US RE50,635 E1
Neural regenerating cells with alterations in DNA methylation
Casey C. Case, San Mateo, CA (US)
Assigned to SanBio, Inc., Oakland, CA (US)
Appl. No. 17/339,167
Filed by SanBio, Inc., Mountain View, CA (US)
PCT Filed Apr. 30, 2009, PCT No. PCT/US2009/002664
§ 371(c)(1), (2), (4) Date Feb. 9, 2011,
PCT Pub. No. WO2009/134409, PCT Pub. Date Nov. 5, 2009.
Application 17/339,167 is a reissue of application No. 12/736,665, filed on Apr. 30, 2009, granted, now 10,316,288, issued on Jun. 11, 2019.
Claims priority of provisional application 61/125,978, filed on Apr. 30, 2008.
Int. Cl. C12N 5/079 (2010.01); C12N 5/0775 (2010.01); C12Q 1/6881 (2018.01); C12Q 1/6883 (2018.01)
CPC C12N 5/0618 (2013.01) [C12N 5/0663 (2013.01); C12Q 1/6881 (2013.01); C12Q 1/6883 (2013.01); C12N 2501/42 (2013.01); C12N 2506/1353 (2013.01); C12N 2510/00 (2013.01); C12Q 2600/156 (2013.01)] 5 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 [ ;
wherein the method does not include transfection of the MASC with a polynucleotide comprising sequences encoding a Notch intracellular domain] .
 
5. The neural regenerating cell of claim 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, ]
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 [ 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, IGFZR and SDF4 genes, and
(ii) decreased methylation of the ROPNIL 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,]
wherein the methylation state of the ROPNIL 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.