Phenylketonuria as a paradigm disease to understand protein instability in conformational disorders and to develop chaperoning therapies

Phenylketonuria (PKU) is the most common inborn error of metabolism (IEM), caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). This enzyme degrades Phe and in PKU, due to the enzymatic loss of function, Phe is accumulated, which can cause mental- and physical disabilities if the disease is not treated. Treatment includes a strict Phe-free diet, which means that patients cannot eat proteins. Development of new effective therapies for PKU, and IEM in general, has shown to be difficult. This project aimed to increase the knowledge of the molecular and cellular mechanisms leading to destabilization of PHA in PKU, and the methodological competence for development of a new treatment that corrects unstable, wrongly folded mutant PAH. One of the most important tools in the development of new treatments are animal models for the disease. In this project a new mouse model for PKU, the first with a human mutation, has been selected and used. It represents one of the most common mutations for PKU, including a large group of patients. Before using a new mouse model in the development of medicines, it is important to subject it to very thorough characterization at different ages. This is done to confirm that the mice are suitable models for the human patients. During the characterization this mouse has been found to suffer from oxidative stress due to the presence of large PAH aggregates in liver, which have never been seen before in a PKU model. This aggregation and its effect on the mice are expected to get worse with age, and the characterization was therefore extended to include old mice as well. But the expected increase in oxidative stress was not detected in the old mice who turned out to show a degree of normalization and adaptation to the large PAH aggregates. This Industrial-PhD project has increased the candidate's competence in the use of mice as disease models in general and the characterization of the effect of PAH mutations in a PKU mouse model in particular, providing advanced knowledge for their use in the study of therapies. This combined expert knowledge will pave the way for optimization of corrective therapies, such as pharmacological chaperones, for PKU and other IEM and genetic cancers. The research group at the University of Bergen and Pluvia collaborate in the guidance of the candidate.

The PhD project has generated increased knowledge on animal models and the generation of a systems medicine model that will benefit the development of new treatment for phenylketonuria (PKU)-patients. This knowledge has contributed to decision making and will increase the assets of the company. The research can enable selection of therapies including combination therapies that increase the functionality in a mutant dependent manner and opens for patient-tailored therapies. Additional outcomes include novel insights in the proteostasis network in PKU. The knowledge can be transferred to development of treatment for other genetic diseases. Application of the research results in this PhD project and education of high skilled researchers within the field of genetic diseases and animal models contribute to developing an internationally competitive life science ecosystem.

Phenylketonuria (PKU) is an inborn error of metabolism (IEM) caused by mutations in the enzyme phenylalanine hydroxylase. Left untreated, PKU leads to elevated, neurotoxic levels of phenylalanine. PKU has the highest incidence among IEMs (1 in 10000 births). Developing effective therapies for IEMs is challenging and the present project has been designed to increase knowledge and methodological expertise to facilitate the generic development of drug candidates for next generation chaperoning therapies. As for PKU, most IEMs lead to instability and misfolding of the mutated enzymes with consequent loss-of-function. The cell tries to limit the accumulation of misfolded proteins using folding assistance by molecular chaperones and degradation through the ubiquitin-dependent proteasome and/or autophagy, collectively constituting the protein quality control (proteostasis system). There is a need for highly trained scientists with competence in integrative and innovative research with commercialization potential. The purpose of the present PhD project is to increase the proficiency of the PhD candidate on the characterization on the effect of mutations in cells and in PKU-model mice, on proteostasis mechanisms in PKU, and on corrective therapies, notably pharmacological chaperones (PCs). These are small compounds that prevent and correct instability, and are the trademark of Pluvia AS. This combined knowledge will pave the way to the optimization of PC-therapies for PKU and to develop a generic translational workflow for PC selection for other IEMs and genetic forms of cancer. The project benefits from both the cutting-edge expertise of the Martinez Lab, UiB, in biophysical, cellular and animal methods and the unique expertise of Pluvia on protocols for the discovery, development and proof-of-concept of PCs. UiB and Pluvia will collaborate on the supervision of the candidate, resulting in a high added value to the PhD project and the commercialization of PC-based drugs.