# A multiple animal and cellular models approach to study frataxin deficiency in Friedreich Ataxia

Hélène Puccio, Valentine Mosbach

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Hélène Puccio, Valentine Mosbach. A multiple animal and cellular models approach to study frataxin deficiency in Friedreich Ataxia. Biochimica et Biophysica Acta - Molecular Cell Research, 2024, 1871 (7), pp.119809. 10.1016/j.bbamcr.2024.119809. hal-04831034

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Submitted on 11 Dec 2024

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# A multiple animal and cellular models approach to study frataxin deficiency in Friedreich Ataxia

Valentine Mosbach, Hélène Puccio*

Institut NeuroMyoGene-PGNM UCBL-CNRS UMR5261 INSERM U1315, Lyon, France

## ABSTRACT

Friedreich's ataxia (FA) is one of the most frequent inherited recessive ataxias characterized by a progressive sensory and spinocerebellar ataxia. The main causative mutation is a GAA repeat expansion in the first intron of the frataxin (FXN) gene which leads to a transcriptional silencing of the gene resulting in a deficit in FXN protein. The nature of the mutation (an unstable GAA expansion), as well as the multi-systemic nature of the disease (with neural and non-neural sites affected) make the generation of models for Friedreich's ataxia quite challenging. Over the years, several cellular and animal models for FA have been developed. These models are all complementary and possess their own strengths to investigate different aspects of the disease, such as the epigenetics of the locus or the pathophysiology of the disease, as well as being used to develop novel therapeutic approaches. This review will explore the recent advancements in the different mammalian models developed for FA.

## 1. Friedreich's Ataxia (FA) a neurodegenerative disease due to a deficit in FXN protein

### 1.1. Clinical aspect

Friedreich's Ataxia (FA) is one of the most frequent inherited recessive ataxias affecting 1 in 20,000 individuals in the Caucasian population [1,2]. It is a multisystemic neurodegenerative disorder characterized by progressive sensory and spinocerebellar ataxia leading notably to gait disturbance, loss of coordination, and tremor of the upper limbs [1-3]. The main neuronal sites affected in FA are the dorsal root ganglia, the dentate nuclei of the cerebellum, posterior columns, spinocerebellar tracts and corticospinal tracts of the spinal cord, and peripheral nerves. Hypertrophic cardiomyopathy (75%) and diabetes (10%) are established comorbidities of FA [4-6]. The FA spectrum varies among patients and is strongly influenced by the genotype. In a typical case, age of onset is around the teenage years, followed by the use of a wheelchair generally required within the 10-15 years after the first symptoms, and a reduced lifespan with an average time of death being 35 years [7]. There was no treatment for FA until the recently FDA-approved Skyclarys (Omaveloxolone), which targets the NRF2 pathway involved in oxidative stress response (see part IV) [8,9].

### 1.2. Genetic cause of FA

The main causative mutation of FA is a GAA repeat expansion

localized in the 1st intron of frataxin (FXN) gene [10]. While the majority of patients are homozygous for the GAA expansion, 4% are compound heterozygote with one allele carrying a GAA expansion and the other one a loss of function mutation [11,12](Fig. 1). Only one family with a Charcot-Marie-Tooth-like phenotype has been reported with a missense mutation in homozygosity [13]. The GAA repeat size of non-pathogenic alleles is shorter than 30 triplets, while pathogenic GAA expansions can reach >1000 repeats. Among the well-described point mutations encountered, we can mention the amino acid substitution G130V, which cause a mild atypical clinical form of FA, and the amino acid substitution I154F, which is also associated with mild clinical features but more severe than for G130V patients [12,14,15](Fig. 1).

The presence of the GAA expansion in the 1st intron leads to epigenetic partial transcriptional silencing leading to decreased protein levels [16-18](Fig. 1). Heterochromatin marks of histone di- or trimethylation H3K9me2 and H3K9me3 are found enriched nearby the expanded GAA repeats and spread upstream [19-21]. Unusual DNA methylation of CpG island is also observed upstream the GAA tract in intron 1 [19,22,23]. However, molecular mechanisms by which GAA-expanded mediate gene silencing are still poorly understood. Expanded GAA repeats have been shown, in vitro, to form DNA triplexes and R-loop structures (DNA-RNA hybrid) that will interfere with transcription, triggering repressive chromatin formation [16,24-26]. Nevertheless, there is no evidence yet that the similar structures could form in vivo, and the chromatin remodeler(s) involved in the deposition of the repressive marks are still unknown. FA, like many genetic diseases

<footnote>* Corresponding author.
E-mail address: helene.puccio@inserm.fr (H. Puccio).</footnote>
due to a microsatellite instability, presents an inverse correlation between the number of repeats, the age of onset of symptoms and the severity of the disease [27,28]. Instability of the GAA expansion in the *FXN* gene is present at the meiotic level, showing a tendency toward contractions during paternal transmission, while both expansions and contractions occur in alleles during maternal transmission [29,30]. Some instability of the GAA-expanded repeats is also observed at a variable rate among tissues and increases as FA patients get older. Notably, while it is present in the central nervous system, instability of the GAA-expanded tract is much higher in the heart and pancreas [28,31,32].

