Delivery of the 135 kb human frataxin genomic DNA locus gives rise to different frataxin isoforms

Abstract

Friedreich's ataxia (FRDA) is the most common form of hereditary ataxia caused by recessive mutations in the FXN gene. Recent results have indicated the presence of different frataxin isoforms due to alternative gene expression mechanisms. Our previous studies demonstrated the advantages of using high-capacity herpes simplex virus type 1 (HSV-1) amplicon vectors containing the entire FXN genomic locus (iBAC-FXN) as a gene-delivery vehicle capable of ensuring physiologically-regulated and long-term persistence. Here we describe how expression from the 135 kb human FXN genomic locus produces the three frataxin isoforms both in cultured neuronal cells and also in vivo. Moreover, we also observed the correct expression of these frataxin isoforms in patient-derived cells after delivery of the iBAC-FXN. These results lend further support to the potential use of HSV-1 vectors containing entire genomic loci whose expression is mediated by complex transcriptional and posttranscriptional mechanisms for gene therapy applications.

Peptide SS-31 upregulates frataxin expression and improves the quality of mitochondria: implications in the treatment of Friedreich ataxia

“…For long term use, this might not be a suitable choice because iron deprivation can downregulate FXN expression 21 . At present, gene therapy in animal and cell models showed some beneficial effect 22 , 23 . More recently, researchers used synthetic DNA or RNA to block R-loop formation, thereby triggering FXN gene activation to levels similar to analogous wild-type cells 24 .…”

Sustained FXN expression in dorsal root ganglia from a nonreplicative genomic HSV‐1 vector

“…We next analyzed human frataxin protein expression in mouse Neuro‐2a cells transfected with our modified FXN transgenes (Figure c), comparing them with the WT FXN genomic locus transgene that was previously demonstrated to direct physiological expression of human frataxin . Western blots of lysates from cells transfected with FXN‐WT and FXNlac revealed three forms of frataxin not present in nontransfected Neuro‐2a cells (Mock) corresponding to the precursor, intermediate and mature forms; as expected, migration of the bands in lysates from FXNlac‐transfected cells was retarded with respect to those in the FXN‐WT samples as a result of the 22 amino acid C‐terminal fusion to the 2A peptide sequence.…”

“…Enzymatic β-galactosidase activity is clearly evident in cells transduced by FXNlac (right) but not by FXN-WT (left).human neuroblastoma cell line SH-SY5Y as a positive control for human neuronal frataxin isoforms. Using human isoform-specific primer pairs for RT-PCR analysis, we were able to detect both the major frataxin splice isoform (FXN-I), as well as the neuron-specific splice isoform (FXN-II) in Neuro-2a cells transfected by the three FXN constructs(Figure 1b), confirming that our reduced transgenes containing intron 1 maintain the desired splicing regulation for neuronal gene therapy.We next analyzed human frataxin protein expression in mouse Neuro-2a cells transfected with our modified FXN transgenes(Figure 1c), comparing them with the WT FXN genomic locus transgene that was previously demonstrated to direct physiological expression of human frataxin 15,16,40. Western blots of lysates from cells transfected with FXN-WT and FXNlac revealed three forms of frataxin not present in nontransfected Neuro-2a cells (Mock) corresponding to the precursor, intermediate and mature forms; as expected, migration of the bands in lysates from FXNlac-transfected cells was retarded with respect to those in the FXN-WT samples as a result of the 22 amino acid C-terminal fusion to the 2A peptide sequence.…”

Altered Secretome and ROS Production in Olfactory Mucosa Stem Cells Derived from Friedreich’s Ataxia Patients

“…Human olfactory mucosa stem cells were obtained from biopsies of healthy subjects (C) and FRDA patients (FA). Cells were cultured in cancer stem cell (CSC) medium composed of Dulbecco´s Modified Eagle Medium (DMEM)/F12 medium supplemented with: GlutaMAX, 0.5% Albumax I, 0.5% Hepes 10 mM, 1% N2 (all from Gibco, Barcelona, Spain), 0.6% Glucose (Sigma-Aldrich, Madrid, Spain), 2% foetal bovine serum (FBS), non-essential amino acids (NEAA, L-Ala 44 mM, L-Asn 45 mM, L-Asp 40 mM, L-Glu 40 mM, L-Pro 30 mM), 0.1% penicillin/streptomycin and freshly added 8 ng/mL recombinant human fibroblast growth factor-2 (rhFGF-2, PeproTech, Rocky Hill, NJ, USA), and 50 ng/mL nerve growth factor (NGF, Sigma-Aldrich) [ 85 ]. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.…”