnight incubation following each transfection.

[0209] Following the gene transfer procedure, each well was trypsinized, and the cells were transferred into one well of a 6-well tissue culture plate for further expansion in FEM. As shown in Figure 17, one week after the final gene transfer, only 2 out of 10 wells of untreated cells had successfully expanded and could be harvested for gDNA extraction, whereas 9 out of 10 wells of treated fibroblasts had expanded and could be harvested.

[0210] Summary of Example 7: The compositions and methods of the present disclosure can promote proliferation of even single cells.

[0211] Example 8: Compositions and methods of the present disclosure increase the migratory activity of high-passage human mesenchymal stem / stromal cells (hMSCs) In this example, human mesenchymal stem / stromal cells (hMSCs) were transfected with compositions of the present disclosure to determine whether the compositions and methods of the present disclosure can increase the migratory activity of high-passage hMSCs.

[0212] Transendothelial migration (TEM) assays were used to measure the migratory activity of treated and untreated hMSCs. A schematic diagram of the TEM assay is shown in Figure 18. Briefly, Corning FluoroBlok cell culture inserts were pre-seeded with human endothelial cells (HUVECs). GFP-expressing hMSCs were coated, and their migration through the HUVEC layer and the pores of the FluoroBlok membrane was quantified over time using a bottom-reading fluorescent microscope such as the CellInsight CX7 High-Content Screening (HCS) Platform.

[0213] 24-well Corning FluoroBlok™ inserts were coated with collagen. After coating, human umbilical vein endothelial cells (HUVECs) were plated at 80K / cm in ECM-2MV BulletKit™ medium (Lonza). 2 The cells were seeded at 100°C and cultured overnight in 5% CO2 to allow attachment. After successful overnight culture, the medium in the basal chamber was changed to human mesenchymal stem cell growth medium (Cyagen) supplemented with human recombinant EGF [10 ng / mL] (Stemcell Technologies). Green fluorescent protein (GFP)-labeled human mesenchymal stem / stromal cells purchased from Cyagen were cultured up to passage 12 (P12) and treated with the rejuvenation composition of the present disclosure as described in Figure 11 and Example 5, while control hMSCs were cultured without mRNA treatment. The rejuvenation procedure continued for two passages, resulting in passage number P14. A portion of the rejuvenated hMSCs was frozen overnight in CoolCell LX™ at -80°C, while the remaining cells remained in culture. After two passages, the frozen cells were thawed and cultured for two additional passages.

[0214] The four conditions were: old, high-passage hMSCs (P20) that had not been rejuvenated or frozen; young, low-passage hMSCs (P5) that were a fresh vial of GFP-labeled hMSCs (Cyagen) that were thawed and allowed to adhere overnight before being lifted for transendothelial migration assays; frozen and rejuvenated hMSCs (P17) that had been frozen at P15, thawed, and cultured for two passages; and rejuvenated hMSCs that had not been frozen at all but had undergone five passages according to the rejuvenation procedure. Samples from these four conditions were then added to the apical chamber of a FluoroBlok on top of the adherent HUVEC layer. Four fields were acquired in triplicate on a ThermoScientific CellInsight CX7 LED High-Content Screening (HCS) Platform. The CX7 HCS is designed to rapidly acquire and quantify high-content data, such as kinetic analyses performed in transendothelial migration assays (TEM), using brightfield, widefield, and confocal microscopy across the entire fluorescence spectrum. As shown in Figure 19, rejuvenated high-passage hMSCs reached saturation significantly faster than untreated young hMSCs (40-50 hours vs. 130 hours). Meanwhile, aged high-passage hMSCs (P20) exhibited reduced migration capacity. One-way ANOVA showed a significant difference (p<0.0001) between young and rejuvenated hMSCs compared to high-passage hMSCs.

[0215] Summary of Example 8: The compositions and methods of the present disclosure can rejuvenate hMSCs, as evidenced by the increased migratory activity of high passage hMSCs treated with the compositions and methods of the present disclosure.