### 1.3. Frataxin protein and function(s)

Frataxin, a highly conserved protein from bacteria to human, is nuclear-encoded in eukaryotes as a protein precursor of 210 amino acid [33,34]. This precursor carries an N-terminal mitochondrial targeting sequence (MTS) allowing import into mitochondria where frataxin will undergo two maturation steps producing an intermediate form (FXN$^{42-210}$) then a full mature and functional form (FXN$^{81-210}$) [33,34]. In eukaryotes, FXN has been shown to be essential, its absence leading to embryonic lethality in mice [35]. FXN has been historically described as an iron-binding protein and thereby has been suggested to be involved in a variety of pathways linked to iron metabolism, transport, storage or heme synthesis [36-38]. However, its precise role in these pathways remains unclear and has never been fully characterized. The only function well described and widely accepted so far for frataxin is its essential role in the regulation of Fe-S clusters biogenesis. Fe-S clusters are inorganic protein cofactors essential for the functioning of a variety of proteins involved in key cellular processes including central metabolism and respiration, DNA replication and repair, ribosome biogenesis or tRNAs modification [39,40]. Fe-S clusters biogenesis is

performed in eukaryotes by the mitochondrial multi-protein ISC machinery that will *de novo* assembles the inorganic sulfur produced by a cysteine desulfurase (NFS1) with iron on a scaffold protein (ISCU) [41-43]. The FXN protein plays a regulatory role in this machinery. By interacting with the NFS1/ISD11/ACP/ISCU complex, FXN will stimulate the reaction by specifically accelerating the persulfide formation and sulfur delivery to the scaffold protein ISCU [43-46] (Fig. 1).

However, there is recent evidence that points toward additional function(s) of FXN. A study highlights a possible role of FXN protein in the regulation of ketone body metabolism by preventing degradation of OXCT1 enzyme which catalyzes the conversion of ketone bodies to acetyl-CoA used by the Krebs cycle [47](Fig. 1). A decrease in OXCT1 protein levels in multiple cell types under FXN deficiency was observed, while overexpression of FXN protein in fibroblasts increased OXCT1 level [47]. In addition, the KIKO mouse model (see below), which present partial FXN deficiency, failed to metabolized ketone bodies to acetyl-CoA [47]. More recently, a study suggests that human FXN protein may have a dedicated role in the respiratory chain complex I [48]. In addition to its localization at the mitochondria cristae in close proximity to respiratory chain complexes, the FXN protein was shown to interact with the complexes, and in particular, with complex I [48]. Moreover, expression of the complex I subunit Nqo15 from *T. thermophilus*, a protein that shares structural similarities to human FXN protein and though to play a role in regeneration of Fe-S clusters of complex I, is able to increase maximal respiration defect in FA patient fibroblasts [48](Fig. 1). However, further investigation is required to elucidate the exact molecular involvement of FXN protein in these pathways.

### 1.4. FA pathophysiology

The pathophysiology of FA was initially characterized by mitochondrial dysfunction, dysregulation of iron homeostasis, impaired

<figure><img src="image_2.png" /><figcaption>Fig. 1. Pathophysiology of Friedreich Ataxia (FA).</figcaption></figure>
antioxidant defense pathways, oxidative stress and inflammation [49–51](Fig. 1). Deficits in Fe-S proteins involved in OXPHOS, Krebs cycle and iron homeostasis have been described in FA tissues, cell lines and mouse models. This can be directly linked to FXN function in Fe-S cluster biogenesis. However, determining the primary causes of FA is complex due to the interplay between mitochondrial dysfunction, oxidative stress and iron regulation, each exacerbating the other (Fig. 1). Whether other essential pathways depending on Fe-S clusters, such as DNA metabolism, contribute to FA pathophysiology is less clear. Furthermore, there is a tissue specificity observed in FA that does not entirely correlate with mitochondrial content and FXN expression. Specifically, the sensory component in FA primarily results from the degeneration of proprioceptive neurons located in the dorsal root ganglia (DRG), which express lower levels of FXN compared to other tissues, such as the heart, liver, skeletal muscle and pancreas [52,53]. Recent research has identified additional pathways contributing to the pathophysiology of FA including ferroptosis, a form of cell death triggered by iron-dependent ROS overload leading to lipid peroxidation, and the NRF2 pathway, a master regulator of the expression of at least 250 genes in response to stress that is impaired in FA models [54–57] (Fig. 1). Over the years, various mouse and cellular models have been developed to investigate the pathophysiology of FA, particularly in neuronal and heart tissues, as well as mutation mechanisms such as the epigenetics of the locus and triplet repeat instability. This review will discuss the diverse mammalian models utilized for studying FA, with a particular emphasis on the recently developed ones.

