NEURAL REGENERATING CELLS WITH ALTERATIONS IN DNA METHYLATION

• US 20110136114A1
• Filed: 04/30/2009
• Published: 06/09/2011
• Est. Priority Date: 04/30/2008
• Status: Active Grant

• Current Assignee: Sanbio, Inc. (SanBio Co., Ltd.)
• Original Assignee: Sanbio, Inc. (SanBio Co., Ltd.)
• Inventors: Case, Casey
• Granted Patent: US 10,316,288 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2506/1353 from bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: C12N 5/0663 Bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Disclosed herein are cells, that are descendents of marrow adherent stem cells (MASCs), capable of rescuing and/or reversing various neural disorders after transplantation into sites of central nervous system (CNS) or peripheral nervous system (PNS) injury. The cells contain alterations in the methylation state of certain genes, compared to their methylation state in MASCs. Methods of making cells capable of rescuing and/or reversing various neural disorders after transplantation into sites of CNS or PNS injury, by alteration of the methylation status of certain genes, are also provided.

Claims

1. A method for altering the methylation state of a gene in a cell, the method comprising:

(a) transfecting the cell with a polynucleotide comprising sequences encoding a Notch intracellular domain; and

(b) culturing the transfected cell such that the methylation state of a gene in the cell or in one or more descendents of the cell is altered as compared to the gene in an untransfected cell, thereby altering the methylation state of the gene.

5. The method of claim 1, wherein the gene is selected from the group consisting of PITX2, DNMT3b, IGF2R and SDF4.

6. The method of claim 1, wherein the gene is selected from the group consisting of ROPN1L and TMEM179.

7. The method of claim 1, wherein methylation of the gene is increased in the descendent cell.

8. The method of claim 1, wherein methylation of the gene is decreased in the descendent cell.

9. The method of claim 1, wherein the sequence C-A-T-C^me-G-C-C-C is converted to C-A-T-C-G-C-C-C.

10. The method of claim 1, wherein the cell is a marrow adherent stromal cell (MASC).

11. The method of claim 1, wherein the progenitor cell is a marrow adherent stromal cell (MASC).

13. The method of claim 7, wherein the methylation state of the gene is altered by contacting the progenitor cell with a fusion protein comprising a methylation domain and a DNA-binding domain, or with a nucleic acid encoding a fusion protein comprising a methylation domain and a DNA-binding domain, wherein the DNA binding domain is engineered to bind to one or more sequences in the gene.

14. The method of claim 8, wherein the methylation state of the gene is altered by contacting the progenitor cell with a fusion protein comprising a demethylation domain and a DNA-binding domain, or with a nucleic acid encoding a fusion protein comprising a demethylation domain and a DNA-binding domain, wherein the DNA binding domain is engineered to bind to one or more sequences in the gene.

21. The method of claim 1, wherein the one or more genes is/are selected from the group consisting of PITX2, ROPN1 L, DNMT3b, IGF2R, TMEM 179 and SDF4.

2. A method for making a descendent cell in which the methylation state of a gene is altered, the method comprising:

(a) transfecting a progenitor cell with a polynucleotide comprising sequences encoding a Notch intracellular domain;

(b) culturing the transfected cell; and

(c) obtaining, among the progeny of the transfected cell, one or more descendent cells in which the methylation state of the gene is altered.

3. A method for converting a progenitor cell to a neural regenerating cell, the method comprising altering the methylation state of one or more genes in the progenitor cell.

12. The method of claim 3, wherein the methylation state of the gene is altered by:

(a) transfecting the progenitor cell with a polynucleotide comprising sequences encoding a Notch intracellular domain;

(b) culturing the transfected cell; and

(c) obtaining, among the progeny of the transfected cell, one or more descendent cells in which the methylation state of the gene is altered;

wherein said descendent cell in which the methylation state of the gene is altered is a neural regenerating cell.

4. A cell that is descended from a progenitor cell through culture in vitro, wherein:

(a) the cell supports the growth and/or regeneration of neural tissue;

(b) the methylation state of one or more genes in the cell is altered compared to the progenitor cell; and

(c) during the culture in vitro, neither the progenitor cell nor any of its descendents were transfected with a polynucleotide comprising sequences encoding the Notch intracellular domain (NICD).

15. The cell of claim 4, wherein the methylation state of the gene is altered by contacting the progenitor cell with a fusion protein comprising a methylation domain and a DNA-binding domain, or with a nucleic acid encoding a fusion protein comprising a methylation domain and a DNA-binding domain, wherein the DNA binding domain is engineered to bind to one or more sequences in the gene.

16. The cell of claim 4, wherein the methylation state of the gene is altered by contacting the progenitor cell with a fusion protein comprising a demethylation domain and a DNA-binding domain, or with a nucleic acid encoding a fusion protein comprising a demethylation domain and a DNA-binding domain, wherein the DNA binding domain is engineered to bind to one or more sequences in the gene.

17. A method for identifying a neural regenerating cell, the method comprising assaying the methylation state of one or more genes in the cell, wherein a change in the methylation state of the assayed genes is indicative of a neural regenerating cell.

18. The method of claim 17, wherein the assay is for increased methylation of the one or more genes.

19. The method of claim 17, wherein the assay is for decreased methylation of the one or more genes.

20. The method of claim 17, wherein the assay is for increased methylation of one or more first genes and for decreased methylation of one or more second genes.