Frataxin Research Articles

Proprioceptors-enriched neuronal cultures from induced pluripotent stem cells from Friedreich ataxia patients show altered transcriptomic and proteomic profiles, abnormal neurite extension, and impaired electrophysiological properties.

Friedreich ataxia is an autosomal recessive multisystem disorder with prominent neurological manifestations and cardiac involvement. The disease is caused by large GAA expansions in the first intron of the FXN gene, encoding the mitochondrial protein frataxin, resulting in downregulation of gene expression and reduced synthesis of frataxin. The selective loss of proprioceptive neurons is a hallmark of Friedreich ataxia, but the cause of the specific vulnerability of these cells is still unknown. We herein perform an in vitro characterization of human induced pluripotent stem cell-derived sensory neuronal cultures highly enriched for primary proprioceptive neurons. We employ neurons differentiated from healthy donors, Friedreich ataxia patients and Friedreich ataxia sibling isogenic control lines. The analysis of the transcriptomic and proteomic profile suggests an impairment of cytoskeleton organization at the growth cone, neurite extension and, at later stages of maturation, synaptic plasticity. Alterations in the spiking profile of tonic neurons are also observed at the electrophysiological analysis of mature neurons. Despite the reversal of the repressive epigenetic state at the FXN locus and the restoration of FXN expression, isogenic control neurons retain many features of Friedreich ataxia neurons. Our study suggests the existence of abnormalities affecting proprioceptors in Friedreich ataxia, particularly their ability to extend towards their targets and transmit proper synaptic signals. It also highlights the need for further investigations to better understand the mechanistic link between FXN silencing and proprioceptive degeneration in Friedreich ataxia.

Retinal and Visual Pathways Involvement in Carriers of Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is a rare autosomal recessive neurodegenerative disorder due to the homozygous pathological expansion of guanine-adenine-adenine (GAA) triplet repeats in the first intron of the FXN gene, which encodes for the mitochondrial protein frataxin. In the visual system, the typical manifestations are ocular motility abnormality, optic neuropathy, and retinopathy. Despite the evidence of ophthalmological impairment in FRDA patients, there is a lack of information about the morpho-functional condition of the retina and of the optic pathways in healthy heterozygous carriers of Friedreich's ataxia (C-FRDA). Ten C-FRDA subjects (providing 20 eyes) and thirty-five Controls (providing 70 eyes) underwent a complete neurological and ophthalmological examination comprehensive of functional (full-field Electroretinogram (ffERG), multifocal Electroretinogram (mfERG), Visual Evoked Potential (VEP), and Pattern Reversal Electroretinogram (PERG)) and morphological assessments (Optical Coherence Tomography, OCT) of the retina, macula, retinal ganglion cells, and visual pathways. The groups' data were compared using a two-sample t-test. Pearson's test was used to investigate the morpho-functional correlations. Statistically significant differences (p < 0.01) between C-FRDA and Control eyes for the values of the following parameters were found: ffERG b-wave amplitude, mfERG Response Amplitude Densities, PERG P50 implicit time and P50-N95 amplitude, VEP P100 implicit time, Retinal Nerve Fiber Layer (RNFL) Overall, and Nasal thickness. The values of the OCT macular volume were not statistically different (p > 0.01) between the two Groups. Therefore, our data suggest that, in C-FRDA, a dysfunction of retinal elements without morphological macular impairment may occur. In addition, a morphological impairment of RNFL associated with an abnormal neural conduction along the visual pathways can be also detected.

G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of FXN expression through indirect effects.

