	US 12,228,571 B2
	Cell-based assay for measuring drug product potency
Brian Kaspar, New Albany, OH (US); Allan Kaspar, Carlsbad, CA (US); Kevin Foust, Poway, CA (US); Martin Fugere, San Diego, CA (US); Eunhye Park, San Diego, CA (US); Gretchen Thomsen, San Diego, CA (US); and Fengrong Zuo, San Diego, CA (US)
Assigned to Novartis AG, Basel (CH)
Appl. No. 16/972,956
Filed by Novartis AG, Basel (CH)
PCT Filed Jun. 7, 2019, PCT No. PCT/US2019/035963 § 371(c)(1), (2) Date Dec. 7, 2020, PCT Pub. No. WO2019/236949, PCT Pub. Date Dec. 12, 2019.
Claims priority of provisional application 62/682,263, filed on Jun. 8, 2018.
Prior Publication US 2021/0255185 A1, Aug. 19, 2021
Int. Cl. G01N 33/569 (2006.01); A61K 48/00 (2006.01); C12N 5/0793 (2010.01); C12N 15/52 (2006.01); C12N 15/63 (2006.01); C12N 15/86 (2006.01); C12N 15/87 (2006.01); G01N 1/30 (2006.01)

CPC G01N 33/56966 (2013.01) [A61K 48/005 (2013.01); C12N 5/0619 (2013.01); C12N 15/52 (2013.01); C12N 15/63 (2013.01); C12N 15/86 (2013.01); C12N 15/87 (2013.01); G01N 1/30 (2013.01); A61K 48/00 (2013.01); C12N 2501/11 (2013.01); C12N 2501/115 (2013.01); C12N 2750/14143 (2013.01)]

24 Claims

1. An in vitro method for measuring transgene expression, comprising:

a) providing a first plurality of terminally differentiated neural progenitor cells (NPCs) comprising a homozygous Survival Motor Neuron (SMN1)−/− mutation;

b) transducing the first plurality of terminally differentiated NPCs with a test sample comprising an AAV9 viral vector comprising a sequence encoding a Survival Motor Neuron (SMN1) protein;

c) incubating the transduced first plurality of terminally differentiated NPCs under conditions sufficient to express the SMN1 protein;

d) contacting the first plurality of terminally differentiated NPCs from (c) with a molecule specific for the SMN1 protein;

e) imaging the first plurality of terminally differentiated NPCs to obtain an integrated fluorescent intensity per cell (IFI-C) assay readout; and

f) determining the expression of the SMN1 protein based on the IFI-C readout,

wherein said first plurality of terminally differentiated NPCs is produced by terminally differentiating neural progenitor cells isolated from the cortex of an SMN1−/− mouse embryo, and wherein the SMN1−/− mutation comprises a deletion of SMN1 exon 7 (Δ7).