1. A Clinical Trial of Betaine for Peroxisomal Biogenesis Disorders Nancy Braverman 1, Ann Moser 2, William Rizzo 3 1 McGill University, Montreal 2 Kennedy Krieger Institute, Baltimore 3 University of Nebraska Medical Center, Omaha

2. Peroxisomal Biogenesis Disorders Clinical features – Neonatal hypotonia, feeding problems – Developmental delay – Dysmorphic facial features – Seizures – Sensorineural deafness – Hepatic dysfunction, adrenal insufficiency Zellweger syndrome, neonatal ALD, infantile Refsum disease

3. Biochemical Abnormalities PEX gene defects – impaired peroxisomal protein import & peroxisome assembly Biochemical abnormalities – Peroxisomal β-oxidation - VLCFA, pristanic acid – Plasmalogen synthesis - RBC plasmalogens – Bile acid synthesis - bile acid precursors (DHCA, THCA) – Lysine degradation - pipecholic acid

4. Rationale for Betaine Betaine acts as a molecular chaperone to improve peroxisomal protein import of GFP-PTS1 reporter. Control100 mM betaine

5. Concentration Dependence of Betaine

6. Concentration and Time Dependence of Betaine in G843D/fs1700 Fibroblasts

7. Betaine has Additive Effect with Flavonoids for GFP-PTS1 import Betaine 25 mM Flavonoids 5μM

8. Betaine FDA approved for homocystinuria Good safety profile - rare GI problems Usual dose: up to 6 g per day (divided TID) Used in clinical studies of Rett and Angelman syndrome at up to 12 g per day w/o side effects

9. Clinical Trial of Betaine for PBD Study sites: Omaha and Montreal Open study design, non-blinded 12 subjects Enrollment criteria: – PEX1 genotype: G843D/null mutation or homozygous G843D/G843D – Expected survival >6 mo Endpoints: biochemical response after 6 mo

10. Clinical Trial of Betaine for PBD Betaine daily dose (÷ 3 times per day) – Age < 3 years: 3 g – Age > 3 years: 6 g – Dose escalation over 4-6 weeks Increase dose at 3 months – Age < 3 years: 6 g – Age > 3 years: 12 g

11. Clinical Trial of Betaine for PBD Peroxisome biochemical tests at: – Baseline, 3 mo and 6 mo Plasma VLCFA, pristanic acid RBC plasmalogens Plasma and stool bile acids Plasma pipecholic acid Safety monitoring: CBC, chemistry panel (LFTs), UA, plasma methionine, betaine

12. Clinical Trial of Betaine for PBD Betaine supplied by Rare Disease Therapeutics and Orphan Europe Funded by Global Foundation for Peroxisomal Disorders