Neural regenerating cells with alterations in DNA methylation

• US 10,316,288 B2
• Filed: 04/30/2009
• Issued: 06/11/2019
• 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
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US12/736,665
• Publication Number:
  US 20110136114A1
• Time in Patent Office: 3,694 Days
• 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 converting a marrow adherent stromal cell (MASC) to a neural regenerating cell (NRC);

the method comprising;

altering the methylation state of one or more genes in the MASC, wherein the alterations comprise;

(a) increased methylation of the PITX2, DNMT3b, IGF2R and SDF4 genes; and

(b) decreased methylation of the ROPN1L and TMEM179 genes.

2. The method of claim 1, wherein the methylation state of the PITX2, DNMT3b, IGF2R and SDF4 genes is increased by contacting the MASC with one or more fusion proteins comprising a methylation domain and a DNA-binding domain, or with one or more nucleic acids encoding a fusion protein comprising a methylation domain and a DNA-binding domain, wherein the DNA-binding domains are engineered to bind to one or more sequences in each of the PITX2, DNMT3b, IGF2R and SDF4 genes.

3. The method of claim 1, wherein the methylation state of the ROPN1L and TMEM179 genes is decreased by contacting the MASC with one or more fusion proteins comprising a demethylation domain and a DNA-binding domain, or with one or more nucleic acids encoding a fusion protein comprising a demethylation domain and a DNA-binding domain, wherein the DNA-binding domains are engineered to bind to one or more sequences in each of the RPON1L and TMEM179 genes.

4. A neural regenerating cell that is descended from a MASC 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 MASC, wherein the alterations in methylation comprise;

(i) increased methylation of the PITX2, DNMT3b, IGF2R and SDF4 genes, and

(ii) decreased methylation of the ROPN1L and TMEM179 genes; and

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

5. The neural regenerating cell of claim 4, wherein the methylation state of the PITX2, DNMT3b, IGF2R and SDF4 genes is increased by contacting the MASC with one or more fusion proteins comprising a methylation domain and a DNA-binding domain, or with one or more nucleic acids encoding a fusion protein comprising a methylation domain and a DNA-binding domain, wherein the DNA-binding domains are engineered to bind to one or more sequences in each of the PITX2, DNMT3b, IGF2R and SDF4 genes.

6. The neural regenerating cell of claim 4, wherein the methylation state of the ROPN1L and TMEM179 genes is decreased by contacting the MASC with one or more fusion proteins comprising a demethylation domain and a DNA-binding domain, or with one or more nucleic acids encoding a fusion protein comprising a demethylation domain and a DNA-binding domain, wherein the DNA-binding domains are engineered to bind to one or more sequences in each of the RPON1L and TMEM179 genes.