阅读说明：本技术 用于哺乳动物物种中诱导性组织再生的组合物和方法 (Compositions and methods for induced tissue regeneration in mammalian species ) 是由 M·韦斯特 K·查普曼 H·斯滕伯格 于 2014-06-03 设计创作，主要内容包括：本发明的多个方面包括涉及调控如下分子的方法和组合物,所述分子调节胚胎状态组织和细胞的再生潜能和在发育上较晚的胎儿期与成熟期中该潜能的丧失。所述方法和组合物可用于干细胞生物学和在原本不能再生的胎儿和成熟组织中增强再生潜能。(Aspects of the invention include methods and compositions relating to the modulation of molecules that modulate the regenerative potential of tissues and cells in an embryonic state and the loss of this potential in the later fetal and mature stages of development. The methods and compositions are useful in stem cell biology and to enhance regenerative potential in fetal and mature tissues that would otherwise not be regenerative.)

1. A pharmaceutical composition for enhancing wound healing in a subject, the composition comprising one or more agents that inhibit COX7a1, wherein the one or more agents that inhibit COX7a1 are selected from the group consisting of: RNAi agents targeting COX7a1, antisense oligonucleotides targeting COX7a1, inhibitory antibodies targeting COX7a1, and ribozymes and triplex nucleic acids that inhibit expression of the COX7a1 gene;

wherein the RNAi agent is selected from: (a) siRNA, (b) double-stranded RNA, (c) shRNA, (d) miRNA precursor, and (e) a plasmid or viral vector encoding the siRNA, shRNA or miRNA precursor.

2. The pharmaceutical composition of claim 1, wherein the subject is a mammal.

3. The pharmaceutical composition of claim 2, wherein the mammal is a human.

4. The pharmaceutical composition of any one of claims 1 to 3, which is a pharmaceutical composition for administration to a wound site.

5. The pharmaceutical composition of any one of claims 1-4, which inhibits expression of the ACTA2 and COL1A1 genes at the site of administration to a subject.

6. The pharmaceutical composition of any one of claims 1 to 5, further comprising one or more agents that inhibit one or more genes or gene products selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2, and MAOA.

7. The pharmaceutical composition of claim 6, wherein the one or more agents that inhibit one or more genes or gene products selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2, and MAOA.

8. The pharmaceutical composition of claim 7, wherein: (a) the nucleic acid is RNA; (b) the nucleic acid is double-stranded RNA, siRNA, shRNA or miRNA precursor; or (c) the protein is an antibody.

9. The pharmaceutical composition of any one of claims 1 to 8, which is a solution.

10. The pharmaceutical composition of any one of claims 1 to 9, further comprising one or more compounds that form a hydrogel in situ.

11. The pharmaceutical composition according to claim 10, wherein the hydrogel is a hydrogel comprising hyaluronic acid or a hydrogel comprising hyaluronic acid and collagen I.

12. The pharmaceutical composition of any one of claims 1 to 11, further comprising one or more cells, a vector expressing a telomerase catalytic component, or both one or more cells and a vector expressing a telomerase catalytic component.

13. The pharmaceutical composition of claim 12, wherein the one or more cells comprise progenitor cells or differentiated cells.

14. The pharmaceutical composition of claim 13, wherein the progenitor cells or the differentiated cells comprise mesenchymal progenitor cells, neural progenitor cells, endothelial progenitor cells, hair follicle progenitor cells, neural crest progenitor cells, mammary stem cells, lung progenitor cells, muscle progenitor cells, adipose-derived progenitor cells, epithelial progenitor cells, hematopoietic progenitor cells, chondrocyte cells, osteoblast cells, keratinocytes, hepatocytes, myoblasts, or a combination thereof.

15. A kit for enhancing wound healing in a subject comprising one or more agents that inhibit COX7a1, wherein the one or more agents that inhibit COX7a1 are selected from the group consisting of: RNAi agents targeting COX7a1, antisense oligonucleotides targeting COX7a1, inhibitory antibodies targeting COX7a1, and ribozymes and triplex nucleic acids that inhibit expression of the COX7a1 gene;

wherein the RNAi agent is selected from: (a) siRNA, (b) double-stranded RNA, (c) shRNA, (d) miRNA precursor, and (e) a plasmid or viral vector encoding the siRNA, shRNA or miRNA precursor.

16. The kit of claim 15, further comprising one or more agents that inhibit one or more genes or gene products selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2, and MAOA.

17. The kit of claim 16, wherein the one or more agents that inhibit one or more genes or gene products selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2, and MAOA.

18. The kit of claim 17, wherein: (a) the nucleic acid is RNA; (b) the nucleic acid is double-stranded RNA, siRNA, shRNA or miRNA precursor; or (c) the protein is an antibody.

19. The kit of any one of claims 15 to 18, further comprising one or more compounds that form a hydrogel in situ.

20. The kit according to claim 19, wherein the hydrogel is a hydrogel comprising hyaluronic acid or a hydrogel comprising hyaluronic acid and collagen I.

21. The kit of any one of claims 15 to 20, further comprising one or more cells, a vector expressing a telomerase catalytic component, or both one or more cells and a vector expressing a telomerase catalytic component.

22. The kit of claim 21, wherein the one or more cells comprise progenitor cells or differentiated cells.

23. The kit of claim 22, wherein the progenitor cells or the differentiated cells comprise mesenchymal progenitor cells, neural progenitor cells, endothelial progenitor cells, hair follicle progenitor cells, neural crest progenitor cells, breast stem cells, lung progenitor cells, muscle progenitor cells, adipose-derived progenitor cells, epithelial progenitor cells, hematopoietic progenitor cells, chondrocyte cells, osteoblast cells, keratinocytes, hepatocytes, myoblasts, or a combination thereof.

Technical Field

The field of the invention relates to the field of tissue regeneration and reprogramming of somatic cells to obtain tissue regeneration capability.

Background

Advances in stem cell technology, such as the isolation and propagation of primitive stem cells (PS cells) in vitro, including embryonic stem cells ("ES" cells, including human ES cells ("hES" cells) and related primitive stem cells, including but not limited to iPS, EG, EC, ICM, ectodermal cells or ED cells (including human iPS, EG, EC, ICM, ectodermal cells or ED cells), constitute an important new area of medical research. PS cells have been demonstrated to be capable of proliferating in an undifferentiated state and subsequently being induced to differentiate into any or all of the cell types of the human body, including complex tissues. Many of these PS cells are naturally telomerase positive in an undifferentiated state, thus allowing these cells to proliferate fully and subsequently be genetically modified and clonally expanded. The telomere length of many of these cells is comparable to that seen in sperm DNA (about 10-18kb TRF length). Differentiated cells derived from these immortalized cell lines begin to exhibit inhibition of telomerase catalytic component (TERT) expression upon differentiation, but nevertheless exhibit long initial telomere lengths, providing the cells with a longer replicative capacity compared to fetal or adult derived tissues. This leads to, for example, some predictions: many diseases caused by cellular dysfunction may be amenable to treatment by administration of hES-derived cells of various differentiated types (Thomson et al, Science 282:1145-1147 (1998)).

Nuclear transfer studies have demonstrated that it is possible to transform differentiated somatic cells back into a PS cell-like state, such as an embryonic stem ("ES") cell state (Cibeli et al, Nature Biotech 16:642-646(1998)) or an embryonic derived ("ED") cell state. The development of techniques for reprogramming somatic cells back to a totipotent ES cell-like state has been described, such as transferring the genome of a somatic cell to an enucleated oocyte and then culturing the reconstituted embryo to produce an ES-like cell, which is often referred to as somatic cell nuclear transfer ("SCNT"); or Somatic Cells can be reprogrammed with transcriptional regulators by analytical Reprogramming (see PCT application Ser. No. PCT/US2006/030632, filed as 8/3.2006, entitled "Improved Methods of Reprogramming Animal Somatic Cells", incorporated herein by reference). These methods provide a potential method for transplantation of primary-derived somatocells with the patient's nuclear genotype (Lanza et al, Nature Medicine 5:975-977(1999)), which may be able to address the problem of transplant rejection.

In addition to SCNT and analytical reprogramming techniques, there are other techniques that focus on the problem of transplant rejection, including the use of gynogenesis and androgenesis (see U.S. application Ser. No.: 60/161,987, filed 28.10/1999; application Ser. No. 09/697,297, filed 27.10/2000; application Ser. No. 09/995,659, filed 29.11/2001; application Ser. No. 10/374,512, filed 27.2/2003; and PCT application Ser. No. PCT/USOO/29551, filed 27.10/2000; the disclosures of which are incorporated herein by reference in their entirety). In a class of gynogenesis known as parthenogenesis, pluripotent stem cells (plurative stem cells) can be prepared without antigens foreign to the gamete donor and are therefore useful for preparing cells capable of rejection-free transplantation. In addition, parthenogenetic stem cells can be assembled into cell line banks that are homozygous for HLA regions (or corresponding MHC regions of non-human animals) to reduce the complexity of the stem cell banks with respect to HLA haplotypes.

Furthermore, cell lines Hemizygous in the HLA region (or the corresponding MHC region of a non-human animal) or a pool of such cell lines can be generated (see PCT application No. PCT/US2006/040985, application date 2006, 10/20, entitled "Totipentless maize Cells or Hemizygou for One or More horse tissue compatibility Genes" (Totipotent, near-to or Pluripotent Mammalian Cells Homozygous or Hemizygous for One or More Histocompatibility Antigen Genes), incorporated herein by reference).

The repertoire of hemizygous cell lines provides the advantage of not only reducing the complexity inherent in the conventional mammalian MHC gene pool, but also reducing the gene dose of the antigens, allowing the expression of these antigens to be reduced without completely abolishing their expression and thus not stimulating natural killer responses.

The potential for clonal isolation of human embryonic progenitor cell lines for differentiation of PS cells into desired cell types provides a means of proliferation of novel highly purified cell lines with a prenatal gene expression pattern that is beneficial for regenerating tissues such as skin in a traceless manner. These Cell types have important applications in research and Cell-based therapy preparation (see PCT application No. PCT/US2006/013519, application date 2006, 4, 11, entitled "Novel use of Cells With a Cell characteristic pattern of Gene Expression"; U.S. patent application No. 11/604,047, application date 2006, 11, 21, entitled "Methods to access the Isolation of Novel Cell Strains from a plurality of Pluripotent Stem Cells" and Cells Obtained therefrom "; and U.S. patent application No. 12/504,630, application date 2009, 7, 16, entitled" Methods to access the Isolation of Novel Cell Strains from a plurality of Pluripotent Stem Cells "and Novel Cell extract from a plurality of Pluripotent Stem Cells isolated therefrom" (see PCT application No. PCT/US2006/013519, application date 2006, 4, 11, entitled "Novel use of Cells With a protein Expression of Gene Expression" and Novel use of Cells With a Prenatal Gene Expression ", U.S. patent application No. 11/604,047, application date 2006, entitled" Methods to access the Isolation of Novel Cell Strains from a plurality of Pluripotent Stem Cells "and Cells isolated therefrom", and U.S. patent application No. 26, entitled "method to a Novel Cell extract from a Novel Cell strain and Cell extract from a Novel Cell strain isolated therefrom, each herein incorporated by reference).

Nevertheless, there remains a need for improved methods of regenerating tissue in mammals where administration of exogenous cells is ineffective.

In contrast to various mammals, some animal species show a prominent ability to regenerate native Tissue (TR). In the case of multicellular animals such as vortexes, starfish and some amphibians such as salamanders, there is a prominent potential for regeneration within these animals, such that many traumas do not result in immediate death of the organism, which has the potential to effect repair by regenerating the target tissue from the remaining cells of the tissue, usually in a traceless or relatively traceless manner, even though the tissue is composed primarily of postmitotic cells, such as brain or cardiac muscle. The molecular mechanism by which such regeneration occurs in some animals but not in normal mammalian species is not clear. The confirmation of such molecular mechanisms contributes to the invention of the following novel methods: these mechanisms are introduced into cells and tissues in vivo, thereby leading to Induced Tissue Regeneration (iTR), which can help repair tissues with trauma or degenerative diseases including, but not limited to, age-related degenerative diseases and can help in tissue regeneration studies.

Mammalian models that can study the effects of iTR in the context of tissue damage and regeneration are envisioned, as well as transgenic mammalian models that employ different genetic backgrounds, including mutant genetic backgrounds that generate different disease models in animals in which methods for iTR can be applied to study the potential of iTR as a therapeutic strategy for such diseases.

Disclosure of Invention

In certain embodiments, the present invention provides methods and compositions useful for enhancing the regeneration of a tissue or organ in a subject or in vitro. In other embodiments of the invention, methods and compositions are provided that can be used to inhibit regeneration of a tissue or organ in a subject or in vitro.

In some embodiments, the present invention provides a method of enhancing tissue or organ regeneration in a subject, comprising administering to the subject one or more genes or gene products selected from the group consisting of: PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR and WSB 1.

In other embodiments, the present invention provides methods of inhibiting tissue or organ regeneration in a subject comprising administering to the subject one or more agents that inhibit the expression of one or more genes selected from the group consisting of: PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR and WSB 1.

In other embodiments, the present invention provides a method of inhibiting tissue or organ regeneration in a subject, comprising administering to the subject one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In other embodiments, the present invention provides methods of enhancing tissue or organ regeneration in a subject comprising administering to the subject one or more agents that inhibit the expression of one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In certain embodiments, the present invention provides a method of inhibiting tissue or organ regeneration in a subject comprising administering to said subject a gene product or gene encoded by COX7a 1.

In other embodiments, the present invention provides methods of enhancing tissue or organ regeneration in a subject comprising administering to the subject one or more agents that inhibit COX7a 1.

In other embodiments, the present invention provides a method of inhibiting tissue or organ regeneration in a subject, comprising administering to said subject a gene product or gene encoded by COX7a1 and one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In other embodiments, the present invention provides methods of enhancing tissue or organ regeneration in a subject comprising administering to the subject one or more agents that inhibit COX7a1 and one or more agents that inhibit one or more genes or gene products selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In some embodiments, the present invention provides a method of enhancing tissue or organ regeneration in vitro, comprising contacting cells in vitro with one or more genes or gene products selected from the group consisting of: PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR and WSB 1.

In other embodiments, the invention provides a method of inhibiting tissue or organ regeneration in vitro comprising contacting cells in vitro with one or more agents that inhibit the expression of one or more genes or genes selected from the group consisting of: PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR and WSB 1.

In other embodiments, the invention provides a method of inhibiting tissue or organ regeneration in vitro comprising contacting cells in vitro with one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In other embodiments, the invention provides a method of enhancing tissue or organ regeneration in vitro, comprising contacting cells in vitro with one or more agents that inhibit the expression of one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In certain embodiments, the present invention provides a method of inhibiting tissue or organ regeneration in vitro comprising contacting a cell in vitro with a gene product or gene encoded by COX7a 1.

In other embodiments, the invention provides methods of enhancing tissue or organ regeneration in vitro comprising contacting cells in vitro with one or more agents that inhibit COX7a 1.

In other embodiments, the present invention provides a method of inhibiting tissue or organ regeneration in vitro comprising contacting a cell in vitro with a gene product or gene encoded by COX7a1 and one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In other embodiments, the invention provides a method of enhancing tissue or organ regeneration in vitro, comprising contacting cells in vitro with one or more agents that inhibit COX7a1 and one or more agents that inhibit one or more genes or gene products selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In some embodiments, the present invention provides a method of regenerating skin in a subject comprising administering to the subject an inhibitor of COX7a 1.

In still other embodiments, the present invention provides methods of rejuvenating skin in a subject comprising administering to the subject an siRNA molecule that inhibits COX7a 1.

In some embodiments, the present invention provides methods of enhancing skin regeneration in vitro comprising administering to a cell (e.g., an epithelial cell) an siRNA molecule that inhibits COX7a1 in a subject.

In other embodiments, the invention provides methods of enhancing the expression of one or more genes expressed in an embryonic cell in a cell comprising contacting the cell with one or more agents that inhibit COX7a 1.

In other embodiments, the invention provides methods of enhancing the expression of one or more genes expressed in an embryonic cell in a cell comprising contacting the cell with an siRNA that inhibits COX7a 1.

In certain embodiments, the present invention provides methods of enhancing expression of KRT17 in a cell, comprising contacting the cell with one or more agents that inhibit COX7a 1.

In still other embodiments, the present invention provides methods of enhancing expression of KRT17 in a cell, comprising contacting the cell with an siRNA that inhibits COX7a 1.

In certain embodiments, the invention provides methods of enhancing expression of ACTA2 and COL1a1 in a cell comprising contacting the cell with one or more agents that inhibit COX7a 1.

In still other embodiments, the invention provides methods of enhancing expression of ACTA2 and COL1a1 in a cell comprising contacting the cell with an siRNA that inhibits COX7a 1.

In still other embodiments, the present invention provides methods of treating cancer comprising administering to a subject a COX7a1 gene or gene product.

In some embodiments, the present invention provides methods of enhancing wound healing in a subject, the methods comprising administering to the subject one or more genes or gene products selected from PCDHHB2, PCDHB17, Nbla110527, RAB3IP, DLXl, DRDl lP, FOXDl, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR, and WSB 1.

In other embodiments, the present invention provides methods of inhibiting wound healing in a subject comprising administering to the subject one or more agents that inhibit the expression of one or more genes selected from the group consisting of: PCDHHB2, PCDHB17, Nblal0527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR and WSB 1.

