Cell-Based Approaches For Modeling and Treating Ataxia-Telangiectasia

Ataxia-Telangiectasia (A-T) is a devastating genetic syndrome of neurodegeneration, immunodeficiency and cancer predisposition caused by mutations in the locus encoding ATM (Ataxia-Telangiectasia Mutated). The current standard of care for A-T consists of aggressive supportive measures, and the prognosis remains poor. There is therefore a pressing need to develop novel experimental approaches and treatments for this disease. In this application, we propose to address this need by developing for the first time human stem cell-based technologies to: 1) generate novel experimental models for A-T that faithfully recapitulate the features of the disease across its complex spectrum of clinical manifestations (Aim 1); and 2) start to test the feasibility of regenerative therapies for A-T, via generation of autologous stem cells that have been rendered disease-free by correction of the mutation (Aim 2). Mutations causing A-T are private, resulting in variable reduction in ATM activity and, correspondingly, a wide spectrum of clinical manifestations. Although the most severe form of the disease ("classical" A-T, with no detectable ATM) has been modeled in the mouse (ATM "knock out"), this approach fails to recapitulate the neurological symptoms of the disease and its characteristic tumor spectrum. Moreover, we are currently lacking experimental models for those patients whose mutations result in reduced ATM activity ("variant" A-T). To address these issues, experiments in Aim 1 will test the hypothesis that the genotype-phenotype correlation in A-T is maintained in patient-derived induced pluripotent stem cells (iPSCs). To test this hypothesis, we will reprogram fibroblasts from A-T patients with variable reduction of ATM levels and determine whether: 1) ATM expression and activity in the iPSCs correlate directly with those observed in the patient fibroblasts they are derived from; 2) the iPSCs recapitulate the phenotypes observed in the fibroblasts, including impaired cell cycle checkpoint activation, defective DNA double-strand break (DSB) repair, radiosensitivity and genomic instability; and 3) these phenotypes in the iPSCs directly correlate with their level of ATM expression/activity. If we find that the genotype-phenotype correlation is maintained in A-T iPSCs, this work would validate a more general use of autologous iPSCs for preclinical studies of A-T, including the evaluation of disease biomarkers, drug testing or genetic screening. The clinical manifestations of A-T result from progressive cell loss and tissue degeneration, making A-T a candidate disease for regenerative therapies. Experiments in Aim 2 will test the hypothesis that correction of the ATM mutation in A-T somatic cells will rescue their severe reprogramming defect and allow the generation of disease-free iPSCs. To test this hypothesis, we propose a series of proof-of-principle experiments using a well-characterized compound heterozygous A-T fibroblast cell line. First, we will "repair" either one or the two ATM mutations in this line by recombination with an exogenous donor plasmid carrying the intact sequence, to generate either "carriers" (one normal allele and one mutated allele) or "intact" cells (two normal alleles). To increase the efficiency of recombination, we will introduce a DSB in close proximity to the mutation using Transcription Activator-Like Effector Nucleases (TALENS) that bind specifically to the mutated region. In Preliminary Experiments, we find that we can successfully induce DSBs and site-specific recombination at a human "safe harbor" locus as well as at the ATM locus itself. After verifying that recombination restores ATM expression and function, we will reprogram the corrected cells into iPSCs and characterize their level of ATM expression, activity and function with passage. Because the "null", "carrier" and "intact" lines are isogenic, the effect of ATM gene dose on reprogramming and iPSC function can be evaluated in these experiments. In this regard, approximately 1% of the US general population is an A-T "carrier", extending the significance of this work well beyond A-T patients. Overall, completion of this Exploratory Project will provide the rationale, expertise and reagents for longer-term studies aimed at modeling and treating A-T with autologous iPSCs and/or their derived products and optimizing the use of regenerative therapies for the general population.