[0216] Example 9: Compositions and methods of the present disclosure restore protein thiol oxidation levels in high-passage, aged human mesenchymal stem / stromal cells (hMSCs) to levels observed in young, low-passage hMSCs In this example, we transfected aged, high-passage human mesenchymal stem / stromal cells (hMSCs) with a composition of the present disclosure to determine whether the compositions and methods of the present disclosure could restore the oxidation levels of protein thiol groups in high-passage, aged human mesenchymal stem / stromal cells (hMSCs) to the levels observed in young, low-passage hMSCs. Among amino acids, the sulfur-containing cysteine ​​(Cys) is particularly susceptible to oxidation. This is due to the presence of a thiol moiety (-SH) in the side chain of Cys, which readily forms a disulfide bond with another thiol moiety in response to oxidation. Reversible oxidation of Cys thiols regulates enzyme activity and ligand binding, as well as redox signaling, and deregulation of this regulation plays an essential role in the development of many human diseases and aging.

[0217] Human mesenchymal stem / stromal cells (hMSCs) were cultured in low oxygen (5%) conditions using Mesenchymal Stem Cell Growth Medium (MSCGM) (available as a kit from Cyagen). Three conditions were used: old, high-passage hMSCs that had not been rejuvenated at all (P14); young, low-passage hMSCs (P5) that were a fresh vial of hMSCs that had been thawed and allowed to attach overnight before being lifted and processed for peptide analysis; and old, high-passage hMSCs that had been treated with the disclosed rejuvenation composition at passage 12. The rejuvenation procedure continued for two passages, so that the passage number of the treated old hMSCs was P14 before peptide analysis was performed, matching that of the untreated old hMSCs.

[0218] hMSCs were pre-transfected three times with 500 ng of mod-mRNA encoding human TERT using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). After pre-transfection with hTERT mod-mRNA, three sequential transfections were performed with 500 ng of mod-mRNA encoding hTERT, followed the next day by 500 ng of mod-mRNA encoding dCas9-VPR and 500 ng of hTERC guide RNA (gRNA) (one selection guide) using Lipofectamine® RNAiMAX™ (RNAiMAX) (Thermo Fisher Scientific). RNA and RNAiMAX were first diluted in Opti-MEM pH 8.2. For mod-mRNA transfection, 100ng / μL of RNA was diluted 5-fold, and 5μL of RNAiMAX per 1μg of mod-mRNA was diluted 10-fold using Opti-MEM pH 8.2. After dilution, these components were combined and incubated at room temperature for 15 minutes. After incubation at room temperature, the mod-RNA mix and RNAiMAX transfection mixtures were applied to cell cultures in their respective media supplemented with 200ng / mL B18R. The media was changed after overnight incubation following each transfection.

[0219] Transfected, non-transfected, and low-passage young hMSCs were treated with the iodinated TMT sixplex Isobaric Mass Tag Labeling Kit (Thermo Scientific). The resulting iodinated TMT-labeled peptide mixture was analyzed using a QExactive HF Orbitrap mass spectrometer equipped with an Easy nLC 1000 UPLC system (Thermo Fischer Scientific). Peptide identification was performed using the MaxQuant program. Each MS / MS spectrum was analyzed against a human-specific database (Uniprot). After this analysis, the data file was exported and further analyzed for the desired data using Perseus software. Each experiment was repeated twice. The MaxQuant and Perseus software were downloaded from the Max Planck Institute for Biochemistry website.

[0220] Thiol oxidation levels in aged high-passage hMSCs were increased in 88 proteins and decreased in 31 proteins compared to young hMSCs. Transfection of aged hMSCs with the rejuvenating composition of the present disclosure resulted in the restoration of thiol oxidation levels in approximately 90% of target proteins to levels observed in young cells. Figure 20 shows representative results of thiol group analysis of proteins with increased (EIF2S1, TM9F3, and USP14) and decreased (IGFB5) thiol oxidation levels in aged high-passage hMSCs, and demonstrates that these thiol oxidation levels are restored to a youthful state in response to treatment with the rejuvenating composition.

