Lecture 35 - Therapeutic Challenges in Muscular Dystrophies Flashcards
What do mutations resulting in muscular dystrophies affect?
Sarcolemmal proteins
Effect of the absence of a sarcolemmal protein
1)
2)
3)
1) Disassembly of the dystrophin-associated complex
2) Increased sarcolemmal fragility
3) Increased Ca2+ entry into muscle fibres, leading to damage
Normal response to damaged muscle fibres
Damaged fibres are replaced or repaired by satellite cells
Response to damaged muscle fibres in muscular dystrophies
1)
2)
3)
1) Satellite cells are exhausted over time
2) Muscle is increasingly replaced with fibrous or adipose tissue
3) Inflammation, release of cytokines (EG: TGF-b)
DMD disease progression 1) 2) 3) 4) 5) 6) 7)
1) Lack of functional dystrophin gene
2) Lack of dystrophin
3) Damage to muscle fibres
4) Death of groups of muscle fibres
5) Satellite cells repair or replace damaged fibres, but are depleted over time
6) Inflammation, release of cytokines (TGF-b)
7) Fibrosis (scar tissue) –> leads to damage of muscle fibres, positive feedback loop
Most abundant tissue in the body
Skeletal muscle
What must MD therapy do?
Restore function in millions of post-mitotic nuclei (muscle cell nuclei can’t divide)
Why doesn’t tissue culture muscle repair necessarily translate into an effective treatment?
1)
2)
1) Local delivery of a therapeutic agent is proof of principle
2) Real clinical benefit only follows systemic delivery
Approaches to MD treatment
1)
2)
3)
1) Gene repair or replacement
2) Upregulation of complementary proteins
3) Blocking downstream effects
Approaches to gene repair or replacement 1) 2) 3) 4)
1) Cell and stem cell repair
2) Gene replacement
3) Translation - stop codon read-through
4) RNA splicing
Downstream effects that can be blocked 1) 2) 3) 4) 5) 6)
1) Fibrosis
2) Block abnormal Ca2+ influx
3) Immune effects
4) Increase NO
5) Increase muscle energy
6) Increase muscle regeneration
Cell replacement therapeutic approaches for MD
1)
2)
1) Myoblast transfer therapy
2) Stem cell therapy
Gene repair approaches for MD 1) 2) 3) 4) 5)
1) Cell replacement
2) Gene replacement
3) Gene repair or upregulation
4) Nonsense mutation skipping
5) Targeted exon skipping
Myoblast transfer therapy
Donor myoblasts are injected into patient muscles
What must myoblasts do for myoblast transfer therapy to work?
Survive, proliferate, migrate away from the injury site , fuse with myofibres, express functional dystrophin
Problems with myoblast transfer therapy
1)
2)
3)
1) After 1 month dystrophin detected in 36% of muscles
2) After 6 months, no dystrphin detected
3) Ascribed to poor cell survival, immune rejection, limited cell distribution after injection
Mouse model of DMD
mdx mice
What happens after an injection of myoblast transfer therapy in humans? 1) 2) 3) 4) 5)
1) After a single injection, intense immune activation
2) Cell-mediated immune response
3) Over 90% of myoblasts killed in under and hour
4) Most killed within a minute of injection
5) No effective dystrophin production
Difficulties with stem cell therapy for MDs
Deliver stem cells systemically, then they need to migrate to muscle, differentiate
Stem cell lines trialled for MD treatment 1) 2) 3) 4) 5) 6)
1) All adult stem cell lines
2) Bone marrow derived
3) Blood and muscle derived CD133+
4) Muscle derived
5) Side population cells
6) Mesoangioblasts
Outcomes of stem cell therapy for MD 1) 2) 3) 4)
1) Variable results. Can be:
2) Incorporated into muscle, but no restoration of wild-type protein
3) Restoration of wild-type protein, but extreme immune response
4) Restoration of wild-type protein, but not enough to have an effect on strength
Novel approach to gene replacement for MD
1)
2)
1) Use microdystrophins
2) Microdystrophins might restore function of smaller dystrophin molecule, partially restore function
Rationale behind using microdystrophins in MD gene therapy
1)
2)
3)
1) Use microdystrophins to repair the absent part of causative protein (EG: dystrophin in DMD)
2) Deletions in N-terminal domain result in milder phenotype
3) Deletions in C-terminal domain result in more severe phenotype (cysteine-rich domain)
Problems with gene therapy for MD 1) 2) 3) 4)
1) Immune response to vector
2) Dystrophin is a very large gene, often doesn’t fit in vector
3) Vectors too large to cross extracellular matrix to target
4) Few adenoviral receptors on myofibre membrane
Problems with adenovirus vectors 1) 2) 3) 4)
1) Immunogenicity
2) Traditional vectors carry up to 8kb, dystrophin cDNA is 14kb
3) Too large to cross ECM to target
4) Few adenoviral receptors on myofibre membrane
Possible solutions to vector issues with gene therapy
1)
2)
1) Adeno-associated vectors are smaller, less immunogenic than traditional adenovirus vectors
2) MIcrodystrophin genes can fit into adeno-associated vectors
Effect of ataluren
Suppresses premature stop codon (nonsense mutation)
Which MDs can ataluren be used to treat?
