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.
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Claim 1: 1. A method for converting a marrow adherent stromal cell (MASC) to a neural regenerating cell (NRC); the method comprising:
Claim 2: 2. The method of, 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.
Claim 3: 3. The method of, 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.
Claim 4: 4. A neural regenerating cell that is descended from a MASC in vitro, wherein:
Claim 5: 5The neural regenerating cell ofA neural regenerating cell that is descended from a MASC in vitro, wherein:
Claim 6: 6The neural regenerating cell ofA neural regenerating cell that is descended from a MASC in vitro, wherein:
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This application is a 35 U.S.C. § 371 filing of PCT/US2009/002664, filed Apr. 30, 2009, PCT/US2009/002664 claims the benefit of U.S. provisional application No. 61/125,978 (filed Apr. 30, 2008), the disclosure of which is incorporated by reference in its entireties for all purposes.
Not applicable.
The instant application contains a sequence listing which has been submitted electronically in ASCII text format and is hereby incorporated by reference in its entirety. Said ASCII text copy, created on Sep. 19, 2023, is named “02_SNBIRZ00900_20230929_sequence_listing.txt” and is 3,434 bytes in size.
The present disclosure is in the fields of cellular therapy for neural disorders and epigenetic regulation of gene expression and differentiation.
Cellular differentiation is controlled, in part, by regulation of gene expression. Regulation of transcription; i.e., use of DNA as a template for the synthesis of a mRNA molecule; is one of the mechanisms by which gene expression is regulated. Transcriptional regulation of gene expression can result, for example, from alteration of chromatin structure and/or binding of transcriptional regulatory proteins to specific DNA sequences in or near the gene.
Another means by which transcriptional control of gene expression is effected is by chemical alteration of DNA. The most widely-studied aspect of this form of regulation is DNA methylation. In eukaryotic genomes, the primary form of DNA methylation is the conversion of cytosine to 5-methyl-cytosine, through the action of one of a number of cellular methyltransferases. In most cases, methylated C residues are located directly upstream of a G residue. In general, methylation of C residues in or near a gene is correlated with reduced expression of the gene. In most cases, CpG methylation is not itself the proximate cause of transcriptional repression of a gene, but appears to be a mechanism to perpetuate transcriptional repression initially mediated by gene regulatory proteins.
The frequency of CG dinucleotide sequences in the upstream regions of certain non-cell-type-specific vertebrate genes (i.e., housekeeping genes) is much higher than would be expected based on the GC content of the genome; such regions are known as CpG Islands. CpG Islands are sites at which the methylation state of the C residues can affect transcription of the associated gene. Conversely, the methylation state of C residues in a CpG island or other region associated with a particular gene can be used as a potential indicator of the transcriptional state of that gene and/or as a diagnostic marker to characterize a particular cell type. See, for example, WO 2006/094836.
Disclosed herein are cells that are capable of stimulating neural recovery and/or neural regeneration after transplantation to sites of nervous system injury or disease. In certain embodiments, the cells are descended from marrow adherent stem cells (MASCs), but have undergone alterations in the methylation status of certain genes after treatment and culture in vitro. Thus, the inventor has discovered that alteration of the methylation state of one or more genes can convert a progenitor cell into a descendent cell having neural regenerative properties not possessed by the progenitor cell.
As a result of this discovery, the present disclosure encompasses, inter alia, the following embodiments:
(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 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.
The method of embodiment 1, wherein the gene is the PITX2 gene.
The method of embodiment 1, wherein the gene is the DNMT3b gene.
The method of embodiment 1, wherein the gene is the IGF2R gene.
The method of embodiment 1, wherein the gene is the SDF4 gene.
The method of embodiment 1, wherein the gene is the ROPN1L gene.
The method of embodiment 1, wherein the gene is the TMEM179 gene.
The method of any of embodiments 1-5, wherein methylation of the gene is increased in the descendent cell.
The method of any of embodiments 1 6 or 7, wherein methylation of the gene is decreased in the descendent cell.
The method of embodiment 9, wherein the sequence C-A-T-C-G-C-C-C is converted to C-A-T-C-G-C-C-C.
The method of any of embodiments 1-10, wherein the cell is a marrow adherent stromal cell (MASC).
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.
The method of embodiment 12, wherein the gene is the PITX2 gene.
The method of embodiment 12, wherein the gene is the DNMT3b gene.
The method of embodiment 12, wherein the gene is the IGF2R gene.
The method of embodiment 12, wherein the gene is the SDF4 gene.
The method of embodiment 12, wherein the gene is the ROPN1L gene.
The method of embodiment 12, wherein the gene is the TMEM179 gene.
The method of any of embodiments 12-16, wherein methylation of the gene is increased in the descendent cell compared to the progenitor cell.
The method of any of embodiments 12 or 17 or 18, wherein methylation of the gene is decreased in the descendent cell compared to the progenitor cell.
The method of embodiment 20, wherein the sequence C-A-T-C-G-C-C-C is converted to C-A-T-C-G-C-C-C.
The method of any of embodiments 12-21, wherein the progenitor cell is a marrow adherent stromal cell (MASC).
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.
The method of embodiment 23, wherein the gene is the PITX2 gene.
The method of embodiment 23, wherein the gene is the DNMT3b gene.
The method of embodiment 23, wherein the gene is the IGF2R gene.
The method of embodiment 23, wherein the gene is the SDF4 gene.
The method of embodiment 23, wherein the gene is the ROPN1L gene.
The method of embodiment 23, wherein the gene is the TMEM179 gene.
The method of any of embodiments 23-27, wherein methylation of the gene is increased in the neural regenerating cell, compared to the progenitor cell.
The method of any of embodiments 23, 28 or 29, wherein methylation of the gene is decreased in the neural regenerating cell, compared to the progenitor cell.
The method of embodiment 31, wherein the sequence C-A-T-C-G-C-C-C is converted to C-A-T-C-G-C-C-C.
The method of any of embodiments 23-32, wherein the progenitor cell is a marrow adherent stromal cell (MASC).
The method of embodiment 23, 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
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October 14, 2025
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