In vivo frataxin expression of a triplex purified minicircle

Abstract Friedreich’s ataxia (FRDA) is a progressive neurodegenerative disorder caused by (GAA • TTC) n repeat expansions in the Frataxin ( FXN) gene, resulting in reduced expression of Frataxin, a mitochondrial protein essential for energy production and iron-sulfur cluster assembly. Minicircles (MCs) are non-viral DNA vectors offering improved safety and efficiency over conventional plasmids. We used a novel method to purify MCs using biotin-conjugated triplex-forming and strand-invading oligonucleotides (ONs). A 21 bp H-DNA-forming sequence was incorporated into the plasmids to enhance binding specificity of the ONs. The ON-plasmid complexes were removed using streptavidin magnetic beads. This approach yielded highly purified MCs. In FRDA patient-derived fibroblasts, the purified MCs expressed FXN mRNA and restored Frataxin protein levels to those observed in healthy cells, demonstrating the therapeutic potential of this approach. More interestingly, MC-FXN treatment of an FRDA mouse model using hydrodynamic tail vein injection resulted in an 18-fold and 3-fold increase of Frataxin in the mice livers and hearts, respectively, supporting the translational relevance of this modality.