Friedreich's ataxia is an incurable disease caused by frataxin (FXN) protein deficiency, which is mostly induced by GAA repeat expansion in intron 1 of the FXN gene. Here, we identified antisense oligonucleotides (ASOs), complementary to two regions within the first intron of FXN pre-mRNA, which could increase FXN mRNA by ∼2-fold in patient fibroblasts. The increase in FXN mRNA was confirmed by the identification of multiple overlapping FXN-activating ASOs at each region, two independent RNA quantification assays, and normalization by multiple housekeeping genes. Experiments on cells with the ASO-binding sites deleted indicate that the ASO-induced FXN activation was driven by indirect effects. RNA sequencing analyses showed that the two ASOs induced similar transcriptome-wide changes, which did not resemble the transcriptome of wild-type cells. This RNA-seq analysis did not identify directly base-paired off-target genes shared across ASOs. Mismatch studies identified two guanosine-rich motifs (CCGG and G4) within the ASOs that were required for FXN activation. The phosphorodiamidate morpholino oligomer analogs of our ASOs did not activate FXN, pointing to a PS-backbone-mediated effect. Our study demonstrates the importance of multiple, detailed control experiments and target validation in oligonucleotide studies employing novel mechanisms such as gene activation.

Clinical, neuroimaging and genetic findings in children with hereditary ataxia: single center study.

The genetics of hereditary ataxia (HA) are complex and multigenic. The diversity of genes that cause ataxia varies considerably between populations. We aimed to investigate the clinical, neuroimaging, and genetic findings of HA in children from a tertiary center in Turkey. The clinical and neuroimaging evaluations of patients, laboratory investigations, and molecular genetic evaluations of those with ataxia were performed at the pediatrics, pediatric neurology, and genetics outpatient clinics between October 2020 and October 2021. With repeated expansions in the ATXN 1, 2, 3, 7, and 8 genes for spinocerebellar ataxia (SCA) and FXN genes for Friedreich's ataxia (FA), whole-exome sequencing (WES) was used to analyze every patient. 25 patients from 24 families had ataxia and an unsteady gait as their main symptoms. The patients had a mean age of 8.5 ± 3.78 years, and the symptoms had begun at a mean age of 2 ± 0.62 years; five of these were males and three were females. A genetic cause of ataxia was found in 8/25 patients (32%). Seven of the eight gene mutations detected in the patients were novel mutations. Spinocerebellar ataxia was found in 16% of cases (n = 4), L-2-Hydroxyglutaric aciduria was found in 12% of cases (n = 3), and ataxia-telangiectasia was found in 4% of cases (n = 1). Our research adds to the body of knowledge by describing the clinical and genetic traits of HA patients in our area and by finding unusual gene changes linked to ataxia.

Frataxin deficiency alters gene expression in Friedreich ataxia derived IPSC-neurons and cardiomyocytes.

Friedreich's ataxia (FRDA) is an autosomal recessive disease, whereby homozygous inheritance of an expanded GAA trinucleotide repeat expansion in the first intron of the FXN gene leads to transcriptional repression of the encoded protein frataxin. FRDA is a progressive neurodegenerative disorder, but the primary cause of death is heart disease which occurs in 60% of the patients. Several functions of frataxin have been proposed, but none of them fully explain why its deficiency causes the FRDA phenotypes nor why the most affected cell types are neurons and cardiomyocytes. To investigate, we generated iPSC-derived neurons (iNs) and cardiomyocytes (iCMs) from an FRDA patient and upregulated FXN expression via lentivirus without altering genomic GAA repeats at the FXN locus. RNA-seq and differential gene expression enrichment analyses demonstrated that frataxin deficiency affected the expression of glycolytic pathway genes in neurons and extracellular matrix pathway genes in cardiomyocytes. Genes in these pathways were differentially expressed when compared to a control and restored to control levels when FRDA cells were supplemented with frataxin. These results offer novel insight into specific roles of frataxin deficiency pathogenesis in neurons and cardiomyocytes.

Abstract 15376: Nrf2 Agonist Omaveloxolone Improves Cardiac Function in Mice With Friedreich's Ataxia

