Improved methods for inducing tissue regeneration and lysis of senescent cells in mammalian cells

Abstract

Aspects of the invention include compositions and methods for discovering novel compositions and applying said compositions to treat diseases including aging, degenerative diseases, wound treatment, and cancer by modulating molecular pathways that regulate regeneration and senescent cell lysis by altering the embryonic-fetal and pre / postnatal transition states of mammalian cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 62 / 661,322, filed April 23, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to compositions and methods for screening new therapeutic agents for treating diseases including aging, degenerative diseases and cancer by modulating molecular pathways that regulate regeneration and senescent cell lysis by altering the embryonic-fetal and pre / postnatal transition states of mammalian cells. Background Art

[0004] The progress of stem cell technology, such as the separation and in vitro propagation of human pluripotent stem (hPS) cells (including but not limited to human embryonic stem (hES) and human induced pluripotent stem (hiPS) cells), constitutes an important new field of medical research. hPS cells have the potential (Thomson et al., Science 282:1145-1147 (1998)) of proliferating or being induced to differentiate into any and all cell types in the human body under a proven undifferentiated state. The unique inherent ability of hPS cells to differentiate into all somatic cell types logically provides a platform for the production of transplantable hPS-derived cells with similar diversity for the treatment of multiple degenerative diseases. Although this pluripotency of hES and hiPS cells has been widely recognized at present, it is less recognized and rarely studied that the hPS cells cultured in vitro produce the unique ability of relatively undifferentiated embryonic primordium.

[0005] Even less studied is the potential of hPS cell-derived cells to differentiate into recognized cell types, such as cardiomyocytes or osteochondrocytes that still display subtle prenatal or even pre-fetal patterns of gene expression that distinguish them from fetal or adult cells. Immediately before the embryo-fetal transition (EFT) in vivo, mammalian differentiated cells and tissues, such as skin, heart, and spinal cord, display a profound potential for scarless regeneration, which is gradually lost after EFT. For some tissues, such as the human heart, the potential for scarless regeneration can be detected about one week after the prenatal-postnatal transition (PPT) period. Given the importance of understanding and regulating tissue regeneration and tissue growth to the fields of regenerative medicine and oncology, improved methods for in vitro and in vivo biological modeling and regulation have great potential utility in research and clinical practice.

[0006] The potential of pluripotent stem cells and derived embryoid bodies to self-assemble into three-dimensional organoids in vitro has attracted interest as a potential pathway for obtaining transplanted tissues (Singh et al., Stem Cells Dev. 2015. 24 (23): 2778-95) and for modeling human embryonic development. The present invention teaches that the formation of the organoids is a reflection of the inherent potential of cells to undergo tissue generation and / or regeneration before EFT. In contrast to embryonic cells, cells of fetal and adult origin often show reduced potential for in vitro organogenesis and in vivo epimorphic regeneration. Epimorphic regeneration, sometimes referred to as "epimorphic", refers to a type of tissue regeneration in which relatively undifferentiated mesenchymal blastema proliferates at the site of injury, and then the cells differentiate to restore the original histology. Although the developmental timing of loss of phenotypic regenerative potential cannot be precisely determined and may vary with tissue type, EFT, which occurs approximately at the end of eight weeks of human development (Carnegie Stage 23; O'Rahilly, R., F. Müller (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) appears to correspond in time 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). Correlations between species show increased regenerative potential in the embryonic or juvenile state (which was reviewed in Morgan, TH (1901). Regeneration (New York: The MacMillan Company); also reviewed in Sanchez Alvarado, A., and Tsonis, PA (2006) Bridging the regeneration gap: genetic insights from diverse animal models. Nat Rev. Genet. 7, 873-884). This suggests that, unlike scarring, tissue regeneration reflects the presence of an embryonic phenotype (e.g., different from a fetal or adult phenotype), although there is currently no consensus in the scientific community that phenotypic tissue regeneration is an embryonic (prenatal, more specifically, pre-fetal) pattern of gene expression.For some species, changes in developmental timing (heterochrony) are associated with profound regenerative potential, such as the developmental arrest (heterochrony) of larval development and the developmental arrest of 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, 293-298).

[0007] Despite these observations, markers of EFT and methods for testing the role of specific molecules in regulating EFT to treat degenerative diseases or cancer remain limited. We have previously disclosed compositions and methods related to markers of EFT in mammalian species, and their use in regulating tissue regeneration and cancer diagnosis, described in "Compositions and Methods for Induced Tissue Regeneration in Mammalian Species" (International Patent Application Publication No. WO 2014 / 197421) (the entire contents of which are incorporated herein by reference) and "Improved Methods for Detecting and Regulating Embryonic-Fetal Transition in Mammalian Species" (International Patent Application Publication No. WO 2017 / 214342, the entire contents of which are incorporated herein by reference). The aforementioned compositions and methods are based in part on methods that allow clonal expansion of hPS cell-derived embryonic progenitor cell lines, which provide a means of proliferating novel, diverse and highly purified cell lines with prenatal gene expression patterns that can be used to regenerate tissues (such as skin) in a scarless manner. Such cell types have important applications in research and in the preparation of cell-based therapies (see PCT Application Serial No. PCT / US2006 / 013519, entitled “Novel Uses of Cells With Prenatal Patterns of Gene Expression,” filed April 11, 2006); U.S. Patent Application Serial No. 11 / 604,047, entitled “Methods to Accelerate the Isolation of Novel Cell Strainsfrom Pluripotent Stem Cells and Cells Obtained Thereby,” filed November 21, 2006; and U.S. Patent Application Serial No. 11 / 604,047, entitled “Methods to Accelerate the Isolation of Novel Cell Strains from Pluripotent Stem Cells and Cells Obtained Thereby,” filed July 16, 2009. No. 12 / 504,630 to Thereby, the entire contents of each of which are incorporated herein by reference). However, there is a need for other improved methods and compositions for modulating EFT to treat degenerative diseases and cancer.The present invention teaches compositions and methods related to the detection and modulation of EFT for scarless mammalian regenerative therapy for a variety of applications including, but not limited to, aging and age-related degenerative diseases and various types of malignancies. Summary of the invention

[0008] The inventors of the present invention teach that the potential for scarless tissue regeneration exists throughout the life course of certain primitive species (e.g., hydra and planarians), but is limited to early development in most mammalian species. The inventors of the present invention further teach that in mammals that benefit the organism by providing tumor suppression, sequential molecular changes occur after embryogenesis is completed, but the above changes have the deleterious effect of inhibiting the potential for scarless regeneration and senescent cell lysis in many tissues thereafter (antagonistic gene pleiotropy). Therefore, it is taught that inhibition of regeneration after embryogenesis interferes with subsequent tumor growth in adults. In another aspect, it is taught herein that senescent cell lysis (defined as programmed cell apoptosis of cells with significant DNA damage (e.g., DNA damage associated with dysfunctional telomeres)) is inhibited after embryogenesis because the tissue is no longer able to regenerate and cannot replace the simple reason of cells that have undergone senescent cell lysis. Therefore, tissues that can regenerate tend to be prone to apoptosis of senescent cells, while those that cannot regenerate tend to inhibit apoptosis. Thus, the inventors of the present invention teach that the intrinsic ability of cells to regenerate or undergo senescent cell lysis is regulated by molecular mechanisms associated with normal development in mammals, and more specifically, those changes associated with EFT.

[0009] Furthermore, the inventors teach altering the molecular regulation of EFT in adult mammalian tissues to restore embryonic gene expression patterns (referred to herein as "induced Tissue Regeneration" or "iTR", which is capable of inducing increased systemic scar-free tissue regeneration while inducing senescent cell lysis).

[0010] Further, the inventors of the present invention teach that although most malignant cancer cells revert to an embryonic gene expression pattern (referred to herein as an "embryonic-oncology phenotype" or "EOP") (characterized, for example, by suppressed expression of COX7A1), the EOP is unstable and a subset of tumor cells shifts to an adult gene expression pattern. As a result, when the tumor is exposed to a genotoxic stimulus, such as when a patient is treated with a chemotherapeutic agent or radiation therapy, the majority of cancer cells with an EOP that are relatively sensitive to apoptosis are destroyed, while the remaining number of cells that are relatively adult in their gene expression pattern (e.g., in expressing COX7A1) are resistant to apoptosis and are able to give rise to further growth of the cancer. Growth and resistance to chemotherapy or radiotherapy. Therefore, unexpectedly, the inventors teach a counterintuitive explanation that cells that are generally considered to be "cancer stem cells" or "CSCs" are not actually immature progenitor cells, but unexpectedly, the situation is just the opposite, and CSCs are more mature adult-like cells (expressing, for example, more COX7A1). This understanding provides a new diagnosis and treatment strategy for monitoring the presence of embryonic markers or adult markers in tumors or blood as a cancer diagnosis and concomitant treatment strategy for using iTR factors to treat cancer (so that cancer cells are returned to embryonic gene expression patterns so that they can once again perform senescent cell lysis in the presence of chemotherapy or radiotherapy).

[0011] The present disclosure provides screening for identifying and utilizing compounds, compositions, and methods that can be used to regulate the embryonic-fetal transition (EFT) in mammalian cells and tissues. More specifically, the present invention provides screening for identifying and utilizing compounds, compositions, and methods that can be used to screen and use agents that can regulate molecular pathways that regulate EFT in mammalian cells, with the goal of causing iTR in cells and tissues that are originally completely unable to cause such scarless regeneration. In addition, the present disclosure provides screening for identifying and utilizing agents that can cause iTR in cancer stem cells (referred to herein as "induced senescent cell lysis of cancer stem cells" or "iS-CSC") to restore CSC to an embryonic phenotype (pre-fetal) gene expression pattern, and detect and target malignant cells that have been restored to an embryonic phenotype to diagnose and treat cancer, and screen agents that can cause iS-CSC.

[0012] Finally, the present disclosure provides screens for identifying and utilizing agents capable of inducing iTR and senescent cell lysis in aged mammalian tissues or generally throughout the mammal to revert the aged tissue to an embryonic (pre-fetal) phenotype having an embryonic gene expression pattern, thereby inducing apoptosis of senescent cells and their removal from the senescent cells (iTR-associated senescent cell lysis), while additionally inducing regenerative potential in the tissue to replace the senescent cells that have undergone apoptosis.

[0013] In one aspect of the present disclosure, a method is provided that allows researchers to identify agents capable of performing iTR, the method comprising the steps of: 1) culturing adult mammalian cells in a form that promotes exposure of the cells to an exogenous agent, 2) adding an exogenous candidate molecule to the cells in juxtaposition, 3) measuring the expression of molecular markers of EFT in the cells to determine whether the agent causes iTR.

[0014] In another aspect of the present disclosure, a method is provided that allows researchers to identify agents capable of performing iTR, the method comprising the steps of: 1) culturing adult human dermal fibroblasts in vitro in a porous format that facilitates exposure of the cells to exogenous agents, 2) adding exogenous candidate molecular regulators of iTR to the cells in different combinations and juxtapositions for different time periods, 3) preparing RNA from the cells and measuring the level of COX7A1 transcripts in the cells to determine whether the agent causes iTR.

[0015] In another aspect of the present disclosure, a method is provided that allows researchers to identify agents capable of iTR, the method comprising the following steps: 1) culturing TERT-immortalized adult dermal fibroblasts carrying a reporter gene that signals EFT, 2) adding exogenous candidate molecular regulators of iTR to the cells in different combinations and at different time periods, 3) measuring the expression of the reporter gene that signals EFT in the cells to determine whether the agent causes iTR.

[0016] In another aspect of the present disclosure, methods of generating iTR in vivo to treat degenerative diseases are provided. The method comprises applying a mixture of active factors to the affected tissue for 17 days, wherein the mixture of active factors is: valproic acid at a concentration of 0.05-5.0 mM, preferably 1.0 mM; GSK-3 inhibitor 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (also known as CHIR99021) at a concentration of 7.0 nM-10 uM, preferably 10 uM; TGFβR-1 / ALK5 inhibitor 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (also known as RepSox) at a concentration of 4.0 nM-10 uM, preferably 10 uM; lysine-specific deoxyribonucleic acid at a concentration of 2.0-10 uM, preferably 10 uM. Parnate (also known as tranylcypromine), an inhibitor of methyltransferase 1; Forskolin, an activator of adenylate cyclase, at a concentration of 0.5-50uM, preferably 50uM; Aromatic retinoic acid (also known as 4-[(E)-2-(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)prop-1-enyl]benzoic acid or TTNPB), a retinoid receptor agonist, at a concentration of 1.0nM-5uM, preferably 5.0uM; and then the following is added to the mixture over an additional 14 days: (1S,2R,5R)-5-(4-aminoimidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (also known as 3-Deazaneplanocin) at a concentration of 0.05-0.24uM, preferably 0.1uM A(DZNep)); then in the last 7 days, the above factors are stopped and the MEK / ERK pathway inhibitor N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoaniline)benzamide (also known as PD0325901) at a concentration of 0.1nM-1.0uM, preferably 1.0uM and the GSK-3 inhibitor 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (also known as CHIR99021) at a concentration of 7.0nM-10uM, preferably 10uM, all of the above factors are formulated in a physiologically compatible carrier.

[0017] In another aspect of the present disclosure, methods are provided together with agents that activate telomerase to produce iTR in vivo to treat degenerative diseases. The method comprises applying a mixture of active factors to the affected tissue for 17 days, wherein the mixture of active factors is: valproic acid at a concentration of 0.05-5.0 mM, preferably 1.0 mM; GSK-3 inhibitor 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (also known as CHIR99021) at a concentration of 7.0 nM-10 uM, preferably 10 uM; TGFβR-1 / ALK5 inhibitor 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (also known as RepSox) at a concentration of 4.0 nM-10 uM, preferably 10 uM; lysine-specific deoxyribonucleic acid at a concentration of 2.0-10 uM, preferably 10 uM. Parnate (also known as tranylcypromine), an inhibitor of methyltransferase 1; Forskolin, an activator of adenylate cyclase, at a concentration of 0.5-50uM, preferably 50uM; Aromatic retinoic acid (also known as 4-[(E)-2-(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)prop-1-enyl]benzoic acid or TTNPB), a retinoid receptor agonist, at a concentration of 1.0nM-5uM, preferably 5.0uM; and then the following is added to the mixture over an additional 14 days: (1S,2R,5R)-5-(4-aminoimidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (also known as 3-Deazaneplanocin) at a concentration of 0.05-0.24uM, preferably 0.1uM A(DZNep)); then in the last 7 days, the above factors are stopped and the MEK / ERK pathway inhibitor N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoaniline)benzamide (also known as PD0325901) at a concentration of 0.1nM-1.0uM, preferably 1.0uM and the GSK-3 inhibitor 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (also known as CHIR99021) at a concentration of 7.0nM-10uM, preferably 10uM, all of the above factors are formulated together with an agent that activates the telomerase catalytic component TERT (e.g., a TERT gene therapy construct expressed in diseased cells or tissues) in a physiologically compatible carrier.

[0018] In another aspect of the present disclosure, a method of generating iTR in vivo to treat cancer, more specifically, to sensitize remaining residual cancer cells after chemotherapy or radiotherapy is provided. The method comprises applying a mixture of active factors to the affected tissue for 17 days, wherein the mixture of active factors is: valproic acid at a concentration of 0.05-5.0 mM, preferably 1.0 mM; GSK-3 inhibitor 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (also known as CHIR99021) at a concentration of 7.0 nM-10 uM, preferably 10 uM; TGFβR-1 / ALK5 inhibitor 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (also known as RepSox) at a concentration of 4.0 nM-10 uM, preferably 10 uM; lysine-specific demethylase at a concentration of 2.0-10 uM, preferably 10 uM an inhibitor of ezetimibe 1 (named Parnate) (also known as tranylcypromine); an activator of adenylate cyclase, Forskolin, at a concentration of 0.5-50uM, preferably 50uM; a retinoid receptor agonist, aromatic retinoic acid (also known as 4-[(E)-2-(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)prop-1-enyl]benzoic acid or TTNPB) at a concentration of 1.0nM-5uM, preferably 5.0uM; and then the following is added to the mixture over an additional 14 days: an inhibitor of EZH2, (1S,2R,5R)-5-(4-aminoimidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (also known as 3-Deazaneplanocin) at a concentration of 0.05-0.24uM, preferably 0.1uM A(DZNep)); then in the last 7 days, the above factors are stopped and the MEK / ERK pathway inhibitor N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoaniline)benzamide (also known as PD0325901) at a concentration of 0.1nM-1.0uM, preferably 1.0uM and the GSK-3 inhibitor 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (also known as CHIR99021) at a concentration of 7.0nM-10uM, preferably 10uM, all of the above factors are formulated in a physiologically compatible carrier, and then other applicable chemotherapy or radiotherapy courses are carried out.

[0019] In another aspect of the present disclosure, methods are provided for generating lysis of senescent cells in vivo to treat diseases associated with aging, and more specifically, to senescent cells to apoptosis. The method comprises applying a mixture of active factors to the affected tissue for 17 days, wherein the mixture of active factors is: valproic acid at a concentration of 0.05-5.0 mM, preferably 1.0 mM; GSK-3 inhibitor 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (also known as CHIR99021) at a concentration of 7.0 nM-10 uM, preferably 10 uM; TGFβR-1 / ALK5 inhibitor 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (also known as RepSox) at a concentration of 4.0 nM-10 uM, preferably 10 uM; lysine-specific demethylase at a concentration of 2.0-10 uM, preferably 10 uM an inhibitor of ezetimibe 1 (named Parnate) (also known as tranylcypromine); an activator of adenylate cyclase, Forskolin, at a concentration of 0.5-50uM, preferably 50uM; a retinoid receptor agonist, aromatic retinoic acid (also known as 4-[(E)-2-(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)prop-1-enyl]benzoic acid or TTNPB) at a concentration of 1.0nM-5uM, preferably 5.0uM; and then the following is added to the mixture over an additional 14 days: an inhibitor of EZH2, (1S,2R,5R)-5-(4-aminoimidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (also known as 3-Deazaneplanocin) at a concentration of 0.05-0.24uM, preferably 0.1uM A(DZNep)); then in the last 7 days, the above factors are stopped and the MEK / ERK pathway inhibitor N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoaniline)benzamide (also known as PD0325901) at a concentration of 0.1nM-1.0uM, preferably 1.0uM and the GSK-3 inhibitor 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (also known as CHIR99021) at a concentration of 7.0nM-10uM, preferably 10uM, all of the above factors are formulated in a physiologically compatible carrier.

[0020] Certain conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant nucleic acid (eg, DNA) techniques, immunology, and the like, which are within the skill of the art, may be employed in aspects of the present invention. Non-limiting descriptions of some 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 John Wiley & Sons, NY, 2008 edition; Sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual, 3.sup.rd 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; Bums, R., Immunochemical Protocols (Methods in Molecular Biology) Humana Press; 3rd edition, 2005, Monoclonal antibodies: a practical approach (P. Shepherd and C Dean (eds.), Oxford University Press; 2003). The entire contents of all patents, patent applications, websites, databases, scientific articles and other publications mentioned herein are incorporated by reference herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Shown is the expression of COX7A1 in hES cells, different hEP lines, human arm fibroblasts during EFT throughout postnatal development, adult arm fibroblasts before and after reprogramming to iPS cells, and adult arm fibroblasts before and after induction of iTR with AGEX1547. Error bars show standard deviation (St. Dev).

[0022] Figure 2 Shown is ADIRF expression in hES cells, different hEP lines, human arm fibroblasts during EFT throughout postnatal development, adult arm fibroblasts before and after reprogramming to iPS cells, and adult arm fibroblasts before and after induction of iTR with AGEX 1547. Error bars show standard deviation.

[0023] Figure 3 Shown is the expression of TNFRSF11B in hES cells, different hEP lines, human arm fibroblasts during EFT throughout postnatal development, adult arm fibroblasts before and after reprogramming to iPS cells, and adult arm fibroblasts before and after induction of iTR with AGEX1547. Error bars show standard deviation.

[0024] Figure 4 Shown is AMH expression in hES cells, different hEP lines, human arm fibroblasts during EFT throughout postnatal development, adult arm fibroblasts before and after reprogramming to iPS cells, and adult arm fibroblasts before and after induction of iTR with AGEX 1547. Error bars show standard deviation.

[0025] Figure 5 Shown is COL1A1 expression in hES cells, different hEP lines, human arm fibroblasts during EFT throughout postnatal development, adult arm fibroblasts before and after reprogramming to iPS cells, and adult arm fibroblasts before and after induction of iTR with AGEX 1547. Error bars show standard deviation.

[0026] Figure 6 Shown is POU5F1 (OCT4) expression in hES cells, different hEP lines, human arm fibroblasts during EFT throughout postnatal development, adult arm fibroblasts before and after reprogramming to iPS cells, and adult arm fibroblasts before and after induction of iTR with AGEX1547. Error bars show standard deviation.

[0027] Figure 7Shown are TUNEL assay results comparing hES cell-derived clonal embryonic progenitor cell lines 4D20.8 and 30MV2-6 with their adult counterparts MSC and HAEC cells exposed to 0 nM, 0.037 nM and 3.7 nM of Thapsigargin.

