Targeting ferredoxin 2 (FDX2) in friedreich ataxia (FRDA)

Abstract

Described herein are compositions and methods for treating subjects with disorders associated with mutations in the FXN gene and / or that have reduced expression of frataxin protein, e.g., Friedreich ataxia (FRDA), using inhibitors of ferredoxin 2 (FDX2) that decreases FDX2 protein expression.

Description

[0001]Attorney Docket No.29539-0796WO1 / MGH 2022-549 TARGETING FERREDOXIN 2 (FDX2) IN FRIEDREICH ATAXIA (FRDA) CLAIM OF PRIORITY This application claims the benefit of U.S. Provisional Patent Application Serial Nos. 63 / 358,807, filed on September 16, 2023, and 63 / 596,910, filed on November 7, 2023. The entire contents of the foregoing are hereby incorporated by reference. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant Nos. S124679, AG016636, and GM140217 awarded by the National Institutes of Health. The Government has certain rights in the invention. TECHNICAL FIELD Described herein are compositions and methods for treating subjects with disorders associated with mutations in the FXN gene and / or that have reduced expression of frataxin protein, e.g., Friedreich ataxia (FRDA), using inhibitors of ferredoxin 2 (FDX2) that decreases FDX2 protein expression. BACKGROUND Friedreich (or Friedreich’s) ataxia (FRDA) is an autosomal recessive inherited disease that causes progressive damage to the nervous system, resulting in movement problems. FRDA affects approximately 1 in 50,000 people, making it the most common Mendelian monogenic mitochondrial disease. Symptoms typically first appear at 5-15 years of age, followed by progressive neurodegeneration. Typically, within 10 years following the onset of symptoms, the patient is wheelchair-bound. FRDA presents with ataxia, cardiomyopathy, and increased incidence of diabetes, and unfortunately patients have an average lifespan of 37.5 years with limited treatment options. SUMMARY Provided herein are methods for treating a subject having a disorder associated with a mutation in the frataxin (FXN) gene and / or having reduced expression of Attorney Docket No.29539-0796WO1 / MGH 2022-549 frataxin protein. The methods comprise administering to the subject a therapeutically effective amount of an inhibitor of human ferredoxin 2 (FDX2) that decreases FDX2 protein expression. In some embodiments, the inhibitor of FDX2 comprises an inhibitory nucleic acid or a CRISPR / Cas9 complex targeting the FDX2 gene. In some embodiments, the inhibitory nucleic acid comprises a small hairpin RNA (shRNA), antisense oligonucleotide, small nuclear RNA (snRNA), small interfering RNA (siRNA), or a single guide RNA (sgRNA) targeting a FDX2 nucleic acid. In some embodiments, the inhibitory nucleic acid comprises a small hairpin RNA (shRNA) targeting the FDX2 nucleic acid. In some embodiments, the inhibitory nucleic acid comprises an antisense oligonucleotide targeting the FDX2 nucleic acid. In some embodiments, the inhibitor comprises a CRISPR / Cas9 complex targeting the FDX2 gene. In some embodiments, the subject has Friedreich ataxia (FRDA). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS FIGs. 1A-I: Mutations in NFS1 / NFS-1 or FDX2 / FDX-2 partially rescue the growth defect caused by Frataxin loss in C. elegans. A, A forward genetic screen using random chemical mutagenesis revealed mutations in other genes that can suppress the loss of Frataxin. B, Multiple sequence alignment of NFS1 (C. elegans residues 239-251) and FDX2 (C. elegans residues 117-127) including homologs from mammals, fish, and invertebrates made using ClustalW. C, Synchronized Frataxin null animals grown at 7% oxygen with or without suppressor mutations in fdx-2 and Attorney Docket No.29539-0796WO1 / MGH 2022-549 nfs-1. d,e,f, Growth of C. elegans at 7% oxygen (4 days) (D), 7% oxygen (2 days) (E), or 1% oxygen (2 days) (F) quantified by body length measurements. For all panels statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test. N.s. = not significant, * = p value <0.05, ** = p value <0.01, *** = p value <0.001. G, Multiple sequence alignment of C. elegans mitochondrial FDX-2 with human FDX2 and FDX1. Sequences necessary and sufficient for the activity of FDX2 are boxed and noted with an asterisk, and a residue critical for the activity of FDX1 is boxed and noted with a plus sign14. H,I, Growth of C. elegans for 2 days (H) or 3 days (I) at indicated oxygen tensions quantified by body length measurements. For all panels statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test. n.s. = not significant, * = p value <0.05, ** = p value <0.01, *** = p value <0.001. FIGs. 2A-D: Frataxin suppressor mutations restore levels of ISC- containing complex I. A, Quantitative TMT proteomics of complex I subunits in wild type animals, Frataxin mutants, and Frataxin mutants with suppressor mutations nfs- 1(R244K) or fdx-2(A126V) grown continuously at 1% oxygen. Values are normalized to wild type; proteins shown from which at least two peptides were quantified. B, SDS-PAGE followed by western blot of whole worm lysate from animals exposed to continuous 1% oxygen. C, Growth of C. elegans at 1% or 10% oxygen for indicated durations. D, SDS-PAGE followed by western blot of whole worm lysate from animals shifted from 1% to 21% for 2 days. Statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test. n.s. = not significant, * = p value <0.05, ** = p value <0.01, *** = p value <0.001. FIGs. 3A-C: Excess FDX2 is detrimental to NFS1 activity and ISC biosynthesis. A, Growth of animals for 3 days at room temperature housed