Introduction: Friedreich’s ataxia (FA) is an inherited, life-shortening, degenerative neuromuscular disorder affecting ~1 in every 50,000 people in the USA and Europe. Initial symptoms, typically seen in children and teenagers, include loss of balance and coordination, muscle weakness placing these patients in wheelchairs. Early death is common and most of FA patients die in their 30s or earlier from cardiac failure, caused by deficient frataxin (FXN) expression in the heart. Omaveloxolone (OMAV), a known Nrf-2 activator, has been recently proposed as the first treatment of the neurological symptoms of FA; however less is known about OMAV effects on cardiac function in FA. Aim: To test the hypothesis that OMAV improves cardiac function in a mouse model of FA via Nfr2 signaling pathway activation. Methods and Results: Cardiospecific FXN knockout (FXN Mck-Cre KO) mice were used as a model of severe cardiomyopathy which is characteristic for the late stage of FA. Animals of either sex were randomly split in vehicle and OMAV-treated groups. Treatment with 24 mg/kg OMAV started at 3 weeks of age and continued for 5 weeks. Cardiac function was examined in-vivo by echocardiography. We found that FXN cKO mice developed severe heart failure with 45% reduction in ejection fraction, increased left ventricular (LV) mass (+80%) and diameter (+308%), decreased stroke volume (-26%) and cardiac output (-38%, n=10) as compared to WT mice (n=12). Cardiac deficits were more pronounced in FA males compared to females. OMAV (n=14) significantly decreased LV mass (-24%), LV diameter (-28%), and partially recovered a decrease in ejection fraction (+40%), stroke volume (+22%) and cardiac output (+35%). Gene expression of NADP(H) quinoline oxidoreductase-1 (NQO1), superoxide dismutase 2 (SOD2), and glutathione S-transferase were decreased by 60-90% in FA and recovered by 60-90% by OMAV. Aconitase activity, used as a surrogate measure of frataxin's iron-sulfur biogenesis function, was decreased in FA by 49% and recovered 33% by OMAV. Conclusions: This is the first study to demonstrate sexual dimorphism in FA hearts and show that OMAV can improve cardiac function in FA mice with severe cardiomyopathy.

Abstract 15690: Bioactive Fumarate Improves Cardiac Function and Expands Lifespan in Friedreich's Ataxia

Introduction: Friedreich ataxia (FA) is a monogenic recessive ataxia caused by reduction of a single mitochondrial protein, frataxin (FXN). Although the name of FA refers to the neurodegenerative ataxia, the lethal event is cardiomyopathy, caused by deficient FXN expression in the heart. Previously, we identified that bioactive fumarates are protective in FA cell models. We have developed a novel monomethyl fumarate prodrug, IMF, which has much improved pharmacokinetic profile and compared its effect to the known fumarate prodrug dimethyl fumarate (DMF, Tecfidera). Aim: To test the hypothesis that novel bioactive fumarate improves cardiac function and survival via Nrf2 and HCA 2 signaling pathways activation. Methods and Results: Cardiospecific FXN knockout (FXN Mck-Cre KO) mice were used as a model of severe cardiomyopathy which is characteristic for the late stage of FA. Animals of either sex were randomly split in vehicle and fumarate-treated groups. Animals were treated by IMF or DMF at concentrations which release equimolar amount of monomethyl fumarate. Treatment started at 3 weeks of age and continued for 5 weeks in the cross-sectional study or until animal death in the survival study. Cardiac function was examined in-vivo by echocardiography. We found that FXN Mck-Cre KO mice developed severe heart failure with 45% reduction in ejection fraction, increased left ventricular (LV) mass (+80%) and diameter (+308%), decreased stroke volume (-26%) and cardiac output (-38%, n=10) as compared to WT mice (n=12). Cardiac deficits were more pronounced in FA males compared to females. Both DMF and IMF partially recovered cardiac deficits in FXN Mck-Cre KO mice but only IMF extended the lifespan in these mice by 13%. Expression of genes in Nrf2 (NQO1, SOD2, glutathione S-transferase) and HCA 2 (HCA 2 and Sirt1) signaling pathways were decreased in FXN Mck-Cre KO and restored to a different degree by DMF and IMF. Aconitase activity, used as a surrogate measure of frataxin's iron-sulfur biogenesis function, was decreased in FA by 49% and recovered 18% by IMF only. Conclusions: Novel fumarate prodrug IMF improved cardiac function and expanded the lifespan in FXN Mck-Cre KO mouse model of FA more effectively than DMF.

RNA as a Major-Groove Ligand: RNA-RNA and RNA-DNA Triplexes Formed by GAA and UUC or TTC Sequences.

