title: Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.
abstract: Sickle cell anemia (SCA) is a genetic disorder of the β-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.
authors.affiliation: 1Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA, USA.
authors.forename: Hu
authors.initials: H
authors.lastname: Li
authors.forename: Wensheng
authors.initials: W
authors.lastname: Xie
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Gore
authors.forename: Monica N
authors.initials: MN
authors.lastname: Montoute
authors.forename: Weilin Tiger
authors.initials: WT
authors.lastname: Bee
authors.forename: Francesca
authors.initials: F
authors.lastname: Zappacosta
authors.forename: Xin
authors.initials: X
authors.lastname: Zeng
authors.forename: Zining
authors.initials: Z
authors.lastname: Wu
authors.forename: Lorena
authors.initials: L
authors.lastname: Kallal
authors.forename: Robert S
authors.initials: RS
authors.lastname: Ames
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Pope
authors.forename: Andrew
authors.initials: A
authors.lastname: Benowitz
authors.forename: Connie L
authors.initials: CL
authors.lastname: Erickson-Miller
mesh_terms: D000755:Anemia, Sickle Cell / Q000188:drug therapy; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism; D020148:Butyric Acid / Q000494:pharmacology; D002470:Cell Survival; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000077209:Decitabine; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D044127:Epigenesis, Genetic / Q000187:drug effects; D015672:Erythroid Precursor Cells / Q000187:drug effects / Q000378:metabolism*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D061251:Primary Cell Culture; D015533:Transcriptional Activation / Q000187:drug effects*; D055545:gamma-Globins / Q000235:genetics* / Q000378:metabolism
chemical_list: D056572:Histone Deacetylase Inhibitors; D055545:gamma-Globins; D020148:Butyric Acid; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine
keywords: ELISA; decitabine; fetal hemoglobin (HbF); hydroxyurea (HU); phenotypic screen; sickle cell anemia; γ-globin