US 11,866,733 B2
Human induced pluripotent stem cells for high efficiency genetic engineering
Alejandro Soto-Gutierrez, Pittsburgh, PA (US); Alexandra Sylvie Collin de l'Hortet, Pittsburgh, PA (US); Kan Handa, Pittsburgh, PA (US); Jorge Guzman Lepe, Pittsburgh, PA (US); Yang Wang, Pittsburgh, PA (US); Kazuki Takeishi, Pittsburgh, PA (US); and Ira Jacob Fox, Pittsburgh, PA (US)
Assigned to University of Pittsburgh—Of the Commonwealth System of Higher Education, Pittsburgh, PA (US)
Appl. No. 16/322,087
Filed by University of Pittsburgh—Of the Commonwealth System of Higher Education, Pittsburgh, PA (US)
PCT Filed Jul. 31, 2017, PCT No. PCT/US2017/044719
§ 371(c)(1), (2) Date Jan. 30, 2019,
PCT Pub. No. WO2018/026723, PCT Pub. Date Feb. 8, 2018.
Claims priority of provisional application 62/369,698, filed on Aug. 1, 2016.
Prior Publication US 2019/0185819 A1, Jun. 20, 2019
Int. Cl. C12N 5/074 (2010.01); C12N 15/11 (2006.01); C12N 9/22 (2006.01)
CPC C12N 5/0696 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 15/111 (2013.01); C12N 2310/20 (2017.05); C12N 2320/12 (2013.01); C12N 2330/51 (2013.01); C12N 2501/602 (2013.01); C12N 2501/603 (2013.01); C12N 2501/604 (2013.01); C12N 2501/608 (2013.01); C12N 2506/1307 (2013.01); C12N 2510/00 (2013.01)] 12 Claims
 
1. An in vitro method of producing a genetically modified human hepatocyte from induced pluripotent stem cells (iPSCs), comprising,
(i) transfecting human somatic fibroblast cells with a lentiviral vector comprising an antibiotic selection cassette and a nucleic acid molecule comprising a) a doxycycline inducible promoter comprising a tetracycline responsive element operably linked to a nucleic acid encoding a Cas9, and b) a constitutive promoter operably linked to a tetracycline transactivator, wherein the tetracycline transactivator binds the tetracycline responsive element in the presence of doxycycline, to produce transfected human fibroblast cells;
(ii) culturing the transfected human fibroblast cells in the presence of the antibiotic for about three weeks to produce a population of stably transfected human fibroblast cells;
(iii) transfecting the stably transfected human fibroblast cells with at least four of: a nucleic acid molecule encoding Klf4, a nucleic acid encoding c-Myc, a nucleic acid encoding Oct4, a nucleic acid encoding Sox2, a nucleic acid encoding Nanog, a nucleic acid encoding Lin28, to produce the human iPSCs;
(iv) culturing the human iPSCs in the presence of the doxycycline to produce human iPSC expressing Cas9, wherein more than 50% of the human iPSCs express the Cas9 upon the exposure to the doxycycline;
(v) differentiating the human iPSCs expressing Cas9 into hepatocytes that express Cas9;
(vi) introducing a heterologous promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas short guide RNAs (sgRNAs) that hybridize with a target gene into the hepatocytes of step (v); and
(vii) culturing the hepatocytes of step (vi) in the presence of doxycycline, thereby producing a genetically modified human hepatocyte.