Friedreich's ataxia is associated with noncanonical nucleic acid structures that emerge when GAA:TTC repeats in the first intron of the FXN gene expand beyond a critical number of repeats. Specifically, the noncanonical repeats are associated with both triplexes and R-loops. Here, we present an in silico investigation of all possible triplexes that form by attaching a third RNA strand to an RNA:RNA or DNA:DNA duplex, complementing previous DNA-based triplex studies. For both new triplexes results are similar. For a pyridimine UUC+ third strand, the parallel orientation is stable while its antiparallel counterpart is unstable. For a neutral GAA third strand, the parallel conformation is stable. A protonated GA+A third strand is stable in both parallel and antiparallel orientations. We have also investigated Na+ and Mg2+ ion distributions around the triplexes. The presence of Mg2+ ions helps stabilize neutral, antiparallel GAA triplexes. These results (along with previous DNA-based studies) allow for the emergence of a complete picture of the stability and structural characteristics of triplexes based on the GAA and TTC/UUC sequences, thereby contributing to the field of trinucleotide repeats and the associated unusual structures that trigger expansion.

Gene Therapy Hits Its Stride in the Clinic

Gene Therapy Hits Its Stride in the Clinic

A promising mouse model for Friedreich Ataxia progressing like human patients

Friedreich Ataxia (FRDA) is a genetic disease caused by an expended GAA repeat in the FXN gene leading to a reduction in frataxin protein production. Frataxin is an essential protein involved in mitochondrial iron-sulfur-cluster formation, its absence affecting numerous cellular rections. In patients, the disease leads to a progressive neuromuscular degeneration and, most of the time, death from heart failure. In order to determine if a treatment is effective or not, it is essential to have the mouse model, which best reflects all of the characteristics of this disease. Many groups were working on the creation of mouse models by decreasing the mouse frataxin or knocking it out, by introducing a transgene with a human frataxin with long GAA repeat. Most of the mouse models are limited to one problem, either neurologic or cardiac symptoms, and, for those who have both, generally these symptoms are too severe and mice have a very short life span, which does not reflect the human disease’s progression. Jackson Laboratories Inc. developed a new mouse model that has 800 GAA repeats. We demonstrate here that these mice accurately reflect the human disease with a progressive neuromuscular degeneration highlighted by the two beam tests and the beginning of heart hypertrophy at 26 weeks. YG8-800 mice are thus currently a promising mouse model for FRDA.

A non-synonymous single nucleotide polymorphism in SIRT6 predicts neurological severity in Friedreich ataxia

Introduction: Friedreich ataxia (FRDA) is a recessive neurodegenerative disease characterized by progressive ataxia, dyscoordination, and loss of vision. The variable length of the pathogenic GAA triplet repeat expansion in the FXN gene in part explains the interindividual variability in the severity of disease. The GAA repeat expansion leads to epigenetic silencing of FXN; therefore, variability in properties of epigenetic effector proteins could also regulate the severity of FRDA. Methods: In an exploratory analysis, DNA from 88 individuals with FRDA was analyzed to determine if any of five non-synonymous SNPs in HDACs/SIRTs predicted FRDA disease severity. Results suggested the need for a full analysis at the rs352493 locus in SIRT6 (p.Asn46Ser). In a cohort of 569 subjects with FRDA, disease features were compared between subjects homozygous for the common thymine SIRT6 variant (TT) and those with the less common cytosine variant on one allele and thymine on the other (CT). The biochemical properties of both variants of SIRT6 were analyzed and compared. Results: Linear regression in the exploratory cohort suggested that an SNP (rs352493) in SIRT6 correlated with neurological severity in FRDA. The follow-up analysis in a larger cohort agreed with the initial result that the genotype of SIRT6 at the locus rs352493 predicted the severity of disease features of FRDA. Those in the CT SIRT6 group performed better on measures of neurological and visual function over time than those in the more common TT SIRT6 group. The Asn to Ser amino acid change resulting from the SNP in SIRT6 did not alter the expression or enzymatic activity of SIRT6 or frataxin, but iPSC-derived neurons from people with FRDA in the CT SIRT6 group showed whole transcriptome differences compared to those in the TT SIRT6 group. Conclusion: People with FRDA in the CT SIRT6 group have less severe neurological and visual dysfunction than those in the TT SIRT6 group. Biochemical analyses indicate that the benefit conferred by T to C SNP in SIRT6 does not come from altered expression or enzymatic activity of SIRT6 or frataxin but is associated with changes in the transcriptome.

