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LGMD Relative Prevalence - 2016

LGMD Relative Prevalence - 2016. LGMD NGS Panel Testing – Results. ~ 5,000 samples to date Initial yield: ~20% have a definitive diagnosis ~25% have a likely diagnosis with 1 pathogenic variant & 1 VOUS About 25% are negative

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LGMD Relative Prevalence - 2016

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  1. LGMD Relative Prevalence - 2016

  2. LGMD NGS Panel Testing – Results • ~ 5,000 samples to date • Initial yield: • ~20% have a definitive diagnosis • ~25% have a likely diagnosis with 1 pathogenic variant & 1 VOUS • About 25% are negative • Recently, received clinical information related to these 5,000 cases

  3. Recommendations Related to Diagnosis

  4. Diagnosis • Old paradigm to left was recommended as evidence based • Currently, more often employing “inverted diagnosis” • In 2017 triennial review of guideline, diagnostic algorithm component most cited for revision

  5. Single Gene vs Panel vs Exome • Diagnostic yields: • Single gene = 15-19% • Panel = 46% • Exome = ~37% • Genome = ~70-90%???

  6. Genome Cost Dropping Precipitously • 1993-2003: first genome, ~$1-3B • 2007: ~$8M • Current genome: ~$750 to run ~$1,500 total • NovaSeq technology: ~$100/genome

  7. Expanded Genetic Testing Past: Future: • Numerator testing • Tautological understanding of disease • Denominator testing • More understand the full breadth and intricacies of disorders

  8. “Whilst looking down the rabbit hole,all I saw was bunnies. But once my eyes gazed ‘bout the glen,the panoply of species I did see.”Old English Fable

  9. Viral Vector Gene Therapy

  10. Confidential • Laboratory Team • Danielle Griffin • Sarah Lewis • Kristin Heller • Ryan Johnson • Rachael Potter • Eric Pozsgai • Patricia Sondergaard • Chrystal Montgomery • Kim Shontz • Darren Murrey • Ellyn Peterson • Clinical Team • Dr. Mendell • Katie Church • Alana Mahley • Beverly Galliers • Stephanie Diemer • Kelly Lehman • Brent Yetter • Markus McColly • Zarife Sahenk • Louis Chicoine • Funding • Jain Foundation • Jesse’s Journey • Families of GFB Onlus • NIH/NIAMS • PPMD • Sarepta • NDF • Kurt and Peter Foundation

  11. Confidential Why Gene Therapy for LGMD? • Monogenic Disorders • Gene Replacement is Not Mutation Specific • Viral vectors can be delivered to muscle and heart efficiently • Muscle cells do not divide

  12. Small packaging capacity 5kb • Non-integrating • Persists as an episome in cells for years • Of over 100 AAV serotypes • Only ~6 widely used (AAV1, AAV5, AAV6, AAV8, AAV9, AAVrh74) Confidential Adeno-Associated Virus (AAV)

  13. Confidential Adeno-associated Virus (AAV) is a Delivery Vehicle Receptor Muscle cell “Mini-chromosome” Does not integrate AAV particle nuclear penetration uncoating dsDNA ssDNA

  14. Confidential Viral Genes are Removed From AAV rep cap Wild-type AAV Remove rep,cap Recombinant (r)AAV LGMD gene LGMD GENE Inserted in Plasmid Plasmid

  15. Need to Target All Skeletal Muscle for Clinical Benefit Diaphragm Cardiac Muscle – some subtypes First AAV Trials Focused on Safety in Isolated Muscles Intermediate Phase – Isolated Limb Perfusion Systemic (Intravenous) Delivery Confidential Goal for Treatment of LGMD

  16. tMCK Intron hSCGA cDNA pA • Design • Double-blind placebo controlled gene transfer of SGCA by IM injection of Extensor digitorum brevis (EDB) muscle • Extremities were randomized for treatment • First Trial Design • Double-blind placebo controlled gene transfer of SGCA by IM injection of Extensor digitorum brevis (EDB) muscle • AAV1.tMCK.SGCA vs saline (placebo) • Extremities were randomized for treatment Confidential Therapeutic Cassette 145 bp 145 bp ITR ITR

  17. Confidential LGMD2D IM Phase 1 Demonstrated ExpressionTreated EDB muscle fiber diameters were equivalent to normal, healthy patients (~50 microns) with no adverse events 6 months saline side 6 months treated side Patient-02-004 EDB Injection 6 months treated side 6 months saline side Patient-02-005 EDB Injection Patients 02-001 thru 02-003 had equivalent responses at 6 weeks and 3 months

  18. Confidential Lessons Learned from Early Trials • High binding antibody titers predispose to poor gene transfer • Account for poor muscle fiber transduction • Gene therapy candidates should be screened for neutralizing antibody titers to AAV • Laboratory studies suggest antibodies can be removed by plasmapheresis

  19. Confidential LGMD2B

  20. LGMD2B Annals of Clinical and Translational Neurology, 2(3), 256-270, March 2015 • Dual cassette viral vector gene therapy ======

  21. AAV Choice of Virus for Gene Replacement • Efficient for gene transfer • “Episomal”- does not disturb other genes • Long term persistence with single injection • AAV size limitation- 4.7 kb 6.5 kb 145 bp 792 bp 148 bp 53 bp 145 bp Mendell, J.. (2015). A Glimpse at the Therapeutic Landscape for NMD.

