Abstract
In Friedreich’s ataxia (FRDA) patients, diminished frataxin (FXN) in sensory neurons is thought to yield the predominant pathology associated with disease. In this study, we demonstrate successful usage of RNA transcript therapy (RTT) as an exogenous human FXN supplementation strategy in vitro and in vivo, specifically to dorsal root ganglia (DRG). Initially, 293 T cells were transfected with codon optimized human FXN mRNA, which was translated to yield FXN protein. Importantly, FXN was rapidly processed into the mature functional form of FXN (mFXN). Next, FXN mRNA, in the form of lipid nanoparticles (LNPs), was administered intravenously in adult mice. Examination of liver homogenates demonstrated efficient FXN LNP uptake in hepatocytes and revealed that the mitochondrial maturation machinery had efficiently processed all FXN protein to mFXN in ~24 h in vivo. Remarkably, greater than 50% mFXN protein derived from LNPs was detected seven days after intravenous administration of FXN LNPs, suggesting that the half-life of mFXN in vivo exceeds one week. Moreover, when FXN LNPs were delivered by intrathecal administration, we detected recombinant human FXN protein in DRG. These observations provide the first demonstration that RTT can be used for the delivery of therapeutic mRNA to DRG.
Highlights
Friedreich’s ataxia (FRDA) is an autosomal recessive disease caused by an intronic trinucleotide (GAA) expansion in intron 1 of the FXN gene[1]
We further tested intrathecal delivery and uptake of human FXN (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 lipid nanoparticles (LNPs)-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
We generated FXN mRNA by in vitro transcription (IVT) using a linearized plasmid containing codon-optimized human FXN (Fig. S2A), which encodes a protein sequence identical to consensus (Fig. S2B). This was followed by the same capping and polyA tailing reactions detailed in Fig. 1A. hFXN mRNA was analyzed following IVT and capping/polyA tailing (Fig. 1B)
Summary
Friedreich’s ataxia (FRDA) is an autosomal recessive disease caused by an intronic trinucleotide (GAA) expansion in intron 1 of the FXN (frataxin) gene[1]. 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 individuals[11] In affected individuals, such a decrease in cellular concentration of FXN yields pathology. 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
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