lecture 36 Flashcards
1
Q
What muscle degeneration occurs in the muscular dystrophies?
A
- mutations affect sarcolemmal proteins, which connect the cytoskeleton and basal lamina
- absence of one protein
→ disassembly of dystrophin-associated complex
→ increased sarcolemmal fragility
→ increased calcium entry, damage to fibres - damaged or dead fibres are repaired or replaced by satellilte cells
- with time, the population of satellite cells is exhausted
- muscle is progressively replaced by connective and adipose tissue
- this causes increasing weakness and contractures
- loss in anyone of the proteins involved
2
Q
What is DMD disease progression?
A
Defective dystrophin gene → lack of dystrophin → damage to individual muscle fibres → death of groups of muscle fibres either → satellite cell activation → muscle fibre repair or → inflammation → release of cytokines (e.g. TGF-beta) → fibrosis (formation of scar tissue)
3
Q
How do we treat MDs?
A
- complex
- skeletal muscle: most abudant tissue of the body
- large multinucleated fibres whose nuclei cannot divide
- Rx must restore gene function in millions of post-mitotic nuclei
- every therapeutic strategy has advantages and limitations
- local delivery of the therapeutic agent is proof of princlple but
- real clinical benefit can only follow systemic delivery
- adverse effects may be apparent only after systemic delivery
4
Q
How can we approach the problem of treatment?
A
- gene repair or replacement
- cell and stem cell transfer
- gene replacement (AAV, myoblast transfer)
- translation: stop codon read through (Ataluren etc)
- RNA splicing - upregulation of compensatory proteins
- utrophin, alpha-dystrobrevin, alpha7integrin, GALNAx, NOS etc - blocking downstream effects
- block abnoraml Ca++ influx: stretch channel blockers, membrane sealers (poloxamer 188)
- fribrosis: anti-fibrotics
- immune: steroids, TNF-alpha antagonists, TGF-beta antagonists
- increase NO: arginine-like drugs
- increase muscle energy: creatine/COQ10
- increase muscle regeneration: MYO-029, IGF, glutamine
combination therapy likely
5
Q
What is an overview of strategies for DMD gene therapy?
A
- adenoviral vetors
- herpes simplex viral vectors
- plasmid vectors
- myoblast transplantation
- stem cell therapy
- chimeric oligonucleotides
- gentamicin therapy
- tAAV vectors
- antisense oligonucleotides
- utrophin upregulation
6
Q
How do treatments for MDs address multiple points in pathophysiology?
A
- downstream effects of decreased dystrophin/a-sarcoglycan
- sometimes have more than one effect
7
Q
What is gene repair/replacement in DMD?
A
- cell replacement:
- myoblast transfer therapy
- stem cell therapy
- gene replacement
- gene repair and/or upregulation
- nonsense mutation suppression
- targeted exon skippin
8
Q
What is MTT?
A
- myoblast transfer therapy
- donor myoblasts (precursor cells) injected into DMD muscles
- fusion with host muscle fibres, contributing nuclei, ? replacing dystrophin
- to work: myoblasts must survive, proliferate, migrate away from the inj, site, fise with myofibres and express functional dystrophin
- promising results in animal models using mdx mice
- evidence of dystrophin transcript expresseion in DMD patients on PCR
- mutliple clinical trials; no objective benefit in DMD patients
- dystrophin-positive fibres in up to 36% of muscles after 1m
- expression undetectable by 6m post-injection
- ascribed to poor cell survival, immune rejection and limited cell distribution after injection
9
Q
What is seen after single injection in myoblast transfer?
A
- intense, cell-mediated host immune response
- > 90% myoblasts are elimated within 1 hour
- most killed within minutes of injection
- no effective dystrophin production
10
Q
What is stem cell therapy?
A
- systemically
- have to get out of blood vessel, migrate into muscle
several adult-derived stem cell lines have been trialled in DMD:
- bone marrow-derived stem cells
- blood- and muscle-derived CD133+ cells
- muscle-derived stem cells
- side population cells
- mesangioblasts
variable results:
- incorporated into muscle but no restoration of expression of wild-type protein
- restoration of wt-protein but extreme immune response
- restoration of wt protein but insuffiecient to affect strength
11
Q
What is gene repalcement?
A
use of microdystrophins:
- deletions of N-termal domain: milkder phenotype
- deletions in cysteine-rich domain: severe disruption of DAPC, severe phenotype
micro and minidystrophins may ameliorate phenotype by restoration of small dystrophin molecule and partial restoration of function
12
Q
What are problems with gene replacements?
A
- those of all genetic disorders i.e. immune response to vector
13
Q
What are adenoviral vectors?
A
- much hope in the past but no longer used
- immunogenicity, size (limited diffusion)
- size of vectors: dystrophin cDNA is 14kb, traditional AAV vectors carry up to *kb
- vectors too large to cross ECM around mature myofibres
- few adenoviral receptors on myofibre membrane
14
Q
What are adeno-associated vectors (AAV)?
A
- smaller, less, immunogenicity
- some effect in other MDs (smaller genes), not yet in DMD
- ? role for microdystrophins (rod domain shortened)
15
Q
What is nonsense mutation supression?
A
- reading through nonsense stop codon
- in about 18%/13% of boys that have DBMD due to a nonsense mutation
- ataluren induces full-length protein production
- increased amount, maybe not full amount
