Improved methods for detecting and modulating the embryo-fetal transition in mammalian species

Abstract

Aspects of the present invention include algorithms, methods, and compositions related to the modulation of molecules that regulate the transition from embryonic to fetal development in mammals. Methods and compositions for the use of such modulation to increase the regenerative capacity in fetal and adult tissues that are otherwise unable to regenerate without scarring are also provided. [Selected Figure] Figure 1

Description

[Technology Field]

[0001] Cross-reference of related applications This application claims priority and benefit of U.S. Provisional Patent Application No. 62 / 347,075, filed June 7, 2016, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to an improved method for detecting and modulating embryo-fetal transfer in mammalian species. [Background technology]

[0003] Advances in stem cell technology, such as the in vitro isolation and proliferation of human pluripotent stem (hPS) cells, constitute a significant new area of ​​medical research. hPS cells have a proven ability to proliferate in an undifferentiated state and subsequently be induced to differentiate into all cell types in the human body, including complex tissues. This suggests that, for example, many diseases caused by cellular dysfunction may be suitable for treatment with the administration of human embryonic stem cells derived from various differentiated forms (Thomson et al., Science 282:1145-1147 (1998)).

[0004] Regarding the differentiation of hPS cells into desired cell types, the possibility of isolating human embryonic progenitor cell lines as clones provides a means to increase novel, highly purified cell lineages that exhibit prenatal patterns of gene expression useful for the regeneration of tissues such as skin in a scar-free manner. Such cell types have important applications in research and for the creation of cell-based therapies (see PCT application PCT / US2006 / 013519, filed April 11, 2006, entitled “Novel Uses of Cells With Prenatal Patterns of Gene Expression,” U.S. Patent Application 11 / 604,047, filed November 21, 2006, entitled “Methods to Accelerate the Isolation of Novel Cell Strains from Pluripotent Stem Cells and Cells Obtained Thereby,” each incorporated herein by reference).

[0005] More recently, the ability of pluripotent stem cells and induced embryoid bodies to self-assemble in vitro into three-dimensional organoids has attracted attention as a potential pathway for both obtaining tissue for transplantation (Singh et al., Stem Cells Dev. 2015. 24(23):2778-95) and modeling human embryonic development. In contrast to germ cells, fetal and adult-derived cells often show reduced capacity for organogenesis in vitro and augmentation in vivo. Augmentation, sometimes referred to as "additional regeneration," is a type of tissue regeneration in which relatively undifferentiated mesenchymal blasts proliferate at the site of injury, and these cells subsequently differentiate to restore the tissue structure of the original tissue. While it is not possible to precisely determine the developmental timing of the loss of additional regenerative capacity, and it likely varies depending on the histological type, it is still thought that embryo-fetal transition (EFT), or the eighth week of human development (Carnegie Developmental Stages 23; O'Rahilly, R., F. Muller (1987) Developmental Stages in Human Embryos, Including a Revision of Streeter's 'Horizons' and a Survey of the Carnegie Collection. Washington, Carnegie Institution of Washington), corresponds temporally to the loss of skin regeneration in placental mammals (Walmsley, GG et al. 2015. Scarless Wound Healing: Chasing the Holy Grail Plast Reconstr Surg. 135(3):907-17).Interspecies correlations indicate increased regenerative capacity in the embryonic or larval stage (Morgan, TH (1901). Regeneration (New York: The MacMillan Company, outlined), and Sanchez Alvarado, A., and Tsonis, PA (2006). Bridging the regeneration gap: genetic insights from diverse animal models (Nat. Rev. Genet. 7, 873-884) suggest that tissue regeneration, in contrast to scarring, reflects the presence of the embryonic phenotype rather than the fetal or adult phenotype. In some species, changes in developmental timing (metachronism) are observed, such as developmental arrest (metachronism) in larval development and limb regeneration observed in the Mexican salamander axolotl (A. mexicanum) (Voss, SR et al., Thyroid hormone responsive QTL and the evolution of paedomorphic salamanders. Heredity (2012) 109). This is associated with remarkable regenerative capacity, as seen in cases 293-298). In contrast, some animals, such as the African spiny mouse (Acomys cahirinus), exhibit remarkable skin regeneration in the absence of apparent metachrony, which likely reflects uncharacterized molecular changes (Gawriluk, TR, 2016. Comparative analysis of ear-hole closure identifies epimorohic regeneration as a discrete trait in mammals. Nature Commun. 7:11164).

[0006] Despite these observations, EFT markers for testing the role of specific molecules in additive regeneration are limited. We have previously disclosed compositions and methods related to EFT markers in mammalian species, as well as their use in modulating tissue regeneration (see, for example, U.S. Provisional Patent Application No. 61 / 831,421 filed June 5, 2013, PCT Patent Application PCT / US2014 / 040601 filed June 3, 2014, and U.S. Patent Application No. 14 / 896,664 filed December 7, 2015 (the disclosures of these documents are incorporated herein by reference in their entirety)). Nevertheless, further molecular modulosers and methods for modulating EFT are needed for research and treatment in regenerative medicine and cancer. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] PCT Application No. PCT / US2006 / 013519 [Patent Document 2] U.S. Patent Application No. 11 / 604,047 [Patent Document 3] U.S. Patent Application No. 12 / 504,630 [Patent Document 4] U.S. Provisional Patent Application No. 61 / 831,421 [Patent Document 5] PCT Patent Application PCT / US2014 / 040601 [Patent Document 6] U.S. Patent Application No. 14 / 896,664 [Non-patent literature]

[0008] [Non-Patent Document 1] Thomson et al., Science 282:1145-1147 (1998) [Non-Patent Document 2] Singh et al., Stem Cells Dev. 2015. 24(23):2778-95 [Non-Patent Document 3] O'Rahilly, R., F. Muller (1987) Developmental Stages in Human Embryos, Including a Revision of Streeter's 'Horizons' and a Survey of the Carnegie Collection. Washington, Carnegie Institution of Washington [Non-Patent Document 4] Walmsley, GG et al. 2015. Scarless Wound Healing: Chasing the Holy Grail Plast Reconstr Surg. 135(3):907-17 [Non-Patent Document 5] Morgan, TH (1901). Regeneration (New York: The MacMillan Company [Non-Patent Document 6] Sanchez Alvarado, A., and Tsonis, PA (2006). Bridging the regeneration gap:genetic insights from diverse animal models(Nat. Rev. Genet. 7, 873-884 [Non-Patent Document 7] Voss, SR et al., Thyroid hormone responsive QTL and the evolution of paedomorphic salamanders. Heredity (2012) 109, 293-298 [Non-Patent Document 8] Gawriluk, TR, 2016. Comparative analysis of ear-hole closure identifies epimorohic regeneration as a discrete trait in mammals. Nature Commun. 7:11164 [Overview of the project]

[0009] Early candidate molecules for metachronic regulatory factors were identified in the nematode Caenorhabditis elegans. These included lin-28 / let-7 (Ambros, V. and Horvitz, HR (1984). Heterochronic mutants of the nematode Caenorhabditis elegans. Science 226, 409-416). More recently, recombinant expression of the paralog Lin28a in mice has been reported to increase skin regeneration after wound formation and regeneration of severed fingers, as well as increase markers of oxidative phosphorylation (Shyh-Chang, N. et al. 2013. Lin28 Enhances Tissue Repair by Reprogramming Cellular Metabolism. Cell 155, 778-792). However, the regenerative capacity in these mice is not comparable to the remarkable augmentative regeneration observed in Cairo spiny mice. The lin-28 / let-7 system has also been observed to be activated in numerous cancer cell types (Jiang, S. and Baltimore, D. 2016. RNA-binding protein Lin28 in cancer and immunity, Cancer Lett. 375(1):108-13). Abnormal expression of LIN28 in cancer suggests that the reason for natural selection, likely the suppression of regenerative capacity during EFT, is that in most vertebrates, the selection of this trait, although potentially limiting post-injury survival, can function as a tumor suppressor mechanism. The well-known observation that many cancers exhibit embryonic reverse mutations such as the Warburg effect is also consistent with this hypothesis.The complete identification of such molecular mechanisms will not only advance fundamental research into tissue regeneration but also facilitate novel methods of the present invention for modulating these molecular mechanisms in cells and tissues in vivo, enabling the screening of drugs that can induce “inducible tissue maturation” (i.e., pre-fetal or prenatal) cells to facilitate the repair of traumatic or degenerative diseases, including but not limited to age-related degenerative diseases, to produce cells exhibiting a relatively more mature phenotype, such as that of a fetal or adult phenotype, and to identify and target malignant or pre-malignant cells that have reverted to the aforementioned embryonic phenotype for diagnostic and therapeutic purposes, and to control the timing of treating such malignant cells with anticancer drugs by maturing those cells to a more mature fetal or adult phenotype, halting their proliferation, and / or modulating apoptosis. Such a diagnosis involves determining the extent to which a carcinoma, adenocarcinoma, or sarcoma has reverted to its embryonic phenotype (embryo-onco phenotype), and subsequently treating the patient's cancer with a drug appropriate to that phenotype, i.e., a drug effective in inhibiting the replication or apoptosis of cancer cells of that particular phenotype. Furthermore, the present invention provides methods and compositions capable of inducing "inducible cancer cell maturation" (iCM), and such drugs, whether used alone or in combination with other therapeutic agents such as commonly used chemotherapeutic agents, have the potential to be useful in treating a very wide range of malignancies.

[0010] overview The present disclosure provides useful compounds, compositions, and methods for creating useful artificial intelligence software (deep learning) for screening agents that can modulate the molecular pathways that regulate embryonic-fetal transition (EFT) in mammalian cells and for using such agents, in identifying whether cells exhibit an embryonic or fetal or adult phenotype, the relative maturity of such fetal or adult cells, regulatory non-coding RNAs and mRNAs involved in embryonic-fetal transition (EFT), the ability of tissue regeneration, and cells and tissues that cannot give rise to regeneration that does not leave the aforementioned scars completely when identifying cancer. By screening and using agents that can induce iTM to produce cells that exhibit an adult phenotype from cells that previously had gene expression of an embryonic or fetal pre-pattern, and further detecting and targeting malignant cells that have returned to an embryonic phenotype for diagnosing and treating cancer, and further screening agents that can induce iCM.

[0011] In certain aspects of the present disclosure, a deep learning algorithm is provided that enables a researcher to identify patterns of gene expression in cells and tissues as belonging to an embryonic or fetal, or adult source. The algorithm describes a method for identifying the developmental state of animal cells, comprising: 1) the step of collating RNA expression data obtained from animal cells; 2) the step of training an artificial intelligence algorithm to identify where in the embryonic developmental time series the cells are normally present based on their RNA expression profile; and 3) the step of testing the RNA expression profile to assign the cells from which the RNA is derived to the time series with at least 90% accuracy.

[0012] In another aspect of the present disclosure, a deep learning algorithm is provided that enables the identification of genes differentially expressed in embryonic and fetal or adult sources.

[0013] In another aspect, a set of screening criteria is described to demonstrate that genes that are candidate regulators of EFT are important in the molecular pathways that regulate EFT by determining whether they are screened against RNA obtained from developing mammals such as mouse or human sources, or malignant counterparts of specific cell types.

[0014] In another aspect, the present disclosure provides a method for identifying genes, RNAs, and proteins that regulate EFT in mammalian species, including primate species, more specifically human species, where the genes are identified by comparing the expression of mRNAs and non-coding RNAs that are differentially expressed at the embryonic stage of development compared to the fetal and adult stages of development, or genes encoding splice variants in such RNAs, using RNA sequencing technology, gene expression array-based analysis of comparative pathway analysis, and deep neural network analysis. More specifically, by this method, genes encoding mRNAs and non-coding RNAs that are differentially expressed in a number of diverse somatic cell types at the embryonic stage of development (prior to EFT) compared to fetal and adult cells (after EFT) at the pre-birth stage of development are identified. In the case of the human species, the transition from embryo to fetus (fetus) during development occurs at approximately 8 weeks of gestational development, in mice at approximately 16 days, and in the rat species at approximately 17.5 days (www.php.med.unsw.edu.au / embryology / index).

[0015] In another aspect of the present disclosure, cloned, oligoclonal, pooled cloned, or pooled oligoclonal embryonic progenitor cell lines derived from pluripotent stem cells that exhibit gene expression patterns specific to the embryonic stage of mammalian development are used as a source of coding and non-coding RNAs and compared to coding and non-coding RNAs in cells and tissues obtained from fetal or adult-derived sources to identify genes encoding mRNAs, non-coding RNAs, or splice variants, as well as transcriptome-based signaling pathway alterations in fetal and adult cells compared to embryonic stage cells.

[0016] In another aspect of this disclosure, gene expression data from cells treated with small molecule drugs or other agents are analyzed to identify factors useful for inducing embryonic pattern gene expression resulting in iTR in fetal or adult-derived cells, or to induce maturation of cells exhibiting embryonic pattern gene expression into corresponding cells exhibiting adult pattern expression (iTM or iCM).

[0017] In another aspect of this disclosure, mammalian cells are fetal or adult-derived cells, and the EFT is modified by modulating the cells in vitro, and the resulting cells are reintroduced into tissue in vivo to increase regenerative capacity.

[0018] In another aspect of this disclosure, transcriptional regulatory genes that are differentially expressed in various types of somatic cells during the embryonic stage of development are compared with various types of somatic cells at post-embryonic developmental stages, such as adult cell types that cannot participate in TR, to identify genes whose altered expression in splice variants causes suppression of tissue regeneration in vivo in adult mammals. In some embodiments, methods for identifying genes whose expression or suppression may result in iTR include comparing the transcriptome of a clone, oligoclone, or pooled clone or pooled oligoclone hPS cell-derived embryonic progenitor cell line with the transcriptome of various types of adult-derived cells or tissues to identify genes commonly expressed in embryonic progenitors, or comparing it with RNA splice variants of embryonic progenitors that are not expressed or expressed at significantly low levels in adult-derived cells, or alternatively, comparing genes or RNA expressed in adult-derived cells with splice variants that are not expressed or expressed at significantly low levels in clone, oligoclone, or pooled clone or pooled oligoclone hPS cell-derived embryonic progenitor cell lines. In another embodiment, candidate iTR genes are identified as those that are expressed at higher levels in embryonic progenitor cells compared to adult-derived cells and are also involved in carcinogenesis, or as genes that are expressed at lower levels in embryonic progenitor cells compared to adult-derived cells and are also involved in tumor suppression.

[0019] In another aspect, the Disclosure provides a method for optimizing a protocol for administering iTR factors, wherein the factors include factors that can induce pluripotency in somatic cell types under other conditions (i.e., when creating iPS cells), and the factors include combinations of genes in diverse cell and tissue types: OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, LIN28A and LIN28B, the RNAs or proteins they encode, for identifying optimized factor and / or repressor combinations for specific cell and tissue types.

[0020] In another aspect, the present disclosure provides a method for administering iTR factors, wherein the factors can induce pluripotency in somatic cells under other conditions (i.e., when creating iPS cells), and the factors include genes: OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, TERT, LIN28A and LIN28B, the RNAs they encode, or combinations of proteins for in vitro to revert fetal or adult-derived cells to their embryonic counterparts or in vivo in diseased tissue without reverting cells in the tissue to pluripotent stem cells.

[0021] In another aspect, the present disclosure provides a method for administering a chemical inducer to iTR, wherein the inducer can improve (enhance) the efficiency of inducing pluripotency to somatic cells under other conditions (i.e., when creating iPS cells), and the inducer is used to convert fetal or adult-derived cells back into their embryonic counterparts in vitro, or in This includes combinations of inhibitors for glycogen synthase 3 (GSK3), TGF-β signaling inhibitors, HDAC inhibitors, H3K4 / 9 histone demethylase LSD1 inhibitors, Dot1L inhibitors, G9a inhibitors, Ezh2 inhibitors, DNA methyltransferase inhibitors, 3' phosphoinositide-dependent kinase 1 activators, glycolysis promoters, RAR agonists, hypoxia-mimicking agents, telomerase activators, or MAPK / ERK pathway inhibitors, administered in vivo to induce tissue regeneration in diseased tissue without converting the cells in the tissue back to pluripotent stem cells.

[0022] In another aspect, the disclosure provides a method for screening iTR gene combinations in a diverse range of cell and tissue types to identify factor and / or repressor combinations optimized for specific cell and tissue types.

[0023] In another aspect, the disclosure provides a method for modifying the expression of iTR genes in cultured cells to restore them to a state in which they can participate in iTR when transplanted into tissues that cannot otherwise produce sufficient TR.

[0024] In another aspect of the disclosure, somatic cell proliferation is enhanced and thereby promotes TR by transiently expressing a telomerase catalytic component, including but not limited to the human gene TERT, in combination with the iTR gene of the disclosure in target cells or tissues in which TR is to be induced. The co-expression of this telomerase activity with the iTR gene (e.g., LIN28A, LIN28B, or the α or β cluster of clustered protocadherin genes expressed at relatively high levels in the embryonic state, such as PCDHA4, PCDHB10, PCDHB2, PCDHB9) is particularly useful in species with short telomeres in which cellular replication senescence occurs during the lifespan of the organism, as is the case with the human species. In another aspect of the disclosure, somatic cell proliferation is enhanced without immortalizing the cells by transiently expressing (rather than constitutively expressing) a telomerase catalytic component, including the human gene TERT, in target cells or tissues.

[0025] In another aspect of the present disclosure, somatic cell proliferation is enhanced and TR is promoted by transiently expressing a telomerase catalytic component, including but not limited to the human gene TERT, in combination with a chemoinducer of iTR of the present disclosure in target cells or tissues in which TR is to be induced. The co-expression of telomerase activity with chemical inducers of iTR (e.g., glycogen synthase 3 (GSK3) inhibitor, TGF-β signaling inhibitor, HDAC inhibitor, H3K4 / 9 histone demethylase LSD1 inhibitor, Dot1L inhibitor, G9a inhibitor, Ezh2 inhibitor, DNA methyltransferase inhibitor, 3' phosphoinositide-dependent kinase 1 activator, glycolysis promoter, RAR agonist, hypoxia mimic, telomerase activator, or MAPK / ERK pathway inhibitor) is particularly useful in species with short telomeres and in which cellular replication senescence occurs during the lifespan of the organism, as in the human species. In another aspect of this disclosure, the proliferative capacity of somatic cells is extended without immortalizing them by transiently expressing (rather than constitutively expressing) a telomerase catalytic component containing the human gene TERT in target cells or tissues.

[0026] In another aspect, the Disclosure provides means for manipulating an animal model, preferably a mouse model, having stable regenerative capacity, wherein the mouse is a common experimental strain, thereby facilitating molecular genetic research and animal preclinical studies. The stable regenerating mouse is produced by creating a mouse in which the transcription, stability, or translation of genes, RNAs, and proteins that express iTR RNA transcripts or inhibit iTR is inhibited, and by crossing the mice with each other such that the significantly regenerating mouse has a sufficient number of gene expressions for embryonic patterns relating to desired tissue regeneration, and / or by treating the mouse, or specific tissues of the mouse, with a drug that can induce an overall change in iTR gene expression.

[0027] In one embodiment, the treatment of aging and age-related diseases is described.

[0028] By inducing metachrony and modifying somatic cell development, it is possible to create larger or smaller animals, or alter their lifespan.

[0029] Metachronism, or alteration of the developmental timeline, is selectively induced in many species to significantly modify the resulting animals, sometimes producing "hopeful monsters." Similarly, RNA-level modifications as disclosed herein can also modulate developmental timing, adult body size, and lifespan.

[0030] Numerous aspects of aging and age-related diseases that can be addressed using iTR therapy are presented in this disclosure. These signs of aging include age-related vascular dysfunction, e.g., peripheral, coronary, and cerebrovascular diseases; musculoskeletal disorders, e.g., osteoarthritis, intervertebral disc degeneration, fractures, tendon and ligament lacerations, and limb regeneration; neurological disorders, e.g., stroke and spinal cord injury; muscle disorders, e.g., muscular dystrophy, sarcopenia, myocardial infarction, and heart failure; endocrine disorders, e.g., type 1 diabetes, Addison's disease, hypothyroidism, and pituitary dysfunction; gastrointestinal disorders, e.g., exocrine pancreatic insufficiency; ocular disorders, e.g., macular degeneration, retinitis pigmentosa, and neuroretinal degeneration; skin conditions, e.g., skin burns, lacerations, surgical incisions, alopecia, graying of hair, and skin aging; lung disorders, e.g., emphysema and pulmonary interstitial fibrosis; hearing impairments, e.g., hearing loss; and hematological disorders, e.g., aplastic anemia and failed hematopoietic stem cell transplants.

[0031] In another aspect, the disclosure provides a method for restoring iTR genes to a state in which they can be involved in iTR by modifying their expression in cells in vivo.

[0032] Another aspect of this disclosure provides a method for inducing iTR in tissue affected by a degenerative disease, including but not limited to osteoarthritis, wherein the means for inducing iTR in the diseased tissue utilize a gene expression vector or a vector resulting in exogenous expression of an iTR gene disclosed herein, including but not limited to a member of the LIN family such as LIN28A or LIN28B, together with a telomerase catalyst component such as human TERT.

[0033] In another aspect, the Disclosure provides a composition comprising (i) and (ii) below (a) to (c): (a) a candidate modulator or plurality of modulators of TR activity, either in a purified state or as a mixture with other molecules; (b) somatic cells that cannot produce TR, wherein the cells exhibit fetal or adult pattern gene expression rather than embryonic pattern gene expression; (c) a reporter construct present in or in an extract of somatic cells that cannot produce TR, wherein a promoter of a gene that is differentially regulated in somatic cells at the embryonic stage of development compared to the fetal and adult stages drives the expression of a reporter gene; and (ii) a method for identifying a candidate global modulator of TR activity, comprising determining whether the candidate modulator or plurality of modulators affects the expression of the reporter gene, wherein the altered expression of the reporter gene compared to the expression of the gene in the absence of the candidate modulator indicates that the compound modulates iTR activity.

[0034] In some embodiments, the method for identifying candidate modulators of TR further includes administering a candidate compound identified as a modulator of TR or a number of compounds.

[0035] In some embodiments, a method for identifying candidate global modulators for TR further includes administering candidate compounds for TR to cells derived from a fetal or adult source and analyzing COX7A1 expression by utilizing readily measurable readout, such as fluorescence generated from GFP driven by the COX7A1 promoter.

[0036] In some embodiments, a method for identifying candidate modulators for TR further includes administering candidate compounds for TR to cells derived from a fetal or adult source and analyzing COX7A1 expression by an assay of the degree of methylation of CpG islands in the COX7A1 gene.

[0037] In some embodiments, the method for identifying a compound further includes administering the compound to a subject. In some embodiments, the subject is a non-human animal, for example, a non-human animal serving as a TR or wound healing model. In some embodiments, the subject is a human.

[0038] In another aspect, the present disclosure provides a pharmaceutical composition comprising (a) and (b) below: (a) an iTR modulator; and (b) a pharmaceutically acceptable carrier.

[0039] In another embodiment, a gene that regulates EFT is modified so that cancer cells are treated with a drug that induces iCM by changing the expression of the gene from embryonic state expression to fetal or adult state expression.

[0040] In another embodiment, the gene that regulates EFT is modified by the use of a histone deacetylase inhibitor so that the expression of the gene in cancer cells is changed from embryonic state expression to fetal or adult state expression, resulting in iCM.

[0041] Certain prior arts, such as cell biology, cell culture, molecular biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, and immunology, are within the scope of the skills of those skilled in the art and may be useful in the embodiments of this disclosure. Some non-exclusive descriptions of these techniques can be found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all published by John Wiley & Sons, NY, editions as of 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies--A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Burns, R., Immunochemical Protocols (Methods in Molecular Biology), Humana Press; 3rd ed., 2005, Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, (Found in Freshney, RI, "Culture of Animal Cells, A Manual of Basic Technique," 5th edition, John Wiley & Sons, Hoboken, NJ, 2005). All patents, patent applications, websites, databases, scientific articles, and other publications mentioned herein are incorporated herein by reference in their entirety.

