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US 10,370,646 B2Granted
Generation of human iPS cells by a synthetic self-replicative RNA
AssigneeThe Regents of the University of California
InventorsSteven F. Dowdy, Naohisa Yoshioka
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Abstract
The disclosure provides methods and compositions useful for obtaining induced stem cells, methods of making and use thereof.
Claims20
A method comprising:
- contacting a somatic cell with an ectopic self-replicating RNA replicon comprising a plurality of non-structural replicase domains from an alphavirus and comprising polynucleotides encoding at least four reprogramming factors (RFs) selected from the group consisting of a (i) KLF4, (ii) OCT4, (iii) SOX2, (iv) c-MYC or n-MYC or L-MYC, (v) GLIS1 and (vi) NANOG, wherein the RNA replicon comprises from 5′ to 3′:
- (i) polynucleotide sequences encoding the plurality of non-structural replicase domains from an alphavirus;
- (ii) a promoter;
- (iii) RF1;
- (iv) a coding sequence for a first self-cleaving peptide;
- (v) RF2;
- (vi) a coding sequence for a second self-cleaving peptide;
- (vii) RF3;
- (viii) an IRES;
- (ix) RF4;
- (x) an optional IRES or an optional promoter;
- (xi) an optional sequence encoding an optional selectable marker;
- and (xii) an alphavirus 3′ UTR and polyA tail,
- culturing the somatic cell to express the at least four reprogramming factors (RFs);
- selecting cells that display a pluripotent stem cell morphology and/or pluripotent stem cell markers;
- and
- subculturing the cells to obtain a population of induced pluripotent stem cells.
14 more elements
Dependencies are inferred from the claim text.