Frataxin sequence for gene therapy

Abstract

A nucleic acid encoding human frataxin protein, a gene therapy vector including the nucleic acid, and genetically modified cells containing the nucleic acid or the vector are disclosed. The encoded human frataxin protein includes upstream cell secretion and cell penetration peptides. The genetically modified cells may be used in a method of treating Friedreich's Ataxia by transplanting a cell genetically modified by a gene therapy vector including the nucleic acid encoding human frataxin protein into a patient.

Description

[0001] FRATAXIN SEQUENCE FOR GENE THERAPY

[0002] Technical Field

[0003] The present invention relates to a nucleic acid encoding human frataxin protein, as well as to a gene therapy vector including the nucleic acid, and to genetically modified cells containing the nucleic acid or the vector. The present invention further relates to the nucleic acid, the vector or the cells for use in a method of medical treatment, in particular for use in a method of treating Friedreich's ataxia.

[0004] Background Art

[0005] Friedreich's ataxia (FR.DA) is an autosomal recessive disease characterised by progressive spinocerebellar neuropathy, ataxia, muscle atrophy, diabetes and cardiomyopathy. Affected individuals show severely reduced cellular levels of the iron-binding, mitochondrial protein frataxin (FXN), which is brought about by the hyperexpansion of a GAA trinucleotide repeat sequence within the first intron of the frataxin gene (FXN) in chromosome 9 that leads to transcriptional repression of frataxin (Bidichandani et al. (1998); Campuzano et al. (1996)). A deficiency in FXN results in elevated oxidative stress and the accumulation of iron within mitochondria, leading to mitochondrial dysfunction (Cook et al. (2006); Gakh et al. (2006)).

[0006] There are an estimated 15,000 Friedreich's ataxia patients worldwide, and it is the most common of the hereditary ataxia syndromes. Currently, an effective disease-modifying therapy for FR.DA remains elusive, and there is no cure. A number of therapeutic approaches for FRDA have been investigated. For example, previous studies by Rocca et al. (2017) and Kemp et al. (2018) observed that the transplantation of allogeneic, wild-type mouse haematopoietic stem cells (HSCs) to the YG8XsR mouse model of FRDA, enabled the delivery of FXN to brains and spinal cords of transplant recipient mice and partially ameliorated disease symptoms.

[0007] However, a potential problem with such a treatment is that transplanted, non-FRDA stem cells may be incapable of restoring FXN contents of cells in affected individuals fully, leading to only partial therapeutic effects being observed, as was seen in the studies mentioned above.

[0008] The more pressing issue greatly hampering the wider use of HSC transplantation therapy, is the high mortality rate resulting from graft versus host disease (GvHD) post-treatment and the subsequent need for histocompatible, human leukocyte antigen (HLA)-matched bone marrow donors.

[0009] Larimar Therapeutics, a US based biotech company, has completed a placebo-controlled phase 2 study in FRDA patients dosed with subcutaneous injections of the frataxin-TAT fusion peptide (Nomlabofusp). They have developed a strategy for Friedreich's ataxia that also uses a therapeutic peptide based on cell-penetrating frataxin. However, the peptide is synthesised in vitro and must be injected into patients. Furthermore, Larimar Therapeutics' frataxin is linked to a penetration peptide derived from the HIV virus. This increases the risk of potentially dangerous immune reactions and / or transplanted cell rejection. Further disadvantages of such a therapy include, risk of acute toxicity posed by systemic injections of high concentrations of the synthetic peptide and Nomlabofusp requiring systemic drug injections that would be necessary throughout the patient's entire lifetime.

[0010] The present invention seeks to provide an improved nucleic acid sequence particularly for use in a method of treating Friedreich's ataxia.

[0011] According to an aspect of the present invention, there is provided an isolated nucleic acid having the sequence:

[0012] ATG TGG ACC CTT GGC AGA AGG GCC GTT GCT GGA CTG CTT GCC TCT CCA TCT CCT GCT CAA GCC CAG ACA CTG ACC AGA GTG CCT AGA CCT GCT GAA CTG GCC CCT CTG TGT GGC AGA AGA GGA CTG AGA ACC GAC ATC GAC GCC ACA TGC ACA CCT AGA AGG GCC AGC AGC AAT CAG AGA GGC CTG AAT CAG ATC TGG AAC GTG AAG AAA CAG AGC GTG TAG CTG ATG AAC CTG AGA AAG AGC GGC ACC CTG GGA CAC CCT GGA AGC CTG GAT GAG ACA ACC TAG GAG AGA CTG GCC GAG GAA ACC CTG GAT TCC CTG GCC GAG TTC TTC GAG GAC CTG GCC GAT AAG CCC TAG ACC TTC GAG GAT TAG GAC GTG TGC TTT GGC AGC GGC GTG CTG ACA GTG AAA CTG GGA GGC GAT CTG GGC ACC TAG GTG ATC AAC AAG CAG ACC CCT AAC AAA CAG ATC TGG CTG AGC AGC CCT AGC AGC GGC CCC AAG AGA TAT GAT TGG ACC GGC AAG AAC TGG GTG TAG AGC CAC GAT GGC GTG TGC CTG CAC GAA CTG CTG GCT GCC GAA CTG ACA AAG GCC CTG AAA ACA AAG CTG GAC CTG TGC AGC CTG GCC TAG TCT GGC AAA GAT GCC (SEQ ID NO: 1) or having at least 95% sequence identity therewith, wherein the nucleic acid encodes human frataxin protein having the sequence: MWTLGRRAVA GLLASPSPAQ AQTLTRVPRP AELAPLCGRR GLRTDIDATC TPRRASSNQR GLNQIWNVKK QSVYLMNLRK SGTLGHPGSL DETTYERLAE ETLDSLAEFF EDLADKPYTF EDYDVSFGSG VLTVKLGGDL GTYVINKQTP NKQIWLSSPS SGPKRYDWTG KNWVYSHDGV SLHELLAAEL TKALKTKLDL SSLAYSGKDA

[0013] (SEQ ID NO: 2).

[0014] In embodiments, the nucleic acid may have at least 95.5% sequence identity, at least 96% sequence identity, at least 96.5% sequence identity, at least 97% sequence identity, at least 97.5% sequence identity, at least 98% sequence identity, at least 98.5% sequence identity, at least 99% sequence identity or at least 99.5% sequence identity.

[0015] The nucleic acid may include a Stop codon, which in an embodiment is TGA.

[0016] The nucleic acid may include a polyadenylation signal, which may be AATAAA.

[0017] In a particular embodiment, the nucleic acid includes the sequence TGA AAT AAA at the 3' end thereof.

[0018] The nucleic acid may encode a cell secretion peptide upstream of the frataxin protein. The encoded cell secretion peptide may have the sequence: MDFQVQIFSF LLISASVI IS RG (SEQ ID NO: 3). Optionally, the nucleic acid sequence encoding the cell secretion peptide has the sequence: ATG GAG TTC GAG GTG GAG ATC TTC AGC TTC CTG CTG ATC TGC GCC AGC GTG ATC ATC AGC AGA GGC (SEQ ID NO: 4). The nucleic acid may encode a cell penetration peptide upstream of the frataxin protein. The cell penetration peptide may be a human cell penetration peptide. The cell penetration peptide may be derived from human annexin 3, which may have the sequence: MASIWVGHRG (SEQ ID NO: 5). In a particular embodiment, the nucleic acid sequence encoding the cell penetration peptide has the sequence: ATG GCC TCT ATC TGG GTC GGA CAC AGA GGA (SEQ ID NO: 6).

[0019] According to an embodiment, the nucleic acid encodes, in N to C order, a cell secretion peptide and a cell penetration peptide fused to the amino-terminus of the human frataxin protein.

[0020] The fusion peptide may have the sequence:

[0021] MDFQVQI FSF LLISASVI IS RG

[0022] MAS IWVGHRG

[0023] MWTLGRRAVA GLLASPSPAQ AQTLTRVPRP AELAPLCGRR GLRTDIDATC TPRRASSNQR GLNQIWNVKK QSVYLMNLRK SGTLGHPGSL DETTYERLAE ETLDSLAEFF EDLADKPYTF EDYDVSFGSG VLTVKLGGDL GTYVINKQTP NKQIWLSSPS SGPKRYDWTG KNWVYSHDGV SLHELLAAEL TKALKTKLDL

[0024] SSLAYSGKDA (SEQ ID NO: 7).

[0025] In an embodiment, the nucleic acid encoding the fusion peptide has the sequence: ATG GAG TTC GAG GTG GAG ATC TTC AGC TTC CTG CTG ATC TGC GCC AGC GTG ATC ATC AGC AGA GGC ATG GCC TCT ATC TGG GTC

[0026] GGA CAC AGA GGA ATG TGG ACC CTT GGC AGA AGG GCC GTT GCT GGA

[0027] CTG CTT GCC TCT CCA TCT CCT GCT CAA GCC CAG ACA CTG ACC AGA

[0028] GTG CCT AGA CCT GCT GAA CTG GCC CCT CTG TGT GGC AGA AGA GGA

[0029] CTG AGA ACC GAC ATC GAC GCC ACA TGC ACA CCT AGA AGG GCC AGC AGC AAT CAG AGA GGC CTG AAT GAG ATC TGG AAC GTG AAG AAA GAG

[0030] AGC GTG TAG CTG ATG AAC CTG AGA AAG AGC GGC ACC CTG GGA CAC CCT GGA AGC CTG GAT GAG ACA ACC TAG GAG AGA CTG GCC GAG GAA ACC CTG GAT TCC CTG GCC GAG TTC TTC GAG GAC CTG GCC GAT AAG CCC TAG ACC TTC GAG GAT TAG GAC GTG TCC TTT GGC AGC GGC GTG CTG ACA GTG AAA CTG GGA GGC GAT CTG GGC ACC TAG GTG ATC AAC AAG CAG ACC CCT AAC AAA CAG ATC TGG CTG AGC AGC CCT AGC AGC GGC CCC AAG AGA TAT GAT TGG ACC GGC AAG AAC TGG GTG TAG AGC CAC GAT GGC GTG TCC CTG CAC GAA CTG CTG GCT GCC GAA CTG ACA AAG GCC CTG AAA ACA AAG CTG GAC CTG TCC AGC CTG GCC TAG TCT GGC AAA GAT GCC TGA AAT AAA (SEQ ID NO: 8).

