1. Lessons from the Mouse: Rett Syndrome is Potentially Treatable John Christodoulou NSW Centre for Rett Syndrome Research Western Sydney Genetics Program, Children’s Hospital at Westmead Disciplines of Paediatrics & Child Health and Medical Genetics, University of Sydney

2. Testing the motor ability of Mecp2-deficient mice

3. Is the brain impairment in Rett syndrome permanent? new research suggests NO!

4. Experimental Design created a mouse model where expression of Mecp2 is blockedcreated a mouse model where expression of Mecp2 is blocked –males have severe neurological abnormalities & reduced lifespan –females have less severe neurological abnormalities & normal survival mouse engineered so that Mecp2 expression is restored on exposure to a specific drugmouse engineered so that Mecp2 expression is restored on exposure to a specific drug Reversal of Neurological Defects in a Mouse Model of Rett Syndrome Guy et al. Science, 2007

5. cen tel MBDTRD3’UTR MECP2 Gene Organisation MBD Methyl Binding Domain TRD Transcription Repression Domain 3’UTR Untranslated Region

6. Western blot – absence of Mecp2 protein In situ hybridisation of dentate gyrus -no Mecp2 protein Survival characteristics typical of null mouse - symptoms develop @ ~ 6 weeks & average survival 11 weeks

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

8. With reactivation of Mecp2, 9/17 male RTT mice developed toxicity and died. The rest showed no toxicity and had normal survival Toxic effect resembled that seen when Mecp2 is overexpressed in mice.

9. Before 12 week old male mouse: Note low stance, inertia, tremor, arrhythmic breathing, and moderate hind limb clasping. Drug treatment was initiated on this day.

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

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

12. 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 treatment and is now indistinguishable from normal. 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 this third mouse.

13. Summary absence of MeCP2 does NOT irreversibly damage brain cellsabsence of MeCP2 does NOT irreversibly damage brain cells there is now real hope that a cure for Rett syndrome might be possiblethere is now real hope that a cure for Rett syndrome might be possible translating this to treatment in humans will be the next trick!translating this to treatment in humans will be the next trick!

14. Background nonsense mutations (in frame UAA, UAG or UGA) cause the production of the MeCP2 protein to stop deadnonsense mutations (in frame UAA, UAG or UGA) cause the production of the MeCP2 protein to stop dead –this is called premature termination gentamicin prompts ribosomes to read through premature termination codons (PTCs)gentamicin prompts ribosomes to read through premature termination codons (PTCs) –but not particularly potent; toxic to the kidneys and inner ear small non-toxic compounds identified through high throughput screening that promote PTC read throughsmall non-toxic compounds identified through high throughput screening that promote PTC read through An Emerging Therapy PTC124 Welch et al. Nature 2007: 447; 87 - 91 http://www.ptcbio.com/3.1.1_genetic_disorders.aspx

15. Structural effects of PTC124 on mdx mouse PTC124 therapy results in the generation of normal dystrophin protein... and was associated with functional improvements... - improved muscle strength - reduced exercise associated muscle damage - reduced CPK levels (marker of muscle damage)

16. no obvious toxicityno obvious toxicity relatively frequent type of mutation (5 – 70%)relatively frequent type of mutation (5 – 70%) –Duchenne dystrophy13% cystic fibrosis 10% –Rett syndrome 30% phase 2 trials in DMD and CF currently under wayphase 2 trials in DMD and CF currently under way The Potential for PTC124 Therapy