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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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Annemieke Aartsma-Rus


Genetic code

Dystrophin RNA 1

79

Dystrophin protein

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Annemieke Aartsma-Rus


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

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79 Genetic code maintained

Dystrophin protein shorter, but functional Department of Human Genetics

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Annemieke Aartsma-Rus


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

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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

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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

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Annemieke Aartsma-Rus


Overview of Duchenne Research Strategies Presentation by Annemieke Aartmsa-Rus, PhD