[0031] According to another aspect of the present invention, there is provided a gene therapy vector including a nucleic acid as specified above.

[0032] The gene therapy vector may a viral vector. In some embodiments, the gene therapy vector is a retroviral vector, such as a lentiviral vector. Other embodiments may use an adeno associated virus based vector.

[0033] According to another aspect of the present invention, there is provided a genetically modified cell containing a nucleic acid or a vector as specified above.

[0034] The cell may be a haematopoietic stem cell. In some embodiments, the cell has been obtained from a Friedreich's Ataxia patient to be treated.

[0035] According to another aspect of the present invention, there is provided a nucleic acid, a vector, or a cell as specified above for use in a method of medical treatment. According to another aspect of the present invention, there is provided a nucleic acid, a vector, or a cell as specified above for use in a method of treating Friedreich's Ataxia.

[0036] According to another aspect of the present invention, there is provided a method of treating Friedreich's ataxia, the method including transplanting a genetically modified cell as specified above into a patient in need thereof.

[0037] In an embodiment, the method includes obtaining cells from the patient for genetic modification. The method may include genetically modifying the cells with a gene therapy vector as specified above.

[0038] Embodiments of the present invention are described below, by way of example only, with reference to the accompanying drawings, in which: Figure 1 schematically illustrates an exemplary fusion peptide;

[0039] Figure 2 schematically illustrates how a fusion peptide encoded by an embodiment of a nucleic acid could be used for treating patients with Friedreich's ataxia;

[0040] Figure 3 shows a Blast sequence alignment of a modified frataxinencoding nucleic acid sequence according to an embodiment (Query) compared to human frataxin (Subject);

[0041] Figure 4 schematically illustrates FXN-TAT and FXN-APP gene constructs;

[0042] Figure 5 shows experimental results from an ELISA revealing human frataxin levels in supernatants from FXN-TAT and FXN-APP transfected HEK293T cell cultures; Figure 6 shows experimental results from a Western blot analysis for frataxin protein;

[0043] Figure 7 schematically illustrates experiments in which fibroblasts were exposed to supernatants from FXN plasmid transfected HEK293 cells and analysed for viability post H2O2 treatment or for aconitase activity levels;

[0044] Figures 8 and 9 show a comparison of the effects of treatment with culture supernatants from transfected HEK293T cells on FR.DA fibroblasts' viability following H2O2 exposure;

[0045] Figure 10 shows that the aconitase activity levels of transfected HEK293T cell are not increased by a truncated frataxin protein, demonstrating specificity of the therapeutic peptide;

[0046] Figure 11 shows experimental results from a Western blot analysis for mature FXN in cell lysates of cultured FR.DA fibroblasts exposed to supernatants from HEK293T cell cultures;

[0047] Figures 12 and 13 show fluorescence microscopy for intracellular FXN in FR.DA / HD fibroblasts;

[0048] Figure 14 shows a Western blot assessment on CD34+cells infected with the pLIG virus expressing frataxin;

[0049] Figure 15 shows a plot comparing the number of colonies in HSCs infected with different lentivirus constructs;

[0050] Figure 16 schematically illustrates an exemplary pCCL PGK FXN transfer vector;

[0051] Figure 17 shows a schematic diagram of an experiment to quantify myeloid colonies;

[0052] Figure 18 shows a plot of the number of white or red blood cells obtained from FR.DA patient vector- infected stem cells;

[0053] Figure 19 shows a plot of aconitase activity in FDRA fibroblasts; Figure 20 shows Western blotting results of frataxin expression from the pCCL PKG vector;

[0054] Figure 21 schematically illustrates transplantation of wild-type HSCs from syngeneic C57BL / 6 male donors into YG8XsR female mice;

[0055] Figure 22 shows percentage of donor HSC engraftment at different times post transplantation;

[0056] Figure 23 shows an ELISA analysis of human FXN levels in blood sera of recipient FR.DA mice;

[0057] Figure 24 shows vector copy numbers in recipient mice;

[0058] Figures 25, 26 and 27 show the results of activity tests;

[0059] Figures 28 to 31 show the results of immunohistochemical analysis and measurement of size of cerebellar dentate nucleus neurons in mice;

[0060] Figure 32 shows aconitase activity in the brain of modified FR.DA mice; Figures 33 and 34 show ELISA analysis of human FXN levels in modified mouse tissues;

[0061] Figures 35 and 36 show mean aconitase activity of modified mouse tissue homogenates; and

[0062] Figure 37 shows mouse genotypes.

[0063] Many diseases occur as a result of genetic defects. Gene therapy seeks to correct the defective gene(s) and restore proper functioning of the encoded protein.

[0064] Approaches investigated for treating Friedreich's ataxia include strategies that promote increased frataxin (FXN) levels, reducing oxidative stress and prevention of cellular iron accumulation. However, the difficulty of getting therapeutic agents into the CNS and across the blood brain barrier (BBB), poses a major obstacle to the effectivity of many of these factors.

[0065] The restoration of FXN levels in patient cells provides a potential therapeutic strategy for Friedreich's ataxia. This work addresses the challenge of ensuring sufficient replenishment of frataxin in FR.DA cells by generating genetically engineered blood stem cells that can secrete frataxin protein capable of penetrating target cells, rescuing their functions.

[0066] It is envisaged that a modified frataxin gene could be used for treating patients with Friedreich's ataxia. Previous work by the applicant has investigated and validated a novel cell and gene therapy approach for Friedreich's Ataxia using a frataxin fusion peptide (Shaban, 2020). The applicant has now demonstrated that a cDNA sequence, codon- optimised for high levels of expression of the therapeutic protein in mammalian cells, represents a promising candidate for treatment of Friedreich's ataxia in humans, and has validated this approach in vivo. It is proposed that a cDNA sequence encodes a fusion peptide (schematically illustrated in Figure 1) including (a) a cell secretion peptide and (b) a cell penetration peptide fused to (c) the human frataxin protein, which can be used in a replacement strategy for patients lacking the frataxin protein. For example, the peptide could be used in a method of treating Friedreich's ataxia.

[0067] As illustrated in Figure 1, the fusion peptide includes a signal secretion peptide (SS peptide), and cell penetration peptide derived from human annexin 3 (APP), fused to the amino terminus of the full-length frataxin precursor protein, frataxin (1-210 amino acids). The fusion peptide containing the full-length form of human frataxin is processed in the mitochondria by peptidases that release the mature, biologically active form, separating it from the secretion and penetration sequences.

[0068] It was envisaged that the cDNA sequence encoding the fusion peptide could be cloned into a clinical lentivirus that could be used to modify patient's haematopoietic stem cells (HSCs) ex vivo. Thus, enhancing the observed beneficial effects of HSC transplantation by modifying the HSCs to continuously secrete a cell penetrating frataxin fusion protein, with the aim of allowing a more efficient replenishment of frataxin within target cells. The modified stem cells could then be transplanted back into Friedreich's ataxia patients, where they would repopulate the bone marrow, thereby replenishing normal levels of frataxin permanently. The rationale of the design was to obtain a secreted version of frataxin that can be released in the environment by the modified cells and penetrate target tissues.

[0069] Figure 2 schematically illustrates steps for use of the fusion peptide in an embodiment of a clinical treatment.

[0070] A construct including nucleic acid sequences encoding a secretion peptide (SS; SEQ ID NO: 4), a cell penetration peptide (APP; SEQ ID NO: 6), and human frataxin (SEQ ID NO: 1) in 5' to 3' order is prepared. Other elements of a clinical lentiviral vector are also shown (AU3R.U5, Pre4 and AU3RU5). Figure 3 shows an alignment of SEQ ID NO: 1 with a downstream TGA Stop codon (Query (SEQ ID NO: 9)) with the human frataxin gene (Subject (SEQ ID NO: 10)). SEQ ID NO: 1 is a codon-optimised sequence encoding the human frataxin peptide of SEQ ID NO: 2, and surprisingly has only 73% identity with the human frataxin gene.

[0071] The skilled person will appreciate that minor changes to the nucleic acid sequences may be made. Nevertheless, the frataxin-encoding sequence should have at least 95% identity with SEQ ID NO: 1, it may have at least 95.5% sequence identity, at least 96% sequence identity, at least 96.5% sequence identity, at least 97% sequence identity, at least 97.5% sequence identity, at least 98% sequence identity, at least 98.5% sequence identity, at least 99% sequence identity or at least 99.5% sequence identity.

[0072] In this embodiment, the nucleic acid sequence also includes at the 3' end of the frataxin-encoding sequence the sequence TGA AAT AAA. This sequence is a Stop codon followed by a polyadenylation signal. While this is the preferred sequence, the skilled person will appreciate that other Stop codons could be used.

