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Introductory video

Animal models - Annemieke Aartsma-Rus

Animal models are useful but they have their limitations. A mouse is not a mini human.

Each step is important, the next step is always more complicated --> no guarantee.


Cells derived from patients

< muscle biopsy (invasive)

< skin biopsy (they convert the cells into muscle cells in a lab)


Mice model

Mostly used in drug development

--> they are cheap (small, cost limited, small amount of medicine needed, easy to breed)


The mdx mouse doesn’t produce dystrophin but the disease isn’t severe because they regenerate better and the utrophin compensates better (the disease is very severe when the mice don’t produce utrophin either).


New mouse: the genetic background is different, with poor regeneration and more fibrosis

BUT also has calcifications (humans don’t), it’s a poor breeder and there’s not a lot of data


Dog models

Problem: moral issues, larger so more expensive

Golden retriever model: big variety in severity. Mutation in exon 6 (they now have a new model with a mutation on exon 50 => new => limited data)


Is it useful to use the mdx mouse?

Almost 10.000 papers on Duchenne, close to 2.000 papers on mdx mouse, only 1 treatment didn’t work. For humans, only 2 treatments (are supposed to) work.

--> There are many trials, but many fail.

--> Very low predictive power.

--> Is it useful to use  the mdx mouse then?


We need a standardisation in pre-clinical trials.

Sometimes, two labs have different results when they test the same thing, that's not good, they really need to have the same results.

A standardisation helps to know if the hypothesis is correct. If so, an optimization is needed to go to clinical trials (dose, frequency, …). Sometimes trials are initiated very quickly (time is ticking).

It’s good to publish the papers even if it doesn’t work, people should definitely do it, to avoid wasting time while trying the same hypothesis another time.


Animal models are useful but have limitations
- We need good outcome measures and natural history for clinical and pre-clinical trials.

Gene therapy - Dr Cohn


  1. Substitution


Can we safely administer DMD genes? It appears so.

--> curative in MDX

--> profound improvement in dogs


  • Relatively effective and safe in animals

  • Long term action in widespread muscles

  • Improved clinical parameters and pathology

  • Prolonged survival (> 4 years for dogs)

  • For al DMD genotypes

    • Clinical trials in the USA + towards clinical trials in France


Challenges ahead:

  • Variability of microdystrophin restoration (on average 40% for dogs, depending on the dog)

  • Product manufacture @ large scale

  • Repetitive dosing may be required


To deliver full gene => 3 AAV (working on that too)


   2. Repair


Dr Cohn “CRISPR as the next generation of precision medicine therapies”

CRISPR = gene theapy 2.0


Discovered 5 years ago (natural process done by bacteria to protect themselves against viruses)

  • Trying to insert a “store-in cassette” to restore the full-length dystrophin!

Recent Developments - Thomas Voit


1. Exon Skipping

  • Efficient skipping of single exon

  • Delivery of AAV9-U7 mediated out-of-frame exon skipping 

-->Dystrophin expression


2. Myostatin inhibition

-->Several trials (anti-myostatin molecules)

* Myostatin inhibits muscle growth

* Follistatin = a protein that inhibits myostatin naturally


If we inhibit myostatin, it should have a positive effect. However, myostatin inhibitors seem to have problems to work and the results are largely disappointing in different muscular diseases.

  • With healthy muscles, it works

  • For neuromuscular diseases, it doesn’t really work


Two questions:


  1. Is it naturally reduced in NM diseases (NMD)?

Circulating levels of myostatin (serum) are lower in Becker and even lower in DMD BUT they have high follistatin levels! So they already do the process spontaneously


  1. Is it because they have less muscles?

Unhealthy muscles don’t synthesise the myostatin oversynthesise the follistatin.

  • If you don’t have the receptor, it cannot work!

Atrophic muscles => anti-myostatin isn’t going to work

BUT if we can correct the gene with gene therapy, then it will have an effect.