Perspectives on current models of Friedreich's ataxia.

Friedreich’s ataxia (FRDA, OMIM#229300) is the most common hereditary ataxia, resulting from the reduction of frataxin protein levels due to the expansion of GAA repeats in the first intron of the FXN gene. Why the triplet repeat expansion causes a decrease in Frataxin protein levels is not entirely known. Generation of effective FRDA disease models is crucial for answering questions regarding the pathophysiology of this disease. There have been considerable efforts to generate in vitro and in vivo models of FRDA. In this perspective article, we highlight studies conducted using FRDA animal models, patient-derived materials, and particularly induced pluripotent stem cell (iPSC)-derived models. We discuss the current challenges in using FRDA animal models and patient-derived cells. Additionally, we provide a brief overview of how iPSC-based models of FRDA were used to investigate the main pathways involved in disease progression and to screen for potential therapeutic agents for FRDA. The specific focus of this perspective article is to discuss the outlook and the remaining challenges in the context of FRDA iPSC-based models.

Replication dependent and independent mechanisms of GAA repeat instability

Trinucleotide repeat instability is a driver of human disease. Large expansions of (GAA)n repeats in the first intron of the FXN gene are the cause Friedreich’s ataxia (FRDA), a progressive degenerative disorder which cannot yet be prevented or treated. (GAA)n repeat instability arises during both replication-dependent processes, such as cell division and intergenerational transmission, as well as in terminally differentiated somatic tissues. Here, we provide a brief historical overview on the discovery of (GAA)n repeat expansions and their association to FRDA, followed by recent advances in the identification of triplex H-DNA formation and replication fork stalling. The main body of this review focuses on the last decade of progress in understanding the mechanism of (GAA)n repeat instability during DNA replication and/or DNA repair. We propose that the discovery of additional mechanisms of (GAA)n repeat instability can be achieved via both comparative approaches to other repeat expansion diseases and genome-wide association studies. Finally, we discuss the advances towards FRDA prevention or amelioration that specifically target (GAA)n repeat expansions.

Metabolomics analysis reveals dysregulation in one carbon metabolism in Friedreich Ataxia.

Friedreich Ataxia (FA) is a rare and often fatal autosomal recessive disease in which a mitochondrial protein, frataxin (FXN), is severely reduced in all tissues. With loss of FXN, mitochondrial metabolism is severely disrupted. Multiple therapeutic approaches are in development, but a key limitation is the lack of biomarkers reflecting the activity of FXN in a timely fashion. We predicted this dysregulated metabolism would present a unique metabolite profile in blood of FA patients versus Controls (Con). Plasma from 10 FA and 11 age and sex matched Con subjects was analyzed by targeted mass spectrometry and untargeted NMR. This combined approach yielded quantitative measurements for 540 metabolites and found 59 unique metabolites (55 from MS and 4 from NMR) that were significantly different between cohorts. Correlation-based network analysis revealed several clusters of pathway related metabolites including a cluster associated with one‑carbon (1C) metabolism composed of formate, sarcosine, hypoxanthine, and homocysteine. Receiver operator characteristics analyses demonstrated an excellent ability to discriminate between Con and FA with AUC values >0.95. These results are the first reported metabolomic analyses of human patients with FA. The metabolic perturbations, especially those related to 1C metabolism, may serve as a valuable biomarker panel of disease progression and response to therapy. The identification of dysregulated 1C metabolism may also inform the search for new therapeutic targets related to this pathway.

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.

Premature transcription termination at the expanded GAA repeats and aberrant alternative polyadenylation contributes to the Frataxin transcriptional deficit in Friedreich's ataxia.