In other embodiments, the present invention provides a method of inhibiting wound healing in a subject comprising administering to the subject one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In other embodiments, the present invention provides methods of wound healing in a subject comprising administering to the subject one or more agents that inhibit the expression of one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In certain embodiments, the present invention provides a method of inhibiting wound healing in a subject comprising administering to said subject a gene product or gene encoded by COX7a 1.

In other embodiments, the present invention provides methods of enhancing wound healing in a subject comprising administering to the subject one or more agents that inhibit COX7a 1.

In other embodiments, the present invention provides a method of inhibiting wound healing in a subject comprising administering to said subject a gene product or gene encoded by COX7a1 and one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In other embodiments, the present invention provides methods of enhancing wound healing in a subject comprising administering to the subject one or more agents that inhibit COX7a1 and one or more agents that inhibit one or more genes or gene products selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In certain embodiments, the present invention provides a pharmaceutical composition comprising one or more gene products or genes encoded by the genes shown in figure 1 and a suitable vector.

In other embodiments, the invention provides pharmaceutical compositions comprising several gene products or genes encoded by the genes shown in figure 1 and a suitable vector.

In still other embodiments, the invention provides transgenic animals expressing one or more heterologous or xenogeneic genes selected from the group consisting of: PCDHHB2, PCDHB17, Nbl 10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR, WSB1, COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADLL, COX7A1, TSL 5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In still other embodiments, the invention provides kits comprising one or more gene products or genes expressed by a gene selected from COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100a6, MGMT, ZNF280D, DYNLT3, NAALADLl, COX7a1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2, and MAOA and at least one container.

In some embodiments, the invention provides kits comprising one or more gene products or genes expressed by a gene selected from the group consisting of PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR, and WSB1, and at least one container.

Brief description of the drawings

FIG. 1 TR suppressor and TR activator genes identified by differential expression of hES-derived clonal embryonic progenitor cells compared to fetal and adult derived soma cells. (A) A TR suppressor gene that is expressed in fetal and adult derived somatic cells but is expressed at a lower level or not expressed in cloned embryonic progenitor cells. (B) Cloning of TR activator genes expressed in embryonic progenitor cells but expressed at lower levels or not in fetal and adult derived somatic cells.

FIG. 2 RFU values for gene PCDHB2, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal Embryonic Progenitor (EP) cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 3 RFU values for gene PCDHB17, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 4 RFU values of gene RAB3IP, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 5 RFU values of gene DLX1, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 6 RFU values of gene SLX1, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 7 RFU values of gene DRD1IP, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 8 RFU values of the gene COMT, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 9 RFU values of gene TRIM4, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 10 RFU value of gene LOC205251, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 11 RFU values for gene ZNF280D were determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 12 RFU values of gene NAALADL1, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 13 RFU values of gene COX7A1, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 115 different somatic cell types from all three germ layers, 545 different clonal EP cell lines, 12 hES cell lines and 17 human iPS cell lines.

FIG. 14 RFU values of Gene CAT, determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 101 different somatic cell types from all three germ layers, 84 different clonal EP cell lines and 4 human ES cell lines.

FIG. 15 Primary neuroblastoma cDNA clone: the RFU value of Nbla10527 gene was determined by Illumina gene expression microarray analysis in different mature or embryonic cell types. From left to right are 14 different blood cell types, 101 different somatic cell types from all three germ layers, 84 different clonal EP cell lines and 4 human ES cell lines.

FIG. 16 is a graph of in vitro regeneration of COX7A 1-expressing neonatal human foreskin fibroblasts following transcriptional silencing of COX7A1, compared to controls, using the in vitro wound repair assay described herein. A) The number of cells in representative regions within the range of injury was counted on days 0 and 1 for the control sample and the sample in which transcription of the iTR suppressor gene COX7a1 was down-regulated. B) Images of representative areas of control samples. C) Image of representative area of COX7A1siRNA area.

Figure 17 in vitro regeneration of COX7a1 transcriptional silencing by neonatal human foreskin fibroblasts expressing COX7a1, compared to controls, using the in vitro wound repair assay described herein. A) Control and COX7a1 down-regulated the relative ACTA2 expression present in the cells. B) Control and COX7a1 down-regulated the relative COL1a1 expression present in the cells.

Detailed Description

Abbreviations

cGMP-current good production Process Specification

CNS-central nervous system

DMEM-Darlington's modified Eagle's medium (Dulbecco's modified Eagle's medium)

DMSO-dimethyl sulfoxide

DPBS-Dardian phosphate buffered saline

EC-embryonal tumor

EC cell-embryoma cell; hEC cells are human embryoma cells

ECM-extracellular matrix

ED cells-embryonic derived cells; hED the cells are human ED cells

EDTA-EDTA

EG cells-embryonic germ cells; hEG cells are human EG cells

ES cell-embryonic stem cell; hES cells are human ES cells

FACS-fluorescence activated cell sorting

FBS-fetal bovine serum

GMP-good manufacturing practice

hED cell-human embryo-derived cell

hEG cells, human embryonic germ cells, are stem cells derived from fetal tissue primordial germ cells.

hiPS cells-human induced pluripotent stem cells, which are cells with hES-like properties, are obtained from somatic cells after exposure to hES-specific transcription factors such as SOX2, KLF4, OCT4, MYC or NANOG, LIN28, OCT4 and SOX 2.

HSE-human skin equivalent, a hybrid of cells and biological or synthetic substrates, prepared for testing purposes or therapeutic applications to promote wound repair.

ICM-inner cell mass at the blastocyst stage of a mammal.

iPS cell-induced pluripotent stem cells, which are cells having hES-like properties, are obtained from somatic cells after exposure to ES-specific transcription factors such as SOX2, KLF4, OCT4, MYC or NANOG, LIN28, OCT4 and SOX 2.

iTR-induced tissue regeneration

LOH-loss of heterozygosity

MEM-Minimal essential Medium (minimum essential medium)

NT-nuclear transplantation

PBS -Phosphate buffered saline

PNS-peripheral nervous System

PS fibroblasts, pre-scar fibroblasts, are fibroblasts derived from skin in the early stages of pregnancy or from ED cells, exhibit a prenatal pattern of gene expression, and promote rapid healing of skin wounds without scar formation.

RFU-relative fluorescence Unit

SCNT-somatic cell nuclear transfer

SFM-serum-free medium

TR-tissue regeneration

Definition of

The term "analytical reprogramming techniques" refers to a variety of methods for reprogramming the gene expression pattern of somatic cells to a more competent state, such as that of iPS, ES, ED, EC, or EG cells, wherein reprogramming occurs in multiple and discrete steps and is not simply dependent on transferring a somatic cell into an oocyte and activating the oocyte (see: U.S. application No. 60/332,510, application date 2001, 11/26/day; 10/304,020, application date 2002, 11/26/day; PCT application No. PCT/US02/37899, application date 2003, 11/26/day; U.S. application No. 60/705625, application date 2005, 8/3/day; U.S. application No. 60/729173, application date 2005, 8/20/day; U.S. application No. 60/818813, application date 2006, 7/5/day; PCT/US06/30632, application date 2006, 8/3/day; the disclosures of each are incorporated herein by reference).

The term "antibody" as used herein refers to an immunoglobulin or portion thereof, and encompasses any polypeptide comprising an antigen binding site, regardless of its origin, method of preparation, or other characteristics. The term includes, for example: polyclonal, monoclonal, monospecific, multispecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant, and CDR grafted antibodies. Portions of antibodies may include any fragment capable of binding an antigen, e.g., Fab, F (ab')2, Fv, scFv.

The term "blastomere/morula cell" refers to a blastomere or morula in mammalian embryos or in vitro cultured blastomere or morula cells, whether or not there are other cells within the differentiated derivatives of such cells.

The term "cell expressing gene X", "gene X is expressed in a cell" (or a population of cells), or equivalent expression thereof means that a positive result is obtained by analyzing said cell with a particular test platform. The reverse is true (i.e., cells that do not express gene X, or their equivalent expression means that analysis of the cells with a particular test platform yields a negative result). Thus, any gene expression result described herein is correlated to one or more probes employed by the test platform(s) for the gene in question.

The term "cell line" refers to a non-immortalized or immortalized population of cells capable of proliferation and expansion in vitro.

The term "cell reconstitution" refers to the transfer of chromatin nuclei into the cytoplasm to obtain functional cells.

The term "clone" refers to a population of cells expanded from a single cell into a population, the cells within the population all being derived from the original single cell and being free of other cells.

The term "colony differentiation in situ" refers to the in situ differentiation of a cell (e.g., hES, hEG, hiPS, hEC, or hED) colony without involving the removal or dispersion of the colony from the culture vessel in which it proliferates as an undifferentiated stem cell line. Colony differentiation in situ does not utilize an intermediate step of forming embryoid bodies, although after a period of colony differentiation in situ may still utilize embryoid body formation or other aggregation techniques such as spinner culture.

The term "cytoplasmic bleb" refers to the cytoplasm of a cell bounded by an intact or permeabilized, but intact, plasma membrane, but without a nucleus.

In reference to cells directed by the methods of the invention from pluripotent stem cells, the term "differentiated cells" refers to cells that have a reduced ability to differentiate as compared to the parental pluripotent stem cells. The differentiated cells of the invention comprise cells that are further differentiable (i.e., not terminally differentiated).

The term "directly differentiating" refers to directly differentiating any of the following cell types using any method known in the art: blastomere cells, morula cells, ICM cells, ED cells, or somatic cells reprogrammed to an undifferentiated state, while undifferentiated stem cells (e.g., hES cells) that have not been isolated and or proliferated are intermediate states of an undifferentiated cell line. A non-limiting example of direct differentiation is the culture of intact Human blastocysts into cultures and derivation of ED cells without production of Human ES cell lines, as described in the literature (Bongso et al, 1994.Human Reproduction 9: 2110).

The term "embryonic stage of development" refers to the prenatal stage of development of a cell, tissue or animal, particularly the embryonic stage of cell development as compared to a fetus or mature cell. In the case of the human species, transformation from embryonic to fetal development occurs at about 8 weeks of prenatal development, approximately 16 days in mice, and about 17.5 days in rat species.

The term "embryonic stem cell" (ES cell) refers to a cell derived from the inner cell mass, blastomere or morula of a blastocyst that is maintained in an undifferentiated state as a cell line through serial passages (e.g., expressing TERT, OCT4 and SSEA and TRA antigens specific to the species ES cell). ES cells can be derived from in vitro fertilization of egg cells with sperm or DNA, nuclear transfer, gynogenesis, or by generating hES cells hemizygous or homozygous for the MHC region by means known in the art. Although ES cells have historically been defined as being capable of differentiating into the full somatic cell type and into germ line cells when implanted into pre-transplantation embryos, candidate ES cultures from many species, including humans, typically do not produce germ line differentiation and are therefore referred to as "ES-like cells". It is generally considered that human ES cells are actually "ES-like," but in this application we use the term ES cells to refer to both ES and ES-like cell lines.

The term "human embryo-derived" ("hED") cell refers to blastomere-derived cells, morula-derived cells, blastocyst-derived cells including cells of the inner cell mass, embryonic shield or ectoderm, or other totipotent or pluripotent stem cells of early embryos including primitive endoderm, ectoderm, mesoderm and neural crest and derivatives thereof, which are upper-limited in their differentiated state relative to the equivalent state of the original 8 weeks of normal human development, but exclude cells derived from hES cells that have been passaged as a cell lineage (see, e.g., Thomsom, U.S. Pat. Nos.: 7,582,479; 7,217,569; 6,887,706; 6,602,711; 6,280,718 and 5,843,780). hED cells can be derived from pre-transplantation embryos prepared by fertilization of an egg cell with sperm or DNA, nuclear transfer or chromatin transfer, induction of parthenote through parthenogenesis, analysis of reprogramming techniques, or production of hES with HLA region hemizygosity or homozygosity.

The term "human embryonic germ cells" (hEG cells) refers to pluripotent stem cells derived from fetal tissue primordial germ cells or mature-type (mate) germ cells such as oocytes and spermatogonial cells that are capable of differentiating into different tissues within the body. hEG cells can also be derived from pluripotent stem cells derived from gynogenesis or androgenesis means, i.e., the pluripotent cells in these methods are derived from oocytes containing only DNA of male or female origin and thus will contain all of the DNA of female (female-derived) or male (male-derived) (see U.S. application No. 60/161,987, application No. 1999, 10/28, 09/697,297, application No. 2000, 10/27, 09/995,659, application No. 2001, 11/29, 10/374,512, application No. 2003, 2/27, PCT application No. PCT/US/00/29551, application No. 2000, 10/27, these descriptions being incorporated herein by reference in their entirety).

The term "human embryonic stem cells" (hES cells) refers to human ES cells (see, e.g., Thomson, U.S. Pat. Nos. 7,582,479, 7,217,569, 6,887,706, 6,602,711, 6,280,718 and 5,843,780).

The term "human iPS cell" refers to a cell with properties similar to hES cells, including the ability to form all three germ layers when implanted in immunocompromised mice, wherein the iPS cell is derived from cells of different somatic lineages upon exposure to dedifferentiating factors exemplified by hES cell-specific transcription factor combinations: KLF4, SOX2, MYC and OCT4 or SOX2, OCT4, NANOG and LIN 28. iPS cells can be generated using any conventional combination of dedifferentiating factors. The iPS cells can be generated by expressing these genes with vectors such as retroviral, lentiviral, or adenoviral vectors known in the art, or by introducing these factors in protein form (e.g., by permeabilization or other techniques). A description of such exemplary methods may be found in: PCT application No. PCT/US2006/030632, filing date 2006, 8/3; U.S. application No. 11/989,988; PCT application PCT/US2000/018063, App. No. 2000, 6/30; U.S. application No. 09,736,268, filed on even 2000, 12/15; U.S. application No. 10/831,599, 2004, 4-23; and U.S. application publication No. 20020142397 (application No. 10/015,824, entitled "Methods for Altering Cell faces"); U.S. application publication No. 20050014258 (application No. 10/910,156 entitled "Methods for Altering Cell faces"); U.S. application publication No. 20030046722 (application No. 10/032,191, entitled "Methods for cloning mammalian chromatin or donor cells with reprogrammed donor chromatin)"); and U.S. application publication No. 20060212952 (application No. 11/439,788 entitled "Methods for cloning mammalian chromatin or donor cells" a method for cloning mammals with reprogrammed donor chromatin or donor cells), all of which are incorporated herein by reference in their entirety.

The term "ICM cells" refers to cells of an embryonic cell mass in a mammal or cells of an inner cell mass cultured in vitro with or without surrounding trophectoderm.

The term "induced tissue regeneration" refers to altering the molecular composition of fetal or mature mammalian cells using the methods of the invention such that the cells are capable of regenerating functional tissue following tissue injury, wherein such regeneration would not otherwise occur without human intervention as described below.

The term "isolated" refers to a substance that is (i) separated from at least some other substance with which it is naturally found in nature, typically by a process involving man power, (ii) artificially produced (e.g., chemically synthesized), and/or (iii) present in an artificial environment or setting (i.e., an environment or setting not found in its natural state).

The term "iTR factor" refers to those molecules that alter the levels of TR activator and TR repressor in a manner that causes TR to be produced by tissues that are not naturally capable of TR.

The term "iTR genes" refers to those genes whose expression is altered to cause induced tissue regeneration in tissues that cannot be regenerated under natural conditions.

The terms "nucleic acid" and "polynucleotide" are used interchangeably and encompass various embodiments, polymers of naturally occurring nucleosides, such as DNA and RNA, and polymers of non-naturally occurring nucleosides or nucleoside analogs. In some embodiments, the nucleic acid comprises a standard nucleoside (abbreviated A, G, C, T, U). In other embodiments, the nucleic acid comprises one or more non-standard nucleosides. In some embodiments, one or more nucleosides are non-naturally occurring nucleosides or nucleotide analogs. Nucleic acids may contain modified bases (e.g., methylated bases), modified sugars (2 '-fluorinated ribose, arabinose, or hexoses), modified phosphate groups, or other linkages between nucleosides or nucleoside analogs (e.g., phosphorothioate or 5' -N-phosphoramidite linkages), locked nucleic acids (locked nucleic acids), or morpholinos. In some embodiments, the nucleic acid contains nucleosides linked by phosphodiester linkages, as in DNA and RNA. In some embodiments, at least some nucleosides are linked by non-phosphodiester linkages. The nucleic acid may be single-stranded, double-stranded or partially double-stranded. Nucleic acids that are at least partially double stranded can have one or more overhangs, e.g., 5 'and/or 3' overhangs. Nucleic acid modifications (e.g., nucleoside and/or backbone modifications, including the use of non-standard nucleosides) of RNA interference (RNAi), aptamers, or antisense-based molecules known in the art to be useful for research or therapeutic purposes are contemplated for use in various embodiments of the present invention. See, for example: crook, S T, Antisense drug technology: principles, stratgies, and applications (Antisense technology: principles, strategies and applications), bocardon (Boca Raton, inc.): CRC press, 2008; kurreck, j. eds, Therapeutic oligonucleotides, RSC biomoleculars, (Therapeutic oligonucleotides, RSC molecular biosciences), cambridge: royal chemical society, 2008. In some embodiments, the modification increases the half-life and/or stability of the nucleic acid (e.g., in vivo) as compared to RNA or DNA of the same length and strand type (Stranddness). In some embodiments, the modification results in increased immunogenicity of the nucleic acid as compared to RNA or DNA of the same length and strand type (Strandedness). In some embodiments, 5% to 95% of the nucleosides in one or both strands of the nucleic acid are modified. The modification positions may be uniform or non-uniform, and the positions of the modifications may be selected (e.g., near the middle, near or at the ends, alternating, etc.) to enhance desired properties. The nucleic acid may comprise a detectable label, such as a fluorescent dye, radioactive atom, or the like.