[0028] FIG8 shows a list of candidate small molecule iTR factors, TR inhibitor genes, and TR activator genes that can be used for screening.

[0029] FIG. 9 shows the expression of COX7A1 for the first group of samples in Example 4. FIG.

[0030] Fig.10 COX7A1 expression for the second group of samples in Example 5 is shown. DETAILED DESCRIPTION

[0031] abbreviation

[0032] AC - Adult-derived cells

[0033] AMH - Anti-Mullerian Hormone

[0034] ASC - Adult Stem Cell

[0035] cGMP - Current Good Manufacturing Process

[0036] CM-Cancer Maturation

[0037] CSC - Cancer Stem Cell

[0038] CNS - Central Nervous System

[0039] DMEM-Dulbecco's Modified Eagle Medium

[0040] DMSO-Dimethyl sulfoxide

[0041] DNAm - Changes in DNA methylation that provide a marker or "clock" for cellular and tissue age.

[0042] DNN - Deep Neural Network

[0043] DPBS - Dulbecco's phosphate buffered saline

[0044] ED cells - cells derived from embryos; hED cells are human ED cells

[0045] EDTA-ethylenediaminetetraacetic acid

[0046] EFT - Embryo-Fetal Transition

[0047] EG cells - embryonic germ cells; hEG cells are human EG cells

[0048] EOP-Embryonic-Tumor Phenotype

[0049] EP-embryonic progenitor cells

[0050] ES cells - embryonic stem cells; hES cells are human ES cells

[0051] ESC - Embryonic Stem Cell

[0052] FACS - Fluorescence Activated Cell Sorting

[0053] FBS - Fetal Bovine Serum

[0054] FPKM - fragments per kilobase of transcript per million mapped reads from RNA sequencing

[0055] GFER-Growth factor and enhancer of liver regeneration (ALR)

[0056] GFP - Green Fluorescent Protein

[0057] GMP - Good Manufacturing Practice

[0058] HAEC - human aortic endothelial cells

[0059] hED cells - cells derived from human embryos

[0060] hEG cells - "human embryonic germ cells" are stem cells derived from primordial germ cells of fetal tissue.

[0061] HESC - Human Embryonic Stem Cell

[0062] hiPS cells - "human induced pluripotent stem cells" are cells with properties similar to hES cells, which are obtained by exposing somatic cells to hES-specific transcription factors (such as SOX2, KLF4, OCT4, MYC or NANOG, L1N28, OCT4 and SOX2).

[0063] HSE - "human skin equivalent" is a mixture of cells and biological or synthetic matrices that is manufactured for testing purposes or for therapeutic applications to promote wound repair.

[0064] iCM-induced cancer maturation.

[0065] iPS cells - "induced pluripotent stem cells" are cells with properties similar to hES cells, which are obtained by exposing somatic cells to hES-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).

[0066] iS-CSC - "induced senescent cell lysis of cancer stem cells" refers to the treatment of cells in malignancies that are refractory to ablative therapy by chemotherapeutic agents or radiation therapy, wherein the iS-CSC treatment causes the refractory cells to revert to a pre-fetal gene expression pattern and become sensitive to the chemotherapeutic agents or radiation therapy.

[0067] iTM-induced tissue maturation

[0068] iTR-induced tissue regeneration

[0069] MEM-Minimum Essential Medium

[0070] MSC-Mesenchymal Stem Cells

[0071] NT-nuclear transfer

[0072] PBS Phosphate Buffered Saline

[0073] PPT-“Prenatal-postnatal transition” refers to the molecular changes that occur in placental mammalian cells within a week or less after birth.

[0074] PS fibroblasts "pre-scarring fibroblasts" are fibroblasts derived from skin of early pregnancy or from ED cells that display a prenatal gene expression pattern that promotes rapid healing of dermal wounds without scarring.

[0075] RFU - relative fluorescence units

[0076] RNA-seq - RNA sequencing

[0077] SFM-Serum Free Medium

[0078] St.Dev.-Standard Deviation

[0079] TR tissue regeneration

[0080] definition

[0081] The term "analytical reprogramming techniques" refers to a variety of methods for reprogramming the gene expression pattern of somatic cells to the gene expression pattern of a more potent state (e.g., the gene expression pattern of iPS, ES, ED, EC or EG cells), wherein the reprogramming occurs in multiple and discrete steps and does not rely solely on the transfer of somatic cells into oocytes and the activation of the oocytes (see U.S. Application Nos. 60 / 332,510, filed November 26, 2001; 10 / 332,510, filed November 26, 2002; and 10 / 332,511, filed November 26, 2003). 0 / 304,020; PCT application number PCT / US02 / 37899 filed on November 26, 2003; U.S. application number 60 / 705625 filed on August 3, 2005; U.S. application number 60 / 729173 filed on August 20, 2005; U.S. application number 60 / 818813 filed on July 5, 2006; PCT / US06 / 30632 filed on August 3, 2006, the disclosures of each of which are incorporated herein by reference).

[0082] The term "induced senescent cell lysis of cancer stem cells" (iS-CSC) refers to the treatment of refractory malignant tumor cells by chemotherapeutic agents or radiation therapy, wherein the iS-CSC treatment causes the refractory cells to revert to a pre-fetal gene expression pattern and become sensitive to the chemotherapeutic agents or radiation therapy.

[0083] The term "cell expressing gene X", "gene X is expressed in a cell" (or cell population), or their equivalents, refers to a cell whose analysis using a particular assay platform provides a positive result. Vice versa (i.e., a cell that does not express gene X, or its equivalent, refers to a cell whose analysis using a particular assay platform provides a negative result). Thus, any gene expression result described herein is associated with one or more specific probes used in one or more assay platforms for that gene.

[0084] The term "cell line" refers to a resident or immortal cell population capable of proliferating and expanding in vitro.

[0085] The term "clone" refers to a cell population that has expanded from a single cell to a cell population that is all derived from the original single cell and contains no other cells.

[0086] The term "differentiated cells" when used to refer to cells prepared from pluripotent stem cells by the method of the present invention refers to cells with reduced differentiation potential compared to the parental pluripotent stem cells. The differentiated cells of the present invention include cells that can further differentiate (ie, they may or may not be terminally differentiated).

[0087] The term "embryonic" or "embryonic development stage" refers to the prenatal development stage of a cell, tissue or animal, particularly the embryonic development stage of a cell compared to a fetal and adult cell. For human species, the transition from embryonic to fetal development occurs at about 8 weeks of prenatal development, in mice, it occurs at or about 16 days, and in rats, it occurs at about 17.5 days after mating. (http: / / php.med.unsw.edu.au / embryology / index.php?title=Mouse_Timeline_Detailed).

[0088] The term "embryonic stem cell" (ES cell) refers to a cell derived from the inner cell mass of a blastocyst, blastomere or morula, which has been continuously passaged as a cell line while maintaining an undifferentiated state (e.g., expressing TERT, OCT4, SSEA and TRA antigens specific to the ES cells of the species). ES cells can be derived by fertilization of an egg cell with a sperm, or DNA, nuclear transfer, parthenogenesis, or by producing hES cells that are hemizygous or homozygous in the MHC region. Historically, ES cells have been defined as cells that can differentiate into all somatic cell types and germ lines when transplanted into preimplantation embryos; candidate ES cultures from many species (including humans) are flatter in appearance in culture and are generally not conducive to germ line differentiation, and are therefore referred to as "ES-like cells." It is generally believed that human ES cells are actually "ES-like," but in this application we will use the term ES cells to refer to both ES and ES-like cell lines.

[0089] The term "global regulator of TR" or "global regulator of iTR" refers to an agent capable of regulating multiple iTR genes or iTM genes, including but not limited to an agent capable of downregulating COX7A1, ADIRF or TNFRSF11B while simultaneously upregulating AMH in cells derived from fetuses or adults and capable of inducing a gene expression pattern that results in increased scarless tissue regeneration in response to tissue injury or degenerative disease.

[0090] The term "human embryonic stem cells" (hES cells) refers to human ES cells.

[0091] The term "human induced pluripotent stem cells" refers to cells having properties similar to hES cells, including the ability to form all three germ layers when transplanted into immunocompromised mice, wherein the iPS cells are derived from cells of a somatic cell lineage that have been altered following exposure to dedifferentiation factors (e.g., a hES cell-specific transcription factor combination (KLF4, SOX2, MYC; OCT4 or various combinations of SOX2, OCT4, NANOG, and LIN28; or OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, LIN28A, and LIN28B)) or other methods of inducing somatic cells to acquire a pluripotent stem cell state having properties similar to those of hES cells. However, reprogramming of somatic cells by somatic cell nuclear transfer (SCNT) is generally referred to as NT-ES cells rather than iPS cells.

[0092] The term "induced cancer maturation" refers to a method that results in a phenotypic change in a pre-malignant or malignant cell such that, following said induction, said cell expresses markers that are normally expressed at fetal or adult developmental stages (rather than embryonic stages) of that cell type.

[0093] The term "induced tissue regeneration" refers to the use of the methods of the present invention to alter the molecular composition of fetal or adult mammalian cells so that the cells are able to regenerate functional tissue after destruction of the tissue, wherein the regeneration would not be a normal outcome in the animal of that species. Although functional iTRs are intended to produce new tissue formation at the site of injury or degenerative disease or to induce senescent cell lysis in CSCs or senescent cells, the inventors of the present invention teach that iTRs actually reverse many aspects of cellular senescence, including markers (e.g., DNAm), but do not include restoration of telomerase activity. Increased telomerase activity is also defined as "iTR" in the present invention together with iTR.

[0094] The term "iTR-associated senescent cell lysis" refers to the induction of apoptosis in cells of senescent tissues with significant DNA damage, including but not limited to damage to DNA caused by cellular senescence (telomere shortening), by reprogramming the damaged cells to an embryonic gene expression pattern.

[0095] The term "isolated" refers to a substance that is (i) separated from at least some of the other substances with which it is normally found in nature, typically by processes involving human hands, (ii) is produced by human effort (e.g., chemical synthesis), and / or (iii) is present in an artificial environment or background (i.e., an environment or background not normally found in nature).

[0096] The term "iS-CSC factor" refers to a molecule that alters the levels of TR activators and TR inhibitors in a manner that results in increased TR and associated sensitivity to apoptosis in cancer cells exposed to chemotherapeutic or radiation therapy.

[0097] The term "iTR factor" refers to a molecule that changes the levels of TR activating factors and TR inhibitory factors in a way that causes TR in tissues that do not have native TR capacity. The iTR factor also refers to a combination of individual factors. Therefore, the mixture of factors described herein (including but not limited to a mixture named AgeX1547) is considered to be an "iTR factor" in this application.

[0098] The term "iTR gene" refers to a gene that can cause such regeneration in tissues that are usually unable to cause induced tissue regeneration when expression changes. The term "nucleic acid" can be used interchangeably with "polynucleotide", and in various embodiments comprises naturally occurring nucleoside polymers (e.g., DNA and RNA) and non-naturally occurring nucleoside polymers or nucleoside analogs. In certain embodiments, nucleic acids comprise standard nucleosides (abbreviated A, G, C, T, U). In other embodiments, nucleic acids comprise one or more non-standard nucleosides. In certain embodiments, one or more nucleosides are non-naturally occurring nucleosides or nucleotide analogs. Nucleic acids may comprise modified bases (e.g., methylated bases), modified sugars (2'-fluororibose, arabinose or hexose), modified phosphate groups or other bonds between nucleosides or nucleoside analogs (e.g., phosphorothioate or 5'-N-phosphoramidite bonds), locked nucleic acids or morpholinos. In certain embodiments, nucleic acids comprise nucleosides connected by phosphodiester bonds, such as in DNA and RNA. In certain embodiments, at least some nucleosides are connected by non-phosphodiester bonds. Nucleic acids can be single-stranded, double-stranded, or partially double-stranded. At least partially double-stranded nucleic acids can have one or more overhangs, such as 5' and / or 3' overhangs. It is contemplated that nucleic acid modifications (e.g., nucleoside and / or backbone modifications, including the use of non-standard nucleosides) known in the art for research or therapeutic purposes in the context of RNA interference (RNAi), aptamers, or antisense molecules are used in various embodiments of the present invention. See, for example, Crooke, ST (editor) Antisense drug technology: principles, strategies, and applications, Boca Raton: CRC Press, 2008; Kurreck, J. (editor) Therapeutic oligonucleotides, RSC biomolecular sciences. Cambridge: Royal Society of Chemistry, 2008. In certain embodiments, the modification increases the half-life and / or stability of the nucleic acid (e.g., in vivo) relative to RNA or DNA of the same length and chain length. In certain embodiments, the modification reduces the immunogenicity of the nucleic acid relative to RNA or DNA of the same length and chain length. In some embodiments, 5% to 95% of the nucleosides in one or both strands of the nucleic acid are modified. The modifications can be distributed evenly or unevenly, and the position of the modification (e.g., near the middle, near the end or end, alternately, etc.) can be selected to enhance the desired characteristics. The nucleic acid can include detectable labels, such as fluorescent dyes, radioactive atoms, etc. "Oligonucleotide" refers to a relatively short nucleic acid, such as a length of generally about 4 to about 60 nucleotides.Where polynucleotides are mentioned herein, it is understood that both DNA, RNA, and in each case single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" may refer to the polynucleotide material itself and / or to sequence information (i.e., a sequence of letters used as abbreviations for bases) that biochemically characterizes a particular nucleic acid. Unless otherwise indicated, the polynucleotide sequences presented herein are presented in a 5' to 3' direction.

[0099] The term "oligoclonal" refers to a population of cells that is derived from a small population of cells, typically 2-1000 cells that appear to have similar characteristics, such as morphology or the presence or absence of differentiation markers that are distinct from those of other cells in the same culture. Oligoclonal cells are isolated from cells that do not share these common characteristics and allowed to proliferate, thereby producing a population of cells that is essentially completely derived from the original population of similar cells.

[0100] The term "pluripotent stem cell" refers to an animal cell that can differentiate into more than one differentiated cell type. Such cells include hES cells, blastomere / morula cells and their derived hED cells, hiPS cells, hEG cells, hEC cells, and adult-derived cells (including mesenchymal stem cells, neuronal stem cells, and stem cells derived from bone marrow). Pluripotent stem cells may or may not be genetically modified. Genetically modified cells may include markers (e.g., fluorescent proteins) to facilitate their identification in eggs.

[0101] 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 between about 2 and 60 amino acids in length. The polypeptides used herein typically contain standard amino acids (i.e., the 20 most common L-amino acids in proteins). However, in certain embodiments, the polypeptide may contain one or more non-standard amino acids (which may be naturally occurring or non-naturally occurring) and / or amino acid analogs known in the art. One or more amino acids in a polypeptide may be modified, for example, by the addition of chemical entities (e.g., carbohydrate groups, phosphate groups, fatty acid groups, linkers) for conjugation, functionalization, etc. A polypeptide having a non-polypeptide portion covalently or non-covalently bound thereto is still considered a "polypeptide". Polypeptides can be purified from natural sources, produced using recombinant DNA technology, and synthesized by chemical means (e.g., conventional solid phase peptide synthesis, etc.). As used herein, the term "polypeptide sequence" or "amino acid sequence" may refer to the polypeptide material itself and / or the sequence information (i.e., a continuous letter or three-letter code used as an abbreviation for the amino acid name) that characterizes the polypeptide biochemically. Unless otherwise indicated, the polypeptide sequences presented herein are presented in the direction of N-terminus to C-terminus. The polypeptide may be cyclic or contain a cyclic portion. When discussing naturally occurring polypeptides herein, it is understood that the present invention includes embodiments related to any isomer thereof (e.g., different proteins produced from the same gene due to selective splicing or editing of mRNA or due to different alleles of a gene (e.g., alleles with one or more single nucleotide polymorphisms (usually such alleles have at least 95%, 96%, 97%, 98%, 99% or more identity with a reference sequence or a common sequence)). The polypeptide may include sequences targeting its secretion or a specific intracellular compartment (e.g., the nucleus) and / or sequences targeting the polypeptide for post-translational modification or degradation. Certain polypeptides may be synthesized as precursors, which are post-translationally cleaved or otherwise processed into mature polypeptides. In some cases, this cleavage may occur only after a specific activation event. In relevant cases, the present invention provides embodiments related to precursor polypeptides and embodiments related to mature versions of polypeptides.

[0102] The term "prenatal" refers to the stage of embryonic development in placental mammals before which the animal cannot survive outside the uterus.

[0103] The term "primitive stem cells" refers to pluripotent stem cells that can differentiate into cells of all three major germ layers (endoderm, mesoderm and ectoderm) and neural crest. Therefore, examples of primordial stem cells include, but are not limited to, ES cells or cell lines, blastomere / morula cells and ED cells derived therefrom, iPS and EG cells of humans or non-human mammals.

[0104] The term "purified" refers to an agent or entity (e.g., a compound) that has been separated from most of the components associated with it in nature or when it was originally produced. Typically, such purification involves the action of a human hand. A purified agent or entity can be partially purified, substantially purified, or pure. Such agents or entities can be, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% pure. In certain embodiments, a nucleic acid or polypeptide is purified so that it constitutes at least 75%, 80%, 855%, 90%, 95%, 96%, 97%, 98%, 99% or more of the total amount of nucleic acid or polypeptide material present in the preparation, respectively. Purity can be based on, for example, dry weight, the size of the peak on the chromatographic trace, molecular abundance, the intensity of the band on the gel, or the intensity of any signal associated with molecular abundance, or any quantitative method recognized in the art. In some embodiments, water, buffer, ions and / or small molecules (e.g., precursors such as nucleotides or amino acids) may optionally be present in the purified preparation. Purified molecules can be prepared by separating them from other substances (e.g., other cellular substances) or by producing them in a manner to reach a desired purity. In some embodiments, purified molecules or compositions refer to molecules or compositions prepared using any art-recognized purification method. In some embodiments, "partial purification" refers to that the molecules produced by cells are no longer present in the cell, for example, the cell has been lysed, and optionally, at least some cellular substances (e.g., cell walls, cell membranes, organelles) have been removed.

[0105] The term "RNA interference" (RNAi) is used herein in accordance with its meaning in the art to refer to a phenomenon whereby a double-stranded RNA (dsRNA) triggers sequence-specific degradation or translational inhibition of a corresponding mRNA that is complementary to the dsRNA strand. It should be understood that the complementarity between the dsRNA strand and the mRNA need not be 100%, but only needs to be sufficient to mediate inhibition of gene expression (also referred to as "silencing" or "knockdown"). For example, the degree of complementarity enables the strand to (i) guide the cleavage of the mRNA in the RNA-induced silencing complex (RISC); or (ii) cause translational inhibition of the mRNA. In certain embodiments, the length of the double-stranded portion of the RNA is less than about 30 nucleotides, for example, between 17 and 29 nucleotides in length. In certain embodiments, the first strand of the dsRNA has at least 80%, 85%, 90%, 95% or 100% complementarity with the target mRNA, and the other strand of the dsRNA has at least 80%, 85%, 90%, 95% or 100% complementarity with the first strand. In mammalian cells, RNAi can be achieved by introducing appropriate double-stranded nucleic acids into cells or expressing nucleic acids in cells (which are then processed in cells to produce dsRNA therein). Nucleic acids capable of mediating RNAi are referred to herein as "RNAi agents". Exemplary nucleic acids capable of mediating RNAi are short hairpin RNA (shRNA), short interfering RNA (siRNA) and microRNA (microRNA) precursors.

[0106] These terms are well known and are used herein with the meaning consistent with their meaning in the art. siRNA generally comprises two independent nucleic acid chains, which are hybridized to form a duplex. They can be synthesized in vitro, for example, by using standard nucleic acid synthesis techniques. siRNA is generally a double-stranded oligonucleotide, and each chain has 16-30 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides (nt)), wherein the double-stranded oligonucleotide comprises a double-stranded portion of length between 15 and 29 nucleotides, and any one or both chains can comprise, for example, a 3' overhang of length between 1-5 nucleotides, or either or both ends can be blunt. In some embodiments, siRNA is included in a chain between 19 and 25nt (e.g., a chain between 21 and 23 nucleotides), wherein one or both chains comprise a 3' overhang of 1-2 nucleotides. One strand of the double-stranded portion of the siRNA (referred to as the "guide strand" or "antisense strand") is substantially complementary (e.g., at least 80% or more (e.g., 85%, 90%, 95% or 100%)) or complementary (e.g., has 3, 2, 1 or 0 mismatched nucleotides) to the target region in the mRNA, while 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 the mRNA, while the other passenger strand is 100% complementary to the first double-stranded portion (it should be understood that in various embodiments, if the 3' overhang of the guide strand is present, the 3' overhang may or may not be complementary to the mRNA when the guide strand hybridizes with the mRNA). In certain embodiments, the shRNA molecule is a nucleic acid molecule comprising a stem-loop, wherein the double-stranded stem is 16-30 nucleotides in length and the loop is about 1-10 nucleotides in length. The siRNA may contain a variety of modified nucleosides, nucleoside analogs, and may contain chemically or biologically modified bases, modified backbones, and the like. Without limitation, any modification recognized in the art as being useful for RNAi can be used. Certain modifications result in increased stability, cellular uptake, efficacy, etc. Certain modifications result in reduced immunogenicity or clearance. In some embodiments, siRNA comprises a duplex of about 19-23 (e.g., 19, 20, 21, 22, or 23) nucleotides in length, and optionally one or two 3' overhangs of 1-5 nucleotides in length, which can be composed of deoxyribonucleotides. shRNA comprises a nucleic acid chain comprising two complementary parts, which are mainly separated by non-self-complementary regions. The complementary parts hybridize to form a duplex structure, and the non-self-complementary regions form a loop connecting the 3' end of a chain of the duplex and the 5' end of another chain. shRNA undergoes intracellular processing to generate siRNA. Typically, the length of the loop is 1 to 8 (e.g., 2-6) nucleotides.