at 21% or 1% oxygen. The nfs-1(R244K); fdx-2(A126V) double mutants are synthetic sick at 21% oxygen and rescued by hypoxia. B, 3 day growth assay with K562 or 293T cells overexpressing GFP or FDX2 cDNA, grown in 21% or 1% oxygen tensions. C, Immunoblots for FDX2, Lipoic Acid, OXPHOS, NFS1 and loading control Tubulin on K562 (left) and 293T (right) cell lysates collected from (B). ns = p > 0.05, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001. Two-way ANOVA with Bonferroni’s post-test. FIGs. 4A-I: Partial loss of wild type FDX2 activity suppresses Frataxin mutants. A, FDX-2 gene model with CRISPR / Cas9-generated 8 bp deletion Attorney Docket No.29539-0796WO1 / MGH 2022-549 indicated, predicted to produce to function protein. B, Growth of animals for 3 days at 21% oxygen. All animals were healthy and fertile except FDX2 null homozygotes. C, Growth of animals for 3 days at 1% oxygen (left) or 6 days at 10% oxygen (right). Frataxin null animals (frh-1 in C. elegans) are rescued by a heterozygous null mutation in FDX2. D, Latency to fall on a rotarod test (left) and grip strength (right) in mice on Doxycycline treatment for 15 weeks. E, Broodsize of wild type and fdx- 2(null) animals grown continuously at 21% or 1% oxygen. F, SDS-PAGE followed by western blot of brain tissue from wild type mice or mice heterozygous for Fdx2 mutation (left), quantified using Fiji (right). G, Latency to fall on a rotarod test (left) and grip strength (right) in mice on Doxycycline treatment for 12 weeks. H, Body weight measurements of male (left) and female (right) mice following Doxycycline treatment. I, Survival of mice following Doxycycline treatment. For all panels statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test. n.s. = not significant, * = p value <0.05, ** = p value <0.01, *** = p value <0.001. DETAILED DESCRIPTION In humans, FRDA is caused by a GAA^TTC triplet repeat expansion in the first intron of the FXN gene (NCBI RefSeqGene NG_008845.2). FXN encodes a protein called frataxin, a ubiquitous, nuclear-encoded mitochondrial protein (Campuzano et al. 1996, Science 271:1423-1427). Unaffected individuals have 6–30 GAA^TTC repeats, whereas affected individuals have approximately 70-1000 repeats (Sharma et al. 2004, Ann Neurol 56:898-901; Pandolfo 2008, Arch Neurol 65:1296- 1303). The GAA^TTC repeat expansion mutation represses FXN expression at the level of transcription, with the extent of FXN repression directly related to the length of the GAA^TTC repeat. Age of onset and disease severity are also correlated with the length of the GAA^TTC repeat (Chutake et al. 2014, Ann Neurol 76:522-528). Importantly, the TRE-FXN gene encodes functional frataxin, albeit at reduced levels. The deficiency of frataxin is associated with impairment of iron-sulfur cluster (ISC)-containing enzymes, deficits of respiratory chain complex activities, and accumulation of iron (Bradley et al. 2000, Hum Mol Genet 9:275-282; Foury and Cazzalini 1997, FEBS Lett 411:373-377; Waldvogel et al. 1999, Ann Neurol 46:123- 125; Koeppen et al. 2007, Acta Neuropathol 114:163-173; Wong et al. 1999, Hum Mol Genet 8:425-430). Attorney Docket No.29539-0796WO1 / MGH 2022-549 Iron sulfur clusters (ISCs) are ancient and essential cofactors necessary for the activity of dozens of proteins in the cell, including those involved in the electron transport chain, TCA cycle, DNA repair and protein translation8. Synthesis of ISCs occurs in the mitochondrial matrix by an enzyme-mediated pathway consisting of the scaffold protein ISCU, the cysteine desulfurase NFS1 which provides sulfur from cysteine, LYRM4 and NDUFAB1 (the acyl carrier protein) which stabilize NFS1, the ferredoxin FDX2 which conveys electrons from FDXR / NADPH, and Frataxin (FXN)9,10. Frataxin is an allosteric activator of NFS12, and accelerates the formation of 2Fe-2S clusters by promoting persulfide transfer from NSF1 to ISCU1,3,4. Reduced levels of Frataxin underlies Friedreich’s ataxia (FA)5, which affects 1 in 50,000 people, making it the most common monogenic mitochondrial disease and the most common recessive ataxia. FA presents with ataxia, cardiomyopathy, and increased incidence of diabetes; patients have an average lifespan of 37.5 years with no effective therapies to date. The growth of yeast, C. elegans, and human cells lacking Frataxin can be rescued by hypoxia due to an increase in ISC synthesis11. Our ability to culture Frataxin null mutant C. elegans in “permissive” hypoxic environments affords us the unique opportunity to identify rare bypass mutations in a whole organism. Our approach is analogous to the use of temperature sensitive mutants that grow well in low temperature environments but then experience fitness defects at non-permissive higher temperatures. Genetic suppressors may inform on the precise molecular action of hypoxia in restoring ISC biosynthesis as well as identify new pathways amenable to therapeutic interventions. Through forward genetics in C. elegans we identified Frataxin suppressor mutations that map to the NFS1 / FDX2 interface: nfs-1(R244K), fdx-2(E117K), fdx- 2(A126V), and fdx-2(P127S) (see Table 1 for homologous residues in humans). Screens in yeast have identified an intra-complex Frataxin suppressor mutation in the scaffold protein ISCU29which bypasses the need for Frataxin by accelerating cluster formation on the glutaredoxin GRX530. However, such bypass mutations have never before been reported in animals. Based on published cryo-EM structures nfs- 1(R244K) and fdx-2(E117K) likely disrupt salt-bridge interactions that