HUMAN GENE THERAPY 25:684–693 (August 2014) ª Mary Ann Liebert, Inc. DOI: 10.1089/hum.2013.180

Review

Cell and Gene Therapy for Friedreich Ataxia: Progress to Date Marguerite V. Evans-Galea,1,2 Alice Pe´bay,3 Mirella Dottori,4 Louise A. Corben,1,5 Sze Hwee Ong,1 Paul J. Lockhart,1,2 and Martin B. Delatycki 1,2,5,6

Abstract

Neurodegenerative disorders such as Friedreich ataxia (FRDA) present significant challenges in developing effective therapeutic intervention. Current treatments aim to manage symptoms and thus improve quality of life, but none can cure, nor are proven to slow, the neurodegeneration inherent to this disease. The primary clinical features of FRDA include progressive ataxia and shortened life span, with complications of cardiomyopathy being the major cause of death. FRDA is most commonly caused by an expanded GAA trinucleotide repeat in the first intron of FXN that leads to reduced levels of frataxin, a mitochondrial protein important for iron metabolism. The GAA expansion in FRDA does not alter the coding sequence of FXN. It results in reduced production of structurally normal frataxin, and hence any increase in protein level is expected to be therapeutically beneficial. Recently, there has been increased interest in developing novel therapeutic applications like cell and/or gene therapies, and these cutting-edge applications could provide effective treatment options for FRDA. Importantly, since individuals with FRDA produce frataxin at low levels, increased expression should not elicit an immune response. Here we review the advances to date and highlight the future potential for cell and gene therapy to treat this debilitating disease.

Introduction

F

riedreich ataxia (FRDA) is an autosomal recessive disease with an average age at onset of 10.0 – 7.4 years (Delatycki et al., 1999). It is the most common inherited ataxia and accounts for 75% of ataxia with onset before 25 years of age (Pandolfo, 2006). Although there is some variation in the prevalence of FRDA in different ethnic backgrounds, it affects 1:29,000 Caucasians (Delatycki et al., 2000; Puccio and Koenig, 2002). Clinical features include progressive ataxia, spasticity, loss of lower limb reflexes, posterior column sensory changes, scoliosis, and foot deformity (Harding, 1981; Delatycki et al., 2000; Cruz-Marino et al., 2010). Reflecting the systemic nature of this disease, multiple studies have also identified progressive visual changes, including optic atrophy (Porter et al., 2007), macular and retinal nerve fiber layer abnormalities (Seyer et al., 2013), eye movement (Fahey et al., 2008) and auditory defects (Rance et al., 2008, 2010), olfactory dysfunction

(Connelly et al., 2003), impaired motor planning (Corben et al., 2010) and higher-order cognitive dysfunction (Fielding et al., 2010), and sleep-disordered breathing (Corben et al., 2013). Though the reported incidence of diabetes mellitus can vary (as reviewed by Cnop et al., 2013), it occurs in approximately 10% of those affected (Lynch et al., 2002). Affected individuals are wheelchair-bound on average 15.5 years after onset (Harding, 1981). They usually develop dysarthria and become progressively dependent over approximately the next 20 years (Delatycki et al., 1999). Affected individuals have a significantly impaired quality of life (Wilson et al., 2007), and while current therapies can provide some symptomatic relief, there are no proven treatments that slow disease progression. Lifespan is markedly reduced with congestive heart failure and arrhythmias being the most commonly reported causes of death (Meyer et al., 2007; Tsou et al., 2011) on average, between 40 and 50 years of age (Schulz et al., 2009).

