1. Steven Steinberg, Ph.D., F.A.C.M.G. Hugo W. Moser Research Institute at KKI & Johns Hopkins University School of Medicine

2. Zellweger Spectrum Disorders MostSevereLeastSevere ZellwegerSyndromeNeonatalAdreno-leukodystrophyInfantileRefsumDisease

3. Peroxisome Assembly: Matrix Protein Receptor Recycling

4. PEX1 Common Mutations Severity ZellwegerSyndrome NeonatalAdreno-leukodystrophyInfantileRefsumDisease I700fs & I700fs I700fs & G843D G843D & G843D LeastSevere MostSevere

5. Biochemical: Summary

6. Why Create a Pex1-G844D Mouse Model? -A model for testing possible therapeutic interventions. e.g.PEX1-G843D is temperature sensitive, indicating it may be responsive to a chaperone-like molecule M 37 30 wild type PEX1- thiolase P P, thiolase precursor, 44 kD M, mature thiolase, 42 kD Pex5 -A means of exploring aspects of pathogenesis not readily explored in humans e.g. RNA expression profiling of multiple tissues

7. Construct Design: Pex1-G844D Construct generated → ES cells electroporation and selection → Blastocyst injection, implantation & chimera birth → Chimera breeding → Germline G844D Transmission

8. Matings of Pex1-G844D Heterozygotes MaleFemaleTotal Wt / Wt91423(25.3%) G844D / Wt192544(48.4%) G844D / G844D131124(26.4%) TOTAL:41 (45%)50 (55%)91 No evidence of neonatal lethality.

9. 3 Week Old Littermates Pex1-G844D / WT Pex1-G844D / Pex1G844D

10. Growth Curve (n = 14) (n = 13) (n = 26) (n = 22) (n = 17) (n = 10)

11. Survival Homozygotes as of June 2013 DIED OF NATURAL CAUSES SexNumberRange (in days)Median Male615-14738 Female87-4516 LIVING COMFORTABLY SexNumberRange (in days)Median Male2026-250106 Female1769-258119

12. Fertility Male homozygotes can be fertile 4 Male Homozygote x Female Heterozygote Matings yielded litters Male homozygote age range at litter birth:95-110 days Female heterozygote age range at litter birth:89-110 days Number of days pair were together at litter birth:25-67 days No evidence that female homozygotes are fertile 5 Male Homozygote x Female Homozygote Matings Male homozygote age range at end of time together:95-135 days Female heterozygote age range at end of time together:95-187 days Number of days pair were together:52-92 days

13. Cultured Fibroblasts Very Long Chain Fatty Acid Content Pex1-G844D AllelesC26:0 µg/mgC26:0/C22:0 Wildtype0.0230.053 Carrier0.0280.073 Homozygote0.4651.298

14. Cultured Fibroblasts Plasmalogen Synthesis Pex1-G844D Alleles 3 H / 14 CC16DMAC18DMA Wildtype0.694.0751.795 Carrier0.664.0972.041 Homozygote0.832.0980.641

15. Cultured Fibroblasts Branched Chain Fatty Acid Metabolism pmol / 48 hours / mg protein Pex1-G844D AllelesPhytanic AcidPristanic Acid Wildtype582487 Carrier406 (70%)509 (105%) Homozygote119 (20%)300 (62%)

16. Cultured Fibroblasts Catalase Solubility Pex1-G844D Alleles% Soluble Wildtype11.6% Carrier11.8% Homozygote83.3%

17. Immunocytochemistry WildtypePex1-G844D / Pex1G844D

18. Chaperone Treatment of Fibroblasts

20. Whole Blood Lipids C26:0-Lysophosphorylcholine Pex1-G844D Alleles% C26:0-LPC Wildtype (n=42)0.052 ± 0.016 Carrier (n=67)0.052 ± 0.018 Homozygote (n=17)0.558 ± 0.363

21. Whole Blood Lipids Phosphatidylethanolamine Plasmalogens Pex1-G844D AllelesSum of Four (pmoles) Wildtype (n=42)5.97 ± 2.34 Carrier (n=67)5.96 ± 2.83 Homozygote (n=17)2.82 ± 2.22

22. Bile Acid Intermediate DHCA Pex1-G844D AllelesPlasma*Feces** Wildtype (n=3,4)0.063 ±0.129 1.064 ±0.378 Carrier (n=5,4)0.073 ±0.0520.971±0.406 Homozygote (n=1,3)8.719.115 ±14.529 *pmol per 10µl plasma; **pmol per mg feces

23. Liver Histology Wildtype Pex1-G844D / G844D

24. Tissue Lipids p = 0.007

25. ERG Wave Forms Visual Testing Electroretinograms b-wave a-wave

26. Visual Testing Electroretinograms Series 1 = Wildtype and Heterozygous Mice, n=4 Series 2 = Pex1-G844D Homozygous Mice, n=4

27. Visual Testing Retinal Histology: Cone Photoreceptor Staining Homozygote, 28 days Heterozygote, 28 days Homozygote, 147 daysWildtype, 149 days inl = inner nuclear layer onl = outer nuclear layer is = inner segment of photoreceptor layer os = outer segment of photoreceptor layer

28. Pex1-G844D Mouse Model Summary -Growth stunted - -Males can be fertile, but not female -Biochemical profile is comparable to human patients homozygous for PEX1-G843D -Compelling evidence of retinal pathology and dysfunction comparable to human patients -Preliminary evidence of liver dysfunction that is likely comparable to human patients

29. Pex1-G844D Mouse Future Plans -Treatment with compounds identified by drug screening using a cell model system to test for in vivo efficacy -Behavioral studies -Generate Pex1 knock out and generate Pex1-KO / Pex1-G844D compound heterozygotes and tissue specific knock outs

30. Collaborators -Amanda Lauer -Tomohiro Masuda -Don Zack -Shandi Hiebler -Ann Moser -Anita Liu -Paul Watkins -Dominik Reisinger -Outside KKI / JHU -Jean Bennett -Nancy Braverman -Gerald Raymond -Joseph Hacia -Michael Paine -Phyllis Faust Sponsored by the Park-Hopkins and Woodbury research funds donations at Kennedy Krieger Institute