"oligonucleotide" refers to a relatively short nucleic acid, for example, generally about 4 to about 60 nucleotides in length. References herein to polynucleotides are to be understood as DNA, RNA, and in each case to provide single-stranded and double-stranded forms (and complements of each single-stranded molecule). "polynucleotide sequence" as used herein may refer to sequence information (i.e., consecutive letters used as base abbreviations) that characterizes a particular nucleic acid, per se and/or biochemically, by polynucleotide material. Unless otherwise indicated, polynucleotide sequences set forth herein are presented in a 5 'to 3' orientation.

The term "oligoclonal" refers to a population of cells derived from a small population of cells, typically 2-1000 cells, that appear to have similar characteristics such as appearance or the presence or absence of differentiation markers that distinguish the cells from other cells in the same culture. Oligoclonal cells are isolated from cells that do not share these same characteristics and are capable of proliferation, resulting in a cell population that is substantially completely derived from an original population of similar cells.

The term "pluripotent stem cell" refers to an animal cell capable of differentiating into more than one differentiated cell type. Such cells include hES cells, blastomere/morula cells and hED cells derived therefrom, hiPS cells, hEG cells, hEC cells, and adult derived cells, including mesenchymal stem cells, neural stem cells, and bone marrow derived stem cells. Pluripotent stem cells may or may not be genetically modified. The genetically modified cells may include a marker, such as a fluorescent protein, to aid in identifying these cells in ovo.

The term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. Peptides are relatively short polypeptides, typically about 2 to 60 amino acids in length. The polypeptides used herein typically contain standard amino acids (i.e., the most common 20L-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 within a polypeptide can be modified, for example, by the addition of chemical entities such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for coupling, functionalization, and the like. Polypeptides having covalently or non-covalently attached non-polypeptide moieties are still considered "polypeptides". The polypeptides may be purified from natural sources, produced using recombinant DNA techniques, synthesized by chemical methods such as conventional solid phase peptide synthesis, and the like. As used herein, "polypeptide sequence" or "amino acid sequence" can refer to the sequence information (i.e., the consecutive letters or three-letter code used as an abbreviation for amino acid name) that characterizes the polypeptide material itself and/or biochemically. Unless otherwise indicated, the polypeptide sequences set forth herein are presented in the N-terminal to C-terminal direction. The polypeptide may be cyclic or contain cyclic moieties. When discussing naturally occurring polypeptides herein, it is to be understood that the invention encompasses various embodiments involving any isoform thereof (e.g., a different protein produced from the same gene as a result of alternative splicing or mRNA editing, or produced from different alleles of a gene, e.g., which may differ from one another by one or more polynucleotide polymorphisms, typically such alleles may be at least 95%, 96%, 97%, 98%, 99% or more identical to a reference or consensus sequence). The polypeptide may contain sequences that target it for secretion or to a particular intracellular compartment (e.g., the nucleus) and/or sequences that target the polypeptide for post-translational modification or degradation. Certain polypeptides may be synthesized as precursors that are post-translationally cleaved or otherwise processed to become mature polypeptides. In some cases, such cleavage may occur only after a particular activation event. In relation thereto, the invention provides various embodiments relating to precursor polypeptides and various embodiments relating to mature forms of polypeptides.

The term "(pooled clones") refers to a cell population obtained by the combination of two or more clonal populations, the resulting cell population having a homogeneous marker, e.g., a gene expression marker, similar to the clonal population, but not all cells of the population being derived from the same original clone. The pooled clonal lines may comprise cells of a single or mixed genotype. The pooled clone is particularly useful in cases where the clone is differentiated earlier or has an unfavorable change early in the propagation period.

The term "prenatal" refers to the stage of embryonic development in a placental mammal before the animal cannot survive leaving the uterus.

The term "primitive stem cell" refers to a collective term for pluripotent stem cells that are capable of differentiating into a variety of cells of all three major germ layers (ectoderm, endoderm and mesoderm) and neural crest. Thus, examples of primitive stem cells would include, but are not limited to, human or non-human mammalian ES cells or cell lines, blastomere/morula cells and their derived ED cells, iPS and EG cells.

The term "purified" refers to an agent or entity (e.g., a compound) that has been separated from most of the components with which it is naturally occurring or coexisting when originally produced. Typically, such purification involves the action of human force. The purified agent or entity may be partially purified, substantially purified, or pure. For example, such an agent or entity may be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure. In some embodiments, the nucleic acid or polypeptide is purified such that it comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the total nucleic acid or polypeptide material in the article. Purity can be based on, for example, dry weight, peak size on chromatographic traces, molecular abundance, intensity of bands on a gel, or intensity of any signal associated with molecular abundance, or any art-accepted quantitative method. In some embodiments, water, buffers, 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 material) or by generating them in such a way as to obtain the desired purity. In some embodiments, a purified molecule or composition refers to a molecule or composition made using any art-accepted purification method. In some embodiments, "partially purified" means that the molecule produced by the cell is no longer present within the cell, e.g., the cell has been lysed, and optionally at least some cellular material (e.g., cell wall, cell membrane, organelle) has been removed.

The term "RNA interference" (RNAi) is used to refer to the phenomenon: double-stranded rna (dsRNA) causes sequence-specific degradation or translational inhibition of the corresponding mRNA with complementarity to one strand of the dsRNA. It is understood that the complementarity between a strand of dsRNA and mRNA need not be 100% but need only be sufficient to mediate inhibition of gene expression (also referred to as "silencing" or "knockdown"). For example, the degree of complementarity is such that the strand is capable of (i) directing cleavage of the mRNA in an RNA-induced silencing complex (RISC); or (ii) results in translation inhibition of the mRNA. In certain embodiments, the double stranded portion of the RNA is less than 30 nucleotides in length, e.g., 17 to 29 nucleotides in length. In certain embodiments, a first strand of the dsRNA is at least 80%, 85%, 90%, 95%, or 100% complementary to the target mRNA, while the other strand of the dsRNA is at least 80%, 85%, 90%, 95%, or 100% complementary to the first strand. In mammalian cells, RNAi can be achieved by introducing into the cell or expressing within the cell a suitable double-stranded nucleic acid, which is subsequently processed intracellularly to produce dsRNA. 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 small rna (microrna) precursors. These terms are well known and used herein consistent with their ordinary meaning in the art. sirnas typically contain two distinct nucleic acid strands that hybridize to each other to form a duplex. They may be synthesized in vitro, for example, using standard nucleic acid synthesis techniques. siRNA are typically double stranded oligonucleotides of 16 to 30 nucleotides, e.g., s16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides (nt), per strand, wherein the double stranded oligonucleotide contains a double stranded portion of 15 to 29 nucleotides in length and one or both strands may contain a 3' overhang (e.g., 1 to 5 nucleotides in length), or one or both strands may be blunt ended. In some embodiments, the siRNA comprises strands between 19 and 25 nt in length, e.g., between 21 and 23 nt in length, wherein one or both strands comprise a 3' overhang of 1-2 nucleotides. One strand (named "guide strand" or "antisense strand") of the double-stranded portion of the siRNA is substantially complementary (e.g., at least 80% or more, such as 85%, 90%, 95%, or 100%) (e.g., there are 3, 2,1, or 0 mismatched nucleotides) to the target region in the mRNA, and the other double-stranded portion is substantially complementary to the first double-stranded portion. In certain embodiments, the guide strand is 100% complementary to the target region of the mRNA, while the other follower strand is 100% complementary to the first double-stranded portion (it being understood that in various embodiments, the 3' overhang of the guide strand, if present, may or may not be complementary to the mRNA when the guide strand is hybridized to the mRNA). In some embodiments, the shRNA molecule is a nucleic acid molecule comprising a stem loop, wherein the double strand has a stem length of 16-30 nucleotides and a loop length of about 1-10 nucleotides. The siRNA can include a variety of different modified nucleosides, nucleoside analogs, and can include chemically or biologically modified bases, modified backbones, and the like. Without limitation, any modification recognized in the art as useful for RNAi can be used. Some modifications result in improvements in stability, cellular uptake, efficacy, and the like. Some modifications result in a reduction in immunogenicity or clearance. In certain embodiments, the siRNA comprises a duplex of about 19-23 (e.g., 19, 20, 21, 22, or 23) nucleotides in length, and optionally has one or two 3' overhangs of 1-5 nucleotides in length, which may consist of deoxyribonucleotides. shRNA comprises a single nucleic acid strand containing two complementary regions separated by a region of predominantly non-self complementarity. These complementary portions hybridize to form a duplex structure rather than self-complementary regions forming a loop connecting the 3 'end of one strand of the duplex to the 5' end of the other strand. The shRNA undergoes intracellular processing to produce siRNA. Typically, the loop length is 1-8 nucleotides, such as 2-6 nucleotides.

Small RNAs (mirnas) are naturally occurring non-coding single stranded small RNAs, about 21-25 nucleotides (in mammalian systems), that inhibit gene expression in a sequence-specific manner. They are produced intracellularly from precursors (precursor-mirnas) that have a characteristic secondary structure with a short hairpin (about 70 nucleotides in length) containing a region that typically includes one or more regions of incomplete complementarity, which in turn is produced from a larger precursor (precursor-miRNA). Naturally occurring mirnas are usually only partially complementary to their target mRNA and often act through translational inhibition. Certain embodiments of the invention utilize RNAi agents modeled on endogenous mirnas or miRNA precursors. For example, the siRNA may be designed such that one strand hybridizes to a target mRNA but has one or more mismatches or bulges, mimicking a duplex formed by the miRNA and its target mRNA. Such sirnas may be referred to as miRNA mimics or miRNA-like molecules. miRNA mimics may be encoded by precursor nucleic acids whose structure mimics naturally occurring miRNA precursors.

In certain embodiments, the RNAi agent is a vector (e.g., a plasmid or virus) containing a template for transcription of siRNA (e.g., two separate strands that can hybridize to each other), shRNA, or small RNA precursors. Typically, the template encoding the siRNA, shRNA, or miRNA precursor is operably linked to an expression control sequence (e.g., a promoter), as is known in the art. Such vectors can be used to introduce a template into a vertebrate cell, e.g., a mammalian cell, and result in transient or stable expression of the siRNA, shRNA, or miRNA precursor.

Typically, small RNAi agents such as sirnas can be chemically synthesized or can be transcribed in vitro or in vivo from a DNA template, either as two separate strands that are later hybridized or as shrnas that are later processed to produce sirnas. RNAi agents, particularly those containing modifications, are often chemically synthesized. Methods for chemically synthesizing oligonucleotides are well known in the art.

The term "small molecule" as used herein is an organic molecule having a molecular weight of less than about 2 KDa. In some embodiments, the small molecule is less than about 1.5KDa or less than about 1 KDa. In some embodiments, the small molecule is less than about 800Da, 600Da, 500Da, 400Da, 300Da, 200Da, or 100 Da. Typically, the small molecules have a mass of at least 50 Da. In some embodiments, the small molecule contains multiple carbon-carbon bonds and may contain one or more heteroatoms and/or one or more functional groups important for structural interaction with (e.g., hydrogen bonding) the protein, e.g., amine, carbonyl, hydroxyl, or carboxyl groups, and in some embodiments at least two functional groups. Small molecules typically contain a structure comprising one or more cyclic carbons or heterocycles and/or aromatic or polyaromatic rings, optionally substituted with one or more of the above 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" may be any multicellular animal. The subject may be a vertebrate, such as a mammal or an avian. Exemplary mammals include, for example: humans, non-human primates, rodents (e.g., mice, rats, rabbits), ungulates (e.g., ovine, bovine, equine, caprine species), canines, and felines. The subject may be an individual to whom the compound is delivered (e.g., for experimental, diagnostic, and/or therapeutic purposes), or from whom a sample is obtained or to whom a diagnostic procedure is performed (e.g., where the sample or procedure is to be used to assess tissue damage and/or to assess the effect of the compound of the invention).

The term "tissue damage" as used herein refers to any type of injury or damage to a cell, tissue, organ, or other bodily result. In various embodiments, the term encompasses degeneration caused by disease, damage caused by physical trauma or surgery, damage caused by exposure to harmful substances, and other disruptions of the structure and/or functionality of cells, tissues, organs, or other bodily structures.

The term "tissue regeneration" or "TR" refers to at least partial regeneration, replacement, restoration of a tissue, organ or other bodily structure or portion thereof following loss, injury or degeneration, wherein said tissue regeneration would not occur if not by the methods described herein. Examples of tissue regeneration include regrowth of severed fingers or limbs, including cartilage, bone, muscle, tendon and ligament, regrowth of bone, cartilage, skin or muscle that has been lost due to injury or disease (which may or may not be scarless), an increase in the size and number of cells of an injured or diseased organ such that the tissue or organ approximates the normal size of the tissue or organ or the size before injury or disease. Depending on the tissue type, tissue regeneration can occur by a number of different mechanisms including (for example): rearrangement of existing cells and/or tissues (e.g., by cell migration), division of mature somatic stem cells or other progenitor cells and differentiation of at least some of their progeny, and/or dedifferentiation, transdifferentiation, and/or proliferation of the cells.

The term "TR activator (daughter) genes" refers to those genes that are not expressed in fetal or mature cells but whose expression in the embryonic stage of development aids TR.

The term "TR suppressor gene" refers to those genes whose expression in fetuses and mature animals suppresses TR.

The terms "treat," "treating," "treatment," "therapy," "therapeutic," and similar terms, with respect to a subject, refer to providing medical and/or surgical treatment to the subject. Treatment can include, but is not limited to, administering a compound or composition (e.g., a pharmaceutical composition such as a cellular composition) to a subject. The treatment of a subject described herein is typically done in an effort to promote regeneration, for example, in a subject suffering from or expected to suffer from tissue damage (e.g., a subject to be operated on). Treatment as used herein includes prophylactic, as well as alleviation of one or more symptoms associated with a disease or condition. The effects of the treatment may generally include: increased regeneration, decreased scarring, and/or improved structural or functional outcome following tissue injury (as compared to the outcome without treatment), and/or may include reversal or reduced severity of degenerative disease.

The term "variant" as applied to a particular polypeptide refers to a polypeptide that differs from the particular polypeptide (sometimes referred to as the "original polypeptide") by one or more amino acid changes such as additions, deletions, and/or substitutions. The original polypeptide is sometimes a naturally occurring polypeptide (e.g., from a human or non-human animal) or a polypeptide identical thereto. Variants may be naturally occurring or may be produced using, for example, recombinant DNA techniques or chemical synthesis. The addition may be an insertion within the polypeptide or an N-or C-terminal addition. In some embodiments, the number of amino acid substitutions, deletions or additions may be, for example, about 1 to 30, e.g., about 1 to 20, e.g., about 1 to 10, e.g., about 1 to 5, e.g., 1, 2,3, 4 or 5. In some embodiments, a variant includes a sequence having a sequence that is homologous to the original polypeptide over at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, or more, up to the full length of the original polypeptide (but is not identical to the sequence of the original polypeptide), e.g., a sequence of a variant polypeptide is at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, or more, up to the full length of the original polypeptide, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, identical to the sequence of the original polypeptide. In some embodiments, a variant comprises at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the original sequence at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the original polypeptide. In some embodiments, the variant comprises at least one functional or structural Domain, such as a Domain identified as such in a Conserved Domain Database (CDD) of the national center for biotechnology information (www.ncbi.nih.gov), e.g., NCBI-labeled Domain (NCBI-curved Domain).

In some embodiments, one, more or all of the biological functions or activities of the variant or fragment are similar to the corresponding biological functions or activities of the original molecule. In some embodiments, the functional change retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more, e.g., approximately equal activity, of the activity of the original polypeptide. 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, the activity of a variant or fragment is considered to be substantially similar to the activity of the original molecule if the amount or concentration of the variant required to produce a particular effect is in the range of 0.5 to 5 times the amount or concentration of the original molecule required to produce that effect.

In some embodiments, an amino acid "substitution" in a variant is the result of a substitution of one amino acid for another with similar structural and/or chemical properties, i.e., a conservative amino acid substitution. "conservative" amino acid substitutions may be made on the basis of similarity in any of a number of properties of the residue in question, such as, for example, side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature. For example, nonpolar (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. Within a particular group, certain substitutions may be of particular interest, for example, a leucine to isoleucine (or vice versa), a serine to threonine (or vice versa), or an alanine to glycine (or vice versa). Of course, non-conservative substitutions are often compatible with preserving function. In some embodiments, the substitution or deletion does not alter or eliminate amino acids important for activity. The size of the insertion or deletion can be in the range of about 1-20 amino acids, e.g., 1-10 amino acids. In some cases, the larger domain can be removed without significantly affecting function. In certain embodiments of the invention, the sequence of the variant may be obtained by insertion, deletion or substitution of a total of no more than 5, 10,15 or 20 amino acids of the sequence of the naturally occurring enzyme. In some embodiments, there are no more than 1%, 5%, 10%, or 20% amino acid insertions, deletions, or substitutions within the polypeptide relative to the original polypeptide.

Guidance in determining which amino acid residues can be substituted, added, or deleted without abolishing or significantly diminishing the activity of interest can be obtained by comparing the sequence of a particular polypeptide to that of a homologous polypeptide (e.g., from another organism) and minimizing the number of amino acid sequence changes within the highly homologous regions (conserved regions), or by substituting amino acids to those found in the homologous sequences, since conserved amino acid residues are more likely to be of importance for activity between different species than non-conserved amino acids.

