Generation of pathogenic TPP1 mutations in human stem cells as a model for neuronal ceroid lipofuscinosis type 2 disease

• Published in: Stem cell research 2021-05, Vol.53, p.102323-102323, Article 102323
• Main Authors: Ma, Li, Prada, Adriana M., Schmidt, Michael, Morrow, Eric M.
• Format: Article
• Language: English
• Subjects: Alleles; CLN2; CRISPR/Cas9; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - genetics; Disease model; Embryonic stem cells; Genome editing; Humans; Mutation; Neuronal Ceroid-Lipofuscinoses - genetics; Stem Cells; TPP1; Tripeptidyl-Peptidase 1
• ISSN: 1873-5061; 1876-7753
• DOI: 10.1016/j.scr.2021.102323

•Creation of stem cell model for neuronal ceroid lipofuscinosis type 2 (CLN2 disease).•Use of CRISPR/Cas9 gene editing to introduce pathogenic TPP1 mutations into H9 cells.•Analysis of mutant and control cells for TPP1 protein and enzyme activity.•Availability of stem cell models for CLN2 disease for wide-spread sharing. Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is an autosomal recessive neurodegenerative disorder generally with onset at 2 to 4 years of age and characterized by seizures, loss of vision, progressive motor and mental decline, and premature death. CLN2 disease is caused by loss-of-function mutations in the tripeptidyl peptidase 1 (TPP1) gene leading to deficiency in TPP1 enzyme activity. Approximately 60% of patients have one of two pathogenic variants (c.509-1G > C or c.622C > T [p.(Arg208*)]). In order to generate a human stem cell model of CLN2 disease, we used CRISPR/Cas9-mediated knock-in technology to introduce these mutations in a homozygous state into H9 human embryonic stem cells. Heterozygous lines of the c.622C > T (p.(Arg208*)) mutation were also generated, which included a heterozygous mutant with a wild-type allele and different compound heterozygous coding mutants resulting from indels on one allele. We describe the methodology that led to the generation of the lines and provide data on the initial validation and characterization of these CLN2 disease models. Notably, both mutant lines (c.509-1G > C and c.622C > T [p.(Arg208*)]) in the homozygous state were shown to have reduced or absent protein, respectively, and deficiency of TPP1 enzyme activity. These models, which we have made available for wide-spread sharing, will be useful for future studies of molecular and cellular mechanisms underlying CLN2 disease and for therapeutic development.