Lecture 36 - Muscular Dystrophies - Therapies Flashcards Preview

BIOM30002 - M2M > Lecture 36 - Muscular Dystrophies - Therapies > Flashcards

Flashcards in Lecture 36 - Muscular Dystrophies - Therapies Deck (41):

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

• 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?

1. Gene repair or replacement

2. Upregulation of compensatory proteins

3. Blocking of downstream effects


Outline ways that the genes can be repaired or replaced

1. Cell / Stem cell transfer

2. Gene replacement
• Myoblast transfer

3. Agents that allow read through of stop codons
• Ataluren

4. RNA splicing - exon skipping


Describe which compensatory proteins could be upregulated

• Utrophin
• alpha-dystrobrevin


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

1. Donor myoblasts (from genetically related individual) injected into DMD muscles

2. Myoblast fusion with host muscle fibres

3. 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)

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:
1. CD4 T cell recognises myoblast as foreign through its expression of foreign antigen in the context of MHC II
2. Activation of CD4 T cell specific for donor myoblast
3. 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?

1. Intra-muscular or intra-vascular injection of stem cells (with WT version of gene)

2. Vectors / cells move into muscle

Stem cells:
• BM-derived
• Muscle-derived stem cells

• 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

1. Micro-dystrophins:
• Deletions in N-terminal domain
• Fewer rod domains in the middle

2. 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

• 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?

1. 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?

• 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)

• 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

• 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

• 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

1. Primary outcome measure:
• 6 min walk distance
• Improvement criteria: > 30m

2. Secondary outcome measure:
• activity levels
• Timed function tests
• Serum CK values

3. 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


What conclusions can be made about Ataluren (PTC124)?

What lessons were learnt?

• Drug may have effect but this effect was insufficient to warrant licensing

• Drug did not meet primary outcome measure of the trial

• Failed trial
- Millions of dollars spent
- Thousands of patient- doctor- hours lost

Lessons learnt
• Re-assessment of primary lab data
• Re-assessment of statistical methods
• Re-assessment of outcome measure

However despite this Phase 3 trial is now underway


What can Antisense oligonucleotides do?

Targeted exon skipping

Act as a 'gene zipper'


Describe Targeted exon skipping as a therapy for DMD

Which agent is used?

Describe how it works

What is the outcome?

Antisense oligonucleotides

1 . Nonsense mutation in one exon
2. Antisense oligonucleotides skip mutated exon
3. Other exons translated
4. Shorter but functional protein produced


What is AON?

Antisense oligonucleotides


Describe clinical trials of AON

1st human trial:
• UK
• 2009

2nd clinical trial:
• International
• Started March 2011

Numerous trials now underway


Describe the structure of the dystrophin protein, including the various domains

Very long protein with various domains:

1. N-terminal domain
• Actin binding

2. Central 'Rod' domain
• Spectrin-like repeats

3. Cysteine rich domain

4. Carboxyl-terminal domain
• Allows assembly of the DAPC


Most new treatment strategies in DMD are...

Mutation specific

e.g. Ataluren:
• Premature STOP codons


Which gene therapies are suitable for gene deletions?

• Gene replacement
• Cell transfer
• Exon skipping


Which gene therapies are suitable for gene duplications?

• Gene replacement
• Cell transfer
• Exon skipping


Which gene therapies are suitable for nonsense mutation?

• Gene replacement
• Cell transfer
• Nonsense mutation read-through
• Exon skipping


Describe the features of Utrophin

Compare it with Dystrophin

Why is it suitable for use as a compensatory protein in the treatment of DMD?

• Structurally very similar to Dystrophin

• An autosomal homologue of dystrophin
• Chromosome 6 (as opposed to X-chromosome)
• Expressed in foetal muscle in the place of dystrophin
• In adults it is expressed only in the neuromuscular and myotendinous junctions
• Can also bind proteins of the DAPC


Compare the structure of Dystrophin and Utrophin

Very structurally similar


Describe the results of Utrophin compensation in studies

Mdx mice:
• Restoration of normal muscle function

No immune response:
• Because utrophin is normally expressed in adult tissues

Human trials:
Phase 2 underway, Phase 3 planned


How is Utrophin over expressed in myofibres?

• Viral vector-mediated delivery
• Transgenesis


What is SMTC1100?

Agent that increases Utrophin expression muscle in patient cells in vitro

Good safety profiles

It has taken a long time to find an agent that can up regulate the expression of Utrophin


Describe various approaches to blocking downstream effects

These agents won't be curative, but may be effective when taken as a cocktail

1. Blockage of abnormal Ca2+ influx
• Stretch channel blockers
• Membrane sealers

2. Fibrosis prevention
• Anti-fibrotics

3. Immune suppression
• Steroid
• TNF-alpha antagonists
• TGF-beta antagonists

4. Increase NO
• Arginine-like drugs

5. Increased muscle energy
• Creatine / CoQ10

6. Increased muscle regeneration
• MYO-029
• Glutamine


Which pathological features of DMD can be targeted to improve symptoms?

• Fibrosis on muscle

• Inflammation

• Abnormal Ca2+ influx

• Muscle regeneration