[0221] Summary of Example 9: The compositions and methods of the present disclosure can rejuvenate hMSCs, as evidenced by the restoration of youthful levels of protein thiol oxidation in high-passage hMSCs treated with the compositions and methods of the present disclosure.

[0222] Example 10: Compositions and methods of the present disclosure reduce senescence-associated DNA methylation in high-passage aged human mesenchymal stem / stromal cells (hMSCs) and human neonatal epidermal keratinocytes (HEKn) In this example, compositions of the present disclosure were transfected into aged high-passage human mesenchymal stem / stromal cells (hMSCs) and aged high-passage human neonatal epidermal keratinocytes (HEKn) to determine whether the compositions and methods of the present disclosure can reduce senescence-associated DNA methylation levels in these cells. Changes in DNA methylation are recognized as one of the most common molecular changes in aging and cellular senescence.

[0223] Human fibroblasts of various origins were cultured in FEM, human keratinocytes of various origins were cultured in EpiLife medium supplemented with EDGS, and human mesenchymal stem cell (hMSC) growth medium (MSCGM) were cultured. The following cell types were used for DNA methylation analysis: young low-passage neonatal fibroblasts (P3), young low-passage adult F50 fibroblasts (P3), young low-passage neonatal HEKn keratinocytes (P3), young low-passage fetal keratinocytes (P2), young low-passage adult keratinocytes (P3), young umbilical cord-derived hMSCs (P2), aged high-passage F50S fibroblasts (P15), aged high-passage hMSCs (P13), and aged high-passage HEKn (P10), all of which were not treated with the rejuvenating composition. The treatment groups included aged high-passage HEKn and aged high-passage hMSCs treated with the rejuvenating composition described in Figure 11 and Example 5. After treatment with the composition was terminated, the treated cells were expanded for an additional 6 days. Genomic DNA (gDNA) was extracted from each cell culture using a Quick-DNA™ Miniprep Kit (Zymo Research) and subjected to DNA methylation analysis using an Illumina Infinium MethylationEPIC BeadChip Kit.

[0224] The DNA methylation data obtained for various cell types was analyzed using the R package "IlluminaHumanMethylationEPICanno.ilm10b2.hg19" and summarized into three groups: young cells, old (high-passage) cells, and old cells treated with the rejuvenation composition of the present disclosure. The young cell group included young low-passage neonatal fibroblasts (P3), young low-passage adult fibroblasts (P3), young low-passage neonatal keratinocytes (P3), young low-passage fetal keratinocytes (P2), young low-passage adult keratinocytes (P3), and young umbilical cord-derived hMSCs (P2). The old cell group included old high-passage F50S fibroblasts (P15), old high-passage hMSCs (P13), and old high-passage HEKn (P10). The treated cell groups included aged, high-passage HEKn and aged, high-passage hMSCs treated with the rejuvenation composition. To eliminate differences in cell type-specific methylation between each group, cells of various types and origins were mixed based on their senescent state. These groups were compared using a two-tailed t-test with unequal variances, and methylation levels were calculated as the percentage of methylated nucleotides at target sites, ranging from 0 to 1. Differentially methylated sites were selected based on the greatest difference in methylation levels between groups, and nine DNA methylation sites were identified as the most highly methylated sites in aged, high-passage cells, regardless of cell type origin.

[0225] 21 shows the locations of the nine identified senescence-associated DNA markers and their associated genomic loci. All nine sites showed elevated DNA methylation levels in the aged high-passage group. Treatment of cells from the aged group with the rejuvenating composition of the present disclosure reduced DNA methylation levels at all nine sites to levels similar to those in the young cell group.

[0226] Summary of Example 10: Compositions and methods of the present disclosure can be used to rejuvenate various cell types, including aged human mesenchymal stem / stromal cells and human epidermal keratinocytes, resulting in a reduction of senescence-associated DNA methylation in the treated cells.

