James Rusche RepliGen Corporation ASENT2010

1. Biomarker Development for HDAC Inhibitor Treatment of Friedreich’s Ataxia James Rusche RepliGen Corporation ASENT2010

2. Clinical Aspects of Friedreich’s Ataxia • Occurs in Caucasian populations with a prevalence of 1 in 50,000 • 98% caused by a (GAA)n expansion in frataxin gene • Normal individuals have 5 to 30 repeats • Affected individuals have 70 to 1000 repeats • Mutation in non-coding region • Onset and course heterogeneous but most cases diagnosed between 5 and 15 years old • Important clinical features include progressive decline in coordination and strength, hypertrophic cardiomyopathy and occasional diabetes - central and peripheral targets • No approved therapy

3. HDAC Rationale for HDAC Inhibitors as Therapeutic • Histone deacetylase enzymes (HDACs) deacetylate histones and are likely involved in FXN suppression through histone deacetylation and associated heterochromatin formation • Observations ~ Histone hypoacetylation around expanded FXN allele1 ~ HDACi reversed hypoacetylation in patient cells1 ~ HDACi increased FXN expression in mouse model of FA2,3 Inactive gene Active gene 1 Nat Chem Biol 2006 Oct;2(10) 2 PLoS One 2008 Apr 9;3(4) 3 PLoS One 2010 Jan 21;5(1)

4. Clinical Development Candidate: RG2833 RG2833 Frataxin mRNA Histone Acetylation FRDA PBMC incubated 48 hr with RG2833 and FXN mRNA measured by RT-QPCR ( n=7) Relative Quantity RG2833 Dose Response (M) • Small molecule inhibitor of Class I histone deacetylases – Selectively HDAC3 (Benzamide compound class) • Preclinical development nearing completion. • Positive biochemical and functional results in animal models • Active in FA patient cells to increase frataxin mRNA and protein PK/PD in transgenic mice (KI/KI) after single sc dose

5. HDAC RG2833 Inactive gene Active gene Deacetylase Inhibition Hyperacetylated Histones Transcription Translation Biomarker Objective • Develop and document methods for RNA, protein, and enzyme activity biomarker measures and applicable use in human dosing studies

6. Time Course of Pharmacodynamic DAC Measures Exceeds Pharmacokinetics of Drug Drug Level and DAC Activity in Dog PBMC % DAC Activity RG2833 (ng/mL)

7. *(<0.0019) Relative Quantity of FXN Transcript FRDA (ng) Control FRDA (RQ) Control Frataxin Transcript and Protein Measured in Whole Blood RNA Protein Frataxin mRNA level increases in response to HDACi treatment, but only in patient cells

8. Microarray Study Design Used to Identify Drug Response Genes Coppola, G. et al., unpublished

9. FA Control Log2 Fold Change FXN STX1A CRIP2 FXYD1 PTRF Drug Response Genes Genes Respond Similarly to RG2833 in FA and Control Cells

10. 1C2 1C1 2C 3C2 3C1 1P2 1P1 2P 3P2 3P1 Frataxin Protein Level is Stable in Carriers and FA Patients Blood samples collected weekly for 15 weeks from 5 patients and 5 carriers Frataxin quantitation in whole blood by Dipstick (Mitosciences) CV= 10-15%

11. Summary and Conclusions Whole cell assay for deacetylase activity in blood observed PK/PD relationship in animal studies Gene Expression Targets Identified drug effect on FXN distinct in patient and controls genes altered equally in all subjects identified methods for collection, transport and storage characterized Frataxin protein measures in whole blood dipstick provides reproducible, quantitative measure longitudinal stability of protein levels in patients and carriers

12. David Lynch Mina Ruggeri Massimo Pandolfo Myriam Rai Jennifer Farmer TSRI The Scripps Research Institute Joel Gottesfeld Acknowledgements Heather Plasterer Matt Belmonte Andrew Cooper Shefali Sharma Jamshid Eshragi Brian Shandra Steven Jones Carly Therkelson