207. Long-Term Therapy for PKU in Mice by rAAV Pseudotype 8 Gene Transfer to Liver

Abstract

Phenylketonuria (PKU) is caused by an autosomal recessive deficiency of the hepatic phenylalanine hydroxylase (PAH) leading to high serum phenylalanine and irreversible damage of the brain. We recently reported on the use of recombinant AAV2 to deliver the PAH gene to the liver of PKU mice (C57Bl/6-Pah-enu2), which is an exceptionally faithful model for the human disease (Ref: Ding, Georgiev and Thony, Gene Ther, advanced online publication Dec 2005). In this study we used an AAV2/8 hybrid vector expressing the murine PAH-cDNA under the control of the composite CMV enhancer-chicken beta-actin promoter and which was pseudotyped with capsids from serotype 8 and delivered to the liver of PKU mice via single injections of portal vein with 5x10e12 vg per mouse or tail vein with 2x10e13 vg per mouse. Untreated PKU mice exhibit normal expression of mutant Pah-mRNA and PAH protein, but no detectable enzyme activity. They consequently had Phe levels between 1500 to 2500 micromol/l (25 to 42 mg/dl), and we (and others) have noticed that Phe concentrations in PKU mice were slightly higher for females (1866 micromol/l) than males (1695 micromol/l) rendering females putatively more resistive to PKU treatment. Two weeks post hepatic or systemic delivery of AAV2/8 a complete phenotypic correction was observed in PKU mice with blood Phe levels decreasing from high to normal values (<120 micromol/l or 2mg/dl). The therapeutic levels of plasma Phe concentrations (360 micromol/l or 6 mg/dl), as determined approximately every four weeks, were observed for a period of at least 7-10 months. Here we report on the follow-up of this gene therapy approach during a period of over a year where animals were successively euthanized for analyzing their hepatic viral load and PAH expression, and only two females were kept in each study until the end of the experiment. For tail vein injection, one female started to rise Phe concentrations after 35 weeks, and at 56 weeks the two females had Phe blood levels of 1203 and 1453 micromol/l. For portal vein infusion, mice remained at therapeutic levels at least until week 61, and three weeks later the remaining two females had Phe values of 284 and 517 micromol/l. Thus far we have collected data on viral titers after 8 and 46 weeks for portal vein injection, and found a decrease from 3400 to below 1000 viral genome copies per diploid hepatocytes genome. PAH expression 8 weeks after portal vein injection was found to be moderate with a 32% increase of Pah-mRNA and a 2.2-fold increase of PAH protein, and PAH enzyme activity comparable to wild-type mice. Expression analysis of PAH and parameter important for the enzymatic activity at different treatment intervals are under way. In conclusion, a 4-fold lower concentration of the AAV2/8 vector directly injected into the portal vein as compared to tail vein resulted in a more efficient long-term correction of serum Phe levels. Furthermore, understanding the mechanism for the re-rise of blood Phe levels might help to improve a gene therapy protocol to eventually perform complete and persistent correction of PKU by hepatic gene transfer with AAV vectors.