Claims

1. a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide; The composition, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

2. a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) a composition comprising at least one DNA targeting polypeptide, wherein said DNA targeting polypeptide increases transcription of telomerase RNA component (TERC).

3. 3. The composition of claim 1 or claim 2, wherein the at least one first polynucleotide molecule comprises an mRNA molecule encoding at least a portion of TERT.

4. 3. The composition of claim 1 or claim 2, wherein the at least one first polynucleotide molecule comprises a plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of at least a portion of TERT.

5. 10. The composition of claim 1, wherein the at least one second polynucleotide molecule comprises an mRNA molecule encoding at least a portion of at least one DNA target polypeptide.

6. 2. The composition of claim 1, wherein the at least one second polynucleotide molecule comprises a plasmid comprising a nucleic acid sequence encoding at least a portion of the at least one DNA target polypeptide operably linked to at least one promoter sufficient to drive expression of at least a portion of the at least one DNA target polypeptide.

7. 7. The composition of any one of claims 1 to 6, wherein the DNA targeting polypeptide comprises at least one Cas9 molecule, at least one Cas9 mutant molecule, at least one Cas9 homologous molecule, or any combination thereof.

8. 8. The composition of claim 7, wherein the Cas9 molecule, Cas9 mutant molecule, or Cas9 homologous molecule is nuclease-deficient or nuclease-inactive.

9. 9. The composition of claim 8, wherein the Cas9 mutant molecule comprises eSpCas9(K855A), eSpCas9(1.0), eSpCas9(1.1), SpCas9-HF1(VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9(VQR), Cas9(EQR), Cas9(VRER), Cas9(D1135E), Cas9(QQR1), SaCas9(KKH), Nme1Cas9, Nme2Cas9, Nme3Cas9, or any combination thereof.

10. The Cas9 homologues include Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria osteomyelitis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006, and the like. Cpf1 (LbCpf1), Streptococcus canis Cas9 (SCCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casφ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum strain F Cas9, Nitratifracter sarsuginis strain DSM 16511 Cas9, Parvibaculum labamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azosopyrirum B510 9. The composition of claim 8, comprising: Sphaerochaeta globus strain buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taphensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudontelmedius Cas9, Filifactor allosis Cas9, Legionella pneumophila strain paris Cas9, Saterella wadswarsensis Cas9, Corynebacter diphtheriae Cas9, or any combination thereof.

11. The Cas9 homologues include Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria osteomyelitis Cas9 (NmCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006, and the like. Cpf1 (LbCpf1), Streptococcus canis Cas9 (SCCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casφ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum strain F Cas9, Nitratifracter sarsuginis strain DSM 16511 Cas9, Parvibaculum labamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azosopyrirum B510 9. The composition of claim 8, comprising a chimeric mutant of Cas9, Sphaerochaeta globus strain buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taphensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudontelmedius Cas9, Filifactor allosis Cas9, Legionella pneumophila strain paris Cas9, Saterella wadswarsensis Cas9, Corynebacter diphtheriae Cas9, or any combination thereof.

12. 12. The composition of any one of claims 1 to 11, wherein the DNA targeting polypeptide comprises at least one TALE molecule, at least one zinc finger molecule, at least one meganuclease molecule, or any combination thereof.

13. The composition of any one of claims 1 to 12, wherein the DNA target polypeptide comprises at least one transactivating molecule.

14. 14. The composition of claim 13, wherein the at least one transactivating molecule comprises at least one P65 molecule, at least one Rta molecule, at least one VP16 molecule, at least one VP64 molecule, at least one VP160 molecule, at least one VP64-P65-Rta (VPR) molecule, at least one SunTag peptide, at least one single guide RNA-MS2 (sgRNA-MS2) molecule, or any combination thereof.

15. The composition of any one of claims 1 to 14, wherein the DNA-targeting polypeptide comprises a DNA-targeting ribonucleoprotein (RNP) complex.