[0042] Brief explanation of the drawing This patent or application file includes at least one drawing drawn in color. A copy of this patent or patent application publication with the color drawing attached will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0043] [Figure 1] Figure 1 shows the experimental design. Using labeled transcriptome data, we train an ensemble of 20 deep neural networks, each with one output neuron, and a multi-class deep neural network with five neurons, one neuron per class, to predict the sample's "embryo score". [Figure 2A] Figures 2A–2D show the training and validation performance of the classifier. Gene (Figures 2A and 2B) and pathway (Figures 2C and 2D) level input data are obtained from Affymetrix (left panel) and Illumina (right panel) data using labeled cross-validation. The F1 scores presented are obtained based on training, internal validation, and external validation sets (see the "Methods" chapter for a detailed description of the nested cross-validation protocol used). [Figure 2B] Figures 2A–2D show the training and validation performance of the classifier. Gene (Figures 2A and 2B) and pathway (Figures 2C and 2D) level input data are obtained from Affymetrix (left panel) and Illumina (right panel) data using labeled cross-validation. The F1 scores presented are obtained based on training, internal validation, and external validation sets (see the "Methods" chapter for a detailed description of the nested cross-validation protocol used). [Figure 2C] Figures 2A–2D show the training and validation performance of the classifier. Gene (Figures 2A and 2B) and pathway (Figures 2C and 2D) level input data are obtained from Affymetrix (left panel) and Illumina (right panel) data using labeled cross-validation. The F1 scores presented are obtained based on training, internal validation, and external validation sets (see the "Methods" chapter for a detailed description of the nested cross-validation protocol used). [Figure 2D] Figures 2A–2D show the training and validation performance of the classifier. Gene (Figures 2A and 2B) and pathway (Figures 2C and 2D) level input data are obtained from Affymetrix (left panel) and Illumina (right panel) data using labeled cross-validation. The F1 scores presented are obtained based on training, internal validation, and external validation sets (see the "Methods" chapter for a detailed description of the nested cross-validation protocol used). [Figure 3A] Figure 3 shows the prediction of embryo state by a DNN ensemble. Figure 3A shows the validation confusion matrix performance of a DNN ensemble trained on Illumina data. [Figure 3B] Figure 3 shows the prediction of embryo state by a DNN ensemble. Figure 3B shows the validation confusion matrix performance of a DNN ensemble trained on Affymetrix data. [Figure 3C] Figure 3 shows the prediction of embryonic state using a DNN ensemble. Figure 3C shows the expression levels across five groups of four key genes obtained from the top 15 classifiers of both GBM and DNN. [Figure 3D] Figure 3 shows the prediction of embryonic state by DNN ensembles. (Figure 3D) shows embryo scores obtained through Affymetrix and Illumina DNN ensembles on the Affymetrix platform dataset GSE65369, which consists of samples obtained from various stages of neural lineage differentiation from ESC cells. [Figure 4] Figure 4 shows the top 50 genes that were confirmed to be differentially expressed in embryonic and adult cell types by DNNs. [Figure 5] Figure 5 shows the genes that were confirmed to be differentially expressed in embryonic and adult cell types by DNN (FDR P value < 0.005). [Figure 6] Figures 6A to 6D show the RNA expression levels of Cox7a1, Naaladl1, Plpp7, and Lin28b measured by RNA-seq in all mouse extracts during mouse development. The time points represent days after mating (dpc). [Figure 7A] Figures 7A to 7D show the mean values ​​for RNA expression measured by Illumina gene expression bead arrays for COX7A1(a), NAALADL1(b), PLPP7(c), and LIN28B(d) in human ES cells (ES), clonal embryonic progenitor cell lines (EP), early passaged fibroblasts cultured in vitro from the upper arm of fetuses at different developmental stages, early passaged fibroblasts cultured in vitro from the upper arm of humans at different postnatal ages, and adult-derived dermal fibroblasts before or after transcriptional reprogramming to pluripotency (iPS cells). [Figure 7B] Figures 7A to 7D show the mean values ​​for RNA expression measured by Illumina gene expression bead arrays for COX7A1(a), NAALADL1(b), PLPP7(c), and LIN28B(d) in human ES cells (ES), clonal embryonic progenitor cell lines (EP), early passaged fibroblasts cultured in vitro from the upper arm of fetuses at different developmental stages, early passaged fibroblasts cultured in vitro from the upper arm of humans at different postnatal ages, and adult-derived dermal fibroblasts before or after transcriptional reprogramming to pluripotency (iPS cells). [Figure 7C] Figures 7A to 7D show the mean values ​​for RNA expression measured by Illumina gene expression bead arrays for COX7A1(a), NAALADL1(b), PLPP7(c), and LIN28B(d) in human ES cells (ES), clonal embryonic progenitor cell lines (EP), early passaged fibroblasts cultured in vitro from the upper arm of fetuses at different developmental stages, early passaged fibroblasts cultured in vitro from the upper arm of humans at different postnatal ages, and adult-derived dermal fibroblasts before or after transcriptional reprogramming to pluripotency (iPS cells). [Figure 7D] Figures 7A to 7D show the mean values ​​for RNA expression measured by Illumina gene expression bead arrays for COX7A1(a), NAALADL1(b), PLPP7(c), and LIN28B(d) in human ES cells (ES), clonal embryonic progenitor cell lines (EP), early passaged fibroblasts cultured in vitro from the upper arm of fetuses at different developmental stages, early passaged fibroblasts cultured in vitro from the upper arm of humans at different postnatal ages, and adult-derived dermal fibroblasts before or after transcriptional reprogramming to pluripotency (iPS cells). [Figure 8A] Figures 8A to 8D show the RNA expression levels of COX7A1(a), NAALADL1(b), PLPP7(c), and LIN28B(d) measured by Illumina gene expression bead arrays in human ES cells (ES), clonal embryonic progenitor cell lines 4D20.8, E3, and SK5, their adult counterparts (MSCs, preadipocytes, and skeletal myoblasts, respectively), and sarcomas corresponding to the aforementioned histological types. [Figure 8B] Figures 8A to 8D show the RNA expression levels of COX7A1(a), NAALADL1(b), PLPP7(c), and LIN28B(d) measured by Illumina gene expression bead arrays in human ES cells (ES), clonal embryonic progenitor cell lines 4D20.8, E3, and SK5, their adult counterparts (MSCs, preadipocytes, and skeletal myoblasts, respectively), and sarcomas corresponding to the aforementioned histological types. [Figure 8C] Figures 8A to 8D show the RNA expression levels of COX7A1(a), NAALADL1(b), PLPP7(c), and LIN28B(d) measured by Illumina gene expression bead arrays in human ES cells (ES), clonal embryonic progenitor cell lines 4D20.8, E3, and SK5, their adult counterparts (MSCs, preadipocytes, and skeletal myoblasts, respectively), and sarcomas corresponding to the aforementioned histological types. [Figure 8D] Figures 8A to 8D show the RNA expression levels of COX7A1(a), NAALADL1(b), PLPP7(c), and LIN28B(d) measured by Illumina gene expression bead arrays in human ES cells (ES), clonal embryonic progenitor cell lines 4D20.8, E3, and SK5, their adult counterparts (MSCs, preadipocytes, and skeletal myoblasts, respectively), and sarcomas corresponding to the aforementioned histological types. [Figure 9] Figure 9 shows the inverse correlation between the embryonic marker LIN28B and the adult markers COX7A1(a), PLPP7(c), and NAALADL1(b) in various sarcomas. [Figure 10] Figure 10 shows novel markers in embryonic and adult cells identified by RNA sequencing. [Figure 11-1] Figure 11 shows AMH expression in various embryonic and adult cell types. [Figure 11-2] Figure 11 shows AMH expression in various embryonic and adult cell types. [Figure 12-1] Figure 12 shows LINC01021 expression in various embryonic and adult cell types. [Figure 12-2] Figure 12 shows LINC01021 expression in various embryonic and adult cell types. [Figure 13-1] Figure 13 shows RGPD1 expression in various embryonic and adult cell types. [Figure 13-2] Figure 13 shows RGPD1 expression in various embryonic and adult cell types. [Figure 14-1] Figure 14 shows ZNF300P1 expression in various embryonic and adult cell types. [Figure 14-2] Figure 14 shows ZNF300P1 expression in various embryonic and adult cell types. [Figure 15-1] Figure 15 shows LINC00654 expression in various embryonic and adult cell types. [Figure 15-2] Figure 15 shows LINC00654 expression in various embryonic and adult cell types. [Figure 16-1] Figure 16 shows PCDHGA12 expression in various embryonic and adult cell types. [Figure 16-2] Figure 16 shows PCDHGA12 expression in various embryonic and adult cell types. [Figure 17-1] Figure 17 shows reads determined by RNA sequencing at clustered protocadherin loci. RS-27 is an adult MSC cell line, and RS-77 is an embryonic vascular endothelial progenitor cell line. [Figure 17-2] Figure 17 shows reads determined by RNA sequencing at clustered protocadherin loci. RS-27 is an adult MSC cell line, and RS-77 is an embryonic vascular endothelial progenitor cell line. [Figure 17-3] Figure 17 shows reads determined by RNA sequencing at clustered protocadherin loci. RS-27 is an adult MSC cell line, and RS-77 is an embryonic vascular endothelial progenitor cell line. [Figure 17-4] Figure 17 shows reads determined by RNA sequencing at clustered protocadherin loci. RS-27 is an adult MSC cell line, and RS-77 is an embryonic vascular endothelial progenitor cell line. [Figure 17-5] Figure 17 shows reads determined by RNA sequencing at clustered protocadherin loci. RS-27 is an adult MSC cell line, and RS-77 is an embryonic vascular endothelial progenitor cell line. [Figure 17-6] Figure 17 shows reads determined by RNA sequencing at clustered protocadherin loci. RS-27 is an adult MSC cell line, and RS-77 is an embryonic vascular endothelial progenitor cell line. [Figure 17-7] Figure 17 shows reads determined by RNA sequencing at clustered protocadherin loci. RS-27 is an adult MSC cell line, and RS-77 is an embryonic vascular endothelial progenitor cell line. [Figure 17-8] Figure 17 shows reads determined by RNA sequencing at clustered protocadherin loci. RS-27 is an adult MSC cell line, and RS-77 is an embryonic vascular endothelial progenitor cell line. [Figure 17-9] Figure 17 shows reads determined by RNA sequencing at clustered protocadherin loci. RS-27 is an adult MSC cell line, and RS-77 is an embryonic vascular endothelial progenitor cell line. [Figure 18] Figure 18 shows drugs that can promote iTR, identified through screening using the COX7A1 marker. The values ​​below the cell line represent the proportional decrease in COX7A1 transcript in that cell line. [Figure 19] Figure 19 shows drugs that can promote iCM, identified through screening using the COX7A1 marker. The values ​​below the cell line represent the proportional increase in COX7A1 transcript in that cell line. [Figure 20] Figure 20 shows additional agents that can promote iCM, identified through screening using the COX7A1 marker. The values ​​below the cell line represent the proportional increase in COX7A1 transcript in that cell line. [Figure 21] Figure 21 shows the formula used to calculate the embryo score (ES). [Figure 22] Figure 22 shows the equation for a multilayer neural network. [Figure 23-1] Figure 23 shows the methylation status of the fetal and adult-specific gene COX7A1 and PCDHB2, which is expressed at relatively high levels in embryonic cells, in clonal embryonic precursor (EP) cell lines 30MV2 and 4D20.8, as well as similar adult-derived human aortic endothelial cells (HAECs) and adult-derived mesenchymal stem cells (MSCs). The height of the histogram corresponds to the percentage of methylated CpG. [Figure 23-2] Figure 23 shows the methylation status of the fetal and adult-specific gene COX7A1 and PCDHB2, which is expressed at relatively high levels in embryonic cells, in clonal embryonic precursor (EP) cell lines 30MV2 and 4D20.8, as well as similar adult-derived human aortic endothelial cells (HAECs) and adult-derived mesenchymal stem cells (MSCs). The height of the histogram corresponds to the percentage of methylated CpG. [Figure 23-3] Figure 23 shows the methylation status of the fetal and adult-specific gene COX7A1 and PCDHB2, which is expressed at relatively high levels in embryonic cells, in clonal embryonic precursor (EP) cell lines 30MV2 and 4D20.8, as well as similar adult-derived human aortic endothelial cells (HAECs) and adult-derived mesenchymal stem cells (MSCs). The height of the histogram corresponds to the percentage of methylated CpG. [Figure 23-4] Figure 23 shows the methylation status of the fetal and adult-specific gene COX7A1 and PCDHB2, which is expressed at relatively high levels in embryonic cells, in clonal embryonic precursor (EP) cell lines 30MV2 and 4D20.8, as well as similar adult-derived human aortic endothelial cells (HAECs) and adult-derived mesenchymal stem cells (MSCs). The height of the histogram corresponds to the percentage of methylated CpG. [Figure 24A] Figures 24A–24D show RNA sequencing of adult-derived dermal fibroblasts (MDWs) treated with various lentiviral constructs expressing the genes shown in the figures. The values ​​shown for the transcript of LIN28A, which is exogenously expressed in these cells along with the iTR markers COX7A1 and CAT, were downregulated in a manner similar to, but to a lower degree than, that of cells fully reprogrammed into iPS cells. The gene GFER was upregulated in a manner proportional to LIN28A expression. [Figure 24B] Figures 24A–24D show RNA sequencing of adult-derived dermal fibroblasts (MDWs) treated with various lentiviral constructs expressing the genes shown in the figures. The values ​​shown for the transcript of LIN28A, which is exogenously expressed in these cells along with the iTR markers COX7A1 and CAT, were downregulated in a manner similar to, but to a lower degree than, that of cells fully reprogrammed into iPS cells. The gene GFER was upregulated in a manner proportional to LIN28A expression. [Figure 24C] Figures 24A–24D show RNA sequencing of adult-derived dermal fibroblasts (MDWs) treated with various lentiviral constructs expressing the genes shown in the figures. The values ​​shown for the transcript of LIN28A, which is exogenously expressed in these cells along with the iTR markers COX7A1 and CAT, were downregulated in a manner similar to, but to a lower degree than, that of cells fully reprogrammed into iPS cells. The gene GFER was upregulated in a manner proportional to LIN28A expression. [Figure 24D] Figures 24A–24D show RNA sequencing of adult-derived dermal fibroblasts (MDWs) treated with various lentiviral constructs expressing the genes shown in the figures. The values ​​shown for the transcript of LIN28A, which is exogenously expressed in these cells along with the iTR markers COX7A1 and CAT, were downregulated in a manner similar to, but to a lower degree than, that of cells fully reprogrammed into iPS cells. The gene GFER was upregulated in a manner proportional to LIN28A expression. [Figure 25] Figure 25 shows the expression of LIN28A and LIN28B in fetal liver-derived CD34+ hematopoietic stem cells and CD36+ erythrocyte progenitor cells compared with various types of adult-derived bone marrow (BM) and peripheral blood (PB) blood cells, assayed using an Illumina gene expression bead array. Relative fluorescence units (RFU) values ​​greater than 130 are considered positive, and RFU values ​​less than 100 are considered negative. [Figure 26] Figures 26A and 26B show the expression of COX7A1 and LIN28B in control embryonic stem (ES) cells, normal adult mesenchymal stem cells (MSCs), and normal blood cells compared to diverse normal and malignant epithelium obtained from bronchi, lungs, epidermis, kidneys, livers, mammary glands, and dermal melanocytes, assayed using an Illumina gene expression bead array. Relative fluorescence units (RFU) values ​​greater than 130 were considered positive, and RFU values ​​less than 100 were considered negative. [Figure 27-1] Figure 27 shows the expression of COX7A1 in various sarcoma strains, including adult-derived human mesenchymal stem cells (hMSCs), normal human articular chondrocytes (NHACs), normal human diploid fibroblasts (NHDFs), normal human osteoblasts (NHOsts), and skeletal muscle cells (SkMCs). For expression, RFU values ​​below 75 are considered negative, and values ​​above 90 are considered positive. [Figure 27-2] Figure 27 shows the expression of COX7A1 in various sarcoma strains, including adult-derived human mesenchymal stem cells (hMSCs), normal human articular chondrocytes (NHACs), normal human diploid fibroblasts (NHDFs), normal human osteoblasts (NHOsts), and skeletal muscle cells (SkMCs). For expression, RFU values ​​below 75 are considered negative, and values ​​above 90 are considered positive. [Figure 28] Figure 28 shows the results of the glycolysis stress test. Clonal EP cells were subjected to the glycolysis stress test in parallel with undifferentiated embryonic adipogenic cells, i.e., white adipocytes labeled E3, adult-derived preadipocytes were subjected to the test with subcutaneous adipose tissue (SAT), and liposarcoma cell lines CRL3043 and CRL3044. The extracellular acidification rate (ECAR) after glycolysis stress is shown. [Figure 29] Figure 29 shows the density percentage achieved by seeding the same number of umbilical cord-derived and adult skin-derived stromal fibroblasts, all of which expressed the fetal / adult marker COX7A1 80 hours after treatment with the same medium and serum supplemented with 10% FBS in DMEM(CTRL) or 10 ng / mL or 100 ng / mL of secreted GFER. [Figure 30] Figure 30 shows the Illumina gene expression bead array values ​​for the expression of fetal / adult marker COX7A1 and fibrosis marker COL1A1 in MDW adult-derived dermal fibroblasts under the following conditions: normal growth medium (DMEM(Ctrl) supplemented with 10% FBS), the same medium supplemented with cMYC or SOX2 mRNA, or the same medium supplemented with cMYC or SOX2 mRNA and 0.5 mM valproic acid. [Modes for carrying out the invention]

[0044] Detailed explanation Abbreviation AC - Adult-derived cells AMH - Anti-Müllerian hormone ASC - Adult Stem Cell cGMP - Current Good Manufacturing Practices for Pharmaceuticals CM - Cancer Maturation CNS - Central Nervous System DMEM - Dulbecco's modified Eagle medium DMSO - Dimethyl sulfoxide DNN - Deep Neural Network DPBS - Dulbecco's phosphate-buffered saline ED cells - embryonic cells; hED cells are human ED cells. EDTA - Ethylenediaminetetraacetic acid EFT - Embryo-Fetal Transfer EG cells - Embryonic germ cells; hEG cells are human EG cells. EP - embryonic precursor ES Cells - Embryonic stem cells; hES cells are human ES cells. ESC - Embryonic stem cells FACS - Fluorescence-Activated Cell Sorting FBS - Fetal Bovine Serum FPKM - Fragments per kilobase transcript per million mapped reads obtained by RNA sequencing. GFER - Growth Factor, Liver Regeneration Enhancer (ALR) GFP - Green fluorescent protein GMP - Good Manufacturing Practices for Pharmaceuticals hED cells - Human embryo-derived cells hEG cells - Human embryonic germ cells are stem cells derived from primordial germ cells in fetal tissue. HESC - Human Embryonic Stem Cells hiPS cells - human induced pluripotent stem cells are cells that possess properties similar to hES cells, obtained from somatic cells after exposure to hES-specific transcription factors, such as SOX2, KLF4, OCT4, MYC, or NANOG, LIN28, OCT4, and SOX2. HSE - Human Skin Equivalents are mixtures of cells and a biological or synthetic matrix, manufactured for therapeutic purposes, or to promote wound healing, depending on the test purpose. iCM - Induced Cancer Maturation. iPS cells - induced pluripotent stem cells are cells that have properties similar to hES cells obtained from somatic cells after exposure to ES-specific transcription factors, such as SOX2, KLF4, OCT4, MYC, or NANOG, LIN28, OCT4, and SOX2, SOX2, KLF4, OCT4, MYC, and (LIN28A or LIN28B), or other combinations of OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, LIN28A, and LIN28B. iTM - Induced Tissue Maturation iTR - Induced Tissue Regeneration MEM - Minimum Essential Medium MSC - Mesenchymal Stem Cells NT - Nuclear transfer PBS - Phosphate-buffered saline PS fibroblasts - Pre-scarring fibroblasts are fibroblasts derived from skin in early pregnancy or from ED cells that exhibit prenatal pattern gene expression in that they promote rapid healing of skin wounds without scarring. RFU - Relative Fluorescence Unit RNA-seq - RNA sequencing SFM - Serum-free medium TR - Tissue Regeneration

[0045] definition The term "analytical reprogramming techniques" refers to various methods for reprogramming the gene expression patterns of somatic cells to more pluripotent states, such as the patterns of iPS, ES, ED, EC, or EG cells, where reprogramming occurs in multiple separate steps and does not rely solely on the introduction of somatic cells into oocytes and the activation of those oocytes (US application 60 / 332,510 filed November 26, 2001, and filed November 26, 2002). See also PCT application No. 10 / 304,020, PCT application No. PCT / US02 / 37899 filed on 26 November 2003, US application No. 60 / 705625 filed on 3 August 2005, US application No. 60 / 729173 filed on 20 August 2005, US application No. 60 / 818813 filed on 5 July 2006, and PCT / US06 / 30632 filed on 3 August 2006 (the disclosures of each of these documents are incorporated herein by reference).

[0046] The term "blastomeres / morula cells" refers to blastomeres or morula cells from mammalian embryos, or blastomeres or morula cells cultured in vitro together with further cells containing differentiated derivatives of these cells, or without such cells.

[0047] The term "cancer maturation" refers to the alteration of gene expression in pre-malignant or malignant cancer cells, where pre-malignant or malignant cancer cells, which initially express germ cell markers, are modified to express fetal or adult cell markers.

[0048] The terms “cells expressing gene X,” “gene X is expressed in cells (or a population of cells),” or their equivalents, mean that analysis of cells using a particular assay platform yielded a positive result. The reverse is also true (i.e., cells not expressing gene X, or their equivalent, mean that analysis of cells using a particular assay platform yielded a negative result). Therefore, any gene expression results described herein are related to the specific probes or probes used with the assay platform(s)(s) for the gene indicated.

[0049] The term "cell line" refers to a population of cells that can proliferate and expand in vitro, whether mortal or immortal.

[0050] The term "cell reconstitution" refers to the process of introducing nuclear chromatin into the cytoplasm of a cell in order to obtain a functional cell.

[0051] The term "clone (clonal)" refers to a group of cells obtained by extending a single cell into a group of cells that all originate from the original single cell and do not contain any other cells.

[0052] The term "colony-in situ differentiation" refers to the differentiation of cell colonies (e.g., hES, hEG, hiPS, hEC, or hED) in situ, where the colonies are grown as undifferentiated stem cell lines in a culture vessel without being removed or disaggregated. Although colony-in situ differentiation does not utilize the intermediate step of embryoid body formation, embryoid body formation or other aggregation techniques, such as the use of agitated culture, may be performed following the colony-in situ differentiation period.

[0053] The term "cytoplasmic bleb" refers to the cytoplasm of a cell lacking a nucleus, which is either untreated or permeabilized but otherwise untreated, and surrounded by a plasma membrane.

[0054] The term "differentiated cells," when used in reference to cells produced from pluripotent stem cells by the method of this disclosure, refers to cells that have a reduced ability to differentiate compared to the parental pluripotent stem cells. Differentiated cells in this disclosure include cells that can further differentiate (i.e., they do not have to be terminally differentiated).

[0055] The term "embryonic" or "embryonic stage of development" refers to the prenatal stage of development of cells, tissues, or animals, specifically the embryonic stage of cell development compared to fetal and adult cells. In humans, the transition from embryo to fetus occurs at approximately 8 weeks of prenatal development, at 16 days or approximately 16 days in mice, and at approximately 17.5 days after mating in rats. (www.php.med.unsw.edu.au / embryology / index.php?title=Mouse_Timeline_Detailed).

[0056] The term “embryonic stem cell” (ES cell) refers to cells derived from the inner cell mass of a blastocyst, blastomeres, or morula that have been successively passaged as a cell line while maintaining an undifferentiated state (e.g., expressing TERT, OCT4, and SSEA, as well as TRA antigens specific to that species of ES cell). ES cells may be induced by fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means of generating hES cells that are hemizygous or isozygous in the MHC region. Historically, ES cells have been defined as cells that can differentiate into all somatic cell types and germline cells when implanted into a preimplantation embryo. However, candidate ES cultures from many species, including humans, have a flatter appearance in culture and typically do not contribute to germline differentiation, and are therefore referred to as “ES-like cells.” While human ES cells are generally considered to be “ES-like,” in this application, the inventors use the term ES cells to refer to both ES cell lines and ES-like cell lines.

[0057] The term "global modulator of TR" or "global modulator of iTR" refers to a drug that can modulate multiple iTR or iTM genes, including but not limited to drugs that can downregulate COX7A1 while simultaneously upregulating PCDHB2, or downregulate NAALADL1 while simultaneously upregulating AMH, in cells derived from fetal or adult sources, and drugs that can induce gene expression patterns that result in increased scar-free tissue regeneration in response to tissue damage or degenerative disease.

[0058] The term “human embryo-derived” (hED) cells refers to blastomeres, morula, blastocysts, or other totipotent or pluripotent stem cells of the early embryo, including the inner cell mass, fetal shield, or epiblastoid, as well as their derivatives corresponding to a state of differentiation that correlates with the equivalent of the first eight weeks of normal human development, but excluding cells derived from hES cells that have been passaged as cell lines (see, for example, Thomson U.S. Patents 7,582,479; 7,217,569; 6,887,706; 6,602,711; 6,280,718; and 5,843,780). hED cells may be derived from preimplantation embryos produced by fertilization of an egg cell with sperm or DNA, nuclear transfer, or chromatin transfer, egg cells induced to form a parthenogenetic organism through parthenogenesis, analytical reprogramming techniques, or means for creating hES cells with hemizygous or isozygous HLA regions. The term "human embryonic germ cells" (hEG cells) refers to pluripotent stem cells derived from maturing or mature germ cells such as primordial germ cells or oocytes and spermatogonial cells of fetal tissue that can differentiate into various tissues in the body. hEG cells may also be derived from pluripotent stem cells produced by gynogenesis or androgenesis, i.e., by methods in which pluripotent cells are derived from oocytes containing only male or female-derived DNA, and thus all contain female or male-derived DNA.

[0059] The term "human embryonic stem cell" (hES cell) refers to human ES cells.

[0060] The term "human induced pluripotent stem cells" refers to cells with properties similar to hES cells, including the ability to form all three germ layers when transplanted into immunodeficient mice. These iPS cells are derived from altered somatic cell lineages after exposure to various combinations of dedifferentiation factors, such as combinations of hES cell-specific transcription factors: KLF4, SOX2, MYC; OCT4 or SOX2, OCT4, NANOG, and LIN28; or OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, LIN28A, and LIN28B, or after other methods of inducing somatic cells to obtain a pluripotent stem cell state with properties similar to hES cells. However, somatic cell reprogramming via somatic cell nuclear transfer (SCNT) is typically referred to as NT-ES cells rather than iPS cells.

[0061] The term "induced cancer maturation" refers to a method by which a change in the phenotype of premalignant or malignant cells occurs, resulting in the cells subsequently expressing markers that are normally expressed in their cell type at the fetal or adult stage, rather than at the embryonic stage of development.

[0062] The term "induced tissue regeneration" refers to the use of the methods of this disclosure to alter the molecular composition of fetal or adult mammalian cells so that, as a result, the cells can regenerate functional tissue after damage to that tissue, where such regeneration is not a normal outcome in that species of animal.

[0063] The term “isolated” means (i) a substance that has been separated from at least some other substances that are normally found together with it in nature, usually by a process that requires human intervention; (ii) a substance that has been artificially produced (e.g., chemically synthesized); and / or (iii) a substance that exists in an artificial environment or situation (i.e., an environment or situation that is not normally found in nature).

[0064] The term "iCM factor" refers to molecules that alter the levels of CM activators and CM inhibitors in a manner that brings about CM in tumors for therapeutic purposes.

[0065] The term "iCM gene" refers to a gene whose expression, when altered, can cause tumor malignancy (CM) in order to enhance therapeutic effects.

[0066] The term "iTR factor" refers to molecules that alter the levels of TR activators and TR inhibitors in a way that induces TR in tissues that cannot naturally produce TR.

[0067] The term "iTR gene" refers to a gene whose expression, when altered, can induce induced tissue regeneration in tissues that would not normally be able to regenerate.

[0068] The term “nucleic acid” is used interchangeably with “polynucleotide” and, in various embodiments, encompasses naturally occurring polymers of nucleosides such as DNA and RNA, as well as polymers of nucleosides or nucleoside analogs that do not exist in nature. In some embodiments, nucleic acids include standard nucleosides (abbreviated as A, G, C, T, U). In other embodiments, nucleic acids include one or more non-standard nucleosides. In some embodiments, one or more nucleosides are nucleosides or nucleotide analogs that do not exist in nature. Nucleic acids may include modified bases (e.g., methylated bases), modified sugars (2'-fluororibose, arabinose, or hexose), modified phosphate groups or other internucleoside or internucleoside analog bonds (e.g., phosphorothioates or 5'-N-phosphoamidite bonds), cross-linked nucleic acids, or morpholinos. In some embodiments, nucleic acids include nucleosides linked by phosphodiester bonds, as found in DNA and RNA. In some embodiments, at least some nucleosides are linked by non-phosphodiester bonds. Nucleic acids can be single-stranded, double-stranded, or partially double-stranded. Nucleic acids that are at least partially double-stranded may have one or more overhangs, e.g., 5' and / or 3' overhangs. Nucleic acid modifications (e.g., nucleoside and / or backbone modifications, including the use of non-standard nucleosides), aptamers, or antisense-based molecules for research or therapeutic purposes that are known in the art to be useful in relation to RNA interference (RNAi) are intended for use in various embodiments of this disclosure. See, for example, Crooke, ST (ed.), Antisense drug technology: principles, strategies, and applications, Boca Raton: CRC Press, 2008; Kurreck, J. (ed.), Therapeutic oligonucleotides, RSC biomolecular sciences. Cambridge: Royal Society of Chemistry, 2008.In some embodiments, modification increases the half-life and / or stability of the nucleic acid, for example, in vivo, compared to RNA or DNA of the same length and strandedness. In some embodiments, modification decreases the immunogenicity of the nucleic acid compared to RNA or DNA of the same length and strandedness. In some embodiments, 5% to 95% of the nucleosides in one or both strands of the nucleic acid are modified. The modifications may be uniform or heterogeneous, and the location of the modifications (e.g., near the center, near the end or at the end, alternating, etc.) can be selected to enhance the desired property(s). The nucleic acid may contain detectable labels, such as fluorescent dyes, radioactive atoms, etc. "Oligononucleotide" refers to relatively short nucleic acids, typically about 4 to about 60 nucleotides in length. When polynucleotides are referred to herein, it will be understood that both DNA and RNA, and in either case both single-stranded and double-stranded forms (and complements of each single-stranded molecule) are provided. Where used herein, "polynucleotide sequence" may refer to sequence information (i.e., sequences of letters used as abbreviations for bases) that biochemically characterize the polynucleotide material itself and / or a particular nucleic acid. Polynucleotide sequences presented herein are presented in the 5' to 3' direction unless otherwise indicated.

