Gene Therapy for PKU

Cary O. Harding, MD Molecular and Medical Genetics Gene therapy approaches to PKU

• Gene addition – Adding a functional gene that codes for phenylalanine hydroxylase (PAH) to liver cells • Gene correction or editing – Directly correcting the disease-causing mutation in the PAH gene Gene Minigene

Heterologous PolyA signal Promoter cDNA Weak promoters: -PGK The stability of the RNA -Pol2 depends on this sequence: Strong promoters -Bovine growth hormone -LTR -SV40 -humanCMV -SV40

Note: Some introns are kept for efficient splicing and export to the cytoplasm. Also, some enhancer sequences are added (e.g.. alpha1 antitrypsin in liver gene therapy)

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

Liver-directed rAAV2/8 gene therapy

• 10 adults with Hemophilia B (Factor IX) • Single PIV administration, escalating doses • 1.5-6% serum Factor IX activity • No acute toxicity • Transient transaminitis 2-3 weeks after injection in patient receiving high doses • 90% reduction in bleeding episodes and use of prophylactic Factor IX infusions

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 genome 9.8-15.1% PAH activity 1.2 X 1010 vg 1.2 X 1011 vg

1.2 X 1012 vg AAV-mediated liver-directed gene therapy trials • Ongoing: – Hemophilia A – Hemophilia B – Acute Intermittent Porphyria – Familial hypercholesterolemia • Planned: – Ornithine transcarbamylase (OTC) deficiency – Glycogen storage disease type 1a – Phenylketonuria The Holy Grail

• A cell-directed therapy that: – Is safe – Yields a physiologically-relevant permanent population of PAH+ cells • Selective growth advantage for PAH+ cells • Increased integration frequency of AAV genomes • Stable episomes in other tissues • Or: – Can be repetitively administered CRISPR/Cas9 gene editing Acknowledgements

• Grompe Lab - OHSU • Harding lab – OHSU – Markus Grompe – Shelley Winn – Nick Morcinek – Sandy Dudley – Zhongya Wang – Daelyn Richards – Laura Roy – Kathryn Baker • Koeberl lab – Duke – Katherine Cobb – Dwight Koeberl – Lindsey Stetson – Andy Bird – Baoyu Lin • Thöny lab – Zurich – Kelly Hamman – Beat Thöny • Funding – Alexander Rebuffat – NIDDK, NHLBI – Hiu Man Viecelli – NPKUA