138. Potential Therapeutic Treatment of Friedrich's Ataxia Using Highly Specific Engineered Meganucleases

Abstract

Friedreich's ataxia (FRDA) is a recessive genetic disorder that results in progressive neuromuscular deterioration. The most common causative genetic alteration of this disorder is an expansion of a trinucleotide (GAA/TTC) repeat within the first intron of the Frataxin (FXN) gene. This triplet expansion causes transcriptional defects, resulting in low FXN mRNA and protein levels. In patients, the correlation between number of repeats and severity of the disease, age of onset and cardiomyopathy indicates that the repeat expansion is the primary cause of FRDA, thus making it a potential therapeutic target. We are investigating the potential use of our engineered meganuclease technology to precisely remove these trinucleotide repeats as a therapeutic treatment for FRDA. We have designed a pair of meganucleases that introduce double-strand breaks on either side of the FXN trinucleotide repeat region, generating compatible 3’ overhangs that can be repaired by direct ligation, resulting in precise excision of the intervening region. Introduction of this pair of meganucleases into FRDA patient fibroblasts in cell culture results in the successful deletion of the causative repeat region in greater than 15% of the transfected cells. This removal of the FRDA repeat region has been confirmed using both digital PCR-based analytics and deep sequencing. Additional analysis of Frataxin mRNA production, Frataxin protein levels, and cellular metabolism will further demonstrate the utility of the repeat excision approach for FRDA gene therapy.