advances in gene therapy for phenylketonuria (pku) cary o. harding, md department of molecular &...
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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
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
• 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
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
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?
LSPmPAH rAAV2/8
LSP promoter = strong Liver Specific Promoter
Chimeric human 1-microglobulin/bikunin enhancer (2 copies) and human thyroglobulin promoter
LSPmPAH rAAV2/8• Portal vein injection• 5 X 1011 vg/mouse• 8 weeks post injection
13-100 vg/haploid genome9.8-15.1% PAH activity
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
rDNA-LSPmPAH rAAV2/8
2.5 X 1011 vg/mousePortal 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
Maximum Integration Frequency
Vector Integration 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.
Non-viral gene therapy
Minicircle DNA
Courtesy of Hiu Man Viecelli and Beat Thöny, Zurich, Switzerland
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
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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