[0073] As illustrated in Figure 2, in a first step, the cDNA sequence (10) encoding the fusion peptide is cloned into a clinical lentivirus (20). In a second step, the lentivirus including the fusion peptide is introduced to hematopoietic stem cells (30) which were, in this embodiment, previously taken from the patient (40) to be treated. In a third step, the modified stem cells are transplanted back (50) into the Friedreich's ataxia patient by intravenous injection.

[0074] Once internalised in the hematopoietic stem cells, the fusion peptide containing the full-length form of human frataxin (23kD) is processed in the mitochondria by peptidases that release the mature, biologically active form (81-210 amino acids, 18kD) (Weng et al. (2019), Schmucker et al. (2008)), separating it from the secretion and penetration sequences. The mature form of frataxin is therefore replenished in the patient and delivered to target tissues (Figure 2).

[0075] This strategy has been validated by subcloning the cDNA encoding the engineered frataxin into lentiviral vectors which were used in Friedreich's ataxia models in vitro and in vivo.

[0076] Although this embodiment is described using lentiviral vectors, other examples of delivery vehicles could be used. For example, non-viral vectors such as lipid nanoparticles Other viral vectors such as adeno associated viruses (AAVs) could also be used, though lentiviral vectors are preferred.

[0077] Although the use of modified hematopoietic stem cells is preferred, other cell-based delivery systems can be used to deliver the mature frataxin protein to target tissues. For example, delivery could be achieved by infection of target organs (for example, brain or heart). Adeno associated virus may conveniently be used in such embodiments, with Adeno Associated Virus encoding the therapeutic peptide.

[0078] Once the cDNA encoding the fusion peptide is prepared, viral vectors or stem cells containing the modified frataxin gene can be injected intravenously into the patient.

[0079] In the embodiment illustrated in Figure 2, the therapy is provided by cell-based delivery (ex vivo cell therapy). For example, the stem-, progenitor-, or differentiated cells are taken from the patient and are modified to include a nucleic acid encoding the human frataxin precursor protein and administered back into the patient.

[0080] In other embodiments, the therapy could be provided by direct delivery. For example, a nucleic acid encoding the human frataxin gene could be packed into a suitable delivery vehicle such as a virus (for example, adeno associated virus) and directly returned into the patient.

[0081] Compared to earlier strategies, the present gene therapy method provides several advantages: (1) there is no risk of acute toxicity posed by systemic injections of high concentrations of the synthetic peptide; (2) the delivery of frataxin will be continuous and long term; (3) fusing frataxin to a penetrating peptide of human origin should minimise the risk of potentially dangerous immune reactions and / or rejection of transplanted cells. Furthermore, bone marrow derived haematopoietic stem cells (HSCs), have a capacity for self-renewal and ability to penetrate many organs of the body following their differentiation into mature leukocytes. Genetically modified haematopoietic stem cells survive in the bone marrow for many years, as shown in successful gene therapy protocols for immunodeficiencies. This approach could provide a means of delivering the much-needed FXN proteins to affected tissues for an extended period. Therefore, the present approach is likely to be safer, more long-lasting and cost-effective than previously described therapies.

[0082] Experimental validation of the gene therapy strategy described above shows autologous transplantation of the modified HSCs resulted in stable secretion of the peptide in the blood stream, impacting disease progression by delaying the manifestation of motor- coordination / sensory symptoms, paralleled by improved biochemical and anatomical parameters. The cell and gene therapy strategy validated by the present inventors demonstrates that it should be feasible to achieve long term stabilisation or correction of the symptoms in Friedreich's ataxia patients.

[0083] The skilled person will appreciate that the modified frataxin precursor protein could be prescribed or administered by a medical practitioner as a treatment in various gene therapy protocols. They would also understand that due to the degeneracy of the genetic code, altering the genetic sequence can result in the same amino acids being encoded and therefore not alter the function of the resulting frataxin protein. Therefore, although SEQ ID NO: 1 is the preferred frataxin-encoding nucleic acid, in some embodiments variations of this sequence may also be used.

[0084] Some embodiments can involve adding the cDNA sequence encoding the fusion peptide to existing gene therapies for treating Friedreich's ataxia. The frataxin-encoding nucleic acid sequence can be provided in the form of a construct for use in a method of medical treatment. For example, it could be provided in a suitable gene therapy vector such as a lentiviral or other viral or retroviral vector.

[0085] All optional and preferred features and modifications of the described embodiments and dependent claims are usable in all aspects of the invention taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another. ell- frataxin with functional

[0086] Modified versions of the frataxin precursor protein, consisting of 210 amino acids, were prepared by fusing at its amino terminus signal secretion (SS) and penetration peptides derived either from the HIV transactivator of transcription (TAT) or human annexin 3 (APP) (Young Kim et al. (2015)).

[0087] A codon optimisation strategy was used to determine the optimal DNA sequence for the chimeric fusion protein. The sequence was obtained using the commercially available algorithm, GeneArt Custom Gene Synthesis. A Blast sequence alignment, shown in Figure 3, confirms a 73% identity match between the optimised modified frataxin gene (Query) and the human frataxin gene (Subject).

[0088] Codon optimised sequences encoding the fusion proteins can be synthesised and subcloned into the lentiviral pLIG vector (schematically illustrated in Figure 4). HEK293T cells were transfected with the pLIG- Frataxin constructs (TAT-FXN and APP-FXN) to quantify the secretion of FXN in supernatants by ELISA (Figure 5) and the overall levels of FXN in cell lysates by Western blotting (Figure 6, which compares lysates of cells transfected with either empty (EMP) plasmid, TAT-FXN or APP-FXN plasmid constructs). Both assays revealed increased levels of frataxin upon transfection with the engineered FXN proteins. Of note, transfected HEK293T cells displayed increased levels of both the 23kDa immature and the 14kDa mature form of FXN, suggesting correct processing of the precursor protein (Figure 6).

[0089] FRDA fibroblasts are abnormally sensitive to reactive oxygen species (ROS) and have a deficit in aconitase activity (Wong et al. (1999), Campanella et al. (2009), Walden (2002)). To assess whether tissue penetrating frataxin could rescue FRDA fibroblasts defects, supernatants from 293 cells transfected with the different frataxin constructs were used. Healthy (HD) and FRDA fibroblasts were exposed to supernatants from empty (EMP), TAT- FXN or APP- FXN plasmid transfected HEK293 cells and analysed for viability post H2O2 treatment or aconitase activity levels (as illustrated schematically in Figure 7). Fibroblasts treated with supernatants containing APP-FXN and, to a lesser extent, TAT-FXN were partially protected from H2O2-induced death, behaving similarly to fibroblasts from healthy donors (Figure 8, which shows a comparison of the effects of treatment with culture supernatants from EMP, TAT- FXN or APP-FXN plasmid transfected HEK293T cells on FRDA fibroblasts' viability following H2O2 exposure). Since the APP peptide was more efficient than TAT in this assay and is of human origin, it was prioritised in subsequent experiments.

[0090] To demonstrate that the protective effect was caused by the frataxin sequence and not the penetration or secretion peptides, a truncated fusion protein in which frataxin was almost totally deleted was used. Full length, but not truncated, frataxin was able to rescue ROS-induced cell death, demonstrating that frataxin, but not the signal or APP peptides, is required for protection from cell death (Figure 9, which shows a comparison of the effects of treatment with culture supernatants from EMP, truncated (TRU)-FXN, or APP-FXN plasmid transfected HEK293T cells on FR.DA fibroblasts' viability following H2O2 exposure).

[0091] Deletion of the frataxin gene has been associated with accumulation of mitochondrial iron deposits and reduced Fe-S cluster levels (Llorens et al. 2019) and results in deficiencies in the Fe-S cluster containing aconitase mitochondrial enzyme (Walden (2002), Whitnail et al. (2008), Vaubel, Isaya (2013)). Exposure of FR.DA fibroblasts to APP-FXN containing supernatants significantly increased aconitase activity. Supernatants containing the peptide with truncated frataxin were indistinguishable from control supernatants, indicating specificity of the effect (Figure 10, which shows the aconitase activity levels of EMP, truncated (TRU)-FXN, and APP-FXN plasmid transfected HEK293T cells on FR.DA fibroblasts).

[0092] Example 2: Uptake of secreted APP-FXN fusion peptide from the supernatants of HEK293T cells transfected with the APP-FXN plasmid and their localisation to the mitochondria

[0093] The ability of the secreted FXN fusion proteins to enter fibroblasts isolated from a Friedrich's ataxia (FR.DA) patient was tested. FR.DA fibroblast Western blotting for mature (14kDa) FXN in cell lysates of cultured FR.DA fibroblasts exposed to supernatants from HEK293T cell cultures revealed higher FXN in those treated with APP-FXN plasmid construct transfected cell supernatants (FR.DA: APP-FXN) vs. empty (FR.DA: EMP) plasmid transfected controls, untreated FR.DA fibroblasts and healthy (HD) fibroblasts as shown in Figure 11. The APP-FXN protein was incorporated and correctly processed in FR.DA fibroblasts (Figure 11). Fluorescence microscopy results for intracellular FXN in FR.DA fibroblasts (DAPI) treated with supernatants from APP-FXN plasmid transfected vs. EMP plasmid transfected HEK293T cell cultures or healthy (HD) and FR.DA untreated fibroblasts are shown in Figure 12. Fluorescence microscopy results for intracellular FXN, mitotracker (red) in FRDA / HD fibroblasts (DAPI) treated with supernatants from APP-FXN plasmid transfected cells are shown in Figure 13. Immunofluorescence staining and microscopy revealed that exogenous frataxin localised in mitochondria, demonstrating that the fusion protein could reach its physiological target within the cell. of secreted frataxin in normal or cells does not differentiation in vitro

[0094] It was assessed whether expression of the frataxin fusion proteins in human CD34+progenitors could disturb haematopoietic differentiation, which would be undesirable in view of potential clinical trials.

