Innovation Grant to Nurture Initial Translational Efforts (IGNITE) to Neurotherapeutic Approaches in Minipig Models of PKU Disorders

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Phenylketonuria (PKU) arises from phenylalanine hydroxylase (PAH) deficiency, with excess phenylalanine (PHE) in the blood leading to neurotoxicity and severe developmental disability (DD) if untreated. Although dietary treatment prevents major clinical features of PKU, poor adherence to a low PHE diet leads to neurodevelopmental, cognitive, and psychological problems, and deficits in executive functioning, psychiatric symptoms, ADHD, white matter degeneration, gait disturbances, and seizures. Indeed, most adolescents and adults are out of the therapeutic range for blood PHE (≤ 360 µMol/L). Additionally, in utero PHE exposure in Maternal PKU Syndrome (MPKUS) leads to DD, microcephaly, and heart defects. Thus, more effective therapeutic modalities are needed to increase PHE tolerance and reduce reliance on dietary PHE restriction. PKU mouse models poorly reflect the neurobehavioral phenotype of human patients. In contrast, pigs' physiology, anatomy, and genome are more similar to human, with both species gyrencephalic (folded cerebral cortex) in contrast to the lissencephalic (smooth) brains of rodents. Thus, a PAH-deficient pig should be a more faithful model of human disease. Using CRISPR-Cas9 genome editing, we efficiently generated deletions and inversions of porcine PAH exon 6 (PAHΔex6H170X null alleles) in cultured cells and zygotes. Subsequently, we generated the first PKU pig and a heterozygous carrier (both females) having two or one deletion alleles of PAH-exon 6, respectively. The PKU pig represents classic PKU (blood PHE >1200 µMol/L), with growth retardation and hypopigmentation. To establish a pre-clinical model for PKU, we propose to generate a minipig model of PAH deficiency (R21 phase) and to fully characterize biochemical, neurodevelopmental and behavioral phenotypes to demonstrate equivalence to human PKU and to identify biological markers as reliable endpoints for therapeutic testing (R33 phase). In Aim 1 [R21 phase], we will breed male and female F2 heterozygous pigs (with one PAH-deletion allele) to generate experimental cohorts; untreated PKU females will be bred to generate MPKUS animals. Clinical, neurological and behavioral evaluation will identify compromised development and behavior, with neuropathological studies marking the R21 endpoint. In Aim 2 [R33 phase], dietary-treated PKU (to manage neurologic presentation), dietary-untreated PKU, and control piglets will undergo neurodevelopmental phenotyping by neurological exam, cognitive and memory testing, MRI, and neuropathology, with a focus on myelination, structure and connections of the cerebral cortex, and presence of leukodystrophy. These and other biological markers (whole body PHE oxidation) will identify new outcome measures beyond PHE levels, as required by the FDA for verifying therapeutic testing in PKU. In summary, this study will provide an optimal pre-clinical model for PKU to allow new opportunities to optimize therapy, providing a model system for discovery of next generation neurotherapeutics of brain disorders.