[0107] MicroRNA (miRNA) is a small natural non-coding single-stranded RNA of about 21 to 25 nucleotides (in mammalian systems) that inhibits gene expression in a sequence-specific manner. They are produced in cells from precursors (pre-miRNA) that have a characteristic secondary structure consisting of a short hairpin (about 70 nucleotides in length), which contains a duplex that typically contains one or more regions of imperfect complementarity, which in turn is produced by a larger precursor (pri-miRNA). Naturally occurring miRNAs are typically only partially complementary to their target mRNAs and typically work by translational inhibition. RNAi agents modeled after endogenous miRNAs or miRNA precursors can be used in certain embodiments of the present invention. For example, siRNAs can be designed to hybridize one strand with a target mRNA, with one or more mismatches or protrusions that mimic the duplex formed by the miRNA and its target mRNA. Such siRNAs can be referred to as miRNA mimics or miRNA-like molecules. MiRNA mimics can be encoded by a precursor nucleic acid whose structure mimics the structure of a naturally occurring miRNA precursor.

[0108] In certain embodiments, the RNAi agent is a vector (e.g., a plasmid or a virus) comprising a template for the transcription of a siRNA (e.g., as two independent chains that can hybridize to each other), a shRNA, or a microRNA precursor. As known in the art, typically, the template encoding the siRNA, shRNA, or miRNA precursor is operably connected to an expression control sequence (e.g., a promoter). Such vectors can be used to introduce the template into vertebrate cells (e.g., mammalian cells) and cause transient or stable expression of siRNA, shRNA, or miRNA precursors. The precursor (shRNA or miRNA precursor) is processed in the cell to produce siRNA or miRNA.

[0109] Typically, small RNAi agents (e.g., siRNA) can be chemically synthesized, or can be transcribed in vivo or in vitro from a DNA template into two independent strands that are then hybridized, or processed as shRNA to generate siRNA. Typically, RNAi agents, particularly those containing modified RNAi agents, are chemically synthesized. Chemical synthesis methods for oligonucleotides are well known in the art.

[0110] As used herein, the term "small molecule" is an organic molecule with a mass less than about 2 kilodaltons (KDa). In some embodiments, the small molecule is less than about 1.5KDa, or less than about 1KDa. In some embodiments, the small molecule is less than about 800 Daltons (Da), 600Da, 500Da, 400Da, 300Da, 200Da or 100Da. Generally, the mass of a small molecule is at least 50Da. In some embodiments, the small molecule comprises a plurality of carbon-carbon bonds and may comprise one or more heteroatoms and / or one or more functional groups important for structural interactions (e.g., hydrogen bonds) with proteins, such as amines, carbonyls, hydroxyls or carboxyls, and in some embodiments, at least two functional groups. The small molecule typically comprises one or more cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures optionally replaced by one or more of the above-mentioned functional groups. In some embodiments, the small molecule is non-polymeric. 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. The term "subject" can be any multicellular animal. The subject is typically a vertebrate, such as a mammal or bird. 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. Typically, a subject is an individual to whom a compound is delivered (e.g., for experimental, diagnostic and / or therapeutic purposes) or from whom a sample is obtained or a diagnostic procedure is performed (e.g., a sample or procedure to be used to assess tissue damage and / or assess the effects of the compounds of the invention). The term "tissue damage" as used herein refers to any type of damage or injury to a cell, tissue, organ, or other body structure. The term includes, in various embodiments, degeneration due to disease, damage due to physical trauma or surgery, damage due to exposure to harmful substances, and other destruction of the structure and / or function of a cell, tissue, organ, or other body structure.

[0111] The term "tissue regeneration" or "TR" refers to the at least partial regeneration, replacement, restoration or regrowth of a tissue, organ or other body structure or a portion thereof after loss, damage or deformation, wherein the tissue regeneration would not occur unless it occurred in the manner described in the present invention. Examples of tissue regeneration include the regrowth of severed fingers or limbs, including the regrowth of cartilage, bone, muscle, tendons and ligaments, scarless regrowth of bone, cartilage, skin or muscle lost due to injury or disease, which is accompanied by an increase in the size and number of cells of the injured or diseased organ so that the tissue or organ approaches the normal size of the tissue or organ or its size before the injury or disease. Depending on the tissue type, tissue regeneration can occur by a variety of different mechanisms (e.g., rearrangement of pre-existing cells and / or tissues (e.g., by cell migration), division of adult somatic stem cells or other progenitor cells and differentiation of at least some of their progeny, and / or dedifferentiation, transdifferentiation and / or proliferation of cells).

[0112] The term "TR activator gene" refers to a gene that lacks expression in fetal and adult cells but whose expression during embryonic development promotes TR.

[0113] The term "TR inhibitor gene" refers to a gene whose expression in fetal and adult animals inhibits TR.

[0114] The terms "treatment," "therapy," "therapeutic," and similar terms with respect to a subject refer to providing medical and / or surgical care to the subject. Treatment may include, but is not limited to, administering a compound or composition (e.g., a pharmaceutical composition) to a subject. Treatment of a subject according to the present invention is typically performed in an effort to promote regeneration, such as in a subject that has suffered tissue damage or is expected to suffer tissue damage (e.g., a subject that will undergo surgery). The effects of treatment may typically include increased regeneration after tissue regeneration, reduced scarring, and / or improved structural or functional outcomes (compared to outcomes without treatment), and / or may include reversal or alleviation of the severity or progression of a degenerative disease.

[0115] The term "variant" as applied to a particular polypeptide refers to a polypeptide that is different from the polypeptide (sometimes referred to as the "original polypeptide") by changes (e.g., additions, deletions and / or substitutions) of one or more amino acids. Sometimes, the original polypeptide is a naturally occurring polypeptide (e.g., a polypeptide from a human or non-human animal) or a polypeptide identical thereto. Variants may be naturally occurring or produced using, for example, recombinant DNA technology or chemical synthesis. Additions may be insertions within the polypeptide or additions at the N- or C-termini. In certain embodiments, the number of amino acids substituted, deleted or added may be, for example, about 1 to 30, such as about 1 to 20, such as about 1 to 10, such as about 1 to 5, such as 1, 2, 3, 4 or 5. In certain embodiments, the variant comprises a polypeptide whose sequence is homologous to the sequence of the original polypeptide (but not identical in sequence to the original polypeptide) over at least 50 amino acids, at least 100 amino acids, at least 150 amino acids or more over the entire length of the original polypeptide, for example, the sequence of the variant polypeptide has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of the original polypeptide over at least 50 amino acids, at least 100 amino acids, at least 150 amino acids or more over the entire length of the original polypeptide. In some embodiments, variants comprise a sequence with the original polypeptide having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the original polypeptide length, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5% or higher sequence identity. In some embodiments, variants comprise at least one function or domain, such as a domain so identified in the Conserved Domain Database (CDD) of the National Center for Biotechnology Information (www.ncbi.nih.gov) (e.g., a domain curated by NCBI).

[0116] In some embodiments, one, more than one or all biological functions or activities of variants or fragments are basically similar to the corresponding biological functions or activities of the original molecule. In some embodiments, functional variants retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher activity of the original polypeptide, such as approximately equal activity. In some embodiments, the activity of the variant is up to about 100%, about 125% or about 150% of the activity of the original molecule. In other non-limiting embodiments, if the amount or concentration of the variant required for a specific effect is within 0.5 to 5 times of the amount or concentration of the original molecule required for the effect, the activity of the variant or fragment is considered to be basically similar to the activity of the original molecule.

[0117] In certain embodiments, the amino acid "replacement" in the variant is the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, i.e., conservative amino acid replacement. "Conservative" amino acid replacement can be performed based on the similarity of any of the various properties (e.g., side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity) of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. Polar (hydrophilic), neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Certain substitutions within a particular group, such as the substitution of isoleucine for leucine (or vice versa), the substitution of threonine for serine (or vice versa), or the substitution of glycine for alanine (or vice versa), may be of particular interest. Of course, non-conservative substitutions are also generally compatible with retained function. In certain embodiments, a substitution or deletion does not alter or delete an amino acid that is important for activity. Insertions or deletions may range in size from about 1 to 20 amino acids, such as 1 to 10 amino acids. In some cases, larger domains may be deleted without substantially affecting function. In certain embodiments of the invention, the sequence of a variant may be obtained by additions, deletions or substitutions of no more than 5, 10, 15 or 20 amino acids in total to the sequence of a native enzyme. In certain embodiments, no more than 1%, 5%, 10% or 20% of the amino acids in a polypeptide are insertions, deletions or substitutions relative to the original polypeptide. Guidance in determining which amino acid residues can be substituted, added or deleted without eliminating or substantially reducing the activity of interest can be obtained by comparing the sequence of a particular polypeptide to sequences of homologous polypeptides (e.g., from other organisms) and minimizing the number of amino acid sequence changes in regions of high homology (conserved regions) or by replacing amino acids with amino acids in homologous sequences, since amino acid residues that are conserved across species are more likely to be important for activity than amino acids that are not conserved.

[0118] In some embodiments, the variant of the polypeptide comprises a heterologous polypeptide portion. The heterologous portion generally has a sequence that is not present in the original polypeptide or is not homologous to the original polypeptide. The length of the heterologous portion can be, for example, between 5 and about 5,000 amino acids or longer. Typically, its length is between 5 and 1,000 amino acids. In some embodiments, the heterologous portion comprises a sequence found in different polypeptides (e.g., a functional domain). In some embodiments, the heterologous portion comprises a sequence for purification, expression, dissolution and / or detection of a polypeptide. In some embodiments, the heterologous portion comprises a polypeptide "tag", such as an affinity tag or an epitope tag. For example, the tag can be an affinity tag (e.g., HA, TAP, Myc, 6xHis, Flag, GST), a fluorescent or luminescent protein (e.g., EGFP, ECFP, EYFP, Cerulean, DsRed, mCherry), a solubility-enhanced tag. (e.g., a monomeric mutant of the Ocr protein of a SUMO tag, a NUSA tag, a SNUT tag, or bacteriophage T7). See, e.g., Esposito D and Chatterjee D K. Curr Opin Biotechnol.; 17(4): 353-8 (2006). In some embodiments, tags can serve multiple functions. Tags are typically relatively small, e.g., from a few amino acids to about 100 amino acids long. In some embodiments, tags are longer than 100 amino acids, e.g., up to about 500 amino acids long or longer. In some embodiments, a polypeptide has a tag located at the N- or C-terminus, e.g., as an N- or C-terminal fusion. A polypeptide can contain multiple tags. In some embodiments, for example, a 6xHis tag and a NUS tag are present at the N-terminus. In some embodiments, the tag is cleavable so that it can be removed from the polypeptide, e.g., by a protease. In some embodiments, this is achieved by including a sequence encoding a protease cleavage site between the sequence encoding a portion homologous to the original polypeptide and the tag. Exemplary proteases include, e.g., thrombin, TEV protease, factor Xa, PreScission protease, etc. In some embodiments, a "self-cleaving" tag is used. See, e.g., PCT / US05 / 05763. The sequence encoding the tag can be located 5' or 3' relative to the polynucleotide encoding the polypeptide (or both). In certain embodiments, the tag or other heterologous sequence is separated from the rest of the polypeptide by a polypeptide linker. For example, the linker can be a short polypeptide (e.g., 15-25 amino acids). The linker is typically composed of small amino acid residues (e.g., serine, glycine, and / or alanine). The heterologous domain can include a transmembrane domain, a secretion signal domain, and the like.

[0119] In certain embodiments of the present invention, if a fragment or variant (optionally excluding a heterologous portion) exists, it has enough 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 overlapping volume is at least 70%, preferably at least 80%, and more preferably at least 90% of the total volume of the original polypeptide structure. The partial or entire three-dimensional structure of a fragment or variant can be determined by using standard methods to crystallize the protein. Alternatively, standard methods can also be used to generate NMR solution structures. Modeling programs such as MODELER (Sali, A. and Blundell, TL, J. Mol. Biol., 234, 779-815, 1993) or any other modeling programs can be used to generate predicted structures. If there is a structure or predicted structure of a related polypeptide, the model can be based on the structure. The PROSPECT-PSPP program suite can be used (Guo, JT et al., Nucleic Acids Res. 32 (Web Server issue): W522-5, July 1, 2004). When an embodiment of the present invention relates to a variant of a polypeptide, it is understood that a polynucleotide encoding the variant is provided.

[0120] The term "vector" as used herein refers to a nucleic acid or virus or part thereof (e.g., a viral capsid or genome) that can mediate the entry of a nucleic acid molecule into (e.g., transfer, transport, etc.) a cell. When the vector is a nucleic acid, the nucleic acid molecule to be transferred is usually connected to the vector nucleic acid molecule, for example, inserted into the vector nucleic acid molecule. The nucleic acid vector may include a sequence that directs autonomous replication (e.g., an origin of replication), or may include a sequence sufficient to allow some or all of the nucleic acid to be integrated into the host cell DNA. Useful nucleic acid vectors include, for example, DNA or RNA plasmids, cosmids, and naturally occurring or modified viral genomes or parts thereof or nucleic acids (DNA or RNA) that can be packaged into viral capsids. Plasmid vectors typically include an origin of replication and one or more selectable markers. Plasmids may include part or all of the viral genome (e.g., viral promoters, enhancers, processing or packaging signals, etc.). Viruses or parts thereof that can be used to introduce nucleic acid molecules into cells are referred to as viral vectors. Useful viral vectors include adenoviruses, adeno-associated viruses (e.g., AAV8, retroviruses, lentiviruses, vaccinia viruses, and other poxviruses), herpes viruses (e.g., herpes simplex viruses), and others. When introduced into a host cell, a viral vector may or may not contain enough viral genetic information to produce infectious viruses, i.e., the viral vector may be replication-defective, and such replication-defective viral vectors may be preferred for therapeutic use. In the absence of sufficient information, it may be provided by a host cell or another vector introduced into the cell, but not necessarily. The nucleic acid to be transferred may be introduced into a naturally occurring or modified viral genome or part thereof, or it may be present in a virus or viral capsid as a separate nucleic acid molecule. It should be understood that some plasmid vectors comprising part or all of the viral genome (generally comprising enough to instruct the transcription of nucleic acids that can be packaged into the viral capsid and / or enough to produce viral genetic information that can be integrated into the host cell genome and / or cause infectious viruses) are sometimes also referred to as viral vectors in the art. The vector may include one or more nucleic acids encoding markers suitable for use in identifying and / or selecting cells that have or have not been transformed or transfected by the vector. Markers include, for example, proteins that increase or decrease resistance or sensitivity to antibiotics (e.g., antibiotic resistance genes encoding proteins that confer resistance to antibiotics, such as puromycin, hygromycin, or blasticidin) or other compounds, enzymes (e.g., beta-galactosidase or alkaline phosphatase) that are active and can be determined by assays known in the art, and proteins or RNAs (e.g., fluorescent proteins) that detectably affect the phenotype of transformed or transfected cells. An expression vector is a vector comprising a regulatory sequence (e.g., an expression control sequence (e.g., a promoter)) sufficient to direct transcription of an operably linked nucleic acid. Regulatory sequences may also include enhancer sequences or upstream activator sequences. The vector may optionally include a 5' leader sequence or a signal sequence. The vector may optionally include a cleavage and / or polyadenylation signal and / or a 3' untranslated region.The vector generally includes one or more appropriately positioned restriction enzyme sites to facilitate the introduction of the nucleic acid to be expressed into the vector. The expression vector contains sufficient cis-acting elements for expression. The host cell or in vitro expression system may provide other elements required for or conducive to expression.

[0121] Nucleic acid molecules can be introduced into cells using various techniques. Such techniques include chemically promoted transfection using compounds (e.g., calcium phosphate, cationic lipids, cationic polymers), liposome-mediated transfection, non-chemical methods (e.g., electroporation, particle bombardment, or microinjection), and infection using viruses containing nucleic acid molecules of interest (sometimes referred to as "transduction"). Markers can be used to identify and / or select cells that have taken up the vector and generally express the nucleic acid. Cells can be cultured in a suitable culture medium to select such cells, and optionally establish stable cell lines.

[0122] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as they can certainly vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention, as the scope of the present invention is limited only by the appended claims.

[0123] When providing a numerical range, it is understood that unless the context clearly dictates otherwise, each intermediate value should be up to one-tenth of the lower limit unit and between the upper and lower limits of the range, and any other stated values ​​or intermediate values ​​within the range are included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also included in the present invention, subject to any explicitly excluded limitations in the range. Where the range includes one or two limits, ranges excluding one or both of those included limits are also included in the present invention.

[0124] Provided herein are certain ranges with numerical values ​​preceded by the term "about". The term "about" is used herein to provide textual support for the exact number that follows and the number that is close to or approximate to the number that follows the term. When determining whether a number is close to or approximate to a specifically listed number, the close or approximate unlisted number can be a number that provides a number that is substantially equivalent to the specifically listed number in the context in which the number is given. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by a person of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar to or equivalent to those methods and materials described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0125] All publications and patents cited in this specification are incorporated herein by reference, just as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications. The citation of any publication is for the purpose of its disclosure prior to the filing date, and should not be construed as an admission that the present invention is not entitled to antedate the publication by virtue of prior invention. In addition, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed.

[0126] It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for using exclusive terminology such as "soley" or "only" in connection with the recitation of claim elements or for using a "negative" limitation.

[0127] It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0128] method

[0129] In addition to the methods described below, methods for producing and using cells having embryonic gene expression patterns corresponding to scarless regenerative potential can be found in: PCT Application Serial No. PCT / US2006 / 013519, entitled "Novel Uses of Cells With Prenatal Patterns of Gene Expression," filed April 11, 2006; and PCT Application Serial No. PCT / US2006 / 013519, entitled "Methods to Accelerate the Isolation of Novel Cell Strains from Pluripotent Stem Cells and Cells Obtained Therefrom," filed November 21, 2006. No. 11 / 604,475, filed on July 16, 2009, entitled "Methods for Accelerating the Isolation of Novel Bacterial Strains from Pluripotent Stem Cells and Cells Derived Therefrom" (see, e.g., U.S. Provisional Patent Application No. 61 / 831,421, filed on June 5, 2013, PCT Patent Application No. PCT / US2014 / 040601, filed on June 3, 2014, and U.S. Patent Application No. 14 / 896,664, filed on December 7, 2015, the entire contents of which are incorporated herein by reference); "Compositions and Methods for Induced Tissue Regeneration in Mammalian Species" (International Patent Application Publication No. WO 2014 / 197421) (the entire contents of which are incorporated herein by reference); and "Improved Methods for Detecting and Regulating Embryonic-Fetal Transition in Mammalian Species" (International Patent Application Publication No. WO 2014 / 197421). 2017 / 214342, the entire contents of which are incorporated herein by reference), the entire contents of each of which are incorporated herein by reference.

[0130] RNAi

[0131] As a non-limiting example, dsRNA was prepared by in vitro transcription reaction (Promega) using a PCR generated template with flanking T7 promoters, purified by phenol extraction and ethanol precipitation, and annealed after resuspension in water. Starting with the first injection two hours after surgery, 4 x 30 nL of dsRNA was injected into intact experimental animals for three consecutive days after induced tissue injury.

[0132] TR regulator and iTR regulator

[0133] The present disclosure provides novel iTR modulators and methods of using the same. In certain aspects, the present invention provides novel methods of enhancing regeneration, comprising administering to a multicellular organism in need thereof an agent that alters the concentration of the iTR modulator.

[0134] Applicants teach that primitive animals that display profound TR potential (e.g., amputation regeneration in axolotls, skin regeneration in MRL or African spiny mice, or regeneration of whole body segments in planarians) do so by simply recapitulating normal embryonic development of the respective tissues. In addition, Applicants teach that the reason for the inability to regenerate damaged tissues in TR-resistant mammals (e.g., most mouse species and humans) is that certain embryonic gene transcription in the EFT in these TR-resistant animals is altered. Applicants further teach that restoration of certain of these altered embryo-specific gene expression patterns in the EFT in TR-resistant animals can induce regenerative capacity in any tissue, including responsiveness to organizing central factors, resulting in a reduction in complex tissue regeneration and attendant scarring. Finally, Applicants teach novel agents and related methods for inducing TR in mammalian species. The methods promote TR in mammalian species, particularly in Homo sapiens species.

