Research Project
2519606
The goal of Oncogene Science Inc. is to discover patentable, orally active, small molecular weight compounds that promote increased fetal gamma-globin gene expression in adult red blood cells for the treatment of sickle cell disease and beta-thalassemia. We propose to perform a high throughput screen using an erythroid cell line containing a yeast artificial chromosome (YAC) tagged' with the luciferase reporter gene. The gammaA- luciferase 'tagged' human beta-globin YAC already has been engineered by homologous recombination where the luciferase reporter gene was inserted into exon 1 of the gammaA-globin gene. Luciferase is now under the transcriptional control of the gammaA promoter within the context of the entire beta-globin locus. It is our belief that this YAC system represents closely the physiological regulation of the gamma-globin gene and should increase the probability of identifying compounds which modulate the expression of the gamma-globin gene in vivo. Currently this 'tagged' YAC is being stably introduced into erythroid cell lines (K562, HEL and MEL) to generate a well characterized cell line suitable for high throughput screening. We propose to rapidly screen more than 100,000 compounds and/or fungal extracts in the erythroid cell line containing the human gamma-luci YAC, seeking compounds which selectively upregulate gamma-globin gene expression. Active compounds identified in the YAC-based screen will be prioritized by (a) specificity of transcriptional modulation relative to control genes; (b) potency and cytotoxicity; (c) effect on endogenous gamma-globin expression in k562 cells and primary human progenitor cells. Compounds identified in the high throughput screen that demonstrate more than 5 fold increase in gamma-globin gene expression in the primary progenitor cell assay with a potency of lesser than 10uM will be evaluated in vivo in gamma-luci YAC transgenic mice. In addition, selected compounds will be optimized for in vivo efficacy and safety characteristics by standard structure activity relationship (SAR) chemistry, Transgenic animal studies will performed by the Grosveld laboratory (Erasmus University, Rotterdam) and additional human primary erythroid progenitor cell assays performed by the Schechter laboratory (NIDDK, NIH).
