Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is an autosomal recessive disorder caused by mutations in the CLN3 gene with a worldwide incidence of 1 in 12,500 live births. It is part of a group of lysosomal storage disorders - the neuronal ceroid lipofuscinoses (NCLs) - that together constitute the most common neurodegenerative diseases of childhood. JNCL is a devastating disease that results in vision loss, motor and cognitive deficits, seizures, autoimmune irregularities, and premature death. Currently there is no cure for JNCL, and treatments are limited to alleviating symptoms of disease. The development of improved therapies for JNCL is currently limited by the lack of an animal model that accurately recreates the multi-systemic nature of this disease. Several mouse models of JNCL have been developed via targeted disruption of the murine Cln3 gene, and these models have been valuable for understanding CLN3 function and JNCL disease. However, none of the mouse models fully replicate the complex clinical manifestations observed in the human disease, and more importantly, none develop the severe neurological phenotype that is the hallmark of human JNCL disease. This is likely the result of species differences in physiology, anatomy, and development. In contrast, pigs may serve as a better model in which to study JNCL because of their similarity to humans. Pigs have long been used to model human diseases and are being studied as a source of organs for human xenotransplantation. Because the development and anatomy of the porcine brain more closely resembles that of humans than mice, mutations in the porcine CLN3 gene may result in many of the same neurological changes that are observed in patients with JNCL. The similarities of the porcine visual system offer advantages for modeling the vision phenotype, as well. Therefore, the ultimate goal of this proposal is to develop a porcine model of JNCL by disrupting the CLN3 gene. We intend to accomplish this in two steps by combining gene-targeting and somatic cell nuclear transfer (SCNT). This proposal outlines the development of porcine fibroblasts with mutated CLN3 alleles. Gene targeting vectors will be developed to delete exons 7 and 8 (the most common mutation in JNCL patients) from both copies of the endogenous CLN3 gene. Porcine fetal fibroblasts will be infected with a virus carrying the CLN3 targeting vector. Our plans for generating properly targeted cells are designed to maximize the frequency of homologous recombination, minimize random integration, and minimize the number of cell passages before targeted cells are harvested. A subsequent project will use these cells for somatic cell nuclear transfer to produce CLN3-targeted pigs followed by characterization and validation activities. The JNCL porcine model will provide academic and industry researchers an opportunity to better understand the consequences of CLN3 dysfunction and the pathogenesis of JNCL, and to develop and test new therapeutic and preventative strategies.
This proposal specifically outlines the development of porcine fibroblasts with mutated CLN3 alleles as a first step towards a new model of human juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease. Subsequent work will use these cells for somatic cell nuclear transfer to produce JNCL pigs that will then be characterized and validated as an appropriate model. This project is relevant to the NIH's mission because it will provide a resource to stimulate discovery and the development of new therapeutic applications.