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Mark S. Sands, Ph.D. Washington University School of Medicine Departments of Medicine and Genetics

Multimodal Therapies for Lysosomal Storage Diseases. Mark S. Sands, Ph.D. Washington University School of Medicine Departments of Medicine and Genetics. Financial Disclosures:. Research Support/Grants: BioMarin Pharmaceuticals - M. Sands, PI

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Mark S. Sands, Ph.D. Washington University School of Medicine Departments of Medicine and Genetics

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  1. Multimodal Therapies for Lysosomal Storage Diseases Mark S. Sands, Ph.D. Washington University School of Medicine Departments of Medicine and Genetics

  2. Financial Disclosures: Research Support/Grants:BioMarin Pharmaceuticals - M. Sands, PI Consulting:Consultant - BioMarin PharmaceuticalsConsultant - LA BioMedConsultant - University of Minnesota

  3. Lysosomal Storage Diseases - Rare pediatric disorders ≥ 50 distinct diseases - Collectively: ≈1 in 5,000 live births - Inherited deficiency in a lysosomal enzyme - Intralysosomal accumulation of undegraded material - Affects many cell types - Common clinical signs: CNS involvement* Organomegally Skeletal defects Cardiac defects Auditory deficits Retinal degeneration

  4. Globoid-Cell Leukodystrophy (Krabbe Disease) - Autosomal recessive, neurodegenerative disease - Galactocerebrosidase deficiency (lysosomal enzyme) - Removes galactose from galactosylated lipids • galactosylceramide • galactosylsphingosine (psychosine, cytotoxic) - Demyelinating/Dysmyelinating disease - Progressive neuroinflammation - CNS and PNS involvement - Seizures - Death – 2-4 years of age

  5. Murine Model of Krabbe Disease (Twitcher Mouse) • Galactocerebrosidase-deficient • De/Dysmyelinating disease • CNS and PNS involvement • Rapid progression (life span ~40d) • Severe tremor • Hind limb ataxia • Progressive neuroinflammation

  6. Treatment of Krabbe Disease 1) 1st BMT performed in 1984 (Yeager et al., Science, 1984) -Life span ~80 d 2) Similar response in children (standard-of-care) 3) Refractory to nearly every treatment attempted • Stem cell transplantation • Anti-inflammatories • Anti-oxidents • Substrate reduction • Dietary intervention • CNS-directed gene therapy • etc.

  7. Krabbe Disease 1) Simple monogenic disease 2) Complicated disease presentation • Enzyme deficiency • Cytotoxic metabolite accumulation • Oligodendrocyte death • Neuroinflammation • Oxidative stress • CNS & PNS involvement • etc.

  8. Combination Therapy: Targeting Different Pathogenic Mechanisms CNS-Directed AAV-Mediated Gene Therapy + Bone Marrow Transplantation

  9. Double Combination(Rationale) Primary Genetic Defect Gene Therapy Accumulation of substrates ? ? Bone Marrow Transplant Inflammation Death and Destruction

  10. Experimental Design(AAV2/5 + BMT) 400rads radiation IV injection of GFP+ BM cells Genotype PND2 PND4 PND3 IC and IT injection of GALC-AAV2/5

  11. GALC Activity Reddy AS, et al., (2011) J. Neurosci31:9945

  12. Psychosine Reddy et al., (2011) J. Neurosci31:9945

  13. Life Span (≈130 d) (≈70 d) Reddy et al., (2011) J. Neurosci31:9945

  14. BMT-Immunomodulation Reddy et al., (2011) J. Neurosci31:9945

  15. Combination Therapy: Targeting Different Pathogenic Mechanisms CNS-Directed AAV2/5-Mediated Gene Therapy + Bone Marrow Transplantation + Substrate Reduction Therapy

  16. Triple Combination(Rationale) Primary Genetic Defect Gene Therapy Substrate Reduction Therapy Accumulation of substrates L-cycloserine Bone Marrow Transplant Inflammation Death and Destruction

