1. Advances in gene therapy for phenylketonuria (PKU) Cary O. Harding, MD Department of Molecular & Medical Genetics

2. Disclosures BioMarin Corporation –Funds for participation in clinical trials Sapropterin dihydrochloride rAvPAL-PEG National PKU Alliance –Funds for PKU gene therapy research

3. Salt Lake City 2002

4. Outline Physiologic requirements for successful PKU gene therapy Liver-directed recombinant adeno- associated virus gene therapy Design and evaluation of novel gene therapy vectors containing the human PAH cDNA

5. Gene therapy Genetic manipulation for therapeutic purposes

6. 6 adults with Hemophilia B (Factor IX) Single PIV administration, escalating doses 1.5-5% serum Factor IX activity No exogenous clotting factors required No acute toxicity Transient transaminitis 2-3 weeks after injection

7. Adeno-associated virus (AAV) Parvovirus family Nonpathogenic Replicates only in presence of Ad High titers Wild type integrates into the host genome Vectors integrate only rarely

8. Retrovirus life cycle

10. PHE TYR

11. Phenylalanine hydroxylase (PAH) Phenylalanine Tyrosine qBH 2 BH 4 DHPRPCD GTP GTPCH PTPS SR

12. What are the physiologic requirements for gene therapy? Which organ? How many cells must express the therapeutic transgene? How much expression per cell? Is permanent expression needed? Does gene expression need to be regulated?

13. Therapeutic liver repopulation Hamman, et al, Molec Med Genet, 2011

14. LSPmPAH rAAV2/8 LSP promoter = strong Liver Specific Promoter Chimeric human  1-microglobulin/bikunin enhancer (2 copies) and human thyroglobulin promoter

15. LSPmPAH rAAV2/8 Portal vein injection 5 X 10 11 vg/mouse 8 weeks post injection 13-100 vg/haploid genome 9.8-15.1% PAH activity

16. 1.2 X 10 10 vg 1.2 X 10 11 vg 1.2 X 10 12 vg 823 ± 80 vg 190 ± 16 vg 109 ± 6 vg

17. Targeted rAAV integration Grompe lab –Permanent integration in up to 5% of hepatocytes Kay lab –FIX expression resistant to partial hepatectomy in Hemophilia B mice –95% of integrations are site specific

18. rDNA-LSPmPAH rAAV2/8 2.5 X 10 11 vg/mouse Portal vein injection Six week evaluation Single male mouse 18% wild type PAH activity Terminal evaluation 2 remaining mice 3-5% wild type PAH activity Site specific integration detected

19. Maximum Integration Frequency VectorIntegration frequency rDNA-LSPmPAH (n = 10) 0.217 ± 0.305 LSPmPAH (n = 2) 0.016 ± 0.004 Conclusion: The maximum permanent integration frequency is 13 fold greater with rDNA-LSPmPAH rAAV2/8 vector.

20. Non-viral gene therapy Minicircle DNA Courtesy of Hiu Man Viecelli and Beat Thöny, Zurich, Switzerland

21. hPAH vector development Full length and truncated versions of codon optimized human PAH cDNA Plan to incorporate best human PAH cDNA into self complementary rAAV2/8 vector

22. Acknowledgements Grompe Lab - OHSU –Markus Grompe –Nick Morcinek –Zhongya Wang –Laura Roy Koeberl lab – Duke –Dwight Koeberl –Andy Bird Thöny lab – Zurich –Beat Thöny –Hiu Man Viecelli –Alex Rebuffat Harding lab – OHSU –Shelley Winn –Katie Cobb –Kevin Watanabe-Smith –Lindsey Stetson –Baoyu Lin –Gloria Baca –Kelly Hamman Funding –NPKUA –NIH