--> We have to check if the myostatin levels are high enough, otherwise the treatment won’t be useful.

3. Sarconeos (new)

First-in-class drug candidate based on the activation of the MAS - receptor.

If you activate this receptor:

  • pro-regenerative effect

  • growth effect

  • fibrosis lowers

  • cardiac fibrosis lowers

Data looks promising but of course, it’s with the mdx. Moving towards the clinical trials.

Early clinical Trials


 1. Wave

Phase 1 clinical trial for the treatment of DMD patients amenable to exon 51 skipping.

New technology, depending on how you connect anti-sense they will act differently and you can obtain different, and better, results.

  • moving towards phase 1 study (Q4 2017). WVE- DMDx51-001

  • Exon 51, ≥ 5 - ≤ 18, no previous 51 study


2. Eteplirsen (6-48 months)


3. NS-065 / NC NP-01-201 (Japan/USA) – exon skipping 53


4. Pfizer – Domagrozumab

 Phase 2: Anti-myostatin, extension study


5. Fibrogen FGCL-3019-079

Pamrevlumab – not only for DMD, treatment in fibrotic and fibro-proliferative diseases

Open-label, 12 years old and older (for 2 years)


6. CAP-1002 – cardiosphere-derived cells

= biologic product consisting of allogenic cardiosphere derived cells (CDCs) derived from donated heart muscle

  • do not engraft into host tissue

  • secrete extracellular vesicles (CDs exosomes) that contain a variety of signalling molecules

  • HOPE-Duchenne trial – age 12+ (LV scars in 4+ segments + EF > 35%)

  • Proof of principle study, cells delivered in the coronary

    • safe and well tolerated

    • significant improvement in LV,reduction in cardiac scars

    • Good surprise: Ú in the PUL

  • Follow-up, hope2 trial, intravenous this time, every 3 months


7. AVV Microdystrophyn – Gene therapy for DMD


o Nationwide children's + Sarepta (start end 2017)


3m-3y (1st cohort),4-7y (2nd cohort)


o Genethon + Sarepta (AAV8, 2018)


o Solid – SGT-001 (start end 2017)

US + exploring sites


o Pfizer (Bamboo)

pre-clinical finished, orphan drug designation (phase 1b human trial in 12 months)

First trials for young and older boys

Combinatorial therapies are still not in trial.

Repurposing drugs


Repurposing drugs is interesting => For 1 drug approved, 5.000 are thrown away!
DMD therapeutic pipeline => Too many drugs?

Promising drugs

• Blocking calcium entry

New compounds (bad problems with old ones)

o Rycal - armgo pharma, ARM210
= calcium release channel stabilizer

- Ryanodine receptors (RYR1) = major calcium release channels on the sarcoplasmic reticulum (SR) of striated muscles

- Improve force generation and exercise capacity

o Akashi Therapeutics
Still a long way to go, ok in mice but no trial

Enhancing muscle size and regeneration

o Creatine: conflicting data in trials

o Myostatin (cfr. T. Voit)

o Givinostat: already approved for another disease

Inhibiting oxidative stress

o Antioxidant – EGCG (Berlin, Germany)

• Inhibiting inflammation

o NF-Kß => new drugs

o Anti-inflammatory drugs => huge reservoir of drugs

• Inhibiting fibrosis

o Halofuginone (natural product) – trial suspended (Akashi


= SERM (selective estrogen receptor modulators)

o Used for breast cancer and precocious puberty

o In mdx mouse: normalized whole body force

o ➚ resistance to repeated contraction

Long term treatment had a positive effect
TAMDMD phase 3 trial 2018
(1 year, 20mg of Tamoxifen, 6 ½ > 12 years old, on stable
steroids regime, 50% Tamoxifen, 50% placebo, ambulant + nonambulant,
80 patients, ≠ study sites (not in Belgium)

