US 12,247,226 B2
Cellular reprogramming using temporal and transient plasmid vector expression system
Bahram Valamehr, San Diego, CA (US); and Megan Robinson, San Diego, CA (US)
Assigned to FATE THERAPEUTICS, INC., San Diego, CA (US)
Appl. No. 16/649,084
Filed by FATE THERAPEUTICS, INC., San Diego, CA (US)
PCT Filed Oct. 10, 2018, PCT No. PCT/US2018/055208
§ 371(c)(1), (2) Date Mar. 19, 2020,
PCT Pub. No. WO2019/075057, PCT Pub. Date Apr. 18, 2019.
Claims priority of provisional application 62/571,105, filed on Oct. 11, 2017.
Prior Publication US 2020/0270581 A1, Aug. 27, 2020
Int. Cl. C12N 5/074 (2010.01); A61K 35/12 (2015.01); C12N 15/85 (2006.01)
CPC C12N 5/0696 (2013.01) [A61K 35/12 (2013.01); C12N 15/85 (2013.01); C12N 2800/10 (2013.01)] 39 Claims
 
1. A method of reprogramming non-pluripotent cells to generate pluripotent cells, a cell line or a population thereof, comprising:
(a) introducing to first cells, wherein the first cells are non-pluripotent cells:
one or more first plasmids, wherein each of the one or more first plasmids comprises a replication origin comprising an Epstein-Barr nuclear antigen (EBNA) binding site, and a polynucleotide encoding one or more reprogramming factors but does not encode an EBNA; wherein the one or more first plasmids comprises polynucleotides encoding reprogramming factor(s) comprising any combination of OCT4, SOX2, NANOG, KLF, LIN28, c-MYC, ECAT1, UTF1, ESRRB, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, and L1TD1; wherein introduction of the one or more first plasmids induces a reprogramming process; and
a second plasmid comprising a nucleotide sequence encoding an EBNA, wherein the second plasmid does not comprise a replication origin comprising an EBNA binding site or polynucleotide(s) encoding reprogramming factor(s);
(b) culturing the cells from step (a) to generate second cells, wherein the second cells are reprogramming cells, wherein the reprogramming cells comprise a morphological change from the first cells and are essentially free of EBNA; and wherein the reprogramming cells do not comprise:
(1) pluripotent cell morphology; and
(2) endogenous OCT4 expression; and,
(c) further culturing the second cells obtained in step (b) for a sufficient amount of time to generate pluripotent cells;
wherein the method does not comprise introducing a third plasmid that comprises both of (1) a replication origin comprising an EBNA binding site, and (2) a polynucleotide encoding an EBNA; and
wherein the pluripotent cells exhibit a reduced rate of spontaneous differentiation as compared to reprogramming non-pluripotent cells with the one or more first plasmids, the second plasmid, and the third plasmid.