[0069] The term "oligoclone (oligoclonal)" refers to a small group of cells, typically originating from 2 to 1000 cells, that are thought to share similar characteristics, such as the presence or absence of morphological or differentiation markers, that differ from other cells in the same culture. Oligoclonal cells can be isolated and grown from cells that do not share these common characteristics to produce a group of cells that are essentially entirely derived from the original group of similar cells.

[0070] The term "pluripotent stem cell" refers to animal cells that can differentiate into two or more differentiated cell types. Such cells include hES cells, blastomeres / morula cells and their induced hED cells, hiPS cells, hEG cells, hEC cells, and adult-derived cells such as mesenchymal stem cells, neural stem cells, and bone marrow-derived stem cells. Pluripotent stem cells may or may not be genetically modified. Genetically modified cells may contain markers, such as fluorescent proteins, to facilitate their identification in the egg.

[0071] The term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. Peptides are relatively short polypeptides, typically about 2 to 60 amino acids in length. Polypeptides as used herein typically contain standard amino acids (i.e., the 20 most commonly found L-amino acids in proteins). However, in certain embodiments, polypeptides may contain one or more non-standard amino acids (which may or may not be naturally occurring) and / or amino acid analogs known in the art. One or more amino acids in a polypeptide may be modified by the addition of chemicals, such as carbohydrate groups, phosphate groups, fatty acid groups, conjugations, or linkers for functionalization. Polypeptides having non-polypeptide moieties linked covalently or non-covalently are still considered "polypeptides." Polypeptides may be purified from natural sources, produced using recombinant DNA technology, or synthesized through chemical means such as conventional solid-phase peptide synthesis. The terms “polypeptide sequence” or “amino acid sequence,” as used herein, may refer to the polypeptide material itself and / or sequence information (i.e., sequences of letters or three-letter abbreviations used as abbreviations for amino acid names) that biochemically characterize the polypeptide. Polypeptide sequences presented herein are presented in the N-terminal to C-terminal direction unless otherwise indicated. Polypeptides may be cyclic or contain cyclic moieties. When discussing naturally occurring polypeptides herein, it should be understood that this disclosure encompasses embodiments relating to any isoform thereof (e.g., different proteins arising from the same gene as a result of alternative splicing or editing of mRNA, or as a result of different alleles of a single gene, e.g., different alleles by one or more single nucleotide polymorphisms (typically, such alleles are at least 95%, 96%, 97%, 98%, 99%, or more identical to the reference or consensus sequence)).A polypeptide may contain sequences that target itself for secretion or to a specific intracellular compartment (e.g., the nucleus) and / or sequences that target the polypeptide for post-translational modification or degradation. A particular polypeptide may be synthesized as a precursor that undergoes post-translational cleavage or other processing to become a mature polypeptide. In some cases, such cleavage may occur only during a specific activation event. Where relevant, this disclosure provides embodiments relating to precursor polypeptides and embodiments relating to mature polypeptides.

[0072] The term "pooled clone" refers to a population of cells obtained by mixing two or more clonal populations to produce a population of cells that is similar to a clonal population but in which not all cells originate from the same original clone, and which exhibits homogeneity in markers such as gene expression markers. This pooled clonal line may contain cells of a single genotype or a mixed genotype. Pooled clonal lines are particularly useful when clonal lines differentiate relatively early or undergo undesirable changes early in their proliferative lifespan.

[0073] The term "prenatal" refers to the stage of embryonic development in placental mammals; before this stage, the animal cannot survive away from the uterus.

[0074] The term "primordial stem cells" collectively refers to pluripotent stem cells capable of differentiating into all three primary germ layers—endoderm, mesoderm, and ectoderm—as well as neural crest cells. Therefore, examples of primordial stem cells include, but are not limited to, human or non-human mammalian ES cells or cell lines, blastomeres / morula cells and their induced ED cells, iPS cells, and EG cells.

[0075] The term “purified” refers to a drug or substance (e.g., a compound) that has been separated from the majority of the components it originally contained or was originally composed of. Generally, such purification requires manual labor. A purified drug or substance may be partially purified, substantially purified, or pure. Such a drug or substance may be, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% or more pure. In some embodiments, a nucleic acid or polypeptide is purified such that the nucleic acid or polypeptide constitutes at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the total nucleic acid or polypeptide material present in the preparation. Purity may be based, for example, on dry weight, peak size in chromatographic tracing, molecular abundance, band intensity on a gel, or intensity of any signal correlated with molecular abundance, or any quantitative method accepted in the art. In some embodiments, water, buffers, ions, and / or small molecules (e.g., precursors such as nucleotides or amino acids) may be present in the purified preparation. The purified molecule may be prepared by separating the molecule from other substances (e.g., other cellular materials) or by producing the molecule in a manner that achieves a desired degree of purity. In some embodiments, the purified molecule or composition refers to a molecule or composition prepared using any purification method accepted in the art. In some embodiments, “partially purified” means that the molecule produced by the cell is no longer present in the cell, for example, the cell is lysed and, in some cases, at least some cellular materials (e.g., cell wall, cell membrane(s), organelle(s)) have been removed.

[0076] The term “RNA interference” (RNAi) is used herein in its art sense to consistently refer to the phenomenon in which double-stranded RNA (dsRNA) causes sequence-specific degradation or translational repression of a corresponding mRNA complementary to the dsRNA strand. It will be understood that the complementarity between the dsRNA strand and the mRNA does not need to be 100%, but it does need to be sufficient to mediate the inhibition of gene expression (also referred to as “silencing” or “knockdown”). For example, the degree of complementarity is such that the strand can (i) lead to the cleavage of mRNA in the RNA-induced silencing complex (RISC), or (ii) cause translational repression of mRNA. In certain embodiments, the double-stranded portion of the RNA is less than about 30 nucleotides long, for example, 17–29 nucleotides long. In certain embodiments, the first strand of the dsRNA is at least 80%, 85%, 90%, 95%, or 100% complementary to the target mRNA, and the other strand of the dsRNA is at least 80%, 85%, 90%, 95%, or 100% complementary to the first strand. In mammalian cells, RNAi may be achieved by introducing a suitable double-stranded nucleic acid into the cell or by expressing a nucleic acid in the cell that is subsequently processed intracellularly to produce dsRNA. A nucleic acid capable of mediating RNAi is referred to herein as an "RNAi agent." Exemplary nucleic acids capable of mediating RNAi are small hairpin RNA (shRNA), small interfering RNA (siRNA), and microRNA precursors. These terms are well known and are used consistently herein in their art-specific sense. siRNA typically consists of two distinct nucleic acid strands that hybridize to form a double helix. They can be synthesized in vitro, for example, using standard nucleic acid synthesis techniques.siRNA is typically a double-stranded oligonucleotide having 16 to 30 nucleotides (nt) per strand, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides (nt), where the double-stranded oligonucleotide includes a double-stranded portion of 15 to 29 nucleotides in length, and one or both strands may include a 3' overhang of, for example, 1 to 5 nucleotides in length, or one or both ends may be blunt. In some embodiments, the siRNA includes a strand of 19 to 25 nt, for example, 21 to 23 nucleotides in length, where one or both strands include a 3' overhang of 1 to 2 nucleotides. One strand of the siRNA double-stranded portion (referred to as the “guide strand” or “antisense strand”) is substantially complementary (e.g., at least 80%, e.g., 85%, 90%, 95%, or 100%) to the target region in mRNA (e.g., having 3, 2, 1, or 0 mismatched nucleotides), and the other double-stranded portion is substantially complementary to the first double-stranded portion. In many embodiments, the guide strand is 100% complementary to the target region in mRNA, and the other passenger strand is 100% complementary to the first double-stranded portion (in various embodiments, the 3' overhang of the guide strand, if present, may or may not be complementary to the mRNA when the guide strand hybridizes to the mRNA). In some embodiments, the shRNA molecule is a nucleic acid molecule containing a stem-loop, where the stem of the double-stranded molecule is 16–30 nucleotides long and the loop is approximately 1–10 nucleotides long. siRNA can contain a wide variety of modified nucleosides and nucleoside analogs, as well as chemically or biologically modified bases, modified skeletons, etc. Any modification recognized in this field as useful for RNAi can be used without restriction. Some modifications lead to increased stability, cellular uptake, and efficacy. Other modifications lead to decreased immunogenicity or clearance.In certain embodiments, siRNA comprises a double helix approximately 19–23 (e.g., 19, 20, 21, 22, or 23) nucleotides long, and optionally includes one or two 1–5 nucleotide-long 3' overhangs, which may consist of deoxyribonucleotides. shRNA comprises a single nucleic acid strand containing two complementary regions separated primarily by a non-complementary region. These complementary regions hybridize to form a double helix structure, and the non-complementary region forms a loop connecting the 3' end of one strand of the double helix to the 5' end of the other strand. The shRNA undergoes intracellular processing to produce siRNA. Typically, the loop is 1–8, for example, 2–6 nucleotides long.

[0077] MicroRNAs (miRNAs) are small, naturally occurring, non-coding, single-stranded RNAs, approximately 21–25 nucleotides (mammalian), that inhibit gene expression in a sequence-specific manner. They are generated intracellularly from precursors (pre-miRNAs) that have a characteristic secondary structure consisting of short hairpins (approximately 70 nucleotides long) containing a double helix, often with one or more incomplete complementary regions, which in turn are generated from larger precursors (pri-miRNAs). Naturally occurring miRNAs are typically only partially complementary to their target mRNA and often act via translational repression. RNAi agents modeled after endogenous miRNAs or miRNA precursors are useful in certain embodiments of this disclosure. For example, siRNAs can be designed to hybridize one strand to a target mRNA having one or more mismatches or bulges, mimicking the double helix formed by the miRNA and its target mRNA. Such siRNAs may be referred to as miRNA mimes or miRNA-like molecules. The miRNA mime may be encoded by a precursor nucleic acid whose structure mimics that of a naturally occurring miRNA precursor.

[0078] In certain embodiments, the RNAi agent is a vector (e.g., a plasmid or virus) containing a template for transcription of siRNA (e.g., as two distinct strands that can hybridize with each other), shRNA, or a microRNA precursor. Typically, the template encoding the siRNA, shRNA, or miRNA precursor is operably ligated to an expression control sequence (e.g., a promoter), as known in the art. Using such a vector, the template can be introduced into vertebrate cells, e.g., mammalian cells, resulting in transient or stable expression of the siRNA, shRNA, or miRNA precursor. The precursor (shRNA or miRNA precursor) is processed within the cell to produce siRNA or miRNA.

[0079] Generally, small RNAi agents such as siRNA can be chemically synthesized or transcribed in vitro or in vivo from a DNA template as two separate strands that later hybridize, or as shRNA that is later processed to produce siRNA. In many cases, RNAi agents, especially modified RNAi agents, are chemically synthesized. Chemical synthesis methods for oligonucleotides are well known in this field.

[0080] The term “small molecule,” as used herein, refers to an organic molecule with a mass of less than about 2 kilodaltons (KDa). In some embodiments, small molecules are less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, small molecules are less than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, small molecules have a mass of at least 50 Da. In some embodiments, small molecules contain multiple carbon-carbon bonds and may also contain one or more heteroatoms and / or one or more functional groups important for structural interactions with proteins (e.g., hydrogen bonding), such as an amine group, carbonyl group, hydroxyl group, or carboxyl group, and in some embodiments, at least two functional groups. Small molecules often contain one or more cyclic carbon or heterocyclic structures and / or aromatic or polycyclic aromatic structures, which may be substituted with one or more of the above functional groups. In some embodiments, small molecules are nonpolymerizable. In some embodiments, the small molecule is not an amino acid. In some embodiments, the small molecule is not a nucleotide. In some embodiments, the small molecule is not a sugar.

[0081] The term “subject” can be any multicellular animal. In many cases, subjects are vertebrates, e.g., mammals or birds. Exemplary mammals include, for example, humans, non-human primates, rodents (e.g., mice, rats, rabbits), ungulates (e.g., sheep, cattle, horses, goats), dogs, and cats. In many cases, subjects are, for example, individuals to which compounds should be delivered for experimental, diagnostic, and / or therapeutic purposes, individuals from which samples are taken or diagnostic procedures are performed (e.g., samples or procedures used to assess tissue damage and / or to evaluate the effects of compounds described herein).

[0082] The term “tissue damage” is used herein to mean any type of damage or injury to cells, tissues, organs, or other body structures. In various embodiments, the term encompasses degeneration due to disease, injury due to physical trauma or surgery, injury caused by exposure to toxic substances, and other destruction of the structure and / or functionality of cells, tissues, organs, or other body structures.

[0083] The term “tissue regeneration” or “TR” means the regeneration, replacement, recovery, or regrowth of at least a portion of a tissue, organ, or other body structure, or part thereof, after loss, injury, or degeneration, provided that such tissue regeneration does not occur by means of the methods described herein. Examples of tissue regeneration include the regrowth of a severed finger or limb, including the regrowth of cartilage, bone, muscle, tendon, and ligament, with an increase in size and cell number of the injured or affected organ, such that the tissue or organ approaches its normal size or the size before injury or disease; and the scar-free regrowth of bone, cartilage, skin, or muscle lost due to injury or disease. Depending on the tissue type, tissue regeneration may occur through a variety of different mechanisms, such as the rearrangement (e.g., by cell migration) of existing cells and / or tissues; the division of adult somatic stem cells or other progenitor cells and the differentiation of at least some of their offspring; and / or the dedifferentiation, transdifferentiation, and / or proliferation of cells.

[0084] The term "TR activator gene" refers to a gene that promotes TR, whose absence in fetal and adult cells, but whose expression during the embryonic stage of development, is observed.

[0085] The term "TR inhibitor gene" refers to a gene whose expression inhibits TR in both fetuses and adult animals.

[0086] The terms “to treat,” “to treat,” “treatment,” “therapeutic,” and similar terms relating to the subject refer to providing medical and / or surgical management of the subject. Treatments include, but are not limited to, the administration of compounds or compositions (e.g., pharmaceutical compositions) to the subject. Treatments of subjects according to this disclosure are typically performed, for example, to promote regeneration in subjects that have suffered or are expected to suffer tissue damage (e.g., subjects undergoing surgery). The effects of treatments may generally include increased regeneration, reduced scarring, and / or improvement of structural or functional outcomes (compared to outcomes without treatment) after tissue damage, and / or reversal or reduction of the severity or progression of a degenerative disease.

[0087] The term “variant” refers to a polypeptide that, when applied to a particular polypeptide, differs from that polypeptide (sometimes referred to as the “original polypeptide”) due to changes in one or more amino acids, such as additions, deletions, and / or substitutions. Sometimes the original polypeptide is a naturally occurring polypeptide (e.g., from human or non-human animals) or an identical polypeptide. Variants may be naturally occurring or may be produced using, for example, recombinant DNA technology or chemical synthesis. Additions can be insertions into the polypeptide or additions at the N- or C-terminus. In some embodiments, the number of amino acids substituted, deleted, or added may be, for example, about 1 to 30, for example, about 1 to 20, for example, about 1 to 10, for example, about 1 to 5, for example, 1, 2, 3, 4, or 5. In some embodiments, the mutant comprises a polypeptide whose sequence is homologous to the sequence of the original polypeptide by at least 50, at least 100, at least 150, or more amino acids over the full length (but not identical in sequence to the original polypeptide), for example, the sequence of the mutant polypeptide is homologous to the sequence of the original polypeptide by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more amino acids over the full length. In some embodiments, the variant comprises a polypeptide that is identical to the original polypeptide by at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the original polypeptide, and by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more.In some embodiments, the variant includes at least one functional or structural domain, for example, a domain identified as such in the National Center for Biotechnology Information's preserved domains database (CDD) (www.ncbi.nih.gov), such as an NCBI-curated domain.

[0088] In some embodiments, the biological function or activity of one, more than one, or all of the mutants or fragments is substantially similar to that of the corresponding biological function or activity of the original molecule. In some embodiments, the functional mutant retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the activity of the original polypeptide, for example, approximately equal activity. In some embodiments, the activity of the mutant is up to about 100%, about 125%, or about 150% of the activity of the original molecule. In other non-limiting embodiments, the activity of the mutant or fragment is considered substantially similar to that of the original molecule if the amount or concentration of the mutant required to produce a particular effect is within 0.5 to 5 times the amount or concentration of the original molecule required to produce the same effect.

[0089] In some embodiments, amino acid "substitutions" in mutants are the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, i.e., conservative amino acid substitutions. "Conservative" amino acid substitutions may be based on similarities in any of the various properties of the residues involved, such as side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. Examples of nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. Examples of polar (hydrophilic), neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Examples of positively charged (basic) amino acids include arginine, lysine, and histidine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Certain substitutions within specific groups, such as the substitution of leucine with isoleucine (or vice versa), the substitution of serine with threonine (or vice versa), or the substitution of alanine with glycine (or vice versa), may be of particular interest. Naturally, non-conservative substitutions are often compatible with the preservation of function. In some embodiments, substitutions or deletions do not alter or delete any amino acids important to activity. Insertions or deletions may be approximately 1 to 20 amino acids in size, e.g., 1 to 10 amino acids. In some cases, larger domains may be removed without substantially affecting function. In certain embodiments of this disclosure, mutant sequences can be obtained by making additions, deletions, or substitutions of only 5, 10, 15, or 20 amino acids in total from the sequence of a naturally occurring enzyme. In some embodiments, only 1%, 5%, 10%, or 20% of the amino acids in the polypeptide are insertions, deletions, or substitutions from the original polypeptide.Guidelines for determining which amino acid residues can be substituted, added, or deleted without removing or substantially reducing the desired activity may be obtained by comparing the sequence of a particular polypeptide with the sequence of a homologous polypeptide (e.g., from another organism) and minimizing the number of amino acid sequence changes made in regions with high homology (conserved regions), or by replacing amino acids with those found in homologous sequences, because amino acid residues conserved across diverse species are more likely to be important for activity than non-conserved amino acids.

[0090] In some embodiments, the polypeptide variant includes a heterologous polypeptide moiety. This heterologous moiety often has sequences that are not present in the original polypeptide or are not homologous to the original polypeptide. The heterologous moiety may be, for example, 5 to about 5,000 amino acids long, or longer. In many cases, the heterologous moiety is 5 to about 1,000 amino acids long. In some embodiments, the heterologous moiety includes sequences found in different polypeptides, such as functional domains. In some embodiments, the heterologous moiety includes sequences useful for the purification, expression, solubilization, and / or detection of the polypeptide. In some embodiments, the heterologous moiety includes a polypeptide "tag," such as an affinity tag or an epitope tag. For example, the tag may be an affinity tag (e.g., HA, TAP, Myc, 6×His, Flag, GST), a fluorescent or luminescent protein (e.g., EGFP, ECFP, EYFP, Cerulean, DsRed, mCherry), or a solubility-enhancing tag (e.g., SUMO tag, NUS A tag, SNUT tag, or monomeric mutant of the Ocr protein of bacteriophage T7). See, for example, Esposito D and Chatterjee D K. Curr Opin Biotechnol.;17(4):353-8 (2006). In some embodiments, the tag may perform multiple functions. The tag is often relatively small, for example, from a few amino acids to a maximum of about 100 amino acids in length. In some embodiments, the tag is longer than 100 amino acids in length, for example, to a maximum of about 500 amino acids or more. In some embodiments, the polypeptide has the tag located at the N- or C-terminus, for example, as an N- or C-terminal fusion. A polypeptide may contain multiple tags. In some embodiments, a 6×His tag and a NUS tag are present, for example, at the N-terminus. In some embodiments, the tags are cleavable and can therefore be removed from the polypeptide, for example, by a protease. In some embodiments, this is achieved by including a sequence encoding a protease cleavage site between the tag and a sequence encoding a portion homologous to the original polypeptide.Examples of proteases include thrombin, TEV proteases, factor Xa, and prescision proteases. In some embodiments, a “self-cleaving” tag is used. See, for example, PCT / US05 / 05763. The sequence encoding the tag may be located at 5' or 3' (or both) with respect to the polynucleotide encoding the polypeptide. In some embodiments, the tag or other heterologous sequence is separated from the rest of the polypeptide by a polypeptide linker. For example, the linker may be a short polypeptide (e.g., 15–25 amino acids). Often, the linker consists of small amino acid residues such as serine, glycine, and / or alanine. Heterologous domains may include transmembrane domains, secretory signaling domains, etc.

[0091] In certain embodiments of this disclosure, the fragment or mutant, excluding the heterogeneous portion, if present, has sufficient structural similarity to the original polypeptide, so that when its three-dimensional structure (actual or predicted structure) is superimposed on the structure of the original polypeptide, the overlap volume is at least 70%, preferably at least 80%, and more preferably at least 90% of the total volume of the structure of the original polypeptide. The partial or complete three-dimensional structure of the fragment or mutant may be determined by crystallizing the protein, which can be done using standard methods. Alternatively, an NMR solution structure can be prepared using similarly standard methods. The predicted structure can be prepared using a modeling program such as MODELER (Sali, A. and Blundell, TL, J. Mol. Biol., 234, 779-815, 1993), or any other modeling program. If the structure or predicted structure of the related polypeptide is available, the model can be based on that structure. The PROSPECT-PSPP suite of programs is available (Guo, JT, et al., Nucleic Acids Res. 32 (Web Server issue):W522-5, Jul. 1, 2004). If embodiments of this disclosure relate to polypeptide variants, it will be understood that polynucleotides encoding such variants are provided.

[0092] The term “vector” is used herein to refer to nucleic acids or viruses or parts thereof (e.g., viral capsids or genomes) that can mediate the transfer, e.g., transport, or delivery of nucleic acid molecules into cells. When the vector is a nucleic acid, the transported nucleic acid molecule is generally ligated, e.g., inserted, into the vector nucleic acid molecule. A nucleic acid vector may contain a sequence that directs autonomous replication (e.g., an origin of replication) or a sequence sufficient to enable the incorporation of part or all of the nucleic acid into host cell DNA. Useful nucleic acid vectors include, for example, DNA or RNA plasmids, cosmids, and nucleic acids (DNA or RNA) that can be packaged into naturally occurring or modified viral genomes or parts thereof or viral capsids. Plasmid vectors typically contain an origin of replication and one or more selection markers. Plasmids may contain part or all of the viral genome (e.g., viral promoters, enhancers, processing signals, or packaging signals). A virus or part thereof that can be used to introduce nucleic acid molecules into cells is referred to as a viral vector. Useful viral vectors include adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, vaccinia viruses and other poxviruses, and herpesviruses (e.g., herpes simplex virus). Viral vectors may or may not contain sufficient viral genetic information to produce infectious virus upon introduction into host cells; that is, viral vectors may be replication-deficient, and such replication-deficient viral vectors may be preferable for therapeutic use. If sufficient information is lacking, it may, but is not necessary, be supplied by the host cell or by another vector introduced into the cell. The carried nucleic acid may be naturally occurring or incorporated into a modified viral genome or portion thereof, or it may exist as a separate nucleic acid molecule within the virus or viral capsid.It will be understood that certain plasmid vectors containing part or all of a viral genome (typically containing enough viral genetic information to direct the transcription of nucleic acids that can be packaged into a viral capsid, and / or to produce nucleic acids that can be incorporated into the host cell genome, and / or to produce an infectious virus) are also sometimes referred to as viral vectors in this art. A vector may contain one or more nucleic acids encoding markers suitable for use in identifying and / or selecting cells that have been transformed or transfected with the vector or not. Examples of markers include proteins that increase or decrease resistance or susceptibility to antibiotics (e.g., antibiotic resistance genes encoding proteins that confer resistance to antibiotics such as puromycin, hygromycin, or blastosidine) or other compounds, enzymes whose activity is detectable by assays known in this art (e.g., β-galactosidase or alkaline phosphatase), and proteins or RNAs that detectably affect the phenotype of transformed or transfected cells (e.g., fluorescent proteins). An expression vector is a vector containing regulatory sequences, such as promoters, sufficient to direct the transcription of a functionally linked nucleic acid. The regulatory sequences may also include enhancer sequences or upstream activator sequences. The vector may optionally contain a 5' leader or signal sequence. The vector may optionally contain cleavage and / or polyadenylation signals and / or a 3' untranslated region. The vector often contains one or more appropriately placed restriction enzyme sites to facilitate the introduction of the nucleic acid to be expressed into the vector. The expression vector contains sufficient cis-elements for expression, and other elements necessary for or assisting expression may be supplied by host cells or an in vitro expression system.

[0093] Various techniques can be used to introduce nucleic acid molecules into cells. These techniques include chemically mediated transfection using compounds such as calcium phosphate, cationic lipids, and cationic polymers; non-chemical methods such as liposome-mediated transfection; electroporation; particle guns; or microinjection; and infection with viruses containing the target nucleic acid molecule (sometimes referred to as "transduction"). Markers can be used to identify and / or select cells that have incorporated the vector and typically express the nucleic acid. Such cells can be selected and, in some cases, a stable cell line can be established by culturing the cells in a suitable medium.

[0094] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and is therefore naturally subject to change. Similarly, since the scope of the invention is limited only by the appended claims, it should be understood that the terms used herein are merely for describing specific embodiments and are not intended to limit them.

[0095] Where a range of values ​​is provided, each intervening value between the upper and lower limits of that range, up to one-tenth of the lower limit unit unless otherwise specified in the context, and any other described values ​​or intervening values ​​within that described range, are understood to be included in the present invention. The upper and lower limits of these smaller ranges may independently be included within those smaller ranges, subject to the explicitly excluded limits within the described range, and these too are included in the present invention. Where a described range includes one or both limits, ranges excluding either or both of the limits they include are also included in the present invention.

[0096] In this specification, certain ranges are presented with the term “approximately” preceding a number. The term “approximately” is used in this specification to provide literal support for the exact number preceding the term, as well as for numbers that are close to or approximate the number preceding the term. When determining whether a number is close to or approximates a specifically stated number, the unspecified close or approximate number may, in the context in which it is presented, provide a substantial equivalent of the specifically stated number.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary art in the field to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but representative exemplary methods and materials are described herein.

[0098] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were explicitly and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and explain the methods and / or materials to which the publications are cited in connection. Any citation of a publication is for disclosure prior to the filing date and should not be construed as an acknowledgment that the present invention has no prior rights to such publication by prior invention. Furthermore, the dates of publications provided may differ from the actual publication dates and may need to be verified separately.

[0099] In this specification and the attached claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise specified in the context. Furthermore, the claims may be drafted to exclude any arbitrary element. Thus, this statement is intended to serve as an antecedent for the use of exclusive terms such as "solely" and "only," or for the use of "negative" limitation, relating to the enumeration of elements of the claims.

[0100] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described herein has distinct components and features that can be readily distinguished from or combined with any of several other embodiments without departing from the scope and spirit of the invention. Any of the listed methods can be performed in the order of the listed events, or in any other logically possible order.

