Lessons from the Mouse:Lessons from the Mouse:Rett Syndrome is Potentially Rett Syndrome is Potentially

TreatableTreatable

John ChristodoulouJohn Christodoulou

NSW Centre for Rett Syndrome ResearchNSW Centre for Rett Syndrome Research

Western Sydney Genetics Program, Children’s Hospital at WestmeadWestern Sydney Genetics Program, Children’s Hospital at Westmead

Disciplines of Paediatrics & Child Health and Medical Genetics, Disciplines of Paediatrics & Child Health and Medical Genetics,

University of SydneyUniversity of Sydney

Testing the motor ability of Testing the motor ability of Mecp2-deficient miceMecp2-deficient mice

Is the brain impairment Is the brain impairment

in Rett syndrome in Rett syndrome

permanent?permanent?

new research suggests NO!new research suggests NO!

Experimental DesignExperimental Design• created a mouse model where expression of created a mouse model where expression of

Mecp2Mecp2 is blocked is blocked– males have severe neurological abnormalities & reduced males have severe neurological abnormalities & reduced

lifespanlifespan

– females have less severe neurological abnormalities & normal females have less severe neurological abnormalities & normal survivalsurvival

• mouse engineered so that mouse engineered so that Mecp2Mecp2 expression expression is restored on exposure to a specific drugis restored on exposure to a specific drug

Reversal of Neurological Defects in a Reversal of Neurological Defects in a Mouse Model of Rett SyndromeMouse Model of Rett Syndrome

Guy et al. Science, 2007Guy et al. Science, 2007

cencenteltel

MBDMBD TRDTRD 3’UTR3’UTR

MECP2 MECP2 Gene OrganisationGene Organisation

MBD MBD Methyl Binding DomainMethyl Binding DomainTRD TRD Transcription Repression DomainTranscription Repression Domain3’UTR 3’UTR Untranslated RegionUntranslated Region

Western blot – absence ofMecp2 protein

In situhybridisation

of dentategyrus

-no Mecp2protein

Survival characteristicstypical of null mouse- symptoms develop @ ~ 6 weeks & averagesurvival 11 weeks

The The Stop Stop cassette can be cut out of the gene by a cassette can be cut out of the gene by a specific enzyme to restore the specific enzyme to restore the Mecp2Mecp2 gene allowing gene allowing it to make the normal Mecp2 protein again.it to make the normal Mecp2 protein again.

With reactivation of With reactivation of Mecp2, 9/17 maleMecp2, 9/17 male RTT RTT mice developed mice developed toxicity and died.toxicity and died.

The rest showed no The rest showed no toxicity and had toxicity and had normal survivalnormal survival

Toxic effect Toxic effect resembled that seen resembled that seen when Mecp2 is when Mecp2 is overexpressed in overexpressed in mice.mice.

BeforeBefore

12 week old male mouse:12 week old male mouse:Note low stance, inertia, tremor, arrhythmic breathing, and Note low stance, inertia, tremor, arrhythmic breathing, and moderate hind limb clasping. moderate hind limb clasping.

Drug treatment was initiated on this day. Drug treatment was initiated on this day.

AfterAfter

The same mouse as shown in the previous movie four The same mouse as shown in the previous movie four weeks later after a course of five weekly drug injections. weeks later after a course of five weekly drug injections.

Female mice with the Female mice with the StopStop cassette develop RTT cassette develop RTT features @ 4 – 12 months, have a normal lifespan, features @ 4 – 12 months, have a normal lifespan, and the phenotype appears to stabilise. Often and the phenotype appears to stabilise. Often become obese.become obese.

Female mice that received identical drug treatment regimes 26 weeks prior to filming. Female mice that received identical drug treatment regimes 26 weeks prior to filming.

The first mouse seen is a mutant female that displayed symptoms at the beginning of The first mouse seen is a mutant female that displayed symptoms at the beginning of the treatment and is now indistinguishable from normal.the treatment and is now indistinguishable from normal.

The second mouse entering the frame is a normal female. The second mouse entering the frame is a normal female.

The third mouse to appear is a mutant female not treated. Note inertia and obesity of The third mouse to appear is a mutant female not treated. Note inertia and obesity of this third mouse. this third mouse.

SummarySummary

• absence of MeCP2 does absence of MeCP2 does NOTNOT irreversibly irreversibly damage brain cellsdamage brain cells

• there is now real hope that a cure for Rett there is now real hope that a cure for Rett syndrome might be possiblesyndrome might be possible

• translating this to treatment in humans will translating this to treatment in humans will be the next trick!be the next trick!

BackgroundBackground• nonsense mutations (in frame UAA, UAG or UGA) nonsense mutations (in frame UAA, UAG or UGA)

cause the production of the MeCP2 protein to stop cause the production of the MeCP2 protein to stop deaddead– this is called premature terminationthis is called premature termination

• gentamicin prompts ribosomes to read through gentamicin prompts ribosomes to read through premature termination codons (PTCs)premature termination codons (PTCs)– but not particularly potent; toxic to the kidneys and inner earbut not particularly potent; toxic to the kidneys and inner ear

• small non-toxic compounds identified through high small non-toxic compounds identified through high throughput screening that promote PTC read throughthroughput screening that promote PTC read through

An Emerging Therapy PTC124An Emerging Therapy PTC124Welch et al. Nature 2007: 447; 87 - 91Welch et al. Nature 2007: 447; 87 - 91

http://www.ptcbio.com/3.1.1_genetic_disorders.aspxhttp://www.ptcbio.com/3.1.1_genetic_disorders.aspx

Structural effects of PTC124 Structural effects of PTC124 on on mdxmdx mouse mouse

PTC124 therapy PTC124 therapy results in the results in the generation of generation of normal normal dystrophin dystrophin proteinprotein

... and was associated with functional improvements...

- improved muscle strength

- reduced exercise associated muscle damage

- reduced CPK levels (marker of muscle damage)

• no obvious toxicityno obvious toxicity

• relatively frequent type of mutation (5 – 70%)relatively frequent type of mutation (5 – 70%)– Duchenne dystrophyDuchenne dystrophy 13% 13% cystic fibrosiscystic fibrosis 10% 10%

– Rett syndromeRett syndrome 30% 30%

• phase 2 trials in DMD and CF currently under phase 2 trials in DMD and CF currently under wayway

The Potential for The Potential for PTC124 TherapyPTC124 Therapy