[0135] Genes whose expression inhibits TR in fetal and adult animals are referred to herein as "TR inhibitors", and genes that lack expression in fetal and adult cells but whose expression during embryonic development promotes TR are referred to herein as "TR activators". In general, TR inhibitor genes and TR activator genes are referred to herein as iTR genes. Molecules that change the levels of TR activators and TR inhibitors in a manner that causes TR are referred to herein as "iTR factors". iTR genes and protein products of iTR genes are generally conserved in animals from sea anemones to mammals. The protein sequences encoded by the genes referred to herein and the nucleic acid (e.g., mRNA) sequences encoding the genes (including sequences from many different non-human animal species) are known in the art and can be found in, for example, public databases (e.g., databases provided at the National Center for Biotechnology Information (NCBI) (www.ncbi.nih.gov)).

[0136] The TR inhibitory gene COX7A1 was observed to be expressed primarily in stromal cells rather than epithelial cells in normal tissues, although it was also expressed at lower levels in epithelial cultures. In the case of tumors, the gene is downregulated in many stromal cancers (e.g., osteosarcoma, chondrosarcoma, rhabdomyosarcoma) and certain gliomas, carcinomas, and adenocarcinomas. This is consistent with the observed increase in glycolysis in cancers known as the Warburg effect, although the Warburg effect has not previously been implicated in the absence of COX7A1 expression. Because the applicants propose that changes in the TR gene during the transition from embryonic to fetal development are partly to prevent adult cancers, inhibition of COX7A1 in the stroma and certain central nervous system and epithelial tumors would restore stromal cells to an embryonic state, thereby promoting tumorigenesis. Therefore, exogenous induction of COX7A1 expression in such tumors lacking expression would have a therapeutic effect, in part by altering the activity of p53 and HIF1α and HIF3α, thereby inhibiting cancer cell proliferation and increasing apoptosis.

[0137] In another embodiment, the invention provides a means for detecting cancer cells. Researchers rarely identify abnormal markers associated with most cancer cell types. As described herein, markers that distinguish embryos from their fetuses and adult counterparts can be used to distinguish normal cells showing adult expression patterns from malignant cells showing embryonic patterns. The detection method (including but not limited to COX7A1, NAALADL1, AMH and genes (including but not limited to PCDHA4, PCDHB2 and PCDHGA12) from the protocadherin genes of α, β and γ clusters in embryonic patterns rather than fetal / adult patterns) can be used not only for identifying the expression of malignant cells (except blood cells), but also for identifying tumors resistant to conventional chemotherapeutic agents characterized by fetal / adult expression patterns.

[0138] The present disclosure provides a variety of different methods for regulating iTR genes and a variety of compounds that can be used to regulate iTR genes. Generally, iTR factors can be, for example, small molecules, nucleic acids, oligonucleotides, polypeptides, peptides, lipids, carbohydrates, etc. The iTR factor can be one of the TR factors listed in Figure 8 or a combination thereof. The factor combination can be screened for its effects on mammalian cells (preferably human cells corresponding to fetal or adult developmental stages (e.g., human cells expressing COX7A1)) and determine the iTR preparation that causes significantly reduced COX7A1, ADIRF, TNFRSF11B expression or significantly increased AMH expression to determine the factor concentration and the best combination.

[0139] In certain embodiments of the invention, the iTR factor is inhibited by reducing the amount of TR inhibitor RNA produced by the cell and / or by reducing the activity level of the TR inhibitor gene. In the case of targeting the TR inhibitor, factors that reduce the level of the TR inhibitor gene product are determined and used in research and treatment. The TR inhibitor gene can be any one or combination of TR inhibitor genes previously disclosed in, for example, WO 2017 / 214342 or WO 2014 / 197421. The TR inhibitor gene can be any one or combination of TR inhibitor genes. The amount of TR inhibitor gene RNA can be reduced by inhibiting the synthesis of TR inhibitor RNA of the cell (also referred to as "inhibiting TR inhibitor gene expression"), for example, by reducing the amount of mRNA encoding the TR inhibitor gene or reducing the translation of mRNA encoding the TR inhibitor gene. By way of non-limiting example, the factor may be RNAi targeting a sequence within a TR inhibitor gene.

[0140] In certain embodiments, TR inhibitor gene expression is inhibited by RNA interference (RNAi). As is known in the art, RNAi is a process in which the presence of double-stranded RNA with sequence correspondence to a gene in a cell generally results in sequence-specific inhibition of gene expression, which is generally the result of cleavage or translational inhibition of mRNA transcribed from the gene. Compounds that can be used to cause inhibition of RNAi expression ("RNAi agents") include short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), and miRNA-like molecules.

[0141] Once a mammalian TR inhibitor gene (e.g., a human TR inhibitor gene) has been identified, one skilled in the art can easily design the sequence of an RNAi agent (e.g., siRNA) for inhibiting expression of the TR inhibitor gene. In certain embodiments, such sequences are selected to minimize "off-target" effects. For example, a sequence that is complementary to a sequence present in the TR inhibitor gene mRNA and that is not present in other mRNAs expressed in the species of interest (or a sequence that is not present in the genome of the species of interest) can be used. Chemical modifications at specific positions can be used to reduce potential off-target effects. In certain embodiments, at least two different RNAi agents, such as siRNA, targeting TR inhibitor gene mRNA are used in combination. In certain embodiments, microRNA (which may be an artificially designed microRNA) is used to inhibit TR inhibitor gene expression.

[0142] In certain embodiments of the present invention, an antisense molecule comprising a single-stranded oligonucleotide that is completely or substantially complementary to the mRNA encoding the TR inhibitor gene is used to inhibit the expression of the TR inhibitor gene. The oligonucleotide hybridizes with the TR inhibitor gene mRNA, causing, for example, degradation of the mRNA by RNase H or blocking of translation by steric hindrance. In other embodiments of the present invention, ribozymes or triplex nucleic acids are used to inhibit the expression of the TR inhibitor gene.

[0143] In certain embodiments of the invention, the inhibitor of the TR inhibitor inhibits at least one activity of the TR inhibitor protein. The activity of the TR inhibitor can be reduced by contacting the TR inhibitor protein with a compound that physically interacts with the TR inhibitor protein. Such a compound can, for example, change the structure of the TR inhibitor protein (for example, by covalently modifying it) and / or block the interaction of the TR inhibitor protein with one or more other molecules (for example, cofactors or substrates). In certain embodiments, the inhibition or reduction can 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 reference level (for example, a control level). The control level can be the level of the TR inhibitor that occurs in the absence of the factor. For example, a TR factor may reduce the level of a TR inhibitor protein to no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 25%, 20%, 10%, or 5% of the level that occurs under the conditions tested in the absence of the factor. In certain embodiments, the level of a TR inhibitor is reduced to 75% or less of the level that occurs under the conditions tested in the absence of the factor. In certain embodiments, the level of a TR inhibitor is reduced to 50% or less of the level that occurs under the conditions tested in the absence of the factor. In certain embodiments, the level of a TR inhibitor is reduced to 25% or less of the level that occurs under the conditions tested in the absence of the factor. In certain embodiments, the level of a TR inhibitor is reduced to 10% or less of the level that occurs under the conditions tested in the absence of the factor. In some cases, the modulation (e.g., inhibition or reduction) level is statistically significant compared to the control level. As used herein, "statistically significant" refers to a p-value less than 0.05 (eg, a p-value less than 0.025 or a p-value less than 0.01) using an appropriate statistical test (eg, ANOVA, t-test).

[0144] In certain embodiments of the invention, the compound directly inhibits the TR inhibitor protein, i.e., the compound inhibits the TR inhibitor protein by a mechanism involving physical interaction (binding) between the TR inhibitor and the iTR factor. For example, the binding of the TR inhibitor to the iTR factor can interfere with the ability of the TR inhibitor to catalyze the reaction and / or can block the active site of the TR inhibitor. A variety of compounds can be used to directly inhibit the TR inhibitor. Exemplary compounds that directly inhibit the TR inhibitor can be, for example, small molecules, antibodies, or aptamers.

[0145] In certain embodiments of the present invention, the iTR factor is covalently bound to the TR inhibitor. For example, the compound can modify the amino acid residues required for enzyme activity. In certain embodiments, the iTR factor comprises one or more reactive functional groups that react with the amino acid side chain of the TR inhibitor, such as aldehydes, halogenated alkanes, alkenes, fluorophosphonates (e.g., fluoroalkyls), Michael (Michael) acceptors, phenyl sulfonates, methyl ketones (such as halogenated methyl ketones or diazomethyl ketones), fluorophosphonates, vinyl esters, vinyl sulfones, or vinyl sulfonamides. In certain embodiments, the iTR factor inhibitor comprises a compound that physically interacts with the TR inhibitor, wherein the compound comprises a reactive functional group. In certain embodiments, the structure of the compound that physically interacts with the TR inhibitor is modified to bind to a reactive functional group. In certain embodiments, the compound comprises a TR inhibitor substrate analog or a transition state analog. In certain embodiments, the compound interacts with the TR inhibitor in or near the TR inhibitor active site.

[0146] In other embodiments, the iTR factor non-covalently binds to the TR inhibitor and / or to a complex comprising a TR inhibitor and a TR inhibitor substrate. In certain embodiments, the iTR factor non-covalently binds to the active site of the TR inhibitor and / or competes with the substrate for access to the TR inhibitor active site. In certain embodiments, the iTR factor is present at about 10 -3 M or smaller (e.g. 10 -4 M or smaller, e.g. 10 -5 M or smaller, e.g. 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, or 10 - 9 M or smaller)K dBinding of a TR inhibitor under test conditions, such as in a physiologically acceptable solution such as phosphate buffered saline. Binding affinity can be measured, for example, using surface plasmon resonance (e.g., using a Biacore system), isothermal titration calorimetry, or competitive binding assays, as is known in the art. In certain embodiments, the inhibitor comprises a TR inhibitor substrate analog or transition state analog.

[0147] In the case of increasing the activity of a TR activating factor, any of the combinations of TR activating factors listed in Figure 8 can be used. Levels of factors or products of these genes can be introduced using the vectors described herein.

[0148] In other embodiments, the iTR factor is a construct that introduces RNA into a cell directly or through a gene expression construct, and if the gene expression construct is allowed to react with the cell for a sufficient time, the gene expression construct is capable of inducing pluripotency; if the reaction time is shorter, iTR will be caused. Preferably, the RNA does not contain all RNA required for reprogramming to pluripotency, but only contains LIN28A or LIN28B, optionally together with an agent that increases telomere length, such as RNA of the catalytic component of telomerase (TERT). Most preferably, the agent that induces iTR is a gene / factor induced by a LIN28A or LIN28B encoded protein (e.g., GFER), optionally combined with an agent that increases telomere length (e.g., RNA or gene encoding TERT), and / or in combination with factors disclosed herein that are important for iTR, such as 0.05-5mM valproic acid (preferably 1.0mM valproic acid), 1-100ng / mL AMH (preferably 10ng / mL AMH) and 2-200ng / mL GFER (preferably 20ng / mL). When administered in vivo, these factors are preferably administered in a slow-release hydrogel matrix, such as a hydrogel matrix comprising chemically modified and cross-linked hyaluronic acid and collagen (eg, a HyStem matrix).

[0149] Report-based screening test for iTR factor

[0150] The present invention provides a method for identifying iTR factors using (a) a reporter molecule, the reporter molecule comprising an easily detectable marker (e.g., GFP or β-galactosidase), the expression of which is driven by the promoter of one of the TR activated genes described herein (e.g., the promoter of COX7A1). The present invention provides a screening assay that involves determining whether a test compound affects the expression of a TR activated gene and / or inhibits the expression of a TR inhibited gene. The present invention further provides a reporter molecule and a composition for implementing the method. In general, the compounds identified using the methods of the present invention can act by any mechanism that causes an increase or decrease in TR activator or inhibitor genes, respectively. With respect to the COX7A1 promoter, the promoter sequence flanking the 5' end of the human gene has been characterized as the position of -756 bases of the ATG translation start codon (Yu, M. et al., Biochimica and Biophysica Acta 1574 (2002) 345-353). It was observed that the transcription start site of most cDNAs is at -55 bases of the translation start codon. The promoter, along with the rest of the gene sequence, is located in a CpG island, similar to the promoters of many housekeeping genes, although COX7A1 expression is tissue-specific. CpG islands are characterized by an abundance of CG dinucleotides that exceeds the expected average genomic content over a range of at least 200 bases. The promoter contains several regulatory binding site sequences: MEF2 at position -524, and three E boxes (referred to as E1, E2, and E3) at positions -58, -279, and -585, respectively; the E boxes are DNA binding sites (CAACTG) for members of the myogenic family of regulatory proteins. In addition, there are multiple CG-rich stretches in the region from approximately -95 to -68 bases, similar to the stretch recognized by the transcription factor Sp1.

[0151] The gene itself, characterized in the GRCh38.p7 primary assembly, occupies 1948 bases between positions 36150922 and 36152869 on human chromosome 18 and contains four exons interspersed by three introns. The gene sequence with promoter sequence is curated at NCBI with site identifier AF037372.

[0152] Reporter molecules, cells, and membranes

[0153] Typically, the detectable portion of the reporter molecule that can be used in the present invention comprises a light emitting or light absorbing compound that produces or quenches a detectable fluorescent, chemiluminescent or bioluminescent signal. In certain embodiments, activation of a TR activator gene or inhibition of a TR inhibitor gene results in the release of a detectable portion into a liquid culture medium, and a signal produced or quenched by the released detectable portion present in the culture medium (or a sample thereof) is detected. In certain embodiments, the resulting signal causes a change in the properties of the detectable portion, and this change can be detected, for example, as a light signal. For example, the signal can change the emission or absorption of electromagnetic radiation (e.g., radiation with a wavelength in the infrared, visible or UV portion of the spectrum) by a detectable portion. In certain embodiments, the reporter molecule comprises a fluorescent or luminescent portion, and a second molecule is used as a quencher that quenches the fluorescent or luminescent portion. This change can be detected using devices and methods known in the art.

[0154] In many embodiments of the invention, the reporter molecule is a genetically encoded molecule that can be expressed by a cell, and the detectable portion comprises, for example, a detectable polypeptide. Thus, in certain embodiments, the reporter molecule is a polypeptide comprising a fluorescent polypeptide (e.g., green, blue, sapphire, yellow, red, orange, and cyan fluorescent proteins and their derivatives (e.g., enhanced GFP), monomeric red fluorescent proteins and their derivatives (e.g., derivatives known as "mFruits" (e.g., mCherry, mStrawberry, mTomato, etc.)), and luminescent proteins (e.g., aequorin)). (It will be appreciated that in certain embodiments, the fluorescence or luminescence occurs in the presence of one or more additional molecules, such as ions (e.g., calcium ions) and / or prosthetic groups (e.g., coelenterazine). In certain embodiments, the detectable moiety comprises an enzyme that acts on a substrate to produce a fluorescent, luminescent, colored, or other detectable product. Examples of enzymes that can be used as detectable moieties include luciferase, β-galactosidase, horseradish peroxidase, alkaline phosphatase, and the like. (It will be appreciated that the enzyme is detected by detecting the reaction product.) In certain embodiments, the detectable moiety comprises a polypeptide tag that can be readily detected using a secondary reagent, such as a labeled (e.g., fluorescently labeled) antibody. For example, fluorescently labeled antibodies are available that bind to HA, Myc, or a variety of other peptide tags. Thus, the present invention provides a method for detecting a fluorescent, luminescent, colored, or other detectable product. The invention includes embodiments in which the detectable portion can be directly detected (i.e., it does not need to interact with the second reagent to produce a detectable signal) and embodiments in which the detectable portion interacts with the second reagent (e.g., combines and / or reacts). This interaction, for example, causes the detection of a detectable signal or makes the detectable portion detectable because the second reagent can be directly detected. In embodiments in which the detectable portion interacts with the second reagent to produce a detectable signal, the detectable portion can react with the second reagent and is acted upon by the second reagent to produce a detectable signal. In many embodiments, the signal intensity provides an indication of the amount of the detectable portion (e.g., in a sample to be evaluated or in an area to be imaged). In certain embodiments, the amount of the detectable portion is optionally (e.g., based on relative or absolute signal intensity) quantitatively determined.

[0155] The present invention provides nucleic acids comprising sequences encoding reporter polypeptides of the present invention. In certain embodiments, the nucleic acid encodes a precursor polypeptide of a reporter polypeptide of the present invention. In certain embodiments, the sequence encoding the polypeptide is operably connected to an expression control element (e.g., a promoter or promoter / enhancer sequence) suitable for directing the transcription of the mRNA encoding the polypeptide. The present invention further provides an expression vector comprising the nucleic acid. The selection of suitable expression control elements can be based on, for example, the cell type and species of the nucleic acid to be expressed. One of ordinary skill in the art can easily select suitable expression control elements and / or expression vectors. In certain embodiments, the expression control element is regulatable, e.g., inducible or repressible. Exemplary promoters suitable for bacterial cells include, for example, Lac, Trp, Tac, araBAD (e.g., in a pBAD vector), a bacteriophage promoter (e.g., T7 or T3). Exemplary expression control sequences that can be used to direct expression in mammalian cells include, for example, the early and late promoters of SV40, the very early promoters of adenovirus or cytomegalovirus, or viral promoter / enhancer sequences, retroviral LTRs, promoters or promoter / enhancers from mammalian genes (e.g., actin, EF-1α, phosphoglycerate kinase, etc.), regulatable (e.g., inducible or repressible) expression systems (e.g., Tet-On and Tet-Off systems (regulatable by tetracycline and analogs (e.g., doxycycline)) and other expression systems that can be regulated by small molecules (e.g., hormone receptor ligands (e.g., steroid receptor ligands that may or may not be steroids)), metal-regulated systems (e.g., metallothionein promoters), and the like.

[0156] The present invention further provides cells and cell lines comprising such nucleic acids and / or vectors. In some embodiments, the cell is a eukaryotic cell, such as a fungus, a plant or an animal cell. In some embodiments, the cell is a vertebrate cell, such as a mammalian cell, such as a human cell, a non-human primate cell or a rodent cell. Generally, the cell is a member of a cell line (e.g., an established or immortalized cell line that has obtained the ability to proliferate indefinitely in a culture medium (e.g., due to mutation or genetic manipulation (e.g., constitutive expression of a telomerase catalytic component)). Many cell lines are known in the art and can be used in the present invention. Mammalian cell lines include, for example, 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 cell is non-tumorigenic and / or not derived from a tumor. In some embodiments, the cell is an adherent cell. In some embodiments, non-adherent cells are used. In certain embodiments, the cells used are cell types or cell lines that have been shown to naturally have a subset of expressed TR activator genes or unexpressed TR inhibitor genes. If a cell lacks one or more TR activator or inhibitor genes, the cell can be genetically engineered to express such a protein. In certain embodiments, the cell lines of the invention are 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 a single cell can be selected and expanded in culture. In certain embodiments, cells are transiently transfected with an expression vector encoding a reporter molecule. Cells can be co-transfected with a control plasmid, which optionally expresses a different detectable polypeptide, to control transfection efficiency (e.g., between multiple assays).

[0157] TR activator and TR inhibitor polypeptides and nucleic acids

[0158] TR activator and TR inhibitor genes are listed in Figure 8. Under the headings "Embryonic Markers" and "Fetal / Adult Markers", respectively. TR activator and TR inhibitor polypeptides that can be used in the methods of the present invention can be obtained by a variety of methods. In certain embodiments, polypeptides are produced using recombinant DNA technology. Standard methods for recombinant protein expression can be used. Nucleic acids encoding the genes can be easily obtained, for example, from cells expressing TR activator or TR inhibitor genes (e.g., by PCR or other amplification methods or by cloning) or by in vitro transcription based on cDNA sequence polypeptide sequences. One of ordinary skill in the art will know that due to the degeneracy of gene encoding, genes can be encoded by many different nucleic acid sequences. Optionally, the sequence is codon optimized for expression in a selected host cell. The gene can be expressed in bacteria, fungi, animal or plant cells or organisms. The gene can be isolated from cells that naturally express the gene or cells that have transiently or stably introduced nucleic acids encoding the protein (e.g., cells containing expression vectors encoding the gene). In certain embodiments, the gene is secreted by cells in culture and separated from the culture medium.

[0159] In certain embodiments of the present invention, the sequence of a TR activator or TR inhibitor polypeptide is used in the screening method of the present invention. A naturally occurring TR activator or TR inhibitor polypeptide may be from any species whose genome encodes a TR activator or TR inhibitor polypeptide, such as humans, non-human primates, rodents, etc. A polypeptide having the same sequence as a naturally occurring TR activator or TR inhibitor is sometimes referred to herein as a "natural TR activator / inhibitor". The TR activator 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 comprising or consisting of amino acids 20-496 of a human TR activator or TR inhibitor (or the corresponding amino acids of a TR activator or TR inhibitor of a different species) may be used.

