Friedreich's Ataxia is caused by a deficiency of the protein frataxin. Frataxin is essential for iron metabolism in mitochondria - the cellular components responsible for energy production. Accordingly, mitochondria are especially important in cells with substantial energy requirements, such as nerve cells or heart muscle cells. It is therefore not surprising that these cells are particularly affected in Friedreich's Ataxia: patients with this condition experience degeneration of the large sensory neurons and spinocerebellar tracts, but also cardiomyopathy.

In patients with Friedreich's Ataxia, a nucleotide sequence in the gene coding for frataxin is repeated up to 1000 times, compared with only about 30 times in healthy individuals. Although we know that this repeated sequence causes frataxin gene silencing, the precise mechanism remains enigmatic and there is currently no treatment available for Friedreich's Ataxia patients. Because the genetic basis for this disease is gene silencing, gene activation could be of therapeutic benefit. Therefore it is important to fully understand the mechanisms by which the frataxin gene is silenced in Friedreich’s Ataxia patients.

Our goal is to apply innovative technologies to gain insights into the pathogenic mechanisms of Friedreich's Ataxia and to search for novel therapeutics. Cardiomyocytes and neurons are the cell types most affected in Friedreich's Ataxia, yet mechanistic studies on frataxin silencing have been mainly performed by using peripheral blood lymphocytes obtained from patients. Therefore, we propose to reprogram patient-derived somatic cells into pluripotent cells (iPS), which will subsequently be differentiated into the relevant cell types. This will allow us to study Friedreich's Ataxia in the appropriate cell types and has great potential to yield novel mechanistic insights into the disease. Furthermore, we will generate a patient-cell-based system for drug discovery. Through high-throughput screens we hope to reveal novel drug targets or promising candidate compounds, which could be further developed into therapeutics to treat Friedreich's Ataxia patients.

Although Friedreich's Ataxia affects only about 4 in 100,000 people, at least 18 other human diseases are also known to be caused by repeated sequences (i.g. Huntington’s Disease, Spinocerebellar Ataxia, Myotonic Dystrophy). Therefore, the development of a treatment for Friedreich's Ataxia would not only help to cure a previously incurable disease, but might also be useful for other conditions with a similar pathogenic mechanism.

Thanks to the support of Gebert Rüf Stiftung, we could take advantage of the recent advances in iPS cell generation and set out to study the mechanism of frataxin silencing in the disease-relevant cell types. The GRS support also allowed us to develop new cell-based drug discovery tools, which have a great potential to reveal novel drug targets or promising candidate compounds that could be further developed into therapeutics.

Zinc-finger nucleases and donor constructs were designed and validated. These genome editing tools enable FRDA researchers to knock in any given sequence into the frataxin locus in any given cell type, facilitating the dissection of molecular pathways involved in FRDA.

A reporter cell line that allows an accurate and effortless assessment of endogenous frataxin gene regulation and is compatible with high throughput biology was established. The cell line is easy to transfect and can be expanded to a very large scale at a relatively low cost. Thus, the system is compatible with the technical and financial constraints of most academic and industrial screening platforms.

Novel potential drug targets were discovered in a high throughput genomics screen.

That screening for general regulators of frataxin transcription, irrespective of GAA repeat length, is an appropriate strategy in the quest for novel therapeutics to treat FRDA was demonstrated.

FRDA and control fibroblasts were successfully reprogrammed towards iPS cells.

Patient-derived iPS cells were differentiated into neurons and cardiomyocytes, the cell types most affected in FRDA.

Rodrigo Villaseñor, Loren Miraglia, Angelica Romero, Buu Tu, Tanel Punga, Philip Knuckles, Stephan Duss, Tony Orth, and Marc Bühler. Genome-engineering tools to establish accurate reporter cell lines that enable identification of therapeutic strategies to treat Friedreich’s Ataxia.
(Manuscript in revision);
Punga T, Bühler M (2010). Long intronic GAA repeats causing Friedreich ataxia impede transcription elongation. EMBO Mol Med 4: 120-129.