16. The composition of any one of claims 1 to 15, wherein the DNA target polypeptide comprises at least one guide RNA.

17. 14. The composition of claim 13, wherein the transactivation molecule comprises at least one single guide RNA-MS2 (sgRNA-MS2) molecule.

18. 18. The composition of claim 17, wherein the at least one sgRNA-MS2 molecule comprises a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of an endogenous TERC gene, and at least about 1, or at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10 MS2 RNA aptamers.

19. The composition of any one of claims 1 to 18, wherein the DNA targeting polypeptide comprises a dCas9 molecule and a VPR molecule.

20. 20. The composition of any one of claims 1 to 19, wherein the DNA targeting polypeptide binds upstream, 5', within, downstream, or 3' of an endogenous TERC gene.

21. The composition of any one of claims 1 to 20, wherein the mRNA molecule is a modified mRNA molecule.

22. 22. The composition of claim 21, wherein the modified mRNA molecule comprises at least one modified ribonucleoside base.

23. The modified ribonucleoside bases include pseudouridine (Ψ) residues, 5-methylcytidine (m ５ C) residues, or any combination thereof.

24. 23. The composition of claim 22, wherein the modified mRNA molecule comprises at least one modified nucleoside.

25. The modified nucleoside is 5-methylcytidine (m ５ C), 5-methyluridine (m ５ U), N6-methyladenosine (m ６ A), an inosine 2'-O-methylated nucleoside, or any combination thereof.

26. 26. The composition of any one of claims 1-25, further comprising a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA of the plurality of gRNAs is complementary to a nucleic acid sequence located upstream, within, or downstream of an endogenous TERC gene.

27. 27. The composition of Claim 26, wherein the plurality of gRNA molecules comprises at least about 1, or at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10 distinct species of gRNA molecules, each of which has a different nucleic acid sequence.

28. 28. The composition of any one of claims 1 to 27, further comprising at least one plasmid comprising at least one nucleic acid sequence encoding at least one gRNA operably linked to at least one promoter sufficient to drive expression of the at least one gRNA.

29. 29. The composition of any one of claims 26-28, wherein the plurality of gRNA molecules comprises a plurality of single guide RNA (sgRNA) molecules, crRNA:tracrRNA molecules, truncated sgRNA molecules, high-fidelity scaffold gRNA molecules, or any combination thereof.

30. 30. The composition of any one of claims 26 to 29, wherein the at least one guide RNA molecule is a modified guide RNA (mod gRNA) molecule.

31. 31. The composition of any one of claims 26 to 30, wherein the at least one guide RNA molecule comprises any sequence listed in Table 1 or Table 2.

32. a) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of human telomerase reverse transcriptase (hTERT); b) at least one modified mRNA molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA target polypeptide; and c) comprising a plurality of guide RNA (gRNA) molecules; The composition, wherein the at least one DNA target polypeptide comprises dCas9 and a VP64-P65-Rta (VPR) molecule, and at least one gRNA of the plurality of gRNAs is complementary to a nucleic acid sequence located upstream of an endogenous hTERC gene.

33. The composition according to any one of claims 1 to 32, further comprising at least one mRNA and / or polynucleotide encoding at least one rejuvenating factor.

34. 34. The composition of claim 33, wherein the rejuvenation factor comprises telomerase RNA component (TERC), telomerase-associated reverse transcriptase (TERT), protection of telomeres 1 (POT1), insulin-like growth factor 1 (IGF1), WD repeat-containing antisense to TP53 (WRAP53), nuclear protein family A, member 3 (NOP3), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), shelterin complex subunit and telomerase recruitment factor (ACD / TPP1), TRF-1-interacting ankyrin-associated ADP-ribose polymerase (TNKS), telomeric repeat-binding factor 1 (TRF-1), telomeric repeat-binding factor 2 (TRF-2), TERF1-interacting nuclear factor 2 (TIN2), telomeric repeat-binding factor 2 (Rap1), dyskerin pseudouridine synthase 1 (DKC1), ribonucleoprotein NHP2, or any combination thereof.

