Lecture 35 - Therapeutic Challenges in Muscular Dystrophies

Question 1

What do mutations resulting in muscular dystrophies affect?

Answer 1

Sarcolemmal proteins

Question 2

Effect of the absence of a sarcolemmal protein
1)
2)
3)

Answer 2

1) Disassembly of the dystrophin-associated complex
2) Increased sarcolemmal fragility
3) Increased Ca2+ entry into muscle fibres, leading to damage

Question 3

Normal response to damaged muscle fibres

Answer 3

Damaged fibres are replaced or repaired by satellite cells

Question 4

Response to damaged muscle fibres in muscular dystrophies
1)
2)
3)

Answer 4

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)

Question 5

DMD disease progression
1)
2)
3)
4)
5)
6)
7)

Answer 5

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

Question 6

Most abundant tissue in the body

Answer 6

Skeletal muscle

Question 7

What must MD therapy do?

Answer 7

Restore function in millions of post-mitotic nuclei (muscle cell nuclei can’t divide)

Question 8

Why doesn’t tissue culture muscle repair necessarily translate into an effective treatment?
1)
2)

Answer 8

1) Local delivery of a therapeutic agent is proof of principle
2) Real clinical benefit only follows systemic delivery

Question 9

Approaches to MD treatment
1)
2)
3)

Answer 9

1) Gene repair or replacement
2) Upregulation of complementary proteins
3) Blocking downstream effects

Question 10

Approaches to gene repair or replacement
1) 
2) 
3) 
4)

Answer 10

1) Cell and stem cell repair
2) Gene replacement
3) Translation - stop codon read-through
4) RNA splicing

Question 11

Downstream effects that can be blocked 
1)
2)
3)
4)
5)
6)

Answer 11

1) Fibrosis
2) Block abnormal Ca2+ influx
3) Immune effects
4) Increase NO
5) Increase muscle energy
6) Increase muscle regeneration

Question 12

Cell replacement therapeutic approaches for MD
1)
2)

Answer 12

1) Myoblast transfer therapy

2) Stem cell therapy

Question 13

Gene repair approaches for MD
1)
2)
3)
4)
5)

Answer 13

1) Cell replacement
2) Gene replacement
3) Gene repair or upregulation
4) Nonsense mutation skipping
5) Targeted exon skipping

Question 14

Myoblast transfer therapy

Answer 14

Donor myoblasts are injected into patient muscles

Question 15

What must myoblasts do for myoblast transfer therapy to work?

Answer 15

Survive, proliferate, migrate away from the injury site , fuse with myofibres, express functional dystrophin

Question 16

Problems with myoblast transfer therapy
1)
2)
3)

Answer 16

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

Question 17

Mouse model of DMD

Answer 17

mdx mice

Question 18

What happens after an injection of myoblast transfer therapy in humans?
1)
2)
3)
4)
5)

Answer 18

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

Question 19

Difficulties with stem cell therapy for MDs

Answer 19

Deliver stem cells systemically, then they need to migrate to muscle, differentiate

Question 20

Stem cell lines trialled for MD treatment
1)
2)
3)
4)
5)
6)

Answer 20

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

Question 21

Outcomes of stem cell therapy for MD
1)
2)
3)
4)

Answer 21

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

Question 22

Novel approach to gene replacement for MD
1)
2)

Answer 22

1) Use microdystrophins

2) Microdystrophins might restore function of smaller dystrophin molecule, partially restore function

Question 23

Rationale behind using microdystrophins in MD gene therapy
1)
2)
3)

Answer 23

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)

Question 24

Problems with gene therapy for MD
1) 
2) 
3) 
4)

Answer 24

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

Question 25

Problems with adenovirus vectors 
1) 
2) 
3) 
4)

Answer 25

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

Question 26

Possible solutions to vector issues with gene therapy
1)
2)

Answer 26

1) Adeno-associated vectors are smaller, less immunogenic than traditional adenovirus vectors
2) MIcrodystrophin genes can fit into adeno-associated vectors

Question 27

Effect of ataluren

Answer 27

Suppresses premature stop codon (nonsense mutation)

Question 28

Which MDs can ataluren be used to treat?

Answer 28

Those which have arisen from a nonsense mutation

Question 29

Experimental results of ataluren 
1)
2)
3)
4)

Answer 29

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

Question 30

Results of ataluren phase I clinical trials 
1)
2)
3)
4)

Answer 30

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

Question 31

Phase 2 ataluren clinical trials
1)
2)

Answer 31

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

Question 32

Phase 2b ataluren trials eligibility criteria
1)
2)
3)

Answer 32

1) Nonsense-mediated DMD
2) Male over 5 years
3) Ambulatory (Can walk over 75m)

Question 33

DMD phase 2b ataluren study primary outcome

Answer 33

6 minute walk test, improvement of 30m

Question 34

Results of ataluren 2b study

Answer 34

1) Placebo and high-dose had same effect

2) Low-dose had an increase in 6 minute walk test of 29m

Question 35

Dose-response curve for ataluren
1)
2)
3)

Answer 35

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

Question 36

Outcome of ataluren 2b trial

Answer 36

Failed trial

Now in stage 3 trials in a few countries

Question 37

Targeted exon skipping concept

Answer 37

Skip exon that contains mutation (EG: skip exon with nonsense mutation)

Question 38

Agent used to skip exons

Answer 38

Antisense oligonucleotides, which act as ‘gene zippers’

Question 39

Is creatine kinase concentration a functionally-significant measure?

Answer 39

No.

It is a good indicator of muscle condition, but not necessarily an indication of strength

Question 40

Timed function tests

Answer 40

How long someone takes to get up from lying down, how long it takes to climb three steps

Question 41

Are some exons more likely to have mutations than others?

Answer 41

Yes

Question 42

DMD deletion commonly resulting in frameshift mutation

Answer 42

Exons 48-51

~10% of DMD patients have a deletion here

Question 43

Possible solution to deletion between exons 48 and 51

Answer 43

Exon skipping of exon 51

Question 44

Types of mutations that can be treated with gene replacement or cell transfer

Answer 44

Gene deletions
Gene duplications
Nonsense mutations
Other mutations

Question 45

Types of mutations that can be treated with exon skipping

Answer 45

Gene duplications
Gene deletions
Nonsense mutations

Question 46

Types of mutations that can be treated with nonsense read-through treatment

Answer 46

Nonsense mutations

Question 47

Upregulation of which protein could help DMD patients?

Answer 47

Utrophin

Question 48

What is utrophin?
1)
2)
3)
4)
5)

Answer 48

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

Question 49

Effect of utrophin overexpression in mdx mice

Answer 49

Compensates to restore normal muscle function

Question 50

How can utrophin be overexpressed?

Answer 50

Adenoviral vector or transgenically

Question 51

Why mightn’t utrophin overexpression induce an immune response?

Answer 51

Utrophin is expressed foetally, so the immune system will recognise it as self

Question 52

Drug that can induce utrophin overexpression

Answer 52

SMTC1100

Question 53

SMTC1100

Answer 53

Utrophin overexpression-inducer

Question 54

Drug that blocks excessive Ca2+ influx

Answer 54

Poloxamer 188

Question 55

Drugs that reduce immune activation in DMD

Answer 55

TNFa antagonists, TGFb antagonists, steroids

Question 56

Drugs that can induce muscle regeneration in DMD

Answer 56

IGF, glutamine

Question 57

Drugs that can increase muscle energy in DMD

Answer 57

Creatine, CoQ10

Question 58

How might drugs that block downstream effects be used?

Answer 58

As a combination therapy