1

Bruce Lefroy Centre for Genetic Health Research, Murdoch Children’s Research Institute, Parkville Victoria 3052, Australia. Department of Paediatrics, The University of Melbourne, Royal Children’s Hospital, Victoria 3052, Australia. Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, and Department of Ophthalmology, The University of Melbourne, East Melbourne, Victoria 3010, Australia. 4 Centre for Neural Engineering, The University of Melbourne, Victoria 3010, Australia. 5 School of Psychology and Psychiatry, Monash University, Clayton, Victoria 3168, Australia. 6 Clinical Genetics, Austin Health, Heidelberg, Victoria 3084, Australia. 2 3

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In FRDA, homozygosity for a GAA trinucleotide-repeat expansion in intron 1 of FXN accounts for approximately 98% of affected individuals, with the remainder being compound heterozygous for a GAA expansion and a point/insertion/ deletion mutation (Cossee et al., 1999; Gordon, 2000; EvansGalea et al., 2011). The GAA expansion in FXN results in reduced expression of the encoded mitochondrial protein frataxin, which plays a role in iron–sulfur cluster assembly, heme synthesis, and intracellular iron homeostasis (Rouault and Tong, 2008; Schmucker et al., 2011). Multiple factors contribute to FXN silencing in FRDA, including disrupted transcriptional elongation (Punga and Buhler, 2010; Kumari et al., 2011; Goula et al., 2012) and epigenetic changes affecting chromatin remodeling and DNA methylation (Greene et al., 2007; Al-Mahdawi et al., 2008; Castaldo et al., 2008; Evans-Galea et al., 2012). This leads to a dysregulated iron regulon, mitochondrial iron accumulation, and increased cellular oxidative stress (Huang et al., 2009; Santos et al., 2010). These events impact mitochondria-rich cardiomyocytes and neurons, resulting in two of the clinical features characteristic of FRDA—cardiomyopathy and neurodegeneration (Dedov and Roufogalis, 1999; Puccio, 2007; Sparaco et al., 2009). Major sites of neurodegeneration in FRDA are the dorsal root ganglia (DRG), and the cerebellar and spinocerebellar tracts (De Biase et al., 2007). The DRG are affected in several ways: sensory neurons (small and large) are reduced in size and number; satellite cells proliferate (these are glial precursors that normally adhere to the neuronal body); and there is a loss of large myelinated fibers of the dorsal root (Koeppen et al., 2009). Effective targeting and correction in each of these different tissues in FRDA are significant challenges facing cell and gene therapy researchers today. Developing Novel Treatments for FRDA

Treatments for FRDA are urgently needed. If neurodegeneration could be slowed or even halted, affected indi-

FIG. 1. Increasing frataxin levels using cell and gene therapy has the potential to improve clinical outcome for individuals with FRDA.

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viduals would be more independent, improving their overall quality of life. The identification of specific genes and molecular mechanisms involved in disease offers significant potential for the development of effective therapies. In individuals homozygous for GAA expansions in intron 1 of FXN, the frataxin produced is structurally normal and functional. It is therefore feasible to propose that increasing frataxin levels will be therapeutic (Fig. 1). Indeed, a modest increase in FXN expression after treatment with benzamide histone deacetylase (HDAC) inhibitors, which inhibit class 1 HDACs, resulted in improved locomotor activity and coordination in an FRDA mouse model (Sandi et al., 2011). Evidence in humans also supporting this was shown in two recent studies where disease severity, as measured by the Friedreich Ataxia Rating Scale, inversely correlated with FXN transcript and frataxin protein levels (Evans-Galea et al., 2012; Plasterer et al., 2013). Several pharmacological compounds have been trialed and others are in development to treat FRDA (Perlman, 2012). These include the antioxidant coenzyme Q and its synthetic analog idebenone (Parkinson et al., 2013); the iron chelator deferiprone (Goncalves et al., 2008); the drug pioglitazone that can stimulate mitochondria by activating PGC-1a (Marmolino et al., 2010); and agents aimed at increasing frataxin like the antioxidant resveratrol (Li et al., 2013) and benzamide HDAC inhibitors (Herman et al., 2006; Rai et al., 2010). Success has varied to date with several agents showing promise in animal and cellular models not always effective in clinical trial. There are new data, however, that the ‘‘drug pipeline’’ is starting to yield potential treatment options for FRDA. HDAC inhibitors like RG2833 (Repligen Corporation) increase frataxin levels (Herman et al., 2006; Rai et al., 2010), and it has recently completed early-phase clinical trials. New derivatives to increase safety and efficacy are currently in development (BioMarin Pharmaceuticals). The mitochondrial disease drug EPI-743 (Edison Pharmaceuticals) was also recently fast-tracked by the U.S. Food and Drug Administration to