  22. Package the Gene in Separate Pieces 5’ Vector 3’ Vector Homologous Recombination Mendell, J.. (2015). A Glimpse at the Therapeutic Landscape for NMD.

  23. Intramuscular Delivery - Murine Dysferlin expression levels are maintained for at least 12 months Sondergaard, P. C., Griffin, D. A., Pozsgai, E. R., Johnson, R. W., Grose, W. E., Heller, K. N., ... & Sahenk, Z. (2015). AAV. Dysferlin overlap vectors restore function in dysferlinopathy animal models. Annals of clinical and translational neurology, 2(3), 256-270.

  24. Limb Perfusion - Murine Sondergaard, P. C., Griffin, D. A., Pozsgai, E. R., Johnson, R. W., Grose, W. E., Heller, K. N., ... & Sahenk, Z. (2015). AAV. Dysferlin overlap vectors restore function in dysferlinopathy animal models. Annals of clinical and translational neurology, 2(3), 256-270.

  25. Systemic Delivery - Murine Sondergaard, P. C., Griffin, D. A., Pozsgai, E. R., Johnson, R. W., Grose, W. E., Heller, K. N., ... & Sahenk, Z. (2015). AAV. Dysferlin overlap vectors restore function in dysferlinopathy animal models. Annals of clinical and translational neurology, 2(3), 256-270.

  26. Functional Improvement - Murine Sondergaard, P. C., Griffin, D. A., Pozsgai, E. R., Johnson, R. W., Grose, W. E., Heller, K. N., ... & Sahenk, Z. (2015). AAV. Dysferlin overlap vectors restore function in dysferlinopathy animal models. Annals of clinical and translational neurology, 2(3), 256-270.

  27. Non-Human Primate - Intramuscular Sondergaard, P. C., Griffin, D. A., Pozsgai, E. R., Johnson, R. W., Grose, W. E., Heller, K. N., ... & Sahenk, Z. (2015). AAV. Dysferlin overlap vectors restore function in dysferlinopathy animal models. Annals of clinical and translational neurology, 2(3), 256-270.

  28. LGMD2B Gene Therapy- Gene Replacement for Dysferlin in LGMD2B Human Phase 1 Clinical Trial started 2016 Now on 4th patient

  29. Dose escalation in 6 non-ambulatory LGMD2B patients Mendell, J. (2008). Gene Therapy for Muscular Dystrophy, A Decade of Research and Challenges.

  30. Confidential Viral Vector Gene Therapy –Systemic Delivery Pendulum Swing for Gene Therapy

  31. Confidential Systemic Therapeutic Design Principles • Systemic intravenous delivery is crucial to target all muscles • rh74 AAV chosen due to superior skeletal muscle transduction in preclinical testing • rh74 provides superior systemic delivery, including heart • Preclinical data support single administration hypothesis • Promoters optimized for desired skeletal muscle and cardiac muscle expression levels • > 20% expression as the level required for significant functional benefit • Clinical goal is > 50% expression across all muscles • Rationale being patients that are heterozygous for any of the LGMD2 genes are asymptomatic

  32. Confidential LGMD2E LGMD2E

  33. Confidential AAV.MHCK7.hSGCB Successfully Targets the Diaphragm and Myocardium in SGCB-/- MiceSystemic delivery of AAVrh.74.MHCK7.hSGCB demonstrates expression in the diaphragm and heart AAV.MHCK7.hSGCB AAV.MHCK7.hSGCB BL6 WT BSG KO Diaphragm Healthy Diseased Treated Treated AAV.MHCK7.hSGCB AAV.MHCK7.hSGCB BL6 WT BSG KO Heart Healthy Diseased Treated Treated

  34. Confidential High Levels of SGCB expression across all muscles following IV delivery

  35. Confidential BL6 WT BSG KO AAV.MHCK7.hSGCB LGMD2E Gene Therapy Reduced FibrosisSystemic delivery of AAVrh74.MHCK7.hSGCB prevents fibrosis in addition to improving muscle function in mice GAS Diaphragm Pink: Fibrosis/Scar Tissue Green: Muscle

  36. Confidential LGMD2E IV Efficacy Demonstrated PreclinicallyRestoration of SGCB expression following systemic delivery of AAVrh74.MHCK7.hSGCB restores diaphragm function in mice

  37. Confidential Reversal of scoliosis and high CK levels following IV delivery

  38. First in Human Trial for LGMD2E straight to systemic delivery Trial will begin Early 2018 Class Effect! Confidential FDA Approval Intravenous Phase I/IIa Trial

  39. Confidential Timeline of AAV Gene Therapy for MD (Nationwide Children’s Hospital)

  40. Reason for Optimism – • Safety evidence of AAV continues to grow • AAV trials moving beyond the goal of just safety • Systemic delivery holds great promise • Active research regarding re-dosing and removing antibodies in pre-immune subjects Confidential Viral Vector Gene Therapy: Conclusions

  41. The Future in Genetic Therapies… • Limb regeneration • Patient and war time limb loss… • Herpetologists have now cracked the keys to molecular mechanisms • ? translatable to humans… Nature, 533, 407-422, May2016

  42. Matthew.wicklund@ucdenver.edu

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