[0160] In some embodiments, a polypeptide comprising a variant or fragment of a TR activator or TR inhibitor or consisting thereof is used. A TR activator or TR inhibitor variant includes a polypeptide that differs from a TR activator or TR inhibitor by one or more amino acid substitutions, additions or deletions. In some embodiments, a TR activator or TR inhibitor variant comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acids 20-496 of a human TR activator or TR inhibitor or amino acids 20-503 of a mouse TR activator or TR inhibitor, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with at least amino acids 20-496 of a TR activator or TR inhibitor (e.g., from a human or mouse). In certain embodiments, the TR activator or TR inhibitor variant comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with at least amino acids 20-496 of a human TR activator or TR inhibitor or amino acids 20-503 of a mouse TR activator or TR inhibitor. In certain embodiments, the TR activator or TR inhibitor polypeptide comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with at least amino acids 20-496 of a human TR activator or TR inhibitor or amino acids 20-503 of a mouse TR activator or TR inhibitor. Nucleic acids encoding TR activator or TR inhibitor variants or fragments can be readily generated, for example, by modifying DNA encoding a natural TR activator or TR inhibitor using, for example, site-directed mutagenesis or by other standard methods, and can be used to generate the TR activator 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 providing a sequence encoding a heterologous moiety. In certain embodiments, a tagged TR activator or TR inhibitor is used. For example, in certain embodiments, a TR activator or TR inhibitor polypeptide is used, for example, comprising a 6xHis tag at its C-terminus.

[0161] Test compound

[0162] A variety of test compounds can be used in the methods of the present invention to identify iTR factors and overall regulators of iTR. For example, the test compound can be a small molecule, polypeptide, peptide, nucleic acid, oligonucleotide, lipid, carbohydrate, antibody or hybrid molecule, including but not limited to those described herein, including the mRNA of the following genes: OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, LIN28A and LIN28B alone and different combinations thereof, and different combinations thereof with small molecule compounds, such as combinations of the following compounds: inhibitors of glycogen synthase 3 (GSK3), including but not limited to CHIR99021; inhibitors of TGF-β signaling, including but not limited to SB431542, A-83-01 and E616452 HDAC inhibitors, including but not limited to aliphatic acid compounds, including but not limited to valproic acid, phenylbutyric acid and n-butyric acid, cyclic tetrapeptides (including aflatoxin B and dipeptides), hydroxamic acids (e.g., trichostatin A, vorinostat (SAHA), belostat (PXD101), LAQ824, panobinostat (LBH589) and benzamide entinostat (MS-275), CI994, mocetinostat (MGCD0103)); specific for class I (HDAC1, HDAC2, HDAC3 and HDAC8), class IIA (HDAC4, HDAC5, HDAC7 and HDAC9), class IIB ... DAC6 and HDAC10), those of class III (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 or SIRT7) (including the sirtuin inhibitors nicotinamide, various derivatives of NAD, dihydrocoumarins, naphthopyranones and 2-hydroxynaphthoacetaldehyde), or class IV (HDAC11) deacetylases; inhibitors of the 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 DZNe p); inhibitors of DNA methyltransferase (including but not limited to RG108); 5-aza-2'deoxycytidine (trade names Vidaza and Azadine); vitamin C, which can inhibit DNA methylation and increase Tetl, thereby increasing 5hmC, which is the first step of 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 (activator of phosphofructokinase 1)); agents that promote the activity of the HIF1 transcription complex (including but not limited to quercetin); 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 exogenous expression of telomerase catalytic component (TERT); agents that promote epigenetic modification by downregulating LSD 1 (H3K4-specific histone demethylase) (including but not limited to lithium); or inhibitors of the MAPK / ERK pathway (including but not limited to PD032590). Other compounds include histone deacetylase inhibitor MS-275, histone methyltransferase inhibitor BIX01294, DNA methyltransferase inhibitor RG108, analogs of retinoic acid AM580 or CD437 (retinoic acid receptor agonists) and histone methyltransferase inhibitor EPZ004777 (inhibitor of DOT1L and disruptor of telomere silencing 1-like) and inhibitors of DNA methyltransferase and HDAC RSC133. Such compounds may be administered in various combinations, concentrations, and over various time periods to optimize the effects of iTR on cells cultured in vitro using overall iTR markers, such as by measuring a decrease in the expression of COX7A1, ADIRF, TNFRSF11B, or other iTR inhibitors as described herein, and / or measuring an increase in the expression of AMH or other activators or iTR as described herein, or an increase in expression in injured or diseased tissues in vivo, or modulating the life course of an animal in vivo.;

[0163] In vitro assays for the iTR gene expression pattern of the genes COX7AJ, ADIRF, TNFRSF11B, or AMH and gene expression or protein markers of pluripotency (including OCT4 and HELLS or Tra-1-60, Tra-1-81, and SSEA4) were performed separately to optimize the overall pattern of iTR gene expression without restoring the target cells to pluripotency. Examples of individual agents and combinations of agents 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, and LIN28A. F4, MYC, and LIN28A; and each of the above combinations of agents was used with 0.25mM NaB, 5μM PS48, and 0.5μM A-83-01 for the first four weeks, followed by treatment with 0.25mM sodium butyrate, 5μM PS48, 0.5μM A-83-01, and 0.5μM PD0325901, and each of the above was tested for markers of global regulation of iTR gene expression on days 0, 1, 2, 4, 7, 10, and 14.

[0164] Improved iTR factor screening

[0165] The screening of compounds or combinations of compounds for producing iTR effects in fetal or adult mammalian cells can be performed in vitro or in vivo (e.g., animal models). Preferably, the screening is performed in vitro with low or high throughput. Screening tests are used to increase or decrease embryonic or fetal / adult markers, respectively. The screening can be performed using antibodies, RNA assays, metabolic markers, or other markers described herein or in "Compositions and methods of tissue regeneration induced in mammalian species" (International Patent Application Publication No. WO 2014 / 197421) (the entire contents of which are incorporated herein by reference) and "Improved methods for detecting and regulating embryo-fetal transition in mammalian species" (International Patent Application Publication No. WO 2017 / 214342, the entire contents of which are incorporated herein by reference). Preferably, the screening measures the level of mRNA markers, most preferably measuring the reduction of COX7A1 expression as indicated by iTR. Most preferably, the screening is performed in a porous form, and expression is measured by using a reporter gene (eGFP knocked into the COX7A1 locus in the genome).

[0166] Compounds can be obtained from natural sources or synthetically produced. Compounds can be at least partially pure or can be present in extracts or other types of mixtures whose components are at least partially unknown or uncharacterized. Extracts or their fractions can be produced from, for example, plants, animals, microorganisms, marine organisms, fermentation broths (e.g., soil, bacteria or fungal fermentation broths), etc. In some embodiments, a test compound collection ("library"). The library can include, for example, 100 to 500,000 or more compounds. Compounds are usually arranged in multiwell plates (e.g., 384-well plates, 1596-well plates, etc.). They can be dissolved in solvents (e.g., DMSO) or provided in dry form (e.g., in powder or solid form). A collection of synthetic, semi-synthetic and / or naturally occurring compounds can be tested. Compound libraries can include compounds that are structurally related, structurally diverse or structurally unrelated. Compounds can be artificial (with structures invented by people that cannot be found in nature) or naturally occurring. In some embodiments, the library includes at least some compounds that are identified as "hit" or "lead" in other drug discovery programs and / or their derivatives. Compound libraries can include natural products and / or compounds generated using non-directional or directional synthetic organic chemical methods. Typically, compound libraries are small molecule libraries. Other libraries of interest include peptide or peptoid libraries, cDNA libraries, antibody libraries, and oligonucleotide libraries. Libraries can be focused (e.g., mainly composed of compounds with the same core structure, derived from the same precursor, or having at least one common biochemical activity).

[0167] Compounds selected for screening can be selected from libraries of synthetic or natural product derived small molecules having a variety of chemical structures known to provide potential bioactive motifs. Valproic acid at a concentration of 0.05-5.0 mM, preferably 1.0 mM; GSK-3 inhibitor 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (also known as CHIR99021) at a concentration of 7.0 nM-10 uM, preferably 10 uM; TGFβR-1 / ALK5 inhibitor 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (also known as RepSox) at a concentration of 4.0 nM-10 uM, preferably 10 uM; Lysine-specific demethylase 1 inhibitor at a concentration of 2.0-10 uM, preferably 10 uM The factor Parnate (also known as tranylcypromine); the adenylate cyclase activator Forskolin at a concentration of 0.5-50uM, preferably 50uM; the retinoid receptor agonist aromatic retinoic acid (also known as 4-[(E)-2-(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)prop-1-enyl]benzoic acid or TTNPB) at a concentration of 1.0nM-5uM, preferably 5.0uM; the EZH2 inhibitor (1S,2R,5R)-5-(4-aminoimidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (also known as 3-Deazaneplanocin) at a concentration of 0.05-0.24uM, preferably 0.1uM A (DZNep)); N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoaniline) benzamide (also known as PD0325901), an inhibitor of the MEK / ERK pathway, administered at a concentration of 0.1nM-1.0uM, preferably 1.0uM; SB431542, preferably 10.0uM; A 83-01, preferably 1.0uM; sodium 4-phenylbutyrate, preferably 1.0uM; romidepsin, preferably 10nM; trichostatin A, preferably 250nM A); SAHA (also known as vorinostat) at a concentration of preferably 1.0 uM; LAQ824 at a concentration of preferably 50 nM; Panobionstat at a concentration of preferably 10-100 nM; MS-275 at a concentration of preferably 5.0 uM; Mocetinostat at a concentration of preferably 1.0 uM; CI-994 at a concentration of preferably 40 uM; BEX01294 at a concentration of preferably 2.0 uM; and Panobionstat at a concentration of preferably 10-100 nM.0uM PD032590; preferably 200nM lithium salt; preferably 100nM NVP-BEZ235; preferably 20uM SB216763; preferably 0.6uM SB203580; preferably 0.1nM-1.0mM, preferably 100nM protein kinase R-like ER kinase (PERK) inhibitor GSK2606414; preferably 10nM 2-hydroxy-1-naphthaldehyde; preferably 1.0mM dihydrocoumarin; preferably 7.5mM nicotinamide; preferably 0.25mM sodium butyrate; RG108, preferably at a concentration of 5.0uM; (+)-L-sodium ascorbate, preferably at a concentration of 50ug / mL; quercetin, preferably at a concentration of 1.0uM; resveratrol, preferably at a concentration of 10uM; AM580, preferably at a concentration of 10nM; CD437, preferably at a concentration of 0.1uM; PS48, preferably at a concentration of 5.0uM; DMOG, preferably at a concentration of 10uM; deferoxamine, preferably at a concentration of 0.5uM-0.5mM; 5-azacytidine, preferably at a concentration of 5.0uM; cyclosporine, preferably at a concentration of 400ng / mL; EPZ, preferably at a concentration of 5.0uM 004777; SGC0946 at a concentration of preferably 10 nM; LY-364947 at a concentration of preferably 1.0 uM; Kenpaullone at a concentration of preferably 5.0 uM; Dasatinib at a concentration of preferably 0.5 uM; PP1 at a concentration of preferably 10 uM; 2-Hydroxy-1-naphthaldehyde isonicotinoyl hydrazone at a concentration of preferably 100 uM hydrazone) (also known as AS8351); 1,5-isoquinolinediol at a concentration preferably of 20-50uM; protein factor GFER (growth factor enhancer of liver regeneration) at a concentration preferably of 10ng / mL; protein factor AMH (anti-Mullerian hormone) at a concentration preferably of 20ng / mL; protein factor fibroblast growth factor beta at a concentration preferably of 10ng / mL; B18R at a concentration preferably of 100ug / mL; phorbol (Phorbol ) 12-myristate 13-acetate; LiC at a concentration of preferably 10 mM; Metformin at a concentration of preferably 5.0 uM; Antimycin A at a concentration of preferably 2.5 nM; Protein insulin-like growth factor 1 (IGF1) at a concentration of preferably 100 ng / mL; Protein insulin-like growth factor 2 (IGF2) at a concentration of preferably 100 ng / mL; Dehydroepiandrosterone (DHEA) at a concentration of preferably 50 uM; Protein growth hormone (GH) at a concentration of preferably 100 ng / mL; Protein growth hormone (GH) at a concentration of preferably 5.0 nM steroid 17-b-estradiol; Wortmannin at a concentration preferably of 1.0 uM; PKC activator (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam (PKC activator V) at a concentration preferably of 1.0–100 uM; ketone body 3-hydroxybutyrate (3-HB) at a concentration preferably of 10 mM; steroid testosterone at a concentration preferably of 1.0-10 uM; 1.0 mM L-carnitine; preferably 10 uM RSC-133; preferably 50 uM arginine N-methyltransferase inhibitor-1 (AMI-5); preferably 1.0 uM hedgehog pathway activator Hh-Ag1.5; preferably 10 uM JNJ10198409; preferably 10 nM LDE225; preferably 100 nM–10 uM RasGAP and ERK1 Inhibitor of SC-1; small molecule enhancer of autophagy SMER28 preferably at a concentration of 50uM; PDGFR tyrosine kinase inhibitor VII SU 16f preferably at a concentration of 50uM; inhibitor of tyrosine kinase domain of VEGFR2, FGFR1 and PDGFRβSU5402 preferably at a concentration of 10uM; mRNA of reprogramming factors OCT4, NANOG, LIN28A, LIN28B, SOX2, MYC, KLF4 or DNMT3B preferably at a concentration of 300ng / mL, used alone or in combination with the above factors. .

[0168] Compound libraries are available from many commercial suppliers (e.g., Tocris BioScience, Nanosyn, BioFocus) and government agencies. For example, the Molecular Library Small Molecule Repository (MLSMR), a component of the Molecular Library Program of the National Institutes of Health (NIH), aims to identify, acquire, maintain and distribute more than 300,000 chemically diverse compounds with known and unknown biological activities for analysis, such as high-throughput screening (HTS). (See https: / / mli.nih.gov / mli / ). The NIH Clinical Collection (NCC) is a plating array of approximately 450 small molecules that have a history of use in human clinical trials. These compounds have a high known drug safety profile. In certain embodiments, a collection of compounds containing "approved human drugs" is tested. An "approved human drug" is a compound approved for use in treating humans by a government regulatory agency (e.g., the U.S. Food and Drug Administration, the European Medicines Evaluation Agency, or a similar agency responsible for assessing at least the safety of therapeutic agents before allowing them to be put on the market). The test compound can be, for example, an antineoplastic, antibacterial, antiviral, antifungal, antiprotozoal, antiparasitic, antidepressant, antipsychotic, anesthetic, antianginal, antihypertensive, antiarrhythmic, anti-inflammatory, analgesic, antithrombotic, antiemetic, immunomodulator, antidiabetic, antilipid, cholesterol-lowering (e.g., statin), anticonvulsant, anticoagulant, anxiolytic, hypnotic (sleep induction), hormone or anti-hormone, etc. In certain embodiments, the compound is a compound that has undergone at least some preclinical or clinical development or has been determined or predicted to have "drug-like" properties. For example, the test compound may have completed a Phase I trial or at least one preclinical study on non-human animals and shown evidence of safety and tolerability.

[0169] In some embodiments, the concentration used or in some embodiments the concentration is 10 times, 100 times or 1,000 times higher than the concentration used, and the test compound is substantially nontoxic to the biological cells of the compound administered and / or the cells tested for the compound. For example, in various embodiments, there may be no statistically significant effect on cell viability and / or proliferation, or the reduction in vigor or proliferation may not exceed 1%, 5% or 10%. Cytotoxicity and / or the effect on cell proliferation can be assessed using any of a variety of assays. For example, cell metabolism assays (such as AlamarBlue, MTT, MTS, XTT and CellTitre Glo assays) can be used, cell membrane integrity assays, viability assays based on cell ATP, mitochondrial reductase activity assays, BrdU, EdU or H3-thymidine incorporation assays. In certain embodiments, the test compound is not a compound found in a cell culture medium known or used in the art, e.g., a medium suitable for culturing vertebrate (e.g., mammalian) cells, or if the test compound is a compound found in a cell culture medium known or used in the art, the test compound is used at a different (e.g., higher) concentration when used in the methods of the invention.

[0170] Determination of iTR global regulators

[0171] Implementation and control aspects of the assay

[0172] The above-mentioned various screening assays of the present invention involve determining whether the test compound inhibits the level of active TR inhibitor or increases the level of active TR activator. Suitable cells for expressing reporter molecules are described above. When performing the assay of the present invention, the assay components (e.g., cells, TR activating factors or TR inhibitor polypeptides and test compounds) are usually distributed into multiple containers or other containers. Any type of container or article capable of accommodating cells can be used. In many embodiments of the present invention, the container is a well of a multi-well plate (also known as a "microplate", "microtiter plate", etc.). For the purpose of description, the term "well" will be used to refer to any well. The type of container or article that can be used to perform the screening of the present invention, such as any container or article that can contain assay components, should be understood that the present invention is not limited to the use of wells or multi-well plates. In certain embodiments, any article having multiple physically separated cavities (or other limiting features) in or on a matrix can be used. For example, the assay components can be confined in droplets, the droplets can be optionally arranged on a plane, and optionally separated by a waterproof substance, which confines the droplets to discrete locations in a microfluidic device channel, etc.

[0173] Typically, the 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 of time (e.g., 6 to 48 hours) before the test compound and target TR activator or TR inhibitor polypeptide or cells with expression constructs are added to the wells. In certain embodiments, the compound is added to the wells before the cell polypeptide is added. In certain embodiments, after plating the cells, optionally after adding the test compound to the wells, the expression of the reporter polypeptide is induced. In certain embodiments, the expression of the reporter molecule is achieved by transfecting the cells with an expression vector encoding the reporter polypeptide. In certain embodiments, the cells have previously been genetically modified to express the reporter polypeptide. In certain embodiments, the expression of the reporter molecule is under the control of an adjustable expression control element, and the induction of the reporter molecule expression is achieved by contacting the cells with an agent that induces (or inhibits) expression.

[0174] The detection composition comprising cells, test compounds or polypeptides is maintained for a suitable period of time during which the test compound may (in the absence of a test compound that inhibits its activity) cause the level or activity of the target TR activator or TR inhibitor to increase or decrease. The number of cells, the number of TR activator or TR inhibitor polypeptides, and the number of test compounds to be added will depend on factors such as, for example, container size, cell type, and can be determined by a person of ordinary skill in the art. In some embodiments, the molar concentration ratio of the TR activator or TR inhibitor polypeptide to the test compound is 1:10 to 10:1. In some embodiments, the number of cells, the amount of the test compound, and the length of time the composition is maintained can be selected so that the level signal is easily detected after a selected period of time in the absence of the test compound. In some embodiments, when the compound is added, the cells are at a confluence of about 25%-75%, for example, about 50%. In some embodiments, 1,000 to 10,000 cells / well (e.g., about 5,000 cells / well) are plated in about 100 μl of culture medium per well in a 96-well plate. In other exemplary embodiments, cells are inoculated into 384-well plates with about 30 μl to 50 μl of culture medium at 500 to 2,000 (e.g., about 1000) cells per well. In certain embodiments, compounds are tested at multiple concentrations (e.g., 2-10 different concentrations) and / or in multiple replicates (e.g., 2-10 replicates). Multiple replicates of some or all different concentrations may be performed. In certain embodiments, candidate TR factors are used at a concentration between 0.1 μg / ml and 100 μg / ml (e.g., 1 μg / ml to 10 μg / ml). In certain embodiments, candidate TR factors are used at multiple concentrations. In certain embodiments, the compound is added to the cells between 6 hours and 1 day (24 hours) after inoculation. In certain aspects of any of the present compound screening and / or characterization methods, the test compound is added to the assay composition in an amount sufficient to reach a predetermined concentration. In certain embodiments, the concentration is up to about 1 nM. In certain embodiments, the concentration is between about 1 nM and about 100 nM. In some embodiments, the concentration is between about 100nM and about 10μM. In some embodiments, the concentration is at least 10μM, such as between 10μM and 100μM. After the last component is added, the assay composition can be maintained for different times. In some embodiments, the assay composition is maintained for about 10 minutes to about 4 days, such as 1 hour to 3 days, such as 2 hours to 2 days, or any intermediate range or specific value, such as about 4-8 hours after adding all components. A plurality of different time points can be tested. Other aliquots of the test compound can be added to the assay composition within this time period. In some embodiments, the cells are maintained in a cell culture medium suitable for culturing this type of cell. In some embodiments, serum-free culture medium is used.In certain embodiments, instead of a cell culture medium, the assay composition comprises a physiologically acceptable liquid compatible with maintaining the integrity of the cell membrane and optionally the viability of the cells. Any suitable liquid may be used, provided it has an appropriate osmotic pressure and is compatible with maintaining reasonable integrity of the cell membrane and optionally the viability of the cells for at least a sufficient period of time (to perform the assay). During or after the incubation, one or more measurements indicative of an increase in the level of active TR activating factor or a decrease in TR inhibitory factor may be made.