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expedite its clinical development for FRDA. Potential combination therapies using up to three different compounds have also recently entered open-label clinical trials (Arpa et al., 2013, 2014). Developing Gene and Cell Therapies for FRDA

even a small percentage of cells could have a positive effect on the surrounding microenvironment and improve overall clinical outcome. This review focuses on recent advances in cell and gene therapy for FRDA. Cell Therapy for FRDA

Gene and cell therapies are being developed as promising treatments for several genetic diseases. Pioneering proofof-principle clinical trials for cell and gene therapy have successfully treated patients with the demyelinating disease X-linked adrenoleukodystrophy (Cartier et al., 2009), adenosine-deaminase-deficient severe combined immunodeficiency (SCID) (Aiuti et al., 2002, 2009), chronic granulomatous disease (CGD) (Ott et al., 2006), X-linked SCID (Cavazzana-Calvo et al., 2010), Leber congenital amaurosis (Cideciyan et al., 2009), hemophilia (Nathwani et al., 2011), and, more recently, Wiskott–Aldrich syndrome (Aiuti et al., 2013) and metachromatic leukodystrophy (Biffi et al., 2013). Following these successes, cell and gene therapy for FRDA is being pursued with many approaches currently being tested for potential efficacy (Table 1). A significant advantage in developing cell and gene therapy for FRDA is that affected individuals already produce frataxin, although at very low levels, so therapeutic introduction of frataxin will not illicit an immune response. Restoring function to

Bone marrow cells

Cells derived from the bone marrow (BM) present an appealing source of readily available cells for therapy. The inherent ability of BM-derived cells to correct cardiac and neurodegenerative phenotypes has been observed, but is not fully understood. BM-derived cells in transplanted mice have been detected at low levels in the liver, kidney, spleen, heart, BM, thymus, and lung, as well as the central nervous system, including the forebrain, olfactory bulb, and cerebellar Purkinje neurons (Priller et al., 2001; Corti et al., 2002b; Jang et al., 2004; Nygren et al., 2008; Jones et al., 2010). Donor BM-derived cells were also detected in the cerebellum of parabiotic mice ( Johansson et al., 2008) and adult human brains post-BM transplant (Weimann et al., 2003a). BM cells can fuse with damaged cells to restore function, and the frequency of such events increases upon injury or inflammation (Weimann et al., 2003b; Nygren et al., 2004,

Table 1. Studies That Have Contributed to the Development of Gene and Cell Therapies for FRDA Increasing FXN/frataxin Yeast artificial chromosome expressing human FXN Bacterial artificial chromosome expressing human FXN Lentiviral or adeno-associated vector expression of human FXN HSV-1 amplicon vectors with 135 kb genomic FXN locus

HSV-1 amplicon vectors expressing human FXN cDNA iPSCs derived from fibroblasts of individuals with FRDA BM-derived MSCs TAT-frataxin fusion protein TALE proteins fused to an activation domain targeting the FXN promoter AAV expressing human FXN

Reported outcomes Rescues embryonic lethality of Fxn-deficient mice

References Pook et al. (2001) Sarsero et al. (2004)

Reduced sensitivity to oxidative stress in fibroblasts from individuals with FRDA Reduced sensitivity to oxidative stress in fibroblasts from individuals with FRDA Prolonged FXN expression in the brain of wild-type mice Improved motor coordination in a conditional Fxn knock-out mouse Successful development of iPSCs and differentiation to neural and cardiac cells Increased frataxin expression and resistance to oxidative stress; neuroprotection Increased lifespan and cardiac function in a conditional Fxn knock-out mouse Increased FXN transcript and frataxin protein in human FRDA fibroblasts Increased lifespan and cardiac function in a conditional Fxn knock-out mouse

Fleming et al. (2005) Gomez-Sebastian et al. (2007) Gimenez-Cassina et al. (2011) Lim et al. (2007) Ku et al. (2010); Liu et al. (2011); Hick et al. (2013); Lim et al. (2013) Kemp et al. (2011b); Jones et al. (2012, 2013) Vyas et al. (2012) Chapdelaine et al. (2013) Perdomini et al. (2014)

AAV, adeno-associated virus; BM, bone marrow; FRDA, Friedreich ataxia; iPSCs, induced pluripotent stem cells; MSCs, mesenchymal stem cells.

