Therapeutic approaches for Duchenne muscular dystrophy Annemieke Aartsma-Rus June 26 2009
Lay-out • Introduction • Read through (PTC124) • Gene therapy • Cell therapy • Pharmaceutical therapies
• Exon skipping in separate presentation Department of Human Genetics
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Annemieke Aartsma-Rus
Dystrophin
Connective tissue
Dystrophin connects cell “skeleton� to connective tissue in muscle fibers
Cell skeleton Department of Human Genetics
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Annemieke Aartsma-Rus
Duchenne: dystrophin function lost
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Annemieke Aartsma-Rus
Dystrophin in Duchenne patients • Normal function: maintain muscle fiber stability during exercize • In DMD patients dystrophin function is lost • Patients’ muscle fibers are damaged during normal excercize • Repair system cannot keep up • Muscle tissue is lost • Muscle function is lost Department of Human Genetics
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Annemieke Aartsma-Rus
Becker: dystrophin partly functional
Connection is shorter, but functional
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Annemieke Aartsma-Rus
Dystrophin in Becker patients • Normal: maintain muscle fiber stability during exercize • In BMD patients dystrophin is partially functional • Patients’ muscle fibers are damaged less during normal excercize • Slower loss of muscle fibers and muscle function • Less severe disease Department of Human Genetics
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Annemieke Aartsma-Rus
Dystrophin gene
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Genetic code
Dystrophin RNA 1
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Dystrophin protein
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Genetic code Duchenne patients
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79 Mutation
Genetic code disrupted
Dystrophin protein translation stops halfway
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Annemieke Aartsma-Rus
Genetic code Becker patients
Mutation
1
79 Genetic code maintained
Dystrophin protein shorter, but functional Department of Human Genetics
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PTC124: Stop signal mutations
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79 Cell uses new stop signal Translation into protein stops prematurely
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Annemieke Aartsma-Rus
PTC124
PTC
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79 Cell ignores new stop signal Complete protein is made
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Annemieke Aartsma-Rus
PTC124 • New stopcodons (due to mutations) “weaker” than real stopcodons • PTC124 targets new stopcodons (not real stopcodons) • Restores dystrophin production for new stopcodon mutations • Developed by PTC therapeutics • Tested in mdx mouse Department of Human Genetics
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Annemieke Aartsma-Rus
Mdx mouse model • Natural mouse model • Stopcodon mutation exon 23 • PTC124 restored dystrophin • Cultured cells • Mice Control
Department of Human Genetics
Mdx
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Mdx + PTC124
Annemieke Aartsma-Rus
PTC124 clinical trials • PTC124 can be taken orally • Tested in healthy volunteers: safe • 1st test in patients (28 days) • Safe • Muscle biopsy: increase in dystrophin levels
• 2nd trial in patients (multicenter: Europe & USA) • Longer treatment • Effect on muscle function? Department of Human Genetics
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Annemieke Aartsma-Rus
Gene therapy • Add a functional gene to muscle cells Duchenne patients • Dystrophin protein can be made from this new gene • Applicable to ALL patients • Genes are located in nucleus of the cells • How to get the gene into (the majority of) nuclei of muscle cells? Department of Human Genetics
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Annemieke Aartsma-Rus
Gene therapy Picture from Maaike van Putten
All dark blue spots (arrow for example) are nuclei! Department of Human Genetics
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Annemieke Aartsma-Rus
Gene therapy Virus • Small organism that injects genetic information into cells • Use to deliver dystrophin gene • Adapt • Remove virus genes (pathogenic) • Add new gene (dystrophin) Department of Human Genetics
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Annemieke Aartsma-Rus
Gene therapy Which virus? • Most viruses do not infect muscle tissue • Do not divide often • Lot of connective tissue (“filters” out viruses) • Exception: adeno-associated virus (AAV) • Preference for muscle • Not pathogenic in man Department of Human Genetics
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Annemieke Aartsma-Rus
AAV • Very small (20 nm (0,00002 mm)) • Capacity: 4.500 DNA subunits • DMD gene: 2.200.000 DNA subunits (500x too big!) • Genetice code DMD gene: 14.000 subunits (3.5 times too big) • Remove parts from genetic code Î Keep only what is essential for dystrophin function Department of Human Genetics
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Annemieke Aartsma-Rus
Micro-dystrophin Microdystrophin Genetic code: 4.200 DNA subunits Fits in AAV virus particle
Very small dystrophin Has all functional elements
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Annemieke Aartsma-Rus
Micro-dystrophin • Mdx mice treated with AAV-microdystrophin • Microdystrophin detected in muscle tissues! • Improved muscle function and quality!