  17. AAV2/5 + BMT + L-cycloserine experimental design 400rads radiation i.v. injection of GFP+ BM cells Genotype PND2 PND4 PND3 3X weekly SubQ injections of L-cycloserine (50mg/kg) i.c. and i.t. injection of GALC-AAV2/5

  18. Increased GALC Activity Hawkins-Salsbury J, et al., (2015) J Neurosci 35:6495

  19. Reduced Psychosine - Brain Hawkins-Salsbury J, et al., (2015) J Neurosci 35:6495

  20. Substrate Reduction (L-cycloserine) Hawkins-Salsbury J, et al., (2015) J Neurosci 35:6495

  21. Increased Myelin & Decreased Infiltrates Hawkins-Salsbury J, et al., (2015) J Neurosci 35:6495

  22. Life Span ~175d↑ Hawkins-Salsbury J, et al., (2015) J Neurosci 35:6495

  23. 36d Twi-/- vs. 454d Triple-Treated Twi-/- Li et al., in preparation

  24. Combination Therapy: Targeting Different Compartments Infantile Neuronal Ceroid Lipofuscinosis (Infantile Batten Disease)

  25. Infantile Batten Disease (INCL) - Palmitoyl protein thioesterase 1 (PPT1)-deficient - Accumulation of autofluorescent material - Neurodegeneration - Severe brain atrophy - Neuroinflammation - Retinal degeneration/visual deficits - Intractable Seizures

  26. Infantile Batten Disease (INCL) Vesa et al, (1995) Nature 376:584

  27. cortex HPC 0.25 mV 5 s I II III IV I II V III IV V VI VI NORMAL PPT1-/- Murine Model of INCL PPT1-deficient Autofluorescent accumulation Neurodegeneration Severe brain atrophy Spontaneous seizures Median life span ~8.5mo Neuroinflammation Gupta et al, (2001) Proc NatlAcadSci 98:13566

  28. Brain-Directed Gene Therapy for Murine INCL • Intracranial injections of AAV-PPT1 @ birth • ≥ Normal levels of PPT1 • Modest response • Median life span ≈ 10-12 mo (untreated ≈ 8.5 mo) Griffey et al., (2005) MolTher13:538 Macauley et al., (2014) J Neurosci34:13077

  29. Spinal Cord Disease In INCL *Spinal cord disease is severe and precedes the brain disease Shyng et al., (2017) ProcNatlAcadSci114:E5920

  30. Reciprocal Enzyme Distribution (Intracranial vs. Intrathecal) Shyng et al., (2017) ProcNatlAcadSci114:E5920

  31. Intracranial + Intrathecal AAV-Mediated Gene Therapy Shyng et al., (2017) ProcNatlAcadSci114:E5920

  32. Intracranial + Intrathecal AAV-Mediated Gene Therapy Shyng et al., (2017) ProcNatlAcadSci114:E5920

  33. Conclusions: • Targeting multiple pathogenic mechanisms dramatically increases therapeutic efficacy (Krabbe) a) AAV-mediated gene therapy provides a persistent source of GALC to the CNS b) BMT synergizes with AAV and decreases disease- and AAV-associated inflammation c) Additional synergy observed when SRT is combined with AAV and BMT • Targeting different CNS compartments dramatically increases therapeutic efficacy (INCL) a) Brain- and spinal cord-directed gene therapy dramatically increase efficacy

  34. Sands Lab: Darshong Lin Adarsh Reddy Jacqui Hawkins-Salsbury Yedda Li Lauren Shea Megan Griffey Shannon Macauley Charles Shyng Marie Roberts Support NIH – R01NS43205 & R01HD55461 NRSA Fellowship F31 NS056718 BDSRA Postdoctoral Fellowship Hunter’s Hope Foundation NTSAD Foundation Legacy of Angels Foundation Acknowledgements Washington University: • David Wozniak (Mouse Behavioral Core) • Mike Wong (Electroencephalography) • Daniel Ory (Mass Spec Core) King’s College - London • Catherine Kielar • Hemanth Nelvagal • Jonathan Cooper

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