Those which have arisen from a nonsense mutation
Experimental results of ataluren 1) 2) 3) 4)
1) Dose-dependent readthrough of stop codons in cultured myotubes
2) Full-length dystrophin expression in diaphragm, skeletal, heart muscle in mdx mice
3) Increase dystrophin production in mdx
4) Reduce creatine kinase levels in mdx
Results of ataluren phase I clinical trials 1) 2) 3) 4)
1) Volunteers received ataluren for 2 weeks
2) Good oral availability
3) Well tolerated up to 100mg/Kg per day
4) Nausea, diarrhoea, headache at over 150mg/Kg/day
Phase 2 ataluren clinical trials
1)
2)
1) Three groups - placebo, low dose, high dose
2) Non-placebo groups had decreased muscle fragility, creatine kinase levels, which went back up with cessation of treatment
Phase 2b ataluren trials eligibility criteria
1)
2)
3)
1) Nonsense-mediated DMD
2) Male over 5 years
3) Ambulatory (Can walk over 75m)
DMD phase 2b ataluren study primary outcome
6 minute walk test, improvement of 30m
Results of ataluren 2b study
1) Placebo and high-dose had same effect
2) Low-dose had an increase in 6 minute walk test of 29m
Dose-response curve for ataluren
1)
2)
3)
1) Irregularly shaped
2) Response increases with dose to a point
3) Peaks, then declines after a point
4) From myocytes in culture for 12 days
Outcome of ataluren 2b trial
Failed trial
Now in stage 3 trials in a few countries
Targeted exon skipping concept
Skip exon that contains mutation (EG: skip exon with nonsense mutation)
Agent used to skip exons
Antisense oligonucleotides, which act as ‘gene zippers’
Is creatine kinase concentration a functionally-significant measure?
No.
It is a good indicator of muscle condition, but not necessarily an indication of strength
Timed function tests
How long someone takes to get up from lying down, how long it takes to climb three steps
Are some exons more likely to have mutations than others?
Yes
DMD deletion commonly resulting in frameshift mutation
Exons 48-51
~10% of DMD patients have a deletion here
Possible solution to deletion between exons 48 and 51
Exon skipping of exon 51
Types of mutations that can be treated with gene replacement or cell transfer
Gene deletions
Gene duplications
Nonsense mutations
Other mutations
Types of mutations that can be treated with exon skipping
Gene duplications
Gene deletions
Nonsense mutations
Types of mutations that can be treated with nonsense read-through treatment
Nonsense mutations
Upregulation of which protein could help DMD patients?
Utrophin
What is utrophin? 1) 2) 3) 4) 5)
1) Autosomal homologue of dystrophin
2) Genomic length is 1/3 of dystrophin, but RNA transcript is a similar length (13kb)
3) Can bind proteins of the dystrophin-associated protein complex
4) Shares 74% amino acids with dystrophin
5) Expressed in place of dystrophin in foetal muscles, in adult muscle confined to neuromuscular, myotendinous junctions
Effect of utrophin overexpression in mdx mice
Compensates to restore normal muscle function
How can utrophin be overexpressed?
Adenoviral vector or transgenically
Why mightn’t utrophin overexpression induce an immune response?
Utrophin is expressed foetally, so the immune system will recognise it as self
Drug that can induce utrophin overexpression
SMTC1100
SMTC1100
Utrophin overexpression-inducer
Drug that blocks excessive Ca2+ influx
Poloxamer 188
Drugs that reduce immune activation in DMD
TNFa antagonists, TGFb antagonists, steroids
Drugs that can induce muscle regeneration in DMD
IGF, glutamine
Drugs that can increase muscle energy in DMD
Creatine, CoQ10
How might drugs that block downstream effects be used?
As a combination therapy