## 2. Mouse models to study Friedreich Ataxia

Due to the nature of the mutation, an unstable GAA expansion, the generation of a mouse model that will recapitulate the genetics of the patients (a large GAA expansion) with reduced levels of FXN and showing progressive spinocerebellar sensory ataxia and cardiomyopathy has been quite challenging. Over the years, several mouse models have been established: conditional deletion of *Fxn* in a specific tissue; GAA repeats insertion into the mouse *Fxn* gene; transgenic models with expression of the human gene with GAA repeats; an inducible Fxn-shRNA; point mutation models (Fig. 2). These models are all complementary and possess their own strengths to investigate different aspects of the disease, such as the epigenetics of the locus or the pathophysiology of the disease, as well as being used to develop novel therapeutic approaches. We will focus in this review only on the most widely used and the most recently developed mouse models for FA.

### 2.1. Mouse models to investigate pathophysiology

#### 2.1.1. Conditional knock-out models

Due to the essential role of FXN, the constitutive knock-out of *Fxn* gene is embryonic lethal at E6.5 in mouse [35]. To circumvent this lethality, conditional knock-out (cKO) mouse models based on the Cre-LoxP recombination system have been developed to induce FXN deficiency in a tissue-dependent manner. Since FA is a multisystemic disorder affecting both neuronal and non-neuronal tissues, several cKO have been generated [58]. These models have been extensively studied, providing insights into the pathophysiology of the loss of FXN in specific tissues and aiding in the development of therapeutic approaches.

The cardiac mouse model (cKO-MCK) is based on the expression of the Cre recombinase under the MCK (Muscle creatine kinase) promoter allowing deletion of *Fxn* restricted to the heart and skeletal muscle (Fig. 2). This model mimics the cardiomyopathy observed in FA patients, displaying a cardiac hypertrophy starting at 5 weeks and typically die around 10 weeks of age [59]. Additionally, the cKO-MCK model recapitulates certain molecular characteristics observed in FA patients, such as deficits in mitochondrial Fe-S enzymes activity (e.g. aconitase or SDH), prior to the onset of cardiac symptoms, along with mitochondrial iron accumulation starting from 7 weeks [59,60]. This mouse model was also the first one to be utilized in the development of gene therapy approaches. Indeed, it was demonstrated that intravenous injection of an adeno-associated virus expressing human FXN (AAVrh10. CAG-hFXN-HA) in these mice could prevent the onset of cardiac phenotype when administered pre-symptomatically, and reverse both the cardiomyopathy and the molecular phenotypes when administered post-symptomatic [61]. Furthermore, a dose-response study showed in the same model that 50 % transduction is sufficient for complete recovery of cardiac and molecular phenotypes [62].

Concurrently with advancements in understanding the FA cardiomyopathy, another model to investigate the neurological aspect of FA has been developed. The cKO parvalbumin (cKO-Pvalb) model is based on the expression of the Cre recombinase under the parvalbumin promoter, enabling the deletion of *Fxn* in parvalbumin expressing cells, including proprioceptive neurons of the DRG, the Purkinje neurons and large neurons of the dentate nucleus in the cerebellum [63](Fig. 2). This model mimics the sensory and cerebellar ataxia observed in FA patient, but in a more severe and accelerated manner [63]. Notably, cKO-Pvalb mice exhibit progressive loss of coordination and movement disorder starting at 6.5 weeks, with coordination defect evident by 8.5 weeks and worsening at 13.5 weeks. Molecular phenotypes, including Fe-S enzymes deficit such as SDH and iron dysregulation, are also observed as

<figure><img src="image_3.png" /><figcaption>Fig. 2. Mouse models for Friedreich Ataxia (FA).</figcaption></figure>
early as 5.5 weeks in the DRG and 18.5 weeks in the cerebellum. Administration of AAV-CAG-FXN-HA at both pre-symptomatic stage and post-symptomatic stage results in rapid and complete recovery of the sensory neuronopathy [63].

