lecture 36

Question 1

What muscle degeneration occurs in the muscular dystrophies?

Answer 1

• 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

Question 2

What is DMD disease progression?

Answer 2

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)

Question 3

How do we treat MDs?

Answer 3

• 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

Question 4

How can we approach the problem of treatment?

Answer 4

1. gene repair or replacement
   - cell and stem cell transfer
   - gene replacement (AAV, myoblast transfer)
   - translation: stop codon read through (Ataluren etc)
   - RNA splicing
2. upregulation of compensatory proteins
   - utrophin, alpha-dystrobrevin, alpha7integrin, GALNAx, NOS etc
3. 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

Question 5

What is an overview of strategies for DMD gene therapy?

Answer 5

• adenoviral vetors
• herpes simplex viral vectors
• plasmid vectors
• myoblast transplantation
• stem cell therapy
• chimeric oligonucleotides
• gentamicin therapy
• tAAV vectors
• antisense oligonucleotides
• utrophin upregulation

Question 6

How do treatments for MDs address multiple points in pathophysiology?

Answer 6

• downstream effects of decreased dystrophin/a-sarcoglycan

- sometimes have more than one effect

Question 7

What is gene repair/replacement in DMD?

Answer 7

• cell replacement:
• • myoblast transfer therapy
• • stem cell therapy
• gene replacement
• gene repair and/or upregulation
• nonsense mutation suppression
• targeted exon skippin

Question 8

What is MTT?

Answer 8

• 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

Question 9

What is seen after single injection in myoblast transfer?

Answer 9

• intense, cell-mediated host immune response
• > 90% myoblasts are elimated within 1 hour
• most killed within minutes of injection
• no effective dystrophin production

Question 10

What is stem cell therapy?

Answer 10

• 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

Question 11

What is gene repalcement?

Answer 11

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

Question 12

What are problems with gene replacements?

Answer 12

• those of all genetic disorders i.e. immune response to vector

Question 13

What are adenoviral vectors?

Answer 13

• 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

Question 14

What are adeno-associated vectors (AAV)?

Answer 14

• smaller, less, immunogenicity
• some effect in other MDs (smaller genes), not yet in DMD
• ? role for microdystrophins (rod domain shortened)

Question 15

What is nonsense mutation supression?

Answer 15

• 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

Question 16

What is PTC124?

Answer 16

• 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

Question 17

What was phase 1 trial of ataluren?

Answer 17

• 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

Question 18

What was phase 2?

Answer 18

• 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

Question 19

What was international DMD ataluren phase 2b study?

Answer 19

• 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

Question 20

What does human myocyte data suggest’?

Answer 20

• 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

Question 21

What are conclusions of ataluren trials?

Answer 21

• 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

Question 22

What is targeted exon skipping?

Answer 22

• 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

Question 23

What is a feature of new treatments in DMD?

Answer 23

• 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

Question 24

What is upregulation of compensatory protiens?

Answer 24

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

Question 25

What is utrophin?

Answer 25

- 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

Question 26

What is blocking downstream effects?

Answer 26

- 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

Question 27

Summary?

Answer 27

- 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