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.

Inclusion Criteria:

Patients that meet the classic diagnosis of A-T and for whom the underlying mutation(s) is
known. The diagnosis of A-T has been made by the clinician using the following criteria:

1. Characteristic neurological abnormalities, including but not limited to oculomotor
apraxia, bulbar dysfunction, postural instability, and ataxia.

2. Presence of telangiectasia on the conjunctivae and/or skin.

3. Laboratory abnormalities including but not limited to elevated serum alpha-feto-
protein, level, absence of ATM on western blot, increased x-ray induced chromosomal
breakage in comparison to a control population, mutations in both alleles of the ATM
gene. Parents of the patients above, who are haploinsufficient and whose mutation is
known.

Exclusion Criteria:

Patients under 2 years of age No subjects will be excluded on the basis of age, sex, race,
or socio-economic status.