[0095] CD34+cells were infected with the pLIG virus expressing the TAT or APP frataxin variants and after 72 hours the protein expression was quantified by Western blotting. Results (see Figure 14) showed higher levels of frataxin in cells infected with virus containing the frataxin fusion peptides compared to those treated with empty virus. Western blot assessment of culture supernatants from the same HSCs revealed that infected cells with the FXN fusion protein DNA secreted substantial levels of immature FXN fusion peptide (Figure 14). Subsequently, the HSCs were differentiated for a twenty-day period and the number of erythroid and myeloid colonies generated from CD34 HSCs infected with either lentivirus containing the empty vector (EMP), frataxin-TAT vector (TAT-FXN) or frataxin-APP vector (APP-FXN) was scored by microscopy analysis. There was no difference in the number of colonies derived from the frataxin containing vectors compared to empty lentivirus (Figure 15). cDNA encoding frataxin-APP was then cloned into a clinical grade vector (pCCL) currently used in phase I and II clinical trials and the construct was packaged into lentivirus as the vector delivery system (Fumagalli et al. (2022), Kohn et al. (2020), Kohn et al. (2021), Magnani et al. (2022), Schejtman et al. (2021)). Figure 16 schematically illustrates the pCCL PGK FXN transfer vector, which includes the following elements:

[0096] CMV promoter; R and U5 components of HIV-1 5' LTR; PGK phosphoglycerate kinase promoter FXN; human Frataxin coding sequence; WPRE4, woodchuck-hepatitis virus post-transcriptional regulatory element mut 4; AU3 RU5, self-inactivating 3' LTR.

[0097] The virus was used to infect CD34+cells donated by a FR.DA patient and myeloid colonies were quantified after three weeks as schematically illustrated in Figure 17. The number of white or red blood cells obtained from the FR.DA patient vector- infected stem cells was similar to that of cells derived from an untreated normal donor, suggesting that the vector had no negative impact on haematopoietic differentiation (Figure 18). Notably, the supernatants of myeloid cells transduced with one vector copy number of the therapeutic vector induced an increase in aconitase activity in FDRA fibroblasts (Figure 19). These results together demonstrate that the clinical lentiviral vector expressing the frataxin fusion protein should not have a negative impact on differentiation of blood stem cells derived from a FR.DA patient. of HSCs modified with a lentiviral vector causes secretion of the cell frataxin in the blood of YG8XsR FRDA mice

[0098] It was investigated whether transplantation of genetically modified HSCs could provide a long-term delivery vehicle for functional frataxin, to increase endogenous cellular frataxin levels and ultimately inhibit disease progression and reduce symptoms.

[0099] Bone marrow cells were isolated from the femurs and tibias of male FRDA mice (YG8XsR) (Sandi et al. (2014)) and lineage negative Lin- HSCs purified by magnetic cell sorting (to a purity of ~60% as assessed by flow cytometric analysis for surface cKit expression), infected with the APP-FTX lentivirus at ~6 MOI (frataxin low) or ~12.5 MOI (frataxin high) then transplanted into pre-symptomatic, eight-week-old female FRDA mice. Physiological levels of expression of the frataxin isoforms by the lentiviral vector were validated by Western blotting of infected Lin- cells (Figure 20).

[0100] Female FRDA (YG8XsR) mice at eight weeks of age were transplanted with male HSCs lentiviral transduced with the APP-FXN plasmid (wildtype HSCs from syngeneic C57BL / 6 male donors) and assessed until forty-eight weeks of age for effects of treatment on motor symptoms and disease affected tissues. A schematic illustrating the different steps of the experiment is shown in Figure 21. After transplantation, successful engraftment of male donors on female recipients was assessed by periodic bleeding and purification of white blood cell DNA which was subjected to digital PCR analysis for the detection of male testis specific protein (TSPY) gene on the Y chromosome. TSPY levels revealed that all but one of the recipient mice receiving lentiviral transduced HSCs had been reconstituted with male, TSPY gene expressing donor HSCs averaging 80%, by 39-43 weeks post transplantation. Male Y chromosome PCR. analysis in white blood cells of female recipients to determine the percentage of donor HSC engraftment at different times post transplantation is shown in Figure 22.

[0101] To confirm production and secretion of the frataxin fusion peptide by the transplanted HSCs, blood samples from mice at different time points were subjected to ELISA assays. The highest levels of frataxin were detected in the plasma of mice receiving HSCs transduced with high titre FXN-APP lentivirus (FXN high), followed by mice transplanted with HSCs infected with low titre virus (FXN low). Mice transplanted with C57BL / 6 HSCs (WTX), that do not secrete exogenous human frataxin, as well as the untransplanted YG8XsR control mice had negligible levels of frataxin detected in their blood. Figure 23 shows the results (ELISA analysis of mean (+SEM) human FXN levels in the blood of HSCs recipient FR.DA mice and untreated controls post-transplant). A gradual decrease in blood frataxin was observed from twenty-three weeks post transplantation for the majority of mice. This is not caused by gradual loss of transplanted cells, as indicated by the high percentage of Y chromosome reconstitution after 43 weeks (Figure 22) and normal blood leukocyte cell counts in recipient mice (Tables 1 to 3). Table 1 : Leukocyte cell counts in YG8sR mice (blood; 35 weeks)

[0102] Table 2: Leukocyte cell counts in YG8sR mice (bone marrow; 48 weeks)

[0103] Table 3: Leukocyte cell counts in YG8sR mice (spleen; 48 weeks) An alternative explanation may be that gene therapy-modified HSCs might be unstable and have been outgrown by the untransduced fraction of transplanted HSCs.

[0104] To investigate this possibility, vector copy number (VCN) levels in blood leukocytes of recipient mice by real-time PCR were measured. Figure 24 shows mean virus levels (VCN) in leukocytes of mice transplanted with APP-FXN transduced HSCs at different times post transplantation. VCN analysis revealed that virus plasmid levels in blood leukocytes of recipient mice in both FXN-APP high and low groups remained relatively stable between twenty-three to forty-seven weeks of age, indicating that there was no selective reduction of APP-FXN plasmid positive HSC / leukocyte populations in these transplant recipients. A possible explanation for the age-related reductions in fusion protein levels in the blood of some host mice is that viral expression became less efficient in these donor cells over time.

[0105] No increase in mortality in mice transplanted with high titre FXN-APP- infected HSCs compared to other transplantation groups was detected. Blood counts in YG8sR mice as well as haematopoietic differentiation of human CD34+ cells from a normal donor or a FR.DA patient did not show a negative impact of the lentivira lly expressed frataxin fusion protein (Figures 17, 18 and 19, and Tables 1 to 3).

[0106] These results demonstrate that transplanted HSCs transduced with APP- FXN fusion protein plasmids increased blood FXN levels in recipient YG8sR mice. Example 5: Delayed motor deficits in YG8XsR mice transplanted with

[0107] HSCs secreting cell penetrating frataxin

[0108] The motor function of transplanted mice was tested up to the age of forty-eight weeks old, i.e. forty weeks post HSCs transplantation. The activity (Figure 25), beam walk (Figure 26) and rotarod (Figure 27) tests were undertaken prior to treatment at eight weeks of age, then eight weeks post transplantation and every four weeks thereafter. Previous studies have observed age-related declines in YG8XsR mice performance in these tests compared with non-diseased, C57BL / 6 age- matched control mice (Anjomani Virmouni et al. (2015)). Mean (±SEM) activity levels indicated by distance travelled over four minutes (Figure 25), balance / coordination assessed by beam walk test (Figure 26), as well as by rotarod test (Figure 27), in transplant recipient FRDA mice, and untransplanted YG8sR and C57BL / 6 controls up to forty-eight weeks of age were determined.

[0109] Activity tests highlighted no statistically significant inter-group differences between any of the mouse groups, although untransplanted YG8XsR and FXN-low mice groups were the worst performing from approximately thirty-six weeks of age onwards and may eventually have shown significant deficits if the test was performed beyond fortyeight weeks of age (Figure 25). This result may suggest that the activity test is not sufficiently challenging to induce deterioration in these FRDA mice to manifest at least up to the age of forty-eight weeks. The beam walk balance test results revealed significant ( =0.0017) age-related performance declines in untreated FRDA mice versus wildtype controls which became evident at approximately thirty-six weeks of age onwards. FRDA mice transplanted with FXN-high HSCs, however, did not show declines in beam walk performance compared to C57BL / 6 controls and, more importantly, were significantly (p =0.0075) better at traversing the beam when compared with the untransplanted group, indicating a beneficial effect of the therapy on motor function. HSCs expressing low frataxin levels showed no significant difference in performance with the untransplanted group. The beneficial effects of wild type HSCs transplantation on beam walk performance was also noted, as reported previously (Kemp et al. (2018), Rocca et al. (2017)) (Figure 26).

[0110] Motor co-ordination analysis by rotarod testing revealed significant (p =0.0002) decreases in performance in the untreated FRDA mice compared with healthy controls from twenty-four weeks of age. Notably, the high frataxin HSCs transplanted mice performed significantly better than untransplanted mice in the rotarod assay (p =0.027), further indicating the beneficial effect of genetically modified HSC transplantation on FRDA mice motor function (Figure 27). Similar to the beam walk test, no beneficial effects were seen by transplantation with HSCs expressing low levels of secreted frataxin, indicating the requirement for a minimal level of restoration for benefits on motor function to be achieved.

