-based HTS Platforms for PD, ALS, and HD

The J. David Gladstone Institutes, University of , San INVENTORS Francisco Gladstone Center for Translational Research Steven Finkbeiner 1650 Owens Street, Gaia Skibinski , CA 94158 Siddhartha Mitra [email protected] Sami Barmada Tel. (415) 734-2018; Fax. (415) 355-0930

Gladstone Docket No TBD UMB Docket No TBD

SUMMARY Cell-based screening is a mainstay of the drug discovery process. However, there are major limitations of conventional cell-based screens for the treatments of neurological disorders. Myriad cellular, molecular, and physiologic differences exist between immortalized cell lines and the that they are meant to model. We have developed faithful and robust models of Parkinson’s disease, amyotrophic lateral sclerosis, , and Huntington’s disease with primary neurons. Recently, we have begun to use induced pluripotent stem cells (iPSCs) differentiated into human neurons to create even more relevant cell-based models of disease. Together with our patented longitudinal imaging and analysis system (Arrasate and Finkbeiner, 2005; Daub et al., 2009), these models form powerful and flexible cell-based HTS platforms to detect small molecule modifiers of various disease phenotypes.

APPLICATIONS ♦ Novel methods of drug discovery for human CNS diseases that can be modeled in vitro. ♦ Highly quantitative and sensitive (100-1000 fold over conventional approaches) assays facilitates structure-activity and toxicity analysis.

ADVANTAGES ♦ The invention relates to the establishment and utility of primary neuron models of neurodegenerative disease. Although pharmaceutical companies are eager to develop therapies for neurodegenerative disease, the HTS conducted in industry are typically performed in cell lines that lack key features of neurons. As such, the availability of relevant neuron models of disease would have the potential to be lead to the discovery of novel small molecule or biological hits from primary screens that might have been missed in cell lines. ♦ The use of primary neuron and human iPS-based screening systems, because they are more relevant, may be more predictive of performance in clinicals, which would reduce significantly drug development costs.

Non Confidential Disclosure 5h August 2010 BACKGROUND OF INVENTION

Cell-based screens are a staple of many drug discovery and development programs. Cell- based screens are often used to find small molecules that have desirable effects on specific biological pathways. Faithful cell-based models of disease can be used to directly identify compounds with therapeutic potential.

It is widely acknowledged that the two biggest obstacles to the development of therapies for neurodegenerative diseases are (1) the need for better laboratory models to discover the underlying causes of disease and to test therapies and (2) the high cost of clinical trials. Our laboratory has focused on the development of faithful cell models of major neurodegenerative disease with properties suitable for use in high-throughput screens.

A critical technology that has enabled the development of these model systems is our patented robotic microscope (US 7,139,415). The system is capable of collecting images of live cells in a high-throughput manner, similar to many commercially available systems. However, it has been programmed to give it the unique power to return to precisely the same cell for as often and as long as the experimenter wants, even if the plate containing the cells has been removed from the microscope stage. Programs have been written to orchestrate fully automated image acquisition and analysis. The software programs we have written automatically stitch images of microscope fields from the same well of a micro-titer plate together, organize the montages temporally, and then look through the images and assign each cell a “social security number” for its lifetime. Fluorescent reporter genes are used to detect the levels and conformation of specific- disease-causing proteins, the activity of relevant biological pathways, and the timing of . These data sets are then analyzed in an automated fashion with the same statistical tools that are used in clinical studies. The platform makes it possible to detect neurodegeneration and small molecule modifiers quantitatively and with unprecedented sensitivity.

STAGE OF DEVELOPMENT The program has successfully established primary neuron models of several major neurodegenerative diseases.

