Lecture 35 - Therapeutic Challenges in Muscular Dystrophies Flashcards Preview

Molecule to Malady > Lecture 35 - Therapeutic Challenges in Muscular Dystrophies > Flashcards

Flashcards in Lecture 35 - Therapeutic Challenges in Muscular Dystrophies Deck (58):
1

What do mutations resulting in muscular dystrophies affect?

Sarcolemmal proteins

2

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

3

Normal response to damaged muscle fibres

Damaged fibres are replaced or repaired by satellite cells

4

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)

5

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

6

Most abundant tissue in the body

Skeletal muscle

7

What must MD therapy do?

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

8

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

9

Approaches to MD treatment
1)
2)
3)

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

10

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

11

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

12

Cell replacement therapeutic approaches for MD
1)
2)

1) Myoblast transfer therapy
2) Stem cell therapy

13

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

14

Myoblast transfer therapy

Donor myoblasts are injected into patient muscles

15

What must myoblasts do for myoblast transfer therapy to work?

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

16

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

17

Mouse model of DMD

mdx mice

18

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

19

Difficulties with stem cell therapy for MDs

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

20

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

21

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

22

Novel approach to gene replacement for MD
1)
2)

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

23

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)

24

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

25

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

26

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

27

Effect of ataluren

Suppresses premature stop codon (nonsense mutation)

28

Which MDs can ataluren be used to treat?

Those which have arisen from a nonsense mutation

29

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

30

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

31

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

32

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

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

33

DMD phase 2b ataluren study primary outcome

6 minute walk test, improvement of 30m

34

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

35

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

36

Outcome of ataluren 2b trial

Failed trial
Now in stage 3 trials in a few countries

37

Targeted exon skipping concept

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

38

Agent used to skip exons

Antisense oligonucleotides, which act as 'gene zippers'

39

Is creatine kinase concentration a functionally-significant measure?

No.
It is a good indicator of muscle condition, but not necessarily an indication of strength

40

Timed function tests

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

41

Are some exons more likely to have mutations than others?

Yes

42

DMD deletion commonly resulting in frameshift mutation

Exons 48-51
(~10% of DMD patients have a deletion here)

43

Possible solution to deletion between exons 48 and 51

Exon skipping of exon 51

44

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

Gene deletions
Gene duplications
Nonsense mutations
Other mutations

45

Types of mutations that can be treated with exon skipping

Gene duplications
Gene deletions
Nonsense mutations

46

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

Nonsense mutations

47

Upregulation of which protein could help DMD patients?

Utrophin

48

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

49

Effect of utrophin overexpression in mdx mice

Compensates to restore normal muscle function

50

How can utrophin be overexpressed?

Adenoviral vector or transgenically

51

Why mightn't utrophin overexpression induce an immune response?

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

52

Drug that can induce utrophin overexpression

SMTC1100

53

SMTC1100

Utrophin overexpression-inducer

54

Drug that blocks excessive Ca2+ influx

Poloxamer 188

55

Drugs that reduce immune activation in DMD

TNFa antagonists, TGFb antagonists, steroids

56

Drugs that can induce muscle regeneration in DMD

IGF, glutamine

57

Drugs that can increase muscle energy in DMD

Creatine, CoQ10

58

How might drugs that block downstream effects be used?

As a combination therapy