stabilize the NFS1 / FDX2 interaction. fdx-2(A126V) and fdx-2(P127S) may also disrupt FDX2 binding through conformational changes or destabilize FDX-2 leading to less overall protein, which we observed for the A126V mutation in our TMT proteomics and cell Attorney Docket No.29539-0796WO1 / MGH 2022-549 culture experiments (Supplementary Figs. 2c & 3e). In principle any mutation that lowers FDX-2 levels could be a suppressor in this system, as heterozygous fdx-2(null) also partially rescued Frataxin loss. Comprehensive genetic analysis from C. elegans nominates partial knockdown of FDX2 as a therapeutic approach to treating Friedreich’s ataxia. Genetic therapy approaches for Friedreich’s ataxia would naturally include replacing Frataxin, however overexpression of Frataxin has shown toxicity in mouse studies31,32and paradoxically causes ISC deficiency dependent on Frataxin binding to NFS132. The ability of Frataxin to competitively inhibit FDX2 binding to NFS16explains why Frataxin over-expression is toxic and motivates genetic therapies that restore the stoichiometric balance of Frataxin and FDX2. In this study we demonstrate that even in contexts of no Frataxin protein (e.g. C. elegans null mutant) or extremely low levels of Frataxin (e.g. Dox-inducible shFXN mouse), lowering FDX2 levels to 50% of wild type is beneficial. This may be due to FDX2 actively inhibiting the cysteine desulfurase and / or persulfide transfer activities of NFS1 that are normally activated by Frataxin. Our experiments in normoxia and hypoxia may also further illuminate the mechanism by which hypoxia rescues defects in iron sulfur cluster biosynthesis. In addition to rescuing Frataxin deficiency11, hypoxia rescues growth defects and ISC deficiencies caused by (1) over-expression of FDX2 or (2) weakening of the NFS1- FDX2 interaction (Fig. 3). This rescue suggests that hypoxia circumvents the necessity of the FXN / FDX2 binding site altogether for iron sulfur cluster assembly, possibly through an alternative complex conformation. In support, the FDX2 binding site on the ISC assembly complex may vary with redox state or oxygenation6,33. Alternatively, hypoxia rescue could result from increased stability of ISCs in a low oxygen environment, such that mutations giving rise to very low (non-zero) levels of ISC are now viable. Indeed, the primary cellular defect associated with oxygen toxicity is loss of vulnerable ISC-containing proteins34. The FDX2 protein is required for iron sulfur cluster synthesis, so it was surprising that knocking it down in the background of FXN mutation / deficiency was beneficial. This idea is further supported by the observation that although FDX2 loss causes a recessive disease in humans, the heterozygous parents are healthy, implying that some loss of FDX2 can be tolerated. Attorney Docket No.29539-0796WO1 / MGH 2022-549 Thus, partial knockdown of FDX2, e.g., via inhibitory nucleic acids, e.g., antisense oligos (ASO) or RNAi (e.g., using small interfering RNA (siRNA) or short hairpin RNA (shRNA)); or CRISPR-based gene editing or CRISPRi, can be used as a therapeutic approach in subjects with disorders associated with mutations in the FXN gene, e.g., Friedreich ataxia (FRDA). Methods of Treatment The methods described herein include methods for the treatment of disorders associated with mutations in the FXN gene. In some embodiments, the disorder is Friedreich ataxia (FRDA). Generally, the methods include administering a therapeutically effective amount of an agent that decreases levels of FDX2 protein as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. Methods known in the art can be used to diagnose or identify a subject as having a disorder associated with a mutation in the FXN gene that reduces expression of the frataxin protein, e.g., sequencing or identification of the presence of trinucleotide repeat expansion (e.g., using a commercially available assay such as the Friedreich ataxia (FXN) Repeat Expansion Test (Athena Diagnostics)); a quantitative immunoassay to measure frataxin levels, e.g., as described in Plasterer et al., PLoS One. 2013; 8(5): e63958, or a lateral flow test as described in Willis et al., Mol Genet Metab. 2008 Aug; 94(4): 491–497 or commercially available kits such as the dipstick kit from abcam (ab109881); MRI to detect atrophy of the cervical spinal cord with minimal evidence of cerebellar atrophy; transcranial sonography for assessment of both cerebellar and noncerebellar abnormalities, e.g., dentate hyperechogenicity. Typically, subjects present with gait ataxia (e.g., tabetocerebellar gait), with ataxia progressing to the legs, trunk, and arms; development of tremors; titubation; and trembling. To determine severity, any of three scales can be used, e.g., The International Cooperative Ataxia Rating Scale (ICARS), the Friedreich Ataxia Rating Scale (FARS), and the Scale for the Assessment and Rating of Ataxia (SARA), see, e.g., Bürk et al., J. Neurochem. 126 Suppl. 