In some embodiments, the variant of the polypeptide comprises a heterologous polypeptide moiety. The heterologous moiety often has a sequence that is not present in or is not homologous to the original polypeptide. Heterologous moieties can be, for example, 5 to about 5000 amino acids or longer. In some embodiments, it is between 5 and about 1000 amino acids in length. In some embodiments, the heterologous moiety comprises a sequence found in a different polypeptide, e.g., a functional domain. In some embodiments, the heterologous moiety comprises a sequence useful for purifying, expressing, solubilizing and/or detecting the polypeptide. In some embodiments, the heterologous moiety comprises a polypeptide "tag", such as an affinity tag or epitope tag. For example, the tag can be an affinity tag (e.g., HA, TAP, Myc, 6XHis, Flag, GST), a fluorescent or fluorescent protein (e.g., EGFP, ECFP, EYFP, Cerulean, DsRed, mCherry), a solubilization tag (e.g., SUMO tag, NUS a tag, SNUT tag, or a monomeric mutant of the Ocr protein of bacteriophage T7). See, e.g., Esposito D and Chatterjee D k. curr Opin BiotechnoL; 17(4):353-8(2006). In some embodiments, the tag may have multiple functions. Tags tend to be relatively small, e.g., from a few amino acids up to about 100 amino acids in length. In some embodiments, the tag is more than 100 amino acids in length, e.g., up to about 500 amino acids or longer. In some embodiments, the polypeptide has a tag at the N-or C-terminus, e.g., as an N-or C-terminal fusion. The polypeptide may contain multiple tags. In some embodiments, there is a6 × His tag and a NUS tag, e.g., at the N-terminus. In some embodiments, the tag is cleavable, e.g., cleaved by a protease, such that the tag can be removed from the polypeptide. In some embodiments, this may be achieved by including a sequence encoding a protease cleavage site between the sequence encoding the homologous portion of the original polypeptide and the tag. Exemplary proteases include, for example, thrombin, TEV protease, factor Xa, PreScission protease, and the like. In some embodiments, "self-cutting" labels are employed. See, for example, PCT/US 05/05763. The sequence encoding the tag may be located 5 'or 3' (or both) relative to the polynucleotide encoding the polypeptide. In some embodiments, the tag or other heterologous sequence is separated from the remainder of the polypeptide by a polypeptide linker. For example, the linker can be a short peptide (e.g., 15-25 amino acids). Linkers often consist of small amino acid residues such as serine, glycine and/or alanine. The heterologous domain may contain a transmembrane domain, a secretion signal domain, and the like.

In certain embodiments of the invention, a fragment or variant (optionally excluding heterologous moieties), if present, has sufficient structural similarity to the original polypeptide such that, when its three-dimensional structure (actual or predicted) is superimposed on the original polypeptide structure, the volume of overlap is at least 70%, preferably at least 80%, more preferably at least 90% of the total volume of the original polypeptide structure. Part or all of the three-dimensional structure of a fragment or variant can be determined by protein crystallization, which can be accomplished using standard methods. Alternatively, NMR resolved structures can be generated, again using standard methods. Modeling programs such as MODELLER (Sali, A. and Blundell, T L, J.Mol.biol.,234, 779-. If a structure or predicted structure of spaced polypeptides is available, the modeling can be based on that structure. The PROSPECT-PSPP program suite (Guo, J T, et al, Nucleic Acids Res.32 (network edition): W522-5, 2004, 7/1) can be used. For embodiments of the invention that relate to polypeptide variants, it will be understood that polynucleotides encoding the variants are provided.

The term "vector" is used to refer to a nucleic acid or virus or portion thereof (e.g., viral capsid or genome) capable of mediating entry (e.g., transfer, transport, etc.) of a nucleic acid molecule into a phase. Where the vector is a nucleic acid, the nucleic acid molecule to be transferred is typically linked to (e.g., inserted into) a vector nucleic acid molecule. Nucleic acid vectors can include sequences that direct autonomous replication (e.g., an origin of replication), or can include sequences sufficient to integrate the nucleic acid, in part or in whole, into the DNA of a host cell. Useful nucleic acid vectors include, for example, DNA or RNA plasmids, cosmids, and naturally occurring or modified viral genomes or portions 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. The plasmid may comprise part or all of the viral genome (e.g., viral promoters, enhancers, processing or packaging signals, etc.). Viruses, or portions 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, retroviruses, lentiviruses, vaccinia viruses and other poxviruses, herpes viruses (e.g., herpes simplex virus), and other viruses. The viral vector may or may not contain sufficient genetic information to produce an infectious virus when introduced into a host cell, i.e., the viral vector may be replication-defective, and such replication-defective viral vectors may be preferred for therapeutic applications. In the absence of sufficient information, it may be (but is not required to be) provided by the host cell or other vector introduced into the cell. The nucleic acid to be transferred may be incorporated into the naturally occurring or modified viral genome or portion thereof, or may be present as a separate nucleic acid molecule within the virus or viral capsid. It will be appreciated that certain plasmid vectors, including part or all of the viral genome, typically include viral genetic information sufficient to direct transcription of nucleic acids capable of being packaged into a viral capsid and/or sufficient to produce nucleic acids capable of being integrated into the genome of a host cell and/or sufficient to produce infectious virus, and such vectors are sometimes referred to in the art as viral vectors. The vector may contain one or more nucleic acids encoding a marker for identifying and/or selecting whether a cell is transformed or transfected with the vector. Markers include (for example): proteins that increase or decrease tolerance or sensitivity to antibiotics (e.g., antibiotic resistance genes that encode proteins that provide resistance to antibiotics such as puromycin, hygromycin or blasticidin) or other compounds, enzymes whose activity can be detected using assays known in the art (e.g., beta-galactosidase or alkaline phosphatase), and proteins or RNAs that have a detectable effect on the phenotype of the transformed or transfected cells (e.g., fluorescent proteins). An expression vector is a vector that includes regulatory sequences, exemplified by expression control sequences such as a promoter, sufficient to direct transcription of an operably linked nucleic acid. Regulatory sequences may also include enhancer sequences or upstream activating sequences. The vector may optionally include a 5' leader or signal sequence. The vector may optionally include a cleavage and/or polyadenylation signal and/or a 3' untranslated region. Vectors often include one or more restriction enzyme sites appropriately located to aid in the introduction of the nucleic acid to be expressed into the vector. The expression vector contains sufficient cis-acting elements for expression, and other elements necessary for or to facilitate expression may be provided by the host cell or an in vitro expression system.

Different techniques can be used to introduce nucleic acid molecules into cells. Such techniques include chemically assisted transfection using compounds such as calcium phosphate, cationic lipids, cationic polymers, liposome-mediated transfection, non-chemical methods such as electroporation, ion bombardment or microinjection, and infection with viruses containing the nucleic acid molecule of interest (sometimes referred to as "transduction"). The markers can be used to identify and/or select cells that have acquired the vector and typically express the nucleic acid. Cells may be cultured in a suitable medium to select for such cells, and optionally to establish a stable cell line.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

It is to be understood that when a range of numerical values is given, each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to the limits explicitly excluded from the range. Where a stated range includes one or both of the limits, ranges excluding either or both of those stated are also included in the invention.

Certain ranges of values set forth herein are preceded by the term "about". The term "about" provides literal support for the exact number of digits that follow, as well as numbers that are near or approximate to the digits that follow the term. In determining whether a digit is near or approaching a particular referenced digit, the near or approaching non-referenced digit may be a substantially equivalent digit that provides the particular referenced digit in the content that it represents.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described below.

It should be 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 should also be noted that the claims may be drafted to exclude any optional element. Also, this description should be taken as a prerequisite for the use of such exclusive terminology as "only," "only," etc., or the use of a "negative/no" limitation in relation to the recited claim elements.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the remaining embodiments without departing from the scope and spirit of the present invention. Any mentioned method may be performed in the order of events mentioned or in any other sequence that is logically possible.

Detailed Description

The present invention provides compounds, compositions and methods useful for somatic iTR during fetal, neonatal, infant or mature periods of mammalian species (e.g., humans) development during which the gene expression pattern that confers TR capability has been lost during embryonic periods. In one aspect, the invention provides methods for identifying TR regulatory genes in mammalian species, including primates, and more particularly human species, wherein the genes are identified by comparing gene expression of coding mRNA and non-coding RNA or splice variants of the RNA to genes differentially expressed during the developing embryonic phase as compared to the developing fetal and mature phases. More specifically, the methods identify genes that encode mRNA and non-coding RNA that are differentially expressed in a plurality of different somatic cell types during prenatal development (particularly during the embryonic stage of development, prior to the point of transformation from embryonic to fetal development) as compared to fetal and mature cells (after the point of transformation from embryonic to fetal development). In the case of the human species, transformation from embryonic to fetal development occurs at about 8 weeks of prenatal development, approximately 16 days in mice, and about 17.5 days in rat species.

In another aspect of the invention, pluripotent stem cell-derived clonal, oligoclonal, pooled clonal, or pooled oligoclonal embryonic progenitor cell lines that exhibit a specific gene expression pattern during the embryonic stage of development in a mammal are utilized as a source of coding and non-coding RNA and compared to coding and non-coding RNA in cells and tissues from fetal or mature derived sources to identify genes that modulate TR and inhibit TR in fetal and mature tissues in cells in the embryonic stage of development compared to genes encoding mRNA and non-coding RNA or splice variants.

In another aspect of the invention, transcriptional regulator genes differentially expressed in different types of somatic cells during the embryonic period of development are compared to different types of somatic cells during the post-embryonic period of development, such as mature cell types that are unable to participate in TR, to identify those genes for which altered expression of splice variants results in suppression of tissue regeneration in mature mammals. In some embodiments, methods of identifying genes whose expression or inhibition results in iTR include: comparing the transcriptome of the cloned, oligoclonal, pooled cloned or pooled oligoclonal hPS cell-derived embryonic progenitor cells with the transcriptomes of different types of mature body-derived cells or tissues to identify genes that are ubiquitously expressed in embryonic progenitor cells or have RNA splice variants in embryonic progenitor cells but are not expressed or are expressed at significantly lower levels in mature body-derived cells, or to identify genes that are expressed or have RNA splice variants in mature body-derived cells but are not expressed or are expressed at significantly lower levels in a cloned, oligoclonal, pooled cloned or pooled oligoclonal hPS cell-derived embryonic progenitor cell line. In another embodiment, candidate iTR genes are identified as candidate iTR genes, either as genes that are more highly expressed in embryonic progenitor cells than in mature somatic derived cells and are considered to be associated with carcinogenesis, or as genes that are less expressed in embryonic progenitor cells than in mature somatic derived cells and are considered to be associated with tumor suppression.

In another aspect, the invention provides methods of screening combinations of iTR genes in different cell and tissue types to identify combinations of factors and/or inhibitors that have been optimized for regeneration of a particular cell or tissue type.

In another aspect, the invention provides methods for altering the expression of the iTR gene in cells in culture to restore the cells to a state that can participate in iTR when implanted in a tissue that would otherwise not be able to undergo sufficient TR.

In another aspect of the invention, a telomerase catalytic component, including but not limited to the human gene TERT, is transiently expressed in target cells and tissues in which TR is to be induced to expand the proliferative capacity of somatic cells thereby promoting TR. In another aspect of the invention, the telomerase catalytic component, including the human gene TERT, is transiently expressed (rather than constitutively expressed) in target cells and tissues to expand the proliferative capacity of somatic cells without immortalizing the cells.

In another aspect, the invention provides methods for altering the expression of an iTR gene in cells in vivo, such that the cells are restored to a state in which they are involved in iTR. In some embodiments, iTR gene expression is altered in vitro.

In another aspect, the invention provides a method of identifying a candidate modulator having TR activity, comprising: (i) providing a composition comprising: (a) candidate modulators with TR activity in purified state or in mixtures with other molecules; (b) a TR-incapable somatic cell that expresses a fetal or mature pattern of gene expression but not an embryonic pattern of gene expression; (c) a reporter construct present in said somatic cells or in an extract from said cells incapable of TR, wherein expression of a reporter gene is driven by a gene promoter that is differentially regulated in somatic cells during the developmental embryonic stage as compared to the fetal or mature stage; and (ii) determining whether the candidate modulator affects expression of the reporter gene, wherein an alteration in expression of the reporter gene as compared to expression of the gene in the absence of the candidate modulator indicates that the compound modulates iTR activity.

In some embodiments, the method of identifying a candidate TR modulator further comprises administering to the subject a candidate compound identified as a TR modulator. Suitable subjects include any animal including, for example: mammals such as humans, non-human primates, ungulates and other domestic animals such as, for example, cows, sheep, horses, goats, pigs, domestic mammals such as cats or dogs and rodents such as mice, rats, rabbits, guinea pigs.

In some embodiments, the method of identifying a compound further comprises administering the compound to a subject. In some embodiments, the subject is a non-human animal, e.g., a non-human animal used as a TR or wound healing model. In some embodiments, the subject is a human.

In another aspect, the present invention provides a pharmaceutical composition comprising: (a) an iTR regulator; and (b) a pharmaceutically acceptable carrier.

Certain conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant nucleic acid (e.g., DNA) techniques, immunology, and the like, may be used with certain aspects of the present invention. Some non-limiting descriptions of these techniques can be found in the following documents: authored by Ausubel, f. et al, Current Protocols in Molecular Biology (new compiled Molecular Biology experiments), Current Protocols in Immunology (new compiled Immunology experiments), Current Protocols in Protein Science (new compiled Protein Science experiments) and Current Protocols in Cell Biology (new compiled cytobiology experiments), both 2008 edition, new John Wiley & Sons, inc; sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual), 3 rd edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, 2001; harlow, E.and Lane, D., Antibodies-A Laboratory Manual (Antibodies-Laboratory Manual), Cold spring harbor Laboratory Press, Cold spring harbor, 1988; burns, r., Immunochemical Protocols (Methods in Molecular Biology) (Immunochemical experiments (Methods of Molecular Biology)), homa Press (Humana Press), 3 rd edition, 2005, Monoclonal antibodies: a reactive proproach (Monoclonal antibody: practical method) (p. shepherd and C Dean, oxford university Press, 2000); freshney, R.L, "Culture of Animal Cells, A Manual of Basic Technique" (Animal cell Culture: Basic technical Manual), 5 th edition, John Welch's father, Hopkin, 2005).

TR and iTR regulators

The invention provides an iTR regulator and a practical method thereof. The invention also provides compositions and methods useful for identifying modulators of itrs. In some aspects, the invention provides methods of enhancing regeneration, comprising administering to a multicellular organism in need thereof an agent that alters the concentration of the iTR modulator.

Primary animals exhibiting significant TR potential (e.g. axolotl amputated limb regeneration or whole body segment regeneration of the worm) achieve TR by simply reproducing normal embryonic development. The inability of TR-resistance in mammals such as mice and humans is due to the alteration of transcription of certain embryonic genes that switch their resistance from fetal development back to an embryonic state. Provided herein are methods for restoring certain such embryo-specific genes in TR-resistant animals, thereby inducing regenerative capacity in any tissue, including responsiveness to tissue-centered factors, resulting in complex tissue regeneration while reducing scar formation.

In these species it has been found that certain genes are differentially expressed in normal and regenerating tissues, and that some genes have been identified as essential for regeneration of such tissues, and none have been reported (whether isolated or combined with other genes) to be sufficient to reprogram cells in animal tissues that are otherwise unable to TR back to a state in which the tissues are capable of self-regeneration. Accordingly, there is a need in the art to identify genes whose expression or inhibition is sufficient to result in Induced Tissue Regeneration (iTR) of mammalian cells and tissues, and compositions and methods for inducing or inhibiting such regeneration in mammalian species, particularly Homo sapiens (Homo sapiens) species.

Genes whose expression inhibits TR in fetuses and mature animals are referred to herein as "TR repressors", while genes that are not expressed in fetuses and mature cells but whose expression promotes TR during the developing embryonic stage are referred to herein as "TR activators". The TR repressor gene and the TR activator gene are collectively referred to as iTR genes. Molecules that alter the TR activator and TR repressor in a manner that results in TR are referred to herein as "iTR factors". The iTR gene and the protein product of the iTR gene are often conserved in a variety of animals, from anemonia to mammals. Protein sequences encoded by the iTR gene, and nucleic acid (e.g., mRNA) sequences encoding the iTR gene, from several different animals are inhibited in the art and can be found from publicly available databases, such as the National Center for Biotechnology Information (NCBI) database.

It was observed that the TR suppressor gene COX7a1 was expressed predominantly in the stroma and not in epithelial cells in normal tissues. In the case of tumors, however, the gene was observed to be down-regulated in most stromal tumors such as osteosarcoma and chondrosarcoma. This is consistent with the enhanced glycolysis observed in cancer (known as the Warburg phenomenon). Since the TR gene is altered during transition from embryonic to fetal development (in part to avoid cancer in the mature body), inhibition of COX7a1 in stromal tumors will cause stromal cells to revert back to the embryonic state, which may contribute to carcinogenesis. Thus exogenous expression of COX7a1 in stromal tumors would have therapeutic effect.

The present invention provides several different methods of regulating the iTR gene, and a variety of different compounds that can be used to regulate the iTR gene. In general, an iTR factor can be, for example, a small molecule, nucleic acid, oligonucleotide, polypeptide, peptide, lipid, carbohydrate, and the like. In some embodiments of the invention, the iTR factor is inhibited by reducing the amount of TR repressor RNA produced by the cell and/or reducing the level of activity of the TR repressor gene. In the case of the TR repressor, factors can be identified and used for research and therapy to reduce the product level of the TR repressor gene. The TR repressor gene may be any combination of the TR repressor genes listed in FIG. 1A. The amount of TR repressor gene RNA can be reduced by inhibiting TR repressor RNA synthesis by the cell (also referred to as "inhibiting TR repressor gene expression"), for example, by reducing the amount of mRNA encoding the TR repressor gene or by reducing translation of mRNA encoding the TR repressor gene. Non-limiting examples of such factors are: RNAi targeting sequences within the TR repressor gene listed in FIG. 1A.