[0111] These results demonstrate that transplanted HSCs transduced with APP- FXN fusion protein plasmids delayed the onset of motor symptoms. 6: Anatomical and biochemical in YG8XsR mice frataxin

[0112] It has been shown previously that the number of frataxin-positive cerebellar dentate nucleus neurons is decreased in an inducible mouse model of Friedreich's ataxia (Mercado-Ayon et al. (2022)).

[0113] Cerebellar dentate nucleus neuron staining for beta III tubulin show differences in size between different mouse treatment groups at fortyeight weeks old, as shown by typical microscopic images of dentate nucleus resident neurons for each mouse group (Figure 28, representative microscopic images of multiple histological analysis (at x70 magnification)). Mean surface area (±SEM) of dentate nucleus neurons for different mouse treatment groups are shown in Figure 29 (BL6 n= 5, UN n= 6, FXN high n= 6 and FXN low n= 3).

[0114] It was demonstrated through immunohistochemical analysis of dentate neurons that there was a drastic decrease of frataxin positive dentate neurons in YG8sR mice compared to isogenic, control c57Black6 mice. Notably, there was a significant increase of frataxin-positive neurons in mice transplanted with HSCs transduced with high titre FXN virus compared to untransplanted mice (Figures 28 and 29). In keeping with previous results (Rocca et al. (2017), mice transplanted with unmodified, wild-type HSCs also showed increased numbers of cerebellar neurons positive to frataxin, although in this case the difference was not statistically significant (Figures 28 and 29). Thus, transplantation of APP-FXN fusion protein secreting HSC into YG8XsR mice preventing size reductions of dentate nucleus resident neurons in the cerebellum of FRDA has been demonstrated.

[0115] Lack of frataxin causes progressive atrophy of the dentate nucleus in Friedreich's ataxia patients (Koeppen et al. (2011)). In order to detect morphological changes in dentate nucleus neurons before and after gene therapy, cerebellar sections of FRDA mice were stained with beta III tubulin at forty-eight weeks of age. Microscopic analysis of the cerebellar dentate nucleus showed significant reductions of the size of neurons in FRDA (YG8XsR) mice versus wild type (C57BL6) controls. In contrast, the size of neurons in FRDA mice transplanted with HSCs transduced with the high titre FXN-APP lentivirus was similar to wild type mice, and ~35% larger than those of untransplanted controls. Figure 30 shows representative microscopic images of multiple histological analysis (at x70 magnification). Figure 31 shows mean surface area (±SEM) of dentate nucleus neurons for different mouse treatment groups. Cerebellar neurons of FRDA mice transplanted with low titre FXN-APP or wild type HSCs also showed a ~25% increase in size, although this was not statistically significant. The iron sulphur cluster dependent enzyme aconitase is less active in FRDA, leading to reduction in the enzyme's function in both patients and FRDA models (Rotig et al. (1997), Al-Mahdawi et al. (2006)). In the brain, transplantation of HSCs transduced with high titre, but not low titre, FXN-APP virus increased aconitase activity in transplanted versus untransplanted FRDA mice. Although the difference did not reach statistical significance, Figure 32 shows aconitase activity in the brain of modified FRDA mice transplanted with HSCs transduced with high and low titre FXN-APP lentivirus compared to wild type (C57BL6) controls and untransplanted controls;

[0116] Other tissues relevant to the pathology of Friedreich's ataxia are the heart and muscle (Sival et al. (2011), Nachbauer et al. (2012), Lees et al. (2022)). An increase of human frataxin expression was detected in the heart and skeletal muscle of transplanted mice vs untransplanted controls that was proportional to the virus titre Figures 33 and 34 show frataxin expression in heart and muscle respectively (ELISA analysis of human FXN levels in heart (Figure 33) and muscle (Figure 34) tissues for the different mouse treatment groups).

[0117] The activity of aconitase paralleled frataxin protein levels, suggesting that restoration of frataxin levels by the gene therapy also rescued biochemical function in these organs. Mean aconitase activity are shown for heart (Figure 35) and muscle (Figure 36) tissue homogenates for each mouse treatment group.

[0118] (UN n= 6, FXN high n= 6-7 and FXN low n= 3). * p <0.05, ** p <0.01, by multiple comparisons two-way ANOVA test with post hoc.

[0119] Transplantation of APP-FXN fusion protein secreting HSC into YG8XsR mice thus increases frataxin protein and aconitase activity in affected tissues of YG8XsR mice.

[0120] The lentiviral construct restored frataxin levels in target tissues of transplanted mice such as heart, brain and muscle, at near physiological levels (Figures 28 to 36), suggesting that the lentiviral delivery approach should be also safe for patients. The above experimental results show that following transplantation, efficient engraftment of donor cells was observed within hosts at least thirty weeks after transplantation, with high levels of human FXN in the blood of YG8XsR mice treated with FXN fusion protein producing HSCs, but not in the control groups, indicating donor cells had engrafted and secreted FXN-APP fusion protein. Although it is possible that the therapeutic effects of donor HSCs could gradually wane as their numbers within recipients diminish, or their capacity to secrete therapeutic protein decreases over time, an ability for these modified HSCs to impact disease symptoms as well as anatomical and biochemical changes was nonetheless, apparent at least up to forty weeks after the transplantations.

[0121] Methods

[0122] Generation of frataxin fusion peptide plasmid

[0123] The cDNA for human precursor frataxin or truncated frataxin fused to the secretion and APP or TAT sequences was synthesised by GeneArt (Invitrogen). The synthetic gene consisting of 5'-signal sequence-TAT or APP-FXN or truncated FXN-3' DNA, with BamHl restriction enzyme sites on each end was assembled from synthetic oligonucleotide or PCR. products. The fragments were inserted into either the pLig GFP+ or the pCCL clinical grade plasmids at BamHl sites. The plasmid DNA was expanded and purified from transformed One Shot™ in Stbl3™ competent E. coli bacteria (Thermo Fisher) using the NucleoBond® Xtra Midi kit (Macherey Nagel), as per manufacturer's instructions, and their sequence verified by DNA sequencing. Secretion sequence: ATG GAG TTC GAG GTG GAG ATC TTC AGC TTC CTG CTG ATC TGC GCC AGC GTG ATC ATC AGC AGA GGC (SEQ ID NO: 4); APP peptide: ATG GCC TCT ATC TGG GTC GGA CAC AGA GGA (SEQ ID NO: 6);

[0124] TAT peptide: ATG TAT GGC CGC CAG CGC CGC CGC (SEQ ID NO: 11);

[0125] Frataxin precursor: ATG TGG ACC CTT GGC AGA AGG GCC GTT GCT GGA

[0126] CTG CTT GCC TCT CCA TCT CCT GCT CAA GCC CAG ACA CTG ACC AGA

[0127] GTG CCT AGA CCT GCT GAA CTG GCC CCT CTG TGT GGC AGA AGA GGA

[0128] CTG AGA ACC GAC ATC GAC GCC ACA TGC ACA CCT AGA AGG GCC AGC

[0129] AGC AAT CAG AGA GGC CTG AAT CAG ATC TGG AAC GTG AAG AAA CAG

[0130] AGC GTG TAG CTG ATG AAC CTG AGA AAG AGC GGC ACC CTG GGA CAC

[0131] CCT GGA AGC CTG GAT GAG ACA ACC TAG GAG AGA CTG GCC GAG GAA

[0132] ACC CTG GAT TGC CTG GCC GAG TTC TTC GAG GAC CTG GCC GAT AAG

[0133] CGC TAG ACC TTC GAG GAT TAG GAC GTG TGC TTT GGC AGC GGC GTG

[0134] CTG ACA GTG AAA CTG GGA GGC GAT CTG GGC ACC TAG GTG ATC AAC

[0135] AAG CAG ACC CCT AAC AAA CAG ATC TGG CTG AGC AGC CCT AGC AGC

[0136] GGC CGC AAG AGA TAT GAT TGG ACC GGC AAG AAC TGG GTG TAG AGC

[0137] CAC GAT GGC GTG TGC CTG CAC GAA CTG CTG GCT GCC GAA CTG ACA

[0138] AAG GCC CTG AAA ACA AAG CTG GAC CTG TGC AGC CTG GCC TAG TCT

[0139] GGC AAA GAT GCC TGA AAT AAA (SEQ ID NO: 12);

[0140] Truncated FXN: ATG TGG ACT CTG GGG CGC CGC GCA GAA AAG ATG CTT

[0141] GAA ATA AAG (SEQ ID NO: 13).

[0142] Lentivirus production

[0143] HEK293T cells were co-transfected with empty or the FXN-APP insert containing pCCL plasmids along with pMDG2 (envelope vector) and pCMV-D8.74 (packaging vector) plasmids by polyethylenimine (Sigma Aldrich) and virus harvested from cell culture supernatants by ultracentrifugation and stored at -80°C. The viral titre was determined by infecting 2xl05PLB985 or K562 cells with serial dilutions of the viral vector preparation and analysing vector copy number (VCN) by quantitative PCR. of lentivector DNA (see VCN method below) in the cellular DNA isolates 3 days post transduction.