[0175] In certain embodiments, the compounds screened as possible global regulators of iTR are selected from drugs that can induce pluripotency of somatic cell types under other conditions. Such agents include the following compounds alone or in combination: genes OCT4, SOX2, KLF4, NANOG, ESRRB, NR5A2, CEBPA, MYC, LIN28A and LIN28B alone and in combination with small molecule compounds (e.g., combinations of the following compounds): inhibitors of glycogen synthase 3 (GSK3), including but not limited to CHIR99021; inhibitors of TGF-β signaling, including but not limited to SB431542, A-83-01 and E616452; HDAC inhibitors, including but not limited to aliphatic acid compounds, including but not limited to valproic acid, phenylbutyric acid and n-butyric acid , cyclic tetrapeptides (including aflatoxin B and dipeptides), hydroxamic acids (e.g., trichostatin A, vorinostat (SAHA), belostat (PXD101), LAQ824, panobinostat (LBH589), and benzamide entinostat (MS-275), CI994, mocetinostat (MGCD0103); specifically targeting class I (HDAC1, HDAC2, HDAC3, and HDAC8), class IIA (HDAC4, HDAC5, HDAC7, and HDAC9), class IIB (HDAC6 and HDAC10), class III (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7), class III (SIRT1, SIRT2, SIRT3, SIRT4, and SIRT8), class IV (HIRT5, HDAC7, and HDAC9), class IV (HIRT6, HDAC10), class V (HIRT1, V (HIRT2, V (HIRT3, V (HIRT4, and SIRT7)), class V (HIRT8, and SIRT9)), class V (HIRT9, HDAC11, and HDAC12), class V (HIRT1 T5, SIRT6 or SIRT7) (including the sirtuin inhibitors nicotinamide, various derivatives of NAD, dihydrocoumarins, naphthopyranones and 2-hydroxynaphthoacetaldehyde, or those of class IV (HDAC11) deacetylases; inhibitors of the 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 glucosides (trade names Vidaza and Azadine); vitamin C, which can inhibit DNA methylation and increase Tetl, thereby increasing 5hmC, which is the first step of 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 (activator of phosphofructokinase 1)); agents that promote the activity of the HIF1 transcription complex (including but not limited to quercetin); RAR agonists (including but not limited to AM580, CD437 and TTNPB); agents that simulate hypoxia (including but not limited to resveratrol);Agents that increase telomerase activity, including but not limited to exogenous expression of telomerase catalytic component (TERT); agents that promote epigenetic modification by downregulating LSD 1 (H3K4-specific histone demethylase) (including but not limited to lithium); or inhibitors of the MAPK / ERK pathway (including but not limited to PD032590). Such compounds can be administered in various combinations, concentrations, and over different time periods to optimize the effects of iTR on cells cultured in vitro using overall iTR markers, which can be achieved, for example, by measuring the reduction in expression of COX7A1, ADIRF, TNFRSF11B, or other iTR inhibitors as described herein, and / or measuring the increase in expression of AMH or other activators or iTR as described herein, or the increase in expression in injured or diseased tissues in vivo, or regulating the life course of animals in vivo. ;

[0176] In certain embodiments, individual compounds, typically with known identities (e.g., structures and / or sequences), are added to each of the plurality of wells. In certain embodiments, two or more compounds may be added to one or more wells. In certain embodiments, one or more compounds of unknown identity may be tested. The identities may then be determined using methods known in the art.

[0177] In various embodiments, the aforementioned assay method of the present invention is suitable for high throughput screening (HTS) implementation. In certain embodiments, the screening assay of the present invention is 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) generally involves testing a large number of compounds efficiently, such as tests performed in parallel. For example, tens of thousands of compounds can be routinely screened in a short time (e.g., hours to days). In certain embodiments, HTS refers to testing 1,000 to 100,000 compounds per day. In certain embodiments, ultra high throughput refers to screening more than 100,000 compounds per day, such as up to one million or more compounds per day. The screening assay of the present invention can be performed in a porous format, such as a 96-well, 384-well format, a 1,536-well format, or a 3,456-well format, and is suitable for automation. In certain embodiments, each well of a microplate can be used to perform a separate assay for a different test compound, or, if the effects of concentration or incubation time are to be observed, multiple wells can contain test samples of a single compound, and at least some wells can be selected to be left empty or used as controls or repeats. Typically, the HTS embodiments of the assays disclosed herein involve automated use. In certain embodiments, an integrated robotic system comprising one or more robots transports assay microplates between multiple assay stations for the addition, mixing, incubation, and reading or detection of compounds, cells, and / or reagents. In certain aspects, the HTS system of the present invention can prepare, incubate, and analyze many plates simultaneously. Appropriate data processing and control software can be used. High-throughput screening embodiments are well known in the art. Without limiting the present invention in any way, certain basic principles and techniques that can be applied in embodiments of the HTS of the present invention are described in Macarron R & Hertzberg RP. Design and implementation of high-throughput screening assays. Methods Mol Biol. 565: 1-32, 2009 and / or An W F & Tolliday N J., Introduction: cell-based assays for high-throughput screening. Methods Mol Biol. 486: 1-12, 2009 and / or references therein in either.Exemplary methods are also disclosed in High Throughput Screening: Methods and Protocols (Methods in Molecular Biology) by William P. Janzen (2002) and High-Throughput Screening in Drug Discovery (Methods and Principles in Medicinal Chemistry) (2006). The additional compounds may, for example, have one or more improved pharmacokinetic and / or pharmacodynamic properties compared to the initial hit, or may simply have a different structure. The "improved properties" may, for example, make the compound more effective or more suitable for one or more purposes described herein. In certain embodiments, for example, the compound may have a higher affinity for a molecular target of interest (e.g., a TR activator or TR inhibitor gene product), a lower affinity for non-target molecules, increased solubility (e.g., increased water solubility), increased stability (e.g., stability in blood, plasma and / or the gastrointestinal tract), increased in vivo half-life, improved bioavailability and / or reduced side effects, etc. Optimization can be achieved by empirically modifying the hit structure (e.g., synthesizing a compound with a related structure and testing it in a cell-free or cell-based assay or in a non-human animal) and / or using computational methods. In some embodiments, this modification can predictably change one or more properties using the established principles of medicinal chemistry. In some embodiments, one or more compounds of the "hit" are identified, and a systematic structural change is made to them to produce a second library of compounds structurally related to the hit (e.g., a refined lead compound). The second library can then be screened using any of the methods described herein. In some embodiments, the iTR factor is modified or introduced to enhance stability (e.g., in serum), increase half-life, reduce toxicity or immunogenicity, or impart a desired property to the compound.

[0178] Use of iTR, iTM and ICM factors

[0179] Pharmaceutical composition

[0180] iTR, iTM, senescent cell lysis, iS-CSC and iCM factors have a variety of different 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 limbs, fingers, cartilage, heart, blood vessels, bones, esophagus, stomach, liver, gallbladder, pancreas, intestines, rectum, anus, endocrine glands (e.g., thyroid, parathyroid, adrenal glands, endocrine parts of the pancreas), skin, hair follicles, thymus, spleen, skeletal muscle, focally damaged myocardium, smooth muscle, brain, spinal cord, peripheral nerves, ovaries, fallopian tubes, uterus, vagina, breast, testicles, 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 matrix layers (e.g., connective tissue that supports tissue substance). In some embodiments, iTR factors are used to enhance regeneration after surgery, for example, surgery that requires removal of at least a portion of a diseased or damaged tissue, organ, or other structure, such as a limb, finger, etc. Such surgery can remove at least a portion of the liver, lung, kidney, stomach, pancreas, intestine, breast, ovary, testicle, bone, limb, finger, muscle, skin, etc. In some embodiments, the surgery is to remove a tumor. In some embodiments, iTR factors are used to promote scarless regeneration of skin after trauma, surgery, disease, and burns.

[0181] In various embodiments, enhancing regeneration may include any one or more of the following: (a) increasing the rate of regeneration; (b) increasing the extent of regeneration; (c) promoting the establishment of appropriate structure (e.g., shape, pattern, tissue structure, tissue polarity) in the regenerated tissue or organ or other body structure; (d) promoting the growth of new tissue in a manner that maintains and / or restores function. Although particular attention is paid to the use of iTR factors to enhance regeneration, the present invention generally includes the use of iTR factors to enhance repair or wound healing without necessarily producing detectable regeneration enhancement. Therefore, the present invention provides a method of enhancing repair or wound healing, wherein the iTR factor is administered to a subject in need thereof according to any method described herein.

[0182] Many aspects of aging and age-related diseases are taught in the present invention, which can be addressed with iTR therapy. These manifestations of aging include age-related vascular dysfunction, including peripheral vascular, coronary artery and cerebrovascular disease; musculoskeletal diseases, including osteoarthritis, disc degeneration, fractures, tendon and ligament tears, and limb regeneration; nervous system diseases, including stroke and spinal cord injury; muscle diseases, including muscular dystrophy, sarcopenia, myocardial infarction and heart failure; endocrine diseases, including type I diabetes, Addison's disease, hypothyroidism and pituitary insufficiency; digestive system diseases, including pancreatic exocrine insufficiency; eye diseases, including macular degeneration, retinitis pigmentosa and neuroretinal degenerative diseases; skin diseases, including skin burns, lacerations, surgical incisions, hair loss, hair graying and skin aging; lung diseases, including emphysema and pulmonary interstitial fibrosis; hearing disorders, including hearing loss; and blood system diseases, such as aplastic anemia and hematopoietic stem cell transplant failure.

[0183] In certain embodiments, the present invention provides a method for enhancing regeneration in a subject in need thereof, the method comprising administering an effective amount of an iTR factor to the subject. In certain embodiments, the effective amount of a compound (e.g., an iTR factor) is an amount that increases the rate or degree of regeneration of damaged tissue compared to a reference value (e.g., a suitable control value). In certain embodiments, a reference value is an expected (e.g., average or typical) regeneration rate or degree of regeneration (optionally administered with a placebo) in the absence of a compound. In certain embodiments, the effective amount of an iTR factor is an amount of improved structural and / or functional results compared to an expected (e.g., average or typical) structural or functional result in the absence of a compound. In certain embodiments, an effective amount of a compound, such as an iTR factor, results in enhanced blastema formation and / or reduced scarring. For example, the degree or rate of regeneration can be assessed based on the size or volume of the regenerated tissue. The structural and / or functional outcome can be assessed based on, for example, visual inspection (optionally including the use of a microscope or imaging techniques (e.g., X-rays, CT scans, MRI scans, PET scans)) and / or by assessing the ability of a 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, an improved structural outcome is a structural outcome that is more similar to a normal structure (e.g., a structure that existed before tissue injury or a structure that exists in a normal, healthy individual) than a structural outcome (e.g., an average or typical outcome) that would be expected without treatment with an iTR factor. One of ordinary skill in the art can select an appropriate assay or functional test. In certain embodiments, the increase in the rate or extent of regeneration compared to a control value is statistically significant (e.g., p value <0.05, or p value <0.01) and / or clinically significant. In certain embodiments, the improvement in the structural and / or functional outcome compared to a control value is statistically and / or clinically significant. "Clinically significant improvement" refers to an improvement that confers a meaningful benefit (e.g., a benefit sufficient to make the treatment worthwhile) to a subject within the reasonable judgment of a medical or surgical physician. It will be appreciated that in many embodiments, the iTR modulator (e.g., iTR factor) administered to a subject of a particular species (e.g., for therapeutic purposes) is a compound that modulates (e.g., inhibits) an endogenous TR gene expressed in a subject of that species. For example, if the subject is a human, a compound that inhibits the activity of the human TR inhibitor gene product and activates the activity of the human TR activator gene product will generally be administered.

[0184] In certain embodiments, iTR factors are used to enhance skin regeneration, for example, after a burn (thermal or chemical), a scrape injury, or other conditions involving skin loss, such as an infection (e.g., necrotizing fasciitis or purpura fulminans). In certain embodiments, the burn is a second degree burn or a third degree burn. In certain embodiments, the area of ​​skin loss is at least 10 cm2 On the one hand, iTR factors enhance the regeneration of transplanted skin. On the other hand, iTR factors reduce excessive and / or pathological wound contraction or scar formation.

[0185] In certain embodiments, iTR factors are used to enhance bone regeneration, for example, in cases such as nonunion fractures, implant fixation, periodontal or alveolar eminence, craniofacial surgery, or other conditions where new bone formation is considered appropriate. In certain embodiments, iTR factors are applied to sites where bone regeneration is required. In certain embodiments, iTR factors are incorporated into or used in combination with bone graft materials. Bone graft materials include a variety of ceramics and protein materials. Bone graft materials include autologous bone (e.g., bone collected from ilium, fibula, ribs, etc.), allogeneic bone from corpses, and xenogeneic bone. Synthetic bone graft materials include a variety of ceramics (e.g., calcium phosphates (e.g., hydroxyapatite and tricalcium phosphate)), bioglass and calcium sulfate, and protein materials (e.g., demineralized bone matrix (DBM)). DBM can be prepared by grinding cortical bone tissue (usually to a particle size of 100-500 μm sieved) and then treating the ground tissue with hydrochloric acid (usually 0.5 to 1N). In certain embodiments, iTR factors are administered to a subject together with one or more bone graft materials. The iTR factor can be combined with a bone graft material (in a composition comprising an iTR factor and a bone graft material), or applied alone, for example, after placement of a graft. In certain embodiments, the present invention provides a bone paste comprising an iTR factor. Bone paste is a product of appropriate consistency and composition, so that they can be introduced into bone defects (e.g., voids, gaps, cavities, cracks, etc.) and used to repair or fill such defects, or applied to existing bone structures. Bone paste generally has sufficient ductility to allow the user to manipulate and mold it into various shapes. The desired result of this treatment is that bone formation will occur to replace the paste, such as maintaining the shape in which the paste is applied. Bone paste provides a support structure for new bone formation and may contain substances that promote bone formation. In addition to one or more ceramic or protein bone graft materials (e.g., DBM, hydroxyapatite), it generally also contains one or more ingredients that give the material a paste or putty consistency, such as hyaluronic acid, chitosan, and starch components (such as pullulan). In certain embodiments, iTR factors enhance the formation and / or recruitment of osteoprogenitor cells from undifferentiated mesenchymal cells and / or enhance the differentiation of osteoprogenitor cells into cells that form new bone (osteoblasts). In certain embodiments, iTR factors are administered to subjects with osteopenia or osteoporosis, for example, to enhance bone regeneration in the subject.

[0186] In certain embodiments, iTR factors are used to enhance the regeneration of joints (e.g., fibrous, cartilaginous, or synovial joints). In certain embodiments, the joint is an intervertebral disc. In certain embodiments, the joint is a hip, knee, elbow, or shoulder joint. In certain embodiments, iTR factors are used to enhance the regeneration of teeth and / or periodontal tissues or structures (e.g., dental pulp, periodontal ligament, teeth, periodontal bone). In certain embodiments, iTR factors are used to reduce glial scar formation in CNS and PNS injuries. In certain embodiments, iTR factors are used to reduce adhesion and stenosis formation in internal surgery.

[0187] In certain embodiments, iTR factors are used to reduce scarring in tendon and ligament repairs, thereby improving mobility.

[0188] In certain embodiments, iTR factors are used to reduce vision loss following ocular trauma.

[0189] In certain embodiments, iTR factors are administered to a subject in combination with cells. iTR factors and cells can be administered separately or in the same composition. If administered separately, they can be administered at the same or different locations. In various embodiments, cells can be autologous, allogeneic or xenogeneic. The cells may include progenitor cells or stem cells, such as adult stem cells. As used herein, stem cells are cells with at least the following properties: (i) self-renewal, that is, the ability to undergo many cell division cycles while remaining undifferentiated; (ii) multipotency or multi-differentiation potential, that is, the ability to produce offspring of several different cell types (e.g., many, most or all different cell types of a particular tissue or organ). Adult stem cells are stem cells derived from non-embryonic tissues (e.g., fetal, postnatal or adult tissues). As used herein, the term "progenitor cell" encompasses pluripotent cells that are more differentiated than pluripotent stem cells but not fully differentiated. Compared with embryonic progenitor cells, this more highly differentiated cell (possibly from embryonic progenitor cells) has a lower self-renewal capacity. In certain embodiments, iTR factors are administered in combination with mesenchymal progenitor cells, neural progenitor cells, endothelial progenitor cells, hair follicle progenitor cells, neural crest progenitor cells, mammary stem cells, lung progenitor cells (e.g., bronchial alveolar 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 certain embodiments, cells include induced pluripotent stem cells (iPS cells) or cells that have been at least partially differentiated from iPS cells. In certain embodiments, progenitor cells include adult stem cells. In certain embodiments, at least some cells are differentiated cells, such as chondrocytes, osteoblasts, keratinocytes, hepatocytes. In certain embodiments, the cells include myoblasts.

[0190] In certain embodiments, iTR factors are administered in a composition (e.g., a solution) comprising one or more compounds that polymerize in situ or become cross-linked or undergo a phase change after administration to a subject, typically forming a hydrogel. The composition may comprise monomers, polymers, initiators, cross-linking agents, and the like. The composition may be applied (e.g., using a syringe) to an area in need of regeneration, where it forms a gel in situ, and the iTR factors are released from the area over time. Gelation may be triggered, for example, by contact with ions in body fluids or by changing temperature or pH or by light or by combining reactive precursors (e.g., using a multi-barreled syringe). (See, e.g., 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 certain embodiments, the hydrogel is a hydrogel containing hyaluronic acid or hyaluronic acid and collagen I (e.g., HyStem-C described herein). In certain embodiments, the composition further comprises cells.

[0191] In certain embodiments, the iTR factor is administered to the subject in combination with a vector expressing a telomerase catalytic component. The vector can be administered alone or in the same composition. If administered separately, they can be administered in the same or different locations. The vector can express the telomerase catalytic component from the same species as the tissue being treated or from another species. The co-administration of the iTR factor and the telomerase catalytic component is particularly useful, wherein the target tissue is from an elderly individual, and the individual is from a human species. Other methods of the present invention include using iTR factors in the ex vivo production of living bodies, functional tissues, organs, or cell-containing compositions to repair or replace tissues or organs lost due to damage. For example, cells or tissues removed from an individual (future recipient, individual of the same species, or individual of a different species) can be cultured in vitro, optionally with a matrix, a scaffold (e.g., a three-dimensional scaffold) or a mold (e.g., containing a biocompatible, optionally biodegradable material, such as a polymer (e.g., HyStem-C)), and can be promoted to develop into functional tissues or organs by contacting the iTR factor. The scaffold, matrix or mold can be composed at least in part of naturally occurring proteins (e.g., collagen, hyaluronic acid or alginate (or chemically modified derivatives of any of them), or synthetic polymers or copolymers of lactic acid, caprolactone, glycolic acid, etc., or self-assembled peptides, or decellularized matrices derived from tissues such as heart valves, intestinal mucosa, blood vessels and trachea. In certain embodiments, the scaffold comprises a hydrogel. In certain embodiments, the scaffold can be coated or impregnated with iTR factors, which can diffuse out of the scaffold over time. After ex vivo production, the tissue or organ is transplanted into or onto a subject. For example, the tissue or organ can be implanted, or in the case of certain tissues (e.g., skin), it can be placed on the body surface. The tissue or organ can continue to develop in vivo. In certain embodiments, at least a portion of the tissue produced ex vivo is implanted. The tissue or organ is a bladder, a blood vessel, a bone, a fascia, a liver, a muscle, a skin mass, etc. A suitable scaffold may, for example, simulate an extracellular matrix (ECM). Optionally, the iTR factor is administered to a subject before, during, and / or after the transplantation of the tissue or organ produced in vitro. In certain aspects, the biocompatible material is a material that is substantially nontoxic to in vitro cells at the concentration used, or in the case of a material administered to a living subject, a certain amount of the biocompatible material is substantially nontoxic to the cells of the subject. And it does not cause or produce significant harmful or adverse effects (e.g., immunological or inflammatory reactions, unacceptable scar tissue formation, etc.) on the location used. It should be understood that certain biocompatible materials may cause such adverse reactions in a small portion of subjects (usually less than about 5%, 1%, 0.5%, or 0.1%).

[0192] In certain embodiments, a matrix or scaffold coated or impregnated with an iTR factor or a combination of factors including factors capable of causing a global pattern of iTR gene expression is optionally combined with cells and implanted into a subject in need of regeneration. The matrix or scaffold can be in the shape of a tissue or organ in need of regeneration. The cells can be one or more types of stem cells that give rise to and / or are found in such tissue or organ.

[0193] In certain embodiments, the iTR factor or a combination of factors is directly applied to or near the site of tissue damage. "Directly reaching the site of tissue damage" includes injecting a compound or composition into the site of tissue damage or diffusing, pouring or directly contacting the compound or composition with the site of tissue damage. In certain embodiments, if the application occurs at a position within about 10 cm of the visible or obvious edge of a blood vessel (e.g., an artery) at least partially located in the damaged tissue or organ, it is considered to be applied "near the site of tissue damage." "Applying near the site of tissue damage" is sometimes applied in the damaged organ, but the damage is not obvious. In certain embodiments, after the damage or loss of a tissue, organ, or other structure, the iTR factor is applied to the rest of the tissue, organ, or other structure. In certain embodiments, the iTR factor is applied to the end of a severed finger or limb that is still connected to the body to enhance the regeneration of the lost part. In certain embodiments, the severed part is reconnected by surgery, and the iTR factor is applied to one or two surfaces of the wound. In certain embodiments, the iTR factor is applied to enhance the implantation or healing or regeneration of a transplanted organ or part thereof. In certain embodiments, the iTR factor is used to enhance nerve regeneration. For example, the iTR factor can be injected into a severed nerve (e.g., near the proximal and / or distal stump). In certain embodiments, the iTR factor is placed in an artificial nerve conduit, which is a tube composed of biological or synthetic materials in which nerve endings and intervening gaps are enclosed. One or more factors can be formulated into a matrix to promote controlled release over time. The matrix can include a biocompatible, optionally biodegradable material, such as a polymer, such as a polymer comprising hyaluronic acid, including cross-linked hyaluronic acid or carboxymethyl hyaluronate cross-linked with PEGDA, or a mixture of carboxymethyl hyaluronate cross-linked with PEGDA and carboxymethyl-modified gelatin (HyStem-C).

