James Rusche

RepliGen Corporation

ASENT2010

Biomarker Development forHDAC Inhibitor Treatment

of Friedreich’s Ataxia

RepliGen

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

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Rationale for HDAC Inhibitors as Therapeutic

• Histone deacetylase enzymes (HDACs) deacetylate histones and arelikely involved in FXN suppression through histone deacetylation andassociated 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

1 Nat Chem Biol 2006 Oct;2(10)2 PLoS One 2008 Apr 9;3(4)3 PLoS One 2010 Jan 21;5(1)

Active geneInactive gene

HDAC

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Clinical Development Candidate: RG2833

• 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

RG2833 Dose Response (M)

FRDA PBMC incubated 48 hr with RG2833 and FXN mRNA measured by RT-QPCR ( n=7)

0 . 1 0 . 5 1 2 . 5 5 1 0

0

1

2

3

Re

lati

ve

Qu

an

tity

HistoneAcetylation

RG2833Frataxin mRNA

PK/PD in transgenic mice (KI/KI) after single sc dose

RepliGen

Biomarker Objective

• Develop and document methods for RNA, protein, and enzyme activity biomarker measures and applicable use in human dosing studies

Active geneInactive gene

HDAC

RG2833

DeacetylaseInhibition Transcription Translation

HyperacetylatedHistones

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0 10 20 30 40 500

50

100

150

0

10000

20000

30000

Time Course of Pharmacodynamic DAC MeasuresExceeds Pharmacokinetics of Drug

Drug Level and DAC Activity in Dog PBMC

% D

AC

Act

ivit

y

RG

2833 (n

g/m

L)

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Frataxin Transcript and Protein Measured in Whole Blood

Control FRDA(RQ)

ControlFRDA(ng)

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

Relative Quantity (RQ)

to Control

Protein (ng)

Re

lati

ve

Qu

an

tity

of

FXN

Tra

ns

cri

pt

*(<0.0019)

RNA Protein

Frataxin mRNA level increases in response to HDACi treatment, but only in patient cells

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Microarray Study Design Used to IdentifyDrug Response Genes

Coppola, G. et al., unpublished

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Genes Respond Similarly to RG2833in FA and Control Cells

FA

Control

Log 2

Fol

d C

hang

e

STX1A CRIP2 FXYD1 PTRFFXN

Drug Response Genes

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Dipstick Repeat Measures

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (weeks)

mABS

Frataxin Protein Level is Stable in Carriers andFA Patients

Dipstick Repeat MeasuresBox & Whisker (Median & Range)

BeRu MiRu IvCe FrCa MgCa AlRu DiRu PaCe NaCa TiCa0

20

40

60

80

Carrier Patient

mABS

1C2 1C1 2C 3C13C2 1P2 2P1P1 3P13P2

CV= 10-15%

Blood samples collected weekly for 15 weeks from 5 patients and 5 carriers

Frataxin quantitation in whole blood by Dipstick (Mitosciences)

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Summary and Conclusions

Whole cell assay for deacetylase activity in bloodobserved PK/PD relationship in animal studies

Gene Expression Targets Identifieddrug effect on FXN distinct in patient and controlsgenes altered equally in all subjects identifiedmethods for collection, transport and storage characterized

Frataxin protein measures in whole blood dipstick provides reproducible, quantitative measurelongitudinal stability of protein levels in patients and carriers

RepliGen

Acknowledgements

Heather Plasterer Matt Belmonte Andrew Cooper Shefali SharmaJamshid Eshragi Brian ShandraSteven Jones Carly Therkelson

Massimo PandolfoMyriam Rai

David Lynch

Jennifer Farmer

Mina Ruggeri

Joel Gottesfeld

TSRI The Scripps Research Institute