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2008; Johansson et al., 2008). It has also been reported that BM cells can transdifferentiate to other cell types (Weimann et al., 2003a; Shetty et al., 2009); however, more recent studies show that the trophic factors released by BM-derived cells are the more likely mechanism providing benefit (Schira et al., 2011; Bai et al., 2012). Clearly, these various mechanisms differ and will impact the degree and longevity of potential correction. In a rat model of complete sciatic nerve transection, injection of BM cells at the injury site (midthigh, distant from the DRG) led to satellite cell proliferation in lumbar DRG, and increased neuron survival in the DRG and spinal cord, compared with noninjected rats (Ribeiro-Resende et al., 2009). In mice that received GFP-positive (GFP + ) BM, GFP + cells expressing markers for immature and differentiated neurons were detected 3 months post-BM transplant in the DRG (Corti et al., 2002a). GFP + cells expressing astrocyte markers were also seen in the spinal cord. These data support that the trophic activity of BM can increase neuron survival in the DRG and spinal cord (Schira et al., 2011), but also that BM-derived cells can populate these tissues. Jones and colleagues (2010), followed by Kemp and colleagues (2011), reported that following the injection of BM-derived mesenchymal stem cells (MSCs), 1–1.5% heterokaryon with Purkinje cells were identified ( Jones et al., 2010; Kemp et al., 2011a). Importantly, both studies found increased neurotrophic activity after transplant with improved neuronal survival. Increased frataxin expression and resistance to oxidative stress were also observed in vitro when FRDA patient fibroblasts were exposed to MSCderived soluble factors (Kemp et al., 2011b). Neuroprotection as well as increased frataxin expression and resistance to oxidative stress were also observed when neural crest stem cell-like periodontal ligament cells isolated from individuals with FRDA were cultured in adipose stem cell-conditioned media ( Jones et al., 2012). A similar neuroprotective effect of media, conditioned by culturing with murine MSCs, was also recently observed using DRG isolated from FRDA mice ( Jones et al., 2013). Significant improvement in motor coordination via rotarod assay was also observed following transplant of BM-derived MSCs into the cerebellum of Lurcher mice, a model of cerebellar ataxia ( Jones et al., 2010). Heart failure (hypertrophic cardiomyopathy) is the major cause of death in FRDA. In mice, BM homes to damaged areas and can repair an infarcted heart, improving its function and extending survival (Orlic et al., 2001a,b,c). Similarly to the neural system, cardiac improvement is primarily caused by the trophic activity of BM and also rare heterokaryon formation (Nygren et al., 2004, 2008; Behfar and Terzic, 2006; Lee, 2010), showing that improving the function of even a small percentage of cells can positively affect the surrounding microenvironment and improve overall outcome. Stem cells

Induced pluripotent stem cells (iPSCs) are cells derived from somatic cells, including fibroblasts (Takahashi and Yamanaka, 2006). Like embryonic stem cells, iPSCs can differentiate to other cell types, including neurons (Kim

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et al., 2008) and cardiomyocytes (Yoshida and Yamanaka, 2011), which are the cells primarily affected in FRDA. In addition to avoiding some of the technical issues associated with the use of primary neural or cardiac stem/progenitor cells that can be difficult to obtain, culture, and experimentally manipulate, iPSCs also circumvent the ethical concerns associated with human embryonic stem cells. When iPSCs were first developed, there was much excitement around their therapeutic potential; however, over time, it has become clear that another significant potential is in developing disease models for examining pathogenic mechanisms and testing therapies. FRDA iPSCs have been produced using fibroblasts isolated from individuals with FRDA (Ku et al., 2010; Liu et al., 2011; Hick et al., 2013), and they are already proving invaluable in research. Similar to other FRDA cellular models, FRDA iPSCs have reduced levels of frataxin and unstable trinucleotide GAA expansions (Ku et al., 2010; Liu et al., 2011). Repeat stability increased upon silencing of MSH2 expression, a mismatch repair enzyme (Ku et al., 2010). It has also been shown that FRDA iPSCs can differentiate to neuronal (Liu et al., 2011; Hick et al., 2013) and cardiac lineages (Liu et al., 2011; Hick et al., 2013; Lim et al., 2013), two key tissues affected in this disease. Impaired mitochondrial function has also been described using FRDA iPSCs (Hick et al., 2013). A very recent study reports that iPSCs derived from individuals with FRDA do not increase frataxin expression during neuronal differentiation in comparison to iPSCs derived from control individuals, indicating a developmental component to FRDA (Eigentler et al., 2013). Several studies are now underway to further characterize these cells and correct their frataxin deficiency, and continued refinement of protocols will increase the efficiency of iPSC generation (O’Malley et al., 2009; Seifinejad et al., 2010). In addition to their potential application in cell and gene therapy research, iPSCs are useful tools for drug screening and disease modeling (Wu et al., 2011). Gene Therapy for FRDA