[0144] Verification of fusion protein production by cells transfected with frataxin fusion peptide vectors

[0145] HEK-293T cells were plated in Dulbecco's Modified Eagle Medium (DMEM) (ThermoFisher Scientific), 10% FBS and 1% penicillin / streptomycin and transfected with GFP+ or GFP- plasmids, with or without the frataxin fusion peptide DNA insert (FXN-TAT, FXN- APP or EMP), or with the APP-truncated frataxin fusion peptide insert (APP-TRU), at 80-90% confluence, using Lipofectamine 3000 kit (ThermoFisher Scientific) according to manufacturer's protocol and cells were incubated at 37°C, 5% CO2. The transfection efficiency was determined 48 hours post transfection by flow cytometric quantification of GFP positive cells. Approximately, lxlO6transfected cells were lysed in Laemmli buffer containing complete protease inhibitors (Roche) and acetone precipitated culture supernatants, were collected for Western blot analysis and culture supernatants for FXN ELISA quantification, 72 hours post transfection. Treatment of FR.DA fibroblasts with frataxin fusion peptide containing culture supernatants

[0146] Fibroblasts from healthy and FR.DA patients were were seeded at lxl06 / ml in 1ml of DMEM, 10% FBS and 1% penicillin / streptomycin in 6 well plates and incubated at 37°C, 5% CO2. After 24 hours, the media was replaced with supernatant from the empty or fusion peptide positive plasmid transfected HEK-293T cells, 72 hours post transfection (see above) and further incubated for 2 and 4 hours. Approximately, lxlO6fibroblasts were lysed in Laemmli buffer containing complete protease inhibitors and assessed for their endogenous frataxin by western blot analysis. Similarly, fibroblasts were analysed by immunofluorescence microscopy for intracellular frataxin by staining 2xl04supernatant treated cells seeded on coverslips with 500nM MitoTracker Red CMXRos (Invitrogen) for 30mins at 37°C, fixing them in 4% paraformaldehyde then permeabilising for 15 minutes with PBS containing 0.2% Triton X-100. The samples were then incubated with a blocking solution of PBS 5% BSA for 1 hour at 25°C and stained with anti-frataxin antibody (#113691, AbCam) for 16hr at 4°C. The cells were incubated with appropriate fluorochrome tagged secondary antibodies (Abeam) at room temperature for an hour and then nuclear stained and mounted on slides with vectashield with DAPI (Vector Laboratories). Samples were subsequently viewed using the NIKON Ti-E super research Live Cell imaging system with 100X 1.45 (NA)Plan Apochromat lens. The data sets were deconvolved with NIS Elements AR analysis software (NIKON). Three-dimensional data sets were converted to Maximum Projection in the NIS software. To test for the effects of treatment with frataxin fusion peptide on fibroblast viability following H2O2 induced oxidative stress, the MTS assay was utilised. Briefly, FR.DA and normal fibroblast cells were seeded in a 96-well plate containing lOOpI of DMEM, 10% FBS and 1% penicillin / streptomycin at 20,000 cells per well overnight at 37°C, 5% CO2. The media was changed and replaced with lOOpI of supernatant from the empty or fusion peptide positive plasmid transfected HEK-293T cells (see above). After 1 hour, 0, 50 or 200pM of hydrogen superoxide (H2O2) (ACR.OS Organics) was added to cells in triplicate wells over 6 hours to induce oxidative stress. Cell viability was subsequently assessed by MTS assay using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation (Promega), in accordance with the manufacturer's protocol, with colorimetric readings obtained using ELx808 microplate reader (Biotek).

[0147] In order to analyse the effects of frataxin fusion peptide on aconitase activity levels in FRDA fibroblasts, lxlO6cells from FA and healthy individuals were incubated for 1 hour with supernatant collected from HEK-T293 cells that had been transfected with frataxin fusions peptide constructs or empty vector control. Fibroblast were lysed and aconitase measured using the Aconitase Assay kit (Abeam) as described below.

[0148] Transduction of human bone marrow derived cells

[0149] Human CD34+stem cells from GCSF mobilised apheresis (AllCells ltd) were seeded at lxl06 / ml in StemSpan media (Stem Cell Technologies), 1% penicillin / streptomycin (Thermo fisher) with full cytokine cocktail; hSCF lOOng / ml, hFlt3L lOOng / ml, hTPO 20 ng / ml (all Peprotech). Following cytokine pre-stimulation for 24 hours, cells were seeded in Stem Span medium, 1% penicillin / streptomycin, hSCF 50ng / ml, hFlt3L 50ng / ml, hTPO 10 ng / ml, hIL3 20ng / ml (Peprotech) at a concentration of 2xl06cells / ml in 24-well plates and fusion peptide plasmid construct positive virus (MOI=20) and 4pg / ml protamine sulphate added to the cells. Twenty-four hours post infection, cells were counted and seeded at 0.3xl06cells / ml into IMDM (Gibco), 20% FBS, 1% penicillin / streptomycin (Thermofiser), and GCSF (Peprotech) lOOng / ml. Cells were split weekly, reseeded at 0.3xl06cells / ml and analysed 14 days or 22 days post infection for colony forming units of specific progenitor cell populations, or differentiation into monocytes by flow cytometry for CDllc expression and their frataxin contents by Western blotting (below) respectively.

[0150] Generation of FR.DA mice

[0151] YG8sR mice (Anjomani Virmouni S, et al. (2015)) (typically with ~200 GAA repeats in FXN intron 1) with the highest GAA repeats were bred through multiple generations to obtain mice with expanded GAA repeats (~800) as a result of intergenerational repeat instability / expansion (Al- Mahdawi et al. (2006)) (Figure 37). Up to 5 mice / cage were housed in environmentally enriched individually ventilated cages at 11-hour dark versus 13-hour light cycle, 20-23°C and 45-60% humidity. The mice were nourished with a diet of SDS RM3 expanded food pellets and standard drinking water. One week pre-transplantation and nine weeks thereafter, mice were temporarily given a wet diet in addition and antibiotic treated water. All procedures were carried out in accordance with the UK Home Office 'Animals (Scientific Procedures) Act 1986' and with approval from the Brunel University Animals Welfare and Ethical Review Board. For GAA repeat genotyping, genomic DNA was extracted from mouse ear clips by standard phenol / chloroform extraction and ethanol precipitation, and GAA PCR. amplification was carried out using GAA forward (5'-GGGATTGGTTGCCAGTGCTTAAAAGTTAG-3' (SEQ ID NO: 14)) and GAA reverse (5'-GATCTAAGGACCATCATGGCCACACTTGCC-3' (SEQ ID NO: 15)) primers followed by agarose gel visualisation, as previously described (Al-Mahdawi et al. (2004)).

[0152] Isolation and transduction of mouse HSCs

[0153] Femurs and tibias were dissected from male donor mice (either FR.DA or C57BL / 6) and bone marrow cells obtained by flushing with R.PMI (Gibco) media. Cells were pelleted and enriched for Terll9‘, Grl, Macl-, B220-, CD4-, CD8‘, IL7R.- and Sca+, cKit+cells by magnetic cell sorting using mouse Lineage Cell Depletion Kit (Miltenyi Biotech) according to the manufacturer's protocol. Flow cytometric analysis for cKit expressing cell levels revealed the percentage of cKit+cells increased from approximately 9% to 56% of total population post cell sorting (data not shown). Isolated HSCs were seeded at lxl06 / ml in lx culture StemSpan media (Stem Cell Technologies), 1% penicillin / streptomycin (Thermo fisher) with full cytokine cocktail; hSCF lOOng / ml, hFlt3L lOOng / ml, hTPO 20 ng / ml. Following cytokine pre-stimulation for 4 hours, cells were seeded in Stem Span medium, 1% penicillin / streptomycin, hSCF 50ng / ml, hFlt3L 50ng / ml, hTPO 10 ng / ml, hIL3 20ng / ml at a concentration of 2xl06cells / ml in 24-well plates and fusion peptide plasmid construct positive plasmid containing virus (MOI=6 or 12.5) added to infect the cells for at least 14 hours. HSCs were extensively washed and reconstituted in PBS at 0.5xl06 cells / lOOp I for transplantation. A portion (0.5xl06) of the cells were plated into 48 well plates in Stem Span medium, 1% penicillin / streptomycin, hSCF 50ng / ml, hFlt3L 50ng / ml, hTPO 10 ng / ml, hIL3 20ng / ml and cultured for 5 days and O.lxlO6cells were used for DNA purification using the Monarch Genomic DNA Purification Kit (New England Biolabs) according to manufacturer's instruction to test vector copy numbers as described below. Another 0.5xl06of the cells were lysed to assess their frataxin content by western blot.

[0154] Assessment of transduction efficiency and enqraftment levels

[0155] Average vector copy number / cell (VCN) was analysed by droplet digital PCR. DNA samples were diluted in water to lOng / pl. Reactions were performed in a final volume of 22 pl, containing lx ddPCR Supermix for probes (BioRad, Hercules, CA), 0.8pl of each primer (lOpM) and 0.4pl of each probe (lOpM), 2 I of DNA per sample and 5 I of nuclease free water. 20,000 droplets were generated using the automated droplet generator (BioRad) before amplification with C1000 Touch™ Thermal Cycler; 96-Deep Well Reaction Module (BioRad). Positives and negatives droplets were quantified using the QX200 Droplet reader (BioRad) and analysed by Poisson statistics using QuantaSoft Software (BioRad). Concentration was provided by Quantasoft software as copies of the relevant gene per pl (copies / pl). VCN was calculated as the ratio between the target gene concentration ( FRATAXIN) and the reference gene concentration (mT / t / n) x2: T copies / mTitin copies *2. Primers and probes used in this experiment are listed in Table 4. Table 4: Primer and probe sequences

[0156] Levels of engraftment (male cells into female hosts) were determined by looking at the Y chromosome copies. TSPY male specific DNA assessment by real time PCR was performed using pre-designed TSPY primers / probe set (#4426961, Thermo Fisher) and normalised with b- actin DNA using pre-designed primer / probe set (#4331182 Thermo Fisher) using TaqMan Fast Advanced Master Mix (#4331182 Thermo Fisher) according to manufacturer's protocol. Fold difference for each female mouse DNA sample was determined by comparison with male DNA as a reference via the delta-Ct method (Pido-Lopez J, et al. (2018)) and subsequently converted as a percentage of the male DNA TSPY gene level (at 100%). YG8XsR mice transplantation and behavioural studies