Although these cKO mouse models have enhanced our understanding of FA, they do not fully recapitulate the genetics of the disease nor the low levels of FXN observed in patients. In fact, these models present a complete absence of FXN in a specific tissue early in development, leading to a more severe phenotype. Furthermore, the complete deletion of FXN protein in these tissues is typically restricted to specific cell types, which are primarily affected in the disease, failing to reflect the low levels of FXN present in all cells with the possible dysfunction of the neighboring cells that could participate in exacerbating the pathology. To develop mouse models that more closely resemble the genetics and clinical phenotype in patients, two new transgenic mouse models have recently been generated based on the FXN point mutations found in compound heterozygote patients.

### 2.1.2. Point mutation-based models

Recently, two mouse models based on missense mutations found in FA patients (G130V and I154F) have been established, namely Fxn<sup>G127V/G127V</sup> and Fxn<sup>I151F/I151F</sup> [64–66](Fig. 2). Unlike conditional KO models, the primary advantage of these models is the expression of very low levels of Fxn ubiquitously. Although these models still do not fully replicate the genetics of patients, as they expressed a point mutation in a homozygous state while patients are compound heterozygote with one allele carrying a point mutation and the other a GAA expansion, they offer new insights into deciphering the pathological impact of missense mutations in FA at a multi-systemic level, as well as the consequence of very low levels of FXN during development and throughout life.

The G130V mutation is one of the most common point mutations found in compound heterozygote FA patients, resulting in a milder clinical phenotype and a slower disease progression compared to patients homozygous for GAA expansions [12]. While this point mutation does not prevent mitochondrial localization of the protein, it was shown that FXN<sup>G130V</sup> protein processing from the FXN<sup>42–210</sup> form to the mature FXN<sup>81–210</sup> form is impaired, leading to increased level of the intermediate FXN<sup>42–210</sup> form [14]. It is still unclear if the intermediate FXN<sup>G130V</sup> protein retains residual activity in the Fe-S cluster biogenesis machinery or if there is still a small amount of the intermediate form that is slowly processed into the mature form, potentially explaining the milder clinical phenotype [14].

The mouse equivalent G127V missense mutation was introduced into the endogenous Fxn gene through zygote microinjection with a CRISPR-Cas9 editing mediated homologous repair system [64,65]. A significantly low level of FXN<sup>G127V</sup> protein was detected in cerebral cortex, cerebellum, spinal cord and heart compared to wild-type, KIKO mouse (GAA230/KO, see below). Fxn<sup>G127V/G127V</sup> mice displayed a smaller size compared with wild-type mice, and mild alterations in strength and locomotor function were observed at a relatively late onset. However, earlier phenotypes cannot be completely excluded necessitating further characterization of the model. Bulk RNA sequencing analysis revealed downregulation of genes associated with oxidative phosphorylation and protein synthesis [65]. Embryonic fibroblasts derived from this mouse model exhibited reduced proliferation, increased cell senescence and mitochondrial impairment [64]. Furthermore, recent multi-omics analyses of cardiac mitochondrial stress response conducted in three mouse models with differentially altered FXN level, showed that only the Fxn<sup>G127V/G127V</sup> mouse model exhibited transcriptional changes consistent with a possible late onset of cardiomyopathy and a mitochondrial integrated stress response (ISR<sup>mt</sup>) previously reported in the conditional cardiac KO mouse models [67].

The I154F point mutation, found in FA patients, also result in a mild clinical form, but is more severe than G130V patients. Similarly to the G130V, the I154F point mutation has been shown to impede the processing of the intermediary FXN<sup>42–210</sup> form, resulting in low levels of

mature FXN protein [14,68]. There is also evidence suggesting that this point mutation inhibits the interaction of FXN with ISD11 and decreases the activation of the cysteine desulfurase NFS1 [69,70]. The Fxn<sup>I151F/I151F</sup> mouse model was established with the CRISPR-Cas9 strategy [66]. Low levels of FXN<sup>I151F</sup> protein were detected in cerebellum, heart, spinal cord, liver, pancreas and skeletal muscle. Additionally, this mouse model exhibited decreased protein levels of OXPHOS complexes and decreased aconitase activity in the brain and heart, potentially correlating with a defect in Fe-S clusters biogenesis [66]. Moreover, a recent study showed decreased activity of complex II + III of the respiratory chain in primary sensory neurons isolated from the Fxn<sup>I151F/I151F</sup> mouse, as well as the presence of abnormal mitochondria without cristae and condensed mitochondria in 39-weeks-old mice [71]. The Fxn<sup>I151F/I151F</sup> mice displayed reduced weight gain starting at 15 weeks of age, as well as neurobehavioral deficits such as reduced motor coordination and strength, albeit only with late onset (30–40 weeks of age), similar to the Fxn<sup>G127V/G127V</sup> mouse model [66].