1:118-24 (2013). As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with mutations in the FXN gene that reduce expression. FRDA is an autosomal recessive neurodegenerative disorder that results in progressive gait and limb ataxia with associated limb muscle weakness, absent lower limb reflexes, extensor plantar responses, dysarthria, and decreased vibratory sense and Attorney Docket No.29539-0796WO1 / MGH 2022-549 proprioception, as well as cardiac manifestations including cardiac dysfunction and heart failure; visual field defects; and diabetes. Thus, a treatment can result in a reduction in the severity of these deficits or a reduction in the rate of decline or degeneration. Administration of a therapeutically effective amount of a compound described herein for the treatment of a condition associated with mutations in the FXN gene that reduce expression, e.g., FRDA, will result in improvement in one or more symptoms, e.g., reduction in the severity or rate of decline in gait and limb ataxia, limb muscle weakness, deficits in lower limb reflexes, extensor plantar responses, dysarthria, and vibratory sense and proprioception, and a return or approach to normal gait and limb movements, a return or approach to normal muscle strength and reflexes, a return or approach to normal (flexor) plantar responses, reduced dysarthria / improved speech, and a return or approach to normal vibratory sense and proprioception, as well as a reduction in the severity or risk of cardiac dysfunction and / or heart failure or diabetes. In some embodiments, the treatment results in decreased morbidity or mortality, e.g., a delayed loss of ambulation, an increased life span (average age of death is 37.7 ± 14.4 years, range 21-69, Harding et al., J. Med Genet. 18(4):285-7 (1981)) and / or an improved quality of life. In some embodiments, the treatment results in an improvement or long-term stabilization in the Friedreich Ataxia Rating Scale, see, e.g., Friedman et al., Mov Disord. 2010 Mar 15; 25(4): 426– 432. Therapeutic Agents Agents that decrease expression of FDX2 protein (ferredoxin-2, mitochondrial) as described herein can include inhibitory nucleic acids and CRISPR- based gene editing or CRISPRi reagents. An exemplary sequence for human FDX2 is provided at GenBank RefSeq ID. NM_001397406.1 (nucleic acid) and NP_001384335.1 (protein); an exemplary gene sequence is provided at RefSeqGene NG_034259.1 (Range 5001-10971). The nucleic acid-based inhibitors of FDX2 can be predicted from the human FDX2 DNA sequence, but the FDX-2 protein sequence is also highly conserved between C. elegans and humans, so that chemicals that inhibit ferredoxins could easily be screened for suppression of the frh-1 null mutant in C. elegans or a human Frataxin mutant cell line. ! Attorney Docket No.29539-0796WO1 / MGH 2022-549 Ferredoxin-2, mitochondrial [Homo sapiens] Sequence ID: NP_001384335.1 Length: 183 >Ferredoxin 1-like [Homo sapiens] Sequence ID: AAH63460.1 Length: 183 Range 1: 43 to 183 Score:176 bits(447), Expect:8e-56, Method: Compositional matrix adjust., Identities:82 / 141(58%), Positives:101 / 141(71%), Gaps:0 / 141(0%) C eleg 29 RHFMTSSVRKTGDFEYEDPKSEDEVVNITYVLRDGTERKIRGKVGDNVMFLAHRYDIEME 88 R F + R G+ + P+ +VVN+ +V R G + G+VGDNV+ LA R+ +++E Human 43 RKFQATGSRPAGEEDAGGPERPGDVVNVVFVDRSGQRIPVSGRVGDNVLHLAQRHGVDLE 102 Query 89 GACEASLACSTCHVYVDPAFQNKLPEPLEEEDDMLDMAPALKDNSRLGCQIVLTKELDGI 148 GACEASLACSTCHVYV + LP P E EDDMLDMAP L++NSRLGCQIVLT EL+G Sbjct 103 GACEASLACSTCHVYVSEDHLDLLPPPEEREDDMLDMAPLLQENSRLGCQIVLTPELEGA 162 Query 149 TVTLPTMTRNFYVDGHVPKPH 169 (SEQ ID NO:1) TLP +TRNFYVDGHVPKPH Sbjct 163 EFTLPKITRNFYVDGHVPKPH 183 (SEQ ID NO:2) An exemplary sequence of human FDX1 is at NM_001397406.1, Homo sapiens ferredoxin 2 (FDX2), mRNA; nuclear gene for mitochondrial product 1 gagtcacgtg atgcatgtca tggccgcctc catggcccgg ggaggcgtga gtgccagggt 61 tctactgcag gctgccaggg gcacctggtg gaacagacct gggggcactt ccgggtcggg 121 ggagggggtg gcgctgggga caaccagaaa gtttcaagcg acaggctcgc gcccggctgg 181 agaggaggac gcgggcggcc cggagcggcc cggggacgtg gtgaacgtgg tgttcgtaga 241 ccgctcaggc cagcggatcc cagtgagtgg cagagtcggg gacaatgttc ttcacctggc 301 ccagcgccac ggggtggacc tggaaggggc ctgtgaagcc tccctggcct gctccacctg 361 ccatgtgtat gtgagtgaag accacctgga tctcctgcct cctcccgagg agagggaaga 421 cgacatgcta gacatggccc ccctcctcca ggagaactcg cggctgggct gccagattgt 481 gctgacaccg gagctggaag gagcggaatt caccctgccc aagatcacca ggaacttcta 541 cgtggatggc catgtcccca agccccactg acatgaacac ctggaccatt ccacattgcc 601 atggccccag ggcccagatt gagggaatag ccaggtgcca gccctgccca gagtgcggac 661 aggcccggga gagacgtgga agcccctgtg aaggacaaca cccctgcttg ggagagagtc 721 ccatgtccag gctctggtgg ggacagggcc cctagtgggg tggccttccc caggcccctg 781 agaatcaggg tttgagtagg agtggactca tattggagct gcaataaatc gataacacag 841 gccccagcat gttgagtgtc cttgggggac agatctgggg tctacaggtg gctcacacct 901 gtaatcctag cactttggga agccaagatg ggaggatcac ttgaggccag gagcttaaga 961 ccatcctggg caacatggcg agacctcgtc tctataaaaa cagtaaaaat ta (SEQ ID NO:3) Inhibitory Nucleic Acids Inhibitory nucleic acids useful in the present methods and compositions include antisense oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified Attorney Docket No.29539-0796WO1 / MGH 2022-549 bases / locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that specifically hybridize to at least a portion of a target nucleic acid and modulate its function to reduce expression of the FDX2 protein. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); short, hairpin RNA (shRNA); or combinations thereof. In some embodiments, the inhibitory nucleic acids are 10 to 50, 10 to 20, 10 to 25, 13 to 50, or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having complementary portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin. In some embodiments, the inhibitory nucleic acids are 15 nucleotides in length. In some embodiments, the inhibitory nucleic acids are 12 or 13 to 20, 25, or 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having complementary portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin (complementary portions refers to those portions of the inhibitory nucleic acids that are complementary to the target sequence). The inhibitory nucleic acids useful in the present methods are sufficiently complementary to the target RNA, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. "Complementary" refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a RNA, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required. Routine methods can be used to design an inhibitory nucleic acid that binds to the target sequence with sufficient specificity. In some embodiments, the methods include using bioinformatics methods known in the art to identify regions of secondary structure, e.g., one, two, or more stem-loop structures, or pseudoknots, and selecting those regions to target with an inhibitory nucleic acid. For example, “gene Attorney Docket No.29539-0796WO1 / MGH 2022-549 walk” methods can be used to optimize the inhibitory activity of the nucleic acid; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can be left between the target sequences to reduce the number of oligonucleotides synthesized and tested. GC content is preferably between about 30-60%. Contiguous runs of three or more Gs or Cs should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides). In general, the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within an RNA. For example, an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Inhibitory nucleic acids that hybridize to an RNA can be identified through routine experimentation. In general, the inhibitory nucleic acids must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target. For further disclosure regarding inhibitory nucleic acids, please see US2010 / 0317718 (antisense oligos); US2010 / 0249052 (double-stranded ribonucleic acid (dsRNA)); US2009 / 0181914 and US2010 / 0234451 (LNAs); US2007 / 0191294 (siRNA analogues); US2008 / 0249039 (modified siRNA); and WO2010 / 129746 and WO2010 / 040112 (inhibitory nucleic acids). Antisense In some embodiments, the inhibitory nucleic acids are antisense oligonucleotides. Antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing. Antisense oligonucleotides of the present invention are complementary nucleic acid sequences designed to hybridize under stringent conditions to an RNA. Thus, oligonucleotides are chosen that are Attorney Docket No.29539-0796WO1 / MGH 2022-549 sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity, to give the desired effect. In some embodiments, the ASO comprises a sequence that targets the human FDX2 as shown in Table A. Table A - Exemplary ASOs 5’ to 3’ targeting the human FDX2 transcript siRNA / shRNA In some embodiments, the nucleic acid sequence that is complementary to a target RNA can be an interfering RNA, including but not limited to a small interfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”). Methods for constructing interfering RNAs are well known in the art. For example, the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self- complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, interfering RNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions are linked by means of nucleic acid based or non-nucleic acid-based linker(s). The interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The interfering can be a circular single-stranded polynucleotide having two or more loop structures and a stem Attorney Docket No.29539-0796WO1 / MGH 2022-549 comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference. In some embodiments, the interfering RNA coding region encodes a self- complementary RNA molecule having a sense region, an antisense region and a loop region. Such an RNA molecule when expressed desirably forms a “hairpin” structure and is referred to herein as an “shRNA.” The loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length. In some embodiments, the sense region and the antisense region are between about 15 and about 20 nucleotides in length. Following post-transcriptional processing, the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family. The siRNA is then capable of inhibiting the expression of a gene with which it shares homology. For details, see Brummelkamp et al., Science 296:550-553, (2002); Lee et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishi and Taira, Nature Biotechnol 20:497-500, (2002); Paddison et al. Genes & Dev. 16:948-958, (2002); Paul, Nature Biotechnol, 20, 505-508, (2002); Sui, Proc. Natl. Acad. Sd. USA, 99(6), 5515-5520, (2002); Yu et al. Proc NatlAcadSci USA 99:6047- 6052, (2002). The target RNA cleavage reaction guided by siRNAs is highly sequence specific. In general, siRNA containing a nucleotide sequence identical to a portion of the target nucleic acid are preferred for inhibition. However, 100% sequence identity between the siRNA and the target gene is not required to practice the present invention. The siRNA has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition. In general, the siRNAs must retain Attorney Docket No.29539-0796WO1 / MGH 2022-549 specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target. Modified Inhibitory Nucleic Acids In some embodiments, the inhibitory nucleic acids used in the methods described herein are modified, e.g., comprise one or more modified bonds or bases. A number of modified bases include phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic acid (LNA) molecules. Some inhibitory nucleic acids are fully modified, while others are chimeric and contain two or more chemically distinct regions, each made up of at least one nucleotide. These inhibitory nucleic acids typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric inhibitory nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and / or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference. In some embodiments, the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-O-alkyl, 2'-O- alkyl-O-alkyl or 2'-fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than; 2'-deoxyoligonucleotides against a given target. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide; these modified oligos survive intact Attorney Docket No.29539-0796WO1 / MGH 2022-549 for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2 -NH-O-CH2, CH,~N(CH3)~O~CH2 (known as a methylene(methylimino) or MMI backbone], CH2 --O--N (CH3)-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)- CH2 -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P-- O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366- 374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus- containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5, 177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799; 5,587,361; and 5,625,050. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. Attorney Docket No.29539-0796WO1 / MGH 2022-549 Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602. Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596, 086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference. One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2or O(CH2)n CH3where n is from 1 to about 10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3 ; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'- methoxyethoxy [2'-0-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl)] (Martin et al, HeIv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'- methoxy (2'-0-CH3), 2'-propoxy (2'-OCH2CH2CH3) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' Attorney Docket No.29539-0796WO1 / MGH 2022-549 terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me- C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5- hydroxymethyluracil, 8- azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6- diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp75- 77; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A "universal" base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide. In some embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the Attorney Docket No.29539-0796WO1 / MGH 2022-549 preparation of PNA compounds comprise, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500. Inhibitory nucleic acids can also include one or more nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases comprise other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Further, nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science and Engineering', pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition', 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications', pages 289- 302, Crooke, S.T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2<0>C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds, 'Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos. Attorney Docket No.29539-0796WO1 / MGH 2022-549 3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference. In some embodiments, the inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533- 538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O- hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). See also US patent nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference. Attorney Docket No.29539-0796WO1 / MGH 2022-549 These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT / US92 / 09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5- tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino- carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941. Attorney Docket No.29539-0796WO1 / MGH 2022-549 Locked Nucleic Acids (LNAs) In some embodiments, the modified inhibitory nucleic acids used in the methods described herein comprise locked nucleic acid (LNA) molecules, e.g., including [alpha]-L-LNAs. LNAs comprise ribonucleic acid analogues wherein the ribose ring is “locked” by a methylene bridge between the 2’-oxgygen and the 4’- carbon – i.e., oligonucleotides containing at least one LNA monomer, that is, one 2'- O,4'-C-methylene-β-D-ribofuranosyl nucleotide. LNA bases form standard Watson- Crick base pairs but the locked configuration increases the rate and stability of the basepairing reaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)). LNAs also have increased affinity to base pair with RNA as compared to DNA. These properties render LNAs especially useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as knockdown tools for miRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs, e.g., RNAs as described herien. The LNA molecules can include molecules comprising 10-30, e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the RNA. The LNA molecules can be chemically synthesized using methods known in the art. The LNA molecules can be designed using any method known in the art; a number of algorithms are known, and are commercially available (e.g., on the internet, for example at exiqon.com). See, e.g., You et al., Nuc. Acids. Res. 34:e60 (2006); McTigue et al., Biochemistry 43:5388-405 (2004); and Levin et al., Nuc. Acids. Res. 34:e142 (2006). For example, “gene walk” methods, similar to those used to design antisense oligos, can be used to optimize the inhibitory activity of the LNA; for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target RNA can be prepared, followed by testing for activity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can be left between the LNAs to reduce the number