Enhancing blood supply via non-selective phosphodiesterase (PDE) inhibition

o PTX (Pentoxifylline) – efficacious in mice

o Tadalafil (disappoint, not efficacious)

  • L-citrulline / metformin in DMD


Muscle function preserved longer, especially in younger patients (treated patients more stable) => other trial with + patients


o Oral compounds available


Myopax – muscle stem cells

Mesoangioblast – another way to exon skip

Taurine ➚ grip strength, force of muscles

TACT = TREAT-NMD Advisory Committee for Therapeutics

--> Provides multidisciplinary advice (non-profit)
--> For the Duchenne UK grant,they had to go through TACT

“Established in 2009, the TREAT-NMD Advisory Committee for Therapeutics (TACT) is a unique multi-disciplinary international group of well recognized academic and industry drug development experts as well as representatives of patient foundations and institutional governmental scientific research centers, who meet twice a year to review and provide guidance on the translation and development path of therapeutics programs in rare neuromuscular diseases with large unmet need, such as muscular dystrophies and amyotrophic lateral sclerosis (ALS). The confidential and comprehensive review provides recommendations including go-no-go milestones, is independent of any funding stream however it may enable subsequent funding.”

Outcome measures in DMD


  • 6-minute walking test (6MWT)

  • north star amb. assessment

  • time to climb 4 stairs, run/walk 10 metres, raise from the floor from lying

  • PUL (performance of upperlimb)

  • Respiratory function

Endpoints are different for each study / phase / …
Problem with Duchenne = important variability (mutation, steroids or not, each boy is different, …)


--> We need a better natural history data

--> AFM - natural history study (5 centres, 6-monthly visits, 4 years follow-up – ongoing but closed to recruitment. 2 groups (> 5 years old and 5-18 years old) – check respiration, PUL, handgrip, whole body DEXA scans,…

-->Illustrates the clinical variability! Between patients but also within patients (improvements and decreases)

--> Changes ➚ when become non-ambulant

--> On average, decline after 9 for respiratory functions, 10 for upper limps
Goals: to have less but better outcome measures and to understand the variability

--> It will help to design trials

MCID = minimal clinically important difference

-->The smallest change in an outcome measure that a patient/parent/clinician would identify
as meaningful

-->MCID in DMD study (study for NSAA and PUL, starting soon)

Standards of care for DMD

Treat-nmd, family guide –


• To receive the best care available

• To provide a basis for the introduction of new trials and treatments

o It’s patient centred (“nothing about me without me”)
o Multidisciplinary approach
o New version with more information for the older population


3 parts :


  • Diagnosis, endocrinology, nutritional management, rehabilitation


  • Respiratory, cardiology, endocrine/bone health, orthopaedic


  • Primary care and emergency, psychosocial, transitions

≠ stages


  • Pre-symptomatic

  • NSAA (north star ambulatory assessment) = test that checks 17 steps/items (walk, hop, stand on 1 heel, etc.) 0 = cannot do it, 1 = can do it but adapts, 2 = ok

  • Timed functional tests (time to run 10 metres, time to get up from the floor, time to climb/descend 4 steps)

  • Corticosteroids : age 4-5 years old (= the plateau phase), balance benefits and side effects, Prednisone, Deflazacort or Prednisone 10 days on/off (less effective)

    • After starting steroids (3 months and 6 months), check the benefits (new things? Nothing?) and the side effects + check urine and blood pressure at least once at the beginning (+ make sure has chicken pox immunity)


A few tips


  • Joint contractures: stretching to prevent it (at the beginning only ankles)

  • Regular exercises – especially in young boys (swimming, horse-riding, etc.)

  • Tip-toe walking? => more stretching, they do it for balance

  • Urine very dark? Plenty of fluid, should go back to normal after max. 24 hours, if not => hospital

Plane: SPO2 higher than 95% => ok, lower => resp. team (fit flighter certificate)


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