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Advances in gene therapy for phenylketonuria (PKU)

Advances in gene therapy for phenylketonuria (PKU). Cary O. Harding, MD Department of Molecular & Medical Genetics. Disclosures. BioMarin Corporation Funds for participation in clinical trials Sapropterin dihydrochloride rAvPAL-PEG National PKU Alliance Funds for PKU gene therapy research.

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Advances in gene therapy for phenylketonuria (PKU)

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  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

  9. PHE PHE TYR

  10. Phenylalanine hydroxylase (PAH) Tyrosine Phenylalanine BH4 qBH2 SR PTPS GTPCH DHPR PCD GTP

  11. 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?

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

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

  14. LSPmPAH rAAV2/8 • Portal vein injection • 5 X 1011 vg/mouse • 8 weeks post injection • 13-100 vg/haploid genome • 9.8-15.1% PAH activity

  15. 109 ± 6 vg 1.2 X 1010 vg 1.2 X 1011 vg 1.2 X 1012 vg 823 ± 80 vg 190 ± 16 vg

  16. 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

  17. rDNA-LSPmPAH rAAV2/8 • 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 2.5 X 1011 vg/mouse Portal vein injection

  18. Maximum Integration Frequency Conclusion: The maximum permanent integration frequency is 13 fold greater with rDNA-LSPmPAH rAAV2/8 vector.

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

  20. 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

  21. 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

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