35. The composition of any one of claims 1 to 34, wherein the TERT is human TERT (hTERT).

36. The composition of any one of claims 1 to 35, wherein the TERC is human TERC (hTERC).

37. A composition comprising at least one virus particle, comprising a composition according to any one of claims 1 to 36.

38. 38. The composition of claim 37, wherein the at least one viral particle is an adeno-associated viral (AAV) particle, an adenoviral particle, a lentiviral particle, a foamy viral particle, a herpes simplex viral (HSV) particle, a retroviral particle, an alphaviral particle, a flaviviral particle, a rhabdoviral particle, a measles viral particle, a Newcastle disease viral particle, a poxviral particle, a picornaviral particle, or any combination thereof.

39. 39. The composition of claim 38, wherein the at least one AAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2 / 1, AAV2 / 2, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV-DJ, or AAV-DJ8 particle.

40. 39. The composition of claim 38, wherein the at least one retroviral particle is an MMSV particle or an MSCV particle.

41. 39. The composition of claim 38, wherein the at least one lentiviral particle is an HIV-1 particle or an HIV-2 particle.

42. 39. The composition of claim 38, wherein the at least one alphavirus particle is an SFV particle, a SIN particle, a VEE particle, or an M1 particle.

43. 39. The composition of claim 38, wherein the at least one flavivirus particle is a Kunjin virus particle, a West Nile virus particle, or a Dengue virus particle.

44. 35. A composition comprising at least one exosome, microvesicle, or liposome, wherein said at least one exosome, microvesicle, or liposome comprises the composition of any one of claims 1 to 34.

45. A composition comprising at least one nanoparticle, wherein said at least one nanoparticle comprises the composition of any one of claims 1 to 36.

46. 46. ​​The composition of claim 45, wherein the nanoparticle comprises a liposome, a micelle, a polymeric nanoparticle, a lipid-polymer nanoparticle, a metal-based nanoparticle, a nanocrystal, a carbon nanotube-based nanoparticle, or a polymeric micelle.

47. A kit comprising the composition of any one of claims 1 to 45.

48. 48. A method of rejuvenating at least one cell, comprising contacting said at least one cell with a composition or kit according to any one of claims 1 to 47.

49. 49. The method of claim 48, further comprising expanding the at least one cell contacted with the composition or kit of any one of claims 1 to 47 to generate a plurality of rejuvenated cells.

50. 1. A method of treating and / or preventing a disease in a subject, comprising: a) contacting at least one cell with the composition or kit of any one of claims 1 to 47; b) expanding said at least one cell contacted with the composition or kit of any one of claims 1 to 47 to generate a plurality of rejuvenated cells; and c) administering the plurality of rejuvenated cells to the subject.

51. 1. A method of treating and / or preventing a disease in a subject, comprising: a) contacting at least one cell with the composition or kit of any one of claims 1 to 47; b) expanding said at least one cell contacted with the composition or kit of any one of claims 1 to 47 to generate a plurality of rejuvenated cells; c) culturing said plurality of rejuvenated cells under conditions sufficient to transform said plurality of rejuvenated cells into at least one tissue or organ; and d) administering said at least one tissue or organ to said subject.

52. 1. A method for generating tissue or organs in vitro, comprising: a) contacting at least one cell with the composition or kit of any one of claims 1 to 47; b) expanding said at least one cell contacted with the composition or kit of any one of claims 1 to 47 to generate a plurality of rejuvenated cells; and c) culturing said plurality of rejuvenated cells under conditions sufficient to transform said plurality of rejuvenated cells into at least one tissue or organ.

53. 1. A method for generating a plurality of rejuvenation-edited cells, comprising: a) contacting a plurality of cells with a gene editing system to edit at least one gene in the genome of at least one cell of the plurality of cells, thereby generating at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with the composition or kit of any one of claims 1 to 47; and d) expanding the at least one cell contacted with the composition or kit of any one of claims 1 to 47 to generate a plurality of rejuvenated edited cells.

