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Phenylketonuria (PKU) and related forms of non-PKU hyperphenylalaninemias (HPA) result from deficiencies in phenylalanine hydroxylase (PAH), the hepatic enzyme that catalyses the conversion of phenylalanine (phe) to tyrosine (tyr). Patients are characterised by a metabolic phenotype comprising elevated levels of phe and some of its metabolites, notably phenyllactate (PLA), phenylacetate (PAA) and phenylpyruvate (PPA), in both tissue and body fluids. Treatment from birth with low-phe diet largely prevents the severe mental retardation that is its major consequence. / Mechanisms underlying the pathophysiology of PKU are still not fully understood; to this end, the availability of an orthologous animal model is relevant. A number of N-ethyl-N-nitrosourea (ENU) mutagenized mouse strains have become available. I report a new heteorallelic strain, developed by crossing female ENU1 (with mild non-PKU HPA) with a male ENU2/+ carrier of a 'severe' PKU-causing allele. I describe the new hybrid ENU1/2 strain and compare it with control (BTBR/Pas), ENU1, ENU2 and the heterozygous counterparts. The ENU1, ENU1/2 and ENU2 strains display mild, moderate and severe phenotypes, respectively, relative to the control and heterozygous counterparts. / I describe a novel method using negative ion chemical ionization gas chromatography/mass spectrometry (NICI-GC/MS) to measure the concentration of PLA, PAA and PPA in the brain of normal and mutant mice. Although elevated moderately in HPA and more so in PKU mice, concentrations of these metabolites are not sufficient to explain impaired brain function; however phe is present in brain at levels associated with harm. / Finally, I describe a new modality for treatment of HPA, compatible with better human compliance: it involves enzyme substitution with non-absorbable and protected phenylalanine ammonia lyase (PAL) in the intestinal lumen, to convert L-phenylalanine to the harmless metabolites (trans-cinnamic acid and trace ammonia). PAL, taken orally, substitutes for the deficient PAH enzyme and depletes body pools of excess phe. I describe an efficient recombinant approach to produce PAL enzyme. I also provide proofs of both pharmacologic and physiologic principles by testing PAL in the orthologous mutant mouse strains with HPA. The findings encourage further development of PAL for oral use as an ancillary treatment of human PKU.

Absence of functional phenylalanine hydroxylase results in phenylketonuria (PKU). Viable treatments remain few, expensive and secondary conditions such as osteopenia occur in most PKU patients. Objective 1: Given the recently described roles of gut microbes to aid host digestion, an orally administered genetically engineered probiotic as the delivery vehicle for enzyme replacement therapy was created. The engineered probiotic, pHENOMMenal, produced phenylalanine ammonia lyase with significant production of trans-cinnamate (phenylalanine cleavage product) in vitro and resulted in a reduction of 515 μM in blood phenylalanine when fed to PKU animals for 14 days (from 2307µM ± 264µM to 1792µM ± 261µM, n = 6, P < 0.05). The control probiotic produced no change in blood phenylalanine. Thus, pHENOMMenal treatment in PKU mice demonstrated engineered microbes could compensate for a metabolic deficiency of the host. Objective 2: Evaluate the PAHenu2 mouse model of PKU for a genetic discrepancy causing ocular enlargement and delayed development observed only after the PAHenu2 mutation was crossed to the C57BL/6J mouse. When compared to healthy littermates, ELISA indicated a consistent but insignificant decrease in plasma IGF-1 and an increase in ocular IGF-1 in PKU animals. SNP screening demonstrated a differential inheritance of IGF-1 alleles in healthy and PKU animals based on PAH allele inheritance. Ocular and developmental phenotypes in the PAHenu2 colony match those described in previous IGF-1 studies. Understanding the IGF-1 inheritance discrepancy will enable better osteopenia research using PAHenu2 mice and allow breeding of a healthier mouse colony for continued research. Collectively the results from this work describe a new therapeutic approach for treatment of PKU as well as a better understanding of the PAHenu2 mouse model to study this disease.