Huntington's disease (HD) is a dominant autosomal neurodegenerative disease that is caused by an expansion of CAG repeats in the HTT locus that translates into an increase of poly-glutamine (polyQ) repeat in the Huntingtin protein HTT. It is estimated that 1 in 10,000 Americans suffer from HD. Despite the fact that HTT was among the first disease-causing genes to be cloned over 20 years ago, no therapy yet exists, and HD remains an orphan disease. The discovery of any drug that can prevent, delay the onset, or prolong life will have a major impact for HD patients and their families. In order to generate a human model of HD, we used CRISPR/Cas9 genome-editing tool in human Embryonic Stem Cells (hESCs) to generate 8 distinct HD-mutant isogenic lines that are genetically identical except for the length of their CAG expansion. Global comparative transcriptome analysis in our CAG-expanded isogenic lines, allowed the discovery of a cluster of 5 genes located on the chromosome 8 that are consistently shutdown in all HD lines. We found that the same cluster is also down-regulated in human samples. The presence of this signature in vivo validates the relevance of this readout as a marker of HD. We call this cluster: ?HD-cold-spot?. Moreover, at the multi- cellular level, self-organization of these lines into human neural structures unveiled previously unrecognized tissue-specific differences, providing a unique signature of the neuropathology underlying the disease. The fact that HTT is ubiquitously expressed and has pleiotropic activity has complicated the assignment of critical pathogenic function to mutant-HTT. We make the hypothesis that an ideal therapeutic agent will rescue as many as possible of the different effects induced by mutant HTT. A successful therapeutic candidate will therefore revert the different phenotypic signatures observed in our HD lines, starting with the rescue of HD- specific transcriptomic changes, the ?HD-cold-spot? which is the focus of this Phase I, up to complex multi-cellular phenotypes arising through developmental self-organization into neural structures. The Phase I SBIR is focused on developing the initial High Throughput screening platform based on the ?HD-cold-spot?. In Phase II, we will combine this transcriptomic readout with tissue level defects observed in self-organized neural structures to screen 150 000 molecules for phenotypic rescue and discover new leads for treating HD.
Our proposal will have a transformative impact on the drug discovery landscape, demonstrating how self- organized neural structures from human embryonic stem cells can be used for drug discovery and ultimately ameliorating the life of patients by finding new therapeutics for Huntington's Disease.! !