Gene therapy is experiencing a renaissance with several successful applications in clinical trial renewing interest in this approach to treat FRDA. There are now several lines of evidence clearly indicating that FRDA is amenable to gene therapy. Viral vector-based delivery Retroviral and lentiviral vectors. Oncoretroviral vectors and lentiviral vectors integrate into the host genome. For retroviral vectors, particularly those derived from the murine leukemia virus (MLV), one of the greatest challenges is the risk of insertional mutagenesis upon integration near oncogenes and growth control genes (as reviewed in Baum et al., 2006; Nienhuis et al., 2006). This can lead to vectordependent side effects, such as clonal dominance, as seen during clinical trials for CGD (Ott et al., 2006) and X-SCID (Hacein-Bey-Abina et al., 2003; Howe et al., 2008). Derived from the human immunodeficiency virus (HIV), lentiviral vectors are *80–120 nm replication-defective particles that can spread a relatively short distance in dense tissue (Segura et al., 2013). They are an appealing vector for gene therapy given their flexible packaging capacity (*12 kb) and ease of

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use. The vesicular stomatitis virus glycoprotein (VSV-G) pseudotype is the most commonly used envelope protein since it facilitates efficient transduction of multiple cell types and allows concentration of particles if required (Segura et al., 2013). Use of multiple packaging helper vectors, characterizing the integration pattern, and introducing novel regulatory elements have significantly increased the safety profile of lentiviral vectors (Schambach et al., 2013). Different pseudotypes have also increased cell targeting specificity. Injection of lentiviral particles into the substantia nigra of rats showed that particles with Mokola virus pseudotypes transduced neurons, while those with lymphocytic choriomeningitis or the Moloney MLV pseudotypes transduced astrocytes (Cannon et al., 2011). Fusion envelopes of VSV-G and other virus glycoproteins, such as the rabies virus, are also proving effective in transducing neuronal cells (Hirano et al., 2013; Kato et al., 2014). A key safety feature of lentiviral vectors is their selfinactivating (SIN) design (Zufferey et al., 1998). SIN vectors lack nonessential enhancer and transcriptional control sequences in the long terminal repeats of conventional retroviral vectors (Yu et al., 1986). An encouraging trial demonstrated that symptoms in two boys with neurodegenerative X-linked adrenoleukodystrophy were reduced after autologous transplant of lentiviral-corrected BM (Cartier et al., 2009). Common integration sites identified postcorrection were because of a benign integration bias and not oncogenic selection (Cartier et al., 2009; Biffi et al., 2011), indicating SIN lentiviral vectors have a superior safety profile over conventional oncoretroviral vectors (such as those used in the X-SCID trials). Latest-generation lentiviral vectors represent an appropriate system to potentially treat FRDA, and one study demonstrated that lentiviral vector-dependent expression of human FXN in fibroblasts isolated from FRDA patients increases their resistance to oxidative stress (Fleming et al., 2005). Several groups are currently developing lentiviral vectors for gene therapy of FRDA. Adeno-associated virus. Adeno-associated virus (AAV) is a small, nonenveloped single-strand DNA parvovirus (Choi et al., 2005). Its small size (approximately 26 nm in diameter) allows AAV-derived gene therapy vectors to spread further in some tissues compared with other viral vectors. With 11 naturally occurring serotypes (AAV1-11), a variety of hybrid serotypes (including mixed and/or modified capsids) have also been developed in the last decade (Choi et al., 2005). This has broadened tropism and increased cell targeting specificity; however, serotypes 2 and 5 remain the most commonly used (Choi et al., 2005; Wang et al., 2011). The primary limitation of AAV vectors is their small packaging capacity within the vector genome (*4.8 kb). AAV can also integrate at chromosome breakage sites (Miller et al., 2004) and a specific site in chromosome 19 (Kotin et al., 1990). AAV (serotype 2) has also been demonstrated to preferentially integrate with low efficiency in active genes in mice (Nakai et al., 2003). An important advance has been the development of selfcomplementary AAV (scAAV) vectors that generate a singlestranded genome with both the coding and complementary sequences packaged (McCarty, 2008). The scAAV vector removes the lag time for second-strand synthesis (a required step with AAV), facilitates more rapid expression once in

