214. Dominant-Negative Interference in the Pahenu2 Mouse: Modified Forms of PAH

Abstract

The common human genetic disorder Phenylketonuria (PKU) is primarily caused by defects in the enzyme phenylalanine hydroxylase (PAH). An animal model for PKU, the BTBR Pahenu2 mouse, has a missense mutation (F263S) that inactivates PAH; the mouse exhibits classic PKU, with elevated blood Phe levels, cognitive deficiencies, and maternal PKU syndrome. We have corrected both the serum Phe levels and maternal PKU in Pahenu2 mice using recombinant AAV vectors containing the mouse PAH gene. Although successful, unusually high vector doses were needed to achieve normal serum Phe levels. More critically, there was a gradual loss of therapeutic effect 20-24 weeks after vector delivery in many animals, particularly females. This was accompanied by changes in liver morphology suggestive of increased metabolic activity.Pahenu2 mice express reduced levels of a mutant PAH protein. The normal enzyme is a tetramer and we have reported evidence fordominant-negative interactions between endogenous mutant subunits and vector-introduced functional subunits. Possible approaches to combating a dominant-negative interaction include the production of ribozymes targeting the endogenous mutant mRNA and expression of modified forms of PAH protein that do not form tetramers. We have shown that a serotype 2 AAV vector expressing both a ribozyme directed against the mouse PAH mRNA and a ribozyme-resistant form of mPAH (mPAH-Hd) were effective at reducing serum Phe levels. We now show that modified forms of the PAH enzyme can, in certain cases, reduce dominant-negative interference. A vector expressing an obligate dimer form of mPAH was still subject to negative interference, indicating this inhibition occurs at the level of the monomer-monomer interaction. We will present in vitro and in vivo data examining the effectiveness of both a monomeric PAH enzyme as well as chimeric enzymes expressing a mouse PAH catalytic core linked to the rat tyrosine hydroxylase tetramerization domain in reducing dominant-negative interactions with missense subunits. The common human genetic disorder Phenylketonuria (PKU) is primarily caused by defects in the enzyme phenylalanine hydroxylase (PAH). An animal model for PKU, the BTBR Pahenu2 mouse, has a missense mutation (F263S) that inactivates PAH; the mouse exhibits classic PKU, with elevated blood Phe levels, cognitive deficiencies, and maternal PKU syndrome. We have corrected both the serum Phe levels and maternal PKU in Pahenu2 mice using recombinant AAV vectors containing the mouse PAH gene. Although successful, unusually high vector doses were needed to achieve normal serum Phe levels. More critically, there was a gradual loss of therapeutic effect 20-24 weeks after vector delivery in many animals, particularly females. This was accompanied by changes in liver morphology suggestive of increased metabolic activity. Pahenu2 mice express reduced levels of a mutant PAH protein. The normal enzyme is a tetramer and we have reported evidence fordominant-negative interactions between endogenous mutant subunits and vector-introduced functional subunits. Possible approaches to combating a dominant-negative interaction include the production of ribozymes targeting the endogenous mutant mRNA and expression of modified forms of PAH protein that do not form tetramers. We have shown that a serotype 2 AAV vector expressing both a ribozyme directed against the mouse PAH mRNA and a ribozyme-resistant form of mPAH (mPAH-Hd) were effective at reducing serum Phe levels. We now show that modified forms of the PAH enzyme can, in certain cases, reduce dominant-negative interference. A vector expressing an obligate dimer form of mPAH was still subject to negative interference, indicating this inhibition occurs at the level of the monomer-monomer interaction. We will present in vitro and in vivo data examining the effectiveness of both a monomeric PAH enzyme as well as chimeric enzymes expressing a mouse PAH catalytic core linked to the rat tyrosine hydroxylase tetramerization domain in reducing dominant-negative interactions with missense subunits.