Frataxin deficiency in Friedreich’s ataxia results from transcriptional downregulation of the FXN gene caused by expansion of the intronic trinucleotide guanine-adenine-adenine (GAA) repeats. We used multiple transcriptomic approaches to determine the molecular mechanism of transcription inhibition caused by long GAAs. We uncovered that transcription of FXN in patient cells is prematurely terminated upstream of the expanded repeats leading to the formation of a novel, truncated and stable RNA. This FXN early terminated transcript (FXN-ett) undergoes alternative, non-productive splicing and does not contribute to the synthesis of functional frataxin. The level the FXN-ett RNA directly correlates with the length of the longer of the two expanded GAA tracts. Targeting GAAs with antisense oligonucleotides or excision of the repeats eliminates the transcription impediment, diminishes expression of the aberrant FXN-ett, while increasing levels of FXN mRNA and frataxin. Non-productive transcription may represent a common phenomenon and attractive therapeutic target in diseases caused by repeat-mediated transcription aberrations.

THE SONIC HEDGEHOG AGONIST SAG ATTENUATES MITOCHONDRIAL DYSFUNCTION AND DECREASES THE NEUROTOXOCITY INDUCED BY FRATAXIN-DEFICIENT ASTROCYTES

Friedreich’s ataxia (FRDA) is predominantly a neurodegenerative disease caused by the deficiency of a protein called frataxin (FXN). Although the main pathological alterations are observed in neurons, it is becoming clear that other non-neuronal cells such as astrocytes may be actively involved in the neurodegenerative process associated with the disease. Depending on the stimuli they respond to, astrocytes acquire different activation states in a process called astrogliosis. Neuroinflammatory stimuli induce the formation of A1 reactive astrocytes, which upregulate proinflammatory genes, being harmful for neurons. A1 astrocytes have been detected in post-mortem tissue of patients with different neurodegenerative disorders, being hypothesized that they might have deleterious effects on neurons, exacerbating the neurodegenerative process. Recent studies have demonstrated positive effects of Sonic Hedgehog (SHH) agonists in astrocyte viability and proliferation, astrocyte-mediated neuroprotection, and also positive effects in mitochondrial activity and dynamics. As mitochondrial changes are important components in the etiology of neurodegenerative disorders, the influence of SHH agonists in mitochondrial physiology could be of therapeutic relevance. In this work, we have thoroughly characterized astrocyte reactivity phenotype and mitochondrial status of FXN-deficient human astrocytes, evaluating as well the effect of SHH agonists on astrocyte reactivity, viability, and function. We used an in vitro model based on a short hairpin RNA packaged in a lentiviral vector, which allowed us to decrease FXN levels in human cortical astrocytes, to similar levels as those observed in FRDA patients, and found that FXN-deficient cells had less cell viability and higher expression of several A1 reactive astrocyte markers, than control cells. Both phenomena were prevented by a chronic treatment with the smoothened agonist (SAG), a SHH signaling agonist. Moreover, FXN-deficient astrocytes showed defects in mitochondrial function and dynamics, which were partially rescued by SAG. Regarding the possible neuroprotective effects of SHH agonists, previous results showed that FXN-deficient astrocytes are able to induce neurodegeneration, and we have observed that the chronic treatment with SAG attenuated the neurotoxicity triggered by the treatment of mouse cortical neurons with conditioned medium of FXN-deficient astrocytes. Overall, our results suggest that the treatment of FXN-deficient astrocytes with a SHH agonist like SAG, could be used as a possible target to reduce FRDA-associated neurodegeneration.

Advantages and Limitations of Gene Therapy and Gene Editing for Friedreich's Ataxia.

Friedreich’s ataxia (FRDA) is an inherited, multisystemic disorder predominantly caused by GAA hyper expansion in intron 1 of frataxin (FXN) gene. This expansion mutation transcriptionally represses FXN, a mitochondrial protein that is required for iron metabolism and mitochondrial homeostasis, leading to neurodegerative and cardiac dysfunction. Current therapeutic options for FRDA are focused on improving mitochondrial function and increasing frataxin expression through pharmacological interventions but are not effective in delaying or preventing the neurodegeneration in clinical trials. Recent research on in vivo and ex vivo gene therapy methods in FRDA animal and cell models showcase its promise as a one-time therapy for FRDA. In this review, we provide an overview on the current and emerging prospects of gene therapy for FRDA, with specific focus on advantages of CRISPR/Cas9-mediated gene editing of FXN as a viable option to restore endogenous frataxin expression. We also assess the potential of ex vivo gene editing in hematopoietic stem and progenitor cells as a potential autologous transplantation therapeutic option and discuss its advantages in tackling FRDA-specific safety aspects for clinical translation.