• Test in Duchenne dog model • Immune problems (against virus)! • AAV also induces immune response in humans Department of Human Genetics
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Annemieke Aartsma-Rus
AAV trials in humans • Safety study in Duchenne patients 2006-7, USA: local injection biceps (Mendell, Samulski, Xiao Xiao)
• Prepare for bigger trial: treat leg or arm muscles • Immune problems: • Find other AAV subtypes (there are many) that are not recognized by human immune system
• Suppress immune system • Use only DNA (Jon Wolff, Serge Braun) Department of Human Genetics
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Annemieke Aartsma-Rus
Cell therapy
Muscle stem cells
• Isolate muscle stem cells for healthy donor • Proliferate outside the body (in culture) • Transplant into patients Department of Human Genetics
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Annemieke Aartsma-Rus
Cell therapy Problem • Immune response (suppress) • Muscle stem cells do not migrate from bloodstream into muscle • Muscle stem cells do not migrate in muscle (stay close to injection area) • Multiple injections (Tremblay)
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Annemieke Aartsma-Rus
Cell therapy Other stem cells • Blood, blood vessel and adipose stem cells can become muscle cells • Can migrate from blood into muscle • At low efficiency • Mesangioblasts and CD133+ promising (Cossu/Torrente) •
Disadvantage: need to suppress immune system Department of Human Genetics
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Annemieke Aartsma-Rus
Cell therapy “Ex vivo” cell therapy • Isolate (muscle) stem cells from patients • Expand in the lab • Treat in the lab (e.g. with gene therapy) • Transplant cells back into patients • No immune response (?)
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Annemieke Aartsma-Rus
Pharmacological therapies • “Drugs” • Usual oral administration • Treat disease symptoms, not underlying cause • Examples • Corticosteroids (Prednisone) • Utrophin upregulation • Myostatin inhibition • Many more! Department of Human Genetics
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Annemieke Aartsma-Rus
Utrophin upregulation • Utrophin resembles dystrophin • Dystrophin: muscle, utrophin: nerve cells • Utrophin can take over function dystrophin
Dystrophin
Department of Human Genetics
Utrophin
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Annemieke Aartsma-Rus
Utrophin upregulation • Utrophin gene hardly used in muscle • Levels utrophin too low in muscle • Find drugs than enhance utrophin levels • High levels utrophin compensate for lack of dystrophin Utrofine
“Promoter” (volume switch for genes) Department of Human Genetics
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Annemieke Aartsma-Rus
Utrophin upregulation • Find drugs that “fit” to utrophin promoter (high throughput screening” in cell model)
• Screen thousands of drugs • Promising drugs further tested in mdx • Prepare for clinical trials (Summit PLC, Kay Davies)
Department of Human Genetics
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Annemieke Aartsma-Rus
Utrophin upregulation • Find drugs that “fit” to utrophin promoter (high throughput screening” in cell model)
• Screen thousands of drugs • Promising drugs further tested in mdx • Prepare for clinical trials (Summit PLC, Kay Davies)
Department of Human Genetics
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Annemieke Aartsma-Rus
Myostatin inhibition • Increase muscle mass (to compensate for loss of muscle tissue) • Myostatin inhibits muscle growth • Animals/humans without myostatin have more muscle
Department of Human Genetics
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Annemieke Aartsma-Rus
Myostatin inhibition • Myostatin turns off switch for muscle genes Proteins Î muscle growth
Myostatin binds protein that turns on switch No muscle growth Use antibody against myostatin Switch turned on Muscle growth Department of Human Genetics
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antibody myostatin
Annemieke Aartsma-Rus
Myostatin inhibition • Inhibit myostatin Î increase muscle mass • Clinical trial with myostatin antibodies completed in adult dystrophy patients • Safe • No increased muscle mass (too short?) • New myostatin inhibitor (Acceleron) • Outperforms antibodies in mice • Tested in healthy volunteers • Clinical trial prepared for patients (2010?) Department of Human Genetics
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Annemieke Aartsma-Rus
Summary • Lot of research to many different therapies • Some already in clinical trials • Perhaps possible to combine therapies in the future • Developing therapies takes a lot of time • National and international researchers collaborate (e.g. ALADIN, TREAT-NMD) to accelerate treatment development
Department of Human Genetics
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Annemieke Aartsma-Rus