The first model was established for Huntington’s disease (Arrasate et al., 2004; Miller et al., 2010; Saudou et al., 1998). The current model involves growing neurons from regions of the brain that are susceptible in HD and introducing into them version of human huntingtin that are normal or that are associated with disease. It recapitulates 18 features of the human disease, including the formation of abnormal protein deposits called and polyglutamine-dependent death. In over a half-dozen examples, discoveries were made first with the HD model that were later confirmed in vivo or in HD tissue, indicating that the model provides physiologically relevant data. The model has been in use for over a decade and is being partnered non-exclusively as a platform to screen proprietary libraries for therapeutics. The longitudinal nature of our robotic microscope technology confers tremendous robustness to the assay. We have measured Z´ for our assay and it has >0.9, which is outstanding for a cell-based assay. We also have

Non Confidential Disclosure 5h August 2010 a panel of induced pluripotent stem cell lines derived from HD patients and have developed protocols to differentiate them into primary neurons. These are being used develop a human neuron model of HD.

Recently, we have developed a model of ALS/FTD based on TDP43. Specific mutations in TDP43 have been associated with ALS. Some of these families also have features of FTD. Abnormal accumulations of TDP43 have also been detected in many sporadic AD cases and are associated with a worse outcome, suggesting TDP43 may also have a role in AD. Using four disease-associated familial mutants of TDP43, we established a model of ALS/FTD in primary cortical neurons (Barmada et al., 2010). The model recapitulates key features of FTD/ALS including abnormal protein deposits and mutation-specific neurodegeneration. The model was used to demonstrate that disease-causing mutations cause TDP43 mislocalization and that neurodegeneration can be prevented by correcting mislocalization. The model is being partnered non-exclusively as a platform to screen proprietary libraries for therapeutics.

We have developed two models of PD. One is based on overexpression of synuclein, the protein found in Lewy Bodies, the characteristic protein deposit seen in the brains of people with sporadic PD. The synuclein model manifests dose-dependent neurodegeneration, which parallels observations seen in people with gene duplication or triplication. The model also manifests mitochondrial phenotypes that predict degeneration. The second PD model we have established is based on familial mutations in leucine-rich related kinase 2 (LRRK2), the most common genetic cause of PD. This model recapitulates abnormal deposits of misfolded protein and mutation-specific neurodegeneration seen in vivo. Both PD models have been used to discover and validate genetic suppressors of neurodegeneration and to identify and validate potential therapeutics targets.

PROPOSED R&D No additional R&D is required, although we continued to develop the microscope technology.

LICENSING POTENTIAL Gladstone seeks to develop and commercialize by an exclusive or non-exclusive license agreement and/or sponsored research with a company active in the area.

PATENT STATUS ♦ Invention disclosure filed.

Arrasate, M., and S. Finkbeiner. 2005. Automated microscope system for determining factors that predict neuronal fate. Proc. Natl. Acad. Sci. U.S.A. 102:3840–3845. Arrasate, M., S. Mitra, E.S. Schweitzer, M.R. Segal, and S. Finkbeiner. 2004. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature. 431:805–810.

Non Confidential Disclosure 5h August 2010 Barmada, S.J., G. Skibinski, E. Korb, E.J. Rao, J.Y. Wu, and S. Finkbeiner. 2010. Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. J. Neurosci. 30:639–649. Daub, A., P. Sharma, and S. Finkbeiner. 2009. High-content screening of primary neurons: Ready for prime time. Curr. Opin. Neurobiol. 19:537–543. Miller, J., M. Arrasate, B.A. Shaby, S. Mitra, E. Masliah, and S. Finkbeiner. 2010. Quanitative relationships between huntingtin levels, polyglutamine length, inclusion body formation, and neuronal death provide novel insight into Huntington‘s disease molecular pathogenesis. J. Neurosci. 30:In press. Saudou, F., S. Finkbeiner, D. Devys, and M.E. Greenberg. 1998. Huntingtin acts in the nucleus to induce apoptosis, but death does not correlate with the formation of intranuclear inclusions. Cell. 95:55–66.

Non Confidential Disclosure 5h August 2010