### 2.1.3. Inducible shRNA mouse model

Another model, known as the inducible FRDAkd mouse has been recently developed using a doxycycline-inducible shRNA to knock-down murine Fxn mRNA [72](Fig. 2). This mouse model does not carry GAA repeat in the endogenous Fxn locus but harbors a single copy of the inducible-shRNA inserted at a specific genomic locus (rosa26). The first characterization of this mouse model revealed motor deficits associated with ataxia similar to those observed in FA patients, along with hypertrophic cardiomyopathy and FA molecular features such as mitochondrial abnormalities in cardiomyocytes and DRG neurons [72]. Importantly, the onset and progression of the mouse phenotypes correlated with the concentration of doxycycline used to induce the shRNA against Fxn, and these phenotypes are partially rescued upon restoration of Fxn expression following removal of doxycycline. This feature makes the FRDAkd mouse a valuable model to investigate the temporality of disease progression and identify the optimal therapeutic window [72]. Furthermore, a recent study demonstrated cerebellar degeneration in the FDRAkd mouse model, characterized by loss of Purkinje neurons and large dentate nucleus neurons, as well as decreased levels of VGLUT2 (a marker of climbing fibers) and VGLUT1 (marker of parallel fibers) at 4 and 18 weeks of age [73].

## 2.2. Mouse models based on GAA repeat expansion insertion

Mouse models based on GAA repeat expansion offer the advantage of being more genetically similar to patients. Although they may not fully replicate the ataxic phenotype and molecular features of FA, or at a relatively late onset, these models are particularly valuable for studying the epigenetics of the FXN locus and GAA repeat instability, as well as for developing therapies targeting the epigenetics.

### 2.2.1. GAA repeats inserted into mouse gene (KIKO)

The KIKO mouse model, developed in 2002 [74], is the first mouse model based on GAA expansion (Fig. 2). In this model, one allele of the endogenous Fxn gene contains an inserted expansion of 230 GAA (KI), while the other allele has a deletion of exon 4 (KO), resulting in a FXN residual protein level of 25–36 % compared to wild-type. Unlike FA patients, this mouse model does not exhibit meiotic and somatic instability of the GAA repeats. Neurobehavioral deficit related to muscle strength and gait ataxia were identified after 8 months of age [75]. More recently, a study found that the KIKO mouse model developed exercise and glucose intolerance at 6 months of age, along with moderate cardiac dysfunction, which correlated with a decreased expression of iron regulatory protein 1 (IPR1) [76]. Impairment of complexes I and II of respiratory chain was also observed in KIKO mice cerebellum, as well as dysregulated cerebellar circuitry [77,78]. Additionally, lipid peroxidation and decreased expression of GPX4 were detected in KIKO mice, suggesting sensitivity to ferroptosis. Overall, this model still maintains a
high level of residual FXN protein and exhibits a late-onset phenotype. However, it remains suitable for developing therapeutic approaches based on upregulation of FXN mRNA or protein, as well as investigating the epigenetic silencing mechanisms of GAA repeats.

### 2.2.2. Humanized mouse models (human gene with a GAA sequence inserted)

The humanized mouse models are hemizygous for a yeast artificial chromosome (YAC) carrying the human *FXN* gene with an expanded GAA repeat tract and homozygous for the complete deletion of the endogenous mouse *Fxn* gene (Fig. 2). Initially, two YAC mouse models were generated: the YG8R model derived from founder YG8 (before deletion of the mouse *Fxn* gene), which carries two copies of the human *FXN* gene with approximately 90 and 190 GAA repeats, and the YG22R derived from YG22 founder, which carries a single copy of *FXN* gene with 190 GAA repeats. Subsequently, the YGs8R mouse model, derived from a YG8R carrying a single copy of *FXN* gene with 250–300 GAA repeats, was established [79–81]. All these mouse models exhibit somatic GAA repeat instability in the brain, cerebellum and liver. The YG8R also demonstrates intergenerational instability and recapitulates epigenetic modifications leading to FXN gene silencing. However, these mouse models have limited phenotypes and do not manifest overt ataxia. They are primarily utilized to investigate repeat instability and gene silencing mechanisms as well as to develop therapeutic approaches aimed at increasing *FXN* expression.