of oligonucleotides synthesized and tested. GC content is preferably between about 30-60%. General guidelines for designing LNAs are known in the art; for example, LNA sequences will bind very tightly to other LNA sequences, so it is preferable to avoid significant complementarity within an LNA. Contiguous runs of more than Attorney Docket No.29539-0796WO1 / MGH 2022-549 four LNA residues, should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides). In some embodiments, the LNAs are xylo-LNAs. For additional information regarding LNAs see U.S. Pat. Nos. 6,268,490; 6,734,291; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,060,809; 7,084,125; and 7,572,582; and U.S. Pre-Grant Pub. Nos. 20100267018; 20100261175; and 20100035968; Koshkin et al. Tetrahedron 54, 3607–3630 (1998); Obika et al. Tetrahedron Lett. 39, 5401–5404 (1998); Jepsen et al., Oligonucleotides 14:130–146 (2004); Kauppinen et al., Drug Disc. Today 2(3):287-290 (2005); and Ponting et al., Cell 136(4):629–641 (2009), and references cited therein. CRISPR Gene Editing and Repression Complexes The present methods can also include the delivery a CRISPR gene editing or CRISPRi repression complex. The complex includes a CRISPR Cas (e.g., Cas9) enzyme and one or more guide RNAs directing the editing enzyme to FDX2 and can be delivered as nucleic acids encoding the enzyme and / or the guide RNAs, or as ribonucleoprotein complexes (RNPs). Guide RNAs directing the CRISPR enzyme to FDX2 The gene editing or repression complex also includes guide RNAs directing the editing enzyme to FDX2, i.e., comprising a sequence that is complementary to the sequence of a nucleic acid encoding FDX2, and that include a PAM sequence that is targetable by the co-administered Cas9 editing enzyme. In some embodiments, the precursor sequence is targeted by the guide RNA, i.e., comprising a sequence that is complementary to the sequence of a nucleic acid encoding FDX2. In some embodiments, the precursor sequence is targeted by the guide RNA. CRISPR Cas enzymes The methods can include the delivery of CRISPR Cas enzymes to cells. The enzymes can include one or more of Streptococcus thermophilus (ST) Cas9 (StCas9); Treponema denticola (TD) (TdCas9); Streptococcus pyogenes (SP) (SpCas9); Staphylococcus aureus (SA) Cas9 (SaCas9); or Neisseria haracteriza (NM) Cas9 (NmCas9), as well as variants thereof that are at least 80%, 85%, 90%, 95%, 99% or 100% identical thereto that retain at least one function of the parent protein, e.g., the Attorney Docket No.29539-0796WO1 / MGH 2022-549 ability to complex with a gRNA, bind to target DNA specified by the gRNA, and alter the sequence of the target DNA. Variants include the SpCas9 D1135E variant; SpCas9 VRER variant; SpCas9 EQR variant; and the SpCas9 VQR variant, among others. In some embodiments, base editors and other Cas fusions can also be used to introduce specific edits that abrogate expression of functional protein, or CRISPR interference (CRISPRi) reagents can be used to reduce but not knock out expression (i.e., dCas9, optionally fused to a repressor protein (e.g., SALL1 and / or SDS3), and a guide RNA specifically designed to target the region immediately downstream of the FDX2 gene’s transcriptional start site (TSS), see Jensen et al., Genome Research. 31 (11): 2120–2130. Qi et al., Cell.152 (5): 1173–118 (2013); Larson et al., Nature Protocols 8:2180–2196 (2013)). The sequences of the Cas9s are known in the art; see, e.g., Kleinstiver et al., Nature. 2015 Jul 23; 523(7561): 481–485; WO 2016 / 141224; US 9,512,446; US- 2014-0295557; WO 2014 / 204578; and WO 2014 / 144761. The methods can also include the use of the other previously described variants of the SpCas9 platform (e.g., truncated sgRNAs (Tsai et al., Nat Biotechnol 33, 187-197 (2015); Fu et al., Nat Biotechnol 32, 279-284 (2014)), nickase mutations (Mali et al., Nat Biotechnol 31, 833-838 (2013); Ran et al., Cell 154, 1380-1389 (2013)), FokI-dCas9 fusions (Guilinger et al., Nat Biotechnol 32, 577-582 (2014); Tsai et al., Nat Biotechnol 32, 569-576 (2014); WO2014144288). See also Hou, Z. et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria haracteriza. Proc Natl Acad Sci U S A (2013); Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual- RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res 42, 2577-2590 (2014); Esvelt, K.M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods 10, 1116-1121 (2013); Cong, L. et al. Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Horvath, P. et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol 190, 1401-1412 (2008). The Cas9 can be delivered as a purified protein (e.g., a recombinantly produced purified protein, prefolded and optionally complexed with the sgRNA, e.g., in a ribonucleoprotein (RNP)) or as a nucleic acid encoding the Cas9, e.g., an expression construct. Purified Cas9 proteins can be produced using methods known Attorney Docket No.29539-0796WO1 / MGH 2022-549 in the art, e.g., expressed in prokaryotic or eukaryotic cells and purified using standard methodology. For example, the methods can include delivering the Cas9 protein and guide RNA together, e.g., as a complex. For example, the Cas9 and gRNA can be overexpressed in a host cell and purified, then complexed with the guide RNA (e.g., in a test tube) to form a ribonucleoprotein (RNP) and delivered to cells. In some embodiments, the Cas9 can be expressed in and purified from bacteria through the use of bacterial Cas9 expression plasmids. For example, His-tagged Cas9 proteins can be expressed in bacterial cells and then purified using nickel affinity chromatography. The RNPs can be delivered to the cells in vivo or in vitro, e.g., using lipid-mediated transfection or electroporation. See, e.g., Liang et al., Journal of biotechnology 208 (2015): 44-53; Zuris et al. Nature biotechnology 33.1 (2015): 73-80; Kim et al. Genome research 24.6 (2014): 1012-1019. Efficiency of protein delivery can be enhanced, e.g., using electroporation (see, e.g., Wang et al., Journal of Genetics and Genomics 43(5):319–327 (2016)); cationic or lipophilic carriers (see, e.g., Yu et al., Biotechnol Lett. 2016; 38: 919–929; Zuris et al., Nat Biotechnol. 