Lecture 36 - Muscular Dystrophies - Therapies Flashcards
What are damaged muscle fibres replaced with?
Early on:
• Satellite cells
Later on:
• Satellite cells have been exhausted
• They are replaced with connective and adipose tissue
List the main features of skeletal muscle
Why is this a therapeutic challenge?
- Most abundant tissue in the body
- Large, multinucleate cells
- Nuclei cannot divide
Challenging:
• Treatment must restore gene function in millions of post-mitotic (non dividing) nuclei
Compare success with local and systemic delivery of therapeutic agents
Local delivery:
• Has achieved proof of principle
• However, no real clinical benefit
• No adverse effects
Systemic delivery:
• Real clinical benefit
• Adverse effects experienced
What are the three general ways to approach MD therapy?
- Gene repair or replacement
- Upregulation of compensatory proteins
- Blocking of downstream effects
Outline ways that the genes can be repaired or replaced
- Cell / Stem cell transfer
- Gene replacement
• AAV
• Myoblast transfer - Agents that allow read through of stop codons
• Ataluren - RNA splicing - exon skipping
• AON
Describe which compensatory proteins could be upregulated
• Utrophin
• alpha-dystrobrevin
etc.
Describe the process of Myoblast transfer therapy (MTT)
What are the requirements for this to work?
Treatment for MD through delivery of functional version of gene
- Donor myoblasts (from genetically related individual) injected into DMD muscles
- Myoblast fusion with host muscle fibres
- Nuclei from myoblasts donated to muscle fibres, replacing dystrophin (?)
To work:
• Myoblast survival, proliferation
• Myoblast fusion with myofibres
• Must express functional dystrophin
What is the animal model for muscular dystrophy?
mdx mice
What are the results of MTT in the mdx mice?
What about in people w/ DMD?
What was seen in the clinical trials?
Conclusions from the clinical trials?
Mdx mice: Promising results
People w/ DMD:
• Evidence of dystrophin transcript expression
Clinical trials:
• No benefit demonstrated
• 1m after injection: 36% of muscle fibres were dystrophin positive
• 6m after: undetectable expression (been destroyed by the immune system)
Conclusion: No benefit Myoblasts did not survive • Immune rejection • Limited cell distribution
Describe the host response after MTT
→ Single injection
Intense, CD4 T cell mediated immune response:
- CD4 T cell recognises myoblast as foreign through its expression of foreign antigen in the context of MHC II
- Activation of CD4 T cell specific for donor myoblast
- Cytokine release → inflammation, cell infiltrate
→ 90% myoblasts eliminated in an hour
Describe stem cell therapy for MD
Which stem cells have been used?
What were the results of the trials?
- Intra-muscular or intra-vascular injection of stem cells (with WT version of gene)
- Vectors / cells move into muscle
Stem cells:
• BM-derived
• Muscle-derived stem cells
etc.
Results:
Variable:
• Incorporated into muscle, but no restoration of expression WT protein
• Restoration of WT-protein, w/ extreme immune response
• Restoration of WT protein, but insufficient to affect strength
The stem cells may be incorporated, but there isn’t sufficient expression of dystrophin
Describe the genes used in gene replacement
- Micro-dystrophins:
• Deletions in N-terminal domain
• Fewer rod domains in the middle - Mini-dystrophins:
• Similar to micro-dystrophin
→ Partial restoration of function
→ Milder phenotype
What happens if there are deletions in the following regions: • Actin binding N-terminal domain • Rod domains • C-terminal domain • Cysteine rich domain
Actin binding N-terminal domain
• Loss of function
Rod domains
• Loss of a few and the protein can still be partially functional
Cysteine rich domain
• Loss of function
C-terminus:
• Loss of function
What are the cons of gene replacement?
Immune response to the vector
same for all genetic disorders treated with gene therapy
Compare the various vectors used for gene replacement
What were the problems encountered with each?
- Adenoviral vectors
• Used in the past, but not any more
• Immunogenic
• Size an issue: limits diffusion into muscle tissue and crossing of ECM
• Few adenoviral receptors on myofibre membrane
2. Adeno-associated vectors (AAVs) • Smaller • Less immunogenicity • Can't carry the 14kb dystrophin gene (only can carry 8kb) • Micro-dystrophins may be better
What have AAVs been used for to date?
Used in other MDs, but not in DMD
What is Ataluren?
Drug that is able to overcome nonsense mutations (due to premature STOP codons)
Describe nonsense mutation suppression
In some of the mutations there is a premature STOP codon (13% of DMD and BMD)
The full transcript is not translated, despite being intact
Ataluren designed to overcome this:
• Allows read through and translation of the entire transcript
• Full-length dystrophin translated
How common are nonsense mutations in DMD and BMD?
13% of affected boys
Describe the use of PTC124 (Ataluren) in the treatment of MD
What were the results in mdx mice?
Mechanism:
• Allows read through past premature stop codons
Result in mdx mice:
• Induces full-length dystrophin production in skeletal, diaphragm and cardiac muscles
• Decreased muscle fragility (i.e. decreased muscle injury)
• Reduced serum Creatine Kinase
Describe the PTC124 (Ataluren) trials in humans
(Phase 1, Phase 2, Phase 2b)
Include discussion about the results
Phase 1:
• 61 healthy, young adults
• 2 weeks of PTC124 (Ataluren)
Results:
• Excellent oral bioavailability
• Well tolerated, except at high doses
( • GIT complaints (nausea, diarrhoea) and headache at high doses: >150mg/kg
• Well tolerated at doses of 100mg/kg/day)
Phase 2: 3 groups of patients w/ DMD: • 6 patients, 4 weeks • 20 patients, 4 weeks • 12 patients, 4 weeks
Results:
• Well tolerated
• Significant serum CK reductions at the end of the treatment period
• Increase (back to normal) serum CK at follow up (i.e. after 4 weeks)
Phase 2b:
• 165 boys w/ DMD
• 12 countries represented
• 48 week study period
• 3 groups: (low dose, high dose, placebo)
• Primary outcome measure: 6 min walk distance
Results:
• Greatest efficacy in low dose Ataluren
• No difference between high dose and placebo groups
• Primary outcome measures were not met: improvement in 29m (didn’t reach requirement of 30m)
→ failed trial
Describe how the outcomes of the PTC124 (Ataluren) Phase 2B trial were measured:
• Primary
• Secondary
• Tertiary
What is this?
"6 minute walk test" • Reproducible • Standardised • Sensitive to change • Easy to perform • Represents an improvement (CK levels not relevant to boy)
When a clinical trial is undertaken, the outcome measures need to be defined
In this trial, the outcome measure that has been universally accepted is improvement in ambulation
- Primary outcome measure:
• 6 min walk distance
• Improvement criteria: > 30m - Secondary outcome measure:
• activity levels
• Timed function tests
• Serum CK values - Tertiary outcome measures
• Muscle strength
• Biceps muscle dystrophin expression
Explain the rationale for the benefit of low dose Ataluren in the treatment of DMD
Ataluren dose-response curve for dystrophin expression in Bell-shaped
Trend in dystrophin expression
• Up to 10 mg dose: increase
• Maximum: 10 mg/kg
• 10-20mg: decrease
This was found in human myocyte studies with Ataluren
What is PTC124 also known as?
aka Ataluren