[0101] method In addition to the methods described below, methods for finding applications in the production and use of cells exhibiting embryonic patterns of gene expression consistent with scar-free regenerative ability are as follows: PCT application PCT / US2006 / 013519, filed on April 11, 2006, entitled "Novel Uses of Cells With Prenatal Patterns of Gene Expression"; U.S. Patent Application 11 / 604,047, filed on November 21, 2006, entitled "Methods to Accelerate the Isolation of Novel Cell Strains from Pluripotent Stem Cells and Cells Obtained Thereby"; and U.S. Patent Application 11 / 604,047, filed on July 16, 2009, entitled "Methods to Accelerate the Isolation of Novel Cell Strains from Pluripotent Stem Cells and Cells Obtained Thereby". This can be found in U.S. Patent Application No. 12 / 504,630, entitled “Thereby” (see, for example, U.S. Provisional Patent Application No. 61 / 831,421 filed June 5, 2013, PCT Patent Application PCT / US2014 / 040601 filed June 3, 2014, and U.S. Patent Application No. 14 / 896,664 filed December 7, 2015, the entirety of each of these documents is incorporated herein by reference).

[0102] Designing Deep Learning Algorithms: Data Collection and Integration Microarray data from diverse platforms can be created or downloaded from Gene Expression Omnibus (GEO) and ArrayExpress. Collected samples may, as an example without limitation, belong to one of the following broad cell classes: embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic progenitor cells (EPCs), adult stem cells (ASCs), and diverse adult cell types (ACs). As a non-limiting example, the samples include the following microarray platforms: Illumina HumanHT-12 V4.0 (GPL10558), Illumina HumanHT-12 V3.0 (GPL6947), Affymetrix HT Human Genome U133A Array (GPL3921), Affymetrix GeneChip Human Genome U133 Array Set HG-U133A (GPL4557), Affymetrix Human Exon 1.0 ST Array (GPL5188), Affymetrix Human Genome U133 Plus 2.0 Array (GPL570), Affymetrix Human Genome U133A 2.0 Array (GPL571), Affymetrix Human Gene 1.0 ST Array (GPL6244), and Affymetrix Human Genome U133A Array. The dataset may include data obtained from (GPL96), Affymetrix Human Genome U133 Plus 2.0 Array (GPL11670), or RNA-seq data.

[0103] Data processing Separate processing pipelines may be used for Affymetrix and Illumina data. For each dataset, probe data can be extracted from raw files and then converted into gene expression values. For processing Affymetrix-related datasets, the Frozen RMA (fRMA) method is used (McCall, MN, et al., Frozen robust multiarray analysis (fRMA). Biostatistics 11, 242-253 (2010); McCall, MN, et al., The Gene Expression Barcode: leveraging public data repositories to begin cataloging the human and murine transcriptomes. Nucleic Acids Res. 39, D10-115 (2011); McCall, MN, et al., Assessing affymetrix GeneChip microarray quality. BMC Bioinformatics 12, 137 (2011); McCall, MN, et al., fRMA ST: frozen robust multiarray analysis for Affymetrix Exon and Gene ST arrays. Bioinformatics 28, 3153-3154). It is possible to use (2012)), and this method allows for the analysis of microarrays individually or in small increments, after which the data can be combined for analysis. For Illumina data, denormalized files can be used in conjunction with subsequent quantile normalization.

[0104] After obtaining probe expression data, this data can be converted to gene expression data using annotation tables available from GEO for the Illumina platform, or the "AnnotationDbi" package from Bioconductor for the Affymetrix platform. These tables include probe-gene mappings for specific microarray platforms. If multiple probes map to the same gene, a geometric mean can be used to average their signals. After conversion to genes, the entire dataset can be processed (separately for Affymetrix and Illumina platforms) using quantile normalization algorithms. The samples to be classified can be normalized using the same set of quantiles determined for the training dataset. Genes included in all target platform sets (Affymetrix and Illumina) can be used as input features for each classifier. Several machine learning methods can be compared in terms of their performance.

[0105] Pathway analysis. For pathway-level analysis, each case sample group can be independently analyzed using an algorithm called OncoFinder (Buzdin, AA et al., Oncofinder, a new method for the analysis of intracellular signaling pathway activation using transcriptomic data. Front. Genet. 5, 55 (2014)). By using pre-processed gene expression data as input, this algorithm enables cross-platform dataset comparison with a low error rate, and it also has the ability to acquire functional characteristics of intracellular regulation using mathematical estimation. For each sample group examined, the algorithm performs case-reference comparison using Student's t-test to generate a list of significantly differentially expressed genes, and further calculates pathway activation intensity (PAS), a value that serves as a qualitative measure of pathway activation. Positive and negative PAS values ​​indicate pathway upregulation and downregulation, respectively.

[0106] k-Nearest Neighbors (kNN): The k-nearest neighbors method is a simple nonparametric method (non-parameter method) that can be used for regression. The concept underlying this method is to predict the value of a given object as the mean of the values ​​of its k nearest neighbors. The optimal choice of k is defined by the nature of the data. In this invention, a Scikit-learn implementation of the method can be used (Pedregosa, F. et al. Scikit-learn: Machine Learning in Python. J. Mach. Learn. Res. 12, 2825-2830 (2011)). Hyperparameters are adjusted, which are the number of neighbors to use (5 to 20), the neighbor weighting (uniform or inversely proportional to the distance), and the metric (Manhattan, Euclidean, or Minkowski, p=3).

[0107] Logistic regression (LR). Logistic regression involves a series of independent variables X i This is a widely used direct approach to model the dependency of a given variable Y on a given variable. This invention utilizes a Scikit-learn implementation (Pedregosa, F. et al. Scikit-learn: Machine Learning in Python. J. Mach. Learn. Res. 12, 2825-2830 (2011)). First, the dimensionality of the data was reduced using principal component analysis with whitening, and then a multi-class classifier was trained by L2 regularization. The tuned hyperparameters were the number of principal components (100–500) and the regularization strength (0.1–100).

[0108] Support Vector Machine (SVM). SVM is another classic machine learning algorithm that, in its basic form, constructs a set of hyperplanes that separate multidimensional data into multiple classes. By using a nonlinear kernel, SVM can perform nonlinear classification. In this study, the inventors utilized a Psykit-Learn implementation of this method (Edgar, R. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207-210 (2002)). The tuned hyperparameters were the kernel type (linear, sigmoid, cubic polynomial, and radial basis function (Gaussian) kernel) and the regularization intensity (0.1–100).

[0109] Gradient boosting machine (GBM). Gradient boosting is a machine learning method used for classification and regression problems. This method utilizes an ensemble of weak models, such as a classification tree in this case, to generate predictions. The inventors used the XGBoost library (Kolesnikov, N. et al. ArrayExpress update--simplifying data submissions. Nucleic Acids Res. 43, D1113-6 (2015)) to implement a gradient boosting classifier. The tuned hyperparameters were the number of trees to grow (10–100), the maximum depth of each tree (3–8), the subsampling ratio (0.5–1.0), the regularization parameter γ (threshold for further splitting, 0.5–1), the minimum sum of leaf weights (minimum leaf weight, minimal child weight) (1–5), and the step size reduction (0.005–0.05).

[0110] A multi-class deep neural network (DNN). The number of neurons in the input layer was the same as the number of genes used. The tuned hyperparameters were the number of hidden layers (2–4), the number of neurons in each hidden layer (100–500), the activation function of all layers except the output layer (ReLU, sigmoid, or tanh), the L2 weight-regularization intensity (0.01–0.05), and the dropout value (0.0–0.5). The output layer utilizes softmax activation. This neural network was trained for 200 epochs using the Adam optimizer (McCall, MN, Bolstad, BM & Irizarry, RA Frozen robust multiarray analysis (fRMA). Biostatistics 11, 242-253 (2010)).

[0111] An ensemble of deep neural networks (DNN ens.). The design of each network is similar to that of a multi-class network, except that the output layer has only one neuron using sigmoid activation. Because optimizing the hyperparameters of a DNN ensemble is computationally expensive, each network used a set of hyperparameters that had been confirmed to be optimal for a multi-class network: two layers of 200 neurons, ReLU activation, dropout of 0.2, and L2 weight regularization intensity of 0.03. The inventors trained 20 binary networks on each target platform set (Affymetrix and Illumina) to perform pairwise (one-to-one) classification. The inventors then evaluated the total ensemble votes for each class as the sum of four one-to-one networks (which perform pairwise discrimination of this class from the other four classes).

[0112] Training of a classifier To utilize any of the above neural networks, the inventors need to train the network on a selected dataset. To do this, the inventors employ the following scheme.

[0113] First, the inventors preprocess the datasets (collected from public data repositories and provided by BioTime, Inc.) to convert the probe data into genes and apply quantile normalization.

[0114] Subsequently, the inventors tuned the hyperparameters by employing a nested cross-validation approach to obtain an unbiased estimate of classifier performance. Both the outer and inner loops utilize stratified labeled 3-fold cross-validation using samples from the same dataset belonging to either the training set or the validation set (but not both).

[0115] In the outer loop, the inventors retain a portion of the data and use the remaining samples to optimize the hyperparameters of the classifier. They then train the classifier using the found optimal hyperparameters and further test the classifier on the retained data to ensure that the hyperparameters are not overfitting. The hyperparameter tuning is repeated for each split. This result is referred to as "external validation (Ext. validation)".

[0116] The inventors utilize the Tree of Parzen Estimators (TPE) algorithm (implemented in the hyperopt package (Bergstra, J., Yamins, D. & Cox, DD Hyperopt: A Python library for optimizing machine learning algorithms; SciPy 2013. in Proceedings of the 12th Python In Science Conference 13-20 (2013))) to optimize hyperparameters. For each parameter set attempted by the algorithm, the inventors perform 3-fold cross-validation and use the average validation score as the target for optimization. For the best hyperparameter set, the inventors present its average performance for the training set ("Training") and the validation set within the inner cross-validation loop ("Int. validation").

[0117] Only the training and validation scores of the DNN ensemble are presented, because the inventors have not performed hyperparameter estimation of the DNN ensemble due to the high computational cost.

[0118] [Table 1]

[0119] Determining the embryo score of the sample To investigate how close a sample is to the embryonic state, the inventors utilize an ensemble of deep neural network predictors constructed based on one of the proposed approaches. The samples to be classified are subjected to the same preprocessing protocol as the training samples from a suitable platform. The genes are fed into the input of the trained deep neural network predictors. The ensemble generates five scores (one for each class) that are used by the inventors to calculate the embryo score (ES) shown by the formula in FIG. 21. (Here, Class 1-5 is the output of the predictor for each class, and w 1-5 is any degree of embryogenesis for the selected class (the inventors assigned w ESC = 1.0, w iPSC = 0.9, w EPC = 0.7, w ASC = 0.5, w AC = 0.0)).

[0120] As a result, the output of the said system calculates the embryo score for each sample.

[0121] To investigate which genes are excellent markers for each stage of cell development, the inventors utilized the method proposed in Yacoub, M. & Bennani, Y. HVS: A Heuristic for Variable Selection in Multilayer Artificial Neural Network Classifier. in Intelligent Engineering Systems Through Artificial Neural Networks 527 - 532 (1997), which can directly estimate the importance of each feature from the weight matrix of the DNN. In this method, the magnitude of all input features propagating all the way to the output layer is measured. In the case of a multi - layer neural network, the expression can be described as shown in FIG. 22, where w l is the DNN weight matrix of layer 1, and w ij l is the connection weight value between neurons i and j in layer l, 1|O| These are all vectors representing the size of the output layer, and they fit the vector of importance of the calculated input features.

[0122] For verification, the inventors measured the importance of genes from a trained multi-class GBM classifier by measuring how many times a particular feature was used to split the tree (F score).

[0123] The inventors found significant overlap among key genes scored by GBM or DNN (Figure 4), indicating that both methods respond to a considerable extent to similar sets of genes for prediction.

[0124] RNAi As a non-limiting example, dsRNA was prepared from an in vitro transcript (Promega) using a template generated by PCR with an adjacent T7 promoter, purified by phenol extraction and ethanol precipitation, and then annealed after resuspension in water. Untreated experimental animals were injected with 4 × 30 nL of dsRNA for 3 consecutive days, starting with the first injection 2 hours after surgery following induced tissue injury.

[0125] TR Modulation and iTR Modulator This disclosure provides novel iTR modulators and methods of using them. In some embodiments, the present invention provides novel methods for enhancing regeneration, which include administering a drug that modifies the concentration of the iTR modulator to a multicellular organism in need of such enhancement.

[0126] The applicants teach that primitive animals exhibiting remarkable TR (transgenic regeneration) capabilities, such as limb regeneration in axolotls, skin regeneration in MRLs or African spiny mice, or whole-body regeneration in planarians, do so simply by repeating the normal embryonic development of each tissue. Furthermore, the applicants teach that the inability of TR-resistant mammals, such as most mouse species and humans, to regenerate damaged tissue is due to alterations in certain embryonic gene transcriptions during EFT in these TR-resistant animals. The applicants further teach that the restoration of certain embryonic-specific patterns of these gene expression altered during EFT in TR-resistant animals can induce the ability to regenerate any tissue, including responses to central factors, leading to complex tissue regeneration and a reduction in associated scar formation. Finally, the applicants teach novel agents and related methods for inducing TR in mammalian species. These methods promote TR in vivo in mammalian species, particularly in Homo sapiens.

[0127] Genes whose expression inhibits TR in fetuses and adult animals are referred to herein as "TR inhibitors," and genes whose expression promotes TR not only in fetal and adult cells but also during the embryonic stage of development are referred to herein as "TR activators." Collectively, TR inhibitor genes and TR activator genes are referred to herein as iTR genes. Molecules that alter the levels of TR activators and TR inhibitors in a manner that results in TR are referred to herein as "iTR factors." iTR genes and their protein products are often conserved among animals ranging from sea anemones to mammals. The gene-encoded protein sequences and gene-encoded nucleic acid (e.g., mRNA) sequences referred herein, for example from numerous different non-human animal species, are publicly known in the art and can be found in publicly available databases, such as the database available at the National Center for Biotechnology Information (NCBI) (www.ncbi.nih.gov).

[0128] The TR inhibitor gene COX7A1 was observed to be primarily expressed in stromal cells rather than epithelial cells in normal tissues, although it was also expressed at low levels in epithelial cultures. In neoplasms, this gene was observed to be downregulated in many stromal cancers, including osteosarcoma, chondrosarcoma, rhabdomyosarcoma, and some gliomas, carcinomas, and adenocarcinomas. This is consistent with the observation of enhanced glycolysis in cancer, known as the Warburg effect, although the lack of COX7A1 expression had not previously been included in the Warburg effect. Since we propose that the TR gene is partially modified during the transition from embryo to fetus to prevent cancer in adults, suppression of COX7A1 in stromal tumors and some CNS tumors and epithelial tumors would revert stromal cells to their embryonic state, thereby promoting carcinogenesis. Exogenous induction of COX7A1 expression in such tumors where expression is deficient would therefore have a therapeutic effect, partly by altering the activity of p53 and HIF1α, thereby inhibiting cell proliferation and increasing apoptosis in cancer cells.

[0129] In another embodiment, the present invention provides a means for detecting cancer cells. Researchers have rarely identified markers of abnormalities associated with most cancer cell types. As described herein, by using markers that distinguish between an embryo and its fetal and adult counterparts, it is possible to distinguish between normal cells exhibiting adult pattern expression and malignant cells exhibiting embryonic pattern expression. Such detection methods include, but are not limited to, the detection of gene expression from α, β, and γ clustered protocadherin genes, including but not limited to COX7A1, NAALADL1, AMH, and PCDHA4, PCDHB2, and PCDHGA12, in the embryonic pattern rather than the fetal / adult pattern. This is useful not only for identifying malignant cells (excluding blood cells) but also for identifying tumors that are resistant to commonly used chemotherapy drugs and are characterized by the expression of their fetal / adult pattern.

[0130] This disclosure provides numerous different methods for modulating iTR genes and a variety of different compounds useful for modulating iTR genes. Generally, iTR factors can be, for example, small molecules, nucleic acids, oligonucleotides, polypeptides, peptides, lipids, carbohydrates, etc. In some embodiments of the present invention, iTR factors are inhibited by reducing the amount of TR inhibitor RNA produced by cells and / or by reducing the level of activity of TR inhibitor genes. When targeting TR inhibitors, factors that reduce the level of TR inhibitor gene products are identified and used for research and therapy. The TR inhibitor genes may be any one or a combination thereof of the TR inhibitor genes listed under the “Fetal / Adult Markers” section in Figure 10. The amount of TR inhibitor gene RNA can be reduced by inhibiting the synthesis of TR inhibitor RNA by cells (also referred to as "inhibiting TR inhibitor gene expression"), for example, by reducing the amount of mRNA encoding the TR inhibitor gene or by reducing the translation of mRNA encoding the TR inhibitor gene. Such factors, in non-limiting examples, may be RNAi targeting sequences within the TR inhibitor gene, as listed under the "Fetal / Adult Markers" section in Figure 10.

[0131] In some embodiments, TR inhibitor gene expression is inhibited by RNA interference (RNAi). As is well known in the art, RNAi is a process in which the intracellular presence of double-stranded RNA sequence-identified with a given gene results in sequence-specific inhibition of the gene's expression, typically as a result of cleavage or translational repression of mRNA transcribed from that gene. Compounds useful for inducing RNAi-mediated inhibition of expression ("RNAi agents") include small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs), and miRNA-like molecules.

[0132] Those skilled in the art can easily design sequences for RNAi agents (e.g., siRNA) useful for inhibiting the expression of a mammalian TR inhibitor gene (e.g., the human TR inhibitor gene) after the TR inhibitor gene has been identified. In some embodiments, such sequences are selected to minimize “off-target” effects. For example, sequences complementary to sequences present in the TR inhibitor gene mRNA but not present in other mRNAs expressed in the target species (or not present in the genome of the target species) may be used. Potential off-target effects may be reduced by utilizing site-directed chemical modifications. In some embodiments, at least two different RNAi agents, e.g., siRNA, targeting the TR inhibitor gene mRNA are used in combination. In some embodiments, TR inhibitor gene expression is inhibited by using microRNA (which may be artificially designed microRNA).

[0133] In some embodiments of the present invention, TR inhibitor gene expression is inhibited using an antisense molecule comprising a single-stranded oligonucleotide that is completely or substantially complementary to the mRNA encoding the TR inhibitor gene. The oligonucleotide hybridizes to the TR inhibitor gene mRNA, for example, resulting in degradation of the mRNA by RNase H or inhibition of translation due to steric hindrance. In other embodiments of the present invention, TR inhibitor gene expression is inhibited using a ribozyme or triple-stranded nucleic acid.

[0134] In some embodiments of the present invention, a TR inhibitor inhibits the activity of at least one TR inhibitor protein. TR inhibitor activity can be reduced by bringing the TR inhibitor protein into contact with a compound that physically interacts with the TR inhibitor protein. Such a compound may, for example, alter the structure of the TR inhibitor protein (e.g., by covalently modifying the structure) and / or block the interaction between the TR inhibitor protein and one or more other molecules, such as cofactors or substrates. In some embodiments, the inhibition or reduction may be a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of a baseline level (e.g., a control level). The control level may be the level of TR inhibitor that occurs in the absence of the factor. For example, the TR factor may reduce the level of TR inhibitor protein to 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 25%, 20%, 10%, or 5% or less of the level that occurs in the absence of the factor under the tested conditions. In some embodiments, the level of TR inhibitor is reduced to 75% or less of the level that occurs in the absence of the factor under the tested conditions. In some embodiments, the level of TR inhibitor is reduced to 50% or less of the level that occurs in the absence of the TR factor under the tested conditions. In some embodiments, the level of TR inhibitor is reduced to 25% or less of the level that occurs in the absence of the iTR factor under the tested conditions. In some embodiments, the level of the TR inhibitor is reduced to 10% or less of the level that would occur under the tested conditions in the absence of the iTR factor. In some cases, the level of modulation (e.g., inhibition or reduction) compared to the control level is statistically significant. As used herein, “statistically significant” means a p-value less than 0.05, e.g., less than 0.025 or less than 0.01, using an appropriate statistical test (e.g., ANOVA, t-test, etc.).

[0135] In some embodiments of the present invention, a compound directly inhibits a TR inhibitor protein; that is, the compound inhibits the TR inhibitor protein through a mechanism involving a physical interaction (binding) between the TR inhibitor and an iTR factor. For example, the binding of a TR inhibitor to an iTR factor may interfere with the TR inhibitor's ability to catalyze a reaction and / or block the active site of the TR inhibitor. By using a variety of compounds, TR inhibitors can be directly inhibited. Exemplary compounds that directly inhibit TR inhibitors may be, for example, small molecules, antibodies, or aptamers.

[0136] In some embodiments of the present invention, the iTR factor covalently binds to the TR inhibitor. For example, such a compound may modify amino acid residues required for enzyme activity. In some embodiments, the iTR factor comprises one or more reactive functional groups that react with the amino acid side chains of the TR inhibitor, such as aldehydes, haloalkanes, alkenes, fluorophosphonates (e.g., alkylfluorophosphonates), Michael acceptors, phenylsulfonates, methyl ketones (e.g., halogenated methyl ketones or diazomethyl ketones), fluorophosphonates, vinyl esters, vinyl sulfones, or vinyl sulfonamides. In some embodiments, the iTR factor inhibitor comprises a compound that physically interacts with the TR inhibitor, wherein the compound comprises a reactive functional group. In some embodiments, the structure of the compound that physically interacts with the TR inhibitor is modified to incorporate the reactive functional group. In some embodiments, the compound comprises a substrate analog or transition state analog of the TR inhibitor. In some embodiments, the compound interacts with the TR inhibitor at or near the TR inhibitor active site.

[0137] In other embodiments, the iTR factor non-covalently binds to the TR inhibitor and / or a complex containing the TR inhibitor and a TR inhibitor substrate. In some embodiments, the iTR factor non-covalently binds to the active site of the TR inhibitor and / or competes with the substrate(s) for access to the TR inhibitor active site. In some embodiments, the iTR factor binds to the TR inhibitor under tested conditions, for example, in a physiologically acceptable solution such as phosphate-buffered saline, at a rate of about 10 -3 M or less, for example, 10 -4 M or less, for example, 10 -5 M or less, for example, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, or 10 -9 K below M d The binding affinity can be measured using, for example, surface plasmon resonance (e.g., using the Biacore system), isothermal titration calorimetry, or competitive binding assays, which are known in the art. In some embodiments, the inhibitor comprises a substrate analog or transition state analog of a TR inhibitor.

[0138] To increase the activity of TR activators, any one of the TR activator gene combinations listed under the "Embryo Markers" section in Figure 10 may be used. The levels of the products of these genes may be introduced using the vectors described herein.

[0139] In other embodiments, the iTR factor is a construct that can be introduced into cells by a gene expression construct that can induce iTR in a shorter time, although pluripotency can be induced if the RNA is reacted with the cells for a sufficient period of time. Preferably, the RNA does not contain all of the RNA required for reprogramming to pluripotency, but instead contains only LIN28A or LIN28B, possibly together with a telomere length increasing agent such as the RNA of the catalytic component of telomerase (TERT). Most preferably, the agent for inducing iTR is a gene / factor induced by a LIN28A or LIN28B encoding protein, such as GFER, combined with a telomere length increasing agent, possibly including TERT encoding RNA or a gene, as disclosed herein, for example, a factor important to iTR, such as 0.05–5 mM valproic acid, preferably 0.5 mM valproic acid, 1–100 ng / mL AMH, preferably 10 ng / mL AMH, and 2–200 ng / mL GFER, preferably 20 ng / mL. When administered in vivo, such factors are preferably administered as a sustained-release hydrogel matrix, such as a HyStem matrix, which consists of chemically modified and cross-linked hyaluronic acid and collagen.

[0140] Reporter-based screening assay for iTR factor The present invention provides a method for identifying iTR factors using (a) a reporter molecule containing an easily detectable marker such as GFP or β-galactosidase, whose expression is driven by the promoter of one of the TR activator genes described herein (e.g., the one for COX7A1). The present invention provides a screening assay that requires determining whether a test compound affects the expression of a TR activator gene and / or inhibits the expression of a TR inhibitor gene. The present invention further provides reporter molecules and compositions useful for carrying out this method. Generally, compounds identified using the method of the present invention may act by either increasing or decreasing TR activator or inhibitor genes, respectively. In the case of the COX7A1 promoter, the promoter sequence adjacent to the 5' end of the human gene is characterized by being located -756 bases from the ATG translation start codon (Yu, M., et al. Biochimica and Biophysica Acta 1574 (2002) 345-353). The transcription start site of most cDNAs has been found to be located -55 bases from the translation start codon.

[0141] Although COX7A1 expression is tissue-specific, the promoter, and the rest of the gene sequence, reside in a CpG island, similar to the promoters of many housekeeping genes. The CpG island is characterized by abundant CG dinucleotides, exceeding the average and expected content of dinucleotides for the genome, over a range of at least 200 base pairs. The promoter contains several regulatory binding site sequences, namely MEF2 at position -524, and three E boxes at positions -58, -279, and -585 (characterized as E1, E2, and E3), respectively, where the E boxes are DNA binding sites (CAACTG) that bind to members of the myogenin family of regulatory proteins. Furthermore, there are multiple CG-rich segments in the region of approximately -95 to -68 base pairs, similar to those recognized by the transcription factor Sp1.

[0142] The gene characterized by the GRCh38.p7 primary assembly occupies 1948 base pairs between positions 36150922 and 36152869 on human chromosome 18, and contains four exons and three introns interspersed between them. The gene sequence, along with its promoter sequence, is publicly available on NCBI under the locus identifier AF037372.

[0143] Reporter molecules, cells, and membranes Generally, useful detectable moieties in the reporter molecule of the present invention include luminescent or light-absorbing compounds that generate or quench a detectable fluorescent signal, chemiluminescent signal, or bioluminescent signal. In some embodiments, activation of a TR activator gene or inhibition (suppression) of a TR inhibitor gene (TR repressor gene) results in the release of a detectable moiety into a liquid culture medium, and the signal generated or quenched by the released detectable moiety present in the medium (or a sample thereof) is detected. In some embodiments, the resulting signal causes a change in the properties of the detectable moiety, such a change can be detected, for example, as a light signal. For example, the signal can alter the emission or absorption of electromagnetic radiation (e.g., radiation having wavelengths in the infrared, visible, or UV portion of the spectrum) by the detectable moiety. In some embodiments, the reporter molecule includes a fluorescent or light-emitting moiety, and a second molecule functions as a quencher that quenches the fluorescent or light-emitting moiety. Such a change can be detected using apparatus and methods known in the art.