[0194] In certain embodiments, the iTR factor is AgeX1547 as described herein and may or may not be formulated for localization and slow release in carboxymethyl hyaluronate cross-linked with PEGDA and carboxymethyl modified gelatin (HyStem-C) to induce iTR. iTM and iCM factors (e.g., exosomes from fetal or adult cells) may be administered in a physiological solution (e.g., saline) or may be slowly released in carboxymethyl hyaluronate cross-linked with PEGDA and carboxymethyl modified gelatin (HyStem-C) to induce iTM or iCM.

[0195] In certain embodiments, iTR factors or a combination of factors are used to promote the production of hair follicles and / or the growth of hair. In certain embodiments, iTR factors trigger the regeneration of hair follicles in epithelial cells that do not normally form hair. In certain embodiments, iTR factors are used to treat hair loss, thinning hair, partial or complete baldness in men or women. In certain embodiments, baldness is a state in which there is no or substantially no hair or hair is missing in places where it usually grows (e.g., on the top, back, and / or sides of the head). In certain embodiments, thinning hair is a state in which the hair is less than normal or ordinary hair, or in certain embodiments, the hair is less than the individual's past hair, or in certain embodiments, the hair is less than the individual's ideal hair. In certain embodiments, iTR factors are used to promote the growth of eyebrows or eyelashes. In certain embodiments, iTR factors are used to treat androgenic alopecia or "male pattern baldness" (which can affect both men and women). In certain embodiments, iTR factors are used to treat alopecia areata (which involves patchy hair loss on the scalp), total baldness (which involves the loss of all hair on the head), or universal baldness (which involves the loss of all hair on the head and body). In some embodiments, the iTR factor is applied to a part where hair growth is needed, such as the scalp or eyebrow area. In some embodiments, the iTR factor is applied to the edge or near the edge of the eyelid to promote the growth of eyelashes. In some embodiments, the iTR factor is applied to a liquid preparation. In some embodiments, the iTR factor is applied to a cream, ointment, paste or gel. In some embodiments, the iTR factor is used to enhance hair growth after burns, surgery, chemotherapy or other events that cause hair loss or hair-bearing skin loss. In some embodiments, the iTR factor or a combination of factors is applied to tissues suffering from age-related degenerative changes to regenerate youthful functions. The 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 factor is co-administered with a carrier expressing the catalytic component of telomerase to extend cell life.

[0196] In certain embodiments, one or more iTR factors are administered to enhance the replacement of cells lost or damaged due to injuries such as chemotherapy, radiation or toxins. In certain embodiments, such cells are stromal cells of solid organs and tissues. The therapeutic methods of the present invention may include the step of identifying or providing a subject suffering from a disease or condition or at risk or at risk thereof, wherein enhanced regeneration will be beneficial to the subject. In certain embodiments, the subject has experienced an injury (e.g., physical trauma) or damage to a tissue or organ. In certain embodiments, the injury is to a limb or finger. In certain embodiments, the subject suffers from a disease affecting the cardiovascular, digestive, endocrine, musculoskeletal, gastrointestinal, liver, skin, nervous, respiratory or urinary systems. In certain embodiments, tissue damage refers to damage to a tissue, organ, or structure, 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, breast, testicles, 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).

[0197] In certain 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 damage (e.g., trauma or acute disease-related events, such as myocardial infarction or stroke), and optionally at least once again after that time. In certain embodiments, the compound or composition is administered to the subject at least once within about 1-2 weeks, 2-6 weeks, or 6-12 weeks after the subject suffers tissue damage, and optionally at least once again after that time.

[0198] In certain embodiments of the invention, it may be useful to stimulate or promote the regeneration or de novo development of missing or malformed tissues, organs or structures by, for example, removing skin, removing at least some tissues, rubbing joints or bone surfaces where regeneration or de novo development is needed, and / or creating another type of wound on a subject. In the case of regeneration after tissue damage, it may be desirable to remove (e.g., by surgical resection or debridement) at least some of the damaged tissue. In certain embodiments, iTR factors are administered at or near such removed or worn sites. In certain embodiments, iTR factors are used to enhance the generation of tissues or organs in a subject in which such tissues or organs are at least partially absent due to a congenital disease (e.g., a genetic disease). Many congenital malformations can result in the underdevelopment or lack of various tissues, organs or body structures, such as limbs or fingers. In other cases, developmental disorders that result in the underdevelopment of tissues, organs or other body structures become apparent after birth. In certain embodiments, iTR factors are administered to subjects with underdeveloped or lacking tissues, organs or other body structures to stimulate the growth or development of such tissues, organs or other body structures. In certain aspects, the invention provides a method of enhancing the production of a tissue, organ or other body structure in a subject that is underdeveloped or congenitally lacks such a tissue, organ or other body structure, the method comprising administering an iTR factor to the subject. In certain embodiments, the iTR factor is administered to the subject before birth, i.e., in utero. The various aspects and embodiments of the invention described herein regarding regeneration are applicable to such de novo production of tissues, organs or other body structures and are included in the present invention.

[0199] In certain aspects, iTR factors are used to enhance the production of tissue in any of a variety of situations where new tissue grows where such tissue did not previously exist and such growth is beneficial. For example, in the case of spinal or other joint fusions, it is often useful to generate bone tissue between the joints.

[0200] iTR factors can be tested in a variety of regenerative animal models. On the one hand, iTR regulators have been tested in murine species. For example, mice can be injured (e.g., by incision, amputation, transverse section or removal of tissue fragments). The iTR factor is applied to the wound site and / or the removed tissue fragments, and its effect on regeneration is evaluated. The effect of vertebrate TR regulators can be tested in a variety of vertebrate models for tissue or organ regeneration. For example, as described in (Mathew LK, Unraveling tissue regeneration pathways using chemical genetics. J Biol Chem. 282 (48): 35202-10 (2007)), the regeneration of fins can be evaluated in zebrafish, and it can be used as a model for limb regeneration. Rodents, dogs, horses, goats, fish, amphibians and other animal models that can be used to test the regeneration of tissues and organs (such as heart, lungs, limbs, skeletal muscle, bones, etc.) have been widely used. For example, various animal models for musculoskeletal regeneration are discussed in Tissue Eng Part B Rev. 16(1)(2010). Common animal models for studying liver regeneration include surgical removal of a large portion of a rodent liver. Other models of liver regeneration include acute or chronic liver injury or liver failure caused by toxins (e.g., carbon tetrachloride). In certain embodiments, models for hair regeneration or skin wound healing involve, for example, excising a skin piece from a mouse. Regeneration of hair follicles, hair growth, re-epithelialization, glandularization, etc. can be assessed.

[0201] Compounds and compositions disclosed herein and / or identified using the methods and / or assay systems described herein may be administered by any suitable means, e.g., orally, intranasally, subcutaneously, intramuscularly, intravenously, intraarterially, parenterally, intraperitoneally, intrathecally, intratracheally, intraocularly, sublingually, vaginally, rectally, dermally, or by inhalation (e.g., as an aerosol). Of course, the particular mode selected will depend on the particular compound selected, the particular condition being treated, and the dose required for therapeutic efficacy. In general, the methods of the present invention may be implemented using any medically or veterinarily acceptable mode of administration, which refers to 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, such as substantially pure preparations, may be combined with one or more pharmaceutically acceptable carriers or excipients, etc., to produce a suitable pharmaceutical composition suitable for administration to a subject. Such pharmaceutically acceptable compositions are one aspect of the present invention. The term "pharmaceutically acceptable carrier or excipient" refers to a carrier (the term includes carriers, media, diluents, solvents, vehicles, etc.) or excipients that do not significantly interfere with the biological activity or effectiveness of the active ingredient. At the use or application concentration of the composition, the composition is not overly toxic to the host. Other pharmaceutically acceptable ingredients may also be present in the composition. Suitable substances and their use in the preparation of pharmaceutically active compounds are well known in the art (see, for example, "Remington's Pharmaceutical Sciences", EW Martin, 19th edition, 1995, Mack Publishing Co.: Easton, Pa., and updated versions, such as Remington: The Science and Practice of Pharmacy. 21st edition. Philadelphia, Pa. Lippincott Williams & Wilkins, 2005, which is a further exploration of pharmaceutically acceptable substances and methods for preparing various types of pharmaceutical compositions). In addition, the compounds and compositions of the present invention can be used in combination with any compound or composition used in the art for treating a specific disease or a target condition. The pharmaceutical composition is usually formulated to be compatible with its intended route of administration. For example, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions (including saline and buffered media, such as sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution).Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil) and injectable organic esters (e.g., ethyl oleate), fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; preservatives, such as antibacterial agents (e.g., benzyl alcohol or methyl paraben); antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for adjusting tonicity, such as sodium chloride or glucose. pH can be adjusted with acids or bases (e.g., hydrochloric acid or sodium hydroxide). Such parenteral preparations can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0202] For oral administration, the compounds can be readily formulated by mixing the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers allow the compounds of the invention to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like. Suitable excipients for oral dosage forms are, for example, fillers such as sugars (including lactose, sucrose, mannitol, or sorbitol); cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP).

[0203] In order to be used by inhalation, the composition of the present invention can be delivered from a pressure vessel or a distributor in the form of an aerosol spray, and the pressure vessel or the distributor contains a suitable propellant, such as a gas such as carbon dioxide, a fluorocarbon or a sprayer. Liquid or dry aerosols (such as dry powders, large porous particles, etc.) can be used. The present invention also contemplates the use of nasal sprays or other forms of nasal administration to deliver the composition. For topical application, the pharmaceutical composition can be formulated into a suitable ointment, lotion, gel or cream, wherein containing an active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers suitable for the composition. For local delivery to the eyes, the pharmaceutically acceptable composition can be formulated into a solution or a micronized suspension in an isotonic pH-regulated sterile saline, such as for use in eye drops or ointments, or for intraocular administration, such as by injection. The pharmaceutical composition can be formulated for transmucosal or transdermal delivery. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated can be used in the preparation. Such a penetrant is well known in the art. The pharmaceutical compositions of this invention may be formulated as suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or as enemas for rectal delivery.

[0204] In certain embodiments, the composition comprises one or more agents intended to protect the active agent from rapid removal from the body, such as controlled release formulations, implants, microcapsule delivery systems, etc. The composition may be incorporated with agents to improve stability (e.g., in the gastrointestinal tract or in the blood) and / or enhance absorption. The compound may be encapsulated or incorporated into particles (e.g., microparticles or nanoparticles). Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, PLGA, collagen, polyorthoesters, polyethers, and polylactic acid may be used. The preparation methods of such preparations are obvious to those skilled in the art. For example, but not limited to, many delivery systems based on particles, lipids, and / or polymers are known in the art for delivering siRNA. The present invention contemplates the use of such compositions. Liposomes or other lipid-based particles may also be used as pharmaceutically acceptable carriers.

[0205] Pharmaceutical compositions and compounds for use in such compositions can be manufactured under conditions that meet standards, requirements or guidelines set by regulatory agencies. For example, such compositions and compounds can be produced in accordance with good manufacturing practices (GMP) and / or follow quality control procedures suitable for medicaments administered to humans, and can be labeled with approval by a government regulatory agency responsible for regulating drugs, surgical procedures or other products with beneficial therapeutic properties.

[0206] The pharmaceutical composition of the present invention, when applied to a subject for therapeutic purposes, is preferably applied in a time and amount sufficient to treat the disease or condition to which it is applied. The therapeutic efficacy and toxicity of the active agent can be evaluated by standard pharmaceutical procedures in cell culture or experimental animals. The data obtained from cell culture assays and animal studies can be used to formulate a series of dosages suitable for humans or other subjects. As known in the art, different doses for human administration can be further tested in human clinical trials. The dose used can be a maximum tolerated dose or a lower dose. The therapeutically effective dose of the active agent in the pharmaceutical composition can 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, about 1 to about 10 mg / kg. Other exemplary doses include, for example, about 1 μg / kg to about 500 mg / kg, about 100 mg / kg to about 5 mg / kg. In some embodiments, a single dose is applied, while in other embodiments, multiple doses are applied. It will be understood by those of ordinary skill in the art that the appropriate dose in any particular case depends on the efficacy of the medicament used and can be optionally adjusted for a specific recipient. The specific dosage level of the subject may depend on a variety of factors, including the activity of the specific agent used, the specific disease or condition and its severity, age, weight, the overall health of the subject, etc. For ease of administration and uniform dosage, it may be necessary to formulate the pharmaceutical composition in unit dosage form, particularly for oral or parenteral compositions. As used herein, a unit dosage form refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of an active agent that is calculated to produce the desired therapeutic effect and contains an appropriate pharmaceutically acceptable carrier. It will be understood that a treatment regimen may include administering multiple doses, such as unit dosage forms, over a period of time, which may last for days, weeks, months, or years. The subject may receive one or more doses per day during the treatment period, or one dose every other day or less frequently. For example, it may be administered every two weeks, every week, etc. For example, administration may continue until the proper structure and / or function of the tissue or organ has been at least partially restored and / or until continued administration of the compound does not appear to promote further regeneration or improvement. In certain embodiments, the subject administers one or more doses of the composition of the present invention to him or her.

[0207] In some embodiments, for example, for the purpose of enhancing regeneration, two or more compounds or compositions are administered in combination. The compounds or compositions administered in combination can be administered together or separately in the same composition. In some embodiments, "combination" administration refers to the following administration relative to the administration of the first and second compounds or compositions: (i) administering the second compound dose before more than 90% of the most recently administered dose of the first agent is metabolized to an inactive form or discharged from the body; or (ii) administering the first and second doses within 48, 72, 96, 120 or 168 hours between each other, or (iii) administering the drug in an overlapping time period (e.g., continuous or intermittent infusion); (iv) any combination of the above. In some embodiments, two or more iTR factors or carriers expressing the catalytic components of telomerase and iTR factors are administered. In some embodiments, iTR factors are used in combination with one or more growth factors, growth factor receptor ligands (e.g., agonists), hormones (e.g., steroids or peptide hormones), or signaling molecules to promote regeneration and polarity. Particularly useful are tissue center molecules, which can be used for tissue regeneration competent cells (e.g., competent cells produced using the methods of the present invention). In certain embodiments, the growth factor is a member of the epidermal growth factor family (e.g., EGF, neuregulin), a fibroblast growth factor (e.g., any of FGF1-FGF23), a hepatocyte growth factor (HGF), a nerve growth factor, a bone morphogenetic protein (e.g., any of BMP1-BMP7), a vascular endothelial growth factor (VEGF), a wnt ligand, a wnt antagonist, retinoic acid, NOTUM, follistatin, sonic hedgehog, or other organizing central factors.

[0208] Sources of iTM and iCM factors

[0209] iTM and iCM factors can be identified by exposing embryonic cells lacking EFT markers (e.g., as a non-limiting example, stromal cells that do not express COX7A1) to various agents and detecting the induction of the markers, such as COX7A1 or reporter constructs (e.g., GFP constructs expressed from the COX7A1 gene promoter). Since exosomes carry potent protein and RNA factors that can reprogram cells to confer new growth, migration, and differentiation properties, we examined whether they can reprogram the developmental state of cells, i.e., iTM and iCM. Therefore, we tested the ability of exosomes from adult cells to induce adult genes in embryonic cells. We evaluated the total RNA expression profiles by Illumina microarray analysis of 15 homologous hESC-derived clonal embryonic progenitor cell lines and compared them with 18 primary endothelial cell lines (neonatal to adult) obtained from various anatomical sites (not shown). We determined that exosomes from cells that had undergone EFT were able to induce COX7A1 expression in embryonic cells that previously lacked such expression, as well as to mature the cells using other markers described herein.

[0210] Only through routine experiments, those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the present invention described herein.The scope of the present invention is not intended to be limited to the description or the details set forth therein.Unless otherwise indicated or apparent from the context, articles such as "a, an" and "the" can represent one or more.Usually, certain methods of the present invention are practiced using, for example, cell groups in vitro or in vivo.Therefore, reference to "cell" should be understood to include embodiments in which the cell is a member of a cell group, such as a group comprising genetically substantially identical cells or a group consisting thereof.However, the present invention includes embodiments in which the method of the present invention is applied to a single cell.Therefore, reference to "cell" should be understood to include embodiments in which the cell is a member of a cell group and embodiments in which the cell is applied to a single isolated cell.

[0211] Unless otherwise stated or obvious from the context, claims or descriptions containing "or" between one or more members of the group will be considered to satisfy the condition if one, more than one, or all of the group members are present, used, or associated with a given product or method. The present invention includes embodiments in which exactly one member of the group is present, used, or associated with a given product or method. The present invention also includes embodiments in which one or more of the group members are present, used, or associated with a given product or method. It is contemplated that all embodiments described herein are applicable to all different aspects of the present invention. It is also contemplated that any embodiment can be freely combined with one or more other such embodiments when appropriate. In addition, it should be understood that the present invention covers all changes, combinations, and permutations that introduce one or more limitations, elements, clauses, descriptive terms, etc. from one or more claims (whether original claims or subsequently added claims) into another claim (whether original claims or subsequently added). For example, any claim that is dependent on another claim may be modified to include one or more elements or limitations found in any other claim that is dependent on the same base claim, and any claim that refers to elements present in a different claim may be modified to include one or more elements or limitations found in any other claim that is dependent on the same base claim as the claim. In addition, where a composition is described in the claims, the present invention provides methods for preparing the composition, for example, according to the methods disclosed herein, and methods for using the composition, for example, for the purposes disclosed herein. Where a method is described in the claims, the present invention provides a composition suitable for performing the method and a method for preparing the composition. Similarly, unless otherwise stated or unless a person of ordinary skill in the art is aware of the emergence of a contradiction or inconsistency, where a method for preparing a composition is described in the claims, the present invention provides a composition prepared by the method of the present invention and a method for using the composition.

[0212] Where elements are presented in a list (e.g., in Markush group form), each subgroup of elements is also disclosed, and any element may be deleted from the group. For the sake of brevity, only some of these embodiments are specifically described herein, but the present invention includes all such embodiments. It should also be understood that, in general, where the present invention or aspects of the present invention are referred to as comprising particular elements, features, etc., certain embodiments of the present invention or aspects of the present invention consist of or consist essentially of such elements, features, etc.

[0213] In the case of numerical ranges mentioned herein, the present invention includes embodiments including endpoints, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other endpoint is excluded. Unless otherwise specified, it should be assumed that two endpoints are included. In addition, unless otherwise indicated or apparent from the context and the understanding of those of ordinary skill in the art, in different embodiments of the present invention, the value expressed as a range can adopt any specific value or sub-range within the range, up to one-tenth of the unit of the lower limit of the range. When using phrases such as "less than X", "greater than X" or "at least X" (wherein X is a number or percentage), it should be understood that any reasonable value can be selected as the lower or upper limit of the range. It should also be understood that in the case of listing a numerical list herein (whether or not starting with "at least"), the present invention includes embodiments related to any intermediate value or range defined by any two values ​​in the list, and the minimum value can be taken as the minimum value, and the maximum value can be taken as the maximum value. In addition, in the case of a numerical list (such as a percentage) with "at least" as a prefix, the term applies to each number in the list. For any embodiment of the present invention in which a numerical value starts with "about" or "approximately", the present invention includes embodiments in which the exact value is recorded. For any embodiment of the invention in which a numerical value does not begin with "about" or "approximately", the invention includes embodiments in which the numerical value begins with "about" or "approximately". Unless otherwise specified or obvious from the context (e.g., the number is not allowed to exceed 100% of the possible value), "about" or "approximately" generally includes numbers that fall within 1% of the number in either direction, in some embodiments 5%, and in some embodiments 10% (greater or less than the number). Unless otherwise specified, a "composition" as used herein may include one or more components. For example, a "composition comprising an activator or a TR activator" may consist of or consist essentially of an activator of a TR activator, or may include one or more additional components. It should be understood that, unless otherwise specified, the inhibitor or TR inhibitor (or other compound mentioned herein) in any embodiment of the invention may be used or administered in the form of a composition comprising one or more additional components, the additional components including an activator of a TR activator.

[0214] Example

[0215] Example 1. A low-throughput screen for iTR factors was performed on adult human skin fibroblasts using PCR to measure the reduction in expression of COX7A1 as an iTR marker.

