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
16
Q
What is PTC124?
A
- ataluren
- treatment induces dose-dependent read-through of stop codons in cultures myotubes
- induces full-length dystrophin production in skeletal, diaphragm, and heart muscles of mdx mice
- decreases muscle fragility in the mdx mouse
- reduced level of CK
- does-dependent in vitro readthrough confirmed potential activity in 100% (35/35) of evaluable patients
17
Q
What was phase 1 trial of ataluren?
A
- two studies performed in 61 healthy young adults
- volunteers received PTC124 for up to 2 weeks
- results showed:
- oral bioavailability was excellent, achieving desired blood levels when given with or without food
- nausea, diarrhea, and headache were seen at high dosese (≥ 150 mg/kg)
- PTC124 was well tolerated at doses up to 100mg/kg/day
- passed safety
18
Q
What was phase 2?
A
- studies of PTC124 activity and safety at different doses in patients with DMD
- safety
- PTC124-related serum CK reductions were observed in most patients
- with cessation of PTC124 treatment, mean serum CK concentrations reverted toward baseline, consistent with pharmacological activity
- tolerated well in DMD patients
- significant serum CK reductions suggested decreases in muscle fragility
19
Q
What was international DMD ataluren phase 2b study?
A
- eligibility criteria
- nonse-mediated DMD
- males, ≥ 5 years
- ambulatory (can walk ≥ 75 metres)
- randomisation and stratification
- age
- steroid use
- baseline 6 minute walk distance
- primary outcome measure
- 6-minute walk distance (primary): improvement >30m (defined prior to the start)
- two witches hat 25 m apart
- CK is not a functionally useful measure
- secondary outcome measures
- activity levels, timed function tests, serum CK values etc
- muscle biopsies before and after (invasive)
- tertiary outcome meaures
- muscle strength, biceps muscle dystrophin expression
- safety and exposure
- safety profile (adverse events and lab abnormalities)
- study drug compliance + PTC124 plasma concentrations
- 165 enrolled from 12 countries.
- year long study
- placebo vs low-dose vs high dose
- would have cost millions of millions
- found there was an improvement of 29m in lose dose
- statistical data was initially done on high dose → no change in this group from placebo
20
Q
What does human myocyte data suggest’?
A
- bell-shaped ataluren dose-response curve for dystriphin expression
- myocytes from phase 2a DBMD patients (N=29) culture in vitro with ataluren for 12 days
21
Q
What are conclusions of ataluren trials?
A
- drug may (?) have effect but this effect was insuffiecient to warrant licensing
- drug did not meet primary outcome measure of trial
- this therefore a failed trail
- millions of dollars spent
- thousands of patient- and doctor-hours lost
- lessons learnt:
- re-assessment of primary lab data
- re-assessment of outcome measure
- re-assessment of statistical methods
- plan: phase 3 trial now underway internationall
- result end 2015
22
Q
What is targeted exon skipping?
A
- antisense oligonucleotides
- act as a gene zipper
- skipping hole
- shortened in frame product
- there are certain hotspots
- certain exons that can be skipped to bypass deletions
- only need to design a certain number of agents
- e.g. exon exon 48 - 51
- AON therapy:
1st human trial in UK 2009
2nd large international trial started march 2011 - numerous international trials now underway
23
Q
What is a feature of new treatments in DMD?
A
- most new treatments are mutation specific
- have to know limits of deletion
- have to know what mutation a boy has
- time consuming and expensive to do sequence genome
24
Q
What is upregulation of compensatory protiens?
A
utrophin: an autosomal homologue of dystrophin
- maps to 6q24
- genomic length 1/3 that of dystrophin but transcript 13kb - similar to dystrophin
- can also bind to proteins of the DAPC
- dystrophin and utrophin share 74% amino acid sequence level and very similar domain structures
- expressed in place of dystrophin in foetal muscle, but in normal adult myofibres confined to the neuromuscular and myotendinous junctions
- in DMD, upregulated and expressed in sarcolemma
- significant structural similarities
25
Q
What is utrophin?
A
- in mdx mice, utrophin overexpression in myofibres by viral vector-mediated delivery or by transgeneic means can compensate restore normal muscle function
- because utrophin is expressed in foetal muscle and in adult non-muscle tissues, its over-expression in muscles of people with DMD is unklikely to provoke an immune response
- utrophin upregulation is therefore an attractive therapeutic approach for DMD
- SMTC1100: safe, well-tolerated, achieved plasma levels shown to increase utrophin in DMD patient cells in vitro
- phase 2 trial underway, phase 3 in next 18m
26
Q
What is blocking downstream effects?
A
- block abnormal Ca++ influx: stretch channel blockers, membrane sealers (poloxamer 188)
- fibrosis: 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
none curative, symptomatic
- probably useful in cocktail forms
27
Q
Summary?
A
- newer therapies are aimed at various points in the pathophysiology of MDs
- development of new therapies is difficult, time-consuming and expensive but there are real prospects for better treatment or cures for these conditions