Clinical Evidence for Variegated Silencing in Patients With Friedreich Ataxia.

Background and ObjectivesFriedreich ataxia (FRDA) is a neurodegenerative disease caused by a GAA triplet repeat (GAA-TR) expansion in intron 1 of the FXN gene. Patients have 100–1,300 GAA triplets compared with less than 30 in healthy controls. The GAA-TR expansion leads to FXN silencing, and consequent frataxin protein deficiency results in progressive ataxia, scoliosis, cardiomyopathy, and diabetes. The overt heterogeneity in age at onset and disease severity is explained partly by the length of the GAA-TR, in which shorter repeats correlate with milder disease. Evidence of variegated silencing in FRDA suggests that patients with shorter repeats retain a significant proportion of cells with FXN genes that have escaped GAA-TR expansion–induced silencing, explaining the less severe frataxin deficiency in this subpopulation. In ex vivo experiments, the proportion of spared cells negatively correlates with GAA-TR length until it plateaus at 500 triplets, an indication that the maximal number of silenced cells has been reached. In this study, we assessed whether an analogous ceiling effect occurs in severity of clinical features of FRDA by analyzing clinical outcome data.MethodsThe FRDA Clinical Outcome Measures Study database was used for a cross-sectional analysis of 1,000 patients with FRDA. Frataxin levels were determined by lateral flow immunoassays.ResultsThe length of the GAA-TR in our cohort predicted frataxin level (R2 = 0.38, p < 0.0001) and age at onset (R2 = 0.46, p < 0.0001) but only with GAA-TRs with ≤700 triplets. Age and disease duration predicted performance on clinical outcome measures, and such predictions in linear regression models statistically improved in the subcohort of patients with >700 GAA triplets. The prevalence of cardiomyopathy and scoliosis increased as GAA-TR length increased up to 700 GAA triplets where prevalence plateaued.DiscussionOur data suggest that there is a ceiling effect on the clinical consequences of GAA-TR length in FRDA, as would be predicted by variegated silencing. Patients with GAA-TRs of >700 triplets represent a subgroup in which the severity of clinical manifestations based on GAA-TR length have reached maximal levels and therefore display limited clinical variability in disease progression.

A Comprehensive Triple-Repeat Primed PCR and a Long-Range PCR Agarose-Based Assay for Improved Genotyping of Guanine-Adenine-Adenine Repeats in Friedreich Ataxia

Friedreich ataxia is a rare autosomal recessive, neuromuscular degenerative disease caused by an expansion of a trinucleotide [guanine-adenine-adenine (GAA)] repeat in intron 1 of the FXN gene. It is common in the White population, characterized by progressive gait and limb ataxia, lack of tendon reflexes in the legs, loss of position sense, and hypertrophic cardiomyopathy. Detection and genotyping of the trinucleotide repeat length is important for the diagnosis and prognosis of the disease. A two-tier genotyping assay with an improved triple-repeat primed PCR (TR-PCR) for alleles <200 GAA repeats (±1 to 5 repeats) and an agarose gel-based, long-range PCR (LR-PCR) assay to genotype expanded alleles >200 GAA repeats (±50 repeats) is described. Of the 1236 DNA samples tested using TR-PCR, 31 were identified to have expanded alleles >200 repeats and were reflexed to the LR-PCR procedure for confirmation and quantification. The TR-PCR assay described herein is a diagnostic genotyping assay that reduces the need for further testing. The LR-PCR component is a confirmatory test for true homozygous and heterozygous samples with normal and expanded alleles, as indicated by the TR-PCR assay. The use of this two-tier method offers a comprehensive evaluation to detect and genotype the smallest and largest number of GAA repeats, improving the classification of FXN alleles as normal, mutable normal, borderline, and expanded alleles.