Small Molecular Therapeutics for Friedreich's Ataxia

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Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease caused by a deficiency in the nuclear-encoded mitochondrial protein frataxin. At present there is no effective cure or treatment for FRDA. The DMAabnormality found in 98% of FRDA patients is the unstable hyper-expansion of a GAA triplet in the first intron of the frataxin gene, which adopts a triplex DMAstructure that interferes with gene transcription. In prior studies, we have successfullydeveloped synthetic pyrrole-imidizole polyamides to target GAA repeat DMA.These molecules bind duplex GAA DNA with high affinity and relieve repression of the frataxin gene in lymphoid cells derived from FRDA patients. We now wish to explore whether these molecules will activate the frataxin gene in neuronal cells, and in a mouse knock-in model for FRDA, and to determine the pharmacological properties of these molecules. We will explore the relationship between polyamide composition and function with a new series of molecules. Deconvolution microscopy will be used to monitor the subcellular localization and kinetics of uptake of fluorescent dye-polyamide conjugates in cultured cell lines and in lymphoid cells from FRDA patients. Real-time PCR will be used to determine the effects of polyamides on frataxin mRNA expression in human FRDA cell lines, lymphoid cells isolated from FRDA donor blood, and in neuronal cell lines established from expanded frataxin knock-in mice. The effects of polyamides on cellular frataxin protein will be determined by western blotting, and the genome-wide effects of polyamide treatment will be assessed by DNA microarray analysis. Animal studies will be performed in normal mice to determine the bioavailability, tissue distribution, pharmacokinetics, half-lives of the compounds in serum, toxicity, and maximum tolerated dosage. Expanded GAA allele knock-in mice will be used to determine whether the GAA-specific compounds activate frataxin geneexpression in vivo. If polyamides fail to cross the blood-brain barrier, alternative chemistries and delivery systems will be investigated. This proposal is aimed at the development of new drugs to treat the inherited neurological disease Friedreich's ataxia (FRDA). FRDA is a genetic disease, in which a region of the affected gene, called frataxin, is expanded in size by the addition of repeats of the simple sequence GAA. These repeats inactivate the gene, and we have developed small molecules that reverse this inactivation by targeting the GAA repeats.