In some embodiments of the invention, TR repressor gene expression is inhibited by RNA infection (RNAi). As known in the art, RNAi is a process that: double-stranded RNA is present in the cell, which has a sequence corresponding to a gene resulting in sequence-specific inhibition of the expression of the gene, which inhibition is usually caused by cleavage or translational inhibition of mRNA transcribed from the gene. Compounds useful for causing expression inhibition by RNAi ("RNAi agents") include short interfering rnas (sirnas), short hairpin rnas (shrnas), small rnas (mirnas), and miRNA-like molecules.

Exemplary sequences capable of inhibiting the expression of human and murine TR repressor genes are provided in the examples. Once the TR suppressor gene is identified, one skilled in the art can conveniently design sequences for RNAi agents (e.g., siRNAs) that can be used to inhibit the expression of mammalian TR suppressor genes, such as human TR suppressor genes. In some embodiments, such sequences are selected to minimize an "off-target" effect. For example, the following sequence may be used: its complementary sequence is present in the TR repressor gene mRNA but not in other mrnas expressed in the species of interest (or not in the genome of the species of interest). Site-specific chemical modifications can be used to reduce the potential for off-target effects. In some embodiments, at least two RNAi agents, such as sirnas, targeted to TR inhibitor gene mRNA are used in combination. In some embodiments, small RNAs (which may be artificially designed small RNAs) are used to inhibit TR repressor gene expression.

In some embodiments of the invention, the TR repressor gene expression is inhibited using an antisense molecule comprising a single stranded oligonucleotide that is fully or substantially complementary to the mRNA encoding the TR repressor gene. Hybridization of the oligonucleotide to the TR repressor gene mRNA results in, for example, degradation of the mRNA by rnase H or blocking its translation by steric hindrance. In other embodiments of the invention, the TR repressor gene expression is inhibited using a ribozyme or triplex nucleic acid.

In some embodiments of the invention, the TR inhibitor inhibits at least one activity of the TR inhibitor protein. The TR inhibitor activity can be reduced by contacting the TR inhibitor protein with a compound in physical contact with the TR inhibitor protein. Such compounds may, for example, alter the structure of the TR inhibitor protein (e.g., by covalently modifying the protein) and/or block the interaction of the TR inhibitor protein with one or more other molecules, such as a cofactor or substrate. In some embodiments, inhibition or reduction can be a decrease 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 (e.g., a control level). The control level may be a level at which the TR inhibitor occurs in the absence of the factor. For example, a TR factor can 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 obtained in the absence of the factor under test conditions. In some embodiments, the level of the TR inhibitor is reduced to 75% or less of the level obtained in the absence of the factor under the test conditions. In some embodiments, the level of the TR inhibitor is reduced to 50% or less of the level obtained in the absence of the TR factor under the test conditions. In some embodiments, the level of TR inhibitor is reduced to 25% or less of the level obtained in the absence of the iTR factor under the test conditions. In some embodiments, the level of TR inhibitor is reduced to 10% or less of the level obtained in the absence of the iTR factor under the test conditions. In some cases, the level of modulation (e.g., inhibition or reduction) is statistically significant compared to a control level. As used herein, "statistically significant" refers to p-values below 0.05, e.g., p-values below 0.025 or p-values below 0.01, using appropriate statistical tests (e.g., ANOVA, t-test, etc.).

In some embodiments of the invention, the compounds inhibit the TR inhibitor protein directly, i.e., the mechanism by which the compounds inhibit the TR inhibitor protein involves the physical interaction (binding) of the TR inhibitor and the iTR factor. For example, binding of a TR repressor to an iTR factor can interfere with the ability of the TR repressor to catalyze a reaction and/or can block the active site of the TR repressor. 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.

In some embodiments of the invention, the iTR factor is covalently bound to the TR inhibitor. For example, the compounds may modify amino acid residues required for enzymatic activity. In some embodiments, the iTR factor contains one or more reactive functional groups that react with the amino acid side chain of the TR inhibitor, e.g., an aldehyde, alkyl halide, alkene, fluorophosphonate (e.g., alkyl fluorophosphonate), michael acceptor, phenylsulfonate, methyl ketone such as halomethyl ketone or diazomethyl ketone, fluorophosphonate, vinyl ester, vinyl sulfone, or vinyl sulfonamide. In some embodiments, the iTR factor inhibitor comprises a compound that physically interacts with the TR inhibitor, the compound comprising an active functional group. In some embodiments, the structure of the compound that physically interacts with the TR inhibitor is modified to incorporate an active functional group. In some embodiments, the compound contains a TR inhibitor substrate analog or a transition state analog. In some embodiments, the compound interacts with the TR inhibitor within or adjacent to the TR inhibitor active site.

In other embodiments, the iTR factor is non-covalently bound to the TR inhibitor and/or non-covalently bound to a complex comprising the TR inhibitor and a TR inhibitor substrate. In some embodiments, the iTR factor binds non-covalently to the active site of the TR repressor and/or competes with the substrate for contact with the TR repressor active site. In some embodiments, the ITR factor binds K of the TR inhibitor under test conditions such as in a physiologically acceptable solution such as phosphate buffered saline_dAbout 10^-3M or less, e.g.10^-4M or less, e.g. 10^-5M or less, e.g. 10^-6M or less, 10^-7M or less, 10^-8M or less, 10^-9M or less. 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 are known in the art. In some embodiments, the inhibitor comprises a TR inhibitor substrate analog or a transition state analog.

In the case of increasing the activity of the TR activator, any combination of the TR activator genes listed in FIG. 1B may be used. The levels of the products of these genes can be introduced using the vectors described herein.

Reporter-based iTR factor screening assays

The present invention provides methods for identifying an iTR factor using (a) a reporter molecule containing a marker, such as GFP, whose expression is driven by a TR activator, such as COX7A1, as described herein. The invention provides screening assays that include determining whether a test compound affects the expression of a TR activator gene and/or inhibits the expression of a TR suppressor gene. The invention also provides reporter molecules and compositions useful for practicing these methods. In general, the compounds identified by the methods of the invention may act by any mechanism that results in the enhancement or attenuation of the TR activator or repressor genes, respectively.

Reporter molecules, cells and membranes

In general, detectable moieties useful in the reporter molecules of the invention include light emitting or absorbing compounds that generate or quench a detectable fluorescent, chemiluminescent, or bioluminescent signal. In some embodiments, activation of the TR activator gene or inhibition of the TR repressor gene results in release of the detectable moiety into the liquid matrix, and a signal generated or quenched by the released detectable moiety present in the matrix (or sample thereof) is measured. In some embodiments, the generated signal causes a change in a property of the detectable moiety, which change is detectable, for example, as an optical signal. For example, the signal can alter the electromagnetic radiation (e.g., radiation having a wavelength in the near infrared, visible, or UV portion of the spectrum) emitted or absorbed by the detectable portion. In some embodiments, the reporter molecule contains a fluorescent or luminescent moiety, and the second molecule acts as a quencher to quench the fluorescent or luminescent moiety. Such changes can be determined using devices and methods known in the art.

In some embodiments of the invention, the reporter molecule is a genetically encodable molecule that can be expressed by a cell, and the detectable moiety comprises, for example, a detectable polypeptide. Thus, in some embodiments, the reporter is a polypeptide that contains a fluorescent polypeptide such as green, blue, turquoise, yellow, red, orange, cyan fluorescent protein and derivatives thereof (e.g., enhanced GFP); monomeric red fluorescent proteins and derivatives thereof such as those known as "mfurt (monomeric fruit color)", e.g., mCherry, mStrawberry, mTomato, etc., and luminescent proteins such as aequorin. (it will be understood that in some embodiments, fluorescence or luminescence occurs in the presence of one or more additional molecules (e.g., ions such as calcium ions and/or artificial groups such as coelenterazine). in some embodiments, the detectable moiety comprises an enzyme that acts on a substrate to produce a fluorescent, luminescent, chromogenic or otherwise detectable product.examples of enzymes that can be used as the detectable moiety include luciferase,. beta. -galactosidase, horseradish peroxidase, alkaline phosphatase, etc. (it is understood that the enzyme is determined by detecting the reaction product.) in some embodiments, the detectable moiety comprises a polypeptide tag that can be conveniently determined using a second reagent such as a labeled (e.g., fluorescently labeled) antibody.e.g., a fluorescently labeled antibody binds to available HA, Myc, or a variety of other peptide tags.accordingly, it is contemplated that the detectable moiety can be determined directly (i.e., it produces a detectable signal without reacting with the second reagent), and embodiments in which the detectable moiety interacts (e.g., binds and/or reacts) with the second reagent and the interaction allows the detectable moiety to be detected, e.g., by causing the production of a detectable signal or because the second reagent is directly detectable. In embodiments where the detectable moiety interacts with a second reagent to produce a detectable signal, the detectable moiety, which may react with the second reagent, is activated by the second reagent to produce the detectable signal. In many embodiments, the intensity of the signal gives an indication of the amount of detectable moiety present, e.g., in the sample being evaluated or within the imaged area. In some embodiments, the amount of the detectable moiety can optionally be quantified based on signal intensity, e.g., relative or absolute quantification.

The invention provides nucleic acids comprising a sequence encoding a reporter polypeptide of the invention. In some embodiments, the nucleic acid encodes a precursor polypeptide of a reporter polypeptide of the present invention. In some embodiments, the polypeptide-encoding sequence is operably linked to an expression control element (e.g., a promoter or promoter/enhancer sequence) suitable for directing transcription of the polypeptide-encoding mRNA. The invention also provides expression vectors containing the nucleic acids. Selection of an appropriate expression control element can be based, for example, on the appropriate cell type and species to be expressed. One of ordinary skill in the art can readily select appropriate expression control elements and/or expression vectors. In some embodiments, the expression control element is tunable, e.g., inducible or repressible. Exemplary promoters for administration to bacterial cells include, for example, Lac, Trp, Tac, araBAD (e.g., in a pBAD vector), phage promoters such as 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 adenovirus or cytomegalovirus immediate early promoter, or viral promoter/enhancer sequences, retroviral LTRs, promoters or promoters/enhancers of mammalian genes (e.g., actin, EF-1 α, phosphoglycerate kinase), and the like. Tunable (e.g., inducible or repressible) expression systems such as the Tet-On and Tet-Off systems (which can be modulated by tetracycline and analogs thereof such as doxycycline) and other systems that can be modulated by small molecules such as hormone receptor ligands (e.g., steroid receptor ligands, which may or may not be steroids), metal modulation systems (e.g., metallothionein promoters), and the like.

The invention also provides cells and cell lines containing such nucleic acids and/or vectors. In some embodiments, the cells are eukaryotic cells, such as fungal, plant, or animal cells. In some embodiments, the cell is a vertebrate cell, e.g., a mammalian cell, e.g., a human cell, a non-human cell, or a rodent cell. The cell may be any of the cells described above. In certain embodiments, the cell may be a clonal cell or an oligoclonal cell. In some embodiments, the cells may be progenitor cells obtained from pluripotent stem cells such as ES cells or iPS cells. In some embodiments, the cell is a member of a cell line, e.g., an established or immortalized cell line, that has acquired the ability to proliferate indefinitely in culture (e.g., as a result of mutation or genetic manipulation such as constitutive expression of a catalytic component of telomerase). A number of 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 tumor-derived. In some embodiments, the cell is an adherent cell. In some embodiments, non-adherent cells are used. In some embodiments, the cell is a cell of a cell type or cell line that has been shown to naturally express the TR activator gene set or not express the TR repressor gene. If a cell lacks one or more TR activator or repressor genes, the cell may be genetically engineered to express the protein(s). In some 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 some embodiments, the cell is transiently transfected with an expression vector encoding a reporter. The cells can be co-transfected with a control plasmid, which optionally expresses a different detectable polypeptide, to control transfection efficiency (e.g., multiple runs of overlay assays).

TR activator and TR repressor polypeptides and nucleic acids

The TR activator and TR repressor genes are listed in FIG. 1. TR activator and TR repressor polypeptides that may be used in the methods of the invention may be obtained in a variety of ways. In some embodiments, the polypeptide is produced using recombinant DNA technology. Standard methods for recombinant protein expression can be used. The nucleic acid encoding the TR activator or TR repressor gene is conveniently obtained, for example, from cells expressing the gene (e.g., by PCR or other amplification methods or by cloning), or by chemical synthesis or in vitro transcription of a polypeptide sequence based on the cDNA sequence. One of ordinary skill in the art will recognize that: because of the degeneracy of the genetic code, genes can be encoded by many different nucleic acid sequences. Alternatively, the sequences are codon optimized for expression in the host cell of choice. The gene may be expressed in bacteria, fungi, animals, plant cells or organisms. The genes may be isolated from cells in which they are naturally expressed, or from cells into which a nucleic acid encoding the protein has been transiently or stably introduced, e.g., cells containing an expression vector encoding the gene. In some embodiments, the gene is secreted by the cells in culture and isolated from the culture matrix.

In some embodiments of the invention the sequence of the TR activator or TR repressor polypeptide is used in the screening methods of the invention. The naturally occurring TR activator or TR repressor polypeptide may be from any species whose genome encodes a TR activator or TR repressor polypeptide, e.g., human, non-human primate, rodent, etc. Polypeptides having a sequence identical to a naturally occurring TR activator or TR repressor are sometimes referred to herein as "natural TR activator/repressor". The TR activator or TR repressor polypeptides useful in the present invention may or may not contain a secretion signal sequence or portion thereof. For example, the 20-496 amino acids of the human TR activator or TR repressor (or the corresponding amino acids of the TR activator or TR repressor from other species) or the mature TR activator or TR repressor composed of these amino acids can be used.

In some embodiments, variants or fragments containing or consisting of TR activators or TR inhibitors are used. TR activator or TR repressor variants include polypeptides that differ from the TR activator or TR repressor by one or more amino acid substitutions, additions, or deletions. In some embodiments, a TR activator or TR repressor variant comprises a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to at least 20-496 amino acids of a TR activator or TR repressor (e.g., from a human or mouse) over at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of at least 20-496 amino acids of a human TR activator or TR repressor or at least 20-503 amino acids of a mouse TR activator or TR repressor. In some embodiments, a TR activator or TR repressor variant polypeptide comprises a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to at least 20-496 amino acids of a human TR activator or TR repressor or at least 20-503 amino acids of a mouse TR activator or TR repressor. In some embodiments, the TR activator or TR repressor polypeptide comprises a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to at least 20-496 amino acids of a human TR activator or TR repressor or at least 20-503 amino acids of a mouse TR activator or TR repressor. Nucleic acids encoding a TR activator or TR repressor variant or fragment can be conveniently generated, for example, by modifying DNA encoding the native TR activator or TR repressor using site-directed mutagenesis or other standard methods, and can be used to generate TR activator or TR repressor variants or fragments. For example, a fusion protein can be produced by cloning a sequence encoding a TR activator or TR repressor into a vector that provides the coding sequence for the heterologous portion. In some embodiments, a tagged TR activator or TR repressor is used. For example, in some embodiments, a TR activator or TR repressor polypeptide is used that contains a 6XHis tag, e.g., at its C-terminus.

Test compounds

The methods of the invention allow for the identification of the iTR factor using a variety of different test compounds. For example, the test compound can be, for example, a small molecule, polypeptide, peptide, nucleic acid, oligonucleotide, lipid, carbohydrate, antibody, or hybrid molecule. The compounds may be obtained from natural sources or produced synthetically. The compounds may be at least partially pure or may be present in an extract or other type of mixture, the composition of which is at least partially unknown or unidentified. The extract or portion thereof can be produced, for example, from plants, animals, microorganisms, marine organisms, fermentation broths (e.g., soil, bacterial, or fungal fermentation broths), and the like. In some embodiments, a collection of compounds ("library") is tested. A library may contain, for example, 100 to 500,000 compounds or more. The compounds are often arrayed in multiwell plates (e.g., 384 well plates, 1596 well plates, etc.). They may be dissolved in a solvent (e.g., DMSO) or provided in dry form, e.g., as a powder or solid. A collection of synthetic, semi-synthetic and/or naturally occurring compounds can be tested. The compound libraries may contain structurally related, structurally diverse, or structurally unrelated compounds. The compounds may be artificial (having a structure artificially created and not found in nature) or naturally occurring. In some embodiments, the library contains at least some compounds and/or derivatives thereof that have been identified as "hits" or "leads" in other drug development programs. The compound libraries may contain natural products and/or compounds produced using non-directed or directed synthetic organic chemistry. Often, the library of compounds is a library of small molecules. Other libraries of interest include peptide or peptoid libraries, cDNA libraries, antibody libraries, and oligonucleotide libraries. The library may be focused (e.g., consisting essentially of compounds having the same core structure, derived from the same precursor, or having at least one common biochemical activity).

