Understanding Gene and Cell Therapy Approaches for DMD

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Understanding Gene and Cell Therapy Approaches
for DMD November 6 2015


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Overview

• What does dystrophin do?
• What happens when there is no dystrophin?
• How does drug development work
• What genetic approaches are in development to
  replace dystrophin?
• How do they work
• Challenges/opportunities

Annemieke Aartsma-Rus
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Some basic biology genes proteins

• Proteins are building blocks of our body
• Genes contain blueprint for proteins
• Mistake in gene ? mistake in protein
• Genes have a volume switch (protein only
  produced in proper tissue)
• Dystrophin protein has a function in muscle
• Mistake in dystrophin ? Problems in muscle

Annemieke Aartsma-Rus
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Muscles

• 30-40 of our body is muscle
• gt750 different muscles
• Muscles can grow bigger or smaller
• Muscles use a lot of energy
• Only maintained when needed
• Muscles are damaged when used too much
• Muscles have efficient system to repair damage
  and prevent future damage (grow bigger)

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Muscle contraction
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Muscle fibers
Skeleton
Connective tissue
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Dystrophin

• Dystrophin provides stability to muscle fibers
  during contraction
• Connects skeleton of muscle fibers to
  connective tissues surrounding muscle fibers
• No dystrophin ? Connection lost
• Muscle more sensitive to damage
• Chronic damage repair system cannot keep up
• Loss of muscle tissue and function

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Dystrophin
Dystrophin
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Duchenne no functional dystrophin
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What happens without dystrophin
Muscle damage
Less blood flow
Too much Ca2
Inflammation
Repair
Oxidative stress
Mitochondria damaged
Fibrosis
Loss of muscle tissue Loss of muscle function
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What are muscle cross sections?
Muscle cross sections
HE staining Fibers pink Nuclei blue
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Dystrophin staining
Dystrophin immune staining
Healthy
Mdx mouse
Duchenne
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Revertant fibers trace amounts
Revertant Fibers
Trace amounts
Untreated DMD muscle
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Western blotting

• Western blot
• Proteins isolated from muscle
• Separated by size
• Stain for dystrophin

DMD
Becker (dilutions)
Control (diluted)
Long Short
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Therapy development

• Dystrophin is missing
• Trying to replace dystrophin
• Also many other therapies aiming at improving
  muscle quality/slowing down disease processes

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What are cell models

• Cells derived from patients
• Muscle biopsies
• Skin biopsies ? converted into muscle cells
  (lab)
• Expanded in the lab (limited!!)
• Immortalized cells
• Muscle stem cells treated with viruses
• Keep expanding, but have been modified
• Cell cultures are model systems
• Valuable tool for early stages of research

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What are mdx mice?

• Mdx mouse
• Mutation in mouse dystrophin gene
• No dystrophin protein
• But.disease not very severe
• Very efficient muscle regeneration
• Turns up volume switch utrophin gene in muscle
• No dsytrophin utrophin severe disease
• (Double knockout mouse)

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Other animal models

• Golden retriever muscular dystrophy (GRMD)
• Mutation in dog dystrophin gene
• No dystrophin protein
• Mutation beginning gene
• Severe disease (muscle heart)
• Muscle weakness, remain ambulant
• Most dogs do not live gt year
• Severity is variable
• Rare mild individuals

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Other animal models

• Pig model
• Mutation in pig dystrophin gene
• No dystrophin protein
• Deletion exon 52
• Severe disease (muscle heart)

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Drug testing in patients

• Test compound properties
• Taken up by tissues efficiently?
• How quickly cleared from the body?
• Test compound for efficacy
• Does it work?
• At which dose?
• Test compound for safety
• Are there side effects?
• At which dose?
• Are they tolerable?

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Development of therapies

• Tests from cell and animal models to clinical
  trials
• All steps are important to show
  proof-of-concept (does it work in a model system
  ?)
• Next steps are always more complicated
• Success in one step is no guarantee for success
  in subsequent steps
• Clinical trials are experiments in humans
• May not work, may not be safe

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Cell therapy
Muscle stem cells

• Isolate muscle stem cells from healthy donor
• Expand outside the body (culture in lab)
• Transplant into patients
• Transplanted cells repair muscle
• Transplanted cells make dystrophin

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Muscle stem cells (myoblasts)

• Immune response (suppress)
• Do not exit circulation after injection
• Local injection stay close to injection site
• Tremblay (Canada) multiple local injections
• Local dystrophin restoration
• Not feasible for larger muscles

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Stem cell therapy

• Stem cells from fat, bone and bloodvessel walls
• Can exit bloodstream and migrate into muscle
• Very low efficiency
• Mesangioblasts most promising
• Encouraging results in dog model
• Safety trial ongoing in Italy (Giulio Cossu)
• 3 patients received stem cells
• Some side effects
• Preparing for injection 2 more patients

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Niche
Muscle damage
Inflammation
Repair
Fibrosis

• Dystrophic muscle is damaged (scar
  tissue/fibrosis)
• The few transplanted stem cells that reach muscle
  
• Do not receive proper signals to become muscle
• Receive signals from scar tissue more fibrosis

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Immunity

• Transplanting cells from one person to another
  will elicit an immune response
• Need chronic immune suppression
• Side effects
• Isolate patient stem cells, expand in the lab,
  correct mutation with gene therapy transplant
• No immune response
• Gene therapy more efficient in cultured cells
  than in muscles

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Cell therapy summary

• Opportunities
• Applicable to all patients
• Deliver dystrophin gene and repair muscle
• Currently in very early stage clinical
  development
• Challenges
• Efficiency very low
• Damaged muscle gives wrong signals to cells
• Immunity (only with allogenic transplantation

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Gene Therapy

• Add functional gene to muscle cells patients
• Dystrophin protein made from new gene
• Applicable to ALL patients
• Genes located in nucleus cells
• How to get gene into (majority) nuclei of
  muscle cells?

