Patent ID: 11866733
Assignee: UNIVERSITY OF PITTSBURGH—OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
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.