[0144] In many embodiments of the present invention, the reporter molecule is a genetically encoding molecule that can be expressed by a cell, and the detectable portion includes, for example, a detectable polypeptide. Thus, in some embodiments, the reporter molecule is a polypeptide comprising fluorescent polypeptides, e.g., green fluorescent protein, blue fluorescent protein, sapphire fluorescent protein, yellow fluorescent protein, red fluorescent protein, orange fluorescent protein, and cyan fluorescent protein, and their derivatives (e.g., high-sensitivity GFP); monomeric red fluorescent protein and derivatives, e.g., those known as "mFruits," e.g., mCherry, mStrawberry, mTomato, etc.; and luminescent proteins, e.g., aequorin. (In some embodiments, it will be understood that fluorescence or luminescence occurs in the presence of one or more additional molecules, e.g., ions, e.g., calcium ions, and / or prosthetic groups, e.g., coelenterazine.) In some embodiments, the detectable portion includes an enzyme that acts on a substrate to produce a fluorescent product, a luminescent product, a colored product, or other detectable product. Examples of enzymes that may be useful as detectable parts include luciferase; β-galactosidase; horseradish peroxidase; alkaline phosphatase; and others. (It will be understood that enzymes are detected by the detection of reaction products.) In some embodiments, the detectable part includes a polypeptide tag that can be readily detected using a second agent, such as a labeled (e.g., fluorescently labeled) antibody. For example, fluorescently labeled antibodies that bind to HA, Myc, or various other peptide tags can be utilized. Thus, the present invention encompasses embodiments in which the detectable part can be directly detected (i.e., it generates a detectable signal without requiring interaction with a second agent), as well as embodiments in which the detectable part interacts with a second agent (e.g., binds and / or reacts), and such interaction makes the detectable part detectable, for example, by producing a detectable signal, or because the second agent is directly detectable.In embodiments where a detectable portion interacts with a second drug to generate a detectable signal, a detectable portion capable of reacting with the second drug is affected by the second drug and generates a detectable signal. In many embodiments, the signal intensity provides an indicator of the amount of detectable portion present, for example, in the sample being evaluated or within the region being imaged. In some embodiments, the amount of detectable portion is optionally quantified against a relative or absolute standard based on the signal intensity.

[0145] This description provides nucleic acids comprising a sequence encoding a reporter polypeptide of the present invention. In some embodiments, the nucleic acid encodes a precursor polypeptide of the reporter polypeptide of the present invention. In some embodiments, the sequence encoding the polypeptide is operably ligated to an expression regulatory element (e.g., a promoter or promoter / enhancer sequence) suitable for directing the transcription of mRNA encoding the polypeptide. The present invention further provides expression vectors comprising nucleic acids. The selection of a suitable expression regulatory element may be based, for example, on the cell type and species in which the nucleic acid is expressed. Those skilled in the art can easily select a suitable expression regulatory element and / or expression vector. In some embodiments, the expression regulatory element(s) are regulated, for example, inducible or repressive. Exemplary promoters suitable for use in bacterial cells include, for example, Lac, Trp, Tac, araBAD (e.g., in the pBAD vector), and phage promoters, such as T7 or T3. Examples of exemplary expression regulatory sequences useful for directing expression in mammalian cells include, for example, the early and late promoters of SV40, the earliest promoters of adenoviruses or cytomegaloviruses, or viral promoter / enhancer sequences, retroviral LTRs, promoters or promoter / enhancers derived from mammalian genes, such as actin, EF-1 alpha, and phosphoglycerate kinase. Others can be regulated by modulotable (e.g., inducible or repressive) expression systems, such as Tet-On and Tet-Off systems (modulated by tetracycline and its analogs, e.g., doxycycline), and small molecules, such as hormone receptor ligands (e.g., steroid receptor ligands, which may or may not be steroids), metal-regulated systems (e.g., metallothionein promoters), etc.

[0146] This description further provides cells and cell lines comprising such nucleic acids and / or vectors. In some embodiments, the cells are eukaryotic cells, e.g., fungal cells, plant cells, or animal cells. In some embodiments, the cells are vertebrate cells, e.g., mammalian cells, e.g., human cells, non-human primate cells, or rodent cells. Often, the cells are members of a cell line, e.g., an established or immortalized cell line that has acquired the ability to proliferate indefinitely during culture (e.g., constitutive expression of a catalytic component of telomerase, for example, as a result of mutation or genetic manipulation). Numerous cell lines are known in the art and can be used in the present invention. Examples of mammalian cell lines include HEK-293 (e.g., HEK-293T), CHO, NIH-3T3, COS, and HeLa cell lines. In some embodiments, the cell line is a tumor cell line. In other embodiments, the cells are non-tumoric and / or not tumor-derived. In some embodiments, the cells are adherent cells. In some embodiments, non-adherent cells are used. In some embodiments, cells from a cell type or cell line known to naturally possess a subset of expressed TR activator genes or unexpressed TR inhibitor genes are used. If a cell lacks one or more TR activator or inhibitor genes, it can be genetically engineered to express such proteins. In some embodiments, the cell line of the present invention is derived from a single cell. For example, a population of cells can be transfected with a nucleic acid encoding a reporter polypeptide, and colonies derived from the single cell can be selectively cultured and grown. In some embodiments, cells are transiently transfected with an expression vector encoding a reporter molecule. Cells are co-transfected with control plasmids that optionally express different detectable polypeptides, allowing for control over transfection efficiency (e.g., over multiple runs of the assay).

[0147] TR activator polypeptides and TR inhibitor polypeptides and nucleic acids The TR activator and TR inhibitor genes are listed in Figure 10, under the headings "Embryo Markers" and "Fetal / Adult Markers," respectively. TR activator polypeptides and TR inhibitor polypeptides useful in the methods of the present invention can be obtained by various methods. In some embodiments, the polypeptides are produced using recombinant DNA technology. Standard methods for recombinant protein expression can be used. Nucleic acids encoding the TR activator or TR inhibitor gene can be readily obtained, for example, from cells expressing the gene (e.g., by PCR or other amplification methods, or by cloning), or by chemical synthesis based on cDNA sequence polypeptide sequences, or by in vitro transcription. Those skilled in the art will know that, due to gene coding degeneracy, a gene can be encoded by a number of different nucleic acid sequences. Optionally, the sequences are codon-optimized for expression in the host cells of choice. The genes can be expressed in bacterial, fungal, animal, or plant cells or organisms. Genes can be isolated from cells that naturally express them, or from cells into which protein-coding nucleic acids have been transiently or stably introduced, for example, from cells containing gene-coding expression vectors. In some embodiments, genes are secreted by cells during culture and isolated from the culture medium.

[0148] In some embodiments of the present invention, the sequence of a TR activator polypeptide or TR inhibitor polypeptide is used in the screening method of the present invention. Naturally occurring TR activator polypeptides or TR inhibitor polypeptides may originate from any species whose genome encodes a TR activator polypeptide or TR inhibitor polypeptide, such as humans, non-human primates, rodents, etc. Polypeptides with sequences identical to naturally occurring TR activators or TR inhibitors may, in some cases, be referred to herein as “natural TR activator / inhibitor.” The TR activator polypeptide or TR inhibitor polypeptide used in the present invention may or may not contain a secretion signal sequence or a portion thereof. For example, a mature TR activator or TR inhibitor containing or consisting of amino acids 20 to 496 of a human TR activator or TR inhibitor (or the corresponding amino acids of a TR activator or TR inhibitor from a different species) can be used.

[0149] In some embodiments, polypeptides comprising or consisting of variants or fragments of TR activators or TR inhibitors are used. TR activator variants or TR inhibitor variants comprise polypeptides that differ from TR activators or TR inhibitors due to the substitution, addition, or deletion of one or more amino acids. In some embodiments, the TR activator variant or TR inhibitor variant comprises a polypeptide in which at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of at least amino acids 20–496 of human TR activator or TR inhibitor or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of at least amino acids 20–496 of (e.g., human or mouse-derived) TR activator or TR inhibitor are identical. In some embodiments, the TR activator variant or TR inhibitor variant comprises a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to at least 20-496 amino acids of human TR activator or TR inhibitor or 20-503 amino acids of mouse TR activator or TR inhibitor. Nucleic acids encoding TR activator variants or TR inhibitor variants or fragments can be readily generated, for example, by modifying DNA encoding a natural TR activator or TR inhibitor using site-directed mutagenesis, or by other standard methods, and can be used to generate TR activator variants or TR inhibitor variants or fragments.For example, a fusion protein can be produced by cloning a sequence encoding a TR activator or TR inhibitor into a vector that provides a sequence encoding a heterologous portion. In some embodiments, tagged TR activators or TR inhibitors are used. For example, in some embodiments, a TR activator polypeptide or TR inhibitor polypeptide containing a 6xHis tag at its C-terminus is used.

[0150] Test compound A wide range of test compounds can be used in the method of the present invention for identifying iTR factors and global modulators of iTR. For example, the test compounds may be small molecules, polypeptides, peptides, nucleic acids, oligonucleotides, lipids, carbohydrates, antibodies, or hybrid molecules, including but not limited to those described herein, including the mRNA of the genes OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, LIN28A, and LIN28B individually, as well as various combinations thereof, or various combinations with small molecule compounds, for example, combinations of the following compounds: glycogen synthase 3 (GSK3), including but not limited to CHIR99021; TGF-β signaling inhibitors, including but not limited to SB431542, A-83-01, and E616452; HDAC inhibitors, including but not limited to fatty acid compounds, including but not limited to valproic acid, phenylbutyrate, and n-butyrate; cyclic tetrapeptides, including trapoxin B and depsipeptides; hydroxamic acids, such as trichostatin A and vorinostat (SAHA). , verinostat (PXD101), LAQ824, panobinostat (LBH589), benzamide-based endinostat (MS-275), CI994, mochenostat (MGCD0103); sirtuin inhibitors nicotinamide, various derivatives of NAD, dihydrocoumarin, naphthopyrane, and 2-hydroxynaphthalenealdehyde, or IV (HDAC11) deacetylases, including Class I (HDAC1, HDAC2, HDAC3 and HDAC8), Class IIA (HDAC 4. Inhibitors specifically targeting HDAC5, HDAC7, and HDAC9), Class IIB (HDAC6 and HDAC10), and Class III (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7); inhibitors of H3K4 / 9 histone demethylase LSD1, including but not limited to Parnate; inhibitors of Dot1L, including but not limited to EPZ004777; inhibitors of G9a, including but not limited to Bix01294;EZH2 inhibitors including but not limited to DZNep; DNA methyltransferase inhibitors including but not limited to RG108; 5-aza-2'-deoxycytidine (trade names Vidaza and Azadine); Vitamin C that can inhibit DNA methylation and increase Tet1, which increases 5hmC, the first step in demethylation; 3'-phosphoinositide-dependent kinase 1 activators including but not limited to PS48; glycolysis promoters including but not limited to quercetin and fructose 2,6-bisphosphate (phosphofructokinase 1 activators); quercetin Agents that promote the activity of the HIF1 transcription complex, including but not limited to ; RAR agonists, including but not limited to AM580, CD437, and TTNPB; agents that mimic hypoxia, including but not limited to resveratrol; agents that increase telomerase activity, including but not limited to the exogenous expression of the catalytic component of telomerase (TERT); agents that promote epigenetic modification via the downregulation of LSD1; H3K4-specific histone demethylases, including but not limited to lithium; or MAPK / ERK pathway inhibitors, including but not limited to PD032590. Such compounds can be administered in various combinations, concentrations, and for different durations to optimize the effects of iTR, for example, by assaying the reduced expression of COX7A1 or NAALADL1 or other iTR inhibitors described herein, and / or by assaying the increased expression of PCDHB2 or AMH or other iTR activators described herein, using global iTR markers, to cells cultured in vitro, or in damaged or diseased tissue in vivo, or in modulating the lifespan of animals in vivo.

[0151] In vitro assays are performed on the expression of COX7A1, PLPP7, and NAALADL1 genes, as well as the TR patterns of pluripotency gene expression or gene markers including DNMT3B, HELLS, or Tra-1-60, Tra-1-81, and SSEA4, respectively, to optimize the global pattern of iTR gene expression without returning target cells to pluripotency. Examples of individual drugs and drug combinations screened include OCT4, SOX2, KLF4, MYC, and LIN28A; OCT4; KLF4; OCT4, KLF4; OCT4, KLF4, LIN28A; OCT4, KLF4, LIN28B; SOX2; MYC; NANOG; ESRRB; NT5A2; OCT4, SOX2, KLF4, and LIN28A; OCT4, SOX2, KLF4, and LIN28B; OCT4, KLF4, MYC, and L IN28A; for the first four weeks, each of the aforementioned drug combinations was treated with 0.25 mM NaB, 5 μM PS48, and 0.5 μM A-83-01, followed by treatment with 0.25 mM sodium butyrate, 5 μM PS48, 0.5 μM A-83-01, and 0.5 μM PD0325901, and each of these was assayed on days 0, 1, 2, 4, 7, 10, and 14 for markers of global modulation of iTR gene expression.

[0152] Compounds can be obtained from natural sources or produced synthetically. Compounds may be at least partially pure, or they may be present in extracts or other types of mixtures in which the components are at least partially unknown or uncharacterized. Extracts or fractions thereof can be produced from, for example, plants, animals, microorganisms, marine organisms, fermentation broths (e.g., soil, bacterial, or fungal fermentation broths). In some embodiments, compound collections ("libraries") are tested. Libraries may contain, for example, 100 to 500,000 compounds or more. Compounds are often placed in multi-well plates (e.g., 384-well plates, 1596-well plates, etc.). They may be soluble in a solvent (e.g., DMSO) or provided in a dry form, for example, as a powder or solid. Collections of synthetic, semi-synthetic, and / or natural compounds can be tested. Compound libraries may contain structurally related, structurally diverse, or structurally unrelated compounds. Compounds may be artificial (invented by humans and having structures not found in nature) or naturally occurring. In some embodiments, the library includes at least several compounds identified as “hits” or “leads” in other drug discovery programs and / or their derivatives. The compound library may include natural products and / or compounds produced using non-directed or directed synthetic organic chemistry. Often, the compound library is a small molecule library. Other libraries of interest include peptide or peptoid libraries, cDNA libraries, antibody libraries, and oligonucleotide libraries. The library can be targeted (e.g., composed primarily of compounds having the same core structure, compounds derived from the same precursor, or compounds having at least one common biochemical activity).

[0153] Compound libraries are available from many product manufacturers, such as Tocris Bioscience, Nanosyn, BioFocus, and government agencies. For example, the Molecular Libraries Small Molecule Repository (MLSMR), a component of the National Institutes of Health (NIH) Molecular Library Programme, aims to identify, acquire, maintain, and distribute a collection of over 300,000 chemically diverse compounds with known and unknown bioactivity for use, for example, in high-throughput screening (HTS) assays (see https: / / mli.nih.gov / mli / ). The NIH Clinical Collection (NCC) is a plate array of approximately 450 small molecules with a history of use in human clinical trials. These compounds are highly drug-like with known safety profiles. In some embodiments, collections of compounds containing “approved human drugs” are tested. "Approved human drugs" are compounds that have been authorized for use in human treatment by a government regulatory agency, such as the U.S. Food and Drug Administration, the European Medicines Evaluation Agency, or a similar agency responsible for evaluating the safety of therapeutic agents, before they become marketed. Test compounds may include, for example, antitumor agents, antibacterial agents, antiviral agents, antifungal agents, antiprotozoal agents, antiparasitic agents, antidepressants, antipsychotics, anesthetics, antianginic agents, antihypertensive agents, antiarrhythmic agents, anti-inflammatory agents, analgesics, antithrombotic agents, antiemetics, immunomodulators, antidiabetic agents, lipid-lowering or cholesterol-lowering agents (e.g., statins), anticonvulsants, anticoagulants, anxiolytics, hypnotics (sleep inducers), hormones, or antihormonal agents. In some embodiments, compounds have undergone at least some preclinical or clinical development, or have been determined or predicted to have "drug-like" properties. For example, the test compound may have undergone Phase I or at least preclinical trials in non-human animals, demonstrating evidence of safety and tolerability.

[0154] In some embodiments, the test compound is substantially nontoxic to the cells of the organism to which the compound may be administered and / or to the cells to which the compound may be tested at the concentration used, or in some embodiments, at concentrations up to 10, 100, or 1,000 times higher than the concentration used. For example, there may be no statistically significant effect on cell viability and / or proliferation, or the decrease in viability or proliferation may be 1%, 5, or 10% or less in various embodiments. The effect on cytotoxicity and / or cell proliferation can be evaluated using any variety of assays. For example, cell metabolism assays, such as AlamarBlue, MTT, MTS, XTT, and CellTitle Glo assays, cell membrane integrity assays, cell ATP-based viability assays, mitochondrial reductase activity assays, BrdU, EdU, or H3-thymidine uptake assays can be used. In some embodiments, the test compound is not a compound identified in cell culture media known or used in the art, such as a culture medium suitable for culturing vertebrate, such as mammalian cells, or if the test compound is a compound identified in cell culture media known or used in the art, the test compound is used in different concentrations, for example, at a higher concentration, when used in the method of the present invention.

[0155] iTR Global Modulator Assay: Assay Implementation and Control Methods The various screening assays of the present invention described above involve determining whether a test compound suppresses the level of active TR inhibitors or increases the level of active TR activators. Cells suitable for the expression of the reporter molecule are described above.

[0156] In carrying out the assay of the present invention, the assay components (e.g., cells, TR activator polypeptide or TR inhibitor polypeptide, and the test compound) are typically distributed into multiple containers or other containers. Any type of container or article capable of holding cells can be used. In many embodiments of the present invention, the container is a well of a multiwell plate (also known as a “microwell plate,” “microtiter plate,” etc.). For clarity, the term “well” is used to mean any type of container or article that can be used to carry out the screening of the present invention, such as any container or article that can contain the assay components. It should be understood that the present invention is not limited to the use of wells or multiwell plates. In some embodiments, any product can be used in which multiple physically separated holes (or other containment features) are present in or on the substrate. For example, the assay components can be contained in fluid droplets, which may optionally be arranged on a surface, and optionally separated by a water-resistant material that contains the droplets in separate locations, such as channels in a microfluidic device.

[0157] Generally, assay components can be added to the wells in any order. For example, cells can be added first and maintained in culture for a selected period (e.g., 6–48 hours) before adding cells containing the test compound and the target TR activator or TR inhibitor polypeptide or expression construct to the wells. In some embodiments, the compound is added to the wells before adding the polypeptide to the cells. In some embodiments, reporter polypeptide expression is induced after plating the cells and optionally after adding the test compound to the wells. In some embodiments, reporter molecule expression is achieved by transfecting the cells with an expression vector encoding the reporter polypeptide. In some embodiments, the cells are pre-genetically engineered to express the reporter polypeptide. In some embodiments, reporter molecule expression is under the control of a regulating expression regulatory element, and induction of reporter molecule expression is achieved by contacting the cells with a drug that induces (or deinhibits) expression.

[0158] An assay composition comprising cells, a test compound, or a polypeptide is maintained for a suitable period of time during which the test compound can cause an increase or decrease in the level or activity of a target TR activator or TR inhibitor (in the absence of a test compound that inhibits its activity). The number of cells, the amount of TR activator polypeptide or TR inhibitor polypeptide, and the amount of test compound added can be determined by those skilled in the art, depending on factors such as container size and cell type. In some embodiments, the molar concentration ratio of TR activator polypeptide or TR inhibitor polypeptide to the test compound is 1:10 to 10:1. In some embodiments, the number of cells, the amount of test compound, and the length of time during which the composition is maintained can be selected so that an easily detectable level of signal is obtained after a selected time in the absence of the test compound. In some embodiments, the cells are at confluence of about 25% to 75%, for example, about 50%, upon addition of the compound. In some embodiments, 1,000 to 10,000 cells / well (e.g., about 5,000 cells / well) are plated in about 100 μl of medium per well in a 96-well plate. In other exemplary embodiments, cells are seeded in a 384-well plate with 500 to 2,000 (e.g., about 1,000) cells per well in about 30 to 50 μl of medium. In some embodiments, the compound is tested at multiple concentrations (e.g., 2 to 10 different concentrations) and / or multiple replications (e.g., 2 to 10 replications). Multiple replications at some or all different concentrations can be performed. In some embodiments, the candidate TR factor is used at concentrations between 0.1 μg / ml and 100 μg / ml, e.g., between 1 μg / ml and 10 μg / ml. In some embodiments, the candidate TR factor is used at multiple concentrations. In some embodiments, the compound is added to the cells between 6 hours and 1 day (24 hours) after seeding.

[0159] In some embodiments of the present invention's method for screening and / or characterizing compounds, the test compound is added to the assay composition in an amount sufficient to achieve a predetermined concentration. In some embodiments, the concentration is up to about 1 nM. In some embodiments, the concentration is about 1 nM to about 100 nM. In some embodiments, the concentration is about 100 nM to about 10 μM. In some embodiments, the concentration is at least 10 μM, for example, 10 μM to 100 μM. The assay composition may be maintained for a variety of periods after the addition of its last component. In certain embodiments, the assay composition is maintained after the addition of all components for about 10 minutes to about 4 days, for example, 1 hour to 3 days, for example, 2 hours to 2 days, or any range in between or a specific value, for example, about 4 to 8 hours. Multiple different time points can be tested. Additional aliquots of the test compound may be added to the assay composition during such periods. In some embodiments, cells are maintained in a cell culture medium suitable for culturing that type of cell. In some embodiments, serum-free medium is used. In some embodiments, the assay composition comprises a physiologically acceptable liquid, instead of cell culture medium, that is suitable for maintaining cell membrane integrity and, optionally, cell viability. Any suitable liquid can be used as long as it has an appropriate osmotic pressure and is suitable for maintaining adequate cell membrane integrity and, optionally, cell viability for at least a sufficient time to perform the assay. One or more measurements indicating an increase in the level of active TR activator or a decrease in TR inhibitor can be performed during or after the incubation period.

[0160] In some embodiments, compounds screened for potential global modulators of iTR are selected from agents capable of inducing pluripotency in somatic cells under other conditions. Such agents include, individually or in combination, the following compounds: the genes OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, LIN28A, and LIN28B, either alone or in combination with small molecule compounds, for example, combinations with the following compounds: inhibitors of glycogen synthase 3 (GSK3), including but not limited to CHIR99021; and TGF-, including but not limited to SB431542, A-83-01, and E616452. β-signaling inhibitors; HDAC inhibitors including but not limited to valproic acid, phenylbutyrate, and n-butyrate, and fatty acid compounds; cyclic tetrapeptides including trapoxin B and depsipeptides; hydroxamic acids, e.g., trichostatin A, vorinostat (SAHA), bellinostat (PXDIOI), LAQ824, panobinostat (LBH589), benzamide-based endinostat (MS-275), CI994, motenostat (MGCD0103); containing sirtuin inhibitors such as nicotinamide, various derivatives of NAD, dihydrocoumarin, naphthopyrane, and 2-hydroxynaphthalenealdehyde, or IV (HDAC11) deacetylase, class I (HDAC1, HDAC2, HDAC3 and HDAC8), class IIA (HDAC4, HDAC5, HDAC7 and HDAC9), class IIB (HDAC6 and HDAC10), and class III (SIRT1, SIRT2, SIRT3, SIR Inhibitors specifically targeting T4, SIRT5, SIRT6, or SIRT7; inhibitors of H3K4 / 9 histone demethylase LSD1, including but not limited to Parnate; inhibitors of Dot1L, including but not limited to EPZ004777; inhibitors of G9a, including but not limited to Bix01294; inhibitors of EZH2, including but not limited to DZNep; inhibitors of DNA methyltransferases, including but not limited to RG108;5-Aza-2'-deoxycytidine (trade names Vidaza and Azadine); Vitamin C that can increase Tet1, which inhibits DNA methylation and increases 5hmC, the first step in demethylation; Activators of 3'-phosphoinositide-dependent kinase 1, including but not limited to PS48; Promoters of glycolysis, including but not limited to quercetin and fructose 2,6-bisphosphate (activators of phosphofructokinase 1); Agents that promote the activity of the HIF1 transcription complex, including but not limited to quercetin; AM RAR agonists including but not limited to 580, CD437, and TTNPB; hypoxic mimics including but not limited to resveratrol; telomerase activity enhancers including but not limited to exogenous expression of telomerase catalytic component (TERT); agents that promote epigenetic modification via LSD1 downregulation; H3K4-specific histone demethylases including but not limited to lithium; or MAPK / ERK pathway inhibitors including but not limited to PD032590. Such compounds can be administered in various combinations, concentrations, and for different durations to optimize the effects of iTR in in vitro cultured cells, in in vivo damaged or diseased tissue, or in in vivo modulation of animal lifespan, using global iTR markers, for example, by assaying the reduced expression of COX7A1 or NAALADL1 or other iTR inhibitors described herein, and / or by assaying the increased expression of PCDHB2 or AMH or other iTR activators described herein.

[0161] In some embodiments, individual compounds, each typically having a known identity (e.g., structure and / or sequence), are added to each of a plurality of wells. In some embodiments, two or more compounds may be added to one or more wells. In some embodiments, one or more compounds of unknown identity can be tested. The identity can then be determined using methods known in the art.

[0162] In various embodiments, the aforementioned assay methods of the present invention are suitable for performing high-throughput screening (HTS). In some embodiments, the screening assays of the present invention are high-throughput or ultra-high-throughput (see, for example, Fernandes, PB, Curr Opin Chem. Biol. 1998, 2:597; Sundberg, SA, Curr Opin Biotechnol. 2000, 11:47). High-throughput screening (HTS) often involves testing a large number of compounds very efficiently, for example, in parallel. For example, tens of thousands to hundreds of thousands of compounds can be routinely screened in a short time, for example, in a few hours to a few days. In some embodiments, HTS means testing 1,000 to 100,000 compounds per day. In some embodiments, ultra-high throughput means screening more than 100,000 compounds per day, for example, up to 1 million or more compounds per day. The screening assays of the present invention can be performed in multi-well formats, such as 96-well, 384-well, 1,536-well, or 3,456-well formats, and are suitable for automation. In some embodiments, each well of a microwell plate can be used to perform individual assays for different test compounds, or multiple wells can contain samples of a single compound, with at least some wells optionally left empty or used as controls or replicas, in order to observe the effect of concentration or incubation time. Typically, the HTS implementation of the assays disclosed herein involves the use of automation. In some embodiments, an integrated robotic system including one or more robots transfers assay microwell plates between multiple assay stations for the addition, mixing, incubation, and readout or detection of compounds, cells, and / or reagents. In some embodiments, the HTS system of the present invention can prepare, incubate, and analyze a large number of plates simultaneously. Appropriate data processing and control software can be used.The implementation of high-throughput screening is well known in the art. Without limiting the present invention, certain general principles and techniques that can be applied to embodiments of the HTS of the present invention are described in reference to Macarron R & Hertzberg R P., *Design and implementation of high-throughput screening assays*, *Methods Mol Biol.*, 565:1-32, 2009 and / or An WF & Tolliday N J., *Introduction: cell-based assays for high-throughput screening*, *Methods Mol Biol.*, 486:1-12, 2009 and / or any of these. Exemplary methods are also disclosed in William P. Janzen (2002) *High Throughput Screening: Methods and Protocols* (Methods in Molecular Biology) and *High-Throughput Screening in Drug Discovery* (Methods and Principles in Medicinal Chemistry) (2006). Further compounds may have, for example, one or more improved pharmacokinetic and / or pharmacodynamic properties compared to the initial hit, or they may simply have a different structure. The “improved properties” may, for example, make the compound more effective or preferable for one or more purposes described herein. In some embodiments, for example, the compound may have higher affinity for the target molecular product of interest (e.g., TR activator or TR inhibitor gene product), lower affinity for non-target molecules, higher solubility (e.g., increased water solubility), increased stability (e.g., in blood, plasma and / or the gastrointestinal tract), increased half-life in the body, increased bioavailability, and / or reduced side effects.Optimization can be achieved using experimental modifications of hit structures (e.g., synthesis of compounds with the relevant structures and their cell-free or cell-based assays or testing in non-human animals), and / or computational methods. In some embodiments, such modifications can utilize established principles of medicinal chemistry for predictably altering one or more properties. In some embodiments, one or more "hit" compounds are identified and subjected to systematic structural modifications to create a second library of structurally hit-related compounds (e.g., purified lead compounds). The second library can then be screened using any of the methods described herein.

