Congenital Muscular Dystrophy Biomarker Discovery James Collins MD, PhD Assistant Professor Division of Neurology Cincinnati Children’s Hospital Medical Center

Disclosures Research Foundation Grant: Cure Congenital Muscular Dystrophies partnered with S.A.M. (www.curecmd.org)

Biomarker “A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” Biomarkers Definition Working Group, NIH Clin Pharmacol Ther 2001;69:89-95

Biomarker Clinical practice identify risk for or diagnose a disease assess disease severity or progression predict prognosis or guide treatment http://www.biomarkersconsortium.org/index.php?option=com_content&task=view&id=132&Itemid=184

Biomarker http://www.prostateuk.org/psa/psa.htm

CMD Biomarker Discovery Liotta, et al. Nature, 2003. Taniguchi, M. et al., Biochem Biophys Res Commun, 2006. 342(2): p. 489-502.

Biomarker Drug development How does a drug work in the body Is the drug safe or effective What dose of the drug is effective Response to a treatment Treatment trial - FDA regulatory approval process http://www.biomarkersconsortium.org/index.php?option=com_content&task=view&id=132&Itemid=184

Beneficial or Harmful Effects Not Measured by a Biomarker Drug development Measured to/ Substitute for Therapeutic Intervention affects Clinical Endpoint Biomarker Beneficial or Harmful Effects Not Measured by a Biomarker Effects of therapeutic interventions on biomarkers and clinical endpoints in clinical trials. Biomarkers Definition Working Group, NIH. Clin Pharmacol Ther 2001;69:89-95

Biomarker development Discovery phase Proximal fluids, cell lines, animal models, tissue of interest candidates Qualification phase Human plasma Confirm candidate molecules Verification phase Population-based (specificity) Validation and clinical assay development Sensitivity and specificity Rifai, N., M.A. Gillette, and S.A. Carr, Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol, 2006. 24(8): p. 971-83.

Biomarker discovery Genomics Proteomics Metabolomics Imaging Relevant disease genes, expression profiles, signaling pathways Proteomics Protein expression and post-translational modifications Metabolomics small molecule metabolites specific to disease Imaging Imaging changes reflect disease state

RNA Expression Profiling of Blood from Humans Apply RNA to MICROARRAYS Whole Blood GeneSpring, Partek and NCI public software to analyze data DAVID, KEGG and others for pathways STORE -700C Isolate RNA

Cluster analysis of genes [with ≥ 2.5-fold change] blood of DMD compared to healthy age matched males) Healthy controls: note general gene expression in Lower Left generally higher than DMD subjects. DMD cluster seperates out to maybe a couple different types maybe more N=34 (14= steroids and 20=not on steroids). Those on steroids maybe on DFZ or Pred Wong, B., et al., Gene expression in blood of subjects with Duchenne muscular dystrophy. Neurogenetics, 2009. 10(2): p. 117-25.

DMD Gene expression profile Steroid treatment effect Lit L. et al., Pharmacogenomics J, 2009. 9(6): p. 411-8.

Proteomics approach http://biogratech.com/resources/From+blood+withdrawal+to+RBC+proteomics.PNG

Merosin-deficient mice models (dy/dy) muscle Gene expression profiling CMD Merosin-deficient mice models (dy/dy) muscle contractile proteins shift towards embryonic and perinatal myosin forms genes involved in cellular adhesion procollagen genes genes related to immune response and complement activation van Lunteren, E. et al., Physiol Genomics, 2006. 25(1): p. 85-95.

Gene expression profiling CMD Fukuyama-type CMD and Merosin - deficient patients expression profile in muscle up-regulation extracellular matrix and basement membrane component genes unique expression pattern dystrophin-deficient muscle Unique profile of FCMD compared to MDC1A Taniguchi, M., et al.Biochem Biophys Res Commun, 2006. 342(2): p. 489-502

Proteomics - CMD no published proteomic studies 14th international congress WMS society 2009, Geneva, Switzerland Abstract Col6a1-/- mice vs WT using 2D-DGE showed 37 proteins differentially expressed in diaphragm Bovelenta et al. NMD 2009 EM.P.5.01; doi:10.1016/j.nmd.2009.06.268

Going Forward: “Bench to Bedside” merosin deficient CMD (proteomic and gene expression profiling) CMD subtypes Verification: animal models / biobank tissues Qualification: therapeutic interventions Validation Teaming up with multiple academic centers, private and governmental agencies Biomarker correlation with natural history outcome measures, imaging, and functional mobility scales Goal: biomarker profile assay use as surrogate end point

Challenges Access to patients and samples Heterogeneity Progressive disorders defining functional endpoints Funding / incentives

Acknowledgements CMD families Carsten Bonnemann Kate Bushby Ton DeGrauw Prasad Devarajan Andrew Hershey Anne Rutkwoski Brenda Wong CMD families

References Biomarkers Definition Working Group, NIH Clin Pharmacol Ther 2001;69:89-95 Rodland K. Systems biology and biomarker discovery. Disease Markers 2010;28:195-7 http://www.biomarkersconsortium.org/index.php?option=com_content&task=view&id=132&Itemid=184 Sorani et al. Clinical and biological data integration for biomarker discovery Drug Discovery Today 2010, doi:10.1016/j.drudis.2010.06.005 Fuller, H.R., et al., Valproate and Bone Loss: iTRAQ Proteomics Show that Valproate Reduces Collagens and Osteonectin in SMA Cells. J Proteome Res, 2010. Hampel, H., et al., Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives. Nat Rev Drug Discov, 2010. 9(7): p. 560-74. Rifai, N., M.A. Gillette, and S.A. Carr, Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol, 2006. 24(8): p. 971-83. Wong, B., et al., Gene expression in blood of subjects with Duchenne muscular dystrophy. Neurogenetics, 2009. 10(2): p. 117-25. Lit, L., et al., Corticosteroid effects on blood gene expression in Duchenne muscular dystrophy. Pharmacogenomics J, 2009. 9(6): p. 411-8. van Lunteren, E., M. Moyer, and P. Leahy, Gene expression profiling of diaphragm muscle in alpha2-laminin (merosin)-deficient dy/dy dystrophic mice. Physiol Genomics, 2006. 25(1): p. 85-95 Taniguchi, M., et al., Expression profiling of muscles from Fukuyama-type congenital muscular dystrophy and laminin-alpha 2 deficient congenital muscular dystrophy; is congenital muscular dystrophy a primary fibrotic disease? Biochem Biophys Res Commun, 2006. 342(2): p. 489-502. Bovelenta et al. Gene expression and proteome profiles in Col6a1-/- mice, a model of Ullrich congenital muscular dystrophy. NMD 2009 EM.P.5.01; doi:10.1016/j.nmd.2009.06.268 Liotta, L.A., M. Ferrari, and E. Petricoin, Clinical proteomics: written in blood. Nature, 2003. 425(6961): p. 905.