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the nucleus, and increases transduction efficiency. A major disadvantage, however, is that scAAV vectors have only half (*2.3 kb) of the already small packaging capacity of AAV (McCarty, 2008). An early study evaluated AAV correction in human FRDA fibroblasts and demonstrated that cells experienced reduced sensitivity to oxidative stress upon AAV-dependent expression of human FXN (Fleming et al., 2005). A very recent study demonstrated not only correction, but also reversal, of a severe cardiomyopathy phenotype in a conditional FRDA mouse model [where frataxin is deleted in cardiac and skeletal muscle; the muscle creatine kinase-Cre or MCK mouse (Puccio et al., 2001)], following a single intravenous injection of AAV constitutively expressing human FXN (Perdomini et al., 2014). This study reports for the first time that MCK mouse cardiomyocytes that have already undergone severe energy failure, iron–sulfur cluster deficiency, and mitochondria and sarcomere ultrastructural changes can be fully rescued with vector-dependent FXN expression. Importantly, FXN expression also restored compromised ventricular function to wild-type levels (Perdomini et al., 2014). Herpes simplex virus type 1. Herpes simplex virus type 1 (HSV-1) is a well-characterized human DNA virus with a large packaging capacity in comparison to other viral vectors: *160 kb (Neve and Geller, 1995; Lim, 2013). This means that HSV-1 has superior versatility with respect to the size and number of transgenes/regulatory regions that can be introduced, which is a distinct advantage when aiming to recapitulate endogenous expression in the therapeutic context. While HSV-1 is not as user friendly as retro/lentiviral vectors or AAV, bacterial artificial chromosome ‘‘recombineering’’ has overcome some of the technical issues, and packaging helper plasmids have increased its safety (Lim, 2013; Neve and Lim, 2013; Laimbacher and Fraefel, 2014). As a neurotropic virus, HSV-1 gene therapy vectors are primarily developed for neurological diseases such as FRDA. Introduction of the FXN genomic locus to FRDA fibroblasts via HSV1-based amplicon vectors also reduced sensitivity to oxidative stress (Gomez-Sebastian et al., 2007), and this construct resulted in long-term persistent FXN expression in the brain of wild-type mice after injection into the adult mouse cerebellum (Gimenez-Cassina et al., 2011). In 2007, Lim and colleagues developed a localized Fxn knock-out mouse lacking Fxn in the olivary neurons of the brain stem (Lim et al., 2007). Using a rotarod assay, the authors demonstrated this FRDA model developed motor incoordination, a hallmark feature of disease, within 4 weeks of Fxn deletion. Importantly, this neurological phenotype was reversed upon stereotaxic injection of an HSV1 amplicon expressing human FXN into the brain stem (Lim et al., 2007). This proof-of-principle study demonstrates that vector-dependent FXN expression can rescue neurological deficits to restore motor coordination in FRDA mice. Nonviral-based delivery

Introduction of frataxin to cells does not need to be via viral-based vector delivery. Bacterial and yeast artificial chromosomes expressing human FXN can rescue the embryonic lethality of Fxn-deficient mice (Pook et al., 2001;

