Protein-based Therapeutics to treat nucleotide expansion disorders associated with aberrant gene expression

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Nucleotide expansion disorders are comprised of a group of genetic diseases that are classified in two groups depending on whether the repeats (e.g., CCG, CCCCGG, GAA, CTG, and CAG) are located within a coding or non-coding region of the genome. There are no curative therapies for nucleotide expansion disorders; it is only possible to provide palliative measures to manage the clinical symptoms. Over 25 nucleotide disorders, mainly associated with neurodegenerative diseases have been identified, including Huntington’s disease, myotonic dystrophy and Friedreich’s ataxia. Friedreich’s Ataxia (FA), associated with an expanded GAA repeat array (up to 1700 repeats; normal alleles have 10-66 repeats) in the first intron of the frataxin gene, is the only nucleotide expansion disorder that is a recessive mutation. The expanded GAA repeat leads to silencing of frataxin (FXN) expression, presumably due to the formation of a RNA:DNA triplex that halts transcription. FA affects 1:50,000 individuals, making it the most common form of hereditary ataxia. FA is associated with impaired mitochondrial iron handling and makes cells highly susceptible to ROS-mediated bioenergetic dysfunction. Clinical manifestations of Friedreich’s Ataxia occur across organ systems, and include muscle weakness, movement disorders, poor neurological development and function, diabetes, and cardiac complications. There are no FDA approved disease modifying drugs for FA. Therapeutic strategies directly targeting expanded repeats in FXN mRNA, such as antisense oligonucleotides (ASO), have produced promising results. However, difficulties in ASO delivery and need for lifelong administration of the ASO therapeutic remain limiting factors for ASO-based therapies. We seek to develop a protein-based therapeutic approach for FA by designing RNA binding proteins, based on the PUF domain family of RNA binding protein, that recognize GAA repeats [PUF(GAA)] in mutated FXN mRNA and assess the ability of PUF(GAA) to reverse the transcriptional silencing of FXN expression. Sucess will be indicated by restoration of FXN expression to 35-50% of normal levels of FXN expression and rescue of two mitochondrial defects associated with FA: resistance to metabolic stress when propagated in galactose; and resistance to H2O2-induced inhibition of mitochondrial respiration. Once feasibility is demonstrated, Phase II will focus on the development of research grade adenoviral associated vectors (AAV) that constitutively express nuclear targeted PUF(GAA) to develop gene delivery protocols and for initial efficacy and safety studies in animal models of FA before progressing to production of clinical grade AAV for IND enabling safety and efficacy studies of this innovative curative gene therapeutic for FA. In the long term, combined with gene delivery vectors, our innovative gene therapeutic approach may provide a new route for targeted therapy for nucleotide expansion disorders that disrupt gene expression.