[0163] In some embodiments, the iTR factor is modified or incorporated by modifying a portion that enhances stability (e.g., in serum), extends half-life, reduces toxicity or immunogenicity, or imparts a desired property to the compound.

[0164] Use of iTR, iTM, and ICM factors Pharmaceutical composition iTR, iTM, and iCM factors have a variety of uses. Non-limiting examples of such uses are discussed herein. In some embodiments, iTR factors are used to enhance the regeneration of organs or tissues. In some embodiments, iTR factors are used to enhance the regeneration of limbs, fingers, cartilage, heart, blood vessels, bone, esophagus, stomach, liver, gallbladder, pancreas, intestines, rectum, anus, endocrine glands (e.g., thyroid, parathyroid, adrenal glands, endocrine portion of pancreas), skin, hair follicles, thymus, spleen, skeletal muscle, locally damaged cardiac muscle, smooth muscle, brain, spinal cord, peripheral nerves, ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, prostate, penis, pharynx, larynx, trachea, bronchi, lungs, kidneys, ureters, bladder, urethra, eyes (e.g., retina, cornea), or ears (e.g., organ of Corti). In some embodiments, iTR factors are used to enhance the regeneration of the interstitial layer, e.g., connective tissue supporting the parenchymal tissue of tissues. In some embodiments, iTR factors are used to enhance regeneration after surgery, such as surgical removal of at least a portion of affected or damaged tissue, organ, or other structure, such as limbs or fingers. For example, such surgery may involve the removal of at least a portion of the liver, lungs, kidneys, stomach, pancreas, intestines, mammary glands, ovaries, testes, bones, limbs, fingers, muscles, or skin. In some embodiments, the surgery is the removal of a tumor. In some embodiments, iTR factors are used to promote scar-free skin regeneration after trauma, surgery, disease, and burns.

[0165] Enhancement of regeneration may, in various embodiments, include one or more of the following: (a) an increase in the rate of regeneration; (b) an increase in the degree of regeneration; (c) promotion of the establishment of appropriate structure (e.g., shape, pattern, tissue structure, tissue polarity) in the regenerating tissue or organ or other body structure; and (d) promotion of the growth of new tissue in a method of maintaining and / or restoring function. While the use of iTR factors to enhance regeneration is of particular interest, the present invention encompasses the use of iTR factors to enhance repair or wound healing in general, without necessarily producing a detectable enhancement of regeneration. Accordingly, the present invention provides a method for enhancing repair or wound healing, wherein an iTR factor is administered to a subject in need according to any of the methods described herein.

[0166] In some embodiments, the present invention provides a method for enhancing regeneration in a subject requiring it, comprising administering an effective amount of iTR factor to the subject. In some embodiments, the effective amount of the compound (e.g., iTR factor) is an amount that results in an increase in the rate or degree of regeneration of damaged tissue compared to a baseline (e.g., an appropriate control). In some embodiments, the baseline is the expected (e.g., mean or typical) rate or degree of regeneration in the absence of the compound (with optionally administration of a placebo). In some embodiments, the effective amount of iTR factor is an amount that results in improved structural and / or functional outcomes compared to the expected (e.g., mean or typical) structural or functional outcomes in the absence of the compound. In some embodiments, the effective amount of the compound, e.g., iTR factor, results in enhanced blast formation and / or reduced scarring. The degree or rate of regeneration can be evaluated, for example, based on the size(s) or volume of the regenerated tissue. Structural and / or functional outcomes can be assessed, for example, based on visual tests (including, optionally, the use of microscopy or imaging techniques, e.g., X-ray, CT scan, MRI scan, PET scan) and / or by assessing the ability of such tissue, organ, or other body part to perform one or more physiological processes or tasks normally performed by such tissue, organ, or body part. Typically, improved structural outcomes are closer to normal structures (e.g., structures present before tissue injury, or structures present in a normal healthy individual) when compared to the predicted (e.g., mean or typical) structural outcomes in the absence of treatment with the iTR factor. Those skilled in the art can select appropriate assays or tests for function. In some embodiments, the increase in regeneration rate or degree compared to a control is statistically significant (e.g., p < 0.05 or p < 0.01) and / or clinically significant. In some embodiments, the improvement in structural and / or functional outcomes compared to a control is statistically significant and / or clinically significant. "Clinically significant improvement" means an improvement that, within the bounds of the physician's or surgeon's full judgment, provides a useful benefit to the subject (for example, a benefit sufficient to make the treatment worthwhile).In many embodiments, iTR modulators, such as iTR factors, administered to a particular species of subject (e.g., for therapeutic purposes) are understood to be compounds that modulate, for example, repress, endogenous TR genes expressed in that species of subject. For example, if the subject is human, a compound that represses the activity of the human TR inhibitor gene product and activates the activity of the human TR activator gene product may typically be administered.

[0167] In some embodiments, iTR factors are used to enhance skin regeneration after, for example, burns (thermal or chemical burns), abrasion injuries, or other conditions involving skin loss, such as infection, necrotizing fasciitis, or purpura. In some embodiments, the burns are second-degree or third-degree burns. In some embodiments, the area of ​​skin loss is at least 10 cm². 2 It has an area of ​​[amount]. In one embodiment, the iTR factor enhances the regeneration of transplanted skin. In one embodiment, the iTR factor reduces excessive and / or pathological wound shrinkage or scarring.

[0168] In some embodiments, iTR factors are used to enhance bone regeneration in situations such as ununion fractures, implant fixation, periodontal or alveolar ridge augmentation, craniofacial surgery, or other conditions where neobial formation is considered appropriate. In some embodiments, iTR factors are applied to the site where bone regeneration is desired. In some embodiments, iTR factors are incorporated into or used in combination with bone graft materials. Examples of bone graft materials include various ceramic and protein materials. Examples of bone graft materials include autologous bone (e.g., bone harvested from the iliac crest, fibula, ribs, etc.), allogeneic bone from cadavers, and xenogeneic bone. Examples of synthetic bone graft materials include various ceramics, such as calcium phosphate (e.g., hydroxyapatite and tricalcium phosphate), bioglass, and calcium sulfate, as well as protein materials, such as demineralized bone matrix (DBM). DBM can be prepared by grinding cortical bone tissue (generally to a sieve particle size of 100-500 μm) and then treating the ground tissue with hydrochloric acid (generally 0.5-1 N). In some embodiments, the iTR factor is administered to a subject together with one or more bone graft materials. The iTR factor can be combined with the bone graft material (in a composition containing the iTR factor and the bone graft material) or administered separately, for example, after graft placement. In some embodiments, the present invention provides a bone paste containing the iTR factor. The bone paste is a product having appropriate viscosity and composition so that it can be introduced into and used to replenish or fill bone defects, such as gaps, spaces, cavities, or cracks, or to apply to existing bone structures. The bone paste is typically plastic enough to be manipulated and molded into various shapes by the user. A desirable outcome of such treatment is, for example, that bone is formed to replace the paste while maintaining the shape to which the paste was applied. The bone paste may contain substances(s) that provide a supporting structure for new bone formation and promote bone formation.Bone paste often contains, in addition to one or more of the above-mentioned ceramic bone graft materials or protein bone graft materials (e.g., DBM, hydroxyapatite), one or more components that impart a paste-like or putty-like viscosity to the material, such as hyaluronic acid, chitosan, starch components, such as amylopectin.

[0169] In some embodiments, iTR factors enhance the formation and / or replenishment of bone progenitor cells from undifferentiated embryoid cells, and / or enhance the differentiation of bone progenitor cells into cells that form new bone (osteoblasts).

[0170] In some embodiments, the iTR factor is administered to subjects with osteopenia or osteoporosis, for example, to enhance bone regeneration in the subject.

[0171] In some embodiments, iTR factors are used to enhance the regeneration of joints (e.g., fibrous, cartilaginous, or synovial joints). In some embodiments, the joint is an intervertebral disc. In some embodiments, the joint is a hip, knee, elbow, or shoulder joint. In some embodiments, iTR factors are used to enhance the regeneration of the dental system and / or periodontal tissues or structures (e.g., dental pulp, periodontal ligaments, teeth, periodontal bone).

[0172] In some embodiments, the iTR factor is used to reduce glial scarring in CNS and PNS injuries.

[0173] In some embodiments, the iTR factor is used to reduce adhesions and stenosis formation in medical surgery.

[0174] In some embodiments, the iTR factor is used to reduce scarring and improve mobility in tendon and ligament repair.

[0175] In some embodiments, the iTR factor is used to reduce vision loss after eye injury.

[0176] In some embodiments, the iTR factor is administered to a subject in combination with cells. The iTR factor and cells may be administered separately or included in the same composition. When administered separately, they may be administered at the same site or at different sites. The cells may be autologous, allogeneic, or heterogeneic in various embodiments. The cells may include progenitor cells or stem cells, such as adult stem cells. As used herein, stem cells are cells having at least the following characteristics: (i) self-renewal, i.e., the ability to go through many cycles of cell division while maintaining an undifferentiated state; and (ii) pluripotency or multipotency, i.e., the ability to produce offspring of several different cell types (e.g., many, most, or all of different cell types of a particular tissue or organ). Adult stem cells are stem cells derived from non-embryonic tissue (e.g., fetal tissue, postnatal tissue, or adult tissue). As used herein, the term “progenitor cell” includes pluripotent cells, which are more differentiated than pluripotent stem cells but not fully differentiated. Such more differentiated cells (which may arise from embryonic progenitor cells) have reduced self-renewal capacity compared to embryonic progenitor cells. In some embodiments, iTR factor is administered in combination with mesenchymal progenitor cells, neural progenitor cells, endothelial progenitor cells, hair follicle progenitor cells, neural corona progenitor cells, mammary gland stem cells, lung progenitor cells (e.g., bronchoalveolar stem cells), muscle progenitor cells (e.g., satellite cells), adipose-derived progenitor cells, epithelial progenitor cells (e.g., keratinocyte stem cells), and / or hematopoietic progenitor cells (e.g., hematopoietic stem cells). In some embodiments, the cells include induced pluripotent stem cells (iPS cells) or cells at least partially differentiated from iPS cells. In some embodiments, the progenitor cells include adult stem cells. In some embodiments, at least some of the cells are differentiated cells, e.g., chondrocytes, osteoblasts, keratinocytes, hepatocytes. In some embodiments, the cells include myoblasts.

[0177] In some embodiments, the iTR factor is administered in a composition (e.g., a solution) containing one or more compounds that polymerize, crosslink, or undergo a phase transition in situ after administration to a subject, typically forming a hydrogel. The composition may include monomers, polymers, initiators, crosslinkers, etc. The composition is applied (e.g., using a syringe) to a region where regeneration is needed, and a gel may form in situ from which the iTR factor can be released over time. Gelation may be induced, for example, by contact with ions in body fluids, by changes in temperature or pH, by light, or by combination with a reactive precursor (e.g., using a multi-barrel syringe). (See, for example, U.S. Patent No. 6,129,761; Yu L, Ding J. Injectable hydrogels as unique biomedical materials. Chem Soc Rev. 37(8):1473-81 (2008)). In some embodiments, the hydrogel is hyaluronic acid, or a hydrogel containing hyaluronic acid and collagen I, such as HyStem-C as described herein. In some embodiments, the composition further comprises cells.

[0178] In some embodiments, the iTR factor is administered to a subject in combination with a vector expressing a telomerase catalytic component. The vectors may be administered individually or included in the same composition. If administered individually, they may be administered at the same or different locations. The vectors may express telomerase catalytic components from the same species as the treated tissue or from a different species. The co-administration of the iTR factor with the telomerase catalytic component is particularly useful when the target tissue is from an aging individual, and the individual is of human origin.

[0179] Other methods of the present invention involve the use of iTR factors in the ex vivo production of compositions containing viable functional tissues, organs, or cells for repairing or replacing tissues or organs lost due to damage. For example, cells or tissues taken from an individual (either a future recipient, an individual of the same species, or an individual of a different species) are cultured in vitro with optionally a matrix, a scaffold (e.g., a three-dimensional scaffold), or a mold (e.g., a biocompatible, optionally biodegradable material, including polymers such as HyStem-C), and their development into functional tissues or organs can be promoted by contact with iTR factors. The scaffold, matrix, or mold may be at least partially composed of naturally occurring proteins, such as collagen, hyaluronic acid, or alginates (or chemically modified derivatives thereof), or synthetic polymers or copolymers such as lactic acid, caprolactone, or glycolic acid, or self-assembling peptides, or decellularized matrices derived from tissues such as heart valves, intestinal mucosa, blood vessels, and trachea. In some embodiments, the scaffold comprises a hydrogel. In certain embodiments, the scaffold may be coated or impregnated with iTR factors that may diffuse from the scaffold over time. After being produced ex vivo, the tissue or organ is transplanted into or onto a subject. For example, the tissue or organ can be transplanted and, in the case of certain tissues, such as skin, can be placed on the body surface. The tissue or organ can continue to develop in vivo. In some embodiments, the tissue or organ produced at least partially ex vivo is the bladder, blood vessels, bone, fascia, liver, muscle, skin patch, etc. A suitable scaffold can, for example, mimic the extracellular matrix (ECM). Optionally, the iTR factors are administered to the subject before, during, and / or after transplantation of the tissue or organ produced ex vivo.In some embodiments, biocompatible materials are substantially nontoxic to cells in vitro at the concentrations used, or, in the case of materials administered to living subjects, substantially nontoxic to the cells of the subject at the amounts and locations used, and do not induce or cause significant adverse or undesirable effects on the subject, such as immune or inflammatory responses, or the formation of unacceptable scar tissue. It will be understood that certain biocompatible materials may induce such adverse reactions in low percentages of subjects, typically less than about 5%, 1%, 0.5%, or 0.1%.

[0180] In some embodiments, a matrix or scaffold coated or impregnated with iTR factors, or a combination of factors that may induce a global pattern of iTR gene expression, is optionally combined with cells and transplanted into a subject requiring regeneration. The matrix or scaffold may be in the shape of the tissue or organ to which regeneration is desired. The cells may be one or more types of stem cells that give rise to such tissue or organ and / or types identified in such tissue or organ.

[0181] In some embodiments, the iTR factor or combination of factors is administered directly to or near the site of tissue injury. “Directly to the site of tissue injury” includes injecting, applying, or infusing the compound or composition to the site of tissue injury, or bringing the site of tissue injury into direct contact with the compound or composition. In some embodiments, administration is considered “near the site of tissue injury” if it is performed within approximately 10 cm of the visible or other obvious edge of the site of tissue injury, or in a blood vessel (e.g., an artery) located in at least part of the damaged tissue or organ. “Near the site of tissue injury” administration sometimes refers to administration within the damaged organ but where the injury is not obvious. In some embodiments, after injury or loss of tissue, organ, or other structure, the iTR factor is applied to the remaining tissue, organ, or other structure. In some embodiments, the iTR factor is applied to the end of a severed finger or limb that remains attached to the body to enhance regeneration of the lost portion. In some embodiments, the severed portion is surgically reattached, and the iTR factor is applied to either or both sides of the wound. In some embodiments, iTR factors are administered to enhance the engraftment, healing, or regeneration of a transplanted organ or part thereof. In some embodiments, iTR factors are used to enhance nerve regeneration. For example, iTR factors can be injected into a severed nerve, for example, near the proximal and / or distal stump. In some embodiments, iTR factors are placed within an artificial neural tube, a tube composed of a biological or synthetic material that encloses the nerve terminal and intervening gap. One or more factors can be formulated into a matrix to promote their controlled release over time. The matrix may include biocompatible and optionally biodegradable materials, such as cross-linked hyaluronic acid, or polymers containing hyaluronic acid cross-linked with PEGDA carboxymethyl hyaluronic acid, or a mixture of PEGDA-cross-linked carboxymethyl hyaluronic acid and carboxymethyl-modified gelatin (HyStem-C).

[0182] In some embodiments, the iTR factor is an anti-Müllerian hormone (AMH), which may or may not be formulated to be localized and slowly released in carboxymethyl hyaluronic acid crosslinked with PEGDA containing carboxymethyl-modified gelatin (HyStem-C) at a concentration in the range of 0.05–5 mM valproic acid, preferably 1–100 ng / mL, preferably 10 ng / mL, sufficient to expose cells in vitro or in vivo to induce iTR.

[0183] In some embodiments, the iTR factor is a secreted, short or long form of GFER (Augmenter of Liver Regeneration (ALR)) localized in the mitochondrial intermembrane space and expressed at relatively high levels in embryonic tissue, and may or may not be formulated to be localized and slowly released in carboxymethyl hyaluronic acid crosslinked with PEGDA containing carboxymethyl-modified gelatin (HyStem-C) at concentrations typically in the range of 2 to 200 ng / mL, preferably 20 ng / mL, sufficient to expose in vitro cells or in vivo cells in tissue to induce iTR.

[0184] In some embodiments, the iTR factor is valproic acid, which may or may not be formulated to be localized and slowly released in carboxymethyl hyaluronic acid crosslinked with PEGDA containing carboxymethyl-modified gelatin (HyStem-C) at concentrations typically in the range of 0.05–5 mM, preferably 0.5 mM, sufficient to expose in vitro cells or cells in tissue in vivo to induce iTR.

[0185] In some embodiments, the iTR factor is any combination of valproic acid at a concentration of 0.05–5 mM, preferably 0.5 mM, GFER protein (either long or short form) at a concentration of 2–200 ng / mL, preferably 20 ng / mL, and AMH protein at a concentration of 1–100 ng / mL, preferably 10 ng / mL. The aforementioned combination of factors valproic acid, GFER, and AMH may or may not be formulated for localization and sustained release in carboxymethyl hyaluronate crosslinked by PEGDA containing carboxymethyl-modified gelatin (HyStem-C) that induces iTR.

[0186] iTM factor and iCM factor, for example, exosomes derived from fetal or adult cells, can be administered in a physiological solution, such as physiological saline, or can be sustained-released in carboxymethyl hyaluronic acid crosslinked with PEGDA containing carboxymethyl-modified gelatin (HyStem-C) to induce iTM or iCM.

[0187] In some embodiments, the gene LIN28B, which is primarily expressed during the embryonic development stage, is exogenously expressed in blood cell types, including CD34+ hematopoietic cells, and promotes their proliferation and engraftment in bone marrow in vivo, comparable to the proliferative and engraftment capabilities of their fetal liver-derived counterparts.

[0188] In some embodiments, tissue regeneration is enhanced by the administration of platelet-rich plasma-derived factors, including but not limited to prolotherapeutic agents containing hyperosmolar dextrose, glycerin, lidocaine, phenol, local anesthetic phenol, and morpholite; sclerotherapy agents, including but not limited to those used to treat vascular and lymphatic malformations (vascular malformations) including Klippel-Trenaunay syndrome, spider veins, varicose veins, hemorrhoids, and hydrocells, wherein the agent used includes drugs such as tetradecyl sulfate sodium or polidocanol, and the sclerosing agent is injected into the blood vessels; and platelet-rich plasma-derived factors. Here, the prolotherapeutic factors, sclerotherapy factors, or platelet-rich plasma-derived factors are formulated in a matrix to localize their effects or to promote their controlled release over time. The matrix may contain biocompatible and optionally biodegradable materials, such as cross-linked hyaluronic acid, or polymers containing hyaluronic acid including PEGDA-crosslinked carboxymethyl hyaluronic acid, or a mixture of PEGDA-crosslinked carboxymethyl hyaluronic acid and carboxymethyl-modified gelatin (HyStem-C).

[0189] In some embodiments, iTR factors or combinations of factors are used to promote hair follicle generation and / or hair growth. In some embodiments, iTR factors induce the regeneration of hair follicles from epithelial cells that do not normally form hair. In some embodiments, iTR factors are used to treat hair loss, thinning hair, partial or complete alopecia in men or women. In some embodiments, alopecia is a condition in which hair does not grow, such as on the top, back and / or sides of the head, is absent, essentially hairless, or sparse. In some embodiments, thinning hair is a condition in which there is less hair than normal or average, or in some embodiments, less hair than the individual has had in the past, or in some embodiments, less hair than the individual would like. In some embodiments, iTR factors are used to promote eyebrow or eyelash growth. In some embodiments, iTR factors are used to treat androgenic alopecia or "male pattern baldness" (which may affect men and women). In some embodiments, iTR factors are used to treat alopecia areata with patchy hair loss of the scalp, alopecia with total hair loss, or alopecia generalis with loss of all hair from the head and body. In some embodiments, iTR factors are applied to areas where hair growth is desired, such as the scalp or eyebrow area. In some embodiments, iTR factors are applied to or near the eyelid margin to promote eyelash growth. In some embodiments, iTR factors are applied in liquid formulations. In some embodiments, iTR factors are applied in creams, ointments, pastes, or gels. In some embodiments, iTR factors are used to enhance hair growth after burns, surgery, chemotherapy, or other events that cause hair or hairy skin loss.

[0190] In some embodiments, iTR factors or combinations of factors are administered to tissues affected by age-related degenerative changes to regenerate youthful function. Examples of such age-related degenerative changes include, but are not limited to, age-related macular degeneration, coronary artery disease, osteoporosis, osteonecrosis, heart failure, emphysema, peripheral artery disease, vocal cord atrophy, hearing loss, Alzheimer's disease, Parkinson's disease, skin ulcers, and other age-related degenerative diseases. In some embodiments, the iTR factors are administered co-administered with a vector expressing a telomerase catalytic component to extend cell lifespan.

[0191] In some embodiments, one or more iTR factors are administered to enhance the replacement of cells lost or damaged by the cause of injury, such as chemotherapy, radiation, or toxins. In some embodiments, such cells are stromal cells of parenchymal organs and tissues.

[0192] The therapeutic method of the present invention may include the step of identifying or providing a subject that is suffering from, or at risk of suffering from, a disease or condition in which enhanced regeneration would be beneficial to the subject. In some embodiments, the subject has suffered injury (e.g., physical trauma) or damage to a tissue or organ. In some embodiments, the injury is to a limb or finger. In some embodiments, the subject has suffered from a disease affecting the cardiovascular system, digestive system, endocrine system, musculoskeletal system, gastrointestinal system, hepatic system, cutaneous system, nervous system, respiratory system, or urinary system. In some embodiments, tissue damage is to tissues, organs, or structures, such as cartilage, bone, heart, blood vessels, esophagus, stomach, liver, gallbladder, pancreas, intestine, rectum, anus, endocrine glands, skin, hair follicles, teeth, gums, lips, nose, mouth, thymus, spleen, skeletal muscle, smooth muscle, joints, brain, spinal cord, peripheral nerves, ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, prostate gland, penis, pharynx, larynx, trachea, bronchi, lungs, kidneys, ureters, bladder, urethra, eye (e.g., retina, cornea), or ear (e.g., organ of Corti).

[0193] In some embodiments, the compound or composition is administered to the subject at least once within about 2, 4, 8, 12, 24, 48, 72, or 96 hours after the subject suffers tissue injury (e.g., an injury and an event related to an acute disease, e.g., myocardial infarction or stroke), and optionally at least once thereafter. In some embodiments, the compound or composition is administered to the subject at least once within about 1 to 2 weeks, 2 to 6 weeks, or 6 to 12 weeks after the subject suffers tissue injury, and optionally at least once thereafter.

[0194] In some embodiments of the present invention, it may be useful to stimulate or promote the regeneration or de novo development of a missing or malformed tissue, organ, or structure by, for example, removing skin, removing at least a portion of tissue at a site where regeneration or de novo development is desired, scraping a joint or bone surface where regeneration or de novo development is desired, and / or inflicting another type of wound on the subject. In the case of regeneration after tissue damage, it may be desirable to remove at least a portion of the damaged tissue (for example, by surgical excision or debridement). In some embodiments, the iTR factor is administered to or near the site of such removal or scraping.

[0195] In some embodiments, iTR factors are used to enhance the generation of tissue or an organ in a subject in which such tissue or organ is at least partially defective as a result of a congenital disease, such as a genetic disease. Many congenital malformations result in hypoplasia or absence of various tissues, organs or body structures, such as limbs or digits. In other instances, developmental disorders that result in aplasia of a tissue, organ or other body structure become apparent after birth. In some embodiments, the iTR factors are administered to a subject afflicted with hypoplasia or absence of a tissue, organ or other body structure in order to stimulate the growth or development of the tissue, organ or other body structure. In some aspects, the present invention provides a method of enhancing the generation of tissue, an organ or other body structure in a subject afflicted with hypoplasia or congenital absence of a tissue, an organ or other body structure, comprising administering an iTR factor to the subject. In some embodiments, the iTR factor is administered to the subject prenatally, i.e., in utero. The various aspects and embodiments of the invention described herein with respect to regeneration are applicable to such de novo generation of tissue, an organ or other body structure and are encompassed by the present invention.

[0196] In some aspects, iTR factors are used to enhance the generation of tissue in any of a variety of situations where new tissue growth is useful in a location where such tissue did not previously exist. For example, generating bone tissue between joints is often useful in the fusion of vertebrae or other joints.

[0197] The iTR factors can be tested in various animal models of regeneration. In one aspect, modulators of iTR are tested in the mouse species. For example, a mouse can be wounded (e.g., by incision, cutting, severance or removal of tissue fragments). The iTR factor is applied to the site of the wound and / or the removed tissue fragment, and its effect on regeneration is evaluated. The effect of modulators of vertebrate TR can be tested in various vertebrate models for regenerating tissue or organs. For example, fin regeneration can be evaluated in zebrafish as described, for example, in (Mathew L K, Unraveling tissue regeneration pathways using chemical genetics. J Biol Chem. 282(48):35202-10 (2007)) and can be used as a model for limb regeneration. Rodent, dog, horse, goat, fish, amphibian and other animal models useful for testing the therapeutic effect on the regeneration of tissues and organs such as heart, lung, limb, skeletal muscle, bone, etc. are widely available. For example, various animal models for musculoskeletal regeneration are discussed in Tissue Eng Part B Rev. 16(1) (2010). Animal models commonly used to test liver regeneration include surgical removal of most of the rodent liver. Other models for liver regeneration include acute or chronic liver injury or liver failure induced by toxins such as carbon tetrachloride. In some embodiments, models of hair regeneration or healing of skin wounds include excising a patch of skin, for example, from a mouse. Regeneration of hair follicles, hair growth, re-epithelialization, gland formation, etc. can be evaluated.

