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
RepliGen
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
RepliGen
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
RepliGen
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)
RepliGen
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
RepliGen
Microarray Study Design Used to IdentifyDrug Response Genes
Coppola, G. et al., unpublished
RepliGen
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
RepliGen
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)
RepliGen
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
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