Transcript Explaining Therapies – Annemieke Aartsma-Rus

Understanding Gene and Cell Therapy
Approaches for DMD
November 6 2015
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
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
Muscles
• 30-40% of our body is muscle
• >750 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)
Annemieke Aartsma-Rus
Muscle contraction
Annemieke Aartsma-Rus
Muscle fibers
Skeleton
Connective tissue
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
Dystrophin
Dystrophin
Annemieke Aartsma-Rus
Duchenne: no functional dystrophin
Annemieke Aartsma-Rus
What happens without dystrophin
Muscle damage
Repair
Inflammation
Less blood flow
Too much Ca2+
Oxidative stress
Mitochondria
damaged
Fibrosis
Loss of muscle tissue
Loss of muscle function
Annemieke Aartsma-Rus
What are muscle cross sections?
Muscle cross sections
H&E staining
Fibers pink
Nuclei blue
Annemieke Aartsma-Rus
Dystrophin staining
Dystrophin immune staining
Healthy
Annemieke Aartsma-Rus
Mdx mouse
Duchenne
Revertant fibers & trace amounts
Revertant Fibers
Trace amounts
Untreated DMD muscle
Annemieke Aartsma-Rus
Western blotting
Western blot
• Proteins isolated from muscle
• Separated by size
• Stain for dystrophin
DMD Becker (dilutions)
Long
Short
Annemieke Aartsma-Rus
Control (diluted)
Therapy development
• Dystrophin is missing
• Trying to replace dystrophin
• Also many other therapies aiming at improving
muscle quality/slowing down disease processes
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
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)
Annemieke Aartsma-Rus
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 > year
• Severity is variable
• Rare mild individuals
Annemieke Aartsma-Rus
Other animal models
Pig model
• Mutation in pig dystrophin gene
• No dystrophin protein
• Deletion exon 52
• Severe disease (muscle & heart)
Annemieke Aartsma-Rus
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?
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
Niche
Muscle damage
Repair
Inflammation
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
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
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?
Annemieke Aartsma-Rus
Gene Therapy
Maaike van Putten
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)
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
Gene Therapy
Microdystrophin
Only crucial domains
Fits in AAV particle
Annemieke Aartsma-Rus
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)
Annemieke Aartsma-Rus
Upscaling
• Mouse
• Monkey
• Human boy
12 gram muscle
4 kg muscle
10-25 kg muscle
333x
2-6x
• 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
Annemieke Aartsma-Rus
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
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
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
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)
Annemieke Aartsma-Rus
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
DNA scissor system
• DNA scissors can cut DNA at specific location
Scissor cuts at/near mutation
Repair break with template
(Correct small mutations)
Annemieke Aartsma-Rus
Repair break without template
Small mutations will be
introduced (can correct genetic
code like exon skipping)
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)
Annemieke Aartsma-Rus
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
Annemieke Aartsma-Rus
Exon skipping
Normal
Annemieke Aartsma-Rus
Duchenne
Dystrophin gene
Annemieke Aartsma-Rus
Splicing
Exons
3
2
1
6
5
Introns
7
Gene (DNA)
4
1
2
3
Splicing messenger RNA
4
5
6
7
RNA copy (pre mRNA)
Annemieke Aartsma-Rus
1 2 3 4 5 6 7 8
1 - - - - - - - - - 79
dystrophin protein
Duchenne: genetic code disrupted
Annemieke Aartsma-Rus
Duchenne: genetic code disrupted
Exon 46
Exon 47
?
Exon 51
Exon 52
Protein translation stops prematurely
Dystrophin not functional
Annemieke Aartsma-Rus
Becker: genetic code maintained
Annemieke Aartsma-Rus
Becker: genetic code maintained
Exon 46
Exon 47
Exon 52
Exon 53
Protein translation continues
Dystrophin partly functional
Less damage
Annemieke Aartsma-Rus
Exon skipping: restore genetic code
Annemieke Aartsma-Rus
Applicability
hotspot
Annemieke Aartsma-Rus
Aartsma-Rus Hum Mutat 2009, 30:293-9
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
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)
Annemieke Aartsma-Rus
Stop codon readthrough
1
79
Annemieke Aartsma-Rus
PTC124/ataluren
PTC
1
79
Cell ignores new stop signal
Complete protein is made
Annemieke Aartsma-Rus
Stop codon readthrough summary
• Opportunities
• Oral delivery
• Applicable to multiple diseases
• Challenges
• Mutation specific approach (15%)
Annemieke Aartsma-Rus