US 11,655,481 B2
Methods for nuclear reprogramming using synthetic transcription factors
Eytan Abraham, Potomac, MD (US); Thomas Payne, Cambridge (GB); Robert J. Young, London (GB); and Inbar Friedrich Ben Nun, Rockville, MD (US)
Assigned to LONZA WALKERSVILLE, INC., Walkersville, MD (US)
Filed by LONZA WALKERSVILLE, INC., Walkersville, MD (US)
Filed on Apr. 9, 2020, as Appl. No. 16/844,723.
Application 16/844,723 is a continuation of application No. 15/180,190, filed on Jun. 13, 2016, abandoned.
Claims priority of provisional application 62/175,111, filed on Jun. 12, 2015.
Prior Publication US 2020/0385755 A1, Dec. 10, 2020
Int. Cl. C12N 15/85 (2006.01); C12N 5/074 (2010.01); A61K 35/15 (2015.01); A61K 35/33 (2015.01); A61K 35/51 (2015.01); G01N 33/50 (2006.01); A61K 35/12 (2015.01)
CPC C12N 15/85 (2013.01) [A61K 35/12 (2013.01); A61K 35/15 (2013.01); A61K 35/33 (2013.01); A61K 35/51 (2013.01); C12N 5/0696 (2013.01); G01N 33/5073 (2013.01); C12N 2501/40 (2013.01); C12N 2501/602 (2013.01); C12N 2501/603 (2013.01); C12N 2501/604 (2013.01); C12N 2501/606 (2013.01); C12N 2501/608 (2013.01); C12N 2506/11 (2013.01); C12N 2510/00 (2013.01)] 9 Claims
1. A method of nuclear reprogramming a mammalian somatic cell, the method comprising: providing a population of mammalian somatic cells comprising an endogenous pluripotency factor gene with:
a. a first nucleic acid encoding from 2 to 7 distinct guide RNAs (gRNAs), each guide RNA comprising a DNA-binding segment and a polypeptide-binding segment, wherein the DNA-binding segment binds the promoter region of the endogenous pluripotency factor gene; and
b. a second nucleic acid encoding at least one transcriptional modulator which binds the polypeptide-binding segment of the gRNAs, wherein the transcriptional modulator comprises an enzymatically inactive Cas9 polypeptide (dCas9), wherein the dCas9 is fused to a transcriptional activation domain; and
culturing the mammalian somatic cells for a period of from about 2 to about 14 days, under conditions sufficient to (i) reprogram the mammalian somatic cell to an induced pluripotent stem cell (iPSC), and/or (ii) transdifferentiate the mammalian somatic cell to a target cell different in cell type from said mammalian somatic cell.