54. 1. A method of treating and / or preventing a disease in a subject, comprising: a) contacting a plurality of cells with a gene editing system to edit at least one gene in the genome of at least one cell of the plurality of cells, thereby generating at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with the composition or kit of any one of claims 1 to 47; d) expanding the at least one cell contacted with the composition or kit of any one of claims 1 to 47 to generate a plurality of rejuvenated edited cells; and e) administering the plurality of rejuvenation edited cells to the subject.

55. 1. A method of treating epidermolysis bullosa (EB) in a subject, comprising: a) contacting a plurality of cells, the plurality of cells comprising keratinocytes, dermal fibroblasts, mesenchymal stem / stromal cells, or any combination thereof, with a gene editing system to edit at least one gene in the genome of at least one cell of the plurality of cells, thereby generating at least one edited cell; b) isolating the at least one edited cell; c) contacting the isolated at least one edited cell with the composition or kit of any one of claims 1 to 47; d) expanding the at least one cell contacted with the composition or kit of any one of claims 1 to 47 to generate a plurality of rejuvenated edited cells; and e) administering the plurality of rejuvenation edited cells to the subject.

56. 56. The method of any one of claims 48-55, wherein expanding the at least one cell comprises culturing the at least one cell with conditioned Opti-MEM, unconditioned Opti-MEM, human serum, fetal bovine serum (FBS), or any combination thereof.

57. 57. The method of any one of claims 48-56, wherein rejuvenating at least one cell comprises increasing expression of TERC in the at least one cell, increasing expression of TERT in the at least one cell, increasing the total number of population doublings exhibited by the at least one cell, increasing telomere length in the at least one cell, increasing mitochondrial DNA copy number in the at least one cell, increasing the amount of mitochondrial DNA in the at least one cell, increasing the number of mitochondria in the at least one cell, increasing the migration activity of the at least one cell, restoring the oxidation level of thiol groups in proteins in the at least one cell to a youthful state, reducing aging-associated DNA methylation in the at least one cell, or any combination thereof.

58. 58. The method of any one of claims 48-57, wherein the at least one cell is a fibroblast, a keratinocyte, a mesenchymal stem cell / stromal cell, a peripheral blood mononuclear cell, a chimeric antigen receptor T cell (CAR-T cell), an endothelial cell, a chondrocyte, a muscle stem cell, a neural stem cell, a hepatocyte, a limbic stem cell, a retinal pigment epithelial cell, a hematopoietic stem cell, a macrophage, a cardiomyocyte, a pancreatic cell, a beta cell, or a combination thereof.

59. 59. The method of any one of claims 48 to 58, wherein the disease comprises graft versus host disease (GvHD), autoimmune diseases, epidermolysis bullosa (EB), recessive dystrophic EB (RDEB), zygotic EB (JEB), simplex EB (EBS), congenital ichthyosis, dyskeratosis congenita, macular degeneration, Parkinson's disease, Alzheimer's disease, aging, type I and type II diabetes, burns, chronic skin wounds, diabetes-related ulcers / wounds, heart disease, osteoporosis, cancer, connective tissue diseases such as Ehlers-Danlos syndrome (EDS) or Marfan syndrome, liver diseases, lung diseases, and any combination thereof.

60. 60. The method of any one of claims 48 to 59, wherein the step of contacting at least one cell comprises gene transfer, transduction, electroporation, nucleic acid injection, at least one cell-penetrating peptide, or any combination thereof.

61. 47. A method of rejuvenating at least one cell in a subject, comprising administering to the subject a therapeutically effective amount of at least one of the compositions of any one of claims 1 to 46.

62. 47. A method of rejuvenating at least one subject, comprising administering to the subject a therapeutically effective amount of at least one composition according to any one of claims 1 to 46.

63. 63. The method of any one of claims 48 to 62, wherein the subject is a mammal.

64. 64. The method of claim 63, wherein the mammal is a human, a primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, pig, or any other mammal.

65. 65. The method of claim 64, wherein the mammal is a human.

Images