Intrathecal delivery of frataxin mRNA encapsulated in lipid nanoparticles to dorsal root ganglia as a potential therapeutic for Friedreich's ataxia.

Friedreich’s ataxia (FRDA) is an autosomal recessive disease caused by an intronic trinucleotide (GAA) expansion in intron 1 of the FXN (frataxin) gene1. FRDA is predominantly a neurodegenerative disease2, but pathology also manifests in multiple tissues including the heart and pancreas3,4. Expansions in the FXN gene have been shown to cause epigenetic changes5 and formation of R-loops6 that hinder the transcriptional machinery, ultimately yielding diminished levels of FXN transcript and protein. In FRDA patients, FXN protein levels have been shown to be reduced in all tested cell types7. Although FXN knockout is lethal8, diminished levels yield pathology only in specific cell types, including select neurons, cardiomyocytes and pancreatic islets, for reasons that are not understood. In the central nervous system (CNS), progressive degeneration leads to disease, therefore therapies that access the CNS are highly desirable. The function of FXN is still a subject of debate but the protein’s primary role is activation of iron-sulfur (Fe-S) cluster biogenesis in the mitochondrial matrix9. Data have also been reported supporting a role for FXN as an iron chaperone10. Regardless of its precise function, it has been established that in FRDA patients, levels of FXN in peripheral tissues drop to ~5–30% of those in non-carrier healthy individuals11. In affected individuals, such a decrease in cellular concentration of FXN yields pathology. Interestingly, heterozygous patients have ~50% FXN compared to non-carriers and do not show any pathology. There is no disease-modifying therapy for FRDA so treatment options remain limited, but increasing FXN levels to those in carriers of the pathogenic FXN intronic expansion is desirable and could be therapeutic. A number of small molecules, such as histone deacetylase inhibitors12 and nicotinamide13, and large molecules, such as engineered transcription activator-like effectors14,15, were reported as FXN upregulation approaches for FRDA therapy. FXN protein supplementation16 and viral gene transfer17,18 are other potential strategies for therapy that are currently being explored. RNA transcript therapy (RTT) is an mRNA replacement/supplementation approach through encapsulation of exogenous mRNA molecules in nanoparticles. RTT is currently being pursued for multiple therapeutic applications19 and has led to major investments in biopharmaceutical companies20. In proof-of-principle example studies, RTT was shown to successfully rescue a lethal genetic knockout mouse model21 and to be useful for delivery of an mRNA encoding a therapeutic protein in a model of disease22, suggesting that it can be utilized as a replacement approach in inherited recessive diseases such as FRDA. In this study, we explored RTT by intravenous and intrathecal lipid nanoparticle (LNP) based delivery of recombinant human FXN (hFXN) mRNA. We present evidence that hFXN mRNA can be efficiently translated in vitro upon cellular transfection and in vivo when administered systemically, and that the corresponding protein is processed into the mature, functional form (mFXN). Importantly, we find that the maturation machinery is not limiting, and the in vivo half-life of mFXN protein is long, in excess of one week. We further tested intrathecal delivery and uptake of hFXN mRNA in dorsal root ganglia (DRG), a disease-affected and primary site of pathology in FRDA2,23. These results demonstrate the potential utility of LNP-based delivery of hFXN mRNA as a supplementation therapy to treat FRDA and for other diseases of the central nervous system where DRG are implicated in pathology.