understanding gene and cell therapy approaches for dmd november 6 2015

Understanding Gene and Cell Therapy Understanding Gene and Cell Therapy Approaches for DMDApproaches for DMD

November 6 2015November 6 2015

Overview

Annemieke Aartsma-Rus

• 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

Some basic biology: genes & proteins

Annemieke Aartsma-Rus

• 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

Muscles

Annemieke Aartsma-Rus

• 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)

Muscle contraction

Annemieke Aartsma-Rus

Muscle fibers

Annemieke Aartsma-Rus

SkeletonSkeleton

Connective tissueConnective tissue

Dystrophin

Annemieke Aartsma-Rus

• 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

Dystrophin

Annemieke Aartsma-Rus

DystrophinDystrophin

Duchenne: no functional dystrophin

Annemieke Aartsma-Rus

What happens without dystrophin

Annemieke Aartsma-Rus

InflammationRepair

Muscle damage

Fibrosis

Less blood flow Too much Ca2+

Oxidative stress Mitochondria damaged

Loss of muscle tissueLoss of muscle function

What are muscle cross sections?Muscle cross sectionsMuscle cross sections

Annemieke Aartsma-Rus

H&E stainingH&E stainingFibers pinkFibers pinkNuclei blueNuclei blue

Dystrophin stainingDystrophin immune staining

Annemieke Aartsma-Rus

Healthy Mdx mouse Duchenne

Revertant fibers & trace amounts

Annemieke Aartsma-Rus

Revertant Fibers

Trace amounts

Untreated DMD muscle

Western blotting

Western blotWestern blot• Proteins isolated from muscleProteins isolated from muscle• Separated by sizeSeparated by size• Stain for dystrophinStain for dystrophin

Annemieke Aartsma-Rus

DMDDMD Becker (dilutions)Becker (dilutions) Control (diluted)Control (diluted)LongLong

ShortShort

Therapy development

Annemieke Aartsma-Rus

• Dystrophin is missing• Trying to replace dystrophin

• Also many other therapies aiming at improving muscle quality/slowing down disease processes

What are cell models

Annemieke Aartsma-Rus

• 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

What are mdx mice?

Annemieke Aartsma-Rus

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)

Other animal models

Annemieke Aartsma-Rus

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

Other animal models

Annemieke Aartsma-Rus

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

Drug testing in patients

Annemieke Aartsma-Rus

• 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?

Development of therapies

Annemieke Aartsma-Rus

• 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

Cell therapy

Annemieke Aartsma-Rus

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

Muscle stem cells (myoblasts)

Annemieke Aartsma-Rus

• 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

Stem cell therapy

Annemieke Aartsma-Rus

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

Niche

Annemieke Aartsma-Rus

InflammationRepair

Muscle damage

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

Immunity

Annemieke Aartsma-Rus

• 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

Cell therapy summary

Annemieke Aartsma-Rus

• 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

Gene Therapy

Annemieke Aartsma-Rus

• 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?

Gene Therapy

Annemieke Aartsma-Rus

Maaike van Putten

Gene Therapy

Annemieke Aartsma-Rus

Virus

• Small organism that injects genetic information into cells

• Use to deliver dystrophin gene

• Adapt

• Remove virus genes (pathogenic)

• Add new gene (dystrophin)

Gene Therapy

Annemieke Aartsma-Rus

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

Gene Therapy

Annemieke Aartsma-Rus

• 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

Gene Therapy

Annemieke Aartsma-Rus

MicrodystrophinOnly crucial domainsFits in AAV particle

Gene Therapy

Annemieke Aartsma-Rus

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)

Upscaling

Annemieke Aartsma-Rus

• 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

Delivery

Annemieke Aartsma-Rus

• 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)

Limb perfusion

Annemieke Aartsma-Rus

• 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

Gene Therapy Summary

Annemieke Aartsma-Rus

• Opportunities

• Applicable to all patients

• Currently in early clincial development (safety/tolerability tests)

• Challenges

• Microdystrophin only partially functional

• Delivery

• Immunity

Gene/cell therapy: DNA editing

Annemieke Aartsma-Rus

• 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)

DNA editing

Annemieke Aartsma-Rus

• 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

DNA scissor system

Annemieke Aartsma-Rus

• DNA scissors can cut DNA at specific location

Scissor cuts at/near mutation

Repair break with template(Correct small mutations)

Repair break without templateSmall mutations will be introduced (can correct genetic code like exon skipping)

DNA scissor system

Annemieke Aartsma-Rus

Combination of DNA scissors to restore genetic code

Scissors cut around exon break is repaired

Exon is deleted Genetic code restored (like exon skipping)

DNA scissor system

Annemieke Aartsma-Rus

• 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

Exon skipping

Annemieke Aartsma-Rus

NormalNormal DuchenneDuchenne

Dystrophin gene

Annemieke Aartsma-Rus

Splicing

Annemieke Aartsma-Rus

ExonsExons IntronsIntrons1

3

5

6

7 Gene (DNA)Gene (DNA)

RNA copy (pre mRNA)RNA copy (pre mRNA)

messenger RNAmessenger RNA

1 - - - - - - - - - 79

dystrophin proteindystrophin protein

SplicingSplicing

2

4

3 4 56 7

1 21 2 3 4 5 6 7 8

Duchenne: genetic code disrupted

Annemieke Aartsma-Rus

Duchenne: genetic code disrupted

Annemieke Aartsma-Rus

Protein translation stops prematurelyProtein translation stops prematurely

Dystrophin not functionalDystrophin not functional

Exon 46 Exon 47 Exon 51 Exon 52?

Becker: genetic code maintained

Annemieke Aartsma-Rus

Becker: genetic code maintained

Annemieke Aartsma-Rus

Protein translation continuesProtein translation continues

Dystrophin partly functionalDystrophin partly functionalLess damageLess damage

Exon 46 Exon 47 Exon 52 Exon 53

Exon skipping: restore genetic code

Annemieke Aartsma-Rus

Applicability

Annemieke Aartsma-Rus Aartsma-Rus Hum Mutat 2009, 30:293-9

hotspot

Exon skip chemistries

Annemieke Aartsma-Rus

• Two chemistries in clinical development

• GSK/Prosensa: 2’-O-methyl phosphorothioate (drisapirsen)

• AVI-Biopharma/Sarepta: phosphorodiamidate morpholino oligomers (eteplirsen)

• Exon 51

Exon skipping summary

Annemieke Aartsma-Rus

• 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)

Stop codon readthrough

Annemieke Aartsma-Rus

1 79

PTC124/ataluren

Annemieke Aartsma-Rus

1 79

PTC

Cell ignores new stop signalCell ignores new stop signalComplete protein is madeComplete protein is made

Stop codon readthrough summary

Annemieke Aartsma-Rus

• Opportunities

• Oral delivery

• Applicable to multiple diseases

• Challenges

• Mutation specific approach (15%)