US 12,275,957 B2
Next generation designer liver organoids and their methods of preparation and use
Samira Kiani, Scottsdale, AZ (US); Mo R Ebrahimkhani, Scottsdale, AZ (US); Jeremy Velazquez, Pittsburgh, PA (US); Ryan LeGraw, Pittsburgh, PA (US); and Patrick Cahan, Baltimore, MD (US)
Assigned to ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY, Scottsdale, AZ (US); and The Johns Hopkins University, Baltimore, MD (US)
Appl. No. 16/973,421
Filed by ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY, Scottsdale, AZ (US); and The Johns Hopkins University, Baltimore, MD (US)
PCT Filed Jun. 10, 2019, PCT No. PCT/US2019/036410
§ 371(c)(1), (2) Date Dec. 8, 2020,
PCT Pub. No. WO2019/237124, PCT Pub. Date Dec. 12, 2019.
Claims priority of provisional application 62/775,505, filed on Dec. 5, 2018.
Claims priority of provisional application 62/682,916, filed on Jun. 9, 2018.
Prior Publication US 2021/0254012 A1, Aug. 19, 2021
Int. Cl. C12N 5/071 (2010.01); A61K 35/407 (2015.01); C12N 15/113 (2010.01); C12N 15/86 (2006.01); C12N 15/90 (2006.01); G01N 33/50 (2006.01)
CPC C12N 5/0671 (2013.01) [A61K 35/407 (2013.01); C12N 15/86 (2013.01); C12N 15/907 (2013.01); G01N 33/5014 (2013.01); G01N 33/5067 (2013.01); G01N 33/5088 (2013.01); C12N 2310/20 (2017.05); C12N 2501/15 (2013.01); C12N 2501/727 (2013.01); C12N 2506/45 (2013.01); C12N 2740/15043 (2013.01); C12N 2800/80 (2013.01)] 15 Claims
 
1. A method for producing a synthetic mature liver organoid, the method comprising the steps of
introducing into cells of a fetal liver organoid one or more lentiviral constructs comprising an inducible transgene encoding at least one transcription factor selected from ATF5, Prox1, MLXIPL1, and CREB3L3, wherein, prior to introducing the one or more lentiviral constructs, the fetal liver organoid comprises a cell population comprising at least 70% CXCR4+ cells;
inducing expression of the inducible transgene by contacting the fetal liver organoid to a small-molecule inducer of transgene expression;
transducing cells of the fetal liver organoid with one or more CRISPR cassettes comprising a nucleic acid sequence encoding dCas9 and one or more gRNAs that bind to a target site in a human CYP3A4 locus, wherein the one or more gRNAs are selected from SEQ ID NO: 2 and SEQ ID NO: 3; and
culturing the fetal liver organoid, thereby producing the synthetic mature liver organoid comprising albumin+/FGF21+/G6PC+/FXR+ hepatocytes and exhibiting CYP3A4 protein metabolizing activity, hepatic bile acid receptor FXR activity, and bile acid production.