[0157] YG8XsR female mice were treated with approximately 0.5xl06male syngeneic HSCs, transfected with frataxin fusion peptide positive plasmid or the empty plasmid or WT HSCs via intravenous tail vein injection, from eight weeks of age following total body lethal irradiation (2x5 grays with 3-4 hours interval). Two to three days prior irradiation, all mice were assessed for their baseline beam walk, activity and rotarod performance at eight weeks of age and then randomised into their treatment groups (WT untreated [B6] n = 5; YG8XsR untreated / untransplanted [UTX] n = 9; YG8XsR-WT HSC transplanted [WTX] n = 6; high (i.e. infected with ~12.5 MOI lentivirus) YG8XsR- fusion peptide+ plasmid HSC transplanted [FXN-high] n= 9, low (i.e. infected with ~6 MOI lentivirus) YG8XsR-fusion peptide+ plasmid HSC transplanted [FXN-low] n= 7). Body weights were monitored weekly from 7 weeks of age pre-transplantation and twice weekly post transplantation for four weeks and then weekly thereafter to detect potential adverse effects of treatment. Mice displaying reduction in body weight by 20%, with other measures of appearance and behaviour indicating ill-health were euthanised, a total of six mice were euthanised with post-mortem analysis indicating lung infection to be the likely cause of illness. Normal blood cell development and reconstitution post transplantation and no signs of T cell, B cell or monocyte / macrophage / dendritic cell tumorigenesis were observed after flow cytometric analysis of CD3, CD19 or CDllb positive splenic and blood cells respectively from these mice (data not shown). Efficacy of treatment was investigated at eight (pre-treatment) and sixteen weeks of age, and then every four weeks thereafter up to forty-eight weeks of age by measuring beam walk and rotarod performance, and exploratory activity in the open field test as previously described (Anjomani Virmouni et al. (2015); Pido-Lopez et al. (2019)). Different tests were undertaken on separate days with a day rest period between each test. Beam walk test was performed three times, after an initial trial run, by each mouse over a day, with a rest period of at least ten mins between each run. The time taken for the mice to traverse from one end of the 18mm thick beam to the other end, 90cm away, was taken and mean time to traverse were calculated for each treatment group. For each rotarod trial, mice were allowed to acclimatise to the rotating drum for 10 sec before it began to accelerate from 4-40 rpm for a maximum of 300 sec. Mice were tested for four trials per day, with the first trial used as a practice run. Mice were rested for a minimum of 10 mins between each trial. The mean latency to fall times for each mouse group at specified time points were calculated as described previously (Anjomani Virmouni et al. (2015); Pido-Lopez et al. (2019)).

[0158] Exploratory / locomotor activity in the open field test was measured by placing mice individually in a beam-breaker activity monitor box (MEDOFA-510 activity chamber; Med Associates) for 4 mins to assess exploratory behaviour in a novel environment. Mice were assessed for four trials per day, with the first trial used as a practice run. Mice were rested for a minimum of 30 mins between each trial. The average total distance travelled (cm) and mean velocity (cm / s) of each mouse was recorded and means for each mouse treatment group were calculated.

[0159] Blood / plasma, white blood cell and tissue sample collection

[0160] Blood was taken via tail vein puncture into EDTA tubes. Blood samples were spun at 1000 x g for two mins and the upper plasma layer removed for human FXN ELISA quantification. The pelleted blood cells were treated with mouse red blood cell lysis solution (R.&D Systems) in order to remove red blood cells and DNA was extracted from the remaining leukocytes samples using the Monarch Genomic DNA Purification Kit (New England Biolabs) according to the manufacturer's protocol for VCN and TSPY PCR. analysis. Following euthanasia, heart and brain tissues were dissected and snap frozen for subsequent storage at -80°C.

[0161] Flow cytometry

[0162] Transfected HEK293T cells were analysed for GFP expression via flow cytometry using the Novocyte flow cytometer (ACEA). Briefly, 0.5xl06cultured cells were harvested 48 hours post transfection and fixed in 2% paraformaldehyde (PFA). For the viral infected foetal CD34+ cells differentiated over 22 days, 0.5xl06cells were stained with anti-human CDllb-APC antibody (MI / 70, Thermofisher) for 1 hour at 4°C and fixed in 2% PFA. For the magnetic cell sorting (MACS) enriched bone marrow derived mouse HSCs purity analysis, 0.5xl06cells were stained with anti-mouse cKit (CD117)-FITC antibody (Biolegend) for 30 mins on ice and fixed in 2% PFA. Blood, bone marrow and splenic cells were stained with antibodies for 30 mins on ice and fixed in 2% PFA and analysed for T cell (anti-mouse CD3-pacific blue antibody, Biolegend), B cell (antimouse CD19-FITC antibody, Biolegend) and monocyte / macrophage and dendritic cell (anti-mouse CDllb-APC antibody, Biolegend) contents in order to assess leukocyte development of transplanted HSC and detect signs of tumorigenesis in these cells. Fixed / stained cells were ran on the flow cytometer acquiring 20,000 cells (gated according to forward and side scatters with doublets excluded according to FCS-A / FCS-H dot plots). Corresponding untransfected or unstained cell sample controls were acquired to establish the gating areas for positively staining cells.

[0163] Western blotting

[0164] Tissue (Tris HCI lysed) or cell (Laemmli lysed) protein lysates and culture supernatants were sonicated at 4 °C using a vibracell sonicator (10 x 1 s 20 kHz pulses) and denatured for 10 min at 95 °C, loaded onto 15% SDS polyacrylamide gels, transferred onto nitrocellulose membranes and subjected to Western blotting. Membranes were blocked in PBS 5% milk containing Tween 20 (BBT) overnight at 4 °C. Primary antibodies against frataxin (abll0328, Abeam) or actin (C-2, Santa Cruz) or GAPDH (1E6D9, Proteintech) were incubated overnight at 4 °C in BBT. Blots were washed three times for 5 min in PBS Tween 20 (PBST) and incubated for 1 hr at RT with the appropriate secondary antibodies conjugated with horse radish peroxidase (HRP). Blots were washed three times in PBST, and the target protein visualised on Amersham Hyperfilm ECL films (GE Healthcare) using enhanced chemiluminescence (ECL) reagents (BioRad) according to the manufacturer's instructions and a film processing unit (Xograph). Frataxin protein bands were quantified using Image Studio (LI-COR), target protein bands were normalised with their corresponding actin or GAPDH protein bands or with total protein via Ponceau S staining.

[0165] ELISA assay

[0166] Culture supernatant, protein lysate or plasma human frataxin contents were quantified using the frataxin ELISA kit (Abeam) according to the manufacturer's protocol. Colorimetric measurements of ELISA plates were performed using the ELx808 microplate reader.

[0167] Aconitase assay

[0168] Fibroblasts from FR.DA and healthy individuals were incubated for 1 hour with supernatant collected from HEK-T293 cells that had been transfected with frataxin fusions peptide construct or empty vector as a control. Around lxlO6human fibroblast were harvested for the assay. The cells were washed with cold PBS and suspended in lOOpL of cold Assay Buffer (Abeam). This was followed by centrifugation at 800g for 10 mins at 4°C. Activated aconitase in the lysates were quantified using the Aconitase Assay kit (Abeam) according to the manufacturer's instructions.

[0169] Frozen 20g mouse heart, muscle and cerebellum tissues were homogenised in cold Aconitase Assay kit Assay Buffer (Abeam), centrifuged at 800g for lOmins at 4C. Activated aconitase in the lysates were quantified using the Aconitase Assay kit according to the manufacturer's instructions and normalised to citrate synthase activity measured via 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) reduction analysis. Briefly, tissue extracts were added to Tris-HCI 100 mM pH 8.1 with 0.4 mg / ml DTNB and 10 mg / ml Acetyl-CoA. Colorimetric analysis of aconitase and citrate synthase plates at 450nm and 412nm respectively were performed with the ELx808 microplate reader.

[0170] For histological analysis, mice were anesthetised and perfused with PBS. Brains were dissected and submerged in 4% PFA in PBS for 24 hours at 4°C then in PBS sodium azide at 4°C until processing. Tissues were embedded in paraffin for sectioning on a rotary microtome and mounted on glass slides. Slides were deparaffinized, rehydrated, Harris Modified Hematoxylin (H) stained and then blocked with 10% normal goat serum diluted in 0.1% Triton X PBS. For beta III tubulin staining, sections were incubated at 4°C overnight with primary antibody to beta III tubulin (ab78078, Abeam) followed by 1 hour at room temp biotinylated anti-mouse antibody (DAKO). For FXN staining, sections were incubated at room temp for 60 minutes with primary antibody to FXN (ab219414, Abeam) followed by 1 hour at room temp biotinylated anti-rabbit antibody (ab207995, Abeam). Immunohistochemistry staining was performed using the Ventana Discovery XT instrument, using the Ventana DAB Map detection Kit (760-124). For pre-treatment, Roche Cell Conditioning Solution CC2 (950-123) for beta III tubulin analysis and Roche Cell Conditioning Solution CC1 (950-500) for FXN staining were used. Slides were scanned using the Hamamatsu Nanozoomer S360 Digital slide scanner and scanned at x40 magnification and viewed / analysed with NZConnect software (Hamamatsu). All histological work was performed at the IQ-Path Lab, UCL Institute of Neurology, UK.