In the past decade, a new human *FXN* YAC transgenic mouse model has been established derived from the YG8sR strain established by the Jackson laboratory, and harboring >800 GAA repeats [82,83](Fig. 2). This new mouse model, called YG8–800, not only exhibits somatic and intergenerational instability, but also recapitulates epigenetic marks such as DNA methylation in the brain, cerebellum and heart tissues, along with decreased histone H3K9 acetylation and increased H3K9 methylation in the cerebellum. Behavioral tests have revealed progressive locomotion impairment and balance deficits from 11 to 20 weeks of age, as well as heart hypertrophy around 26 weeks of age. However, the presence or severity of phenotypes observed in these mice can vary between laboratories and may depend on the sanitary status or microbiome of the animal facility, necessitating further characterization.

## 3. Cellular models to study Friedreich Ataxia

Cellular models to study FA have made significant progress in the past 15 years, particularly with the advent of induced pluripotent stem cell (iPSC) technology. This breakthrough was made possible by the cellular reprogramming of somatic cells into iPSCs, a technique developed by Yamanaka in 2006 [84]. The first iPSCs derived from FA patients were generated in 2010 and showed a maintenance of *FXN* gene silencing as well as a GAA repeat instability (both expansions and contractions) through passages [85]. Subsequently, the differentiation of FA iPSCs into neurons and cardiomyocytes revealed phenotypes associated with FXN deficiency [86,87]. Since then, several FA iPSCs lines have been established and utilized to investigate molecular mechanisms related to GAA instability and epigenetics of the locus, as well as to model FA phenotypes in disease related cell types, owing to the increasing number of differentiation protocols available for various cellular types [88]. In this context, we will focus on recent advancement using FA iPSC-based models.

### 3.1. New neuronal models from FA iPSCs

In FA, specific cell types in both central nervous system (CNS) and peripheral nervous system (PNS) are affected. Particularly, a primarily degeneration of the large sensory neurons, and more specifically of the proprioceptive neurons, of the dorsal root ganglia (DRG) are observed [52,53]. However, DRG is a complex PNS tissue composed of non-neuronal cells and different types of sensory neurons

(mechanoreceptor, nociceptor and proprioceptor). Proprioceptive neurons represent only a minor population (approximately 7.5 %) in the DRG. Differentiation protocols have primarily focused on generating mixed sensory neurons or nociceptive-enriched cultures. Initial studies using FA iPSCs cells mainly generated either central nervous system-related neurons or mixed sensory neurons cultures [86,88]. Despite the identification of deficit in some Fe-S proteins and reduced mitochondrial potential, the relatively mild FA phenotype observed may be attributed to the presence of cell types less sensitive to reduced FXN levels. Recently, a novel protocol has been established to produce iPSC-derived primary sensory neurons cultures enriched up to 50 % for proprioceptive neurons [89]. Proprioceptive-enriched cultures derived from FA iPSCs were successfully established and presented decreased survival compared to controls. Transcriptomic and proteomic characterization of this model revealed impairments in cytoskeleton organization, abnormal neurite extension, and altered synaptic plasticity at late maturation stages [90]. This model also recapitulated the epigenetic marks observed in patients. Interestingly, neurons derived from an isogenic iPSCs control line, generated from a GAA repeat CRISPR-edited FA line, show a reversal of *FXN* locus repression but still expressed FA phenotypic features [90].

Advancement in iPSCs technology have enabled the production of organoids, self-organized 3D structures that mimic the key functional and spatial organization of specific tissues/organs with their different cell types and their interactions. A protocol has recently been published to generate DRG organoids from iPSCs [91]. Single cell transcriptomic analysis of DRG organoids derived from control iPSCs showed a transcriptional signature similar to *in vivo* DRG, with the presence of the three sensory neuronal subtypes. However, the expression of early neuronal markers was still detectable at late-stage maturation of the organoids, suggesting that the system is not fully mature [91]. DRG organoids from FA iPSCs lines were successfully obtained and exhibited decreased neuronal survival as well as abnormal axonal morphology [91]. These phenotypes were rescued when intron 1 with the GAA repeat was entirely removed, but not when only the GAA repeat tract was removed [91]. This suggests that removing just the GAA expanded repeat might not be sufficient to reverse the epigenetic silencing.

