The genetically engineered minipig as a preclinical disease model for Neurofibromatosis type 1 syndrome

Abstract

Neurofibromatosis type 1 (NF1) syndrome is one of the most common inherited neurological disorders, affecting about one in every three thousand individuals. The disease typically manifests in childhood and can result in significant morbidity and a shortened lifespan. Despite decades of research, there is still no cure for NF1, and treatment is largely symptomatic. This is due in part to the multisystemic nature of the disease and a lack of representative, translational animal models. Murine models of NF1 mimic individual aspects of the syndrome, but none fully represents the complexity of disease seen in human patients. There is a need for new models of NF1 to complement the mouse and improve success in clinical trials. The objective of this dissertation was to fill this need by developing and characterizing the first genetically engineered NF1+/- minipig and determining its utility as a preclinical disease model for human NF1. Using targeted gene editing and somatic cell nuclear transfer cloning, we generated NF1+/- minipigs harboring a specific human disease allele associated with severe phenotypes in NF1 patients. We enrolled cohorts of NF1+/- minipigs and wild-type litter mate controls in a longitudinal phenotyping study and assessed for manifestations of NF1 syndrome. We performed gross assessments by physical examination, radiography, and magnetic resonance imaging over time. We also assessed the histological, molecular and biochemical defects associated with NF1 using tissues and primary cells isolated from lesions in NF1+/- minipigs. We evaluated several targeted therapies currently in clinical trials for NF1-associated neoplasia using pharmacokinetic and pharmacodynamic analyses in blood and clinically relevant tissues from NF1+/- minipigs. The results of these studies show that our NF1 minipig model offers a predictive preclinical disease model that will be crucial for developing and validating new NF1-targeted therapies as well as improved imaging and surgical modalities for early detection of nervous system tumors. Furthermore, the methods presented provide a blueprint for using minipig tissues to evaluate pharmacodynamics of new targeted therapies and using primary cells from minipigs to uncover novel targets and cellular interactions within the neurofibroma microenvironment.