[0171] Infection of FRDA patient haematopoietic stem cells

[0172] Haematopoietic stem and progenitor cells (FRDA CD34+) were isolated from the peripheral blood of a FRDA patient volunteer and grown for one week in the presence of StemRegenin and UM171 to maintain sternness. Cells were then transduced with the lentiviral vector expressing the modified frataxin protein for 24 hrs and cultured in macrophage differentiation medium for further 3 weeks. The medium was then added to FRDA fibroblasts to evaluate the rescue of aconitase activity.

[0173] Statistical analysis

[0174] Differences between specified groups were detected using Student's t- test (Microsoft Excel) or analysis of variance (ANOVA) test with post hoc Bonferroni correction for multiple comparison test where appropriate (IBM SPSS Statistics Ver.22). All data were screened for statistical outliers using Grubbs test (GraphPad Software) and outlier values were excluded from the analysis. P values of <0.05 were considered significant.

[0175] All optional and preferred features and modifications of the described embodiments and dependent claims are usable in all aspects of the invention taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.

[0176] The disclosures in British patent application number 2413430.6, from which this application claims priority, and in the abstract accompanying this application are incorporated herein by reference. References

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Claims

CLAIMS1. An isolated nucleic acid having the sequence:ATG TGG ACC CTT GGC AGA AGG GCC GTT GCT GGA CTG CTT GCC TCT CCA TCT CCT GCT CAA GCC CAG ACA CTG ACC AGA GTG CCT AGA CCT GCT GAA CTG GCC CCT CTG TGT GGC AGA AGA GGA CTG AGA ACC GAC ATC GAC GCC ACA TGC ACA CCT AGA AGG GCC AGC AGC AAT CAG AGA GGC CTG AAT CAG ATC TGG AAC GTG AAG AAA CAG AGC GTG TAG CTG ATG AAC CTG AGA AAG AGC GGC ACC CTG GGA CAC CCT GGA AGC CTG GAT GAG ACA ACC TAG GAG AGA CTG GCC GAG GAA ACC CTG GAT TCC CTG GCC GAG TTC TTC GAG GAC CTG GCC GAT AAG CCC TAG ACC TTC GAG GAT TAG GAC GTG TGC TTT GGC AGC GGC GTG CTG ACA GTG AAA CTG GGA GGC GAT CTG GGC ACC TAG GTG ATC AAC AAG CAG ACC CCT AAC AAA CAG ATC TGG CTG AGC AGC CCT AGC AGC GGC CCC AAG AGA TAT GAT TGG ACC GGC AAG AAC TGG GTG TAG AGC CAC GAT GGC GTG TGC CTG CAC GAA CTG CTG GCT GCC GAA CTG ACA AAG GCC CTG AAA ACA AAG CTG GAC CTG TGC AGC CTG GCC TAG TCT GGC AAA GAT GCC (SEQ ID NO: 1) or having at least 95% sequence identity therewith, wherein the nucleic acid encodes human frataxin protein having the sequence:MWTLGRRAVA GLLASPSPAQ AQTLTRVPRP AELAPLCGRR GLRTDIDATC TPRRASSNQR GLNQIWNVKK QSVYLMNLRK SGTLGHPGSL DETTYERLAE ETLDSLAEFF EDLADKPYTF EDYDVSFGSG VLTVKLGGDL GTYVINKQTPNKQIWLSSPS SGPKRYDWTG KNWVYSHDGV SLHELLAAEL TKALKTKLDL SSLAYSGKDA(SEQ ID NO: 2)2. A nucleic acid as claimed in claim 1 having at least 97.5% sequence identity.

3. A nucleic acid as claimed in claim 1 or 2 having at least 99% sequence identity.

4. A nucleic acid as claimed in claim 1, 2 or 3, further including aStop codon.

5. A nucleic acid as claimed in claim 4, wherein the Stop codon isTGA.

6. A nucleic acid as claimed in any preceding claim further including a polyadenylation signal.

7. A nucleic acid as claimed in claim 6, wherein the polyadenylation signal is AATAAA.

8. A nucleic acid as claimed in claim 1, 2 or 3, further including the sequence TGA AAT AAA at the 3' end thereof.

9. A nucleic acid as claimed in any preceding claim, wherein the nucleic acid encodes a cell secretion peptide upstream of the frataxin protein.

10. A nucleic acid as claimed in any of claims 9, wherein the encoded cell secretion peptide has the sequence:MDFQVQI FS F LLI SASVI I S RG(SEQ ID NO: 3)11. A nucleic acid as claimed in claim 10, wherein the nucleic acid sequence encoding the cell secretion peptide has the sequence:ATG GAG TTC GAG GTG GAG ATC TTC AGC TTC CTG CTG ATC TCC GCC AGC GTG ATC ATC AGC AGA GGC (SEQ ID NO: 4)12. A nucleic acid as claimed in any preceding claim, wherein the nucleic acid encodes a cell penetration peptide upstream of the frataxin protein.

13. A nucleic acid as claimed in claim 12, wherein the cell penetration peptide is a human cell penetration peptide.

14. A nucleic acid as claimed in claim 13, wherein the cell penetration peptide is derived from human annexin 3.

15. A nucleic acid as claimed in claim 14, wherein the encoded cell penetration peptide has the sequence:MAS IWVGHRG(SEQ ID NO: 5)16. A nucleic acid as claimed in claim 15, wherein the nucleic acid sequence encoding the cell penetration peptide has the sequence:ATG GCC TCT ATC TGG GTC GGA CAC AGA GGA(SEQ ID NO: 6)17. A nucleic acid as claimed in any preceding claim, wherein the nucleic acid encodes, in N to C order, a cell secretion peptide and a cell penetration peptide fused to the amino-terminus of the human frataxin protein.

18. A nucleic acid as claimed in any preceding claim, wherein the nucleic acid encodes a peptide having the sequence:MDFQVQI FSF LLISASVI IS RGMAS IWVGHRGMWTLGRRAVA GLLASPSPAQ AQTLTRVPRP AELAPLCGRR GLRTDIDATCTPRRASSNQR GLNQIWNVKK QSVYLMNLRK SGTLGHPGSL DETTYERLAEETLDSLAEFF EDLADKPYTF EDYDVSFGSG VLTVKLGGDL GTYVINKQTPNKQIWLSSPS SGPKRYDWTG KNWVYSHDGV SLHELLAAEL TKALKTKLDLSSLAYSGKDA(SEQ ID NO: 7)19. A nucleic acid as claimed in any preceding claim, having the sequence:ATG GAG TTC GAG GTG GAG ATC TTC AGC TTC CTG CTG ATC TCC GCCAGC GTG ATC ATC AGC AGA GGCATG GCC TCT ATC TGG GTC GGA CAC AGA GGAATG TGG ACC CTT GGC AGA AGG GCC GTT GCT GGA CTG CTT GCC TCTCCA TCT CCT GCT CAA GCC CAG ACA CTG ACC AGA GTG CCT AGA CCTGCT GAA CTG GCC CCT CTG TGT GGC AGA AGA GGA CTG AGA ACC GACATC GAC GCC ACA TGC ACA CCT AGA AGG GCC AGC AGC AAT CAG AGAGGC CTG AAT GAG ATC TGG AAC GTG AAG AAA GAG AGC GTG TAG CTGATG AAC CTG AGA AAG AGC GGC ACC CTG GGA GAG CCT GGA AGC CTGGAT GAG AGA ACC TAG GAG AGA CTG GCC GAG GAA ACC CTG GAT TGCCTG GCC GAG TTC TTC GAG GAG CTG GCC GAT AAG GCC TAG ACC TTCGAG GAT TAG GAG GTG TGC TTT GGC AGC GGC GTG CTG AGA GTG AAACTG GGA GGC GAT CTG GGC ACC TAG GTG ATG AAC AAG GAG ACC CCTAAC AAA GAG ATG TGG CTG AGC AGC CCT AGC AGC GGC GCC AAG AGATAT GAT TGG ACC GGC AAG AAC TGG GTG TAG AGC GAG GAT GGC GTGTGC CTG GAG GAA CTG CTG GCT GCC GAA CTG AGA AAG GCC CTG AAAAGA AAG CTG GAG CTG TGC AGC CTG GCC TAG TCT GGC AAA GAT GCCTGA AAT AAA(SEQ ID NO: 8)20. A gene therapy vector including a nucleic acid as claimed in any preceding claim.

21. A gene therapy vector as claimed in claim 20, wherein the gene therapy vector is a viral vector.

22. A gene therapy vector as claimed in claim 20 or 21, wherein the gene therapy vector is a retroviral vector.

23. A gene therapy vector as claimed in claim 20, 21, or 22, wherein the gene therapy vector is a lentiviral vector.

24. A genetically modified cell containing a nucleic acid as claimed in any of claims 1 to 19 or a vector as claimed in any of claims 20 to 23.

25. A cell as claimed in claim 24, wherein the cell is a haematopoietic stem cell.

26. A cell as claimed in claim 24 or 25, wherein the cell has been obtained from a Friedreich's Ataxia patient to be treated.

27. A nucleic acid as claimed in any of claims 1 to 19, a vector as claimed in any of claims 20 to 23, or a cell as claimed in claim 24, 25 or 26, for use in a method of medical treatment.

28. A nucleic acid as claimed in any of claims 1 to 19, a vector as claimed in any of claims 20 to 23, or a cell as claimed in claim 24, 25 or 26, for use in a method of treating Friedreich's Ataxia.

29. A method of treating Freidreich's Ataxia, wherein the method includes transplanting a cell as claimed in claim 24, 25 or 26, into a patient in need thereof.

30. The method of claim 29, wherein the method includes obtaining cells from a Freidreich's Ataxia patient for genetic modification.

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