### 3.2. Cardiomyocytes from FA iPSCs

FA iPSC-derived cardiomyocytes exhibit mitochondrial dysfunction with OXPHOS impairment and iron accumulation [86,87]. They also display features of hypertrophic cardiac stress response, as well as calcium signaling deficiency and lipid droplet accumulation, which may contribute to ferroptosis [87,92,93]. Correction of *FXN* gene using zinc-finger nuclease (ZFN) to excise the expanded GAA repeats in FA iPSC-derived cardiomyocytes resulted in upregulation of *FXN* expression, reduction in lipid accumulation and a reversal in transcriptomic signatures associated with the disease [93].

## 4. Therapeutic aspect

Given the complexity of FA, addressing its therapeutic aspects requires a multifaceted approach that spans genetic modulation, mitochondrial and metabolic interventions, symptomatic treatments, and cutting-edge emerging therapies. Developing effective treatments involves a delicate balance between targeting the underlying genetic abnormalities, mitigating mitochondrial dysfunction, and providing symptomatic relief. Various therapeutic approaches, including gene therapy, protein replacement therapy, or targeting downstream events, are being developed by leveraging the strengths of each specific models. We will briefly mention a few therapeutic approaches, with an emphasis on the recently first FDA-approved drug. For a full review of therapeutic approaches being developed for FA, please refer to the Friedreich ataxia Research alliance pipeline (https://www.curefa.org/research/research-pipeline) which is always updated with the most recent developments.
## 4.1. Therapeutic approaches aimed to increased FXN (mRNA or protein) levels

Different strategies have been explored to increase FXN levels such as epigenetic remodeling of the *FXN* locus or gene therapy. We already discussed the use of conditional KO mouse models (cKO-MCK and cKO-Pvalb) in developing AAV-based gene therapy (see part II.1.a). Antisense Oligonucleotides (ASOs) designed to target the expanded GAA repeats in the 1st intron and modulate RNA splicing mechanism to increase *FXN* gene expression have been investigated as a therapeutic strategy in the YG22R mouse model. This study showed potential of ASOs to rectify the fundamental genetic aberrations underlying FA, thereby offering a promising avenue for therapeutic advancement [94].

Epigenetic remodeling aimed at removing *FXN* gene repression have been mainly based on the use of Histone Deacetylase Inhibitors (HDACIs) and tested in mouse models engineered to carry GAA repeat expansions [95]. Among them, HDACI 106 and 109 were observed to restore *FXN* expression in YG8R and KIKI (carrying two GAA<sub>230</sub> in the mouse Fxn gene derived from KIKO) mice models [96–98]. Nicotinamide (vitamin B3) was also able to remove *FXN* repression in YG8R mouse model [99]. Cellular models based on iPSC technology offer unique perspective to test epigenetic-based FA therapeutic strategies and to conduct large-scale drug screenings in a human-based model. For example, neurons derived from FA iPSCs treated with a benzamide HDACIs expressed an increase in FXN protein levels and reduced oxidative stress [100]. Recently, a drug screening of commercially available compounds targeting pathways for gene activation or repression were conducted using an engineered FA iPSC line containing a *FXN* fusion reporter gene (*FXN-NLuc*) to assess *FXN* expression [101]. *They identified that only* HDACIs were able to reactivate *FXN* expression [101].

Protein replacement therapy targets frataxin deficiency by delivering exogenous frataxin directly to mitochondria. The TAT protein transduction domain facilitates this process by aiding in the transport of compounds across cell membranes. CTI-1601, a fusion protein of TAT and frataxin, is designed to enter mitochondria where it is cleaved, releasing mature frataxin and effectively “locking” it within the organelle [102]. In preclinical studies, CTI-1601 has shown promise in improving cardiac function and increasing lifespan in animal models of Friedreich’s ataxia (FA), albeit without complete phenotype rescue [103]. CTI-1601 has received various designations from the FDA for FA treatment. Phase 1 trials have shown promising results in terms of safety, tolerability, and increased frataxin levels in participant tissues, and CTI-1601 is currently in phase 2.

## 4.2. Therapeutic approaches targeting downstream events of FXN deficiency

Since the main consequences of FXN deficiency is a mitochondrial deficit, iron dysregulation and sensitivity to oxidative stress, the first developed therapeutic approaches were based on iron chelators or antioxidant drugs. Among them, the antioxidant Idebenone has been investigated for its ability to mitigate oxidative stress and improve mitochondrial function in conditional cardiac model and its neuroprotective effects have been associated with improvements in neurobehavioral parameters, emphasizing its potential as a therapeutic agent for FA [60,104]. However, none of these molecules have been validated as effective in clinical trials. More recently, new active molecules have shown promise in targeting effects of FXN deficiency such as EPI-743, a compound targeting mitochondrial function, that was shown to ameliorate neurological and cardiac parameters in KIKO mouse model, underscoring its potential as a therapeutic intervention for FA [105].