[0216] About 200 combinations of candidate iTR factors described herein were screened under low-throughput conditions, and as the first assay, COX7A1 expression was determined by qPCR. Conditions that showed a significant reduction in COX7A1 expression were used to prepare RNA for transcriptomic analysis based on Illumina bead arrays or RNA sequencing. The best conditions that resulted in the greatest reduction in COX7A1 levels while maintaining viable cells were 0.5 mM valproic acid, 10 uM CHIR99021, 10 uM RepSox, 10 uM Tranylpromine (Parnate), 50 uM Forskolin, 5 uM TTNPB given for the first 17 days, and then 100 nM 3-Deazaneplanocin A (DZNep) was added to the above mixture for an additional 14 days, and then only 1.0 uM PD0325901 and 10 uM CHIR99021 were used for the last 7 days. All factors were used in cell growth medium (high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS)).

[0217] RNA was prepared from cells under this iTR condition (called AgeX1547) and then subjected to Illumina bead array analysis. RFU values ​​< 100 were considered to be absent. Figure 1 As shown, Illumina bead array analysis showed no COX7A1 expression in hES cells or their derived hEP cell lines. However, in early passage dermal skin fibroblast cultures, COX7A1 expression gradually increased from the earliest in vivo experimental date (8 weeks of gestational development) and even gradually increased after PPT to adulthood. After iPS cell reprogramming, COX7A1 levels returned to essentially hES cell levels (no expression), and the above-mentioned iTR mixture AgeX1547 caused COX7A1 levels to be reprogrammed back to levels of approximately 8 weeks (pre-fetal) development. As shown Figure 2 As shown, ADIRF provides a marker for PPT, and AgeX1547 again reverts 59-year-old adult dermal fibroblasts to the pre-fetal ADIRF gene expression pattern. Figure 3 As shown, TNFRSF11B also provides a marker for PPT, and AgeX1547 again reverted 59-year-old adult dermal fibroblasts to a pre-fetal TNFRSF11B gene expression pattern. Since TNFRSF11B expression is associated with the arrest of osteogenesis, the reversal of TNFRSF11B expression is consistent with the restoration of osteogenic potential in cells capable of forming new bone.

[0218] like Figure 4As shown, the embryonic marker AMH is expressed in the hEP cell line but not in fetal or adult skin fibroblasts, but after AgeX1547 treatment as described above, AMH expression is restored to the embryonic expression pattern.

[0219] To determine whether 59-year-old dermal fibroblasts reprogrammed by the iTR factor AgeX1547 had reverted to pluripotency (which is unexpected, since the purpose of iTR is to induce a lytic phenotype in regenerated senescent cells without changing the cell differentiation state), we examined the expression of COL1A1. Figure 5 As shown, while hES cells showed much lower COL1A1 expression than fetal and adult skin fibroblasts, and while 59-year-old skin fibroblasts did suppress COL1A1 expression back to hES cell levels when reprogrammed into their iPSC counterparts, 59-year-old skin fibroblasts treated with AgeX1547 did not suppress COL1A1 to hES cell levels. Instead, the cells retained fibroblast morphology, had no obvious signs of toxicity, and had COL1A1 levels comparable to normal dermal fibroblasts cultured in vitro.

[0220] like Figure 6 As shown, iPS cell reprogramming restored OCT4 expression to hES cell levels, whereas AgeX1547 did not induce the pluripotency marker OCT4 or other markers specific to pluripotency cells (not shown). We therefore conclude that AgeX1547 appears to be tolerated by human cells during treatment and appears to be able to restore adult-derived cells to prenatal, even pre-fetal, patterns of gene expression without altering their differentiation state (iTR).

[0221] Example 2. Screening for iTR factors that can induce lysis of senescent cells in senescent cells or CSCs.

[0222] Optimal conditions were determined to distinguish the increase in apoptosis expected for cells with a pre-fetal gene expression pattern. Mesenchymal (4D20.8 and MSC, respectively) and vascular endothelial cells (30MV2-6 and HAEC, respectively) of embryonic and adult origin were subjected to different apoptotic stimuli, including two concentrations of camptothecin (CPT), H2O2 and thapsigargin (TG) as well as a vehicle control. Compounds were applied for 24 hours before analysis. After treatment, apoptosis was monitored by TUNEL staining using the In Situ Cell Death Detection kit (Roche, catalog number 12156792910) according to the manufacturer's instructions. Figure 7As shown, thapsigargin at 3.7 nM provided a statistically significant increase in apoptosis in both embryonic mesenchymal and endothelial cells compared to their adult counterparts. Therefore, this assay can be used to determine the effectiveness of iTR factors in inducing lysis of senescent cells in both senescent cells and CSCs.

[0223] Example 3. Methods for preparing an EGFP reporter for COX7A1 expression for use in high-throughput iTR screening.

[0224] IRES-EGFP was inserted downstream of the COX7A1 locus in TERT-immortalized human foreskin fibroblasts. To achieve this, two candidate guide RNAs (gRNAs) were designed and cloned to target the COX7A1 locus. gRNA-mediated CRISPR / Cas9 modification resulted in targeted double-strand breaks (DSBs). A donor plasmid carrying an IRES-EGFP cassette was constructed based on the location of the active gRNA to serve as a DNA repair template. The gRNA and donor plasmid were co-transfected into TERT-immortalized human foreskin fibroblasts to mediate homology-directed repair (HDR), thereby knocking IRES-EGFP into the target region of COX7A1 ( Figure 7 ).

[0225] Example 4.

[0226] In the first set of samples, human dermal fibroblasts (MDW1) at passage 5-8 were seeded at approximately 50,000 cells per well in a 6-well Corning tissue culture plate in growth medium DMEM + 10% FBS.

[0227] The next day, cells were treated with various mixtures as indicated below for various durations (27-42 days as indicated).

[0228] mixture (in vehicle: KnockOut DMEM supplemented with 10% KSR, 10% FBS, 2 mM GlutaMAX, 1% NEAA, 0.055 mM 2-mercaptoethanol, 10,000 U / ml penicillin-streptomycin) and described below.

[0229] 1.MDW-1P8 D41 CHIR99021 10uM+RepSox (l0 uM)+tranylcypromine (Parnate) 10uM+Forskolin 50uM+TTNPB 5uM+DZNep 100nM+PD0325901 1uM+FGF2 50ng / ml+EPZ004777 5uM+SGC0946 5uM+RSC133 10uM+CI994 1uM

[0230] 2. MDW-1P5 D39, CHIR99021 at 10 uM + RepSox (10 uM) + Tranylcypromine (Parnate) 10 uM + Forskolin 50 uM + TTNPB 5 uM + DZNep 100 nM + PD0325901 1 uM + FGF2 50 ng / ml + OAC3 1 uM + EPZ004777 5 uM + SGC094 5 uM + RSC133 10 uM + CI994 1 uM + SB216763 20 uM + SAHA 1 uM + OCT4 250 ng / ml + AM580 50 nM

[0231] 3. MDW-1P5 D39, CHIR99021 at 10 uM + RepSox (10 uM) + Tranylcypromine (Parnate) 10 uM + Forskolin 50 uM + TTNPB 5 uM + DZNep 100 nM + PD0325901 1 uM + FGF2 50 ng / ml + OAC3 1 uM + EPZ004777 5 uM + SGC094 5 uM + RSC133 10 uM + CI994 1 uM + SB216763 20 uM + SAHA 1 uM + OCT4 250 ng / ml + AM580 50 nM + PP1 10 uM

[0232] 4. MDW-1P5 D42, CHIR99021 at 10 uM + RepSox (10 uM) + Tranylcypromine (Parnate) 10 uM + Forskolin 50 uM + TTNPB 5 uM + DZNep 100 nM + PD0325901 1 uM + FGF2 50 ng / ml + EPZ004777 5 uM + SGC094 5 uM + RSC133 10 uM + CI994 1 uM + SB216763 20 uM + SAHA 1 uM + AM580 50 nM + PP1 10 uM

[0233] 5. MDW-1P8 D42, MS-275 at 1 uM + CHIR99021 10 uM + RepSox (10 uM) + Tranylcypromine (parnate) 10 uM + Forskolin 50 uM + TTNPB 5 uM + DZNep 100 nM + PD0325901 1 uM + FGF2 50 ng / ml + OAC3 1 uM + EPZ004 5 uM + SGC094 5 uM + RSC133 10 uM + CI994 1 uM

[0234] 6.MDW-1P8 D27 MS-275 1uM+CHIR99021 10uM+RepSox(10uM)+Parnate 10uM+Forskolin 50uM+TTNPB 5uM+DZNep 100nM+PD0325901 1uM+FGF 50ng / ml+OAC3 1uM+EPZ004777 5uM

[0235] Two controls were performed using "fasting medium" (DMEM with 0.5% FBS) (no mixture). The seeded cells were grown to confluence and then fed with fasting medium for 3 days and then with fasting medium for an additional 2 days before being lysed to obtain RNA.

[0236] In a second set of samples, human dermal fibroblasts (MDW1) at passage 6-7 were seeded at approximately 50,000 cells per well in a 6-well Corning tissue culture plate in growth medium DMEM + 10% FBS.

[0237] The next day, cells were treated with a mixture containing BIX01294 2mM, and then after 7 days, Repsox 10uM, Dastinib 0.5uM, and Kenpaullone 5uM were added to the growth medium for a total of 14 days.

[0238] Additionally, a control was performed using "fasting medium" (DMEM with 0.5% FBS) (no mixture). The seeded cells were grown to confluence and then fed with fasting medium for 3 days and then fed with fasting medium for an additional 2 days before being lysed to obtain RNA.

[0239] For both the first and second group samples, cells were lysed with RLT (Qiagen, Valencia CA, catalog number 79216) and total RNA was extracted using the Qiagen RNeasy mini kit (Qiagen, catalog number 74104). cDNA was prepared according to the manufacturer's instructions using the SuperScript III first-strand kit and random hexamers (Invitrogen, Carlsbad CA, catalog number 18080-051). cDNA purification was performed using the QIAquick PCR purification kit (Qiagen, Valencia CA, catalog number 28104) according to the manufacturer's instructions to remove nucleotides, primers, salts and polymerase.

[0240] Test samples (templates) were prepared in standard optical 96-well reaction plates (Applied Biosystems Carlsbad, CA, PN 4306737) consisting of 30 ng RNA equivalents of cDNA, 0.8 uM of each gene-specific custom oligonucleotide primer set (Invitrogen), ultrapure distilled water (Invitrogen catalog number 10977015), and diluted 1:1 with 12.5 ul of Power SYBR Green PCR Master Mix (Applied Biosystems Carlsbad, CA, catalog number 4367659) with AmpliTaq Gold DNA polymerase in a total reaction volume of 25 ul. Real-time qPCR was performed using an Applied Biosystems 7500 Real-Time PCR System with SDSv2.3 software. Amplification conditions were set at 50°C for 2 min (stage 1), 95°C for 10 min (stage 2), 40 cycles of 95°C for 15 s, followed by 60°C for 1 min (stage 3), a dissociation phase at 95°C for 15 s (stage 4), 60°C for 1 min, and 95°C for 15 s. The Ct values ​​of the amplicons were normalized to the average Ct value of GAPDH.

[0241] The results of the first set of samples are shown in Figure 9. The results of the second set of samples are shown in Fig.10 All combinations of iTR factors shown in Figures 9 and 10 significantly reduced COX7A1 and other EFT markers. The best combination was #5 above.

[0242] Example 5. Scratch wound assay of tissue regeneration and cell proliferation after mixture treatment

[0243] BJ-TERT fibroblasts at passage 8 were seeded at 50,000 cells / well in 6-well plates in DMEM 10% FBS. The next day, the cells were treated with the following mixtures for 2 weeks:

[0244] Control: Growth medium only

[0245] A: LIN28A-11R (LD Biopharma HTF 0046) 4ug / ml

[0246] B: Valproic acid (Cayman 13033) 0.5 mM

[0247] C: LIN28A-11R 4ug / ml + valproic acid 0.5mM

[0248] D: MS-275(Cayman 13284)1uM+CHIR99021(Cayman 13122)10uM+RepSox(SigmaR0158)10uM+tranylcypromine (Parnate)(Sigma 616431)10uM+Forskolin(Cayman 11018)50uM+TTNPB(Cayman 16144N)5uM+DZNep(Cayman 13128)100nM+PD0325901(Cayman 13034)1uM+FGF2(Peprotech AF-100-18B)50ng / ml+OAC3(Cayman 14104)1uM+EPZ004777(Cayman16173)5uM+SGC0946(Cayman 13967)5uM+RSC133(Cayman 90018139)10uM

[0249] E: In hES medium (DMEM / F12 10% KSR, 10% FBS, 1% glutamine, 1% NEAA, 0.055mM 2-mercaptoethanol, bFGF 50ng / ml), LIN28A 4ug / ml + VPA 0.5mM, CHIR99021 10uM, RepSox 10uM, Parnate 10uM, Forskolin 50uM, TTNPB 5uM + DZNep 100nM + PD0325901 1uM

[0250] After treatment, 800 μm scratches were made at confluence and the percentage of filling was assessed after 16 hours. Scratch filling in treated cells was compared to that in controls. The data are shown below.

[0251]

[0252]

[0253] This study suggests that the reprogramming factors included here aid in cell migration and may be useful in the regeneration and induction of cell proliferation associated with healing injured or damaged mammalian tissues. In particular, conditions C, D, and E showed 50% or more more population than the control group.

[0254] Example 6. Fibroblasts treated with reprogramming agents showed senescent cell lytic activity against late passage cells and reduced cell numbers.

[0255] Xgene fibroblasts (human foreskin obtained from Xgene, Sausilito CA) were seeded at 20,000 cells / well (0.1% gelatin coated 24-well plates) at early (P9) and late (P33) passages in growth medium (DMEM + 10% FBS). The next day, they were re-fed with medium containing the supplements listed below for 1 week, every two days, and then counted.

[0256] The control group had no additives.

[0257] A.LIN28A-11R(LD Biopharma HTF 0046)4ug / ml,

[0258] B. Valproic acid (Cayman 13033) 0.5 mM,

[0259] C.LIN28A 4ug / ml+valproic acid 0.5mM

[0260] D.LIN28A 4ug / ml+VPA 0.5mM, CHIR99021 10uM (Cayman 13122), Repsox 10uM (Sigma R0158), Parnate 10uM (Sigma 616431), Forskolin 50uM (Cayman 11018), TNTPB5uM (Cayman 16144), DZNep 100nM (Cayman 13128) and PD032590l 1uM (Cayman 13034).

[0261] The data looks like this:

[0262]

[0263]

[0264] As expected, the population doubling time of early passage fibroblasts (P9) was nearly four times faster than that of late passage (P33) in growth medium alone. Compared with the control (growth medium only), late passage cells showed a decrease in cell number within 1 week after exposure to factors in conditions A (LIN28A) and C (LIN28A+VPA). This reduction in late passage cells after exposure to LIN28A with and without valproic acid is at least partly due to senescent specific cell death (senescent cell lysis). In contrast, exposure to these agents during early passages resulted in an increase in cell number. Treating cells with condition D (composed of a more comprehensive drug cocktail) resulted in a substantial reduction in cell number, which was greater during late passages, in part due to senescent cell lysis. Here, we show that the cell number of early passage cells increased 2.8 times over the number of cells seeded, while the cell number of late passage cells increased only 1.1 times over the number of cells seeded.

[0265] The reprogramming additives valproic acid and LIN28A-11R used alone and in combination have different effects on late passage cells due to their senescent cell lysis. Similarly, the treatment of an inclusive reprogramming mixture comprising LIN28A 4ug / ml+VPA 0.5mM, CHIR99021 10uM, Repsox 10uM, Parnate 10uM, Forskolin 50uM, TTNPB, DZNep 100nM and PD0325901 1uM resulted in a significant reduction in late passage cells due to the properties of the senescent cell lysis of the above substances.

[0266] Example 7. iTR was performed using 1 mM valproic acid

[0267] Adult derived dermal fibroblasts were cultured in DMEM 10% FBS in wells of 6-well plates and treated with 0.5 mM and 1 mM valproic acid for 28 days. On day 28, the culture medium was removed, the cells were washed with PBS, and then lysed using RLT with β-mercaptoethanol (Qiagen catalog number 79216). Total RNA was extracted using Qiagen RNeasymini-kit (Qiagen catalog number 74106) according to the manufacturer's instructions. cDNA was then prepared using ThermoFisher superscript III (catalog number 18080044). qPCR was run on an Applied Biosystems 7500Real-Time PCR System using SDSv2.3 software to assess COPD7A1 expression relative to GAPDH in treated cell lines compared to untreated cell lines (a clear indication of an adult phenotype).

[0268] Surprisingly, 0.5 mM concentration of valproate had negligible effect on COX7A1 expression (normalized to GAPDH) compared to the control (untreated) group, but a significant decrease in its expression to near zero (0.001) was observed at 1 mM concentration of valproate.

Claims

1. A composition suitable for altering the genetic profile of a postnatal mammalian cell, comprising: a. A first composition comprising: 0.5-5.0 mM valproic acid, 7-10 uM 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR99021), 4.0-10 uM 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox), 2.0-10 uM tranylcypromine (Parnate), 0.5-50 uM Forskolin and 1.0-5 uM 4-[(E)-2-(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)prop-1-enyl]benzoic acid (TTNPB); b. A second composition comprising: 0.5-5.0 mM valproic acid, 7-10 uM CHIR99021, 4.0-10 uM RepSox, 2.0-10 uM Parnate, 0.5-50 uM Forskolin, 1.0-5 uM TTNPB, and 50 nM-240 nM 3-Deazaneplanocin A (DZNep); and c. A third composition comprising: 0.1-1.0 uM N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoaniline)benzamide (PD0325901) and 7.0-10 uM 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR99021), d. A fourth composition comprising a suitable expression vector for expressing the TERT gene in mammalian cells.

2. The composition of claim 1, wherein the first composition, the second composition, and the third composition are in cell culture medium.

3. The composition of claim 1, wherein the first composition, the second composition, and the third composition are in a hydrogel comprising hyaluronic acid.

4. A composition suitable for altering the genetic profile of a postnatal mammalian cell, comprising: a. A first composition comprising: 0.5-5.0 mM valproic acid, 7-10 uM 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR99021), 4.0-10 uM 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox), 2.0-10 uM tranylcypromine (Parnate), 0.5-50 uM Forskolin and 1.0-5 uM 4-[(E)-2-(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)prop-1-enyl]benzoic acid (TTNPB); b. A second composition comprising: 0.5-5.0 mM valproic acid, 7-10 uM CHIR99021, 4.0-10 uM RepSox, 2.0-10 uM Parnate, 0.5-50 uM Forskolin, 1.0-5 uM TTNPB, and 50 nM-240 nM 3-Deazaneplanocin A (DZNep); and c. A third composition comprising: 0.1-1.0 uM N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoaniline)benzamide (PD0325901) and 7.0-10 uM 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR99021).

5. The composition of claim 4, wherein the first composition, the second composition, and the third composition are in cell culture medium.

6. The composition of claim 5, wherein the first composition, the second composition, and the third composition are in a hydrogel comprising hyaluronic acid.

7. Use of the first composition, the second composition and the third composition for preparing a reagent for changing the genetic profile of postnatal cells, wherein: The first composition comprises 0.5-5.0 mM valproic acid, 7-10 uM CHIR99021, 4.0-10 uM RepSox, 2.0-10 uM Parnate, 0.5-50 uM Forskolin and 1.0-5 uM TTNPB, The second composition comprises 0.5-5.0 mM valproic acid, 7-10 uM CHIR99021, 4.0-10 uM RepSox, 2.0-10 uM Parnate, 0.5-50 uM Forskolin, 1.0-5 uM TTNPB and 50-240 nM 3-Deazaneplanocin A (DZNep), The third composition comprises 0.1-1.0 uM PD0325901 and 7.0-10 uM CHIR99021; The reagent is used in a method comprising the steps of: a. contacting one or more postnatal mammalian cells with a first composition comprising 0.5-5.0 mM valproic acid, 7-10 uM CHIR99021, 4.0-10 uM RepSox, 2.0-10 uM Parnate, 0.5-50 uM Forskolin and 1.0-5 uM TTNPB for about 17 days; b. contacting the one or more cells of step (a) with a second composition comprising 0.5-5.0 mM valproic acid, 7-10 uM CHIR99021, 4.0-10 uM RepSox, 2.0-10 uM Parnate, 0.5-50 uM Forskolin, 1.0-5 uM TTNPB, and 50-240 nM 3-Deazaneplanocin A (DZNep) for about 14 days; and c. contacting the one or more cells of step (b) with a third composition comprising 0.1-1.0 uM PD0325901 and 7.0-10 uM CHIR99021 for about 7 days.

8. The use of claim 7, wherein the one or more cells are considered to have an altered genetic profile when the expression of one or more of COX7A1, ADIRF and TNFRSF11B is reduced to an expression level associated with embryonic stem cells.

9. The use of claim 7, wherein the one or more cells are considered to have an altered genetic profile when the expression of COX7A1 is reduced to an expression level associated with embryonic stem cells.

10. The use of claim 7, wherein the first composition, the second composition, and the third composition are in a cell culture medium.

11. The use of claim 7, wherein the first composition, the second composition, and the third composition are in a hydrogel comprising hyaluronic acid.

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