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Sarsero et al., 2004). In a recent study, human frataxin was directed to the mitochondria via an N-terminal fusion of the transduction domain of the cationic peptide transactivator of the HIV transcription protein TAT (Vyas et al., 2012). TATfrataxin was introduced to fibroblasts isolated from individuals with FRDA and also the severe conditional frataxin knock-out mouse (neuron-specific enolase-Cre; NSE) with deletion of Fxn in cardiac and neural crest-derived tissues (Puccio et al., 2001). This exogenous replacement of frataxin to mitochondria improved cardiac function and increased lifespan in the NSE mouse model (Vyas et al., 2012). A very recent study demonstrated increased FXN expression after nucleoinfection of human FRDA fibroblasts with a transcription activator-like effector protein (TALE), engineered to bind to the FXN promoter, fused to a transcription activation domain (VP64) (Chapdelaine et al., 2013). A 1.6-fold increase in mature FXN transcript and a 1.6–1.8-fold increase in frataxin protein were observed (Chapdelaine et al., 2013). In combination, these studies indicate that increasing frataxin levels can improve phenotype and ultimately clinical outcome, validating further studies testing the corrective potential of gene therapy for FRDA. Several international collaborations that focus on developing gene therapy for FRDA have recently formed—including GENEFA, AAV Life, and Voyager Therapeutics (Third Rock Ventures). The Challenges Faced in Developing Cell and Gene Therapies for FRDA

FRDA is a multifaceted disease that can vary in its age of onset, severity, and progression. FRDA also affects multiple organs and this presents several challenges for the development of effective cell and gene therapies. For gene therapy to be of lifelong clinical benefit, it requires long-term, stable, and effective expression of the therapeutic transgene. Gene therapy vectors ideally need to transduce long-term repopulating cells for stable correction. Progenitor and/or terminally differentiated cells have limited survival requiring multiple sources and treatments. Effective targeting of therapies

Localized delivery of cells and/or gene therapy vectors by direct injection into specific sites could effectively correct enough cells to improve function of the targeted organ and ameliorate phenotype. However, targeting each tissue with significant pathology presents logistical and financial challenges and is especially difficult in a single individual. While systemic delivery of viral vectors can result in high transduction rates, there is limited control of vector copy number and the specific cell types targeted, which can raise additional safety concerns. The delivery of vectors and/or corrected cells presents significant challenges in clinical translation. Specific targeting and distribution of vectors and/or sufficient engraftment of corrected cells in the main sites of pathology (the heart, DRG, and cerebellum) will be essential for therapeutic benefit. While increased tropism of gene therapy vectors will improve overall targeting capacity, correct homing and engraftment of corrected cells could pose the greater challenge. This is especially difficult if there is no inherent selective advantage for frataxin-expressing cells.

689 Safety concerns

With integrating vectors, insertional mutagenesis remains a viable concern. A SIN design for MLV-based vectors has been used to overcome insertional mutagenesis; however, it was shown that SIN-MLV vectors demonstrate residual promoter activity (Xu et al., 2012). SIN-MLV vectors can cause tumor formation in a tumor-prone mouse model in comparison to SIN-lentiviral vectors, indicating that the unique integration pattern of lentiviral vectors also increases their safety profile (Montini et al., 2009). Vector safety can also be significantly increased by incorporating insulators and/or cellular promoters (Evans-Galea et al., 2007; RobertRichard et al., 2007; Ryu et al., 2007; Zychlinski et al., 2008; Zhou et al., 2010). A clinical trial for beta-thalassemia using an insulated lentiviral vector has demonstrated safe therapeutic benefit over 3 years postcorrection (CavazzanaCalvo et al., 2010). New insulated vectors are proving significantly safer and more reliable in both human cells and mice (Zhou et al., 2010). Similarly to other pluripotent stem cells, iPSCs generate teratomas when injected into animals (Miura et al., 2009; Gutierrez-Aranda et al., 2010; Lim et al., 2012). This safety issue would need to be addressed before these cells, or their derivatives, could be used in the clinic, such as selecting predifferentiated progenitor cells or fully differentiated cells before implantation. The ability of iPSCs to differentiate into the appropriate cell type in vivo that can survive, integrate, and function in the host tissue remains a challenge [as reviewed by Gaspard and Vanderhaeghen (2011) and Josowitz et al. (2011)]. A fully developed organ, such as the heart or the brain, lacks the essential developmental signals within its microenvironment to support integration of transplanted donor progenitor cells, and this could be compounded in the disease context. Chromosomal abnormalities have also been observed after long-term culture of pluripotent stem cells, including human embryonic stem cells (Spits et al., 2008). Potential issues linked to the method of iPSC generation also need to be resolved such as the development and validation of small molecules that increase efficiency in reprogramming somatic cells (Chua et al., 2011; Yang et al., 2011). Currently, iPSCs derived from individuals with FRDA provide valuable models for the assessment of preclinical vectors in terminally differentiated neuronal and cardiac cells (Liu et al., 2011; Hick et al., 2013), but there are still several logistical/safety issues to resolve before iPSCs can be developed as a therapy. FRDA-iPSCs would need to be corrected either at the genomic or protein level to obtain higher levels of frataxin. FRDA also has a reproducible disease-related epigenetic profile at the FXN allele, including changes in DNA methylation and histone modification; therefore, it would be essential for reprogrammed cells to reflect the epigenetic features of the primary tissue being modeled. Future Perspectives