[0198] Compounds and compositions disclosed herein, and / or identified using the methods and / or assay systems described herein, can be administered by any suitable means, e.g., orally, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, parenterally, intraperitoneally, intrathecally, intratracheally, intraocularly, sublingually, vaginally, rectally, percutaneously, or by inhalation, e.g., as an aerosol. The specific mode of administration selected naturally depends on the specific compound selected, the specific condition to be treated, and the dosage required for therapeutic efficacy. The methods of the present invention can be carried out using any medically or veterinarily acceptable mode of administration, meaning any mode that produces an acceptable level of efficacy without causing clinically unacceptable (e.g., medically or veterinarily unacceptable) side effects. Suitable preparations of one or more compounds, e.g., substantially pure preparations, can be combined with one or more pharmaceutically acceptable carriers or excipients, etc., to produce suitable pharmaceutical compositions suitable for administration to a subject. Such pharmaceutically acceptable compositions are one aspect of the present invention. The term “pharmaceutically acceptable carrier or excipient” means a carrier (this term encompasses carriers, media, diluents, solvents, vehicles, etc.) or excipient that does not significantly interfere with the biological activity or efficacy of the active ingredient(s) of the composition and is not excessively toxic to the host at the concentrations used or administered. Other pharmaceutically acceptable ingredients may also be present in the composition. Their use for the formulation of suitable substances and pharmaceutically active compounds is well known in the art (see, for example, “Remington's Pharmaceutical Sciences”, EW Martin, 19th Ed., 1995, Mack Publishing Co.: Easton, Pa., and for further discussion of pharmaceutically acceptable substances and methods for preparing various types of pharmaceutical compositions, see newer editions or versions thereof, e.g., Remington: The Science and Practice of Pharmacy. 21st Edition. Philadelphia, Pa. Lippincott Williams & Wilkins, 2005).Furthermore, the compounds and compositions of the present invention can be used in combination with any compounds or compositions used in the art to treat a specific disease or condition of interest.

[0199] In some embodiments, LIN28B is exogenously expressed in blood cell types, including CD34+ hematopoietic cells, and promotes their proliferation and engraftment in bone marrow in vivo, comparable to the proliferation and engraftment capacity of their fetal liver-derived counterparts.

[0200] Pharmaceutical compositions are typically formulated to suit the intended route of administration. For example, formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions containing physiological saline and buffering media, such as sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's dextrose. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate, fixing oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; preservatives, such as antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffering agents, such as acetates, citrates, or phosphoric acid; and tonicity adjusters, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Such parenteral formulations can be enclosed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0201] With regard to oral administration, the compounds can be readily formulated by combining the active compound with a pharmaceutically acceptable carrier well known in the art. With such carriers, the compounds of the present invention can be formulated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc. Suitable excipients for oral dosage forms include, for example, volume extenders, such as sugars containing lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP).

[0202] With regard to administration by inhalation, the compositions of the present invention can be delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant, such as carbon dioxide or fluorocarbon gas. Liquids or dry aerosols (e.g., dry powder, porous coarse particles, etc.) can be used. The present invention is also intended for the delivery of compositions using nasal sprays or other forms of intranasal administration.

[0203] With regard to topical application, the pharmaceutical composition can be formulated into a suitable ointment, lotion, gel, or cream containing an active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers suitable for use in such a composition.

[0204] With regard to local delivery to the eye, pharmaceutically acceptable compositions can be formulated, for example, as a solution or fine powder suspension in pH-adjusted isotonic sterile saline for use in eye drops or ointments, or for intraocular administration by injection.

[0205] The pharmaceutical composition may be formulated for transmucosal or transdermal delivery. For transmucosal or transdermal administration, a suitable penetrating agent for the barrier to be penetrated can be used in the formulation. Such penetrating agents are generally known in the art. The pharmaceutical composition of the present invention may be formulated as a suppository (e.g., using a conventional suppository base, e.g., cocoa butter and other glycerides) or as a retaining enema for rectal delivery.

[0206] In some embodiments, the composition includes one or more agents, such as controlled-release formulations, implants, or microencapsulated delivery systems, intended to protect the active agent from rapid elimination from the body. The composition may incorporate agents that improve stability (e.g., in the gastrointestinal tract or bloodstream) and / or enhance absorption. The compounds may be encapsulated or incorporated into particles, such as microparticles or nanoparticles. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, PLGA, collagen, polyorthoesters, polyethers, and polylactic acid, can be used. Methods for preparing such formulations will be obvious to those skilled in the art. For example, but not limited to, many particle, lipid, and / or polymer-based delivery systems are known in the art for the delivery of siRNA. The present invention intends for the use of such compositions. Liposomes or other lipid-based particles can also be used as pharmaceutically acceptable carriers.

[0207] Pharmaceutical compositions and compounds for use in such compositions may be manufactured under conditions that meet standards, criteria, or guidelines set forth by regulatory bodies. For example, such compositions and compounds may be manufactured in accordance with Good Manufacturing Practices (GMP) and / or subjected to quality control procedures suitable for pharmaceuticals administered to humans, and may be supplied with labels approved by government regulatory bodies responsible for regulating pharmaceuticals, surgical procedures, or other therapeutically useful products.

[0208] When the pharmaceutical compositions of the present invention are administered to a subject for therapeutic purposes, they are preferably administered for a sufficient time and in an amount to treat the disease or condition to which they are administered. The therapeutic efficacy and toxicity of the activators can be evaluated by standard pharmaceutical procedures in cell culture or experimental animals. Data obtained from cell culture assays and animal studies can be used to determine a range of appropriate dosages for use in human or other subjects. Different dosages for human administration can be further tested in human clinical trials known in the art. The dosage used may be the maximum tolerable dose or a lower dose. The therapeutically effective amount of the activator in the pharmaceutical composition may be in the range of about 0.001 mg / kg to about 100 mg / kg body weight, about 0.01 to about 25 mg / kg body weight, about 0.1 to about 20 mg / kg body weight, or about 1 to about 10 mg / kg body weight. Other exemplary dosages include, for example, about 1 μg / kg to about 500 mg / kg and about 100 μg / kg to about 5 mg / kg. In some embodiments, a single dose is administered, and in other embodiments, multiple doses are administered. Those skilled in the art will understand that the appropriate dosage in any particular situation depends on the potency of the drug(s) used and can be arbitrarily adjusted to a specific recipient. The specific dosage level for a subject may depend on various factors, including the activity of the specific drug(s) used, the subject's specific disease or condition and its severity, age, weight, and general health status. To facilitate administration and ensure uniformity of dosage, it may be desirable to formulate pharmaceutical compositions, particularly oral or parenteral compositions, into unit dosage forms. Where the term is used herein, a unit dosage form means a physically distinct unit suitable as a unit dose for the subject to be treated. Each unit contains a predetermined amount of activator(s) calculated to produce the desired therapeutic effect, along with a suitable pharmaceutically acceptable carrier. It will be understood that a treatment regimen may include multiple doses, such as unit dosage forms, over a period that can be extended over days, weeks, months, or years. The subject may receive doses once or more times daily, or every other day or less frequently during the treatment period. For example, administration may be every other week, every week, etc.Administration may be continued, for example, until the proper structure and / or function of the tissue or organ is at least partially restored, and / or until continued administration of the compound no longer appears to promote further regeneration or improvement. In some embodiments, the subject administers one or more doses of the composition of the present invention to itself.

[0209] In some embodiments, two or more compounds or compositions are administered in combination, for example, to enhance regeneration. The compounds or compositions administered in combination may be administered together in the same composition or individually. In some embodiments, “combined” administration means, with respect to the administration of the first and second compounds or compositions, (i) the dose of the second compound is administered before more than 90% of the most recently administered dose of the first agent has been metabolized to an inactive form or eliminated from the body, or (ii) the doses of the first and second compounds are administered within 48, 72, 96, 120, or 168 hours of each other, or (iii) the agents are administered during overlapping periods (e.g., by continuous or intermittent infusions), or (iv) any combination of the above is performed. In some embodiments, two or more iTR factors, or telomerase catalytic components and vectors expressing iTR factors are administered. In some embodiments, iTR factors are administered in combination with one or more growth factors, growth factor receptor ligands (e.g., agonists), hormones (e.g., steroid hormones or peptide hormones), or signaling molecules useful for promoting regeneration and polarity. Particularly useful are organizing center molecules useful for organizing regenerating competent cells, such as those produced using the methods of the present invention. In some embodiments, the growth factors are epidermal growth factor family members (e.g., EGF, neuregulin), fibroblast growth factor (e.g., any of FGF1-FGF23), hepatocyte growth factor (HGF), nerve growth factor, bone morphogenetic protein (e.g., any of BMP1-BMP7), vascular endothelial growth factor (VEGF), wnt ligands, wnt antagonists, retinoic acid, NOTUM, follistatin, Sonic Hedgehog, or other organizing center factors.

[0210] Those skilled in the art will recognize, or can confirm by routine experimentation alone, many equivalents to the specific embodiments of the Invention described herein. The scope of the Invention is not intended to be limited to this Specified Embodiment or the Details Described Therein. Articles such as “a,” “an,” and “the” can mean one or more unless the context makes it clear that they do not mean the opposite or not. Certain methods of the Invention are often carried out using populations of cells, for example, in vitro or in vivo. Thus, references to “cells” should be understood to include embodiments in which cells are members of a population of cells, e.g., a population of substantially genetically identical cells. However, the Invention also encompasses embodiments in which the Method of the Invention is applied to individual cells. Thus, references to “cells” should be understood to include embodiments applicable to individual cells within a population of cells, and embodiments applicable to individual isolated cells.

[0211] Claims or descriptions containing "or" between one or more members of a group are considered satisfied when one, more, or all members of a group are present, used, or related to a given product or process, unless the context expressly indicates otherwise. The present invention includes embodiments in which exactly one member of a group is present, used, or related to a given product or process. The present invention also includes embodiments in which more or all members of a group are present, used, or related to a given product or process. All embodiments described herein are considered applicable to all different aspects of the present invention. Furthermore, any embodiment may be freely combined with one or more other such embodiments where appropriate. In addition, the present invention is understood to encompass all variations, combinations, and substitutions in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more claims (whether original claims or subsequently added claims) are introduced into another claim (whether original claims or subsequently added claims). For example, any claim dependent on another claim may be modified to include one or more elements or limitations confirmed in any other claim dependent on the same basic claim, and any claim referring to an element present in a different claim may be modified to include one or more elements or limitations confirmed in any other claim dependent on the same basic claim as that claim. Furthermore, where a claim details a composition, the present invention provides, for example, a method for producing the composition according to the method disclosed herein, and for example, a method for using the composition for the purposes disclosed herein. Where a claim details a method, the present invention provides a composition suitable for carrying out the method, and a method for producing the composition. Also, where a claim details a method for producing a composition, the present invention provides, unless otherwise indicated, or unless a person skilled in the art would recognize that such a method would result in a contradiction or inconsistency, a composition produced according to the method of the present invention, and a method for using the composition. Where elements are presented as a list, for example in Markush group format, each subgroup of the elements is also disclosed, and any element(s) may be excluded from a group.For the sake of brevity, only some of these embodiments are specifically described herein, but the present invention encompasses all such embodiments. Furthermore, where the present invention or an aspect of the present invention is generally referred to as including certain elements, features, etc., it should be understood that a particular embodiment or aspect of the present invention consists of, or substantially comprises, such elements, features, etc.

[0212] Where numerical ranges are referred to herein, the present invention includes embodiments that include endpoints, embodiments that exclude both endpoints, and embodiments that include one endpoint and exclude the other. Both endpoints are to be considered included unless otherwise indicated. Furthermore, unless otherwise indicated, or unless evident from the context and the understanding of those skilled in the art, values ​​indicated as a range may take any specific value or subrange within the range described in different embodiments of the present invention, and unless the context explicitly indicates otherwise, it shall mean one-tenth of the lower limit unit of that range. Where phrases such as “less than X,” “greater than X,” or “at least X” are used (where X is a number or a percentage), it should be understood that any suitable value may be selected as the lower or upper limit of the range. Also, where a list of numerical values ​​is provided (whether or not it begins with “at least”), it should be understood that the present invention includes embodiments relating to any intervening value or range defined by any two values ​​in the list, and that the lower limit may be the minimum value and the upper limit may be the maximum value. Furthermore, if a list of numbers, such as percentages, begins with “at least,” the term applies to each number in the list. In any embodiment of the Invention where a number begins with “about” or “approximately,” the Invention includes embodiments in which exact values ​​are enumerated. In any embodiment of the Invention where a number does not begin with “about” or “approximately,” the Invention includes embodiments in which the value begins with “about” or “approximately.” “Approximately” or “approximately” generally means a number that is within the range of 1% of a number in either direction (greater than or less than that number), or in some embodiments 5%, or in some embodiments 10%, unless otherwise stated or evident from the context (for example, if such a number exceeds the possible value of 100% to an unacceptable degree). As used herein, “composition” may consist of or essentially consist of activators of TR activators, or may contain one or more further components.Unless otherwise indicated, the inhibitor or TR inhibitor (or other compounds referred to herein) in any embodiment of the present invention may be used or administered in a composition comprising one or more further components, including the presence of a TR activator.

[0213] Origin of iTM factor and iCM factor iTM and iCM factors can be identified by exposing germ cells lacking EFT markers (e.g., stromal cells not expressing COX7A1, as an unspecified example) to various drugs and assaying the induction of GFP expression using the marker, e.g., COX7A1, or a reporter construct, e.g., the COX7A1 gene promoter.

[0214] Since exosomes possess potent protein and RNA factors that can reprogram cells to confer new growth, migration, and differentiation characteristics, the inventors tested whether they can reprogram the growth states of cells, i.e., iTM and iCM. Accordingly, the inventors tested adult cell-derived exosomes for induction of adult genes in embryonic cells. The inventors evaluated the total RNA expression profiles of a series of 15 hESC-derived clonal embryonic progenitor cell lines using Illumina microarray analysis and compared them with 18 primary endothelial cell lines (from neonatal to adult) obtained from various dissection sites (not shown). The inventors determined that exosomes derived from cells that have passed through EFT can induce COX7A1 expression in embryonic cells that previously lacked such expression, and that they can mature cells using other markers described herein.

[0215] (Examples) [Example 1] DNN training using online microarray data and data from cultured clonal embryonic progenitor cell lines. The inventors used data from the publicly available databases Gene Expression Omnibus (GEO) and ArrayExpress, as well as additional datasets provided by BioTime, Inc. Each collected sample belonged to one of the following broad cell classes: embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic progenitor cells (EPCs), adult stem cells (ASCs), and adult cells (ACs). The samples used in this example were obtained from the following microarray platforms: Illumina HumanHT-12 V4.0 (GPL10558), Illumina HumanHT-12 V3.0 (GPL6947), Affymetrix HT Human Genome U133A Array (GPL3921), Affymetrix GeneChip Human Genome U133 Array Set HG-U133A (GPL4557), Affymetrix Human Exon 1.0 ST Array (GPL5188), Affymetrix Human Genome U133 Plus 2.0 Array (GPL570), Affymetrix Human Genome U133A 2.0 Array (GPL571), and Affymetrix Human Gene 1.0 ST array (GPL6244), Affymetrix Human Genome U133A array (GPL96), Affymetrix Human Genome U133 Plus 2.0 array (GPL11670). When working with processed data, no distinction was made between platforms from the same vendor. The selection of cell classes was motivated by the need to obtain the most diverse stages of stem cell development. Adult cells included cells derived from the following tissues: kidney, liver, muscle, blood, and nerve tissue. Adult stem cells included adipose-derived stem cells, epithelial stem cells, hematopoietic stem cells, mesenchymal stem cells, and neural stem cells.

[0216] The inventors collected and preprocessed the transcriptional profiles of 12,404 healthy untreated tissue samples from Affymetrix (4,822 samples) and Illumina (7,582 samples) microarray platforms. The samples collected were assigned to five categories: embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), embryonic progenitor cells (EPC), adult stem cells (ASC), and adult cells (AC). The population of samples collected was substantially homogeneous.

[0217] For each microarray platform, the inventors then separately trained six different classifiers: k-nearest neighbors (kNN), logistic regression (LR) with PCA-based dimensionality reduction, support vector machine (SVM), gradient boosting machine (GBM), multi-class deep neural network (DNN), and an ensemble of 20 deep neural networks (DNN ens.). For all classifiers except the DNN ensemble, the inventors performed hyperparameter search, and for the DNN ensemble, the optimal network hyperparameters obtained for a single DNN were used. Since it was shown that there were many drawbacks to using a single multi-class DNN, the inventors also developed a more computationally intensive but more accurate method that uses an ensemble of two-class deep neural networks (Figure 1). The inventors trained 20 binary networks for each target platform set (Affymetrix and Illumina) to perform pairwise (one-versus-one) classification. The inventors then evaluated the overall ensemble voting for each class as the sum of four one-versus-one networks. This distinguished this class pairwise from the other four classes. This minimized the false positive voting for any class and achieved a smoother distribution of embryo scores. The classification performance is shown in Figure 2.

[0218] With Affymetrix microarray gene-level data, the deep neural network ensemble achieved an average F1 score of 0.99 on the training dataset and 0.75 on the validation dataset, while other methods achieved 0.50–0.64 F1 scores on the external validation dataset. For Illumina microarray gene-level data, the deep neural network achieved an average F1 score of 0.99 on the training dataset and 0.83 on the external validation dataset, while other methods achieved 0.52–0.58 F1 scores on the external validation dataset.

[0219] As is clear, classical methods such as kNN and LR perform significantly worse than SVM, XGB, and DNN methods. By using a DNN ensemble, substantially better performance was achieved (a relative improvement of approximately 12% for Affymetrix and a relative improvement of 36% for Illumina).

[0220] For pathway-level analysis, the inventors used an established pathway analysis method called OncoFinder, as described herein. This method preserves information about biological function and allows for dimensionality reduction. At the pathway level (Figures 3c and 3d), it is clear that the DNN ensemble performance was similar to that achieved at the gene level in the validation set, with F1 scores of 0.74 and 0.81 for the Affymetrix and Illumina platforms, respectively, despite the low accuracy of the training set.

[0221] The inventors would like to draw attention to the importance of using labeled cross-validation procedures. When samples from the same dataset are present in both the training and validation sets, the classifier's performance is significantly overestimated (validation performance above 0.9 for all methods, data not shown). This highlights the need for careful selection of cross-validation procedures to obtain an unbiased estimate of how much batch effects affect transcription data and how well the classifier's behavior is handled on new datasets.

[0222] Based on the output of a DNN ensemble, the inventors developed an integrated embryo score (ES). To investigate the performance of the score based on the DNN ensemble, the inventors used a transcriptional dataset consisting of samples belonging to different stages of neural differentiation. This dataset was profiled on the Affymetrix platform, allowing the inventors to observe a clear decrease in ES with each stage of differentiation (Figure 3d). With the Affymetrix-based DNN ensemble, the first significant decrease in ES score occurred among early (day 14) and mid-stage (day 35) radial glial cells, then decreased again, and plateaued at around ES ~0.65 in late radial glial cells (day 80) and long-term neural progenitor cells (day 220). On the other hand, the Illumina-based DNN ensemble was extremely sensitive, with ES decreasing immediately after the cells differentiated from human ESCs (H9 strain). As shown in Figure 3c, the genes COX7A1, PCDHB2, COMT, CAT, and ADIRF(c10orf116) were expressed differently in embryonic cell types versus adult cell types.

[0223] To validate genes as markers for mammalian EFT, the inventors tested a total of 83 discriminator genes, shown in Figure 4, using RNA-seq in a panel of 15 diverse adult-derived cell types representing endoderm, mesoderm, ectoderm, and neural crest inducers, as well as 17 clonal embryonic progenitor cell lines. Analysis using t-tests revealed 14 markers with FDR P<0.005: CAT, COMT, TRIM4, NAALADL1, MGMT, SPESP1, PLPP7, TSPYL5, PCDHB2, COX7A1, ZNF280D, DYNLT3, CYTH2, and PLEKHA1 (Figure 5). Of these 14 markers, 11 genes—CAT, COMT, TRIM4, NAALADL1, MGMT, SPESP1, PLPP7, TSPYL5, COX7A1, ZNF280D, and DYNLT3—showed increased expression in adult-derived cells, while 2 genes—PCDHB2, CYTH2, and PLEKHA1—showed increased expression in embryonic progenitor cells. LIN28A, already identified as a potential regulator of embryonic regeneration, was not differentially expressed, while LIN28B was expressed in a subset of embryonic progenitor cell lines, particularly those with vascular endothelial markers, but not in their adult counterparts.

[0224] To further investigate 13 genes as markers for EFT, expression levels were measured throughout the entire mouse embryo at embryonic stages spanning the mouse EFT (estimated to roughly correspond to Carnegie Stage 23 / Theiler Stage 24 or E16). As shown in Figure 6, the genes COX7A1, NAALADL1, and PLPP7 showed significant upregulation at stages close to the mouse EFT, while the expression of the gene LIN28B decreased during the same period.

[0225] To verify markers in human development, the inventors used early passaged dermal fibroblasts from the upper arm, all cultured under identical conditions, from the onset of fetal development (8 weeks of gestation). Data based on the Illumina gene expression bead array shown in Figure 7 indicated that the COX7A1, NAALADL1, and PLPP7 genes were again induced from 8 weeks of development, with COX7A1 being perhaps the most prominent marker, which appeared to gradually increase in expression throughout fetal and postnatal development and plateau in adulthood. LIN28B was expressed at the highest levels in ES cells, at low but detectable levels in embryonic progenitor cells and fibroblasts from early fetal development, but not in adult-derived cells. All patterns of EFT gene expression described in this invention were effectively reprogrammed from adult to embryonic gene expression patterns in senescent fibroblast-derived iPS cells. For example, as is evident in Figure 7, normal dermal fibroblasts derived from donors aged 60, 61, and 62 expressed relatively high levels of COX7A1, NAALADL1, and PLPP7 mRNA, but those transcriptionally reprogrammed to be pluripotent (labeled "iPS cells-60 Yr," "iPS cells-61 Yr," and "iPS cells-62 Yr" in Figure 7) expressed embryonic (pre-fetal or prenatal) patterns. Reprogramming to iPS cells was carried out as described herein. Briefly, adult-derived COX7A1, NAALADL1, and PLPP7-expressing human fibroblasts were plated onto a Matrigel-coated 6-well plate and reprogrammed to be pluripotent by transfecting mRNA with OCT4 (POU5F1), SOX2, KLF4, MYC, and LIN28 (day 0). From days 1 to 12, cells were re-transfected with the OCT4(POU5F1), SOX2, KLF4, MYC, and LIN28 cocktail. From days 12 to 14, reprogramming to pluripotency was confirmed using live-stained Tra-1-60 antibody, and iPS cell colonies were selected.

[0226] Next, the inventors investigated gene expression in three types of sarcomas (osteosarcoma, liposarcoma, and rhabdomyosarcoma) (see Figure 8). Embryonic progenitor cells capable of osteochondral differentiation, such as the 4D20.8 cell line, showed no evidence of COX7A1, NAALADL1, or PLPP7 expression in either the progenitor or differentiated state, despite expressing high levels of osteochondral markers. Conversely, adult-derived MSCs expressed COX7A1, NAALADL1, and PLPP7 before and after differentiation. In osteosarcoma, the cell line generally showed an embryonic pattern of gene expression. For example, the 4 / 5 osteosarcoma cell line showed little to no detectable COX7A1 transcripts. Similarly, the adipogenic embryonic progenitor cell line named E3, despite expressing robust markers of adipogenesis, did not induce COX7A1, NAALADL1, or PLPP7, whereas adult-derived subcutaneous adipose tissue (SAT) adipocyte progenitor cells expressed COX7A1, NAALADL1, and PLPP7 in both relatively undifferentiated and fully differentiated adipocytes. However, in the two liposarcoma cell lines tested, the markers appeared to reflect the embryonic pattern of gene expression; for example, both liposarcoma lines either did not express the COX7A1 transcript at all or expressed it at very low levels. Finally, five rhabdomyosarcoma cell lines were similarly tested compared to an embryonic myoblast progenitor cell line named SK5 and adult-derived myoblasts. COX7A1, which has already been described as being highly expressed in skeletal muscle cells and cardiomyocytes, was expressed at high levels in adult-derived cells but not in the embryonic progenitor cell line SK5. It was either not expressed or expressed at low levels in 4 / 5 of the rhabdomyosarcoma cell lines, while LIN28B was expressed at relatively high levels in 3 / 5 of those lines.

[0227] As further evidence for the validation of the genes COX7A1, NAALADL1, and PLPP7 as markers of embryonic transition, and the gene LIN28B as a marker of embryonic state, three gene pairs (i.e., LIN28B vs. COX7A1, NAALADL1, or PLPP7) were identified with strong inverse agreement in various sarcoma cell lines. That is, if one gene was expressed, the other gene was not expressed, or neither gene was expressed. Expression was defined as XYZ greater than 100XYZ. The gene pairs are shown in Figure 9, with the shaded areas highlighting the strong inverse agreement of the genes. The inverse agreement rate between LIN28B and COX7A1 was 83.3% (95% CI: 66.4–92.7), between LIN28B and NAALAD1 was 100% (95% CI: 88.6–100), and between LIN28B and PLPP7 was 73.3% (95% CI: 55.6–85.8).

[0228] [Example 2] Use of embryo marker PCDHB2 and fetal-adult marker COX7A1 for screening of hormones that can induce iTM in hES cell-derived cloned EP cell lines. The clonal EP cell line named 4D20.8 was sequentially passaged in a relatively undifferentiated state, as described in West et al., Regen. Med. (2008) 3(3), 287-308, which is incorporated herein by reference. In the relatively undifferentiated precursor state, the 4D20.8 cell line expresses markers consistent with embryonic (pre-fetal) cells, such as PCDHB2, at relatively high levels, but does not show COX7A1 levels similar to those of the hES cells from which it originates. After sequential in vitro passage to P36 for longer than 8 weeks, no induction of COX7A1 expression was observed, and any PCDHB2 expression deficiency was minimal. Therefore, we concluded that sequential passage and a single time course alone are insufficient to mature EP cells for fetal transition. Similarly, when embryonic progenitor cell line 4D20.8 was differentiated under micromass conditions for 62 days in the presence of TGFb3 and BMP4, COX7A1 induction did not occur, and only a partial decrease in PCDHB2 expression was observed.

[0229] Therefore, the 4D20.8 strain is useful for screening factors that may promote iTM. 4D20.8 or other embryonic progenitor cells are exposed to hormonal factors including the following pool, and RNA is recovered from the cells after 2, 4, or 6 weeks to assay for induction of adult markers, e.g., COX7A1, or reduction of embryonic markers, e.g., PCDHB2. The hormonal factors screened are as follows: Pituitary gland pool: Thyroid-stimulating hormone (TSH) 1nM, adrenocorticotropic hormone (ACTH) 5nM, luteinizing hormone (LH) 100ng / ml, follicle-stimulating hormone (FSH) 10ng / ml, somatotropin / growth hormone (GH) 1ng / ml, prolactin (PRL) 50ng / ml, melanocyte-stimulating hormone (MSH) 1ng / ml, oxytocin 10nM, arginine 0.5mM, vasopressin 1uM. Hypothalamic pool: Tyrotrophin-releasing hormone (TRH) 1 μM, corticotrophin-releasing hormone (CRH) 100 nM, arginine vasopressin (AVP) 1 μM, gonadotropin-releasing hormone (GnRH) 1 μg / ml, growth hormone-releasing hormone (GHRH) 10 nM, somatostatin 1 nM, prolactin-releasing factor (PRF) 10 nM, dopamine 50 μM. Thyroid / parathyroid pool: T3 2nM, T4 10ng / ml, parathyroid hormone 50nM. Pancreatic pool: insulin 5 ng / ml, glucagon 5 ug / ml, somatostatin 1 nM, pancreatic polypeptide 2 ng / ml. Adrenal pool: Cortisol 10 nM, Aldosterone 2 ng / ml, Dehydroepiandrosterone 10 μM, Epinephrine 1 μM, Norepinephrine 1 μM. Gonadal pool: Testosterone 50 ng / ml, estrogen 5 nM, progesterone 100 nM. Placental pool: Chorionic gonadotropin 1 ng / ml, human chorionic somatotropin (hCS), human chorionic corticotropin (hCACTH), chorionic cytotropin (hCT). Pool 1+2+3 Pool 1+2+5 Pools 1+2+3+5.

