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

Salt Lake City 2002

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

Gene therapy

Genetic manipulation for therapeutic purposes

• 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

Retrovirus life cycle

PHE

PHE TYR

Phenylalanine hydroxylase (PAH)

Phenylalanine Tyrosine

qBH2BH4

DHPR PCDGTP

GTPCH

PTPS

SR

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?

Therapeutic liver repopulation

Hamman, et al, Molec Med Genet, 2011

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

1.2 X 1010 vg

1.2 X 1011 vg

1.2 X 1012 vg

823 ± 80 vg 190 ± 16 vg

109 ± 6 vg

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