Compound libraries are available from a number of commercial suppliers such as Tocris Bioscience, Nanosyn, BioFocus, and government departments. For example, one MLSMR (molecular library reproduction) of the National Institutes of Health (NIH) molecular library project is designed to identify, acquire, maintain and distribute >300,000 chemically diverse compounds with known and unknown biological activities for use in High Throughput Screening (HTS) assays (residues https:// mli. NIH. gov/mli /). The NIH clinical bank (NCC) is a plate-like array of about 450 small molecules with a history of human clinical trials. These compounds are drug-like and have known safety profiles. In some embodiments, a collection of compounds comprising "approved human drugs" is tested. An "approved human drug" is a compound that has been approved for use in treating a human by a governmental regulatory agency such as the U.S. FDA, european drug administration, or the like, responsible for at least assessing the safety of a therapeutic agent prior to its sale. The test compound may be, for example, an antineoplastic, antibacterial, antiviral, antifungal, antiprotozoal, antiparasitic, antidepressant, antipsychotic, anesthetic, antianginal, antihypertensive, antiarrhythmic, anti-inflammatory, analgesic, antithrombotic, antiemetic, immunomodulatory, antidiabetic, lipid or cholesterol lowering (e.g., statins), anticonvulsant, anticoagulant, anxiolytic, hypnotic (sleep-inducing), hormonal, or antihormonal agent. In some embodiments, the compound is a compound that has been subjected to at least some antenatal or clinical development or has been determined or expected to have "drug-like" properties. For example, the test compound may have completed a phase I trial or at least completed a preclinical study in a non-human animal and show evidence of safety and tolerability.

In some embodiments, the test compound is substantially non-toxic to cells of the organism to be administered and/or to cells in which the compound can be tested, at the concentrations used, or in some embodiments, at concentrations up to 10-fold, up to 100-fold, or up to 1000-fold the concentrations used. For example, there is no statistically limiting effect on cell viability and/or proliferation, or in various embodiments, the reduction in viability or proliferation may be no more than 1%, 5%, or 10%. Cytotoxicity and/or effect on cell proliferation can be assessed by any of a variety of methods in assays. For example, cell metabolism assays such as AlamarBlue, MTT, MTS, XTT and CellTitre Glo assays, cell membrane integrity assays, ATP-based cell viability assays, mitochondrial reductase activity assays, BrdU, EdU or H3-thymidine incorporation assays can be used. In some embodiments, the test compound is not a compound found in a cell culture medium known or used in the art (e.g., a medium administered to culture vertebrate, such as mammalian cells), or, if the test compound is a compound found in a cell culture medium known or used in the art, is used in different concentrations, e.g., higher concentrations, in the methods described herein.

Aspects and controls of test implementation

Various inventive screening assays are described above, involving determining whether a test compound is known to be at the level of an active TR inhibitor or to provide the level of an active TR activator. Cells suitable for expressing a reporter are described above.

In conducting the assays described herein, the assay components (e.g., cells, TR activator or TR inhibitor polypeptide and test compound) are typically dispensed into a plurality of containers or other receptacles. Any type of container or article capable of holding cells may be used. In many embodiments of the invention, the containers are wells of a multi-well plate (also known as a "microplate," microtiter plate, etc.). For purposes of this specification, the term "well" will be used to refer to any type of container or article that can be used to perform the screening described herein, for example any container or article that can contain a test composition. It should be understood that the present invention is not limited to the use of wells or the use of multi-well plates. In some embodiments, any article having a plurality of physically separated cavities (or other defined structures) therein or on a substrate may be used. For example, the assay components may be defined as droplets, which may optionally be arranged on a surface, and these droplets may optionally be confined to discrete locations with water-resistant material spacers, within channels of a microfluidic device, and the like.

In general, the assay components can be added to the wells in any order. For example, cells can be added to the wells first and maintained in culture for a selected period of time (e.g., 6 to 48 hours), followed by addition of the test compound and the target TR activator or TR inhibitor polypeptide or cells with the expression construct. In some embodiments, the compound is added to the well prior to adding the polypeptide of the cell. In some embodiments, expression of the reporter polypeptide is induced after seeding the wells with cells, optionally after addition of a test compound. In some embodiments, expression of the reporter is achieved by transfecting the cell with an expression vector encoding the reporter polypeptide. In some embodiments, the cell is first genetically engineered to express a reporter polypeptide. In some embodiments, expression of the reporter is controlled by a regulatable expression control element, and induction of expression of the reporter is achieved by contacting the cell with an agent that induces (or derepresses) expression.

The test composition containing the cells, test compound or polypeptide is maintained for a suitable period of time during which the test compound may (in the absence of a test compound inhibiting its activity) result in an increase or decrease in the level of target TR activator or TR inhibitor activity. The number of cells, the amount of TR activator or TR repressor polypeptide, and the amount of test compound added can depend on a variety of factors such as container size, cell type, and can be determined by one of ordinary skill in the art. In some embodiments, the ratio of the molar concentration of the TR activator or TR inhibitor polypeptide to the test compound is between 1:10 and 10: 1. In some embodiments, the number of cells, the amount of test compound, and the duration of time the composition is maintained can be selected to provide a conveniently detectable level of signal after a selected period of time in the absence of the test compound. In some embodiments, the degree of confluence of the cells upon addition of the compound is about 25% to 75%, e.g., about 50%. In some embodiments, a 96-well plate is seeded with 1,000 to 100,000 cells/well (e.g., about 5,000 cells/well) per well in about 100 μ l of culture medium. In other exemplary embodiments, the cells are seeded in 30 μ 1-50 μ l of culture medium at 500 to 2,000 (e.g., about 1000) cells per well into 384-well plates. In some embodiments, the compound is tested at multiple concentrations (e.g., 2-10 different concentrations) and/or multiple replicates (e.g., 2-10 replicates).

Some or all of the replicates at different concentrations may be performed. In some embodiments, the candidate TR factors are used at a concentration of 0.1 μ g/ml to 100 μ g/ml, e.g., 1 μ g/ml and 10 μ g/ml. In some embodiments, the candidate TR factors are used at multiple concentrations. In some embodiments, the compound is added between 6 hours and 1 day (24 hours) after inoculation.

In some aspects of any of the compound screening and/or identification methods of the invention, the test compound is added to the test composition in an amount sufficient to achieve a predetermined concentration. In some embodiments, the concentration is up to about 1 nM. In some embodiments, the concentration is between about 1nM and about 100 nM. In some embodiments, the concentration is between about 100nM to about 10 μ Μ. In some embodiments, the concentration is at least 10 μ Μ, for example between 10 μ Μ and 100 μ Μ. The test composition may be held for various periods of time after the addition of its final ingredients. In certain embodiments, the test composition is maintained for a period of time of about 10 minutes to about 4 days, such as 1 hour to 3 days, such as 2 hours to 2 days, or any intervening range or specified value, such as about 4-8 hours, after addition of all ingredients. A plurality of different time points may be tested. Over such a period of time, additional aliquots of the test compound may be added to the test composition. In some embodiments, the cells are maintained in a cell culture medium suitable for culturing such cells. In some embodiments, serum-free media is employed. In some embodiments, the test composition comprises a physiologically acceptable liquid that is compatible with maintaining cell membrane integrity and (optionally) cell viability, in place of the cell culture medium. Any suitable liquid may be used, provided that the liquid has an appropriate osmotic pressure and is otherwise compatible with maintaining cell membrane integrity and (optionally) cell viability for at least a sufficient period of time for the assay to be performed. One or more assays indicative of an increase in the level of active TR activator or a decrease in TR inhibitor may be made during or after the incubation period.

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

In various embodiments, the foregoing assay methods of the invention are suitable for High Throughput Screening (HTS) implementation. In some embodiments, the screening methods of the invention are high-throughput or ultra-high-throughput (see, e.g., Fernandes, P.B., Curr Opin chem.biol.1998,2: 597; Sundberg, S A, Curr Opin Biotechnol.2000,11: 47). High throughput screening often involves highly efficient testing of large numbers of compounds, e.g., parallel testing. For example, tens or hundreds of thousands of compounds can be routinely screened in a short period of time, e.g., hours to days. In some embodiments, HTS refers to testing 1,000 to 100,000 compounds per day. In some embodiments, ultra-high throughput refers to screening over 100,000 compounds per day, e.g., up to one million or more compounds per day. The screening assay of the invention may be performed in a multi-well format, such as a 96-well, 384-well format, 1536-well format or 3,456-well format, and is amenable to automation. In some embodiments, each well of a microplate may be used to run a separate assay for a different test compound, or if the effect of concentration or incubation time is to be observed, there may be multiple wells containing test samples of a single compound, at least some of the wells optionally being left empty or used as controls or replicates. In general, HTS embodiments of the assays described herein relate to automated applications. In some embodiments, an integrated robotic system comprising one or more robots transports test microplates between multiple test stations for addition of compounds, cells, and/or reagents, mixing, incubation, and reading or detection. In some aspects, HTS systems of the present invention can simultaneously sample, incubate, and analyze multiple plates. Suitable data processing and control software may be employed. High throughput screening embodiments are well known in the art. Without limiting the invention in any way, some of the general principles and techniques that may be employed in the practice of HTS as described herein are documented in the literature: macroron R and Hertzberg R P.design and implementation of high-throughput screening assays (design and implementation of high throughput screening assays), Methods Mol biol.,565:1-32,2009 and/or An W F and Tolliday N J., Introduction to cell-based assays for high-throughput screening assays, Methods Mol biol.486:1-12,2009, and/or the references cited therein. Exemplary methods are also disclosed: high Throughput Screening, Methods and Protocols (High Throughput Screening: Methods and Protocols (Molecular Biology)), William P.Janzen (2002); and High-Throughput Screening in Drug Discovery (High Throughput Screening in Medicinal Chemistry) (2006).

The additional compounds may have one or more improved pharmacokinetic and/or pharmacodynamic properties or simply a different structure than the initial hits. An "improved property" may be, for example, to make a compound more effective or more suitable for one or more of the purposes described herein. In some embodiments, for example, a compound may have a higher affinity for a molecular target of interest (e.g., a TR activator or TR repressor gene product), a lower affinity for a non-target molecule, a higher solubility (e.g., increased water solubility), increased stability (e.g., in the blood, plasma, and/or gastrointestinal tract), increased in vivo half-life, increased bioavailability, and/or reduced side effects, among others. Optimization can be achieved by empirically modifying the hit structure (e.g., synthesizing compounds with related structures and testing in cell-free and cell-based assays or in non-human animals) and/or using computational methods. In some embodiments, such modifications can utilize principles established in medicinal chemistry to predictably alter one or more properties. In some embodiments, one or more compounds are identified as "hits," and systematic structural changes are made to generate a secondary library of compounds (e.g., a refinement lead compound) that are structurally related to the hits. The secondary library can then be screened using any of the methods described herein.

In some embodiments, the iTR factor is modified or incorporated into a moiety to enhance stability (e.g., in serum), increase half-life, reduce toxicity and immunogenicity, or otherwise confer a desired property to the compound.

Application of iTR factor

Pharmaceutical composition

There are a number of different applications for the iTR factor. Non-limiting examples of such applications are described herein. In some embodiments, the iTR factor is used to enhance regeneration of an organ or tissue.

Exemplary organs or tissues suitable for proliferative regeneration include limb, digit, cartilage, heart, blood vessel, bone, esophagus, stomach, liver, gall bladder, pancreas, intestine, rectum, anus, endocrine glands (e.g., thyroid, parathyroid, adrenal, endocrine parts of the pancreas), skin, hair follicles, thymus, spleen, skeletal muscle, locally damaged cardiac muscle, smooth muscle, brain, spinal cord, peripheral nerve, ovary, fallopian tube, uterus, vagina, breast, testis, vas deferens, seminal vesicle, prostate, penis, pharynx, larynx, trachea, bronchus, lung, kidney, ureter, bladder, urethra, eye (e.g., retina, cornea), or ear (e.g., screw machine). In some embodiments, the iTR factor is used to enhance regeneration of a stromal layer (e.g., connective tissue supporting tissue parenchyma). In some embodiments, the iTR factor is used to enhance postoperative regeneration, e.g., surgery requires removal of at least a portion of diseased or damaged tissue, organs, or other structures such as limbs, fingers (toes), etc. For example, such procedures may remove at least a portion of the liver, kidney, stomach, pancreas, intestine, breast, ovary, testis, bone, limb, digit, muscle, skin, and the like. In some embodiments, the surgery is removal of a tumor. In some embodiments, the iTR factor is used to promote scarless regeneration of skin following trauma, surgery, disease, and scald.

In various embodiments, enhancing regeneration may include one or more of the following: (a) the regeneration rate is improved; (b) the regeneration degree is improved; (c) facilitating establishment of appropriate structures (e.g., shape, pattern, tissue architecture, tissue polarity) in the regenerating tissue or organ or other body structure; (d) promoting new tissue growth in a manner that preserves and/or restores function. Although the use of iTR factors to enhance regeneration is of particular interest, the invention also encompasses the general use of iTR factors to enhance repair or wound healing, rather than necessarily to produce detectable regeneration enhancement. Accordingly, the present invention provides a method of enhancing repair or wound healing, according to any of the methods described herein, wherein the iTR factor is administered to a subject in need thereof. In some embodiments, the iTR factor is used to heal a wound or enhance the natural wound healing capabilities of a subject. For example, the iTR factor may be used to heal wounds faster or may be used to heal wounds without scar formation.

In some embodiments, the present invention provides a method of enhancing regeneration in a subject in need thereof, the method comprising administering to the subject an effective amount of an iTR factor. In some embodiments, an effective amount of a compound (e.g., an iTR factor) is an amount that results in an increase in the rate or extent of regeneration of damaged tissue as compared to a reference value (e.g., a suitable control value). In some embodiments, the reference value is the expected (e.g., average or typical) rate or extent of regeneration in the absence of the compound (optionally administered with a placebo). In some embodiments, an effective amount of an iTR factor is an amount that results in an improvement in structural and/or functional outcome as compared to the structural and/or functional outcome expected (e.g., averaged or typical) in the absence of the compound. In some embodiments, an effective amount of a compound, such as an iTR factor, results in enhanced primordial formation and/or reduced scarring. The degree or rate of regeneration can be assessed based on, for example, the dimensions or volume of the regenerated tissue. Structural and/or functional structures may be assessed based on, for example, visual inspection (optionally including use of a microscope or imaging calculations such as X-ray, CT scan, MRI scan, PET scan) and/or by assessing a tissue, organ, or other body part for one or more physiological processes or tasks that the tissue, organ, or other body part routinely performs. Generally, an improved structural result refers to a result that more closely approximates a normal structure (e.g., a structure before tissue damage or a structure in a normal, healthy individual) than would be expected (e.g., an average or typical result) without treatment with an iTR factor. One of ordinary skill in the art can select an appropriate functional assay or test. In some 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 some embodiments, the improvement in structural and/or functional outcome compared to a control value is statistically and/or clinically significant. By "clinically significant improvement" is meant an improvement that, at the discretion of a medical or surgical practitioner, brings a meaningful benefit to the patient (e.g., a benefit that makes the treatment worthwhile). It will be appreciated that in many embodiments, the iTR modulator administered to a subject of a particular substance (e.g., for therapeutic purposes) is a compound that modulates (e.g., inhibits) the expression of an endogenous TR gene in a subject of that species. For example, if the subject is a human, a compound that inhibits the activity of a human TR repressor gene product and activates the activity of a human TR activator gene product is typically administered.

In some embodiments, the iTR factor is used to enhance skin regeneration, for example, following burns (temperature or chemical), scratches, or other conditions involving skin loss, such as infection, e.g., necrotizing fasciitis, or purpura fulminans. In some embodiments, the scald is a secondary or tertiary scald. In some embodiments, the area of the skin loss region is at least 10cm². In one aspect, the iTR factor is enhancedRegeneration of the skin graft. In one aspect, the iTR factor reduces excessive and/or pathological wound contraction or scarring.

In some embodiments, the iTR factor is used to enhance bone regeneration, for example, in the case of non-union fractures, implant fixation, periodontal or alveolar ridge augmentation, craniofacial surgery, or other situations where new bone formation is deemed appropriate. In some embodiments, the iTR factor is applied to a site in need of bone regeneration. In some embodiments, the iTR factor is incorporated into or used in conjunction with a bone graft material. Bone graft materials include a variety of ceramic and proteinaceous materials. Bone graft materials include autologous bone (e.g., bone harvested from the ilium, fibula, ribs, etc.), allogeneic bone from cadavers, and xenogeneic bone. Synthetic bone graft materials include a variety of ceramics, such as calcium phosphates (e.g., hydroxyapatite and tricalcium phosphate), bioglass, and calcium sulfate, as well as proteinaceous materials such as Demineralized Bone Matrix (DBM). DBM can be prepared by milling cortical bone tissue (typically to a sieved particle size of 100-500 μm) and then treating the milled tissue with hydrochloric acid (typically 0.5 to 1N). In some embodiments, the iTR factor is administered to the subject with one or more bone graft materials. The iTR factor can be mixed with the bone graft material (in a composition containing the iTR factor and the bone graft material) or administered separately, for example, after placement of the graft. In some embodiments, the present invention provides bone pastes containing iTR factors. Bone pastes are products of a suitable consistency and composition so that they may be introduced into bone defects, such as pores, gaps, cavities, cracks, etc., and used to repair or fill such defects, or applied to existing bone structures. Bone paste is generally sufficiently malleable to allow it to be manipulated and shaped into various shapes by a user. Such treatment requires that bone formation occur to displace the paste, for example, to maintain the shape of the paste when applied. The bone paste provides a support structure for the formation of new bone and may contain a substance that promotes bone formation. In addition to one or more of the aforementioned ceramic or proteinaceous bone graft materials (e.g., DBM, hydroxyapatite), bone pastes often contain one or more ingredients that impart a paste-like or putty-like consistency to the material, such as hyaluronic acid, chitosan, starch ingredients such as pullulan.