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Gene Therapy
Maaike van Putten
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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)

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Gene Therapy

• Which virus?
• Most viruses do not infect muscle tissue
• Muscle cells do not divide often
• Lot of connective tissue (filters out viruses)
• Exception adeno-associated virus (AAV)
• Preference for muscle
• Not pathogenic in man

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Gene Therapy

• Very small (20 nm, 0.00002 mm)
• Capacity 4.500 DNA subunits
• Dystrophin gene 2.200.000 DNA subunits
• Genetic code gene 14.000 subunits
• Remove part from genetic code
• Only essential parts remain

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Gene Therapy
Microdystrophin Only crucial domains Fits in AAV
particle
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Gene Therapy

• Clinical trials
• Safety study in 6 Duchenne patients
• 2006/7, USA local injection biceps
  (Mendell,Samulski, Xiao Xiao)
• Immune response!
• Dystrophin in 2/6 patients (very low levels)
• Prepare for bigger trial (whole muscle
  treatment)

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Upscaling

• Mouse 12 gram muscle
• Monkey 4 kg muscle
• Human boy 10-25 kg muscle
• Monkeys and humans much larger than mice
• Need much more viruses
• Manufacturing systems optimized to allow
  production of sufficient amounts for treating
  human limbs at clinical grade

333x
2-6x
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Delivery

• Whole animal delivery possible for mouse
• Not feasible (yet) for large animals
• Limited by amount of virus
• Produced
• Injected
• Whole limb delivery in development for human
• Hydrodynamic limb perfusion (most efficient)
• Regional limb perfusion (less damage)

Annemieke Aartsma-Rus
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Limb perfusion

• Tested in monkeys and dogs with color gene
• Delivery to multiple muscles feasible
• Tested in adult MD patients with saline
• Possible for lower leg or arm (less efficient)
• Not yet tested in humans to deliver gene

Annemieke Aartsma-Rus
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Gene Therapy Summary

• Opportunities
• Applicable to all patients
• Currently in early clincial development
  (safety/tolerability tests)
• Challenges
• Microdystrophin only partially functional
• Delivery
• Immunity

Annemieke Aartsma-Rus
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Gene/cell therapy DNA editing

• DNA has a repair system
• Activated upon DNA damage
• Use this system to correct for DNA mistakes?

Mutation
Template with correct DNA information
DNA repair system
Mistake corrected (in one cell)
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DNA editing

• Challenge DNA repair system very inefficient
  (1 in 1,000,000 1,000,000,000 cells)
• Much more efficient when DNA is broken
  (1 in 10-1000 cells)
• ? Have to generate DNA breaks at/close to mutation

Annemieke Aartsma-Rus
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DNA scissor system

• DNA scissors can cut DNA at specific location

Scissor cuts at/near mutation
Repair break with template (Correct small
mutations)
Repair break without template Small mutations
will be introduced (can correct genetic code like
exon skipping)
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DNA scissor system
Combination of DNA scissors to restore genetic
code
Scissors cut around exon ? break is repaired
Exon is deleted ? Genetic code restored (like
exon skipping)
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DNA scissor system

• DNA scissors can make breaks in DNA
• Different types of scissors in development
• Zinc Fingers, TALENs and RGNs
• Challenge deliver scissors and templates to
  muscles

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Exon skipping
Normal
Duchenne
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Dystrophin gene
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Splicing
Exons
Introns
6
3
2
1
5
7
Gene (DNA)
Splicing
messenger RNA
1
2
3
4
5
6
7
8
1 - - - - - - - - - 79
dystrophin protein
RNA copy (pre mRNA)
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Duchenne genetic code disrupted
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Duchenne genetic code disrupted
?
Exon 46
Exon 47
Exon 51
Exon 52
Protein translation stops prematurely
Dystrophin not functional
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Becker genetic code maintained
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Becker genetic code maintained
Exon 46
Exon 47
Exon 52
Exon 53
Protein translation continues
Dystrophin partly functional Less damage
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Exon skipping restore genetic code
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Applicability
hotspot
Aartsma-Rus Hum Mutat 2009, 30293-9
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Exon skip chemistries

• Two chemistries in clinical development
• GSK/Prosensa 2-O-methyl phosphorothioate
  (drisapirsen)
• AVI-Biopharma/Sarepta phosphorodiamidate
  morpholino oligomers (eteplirsen)
• Exon 51

Annemieke Aartsma-Rus
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Exon skipping summary

• Opportunities
• AON delivery easier than genes/cells
• Clinical trial results encouraging
• Challenges
• Mutation specific approach
• Need to develop many AONs to treat majority of
  patients
• Repeated treatment needed (also opportunity)

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Stop codon readthrough
1
79
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PTC124/ataluren
1
79
Cell ignores new stop signal Complete protein is
made
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Stop codon readthrough summary

• Opportunities
• Oral delivery
• Applicable to multiple diseases
• Challenges
• Mutation specific approach (15)

Annemieke Aartsma-Rus