Assays to Detect Small Molecules that Suppress Expression of the Mutant Huntington Disease Gene

ABSTRACT
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin gene (HTT). JHU researchers approach to finding a treatment for HD is two-fold: 1) to find small molecules (that could be developed into clinically useful drugs) that can directly suppress expression of HTT by blocking activity of the HTT promoter, or 2) to find small molecules that indirectly suppress the expression of HTT by increasing activity at the HTT antisense gene (HTTAS) promoter.
 
FEATURES
JHU researchers have engineered commercially obtained HEK293 cell lines by inserting either the promoter of the huntingtin gene or the promoter of the huntingtin antisense gene, which they attached to a reporter construct. HTS design Promoter constructs.
Approach 1: HTT promoter. 1.7 kb HTT promoter region ending at ATG, fused to dual luciferase reporters (firefly, renilla).
Approach 2: HTTAS promoter. 1.5 kb extending into HTTAS exon 1, but ending before the repeat (3’ end corresponds to ~ 50 bp downstream of the HTT CAG repeat). Also fused to dual luciferase reporters (firefly, renilla).
Cell line generation and QC. Both constructs were inserted by homologous recombination into HEK293 cells at pre-exsiting Flp-sites. (Additional control cell lines were generated with no promoter or with a tet-inducible promoter.) Using a 96-well plate format, with ~104 cells/well, and a 72 hour culture, we selected clonal cell lines containing either the HTT or HTTAS promoter constructs based on high quality cell morphology, fast growth rate, comparable basal expression of luc and ren reporters, lack of effect of DMSO up 1% on growth and reporter expression, and no loss of transgene through at least P10.
 
STAGE OF DEVELOPMENT
A pilot screen of the LOPAC 1280 compound library was performed, and 17 compounds that increased HTT-AS promoter activity were chosen to be validate their effect on reducing HTT expression in HD.
 
DISEASE INDICATION
Huntington Disease

ASSOCIATED INVENTORS
Psychiatry & behavioral Sciences, School of Medicine
PI: Russell L. Margolis, M.D.
Dobrilla D. Rudnicki, Ph.D., Wang Zheng, PhD, Li Pan, PhD, Xin Sun, PhD

 
ASSOCIATED PUBLICATIONS
■ None ■
 

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