FRDA is a devastating neurodegenerative disease with both complex pathology and phenotype. When multiple organs are affected, targeting each one individually presents significant and costly challenges. FRDA is highly amenable to cell and gene therapy with multiple, potential cellular

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targets and gene correction strategies currently being explored. Successful correction of frataxin deficiency using these different strategies will be an important achievement. The greater significance, however, will be in determining the minimum number of frataxin-expressing cells required for improvement and the potential for long-term clinical benefit. The true capacity of iPSCs for use in cell therapy remains to be seen. Transplant of terminally differentiated neuronal or cardiac cells derived from iPSCs would still require invasive, risky procedures, localized delivery, and absolute removal of any remaining iPSCs. In addition to the risks, this could be time-consuming and costly, making it less appealing as a treatment on a larger scale. The plasticity and renewable supply of iPSCs may eventually overcome such hurdles particularly as the technology used for their generation improves and the safety issues associated with pluripotency are addressed. If iPSC therapy was proven to be safe and effective for cell replacement/enhancement, it would also provide the long-term benefit of immunologically matched cells. Another potential source of immunologically matched pluripotent stem cells that should not be overlooked is through somatic nuclear cell transfer, which would provide the additional advantage of not reprogramming cells. In developing cell and gene therapy for FRDA, ongoing monitoring of adverse events, including integration site analyses, screening for polyclonal cell populations, and clonal dominance, will be required. Rigorous analysis and testing of vectors in FRDA disease models will be essential to ensure their efficacy and safety. Ultimately, new and established tools must be employed to characterize safety. In tandem with the development of cell and gene therapies, it is critical that the profoundly detrimental effects of FRDA on mobility and function continue to be addressed. This will ensure that people with FRDA will be able to obtain the maximum advantage from potential cell and gene therapies. In the longer term, the knowledge gained and the resources generated in developing cell and gene therapies for FRDA could also facilitate much-needed treatment options for other neurogenetic diseases. Acknowledgments

The authors are funded by the National Health and Medical Research Council of Australia (Project Grants to M.B.D., A.P., P.J.L., and M.V.E-G.; Career Development Fellowships, Level 2, to P.J.L. and A.P.; Early Career Fellowship to L.A.C.); the Australian Research Council (Future Fellowship FT1 to M.D.); Friedreich Ataxia Research Alliance, USA; the Friedreich Ataxia Research Association, Australasia; and the Victorian Government Operational Infrastructure Support Program. Author Disclosure Statement

No competing financial interests exist for any of the authors of this article. References

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Address correspondence to: Dr. Marguerite V. Evans-Galea Bruce Lefroy Centre for Genetic Health Research Murdoch Children’s Research Institute Flemington Road Parkville, Victoria 3052 Australia E-mail: [email protected] Received for publication September 20, 2013; accepted after revision April 14, 2014. Published online: April 21, 2014.