[0230] [Example 3] Novel RNAs that are differentially expressed in embryonic cells versus adult cells when measured by RNA-seq. RNA sequencing was performed on the Illumina HiSeq4000 platform to a minimum depth of 25 million 100-base pair end reads. Data were processed using the Tuxedo suite protocol (Trapnell C. et al., Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks; Nat Protoc. 2012 Mar 1;7(3):562-78. doi: 10.1038 / nprot.2012.016.). Alignment data were visualized using the presented data and FPKM values ​​with Integrative Genomics Viewer (Robinson, J. et al., Integrative Genomics Viewer, Nature Biotechnology 29, 24-26(2011)). Novel embryo-specific and adult-specific markers are shown in Figure 10. As shown in Figure 11, growth factor AMH was expressed at extremely high levels in embryonic cells. As shown in Figure 12, the non-coding RNA LINC01021 was expressed at higher levels in embryonic progenitor cells compared to adult cells, and the RNA's proliferation decreased with progenitor cell differentiation. As shown in Figure 13, the transcript RGPD1 was generally detected at higher levels in embryos compared to adult cells, with the exception of adult hepatocytes expressing extremely high levels of RGPD1 transcript. As shown in Figure 14, the transcript from ZNF300P1 was generally expressed in adult cell types (but not in hepatocytes), but not in most embryonic progenitor cells. As shown in Figure 15, the transcript LINC00654 was generally expressed in adults, but not in embryonic progenitor cells. As shown in Figure 16, the transcript of PCDHGA12 was significantly expressed in adult-derived cells compared to embryonic progenitor cells. As with other genes described herein, increased embryonic patterns promoted iTR, and increased adult patterns promoted iTM and iCM.

[0231] [Example 4] Differential expression of clustered protocadherin genes in embryonic cells vs. adult cells, as measured by RNA-seq. RNA sequencing was performed on the Illumina HiSeq4000 platform to a minimum depth of 25 million 100-base pair end reads. Data were processed using the Tuxedo suite protocol (Trapnell C. et al., Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks; Nat Protoc. 2012 Mar 1;7(3):562-78. doi: 10.1038 / nprot.2012.016.). Alignment data were visualized using Integrative Genomics Viewer (Robinson, J. et al., Integrative Genomics Viewer, Nature Biotechnology 29, 24-26(2011)). As shown in Figure 17, numerous significant differences were observed in the readings from exons of clustered protocadherin loci between embryonic and adult cell types, with embryonic cells generally expressing more transcripts from α and β genes, and adult cells expressing more from γ cluster genes. As with other genes described herein, increased embryonic patterns promoted iTR, and increased adult patterns promoted iTM and iCM.

[0232] [Example 5] Screening of factors that can induce iTR using the marker of the present invention. Lung cancer (A549), breast cancer (MCF7), and prostate cancer (PC3) cell lines were exposed to RNAi targeting various factors, including biologically active small molecules, and specific genes, and the relative decrease in COX7A1 transcript levels was used as a marker for iTR. As shown in Figure 18, HSP90 inhibitors, such as radicicol and alvespimycin, reduce COX7A1 expression, as do RNAi targets HIF1A, curcumin, azacitidine (5-aza-2'-deoxycytidine), and Aurora B / C kinase inhibitors, such as GSK1070916 and MK-5108, and highly selective Aurora-A kinase inhibitors.

[0233] [Example 6] Screening of factors that can induce iCM using the marker of the present invention. Lung cancer (A549), breast cancer (MCF7), and prostate cancer (PC3) cell lines were exposed to a variety of factors, including biologically active small molecules, and RNAi targeting specific genes. The relative increase in COX7A1 transcript levels was used as a marker for iCM. As shown in Figures 19–20, numerous histone deacetylase inhibitors, including trichostatin-A, panobinostat, apicidine, and vidinostat, as well as BI 2536, a potent and selective inhibitor of polo-like kinase 1 reported to have tryptolide and antitumor activity, and wartmannin, a nonspecific covalent inhibitor of phosphoinositide 3-kinase, and dactinomycin, flucloxacillin, gefitinib, mitoxantrone, vitexin, daunorubicin, carbenoxolone, sulmazole, albocidib, SN-38, teniposide, kallicrin, staurosporine, and doxorubicin, increased COX7A1 expression in cancer cell lines. Therefore, these agents are therapeutically useful in treating cancer, particularly those cancers that exhibit embryonic patterns of gene expression, as determined using the markers described herein.

[0234] [Example 7] Screening for optimal conditions for iTR in differentiated fetal or adult-derived cells using iPS cell factors that do not restore pluripotency to cells. As shown in Example 1 above, transcriptional reprogramming of adult-derived differentiated cells to pluripotency, such as by using mRNA of the genes OCT4, SOX2, KLF4, MYC, and LIN28A, produces multiple patterns of gene expression changes associated with iTR as described in the present invention. Therefore, the cocktail itself can be used as an iTR agent to increase tissue regeneration in terms of degenerative diseases, not only in vitro as shown in Example 1, but also in vivo as described herein. However, long-term administration of such a cocktail of reprogramming factors in vivo may carry an inherent risk of the return of cells to pluripotency in vivo, followed by the appearance of teratomas, abnormal ectopic tissues, or malignant tumors. Therefore, it is necessary to identify a method to safely induce iTR patterns of gene expression in embryonic and adult-derived cells in vitro and in vivo without completely reprogramming cells to pluripotency. Such protocols provide a method for epigenetic reprogramming of fetal or adult-derived differentiated cells expressing markers of cells traversing the EFT, such as COX7A1, CAT, and NAALADL1, resulting in relatively high methylation of the promoters of the aforementioned genes, thereby restoring the cells to a pre-fetal (i.e., iTR) pattern of gene expression without the cells reverting to pluripotency or altering their final outcomes from the primary germ layers (i.e., mesoderm, ectoderm, endoderm, neural crest) before exogenous drug administration. Ideally, such screening would further identify downstream regulators that can generate iTR without the use of iPS cell-generating factors to further reduce the risk of malignancy in target tissues.

[0235] This optimization protocol for identifying such conditions is identified as follows: Adult-derived normal dermal fibroblasts, and identical fibroblasts selected by immortalizing them using lentivires (TERT) expressing telomerase catalytic components and using neomycin (G418) resistance, are injected with lentiviral vectors expressing the OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, LIN28A, and LIN28B genes individually and in various combinations, as well as small molecule compounds such as CHIR9902 in combination with the following compounds: Glycogen synthase 3 (GSK3) inhibitors, including but not limited to 1; TGF-β signaling inhibitors, including but not limited to SB431542, A-83-01, and E616452; HDAC inhibitors, including but not limited to fatty acid compounds, including but not limited to valproic acid, phenyl butyrate, and butyrate; cyclic tetrapeptides, including trapoxin B and depsipeptides; hydroxamic acids, e.g., trichostatin A, vorinostat (SAHA), Bellinostat (PXD101), LAQ824, panobinostat (LBH589), benzamide-based endinostat (MS-275), CI994, mochenostat (MGCD0103); sirtuin inhibitors containing nicotinamide, various derivatives of NAD, dihydrocoumarin, naphthopyrane, and 2-hydroxynaphthalenealdehyde, or IV (HDAC11) deacetylase, class I (HDAC1, HDAC2, HDAC3, and HDAC8), class IIA (HDA Inhibitors specifically targeting C4, HDAC5, HDAC7, and HDAC9), Class IIB (HDAC6 and HDACIO), and Class III (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7); inhibitors of H3K4 / 9 histone demethylase LSD1, including but not limited to Parnate; inhibitors of Dot1L, including but not limited to EPZ004777; inhibitors of G9a, including but not limited to Bix01294;DNA methyltransferase inhibitors including but not limited to RG108; EZH2 inhibitors including but not limited to DZNep; 5-aza-2'-deoxycytidine (trade names Vidaza and Azadine); Vitamin C which can suppress DNA methylation and increase Tet1 which increases 5hmC, the first step in demethylation; 3'-phosphoinositide-dependent kinase 1 activators including but not limited to PS48; quercetin and fructose 2,6-bisphosphate (phosphofructokinase 1 activators) These compounds further enhance the effects of iTR in various combinations, concentrations, and durations to optimize the effects of iTR in cells cultured in vitro, in damaged or diseased tissue in vivo, or in modulating the lifespan of animals in vivo, using global iTR markers, for example, by assaying the reduced expression of COX7A1 or CAT or other iTR inhibitors described herein, and / or by assaying the increased expression of PCDHB2 or AMH or other iTR activators described herein.

[0236] MDW cells (normal and TERT-immortalized) were measured in 12-well plates containing DMEM medium with 10% FBS, resulting in 1 × 10⁶ cells. 5Cells were seeded in wells and plated in vitro. Polybrene was added to the medium (0.8 ul / mL of polybrene (10 ug / ul stock) per 1 mL of virus / medium to achieve a final concentration of 8 ug / mL). Virus was added at a rate of 1 ml per well. One well was used for control type 1 ("empty virus" = virus expressing only GFP) and control type 2 (uninfected). Since there were two different types of cells (with or without TERT), two sets of controls were present. The medium was gently stirred and mixed, and the cells were coated. The plates of cells containing the virus were returned to an incubator at 37°C and 5% CO2 overnight. On day 1 after transduction, the viral medium was removed and replaced with standard DMEM medium supplemented with 10% FBS, supplied every other day, and then RNA was collected for RNA sequencing.

[0237] In vitro assays of the expression of the genes COX7A1, CAT, and NAALADL1, as well as iTR patterns of pluripotency gene expression or protein markers including DNMT3B, HELLS, or Tra-1-60, Tra-1-81, and SSEA4, are performed to optimize the global pattern of iTR gene expression without restoring target cells to pluripotency. Examples of individual drugs and drug combinations screened are OCT4, SOX2, KLF4, MYC, and LIN28A; OCT4, KLF4; OCT4, KLF4; OCT4, KLF4, LIN28A; OCT4, KLF4, LIN28B; SOX2, MYC; NANOG; ESRRB; NT5A2; OCT4, SOX2, KLF4, and LIN28A; OCT4, SOX2, KLF4, and LIN28B; OCT4, KLF4, MYC, and LIN28A. For the first four weeks, each of the aforementioned drug combinations was used in combination with 0.25 mM NaB, 5 μM PS48, and 0.5 μM A-83-01, followed by treatment with 0.25 mM sodium butyrate, 5 μM PS48, 0.5 μM A-83-01, and 0.5 μM PD0325901. For each of these combinations, markers for global modulation of iTR gene expression were assayed on days 0, 1, 2, 4, 7, 10, and 14.

[0238] The optimized conditions obtained when combined with increased expression of telomerase catalytic components, such as transient expression of the gene TERT, in cases where telomere length is restricted, provide a means for inducing iTR pattern gene expression in embryonic or adult cells in vitro or in vivo, including but not limited to decreased expression of COX7A1, PLPP7, and NAALADL1 genes, thereby inducing tissue regeneration.

[0239] As shown in Figure 24, LIN28A from two manufacturers, Genecopia (G) and another manufacturer (O), was used with the Genecopia vector to obtain the highest expression levels of LIN28A (indicated by "*"). Correlations with higher levels of LIN28A expression included low levels of COX7A1, high levels of GFER, and low levels of CAT, indicating a shift to iTR. No detectable pluripotency markers, such as HELLS or DNMT3B, were present under these conditions. Therefore, LIN28A alone, LIN28A in combination with TERT, or OCT4, KLF4, LIN28A, or OCT4, KLF4, LIN28A and TERT were able to induce iTR.

[0240] [Example 8] Use of LIN28B in conferring the fetal liver phenotype to adult hematopoietic stem cell and progenitor cell types. As shown in Figure 24, the gene LIN28B is typically expressed only in the early stages of embryonic development in most tissues (minimal expression in early fetal skin cells shown in Figure 7d), and is expressed at relatively high levels in fetal liver-derived CD34+ hematopoietic stem cells and CD36+ erythrocyte progenitor cells compared to various types of adult-derived bone marrow (BM) and peripheral blood (PB) hematopoietic cells when assayed using an Illumina gene expression bead array. The cDNA of LIN28B will be expressed in mouse and human CD34+ candidate hematopoietic supplement cells, and the relative proliferation of cells in vitro will be compared to pseudo-infected cells (mock-infected cells). The relative engraftment of LIN28B-expressing cells will be compared to pseudo-transfected cells to assay the degree of efficacy provided to cells in terms of proliferation and engraftment when LIN28B is expressed.

[0241] Sarcoma strains screened for sensitivity to chemotherapeutic agents show that fetal or adult stage markers, such as COX7A1 expression (ASPS-1 strain and Rh28 PX11 / LPAM strain), are uniquely resistant to apoptosis in response to chemotherapeutic agents, such as teniposide, paclitaxel, etoposide, barrubicin, mitomycin C, floxlysine sulfate, clofarabine, vinorelbine tartrate, and daunorubicin HCl (typically with an IC50 one to two orders of magnitude higher). Therefore, detection of this gene expression or the methylation status of the encoded protein or gene is useful in predicting responses to chemotherapeutic agents across various tumor types.

[0242] [Example 9] Creating a mouse model for iTR The Cox7a1 gene was knocked out in BL6 mice, and the animals were bred to produce homozygous knockout animals (ko / ko). As shown in Figure 28, human undifferentiated lipid-producing cells from Figure 8, namely white adipocytes named E3 from clone EP, subcutaneous adipose tissue (SAT) from adult adipocyte progenitor cells, and liposarcoma cell lines CRL3043 and CRL3044, were simultaneously exposed to a glycolytic stress test. The highest extracellular acidification rate (ECAR) observed was in cell types that did not express COX7A1 (i.e., E3, CRL3043, and CRL3044), while the lowest glycolytic shift was observed in COX7A1 (adult-derived) SAT cells, which was consistent with the highest Warburg shift occurring in cells that did not express COX7A1. Next, heart-derived ko / ko mouse cells were compared to wt cardiac cells in the same glycolytic stress test to measure ECAR. As shown in Figure 28, homozygous Cox7a1 ko / ko cells exhibit a shift to glycolysis with higher levels of extracellular acidification than wt cells, which exhibit a Wahlberg shift in metabolism. When ko / ko mice were subjected to an ear punch assay, the ears of ko / ko mice showed accelerated wound healing compared to the ears of wt mice.

[0243] Next, to enhance the robustness of the TR in the animal model, homozygous ko / ko mice for Cox7a1 are crossed with mice expressing other iTR genes, including Lin28a.

[0244] [Example 10] Modulation of DNA methylation as a mechanism to affect iTR and iCM. The global methylation patterns of genomic DNA from human ES cell-derived clonal embryonic progenitor cells to vascular endothelium (30MV2) were compared with those of adult-derived human aortic endothelial cells (HAECs), and in parallel, the global methylation patterns of genomic DNA from human ES cell-derived clonal embryonic progenitor cells to osteochondrocytes (4D20.8) were compared with those of adult-derived bone marrow mesenchymal stem cells (MSCs). As shown in Figure 23, where the bar height in the histogram corresponds to the percentage of cytosine methylated readings in CpG, the COX7A1 gene was highly methylated in both progenitor cell lines in which COX7A1 expression could not be detected, and relatively demethylated in the corresponding adult-derived cell types in which COX7A1 could be detected. Similar results were measured in the genes COMT, TRIM4, NAALADL1, TSPYL5, and PLPP7, and DNA methyltransferase inhibitors, including but not limited to RG108; 5-aza-2'deoxycytidine (trade names Vidaza and Azadine) and vitamin C (which inhibit DNA methylation and increase Tet1, which increases 5hmC, the first step in demethylation) are useful global activators of iTR, providing novel evidence that these drugs, which increase methylation in these regions of the genome, are effective in inducing iCM.

[0245] [Example 11] Use of GFER protein, AMH protein, and valproic acid to induce iTR. The identification of the secreted proteins iTR factors GFER and AMH, as well as the drug valproic acid, provides a novel cocktail of factors for iTR generation in vitro and in vitro. To determine the effects of the factors, cultured adult-derived MDW fibroblasts and umbilical cord-derived MSCs (MSCwj) were treated with various concentrations of the factors, and the growth and regrowth rates were measured after scratch testing.

[0246] Fibroblast cell line MDW (passage 5) was seeded in multiple wells of a 6-well plate and cultured until confluence. A 1.5 mm "scratch" was introduced onto a monolayer using a 200 μl pipette tip, thereby detaching fibroblasts from the culture surface. Factors including 0.5 mM valproic acid, 10 ng / mL AMH, and 20 ng / mL GFER were added to growth medium as DMEM medium supplemented with 10% FBS. After 24 hours, the percentage of detached surface was measured by phase-contrast microscopy. Control MDW cells in growth medium alone showed 20% coverage of the "wounded" area. Cells treated with valproic acid alone regenerated 25%, indicating improved wound regeneration, while cells treated with supplemented AMH alone showed 68% regeneration. Cells treated with GFER supplement showed 28% regeneration. Cells treated with both valproic acid and AMH showed 50% regeneration. Cells treated with valproic acid, AMH, and GFER showed 50% regeneration. Due to the easy denaturation and diffusion of the factors when administered in vivo, formulations of the factors alone, or formulations of the factors combined in hydrogels, such as cross-linked hyaluronic acid or cross-linked hyaluronic acid and collagen, provide a preferred method for delivering the factors to enhance tissue regeneration in animals. Alternatively, umbilical cord-derived MSCs (MSCwj) and adult dermal fibroblasts (MDWs) expressing fetal / adult markers such as COX7A1, respectively, were treated with secreted GFER alone at concentrations of 0, 10.0, and 100 ng / mL in DMEM medium supplemented with 10% FBS and AMH, cultured for 80 hours, and confluence % was measured. As shown in Figure 29, there was a dose-dependent increase in confluence achieved in the presence of escalating doses of GFER, providing evidence of its usefulness in iTR generation in fetal and adult-derived stromal cells.

[0247] Adult dermal fibroblasts (MDW (passages 6–7)) were plated and incubated for 6 days in the presence of mRNA for GFP, cMYC, SOX2, or cMYC + 0.5 mM valproic acid, or SOX2 + 0.5 mM valproic acid. As shown in Figure 30, the addition of 0.5 mM valproic acid provided evidence of iTR, as determined by the decrease in COX7A1 expression and the decrease in the expression of the fibrosis marker COL1A1.

[0248] Induction of iTR can be achieved by a relative shift in fetal or adult cells from an oxidatively phosphorylated state to an anaerobic glycolytic state through modification of components of the MIA pathway, also known as the mitochondrial intermembrane space disulfide relay system. More specifically, this shift to iTR can be induced in fetal or adult somatic cells by increasing GFER protein levels in cells or decreasing COX7A1 protein levels in cells, more preferably by increasing GFER levels and decreasing COX7A1 levels in cells. This modification can be achieved by LIN28A expression or by exogenous administration of GFER or GFER-increasing agents in cells together with agents that decrease COX7A1 protein levels. Even more preferably, iTR is induced in humans in cells with increased telomerase expression levels, for example, by administration of the TERT gene, which can be combined with the induction of LIN28A expression.

[0249] This disclosure also describes means for intervening in mammalian aging, thereby utilizing iTR to restore function in tissues affected by age-related degenerative diseases. Preferably, iTR is carried out in combination with telomere extension, such as through the reexpression of telomerase activity.

[0250] [Example 12] Use of fetal cell-derived or adult cell-derived exosomes for generating iTM and iCM. Adult cell-derived exosomes were tested for their ability to induce iTM using the hES cell-derived clonal embryonic vascular endothelial cell line 30-MV2-6. (See, for example, International Patent Application No. PCT / 2012 / 054525, published as International Publication No. 2013 / 036969, and U.S. Patent Application No. 14 / 238,160, published as U.S. Patent Application Publication No. 2014-0349396, both incorporated herein by reference.) Total RNA expression was profiled using Illumina microarray analysis of a series of 15 hESC-derived clonal embryonic progenitor cell lines and compared with 18 primary endothelial cell lines (from neonatal to adult) obtained from various anatomical sites (not shown). Differentially expressed genes were re-tested using qPCR to assess the doubling of RNA levels between the embryonic progenitor cell line 30-MV2-6 and the umbilical cord-derived cell line HUVEC. COX7A1 showed the largest double-fold difference in gene expression between the two cell lines, with significantly higher levels in HUVEC compared to the embryonic endothelial cell line 30-MV2-6. CAT and TRIM4 were induced to a lower degree. The inventors extracted 1 × 10⁶ exosomes from HUVEC. 930-MV2-6 cell lines were incubated in exosome-depleted medium, which had been added at a concentration of particles / ml and left for 24 hours. Negative control 30-MV2-6 cells were incubated in exosome-depleted medium with an equal volume of PBS. HUVEC exosome-treated 30-MV2-6 cells showed detectable expression of COX7A1 compared to undetectable expression in the PBS control. Embryonic genes ACP5 and LIN28B appeared to be downregulated after treatment of embryonic progenitor cells with HUVEC exosomes. This result demonstrates the ability of exosomes to reprogram target cells from one developmental stage to a different one. In this case, treatment with adult cell exosomes results in a gene expression pattern in recipient embryonic cells that resembles the adult pattern. Importantly, COX7A1 is the most tightly regulated gene identified by the inventors. It represents an embryonic marker for fetal transition, present in fetal cells to adult cells but absent in a wide variety of embryonic cell lines. The use of fetal or adult-derived somatic cell type exomes in iTM and iCM has practical applications in introducing EFTs into mature cancer cell types in vitro and in vivo, thereby increasing their availability for nuclear translocation of p53 and induction of gene expression such as p21.

[0251] From the description herein, it is understood that this disclosure encompasses multiple embodiments, including but not limited to:

[0252] A method for regenerating damaged or aged tissue in a target by contacting one or more target cells with one or more inducible tissue regeneration (iTR) factors.

[0253] The method of any embodiment wherein one or more of the iTR factors include nucleic acids.

[0254] The method of any embodiment, wherein the nucleic acid comprises RNA.

[0255] The method of any embodiment wherein one or more of the iTR factors include anti-Müllerian hormone (AMH).

[0256] A method according to any embodiment, comprising contacting one or more of the target cells with anti-Müllerian hormone (AMH) at a concentration of 0.05 mM to 5 mM.

[0257] The method of any embodiment wherein one or more of the iTR factors include proteins encoded by the GFER gene.

[0258] A method according to any embodiment, comprising contacting one or more of the target cells with the protein encoded by the GFER gene at a concentration of 2 ng / mL to 200 ng / mL.

[0259] The method of any embodiment wherein one or more of the iTR factors include valproic acid.

[0260] A method according to any embodiment, comprising contacting one or more of the target cells with valproic acid at a concentration of 0.05 mM to 5 mM.

[0261] A method according to any embodiment, comprising combining one or more of the aforementioned iTR factors with a hydrogel.

[0262] The method of any embodiment wherein the iTR factor increases GFER protein levels and decreases COX7A1 protein levels.

[0263] A method according to any embodiment, wherein the iTR factor increases the expression of LIN28A.

[0264] A method of any embodiment, further comprising increasing telomerase expression in one or more of the aforementioned target cells.

[0265] The method of any embodiment wherein administration of the TERT gene to one or more target cells increases telomerase expression.

[0266] The method of any embodiment wherein the iTR factor increases the expression of LIN28A and also increases the expression of telomerase.

[0267] The method of any embodiment, wherein the subject is a human.

[0268] A method for repairing damaged or aged tissue in a target by inducing embryonic pattern gene expression in one or more target cells.

[0269] A kit for regenerating damaged or aging tissue in a subject, comprising one or more of AMH, GFER protein, and valproic acid iTF factor (iTR factor).

[0270] A kit of any embodiment comprising the iTR factor combined with a hydrogel.

[0271] A method for regenerating tissue in a subject by contacting one or more cells of the subject with a drug capable of inducing pluripotency, wherein pluripotency itself is not induced.

Claims

1. A composition for regenerating one or more lesional skin cells without causing the cells to revert to pluripotent stem cells, comprising: The composition comprises one or more induced tissue regeneration (iTR) factors, including (a) a nucleic acid encoding LIN28A, (b) one or more nucleic acids encoding LIN28A and TERT, (c) one or more nucleic acids encoding OCT4, KLF4 and LIN28A, or (d) one or more nucleic acids encoding OCT4, LIN28A, KLF4 and TERT, or any combination thereof, wherein expression of the one or more induced tissue regeneration (iTR) factors from the nucleic acid is for 4 days or more and 7 days or less, the one or more lesional skin cells do not revert to pluripotent stem cells, and the one or more lesional skin cells are not pluripotent stem cells.

2. The composition of claim 1 , wherein the one or more iTR factors comprise RNA.

3. The composition of claim 1 or 2, wherein the one or more iTR factors are transiently expressed in vivo.

4. The composition of any one of claims 1 to 3, wherein the one or more iTR factors are combined with a hydrogel.

5. The composition of any one of claims 1 to 4, wherein the composition is for increasing GFER protein levels and decreasing COX7A1 protein levels in the one or more lesional skin cells in the subject compared to a control.

6. The composition of any one of claims 1 to 5, wherein the composition is for further increasing the expression of LIN28A in the one or more lesional skin cells in the subject as compared to a control.

7. The composition of any one of claims 1 to 6, wherein the composition is for further increasing expression of LIN28A and telomerase in the one or more lesional skin cells in the subject as compared to a control.

8. The composition of any one of claims 1 to 7, wherein the composition is for reducing the expression of PLPP7 in the one or more lesional skin cells in the subject as compared to a control.

9. The composition of claim 2 , wherein the RNA is mRNA.

10. 10. The composition of any one of claims 1 to 9, wherein the one or more lesional skin cells are lesional skin cells affected by ageing or an age-related disease or condition.

11. The composition of claim 1 , wherein the one or more lesional skin cells are lesional skin cells from a wound, a burn, or a skin graft.

12. A composition according to any preceding claim, wherein the one or more lesional skin cells are regenerated without scarring or excessive scarring.

13. The composition of any one of claims 1 to 12, wherein the one or more iTR factors are formulated for controlled release.

14. The composition of any one of claims 1 to 13, wherein the one or more iTR factors are encoded by a viral vector.

15. The composition of claim 14 , wherein the viral vector is an adeno-associated viral vector.

16. 9. The composition of any of claims 1 to 8, wherein the composition is further for measuring expression of HELLS and / or DMNT3B compared to a control, and failure of the one or more lesional skin cells to revert to pluripotent stem cells is determined by the absence of detectable expression of HELLS and / or DMNT3B compared to a control.

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