Developing personalised medicine for CLN3 disease

Brain diseases affect all ages and are a growing burden to health and society. Many are inherited, and caused by changes in our genomic DNA, which specifies the proteins that make up our organs and tissues. For most types, no cure is available, making them a significant priority for research and therapy development. The neuronal ceroid lipofuscinoses (NCL) or Batten disease are the most common of these conditions affecting children. Batten disease is a devastating disease, causing seizures, progressive loss of vision, motor skills, speech and ordered thought, and is life-limiting. About half the cases of Batten disease in children are caused by the same mutation in the gene CLN3; this is a deletion of the genomic DNA that removes a small part of the gene which we call the 1-kb deletion. Recently, some who have lost their vision as adults have been found to have a mutation in CLN3 that affects just one building block of the CLN3 protein.

We have been studying CLN3 and think that the gene produces many different templates for making the protein, even in healthy cells. We have looked at the effect of some of the disease-causing mutations and found that these affect these templates. We have also found that the 1-kb deletion has a very complex effect on the function of the CLN3 protein, such that it both keeps and loses some of its functionalities as well as acquiring new characteristics. To develop effective therapies, we will need to test these in model systems that accurately model these complex effects.

This project will use technology at the cutting edge of research and aims to (1) describe the variety of templates for the CLN3 protein made in patients carrying the 1-kb deletion on both chromosome copies; (2) make different zebrafish strains to model significant mutations including all the consequences of the 1-kb deletion and the mutation causing adult eye disease; (3) identify which new zebrafish strains best match the variety of templates in patients; (4) describe the effect of each mutation on the zebrafish; (5) investigate any new variant CLN3 templates found in patients at a cell level.

By the end of the project we will have made a genetically representative set of cell and zebrafish models to test novel therapies, such as drugs. We may also have new ideas for therapies, such as enhancing the concentration of templates are beneficial and reducing the concentration of templates that are deleterious.

Mutation of the CLN3 gene causes a lysosomal disease with a wide spectrum of symptoms including classic juvenile neuronal ceroid lipofuscinosis (NCL), a fatal devastating neurodegenerative disease beginning with loss of sight as well as adult onset visual failure. CLN3 encodes a highly conserved intracellular membrane protein whose function is not fully defined, although it is important under conditions of stress and the latest understanding is that it may transport ions across the lysosomal membrane. There is evidence for multiple CLN3 transcripts and and further novel isoforms arise in the presence of the 1-kb deletion. This project aims to develop genetically and transcriptionally accurate models of CLN3 disease that can be used to test novel therapies, enable a molecular, cell, and whole body understanding of the consequences of disease-causing mutations and CLN3 dysfunction. Our main objectives are: 1. CLN3 transcript profiling of patients with classic juvenile CLN3 disease; 2. Genetically accurate zebrafish models of CLN3; 3. Transcript profiling of CLN3 disease models; 4. Exhaustive phenotypic characterisation of zebrafish CLN3 models; 5. Functionality of novel CLN3 transcripts. This programme of work will use a range of methodologies, with an emphasis on longread RNAseq supported by bioinformatic analysis, advanced gene editing techniques in zebrafish including CRISPR/cas9 gene editing, specialised phenotypic assays for zebrafish such as electroencephalography to detect seizures and electroretinography to assess eye function, supported by classic molecular and cell biology techniques. Consideration has been given to sample size for RNA extraction and phenotypic analysis.