The NRF2-signaling pathway is one of the major antioxidant defense mechanisms of the cell. Under stress conditions, Nrf2 protein will escape its cytosolic sequestration and degradation by the homodimer protein KEAP1 to translocate to the nucleus where it binds to DNA antioxidant response element (ARE) to activate a multitude of genes involved in

stress response. The first observation of the impairment the NRF2-signaling pathway in FA was made in 2009 in a study conducted on FA patient fibroblasts, where an abnormal location of Nrf2 protein which fails to be translocated in the nuclei in response to oxidative stress was observed [106]. The current understanding is that inflammation, mitochondrial dysfunction and iron accumulation, results in an overload of ROS, triggering the aberrant NRF2 degradation *via* KEAP1 hindering its nucleus translocation. Thereby, the expression of NRF2 target genes is not activated, exacerbating inflammation, ROS production and sensitivity to ferroptosis, forming a detrimental cycle. In 2018, omaveloxolone, a synthetic compound and a second-generation member of oleanane triterpenoid, known to be a NRF2 activator by directly acting on the KEAP1 protein to prevent NRF2 degradation, demonstrated efficacy in preventing mitochondrial defects and oxidative stress on FA patient fibroblasts [107]. Subsequently, Omaveloxolone was tested in clinical trials and has recently received FDA approval as the first treatment for FA, marketed under the name Skylarys [8,9].

In the future, the recently established mouse models based on G127V and I151F point mutations offer the opportunity to explore mutation-specific responses to interventions [64,66]. This may encompass targeted gene therapies designed to restore normal frataxin expression or pharmacological agents aiming to mitigate the downstream effects of these mutations on cellular physiology.

GAA expansion in the 1st intron of *FXN* gene leads to a decrease in FXN protein production. Alternatively, some patients are compound heterozygotes with one allele carrying a GAA expansion and the other one a point mutation. Deficit in FXN protein leads to a global mitochondrial dysfunction with a decrease in Fe-S clusters biogenesis, an iron accumulation in the mitochondria and increase in reactive oxygen species (ROS) production, all these phenomena exacerbating each other. FXN function in Fe-S clusters biogenesis is well defined, it will accelerate each step of the reaction from persulfide formation by NFS1 and sulfur delivery to the scaffold protein ISCU. New evidence suggests two other possible functions of FXN in mitochondria that could contribute to the pathophysiology: 1) FXN may prevent the degradation of OXCT1 enzyme which catalyzes the conversion of ketone bodies to acetyl-CoA. A deficit in FXN leads to a decrease in OXCT1 and, consequently, in acetyl-CoA production, which may affect TCA function. 2) FXN may have a dedicated role in complex I of respiratory chain, which, if lost, could exacerbate the OXPHOS deficiency already present due to the lack of Fe-S clusters. The iron-dependent ROS overload generating lipid peroxidation, in addition to impaired glutathione metabolism, will increase the sensitivity of cells to ferroptosis cell death pathway. Moreover, the mitochondrial dysfunction and iron accumulation that leads to ROS overload will trigger aberrant NRF2 degradation *via* KEAP1, preventing its translocation to the nucleus and consequently the expression of NRF2 target genes, leading to further increases in inflammation, ROS production, and sensitivity to ferroptosis.

The different mouse models developed to study FA are presented in two tables, one focusing on the mouse models mainly used to investigate pathophysiology that do not possess GAA expansions and the other one presenting the mouse models based on GAA expansion insertion either in the *Fxn* mouse gene or in a YAC containing the human *FXN* gene. For each mouse model, it is described whether it expresses a FA-like phenotype, recapitulates the genetics of the disease and if GAA repeat instability as well as chromatin repression of the *Fxn* gene are observed. For each mouse model, it is indicated for which types of developing therapeutic approaches it could be used for, the * indicating that this type of therapeutic strategy has already been tested in the model.

### CRediT authorship contribution statement

**Valentine Mosbach:** Writing – original draft. **Hélène Puccio:** Writing – original draft.
# Declaration of competing interest

The authors declare no conflict of interest.

# Data availability

Review article no original data have been generated

# Acknowledgements

This research was funded by the AFM, as part of the MyoNeurAlp II Strategic grant (#23894) (to H.P.) and by a FARA Postdoctoral fellowship (to V.M.). All the figures were created with BioRender.com

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