In some embodiments, the iTR factor enhances recruitment and/or formation of osteoprogenitor cells from undifferentiated stromal cells and/or enhances differentiation of osteoprogenitor cells into cells forming new bone (osteoblasts).

In some embodiments, the iTR factor is administered to a subject with osteopenia or osteoporosis, e.g., to enhance bone regeneration in the subject.

In some embodiments, the iTR factor is used to enhance regeneration of a joint (e.g., a fibrous, cartilage, or synovial joint). In some embodiments, the joint is an intervertebral disc. In some embodiments, the joint is a hip, knee, elbow, or shoulder joint. In some embodiments, the iTR factor is used to enhance regeneration of dental and/or periodontal tissues or structures (e.g., dental pulp, periodontal ligament, tooth, periodontal bone).

In some embodiments, the iTR factor is administered to the subject in combination with the cells. The iTR factor and the cells can be administered separately or in the same composition. If administered separately, they may be administered to the same or different locations. In various embodiments, the cells may be autologous, allogeneic or xenogeneic. The cells may comprise progenitor cells or stem cells, such as mature stem cells. As used herein, a stem cell is a cell having at least the following properties: (i) self-renewal, i.e., the ability to undergo multiple rounds of cell division while remaining undifferentiated; (ii) pluripotency or multi-differentiation potential, i.e., the ability to produce progeny of a variety of different cell types (e.g., multiple, most, or all of the different cell types of a particular tissue or organ). Mature stem cells are stem cells derived from non-embryonic tissue (e.g., fetal, postpartum, or mature tissue). As used herein, "progenitor cells" encompass pluripotent cells and cells that are more differentiated than pluripotent stem cells, but not yet fully differentiated. These more differentiated cells (which may be produced from embryonic progenitor cells) have a reduced self-renewal capacity compared to embryonic progenitor cells. In some embodiments, the administration of the iTR factor is 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., bronchoalveolar stem cells), muscle progenitor cells (e.g., satellite cells), adipose-derived progenitor cells, epithelial progenitor cells (e.g., keratinocyte stem cells), and/or hematopoietic progenitor cells (e.g., hematopoietic stem cells). In some embodiments, the cell contains an induced pluripotent stem cell (iPS cell) or a cell that has been at least partially differentiated from an iPS cell. In some embodiments, the progenitor cells comprise mature stem cells. In some embodiments, at least some of the cells are differentiated cells, e.g., chondrocytes, osteoblasts, keratinocytes, hepatocytes. In some embodiments, the cell comprises a myoblast.

In some embodiments, the iTR factor is administered in a composition (e.g., in solution) containing one or more compounds that polymerize or become cross-linked or undergo transformation in situ after administration to the subject, typically forming a hydrogel. The composition may contain monomers, polymers, initiators, crosslinking agents, and the like. The composition may be applied (e.g., with a syringe) to the area where regeneration is desired, where the gel is formed in situ, from which the iTR factor is released over time. Gelation may be initiated, for example, by exposure to ions in the body fluid or a change in temperature or pH, or by mixing active precursors (e.g., using a multi-barrel syringe). See, for example, U.S. Pat. nos. 6,129,761; yu L, Ding J. Injectable hydrogels as unique biomedical materials Chem Soc Rev.37(8):1473-81 (2008)). In some embodiments, the hydrogel is a hydrogel comprising hyaluronic acid or hyaluronic acid and collagen I as described herein HyStem-C. In some embodiments, the composition further comprises a cell.

In some embodiments, the iTR factor is administered to a subject in combination with a vector that expresses a telomerase catalytic component. The carriers can be administered separately or in the same composition. If administered separately, they may be administered to the same or different locations. The vector may express telomerase catalytic components from the same species as the tissue being treated or from a different species. Co-administration of iTR factor with the telomerase catalytic component is particularly useful where the target tissue is from an elderly subject and the subject is a human.

Other inventive methods include the use of the iTR factor in ex vivo to produce a viable functional tissue, organ or cell-containing composition to repair or replace a tissue or organ lost to injury. For example, cells or tissues taken from an individual (whether the intended recipient, an individual of the same species, or an individual of a different species) may be cultured in vitro, optionally with a matrix, scaffold (e.g., a three-dimensional scaffold), or mold (e.g., containing a biocompatible (optionally biodegradable) material, e.g., a polymer such as HyStem-C), and their development into functional tissues or organs may be promoted by exposure to iTR factors. The scaffold, matrix or mold may be at least partially composed of naturally occurring proteins such as collagen, hyaluronic acid or alginate (or any chemically modified derivative thereof), or synthetic polymers or copolymers of lactic acid, caprolactone, glycolic acid, etc., or self-assembling peptides, or acellular matrices derived from tissues such as heart valves, intestinal mucosa, blood vessels and trachea. In some embodiments, the scaffold comprises a hydrogel. In certain embodiments, the scaffold may be coated or impregnated with an iTR factor that can diffuse out of the scaffold over time. After ex vivo production, the tissue or organ is transplanted into or onto the subject. For example, in the case of certain tissues, such as skin, the tissue or organ may be placed on the surface of the body. The tissue or organ may continue to develop in vivo. In some embodiments, the tissue or organ to be produced at least partially ex vivo may be a bladder, a blood vessel, bone, fascia, liver, muscle, skin patch, or the like. For example, a suitable scaffold may mimic an extracellular matrix (ECM). Optionally, the iTR factor is administered to the subject before, during and/or after ex vivo tissue or organ transplantation. In some aspects, the biocompatible material is one that is substantially non-toxic to cells at the concentrations used in vitro, or in the case of materials administered to a living subject, in the amounts used and at the location of use, and does not elicit or cause deleterious or adverse effects on the subject, such as an immune or inflammatory response, unacceptable scar tissue formation, and the like. It is understood that certain biocompatible materials may cause such adverse reactions in a small percentage of subjects, typically less than about 5%, 1%, 0.5%, or 0.1%.

In some embodiments, a matrix or scaffold coated or impregnated with an iTR factor is implanted into a subject in need of regeneration, optionally in combination with cells. The matrix or scaffold may be in the shape of a tissue or organ to be regenerated. The cells may be any of the cells described above, for example one or more types of stem cells capable of giving rise to such tissues or organs and/or the types found in such tissues or organs.

In some embodiments, the iTR factor is administered directly to or near the site of tissue injury. "directly … … to the site of tissue injury" encompasses injection of the compound or composition into the site of tissue injury or spreading, pouring, or otherwise directly contacting the injury compound or composition to the site of tissue injury. In some embodiments, administration is considered "administered/given" near the site of tissue damage if it is performed about 10cm furthest from the visible or otherwise apparent edge of the site of tissue damage or a blood vessel (e.g., artery) located at least partially within the damaged tissue or organ. Administration "near the site of tissue damage" is sometimes at a location within the damaged organ where no damage is apparent. In some embodiments, the iTR factor is applied to the remainder of the tissue, organ or other structure following injury or loss of the tissue, organ or other structure. In some embodiments, the iTR factor is applied to the remaining part of the amputation of a finger or limb still connected to the body to enhance regeneration of the deleted part. In some embodiments, the excised portions are surgically reconnected and the iTR factor is applied to either or both sides of the wound. In some embodiments, the iTR factor is administered to enhance implantation or repair or regeneration of the transplanted organ or portion thereof. In some embodiments, the iTR factor is used to enhance nerve regeneration. For example, the iTR factor may be infused into the severed nerve, e.g., near its proximal and/or distal ends. In some embodiments, the iTR factor is located within an artificial nerve conduit, which is a tube constructed of biological or synthetic material that encloses nerve endings and intervening spaces therein.

In some embodiments, the iTR factor is used to promote hair follicle production and/or hair growth. In some embodiments, the iTR factor triggers regeneration of hair follicles from epithelial cells that do not normally form hair. In some embodiments, the iTR factor is used to treat hair loss, thinning, and partial or complete baldness in men or women. In some embodiments, alopecia is a condition where there is no or substantially no hair or a lack of hair on areas where hair is often growing, such as the top, back, and/or sides of the head. In some embodiments, thinning is a condition where there is less than normal or average hair, or in some embodiments, less hair than the individual has had hair in the past, or in some embodiments, less hair than the individual thinks is needed. In some embodiments, the iTR factor is used to enhance growth of eyebrows or eyelashes. In some embodiments, the iTR factor is used to treat androgenetic alopecia or "male pattern baldness" (which can affect both males and females). In some embodiments the iTR factor is used to treat alopecia areata involving alopecia areata on the scalp, alopecia totalis involving all hairs, or alopecia universalis involving all hairs and body hair. In some embodiments, the iTR factor is applied to a site where hair growth is desired, such as the scalp or eyebrow area. In some embodiments, the iTR factor is applied to the eyelid margin to promote eyelash growth. In some embodiments, the iTR factor is applied in a liquid formulation. In some embodiments, the iTR factor is applied in the form of a cream, ointment, salve or gel. In some embodiments, the iTR factor is used to enhance hair growth following burns, surgery, chemotherapy, or other events that result in the loss of hair or hairy skin.

In some embodiments, one or more iTR factors are administered to age-related degenerative tissue to regenerate youthful state function. Non-limiting examples of the age-related degenerative change include: 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 administered in combination with a vector that expresses a telomerase catalytic component to extend cell life.

In some embodiments, one or more iTR factors are administered to enhance replacement of cells lost or damaged by injury, such as chemotherapy, radiation therapy, or toxins. In some embodiments, such cells are stromal cells of solid organs and tissues.

The treatment method of the invention may comprise the steps of: identifying or providing a subject having or at risk of having a disease or condition wherein enhanced regeneration would benefit the subject. In some embodiments, the subject experiences injury or trauma (e.g., physical trauma) to a tissue or organ. In some embodiments, the injury is of a limb or digit. In some embodiments, the subject is suffering from a disease affecting the cardiovascular, cerebrovascular, digestive, endocrine, musculoskeletal, gastrointestinal, hepatic, integumentary, neurological, respiratory, or urinary systems. In some embodiments, the tissue damage is damage to a tissue, organ, or structure as follows: cartilage, bone, heart, blood vessels, esophagus, stomach, liver, gall bladder, 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 nerve, ovary, fallopian tube, uterus, vagina, breast, testis, vas deferens, seminal vesicle, prostate, penis, pharynx, larynx, trachea, bronchus, lung, kidney, ureter, bladder, urethra, eye (e.g., retina, cornea), or ear (e.g., spiral organ).

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

In some embodiments of the invention, it may be advantageous to stimulate or promote regeneration or de novo development of a missing or underdeveloped tissue, organ or structure, for example, by removing skin, removing at least some tissue at a location where regeneration or de novo development is desired, abrading a joint or bone surface where regeneration or de novo development is desired, and/or causing other types of wounds on a subject. In the case of regeneration after tissue injury, it may be desirable to remove (e.g., by surgical resection or debridement) at least some of the damaged tissue. In some embodiments, the iTR factor is administered at or near the site of such removal or abrasion.

In some embodiments, the iTR factor is used to enhance regeneration of a tissue or organ in a subject that is at least partially missing in an injured subject due to an innate disorder, such as a genetic disease. Many congenital malformations result in the hypoplasia or absence of various tissues, organs, or body structures such as limbs or fingers (toes). In other cases, developmental disorders leading to underdevelopment of tissues, organs, or other bodily structures manifest postnatally. In some embodiments, the iTR factor is administered to a subject suffering from an hypoplasia or loss of a tissue, organ or other bodily structure with the aim of stimulating the growth and development of such tissue, organ or other bodily structure. In some aspects, the invention provides methods of enhancing production of a tissue, organ, or other bodily structure in a subject having an hypoplasia or loss of the tissue, organ, or other bodily structure, the method comprising administering to the subject an iTR factor. In some 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 relating to regeneration are applicable to the de novo generation of such tissues, organs, or other body structures and are encompassed within the present invention.

In some aspects, the iTR factor is used to enhance tissue production in a variety of situations where new tissue growth is useful in locations where such tissue was not previously present. For example, it is often useful in the context of fusion of bone tissue between joints in the spine or other joints.

The iTR factor can be tested in a variety of animal models of regeneration. In one aspect, the iTR modulator is tested in murine species. For example, a mouse may be wounded (e.g., by incision, amputation, transection, or removal of a tissue segment). The iTR factor is applied to the wound and/or site of removed tissue fragments and its effect on regeneration is assessed. The effect of modulators on vertebrate TR can be tested in a variety of vertebrate models for tissue or organ regeneration. For example, fin regeneration can be assessed in zebrafish, as described in the literature (horsek, innovative tissue regeneration pathway used). Rodents, canines, equines, caprines, fish, amphibians and other animal models are widely available that can be used to test the effect of treatment on the regeneration of tissues and organs such as heart, lung, limbs, skeletal muscle, bone, etc. For example, various animal models for skeletal muscle regeneration are discussed in Tissue Eng Part B rev.16(1) (2010). One common animal model for studying liver regeneration involves surgical resection of a significant portion of the rodent liver. Other models for liver regeneration include acute or chronic liver injury or liver failure due to toxins such as carbon tetrachloride. In some embodiments, the hair regeneration or skin wound healing model comprises excision of a piece of skin, e.g., from a mouse.

Hair follicle regeneration, hair growth, re-epithelialization, glandular formation, etc. can be evaluated.

Administration of the compounds and compositions disclosed herein and/or identified using the methods and/or assays described herein can be by any suitable means, such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intraarterially, parenterally, intraperitoneally, intrathecally, intratracheally, ocularly, sublingually, vaginally, rectally, transdermally, or by inhalation, e.g., as an aerosol. The particular mode selected will, of course, depend on the particular compound selected, the particular condition to be treated and the dosage required for therapeutic efficacy. In general, the methods of the invention can be practiced with any mode of administration that is medically or veterinarily acceptable, which refers to producing an acceptable level of efficacy without causing clinically unacceptable (e.g., medically or veterinarily unacceptable) side effects. Suitable preparations, e.g., substantially pure preparations, of one or more compounds can be combined with one or more pharmaceutically acceptable carriers or excipients, and the like, to produce suitable pharmaceutical compositions suitable for administration to a subject. Such pharmaceutically acceptable compositions are an aspect of the present invention. The term "pharmaceutically acceptable carrier or excipient" refers to a carrier (which term encompasses a carrier, vehicle, diluent, solvent, carrier, etc.) or excipient that does not significantly interfere with the biological activity or efficacy of one or more of the active ingredients of the composition and that is not unduly toxic to the host at the concentrations employed or administered. Other pharmaceutically acceptable ingredients may also be present in the composition. Suitable materials and their use for The preparation of pharmaceutically active compounds are well known in The art (see, e.g., "Remington's Pharmaceutical Sciences", E.W. Martin, 19 th edition, 1995, Mack publishing company, Iston, Pa., and newer versions thereof, such as Remington: The Science and Practice of Pharmacy (Remington Pharmaceutical Science and Practice, 21 st edition, Philadelphia, LWW publishers, 2005, additional discussion of pharmaceutically acceptable materials and methods of preparation of various types of Pharmaceutical compositions.) furthermore, The compounds and compositions of The present invention may be used in combination with any compound or composition in The art for The treatment of a particular disease or condition of interest.

Pharmaceutical compositions are generally formulated to be compatible with their intended route of administration. For example, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media, for example, sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, ringer's lactate. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; preservatives, for example, antibacterial agents such as 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 tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Such parenteral preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

For oral administration, the compounds can be readily formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. Suitable excipients for oral administration include fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).

For administration by inhalation, the compositions of the present invention may be delivered in the form of an aerosol spray from a pressurized container or a nebulizer with a suitable propellant (e.g., a gas such as carbon dioxide or a fluorocarbon polymer). Liquid or dry aerosols (e.g., dry powders, large porous particles, etc.) may 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 compositions may be formulated in a suitable ointment, lotion, gel, or cream in which the active ingredient is suspended or dissolved in one or more pharmaceutically acceptable carriers suitable for such compositions.

For topical delivery to the eye, the pharmaceutically acceptable compositions may be formulated as solutions or micronised suspensions in isotonic, pH adjusted sterile saline, e.g. for ophthalmic drops, or ointments, or for intraocular administration, e.g. by injection.

The pharmaceutical compositions may be formulated for transmucosal or transdermal administration. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The pharmaceutical compositions of the present invention may be formulated as suppositories (e.g., using conventional suppository bases such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

In some embodiments, the compositions include one or more agents for protecting the active agent from rapid elimination from the body, such as controlled release formulations, implants, microencapsulated delivery systems, and the like. The compositions may incorporate agents to improve stability (e.g., in the gastrointestinal tract or bloodstream) and/or enhance absorption. The compounds may be encapsulated or incorporated into particles, for example, microparticles and nanoparticles. Biodegradable, biocompatible polymers may be utilized, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, PLGA, collagen, polyorthoesters, polyethers, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. By way of example and not limitation, a variety of particle, lipid, and/or polymer-based delivery systems are known in the art for delivering siRNA. The present invention contemplates the use of such compositions. Liposomes or gas lipid-based particles can also be used as pharmaceutically acceptable carriers

Pharmaceutical compositions and compounds for use in such compositions can be prepared under conditions that comply with standards, rules, or guidelines set forth by regulatory agencies. For example, such compositions and compounds may be manufactured in accordance with Good Manufacturing Practice (GMP) and/or in compliance with quality control procedures appropriate for human pharmaceutical use, and may be provided with labeling approved by a governmental regulatory agency responsible for the administration of pharmaceuticals, surgical or other therapeutically useful products.

