1. Phenylbutyrate increases SMN gene expression in vitro and in vivo Christina Brahe Università Cattolica del S.Cuore, Roma

2. SMA is caused by homozygous absence of the SMN1 gene cen tel SMN1 SMN2

3. SMA patients have one or more SMN2 genes which modulate the disease severity SMN2 severe form mild form SMN2 These genes produce an insufficient level of SMN protein

4. Hypothesis Particular drugs may activate the SMN2 genes to produce more SMN protein SMN improvement of muscle strength

5. There are drugs that open up the DNA to allow the expression of genes at a higher level inactive gene condensed chromatin active gene HDAC accessible chromatin HAT

6. Phenylbutyrate belongs to this class of drugs FDA approved for the treatment of another pediatric disease is generally well tolerated crosses the blood-brain barrier

7. Study of the effect of phenylbutyrate on SMN2 expression in cell cultures from SMA patients Increase in SMN transcripts (precursor of protein) in most but not all cultures the increase in transcripts ranged from 50%-400% increase in SMN protein Results

8. patient % increase in SMN2-fl transcripts SMA I SMA II SMA III 50 100 150 200 250 300 350 400 450 500 12345678910111213141516 Maximum increase in SMN2-fl transcript levels

9. % increase in SMN protein 16 h 24 h 20 40 60 80 100 120 SMA I SMA II SMA III Increase in SMN protein

10. Study of the effect of phenylbutyrate on SMN2 expression in leukocytes of SMA patients Phenylbutyrate was administered to 6 patients (4 type II and 2 type III) and 3 parents for 7 days blood samples were taken before, during and after the trial blood samples were taken before, during and after the trial leukocyte SMN2 transcripts were measured leukocyte SMN2 transcripts were measured

11. T1T2 T3 T4 T7 Mean PB increases SMN expression in leukocytes -100 0 100 200 300 400 500 800900123456M2M3F612345 patientsparents healthy untreated controls -100 0 100 200 300 400 500 800 900 SMN-fl transcripts (%) relative to T0 Brahe et al., EJHG 2005

12. Open pilot trial 10 patients with SMA II (age: 30 months-12 years) 10 patients with SMA II (age: 30 months-12 years) Treatment with phenylbutyrate for 13 weeks Treatment with phenylbutyrate for 13 weeks (1 week on, 1 week off) (1 week on, 1 week off) Efficacy was evaluated by using a functional motor scale Efficacy was evaluated by using a functional motor scale

13. Three point scoring system: Three point scoring system: 2 scores for unaided, 1 for assistance and 0 for inability 20 items 20 items Score 2 points Score 1 point Score 0 Score Score 2 points Score 1 point Score 0 Score Touch one hand to head flexes head to hand unable ( ) Touch one hand to head flexes head to hand unable ( ) Functional motor scale

14. 0 5 10 15 20 25 30 35 2.63.53.63.84.16.16.37.79.712.7 Age (years) before 3 weeks 9 weeks Results on the functional scale Mercuri et al., 2004 score

15. 2 4 6 8 10 12 14 16 18 20 22 T1T2 Mean scores in treated patients and control group treated patients untreated controls T0 T3 (6 months ) T0 Mercuri et al., 2004 score

16. Conclusions of the open trial Conclusions of the open trial improvement of functional ability children <5 years had a more obvious improvement great variability of response (as previously observed in vitro)

17. Large randomised, double-blind, placebo controlled trial Enrolled : 106 patients SMA II or non-ambulant type III Age: 30 months -12 years Drop out: 14 (13%) Scheduleintermittent (1 week on, 1 week off) for 13 weeks Schedule: intermittent (1 week on, 1 week off) for 13 weeksAssessments: Hammersmith functional motor scale Hammersmith functional motor scale myometry (>5 years) myometry (>5 years) FVC (>5 years) FVC (>5 years)

18. Problems to be solved More reliable outcome measures (more sensitive functional motor scale and quantitative muscle testing, etc) protocol design to achieve the maximum response (dosage, continuous or pulsed administration, duration) gain information on the mechanisms underlying the difference in response

19. Istituto di Genetica Medica Istituto di Genetica Medica Università Cattolica, Roma Università Cattolica, Roma Danilo Tiziano Carla Angelozzi Federica Borgo Anna Maria Pinto Daria Darelli Tiziana Vitali Giovanni Neri Acknowledgements: Patients and parents FSMA USA FSMA Italy A.S.A.M.S.I. Telethon - Italy Ospedale Bambino Gesù Rome Enrico Bertini Istituto di Neurologia Università Cattolica, Roma Eugenio Mercuri