The pharmaceutical compositions of the present invention, when administered to a subject for therapeutic purposes, are preferably administered for a time and in an amount sufficient to treat the disease or condition to which they are administered. The therapeutic efficacy and toxicity of active agents can be assessed by standard pharmaceutical procedures in cell cultures or experimental animals. Data obtained from cell culture experiments and animal studies can be used to formulate a range of doses suitable for use in humans or other subjects. The different doses administered to humans can be further tested in clinical trials, as is known in the art. The dose used may be the maximum tolerated dose or a lower dose. The therapeutically effective dose of the active agent in the pharmaceutical composition may be in the following range: from about 0.001mg/kg to about 100mg/kg body weight, from about 0.01 to about 25mg/kg body weight, from 0.1 to about 20mg/kg body weight, from 1 to about 10mg/kg body weight. Other exemplary dosages include, for example, from about 1 μ g/kg to about 500mg/kg, from about 100 μ g/kg to about 5 mg/kg. In some embodiments, a single dose is administered, while other embodiments are administered in multiple doses. One of ordinary skill in the art will appreciate that the appropriate dosage in any particular case depends on the potency of the agent used and may optionally be adjusted for the particular recipient. The specific dosage level for a subject may depend upon a variety of factors including the activity of the particular agent employed, the particular disease or condition and its severity, the age, weight, general health of the subject, and the like. It may be desirable to formulate pharmaceutical compositions, particularly for oral or parenteral use, in unit dosage form for ease of administration and uniformity of dosage. The term "unit dosage form" as used herein refers to physically discrete units serving as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutically acceptable carrier. It is understood that a treatment regimen may include the administration of multiple doses, e.g., multiple unit dosage forms, over a period of time, which may extend over several days, weeks, months, or years. The subject may receive one or more doses a day during the treatment period, or may receive doses every other day or less frequently. For example, administration may be every two weeks, weekly, etc. Administration may be continued, for example, until the appropriate structure and/or function of the tissue or organ has at least partially recovered and/or until continued administration of the compound has not been seen to promote further regeneration or improvement. In some embodiments, the subject himself is administered one or more doses of the compositions of the present invention.

In some embodiments, two or more compounds or compositions are administered in combination, e.g., for the purpose of enhancing regeneration. The compounds or compositions for combined administration may be administered in the same composition in combination or may be administered separately. In some embodiments, "combined" administration means that, with respect to the administration of the first and second compounds or compositions, the administration is performed such that (i) the dose of the second compound is administered before more than 90% of the most recently administered dose of the first agent is metabolized to an inactive form or secreted by the body; or (ii) the first and second compounds are administered within 48, 72, 96, 120, or 168 hours of each other, or (iii) the agents are administered within coincident time periods (e.g., by continuous or intermittent infusion); or (iv) any combination of the foregoing. In some embodiments, two or more iTR factors, or a vector expressing a telomerase catalytic component, and iTR factors are administered. In some embodiments, the administration of the iTR factor is combined with one or more growth factors, growth factor receptor ligands (e.g., agonists), hormones (e.g., steroid or peptide hormones), or signaling molecules to help promote regeneration and polarization. Particularly useful are tissue center (organizing center) molecules that aid in the regeneration of tissue competent cells, such as those produced using the methods of the invention. In some embodiments, the growth factor is an epidermal growth factor family member (e.g., epidermal growth factor, neuregulin), a fibroblast growth factor (e.g., any FGF1-FGF23), a Hepatocyte Growth Factor (HGF), a nerve growth factor, a bone morphogenic protein (e.g., any BMP1-BMP7), a Vascular Endothelial Growth Factor (VEGF), a Wnt ligand, a Wnt antagonist, retinoic acid, NOTUM, follistatin, sonic hedgehog (sonic hedgehog) or other tissue center factor.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the details described in the specification or illustrated therein. The articles such as "a", "an" and "the" may mean one or more than one unless otherwise indicated herein or otherwise clearly contradicted by context. Certain of the methods of the invention are often performed using a population of cells, e.g., in vitro or in vivo. Thus, reference to "a cell" should be understood to include embodiments in which the cell is a population of cells, e.g., a population comprising or consisting of a plurality of cells that are substantially genetically identical. However, the invention encompasses embodiments in which the methods of the invention are applied to cells of an individual. Thus, reference to "a cell" should be understood to include embodiments that apply to individual cells within a population of cells and embodiments that apply to isolated cells from an individual.

Claims or descriptions that include an "or" between one or more members of a group are deemed to be satisfied when one, more or all of them are present, used or otherwise relevant in a given product or process in a group member, unless expressly stated otherwise or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The invention also includes embodiments in which multiple or all of the group members are present in, used in, or otherwise associated with a given product or process. It is contemplated that all of the embodiments described herein are applicable to all of the different aspects of the present invention. It is also contemplated that any embodiment may be freely combined with one or more other such embodiments, as appropriate. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more claims (whether original or subsequently added) is introduced into another claim (whether original or subsequently added). For example, any claim dependent on another claim may be modified to include one or more elements or limitations found in any other claim dependent on the same base claim, and any claim whose recitation of a claim is in a different claim may be modified to include one or more elements or limitations found in any other claim to which that claim is dependent on the same base claim. Further, when the claims refer to a composition, the invention provides methods of making the composition, e.g., as disclosed herein, and methods of using the composition, e.g., for the purposes disclosed herein. Where the claims refer to a method, the invention provides compositions suitable for performing the method, and methods of making the compositions. In addition, when the claims are directed to a method of making a composition, the present invention provides compositions made according to the method of the present invention and methods of using the compositions, unless otherwise indicated or unless one of ordinary skill in the art would recognize that a contradiction or inconsistency would arise. Where elements are presented in lists, such as in markush groups, each subset of these elements is also disclosed, and any element can be removed from the group. For the sake of brevity, only some of these embodiments are explicitly mentioned, but the invention encompasses all such embodiments. It will also be understood that in general, when the invention or aspects of the invention are/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist of or consist essentially of such elements, features, etc.

Where numerical ranges are recited herein, the invention includes embodiments having endpoints, embodiments in which both endpoints are excluded, and embodiments in which one of the endpoints is included and the other is excluded. Unless otherwise indicated, two endpoints should be assumed to be included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values expressed as ranges can be assumed in various embodiments of the invention to be any specific value or subrange within the stated range, to the tenth of the lower limit of the range, unless the context clearly dictates otherwise. When phrases such as "less than X", "greater than X" or "at least X" (where X is a number or percentage) are used, it is understood that any reasonable value can be selected as the lower or upper limit of the range. It will also be understood that when a list of numerical values (whether or not preceded by the word "at least") is provided herein, the invention includes embodiments that relate to any intermediate value or range defined by any two of the numerical values listed, and that the lowest value can be taken as the minimum value and the highest value can be taken as the maximum value. Further, when a list of numbers, such as a percentage, is preceded by the word "at least," the term applies to each number in the list. For any embodiment of the invention in which a numerical value is preceded by "about" or "approximately," the invention includes embodiments in which the precise value is mentioned. For any embodiment of the invention in which a numerical value is not preceded by "about" or "approximately," the invention includes embodiments in which the numerical value is preceded by "about" or "approximately. "approximately" or "about" generally includes numbers that are within 1% or, in some embodiments, 5% or, in some embodiments, 10% of any direction (greater than or less than the number) of the value unless otherwise indicated or otherwise evident from the context (e.g., when such number cannot exceed the possible value of 100% d). As used herein, a "composition" may include one or more components, unless otherwise specified. For example, a "composition comprising an activator or TR activator" may consist of or consist essentially of the TR activator or may contain one or more additional components. It is to be understood that, unless otherwise indicated, in any embodiment of the invention, the inhibitor or TR inhibitor (or other compounds mentioned herein) may be used or administered in a composition comprising one or more additional components including an activator in which the TR activator is present.

Reagent kit

Certain embodiments of the invention provide kits comprising one or more genes of figure 1 or one or more gene products encoded by the genes of figure 1. In some embodiments, the kit comprises a gene or gene product encoded by one or more genes selected from PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR, and WSB 1. In other embodiments, the kit comprises a gene or gene product encoded by one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA. In still other embodiments, the kit comprises a gene product or gene encoded by a plurality of genes selected from PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR, and WSB 1. In other embodiments, the kit comprises a gene or gene product encoded by a plurality of genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADL1, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In some embodiments, a kit may comprise one or more agents that inhibit the expression of one or more genes selected from PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR, and WSB 1. In other embodiments, the kit comprises one or more agents that inhibit the expression of one or more genes selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADLl, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA. In still other embodiments, the kit can comprise a plurality of agents that inhibit the expression of a plurality of genes selected from the group consisting of PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR, and WSB 1. In other embodiments, the kit comprises a plurality of agents that inhibit a protein selected from the group consisting of: COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100A6, MGMT, ZNF280D, DYNLT3, NAALADLl, COX7A1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, INSIG1, ACAT2 and MAOA.

In some embodiments, the kit provides an agent that binds to one or more genes or gene products encoded by one or more genes selected from the group consisting of PCDHHB2, PCDHB17, Nbla10527, RAB3IP, DLX1, DRD11P, FOXD1, LOC728755, AFF3, F2RL2, MN1, CBCAQH035, LOC791120, SIX1, OXTR, and WSB 1. In other embodiments, the kit provides reagents that bind to one or more genes or gene products encoded by one or more genes selected from COMT, TRIM4, CAT, PSMD, SHMT, LOC205251, ZNF280D, S100a6, MGMT, ZNF280D, DYNLT3, NAALADLl, COX7a1, TSPYL5, IAH1, C18orf56, RPS7, FDPS, ELOVL6, ig1, ACAT2, and MAOA. An agent that binds to one or more genes can inhibit the expression of the gene or the gene product encoded by the gene. The agent may be a protein, such as an antibody. The agent may be a nucleic acid, such as a DNA molecule, an RNA molecule, such as an mRNA molecule, an siRNA molecule, a dsRNA molecule.

The contents of the kit may be provided in one or more containers. The contents of the kit may be provided in solution, for example in a suitable buffer such as PBS or deionized water. Alternatively, the contents of the kit may be provided in a dry, e.g. lyophilized, form.

Method

In addition to the methods described below, methods for generating and using cells with embryonic pattern gene expression corresponding to scarless regeneration potential are described in the following references: PCT application No. PCT/US2006/013519, application date No. 2006, 4/11/2006, entitled "Novel Uses of Cells With Prestate Patterns of Gene Expression" (New use of Cells With a Prenatal Gene Expression Pattern) "; U.S. patent application No. 11/604, application date 2006, 11/21, entitled "Methods to Accelerate the Isolation of Novel Cell Strains from plura lity of Stem Cells and Cells associated with Thereby" (accelerated method for Isolation of Novel Cell lines from Pluripotent Stem Cells and Cells Obtained therefrom) "; and U.S. patent application No. 12/504,630, application date 2009, 16/7, entitled "Methods to accumulate the Isolation of Novel Cell Strains from Pluripotent Stem Cells and Cells associated with recovery therby," each of which is incorporated herein by reference.

Preparation of hydrogel of hyaluronic acid and collagen

HyStem-C (BioTime, Inc. (BioTime), Alamada, Calif.) was reconstituted according to the manufacturer's instructions. Briefly, HyStem ingredients (thiol-modified hyaluronic acid, 10mg) were dissolved in 1.0ml degassed deionized water for about 20 minutes to prepare a 1% w/v solution. Gelin-S ingredient (thiol-modified gelatin, 10mg) was dissolved in 1.0ml degassed deionized water to make a 1% w/v solution, while PEGDA (PEG diacrylate, 10mg) was dissolved in 0.5ml degassed deionized water to make a 2% w/v solution. Then, HyStem (1ml, 1% w/v) was mixed with Gelin-S (1ml, 1% w/v) just before use. The pelleted cells were suspended in a HyStem: Gelin-S (1:1v/v) mixture, freshly prepared as described above. Adding PEGDA as crosslinking agent to give final concentration of 2.0 × 10⁷After cell suspension of cells/ml, the cell/matrix combination is injected into the target tissue.

RNAi

As a non-limiting example, dsRNA was made via in vitro transcription reaction (Promega) using PCR generated templates, flanked by the T7 promoter, purified by phenol extraction and ethanol precipitation, and annealed after resuspension in water. Intact experimental animals were injected with 4 30nL dsRNA for 3 consecutive days after induction of tissue damage, beginning with the first injection 2 hours post-surgery.

Examples

Example 1.The iTR genes were identified by comparing gene expression of hES, iPS and cloned EP cell lines with different fetal and adult derived soma cell types.

Illumina gene expression microarray analysis was performed in different mature and embryonic cell types, including 14 different blood cell types, 115 different fetal and mature derived soma cell types from all three germ layers, different clonal hES-derived and iPS-derived EP cell lines in 545, average RFU values for each probe in 12 hES cell lines and 17 human iPS cell lines fetal/mature derived cells compared to the corresponding average in clonal EP cell lines, identifying probes with relatively uniform higher expression in one of the two groups. As shown in FIG. 1 and FIGS. 2 to 15, these genes represent factors (COX7A1) having known roles in oxidative phosphorylation in cells, and transcription factors such as SIX1 and DLX1, and other factors having various activities. RFU values below 100 are considered background signals (i.e., no detectable expression).

Example 2.Modification of the iTR gene in transgenic mice and iTR assays in mature animals. Inducing genes separately or in combination: CAT, COMT, COX7A1, DLX1, DRD1IP, LOC205251NAALADL1, PCDHB2, PCDHB17, primary neuroblastoma cDNA clones the embryonic patterns of expression of Nbl 10527, RAB3IP, SIX1, TRIM4 and ZNF280D to record the effect of these genes in cut ear lobes and other somatic tissues on improving tissue regeneration.

Example 3 in vitro assays the regeneration potential of human stromal tissue regeneration by modulating the iTR gene to up-regulate the iTR inducible gene or down-regulate the iTR suppressor gene can be tested using the in vitro wound repair assay described herein. Briefly, the scratch test described In (Nature Protocols 2,329-333(2007) Liang CC et al, "In vitro scratch assay: a continuous and extended method for analysis of cell migration In vitro") was used. The assay utilized neonatal human foreskin fibroblasts (Xgene, Sausarito, Calif.) that express COX7A1 at levels similar to those described elsewhere hereinFetal and adult derived stromal cells were comparable. Fibroblasts were grown to confluence in 6-well plates in DMEM media supplemented with 10% FBS, the plates first coated with 0.1% gelatin, and then in wet incubator with 5% O₂And 10% CO₂And (5) culturing.

The expression of the iTR suppressor gene COX7a1 was altered on day 0 using the following reagents:

SMART pool ON-TARGETplus COX7A1siRNA, 5nmol (Dharmacon, cat # L-013152-02-0005)

5nmol of ON-TARGETplus non-targeting pool (Dharmacon, cat # D-001810-10-05)

0.75mL of DharmaFECT 1 transfection reagent (Dharmacon, cat # T-2001-02)

Serum-free, antibiotic-free, basal medium

Complete medium without antibiotics

Non-targeted pool siRNAs and stock solutions (100uM) in the target (on-target) (COX7A1) siRNA pool were prepared by adding 50ul sterile water to 5 nmol. Then 5uM solution of non-targeted and on-target siRNA was prepared after thinning with sterile water. 50ul of each 5uM siRNA solution was added to 2 correspondingly labeled microcentrifuge tubes containing 450ul of serum-free medium (DMEM medium + glutamax 2mM) to give total volumes of 500ul each.

The transfection reagent was prepared by adding 110ul to 2090ul serum free medium (DMEM + glutamax 2 mM). After shaking, centrifugation and standing for 5 minutes, 500ul of transfection mixture was added to 500ul of (a) non-targeting siRNA mixture (control) and (b) target siRNA mixture. The reagents (1ml each) were mixed by pipetting up and down.

Transfection was initiated after cultured Xgene foreskin fibroblasts were aspirated to remove growth medium, washed with PBS, and 1.6ml of antibiotic-free growth medium was added to each well of the 6-well plate. 400ul of (a) transfection reagent for non-targeted siRNA control, (b) at the target transfection reagent mixture was added to give 2ml per treated well. The final siRNA concentration was 50 nM. The well plates were centrifuged to ensure uniform distribution and placed in a wet incubator at 5% O₂And 10% CO₂The culture was carried out for 6 hours.

After 6 hours, the well plates were removed from the incubator and a "scratch" was made in the center of each well with a 200ul pipette tip. The plates were then washed twice with PBS, fresh growth medium (3 ml/well) was added and returned to the incubator. D0 ("shortly after scratch"), D1 and D2 were photographed at 4x to observe cell movement, proliferation and appearance. RNA was extracted at day 2 and day 4 for subsequent qPCR analysis.

As shown in fig. 16, down-regulation of the iTR suppressor gene COX7a1 resulted in a significant improvement in three-dimensional tissue architecture regeneration, which occurred at a faster rate than control somatic cells. Samples were taken for RNA and analyzed by PCR for relative levels of ACTA2 and COL1a1 transcripts, which were thought to be involved in myofibroblast contractile responses and fibrotic scar responses, respectively, of stromal cells in the scarred wound bed. As shown in figure 17, neonatal foreskin fibroblasts respond to down-regulation of the iTR suppressor gene COX7a1 by reducing expression of ACTA2 and COL1a1, consistent with down-regulation of COX7a1 leading to enhanced regeneration of three-dimensional human tissue and, at the same time, less scarring response.

In addition to COX7a1, other iTR modulators described herein were used, alone or in combination, in place of COX7a1, to test in vitro for regeneration of three-dimensional tissue structures and to reduce fibrotic, targetable responses to tissue injury.