33(1):73-80 (2015)); or even lentiviral packaging particles (see, e.g., Choi et al., Gene Therapy 23, 627-633 (2016)). Exemplary Sequences In some embodiments, the methods and compositions described herein include sequence that are at least 80%, 85%, 90%, 95%, 99% or 100% identical to a reference sequence (e.g., as described herein). To determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (gaps are introduced in one or both of a first and a second amino acid or nucleic acid sequence as required for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% (in some embodiments, about 85%, 90%, 95%, or 100% of the length of the reference sequence) is aligned. The nucleotides or residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions Attorney Docket No.29539-0796WO1 / MGH 2022-549 shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Expression Constructs Expression constructs encoding an inhibitory nucleic acid, or one or both of guide RNAs and / or Cas9 editing or repression enzymes, can be administered in any effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells. Approaches include insertion of the gene in viral vectors, including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered naked or with the help of, for example, cationic liposomes (lipofectamine) or derivatized (e.g., antibody conjugated), polylysine conjugates, haracteri S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation. A preferred approach for introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g., a cDNA. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid. Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host cell. The development of specialized cell lines (termed “packaging cells”) which produce only replication- Attorney Docket No.29539-0796WO1 / MGH 2022-549 defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, Blood 76:271 (1990)). A replication defective retrovirus can be packaged into virions, which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro with such viruses can be found in Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include YCrip, YCre, Y2 and YAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573). Lentiviral vectors transduce dividing as well as quiescent cells. This can be viewed as a major advantage with respect to gene therapy for tumors in general, as within a short treatment window most tumor cells (and especially GSC) do not divide. Therapeutic use of the lentiviral editing approach can be a legitimate alternative to other viral systems, as high viral titers can be produced, nonproliferating cells that are especially abundant in the walls of the tumor cavity after surgery can be transduced, and transduction efficacies are very high. An additional advantage of a locally applied vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus is its inactivation by human serum that would reduce systemic effects. To further reduce Attorney Docket No.29539-0796WO1 / MGH 2022-549 neurotrophism, and enhance selective tropism for glioma and GSC, the commonly bound envelope glycoprotein of VSV can be replaced with a more selective variant glycoprotein of lymphocytic choriomeningitis virus (LCMV-GP). LCMV-GP is not cytotoxic when injected locally or systemically, can be packaged with other components of the CRISPR-Cas9 system, and efficiently transduces solid glioma tissues as well as infiltrating tumor cells. Another viral gene delivery system useful in the present methods utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated, such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, or Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances, in that they are not capable of infecting non- dividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situ, where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986). Vectors based on recombinant Adenovirus-5 (Ad5): Ad5 have many advantages for this purpose, including non-integration, lack of insertional mutagenesis, high-efficiency transduction, and accommodation of large expression cassettes; these vectors have also been utilized in multiple clinical trials. Helper-dependent (HDAd) vectors can also be produced with all adenoviral sequences deleted except the origin of DNA replication at each end of the viral DNA along with packaging signal at 5-prime end of the genome downstream of the left Attorney Docket No.29539-0796WO1 / MGH 2022-549 packaging signal. HDAd vectors are constructed and propagated in the presence of a replication-competent helper adenovirus that provides the required early and late proteins necessary for replication. Yet another viral vector system useful for delivery of nucleic acids is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al., Curr. Topics in Micro. And Immunol.158:97-129 (1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells and exhibits a high frequency of stable integration (see for example Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62:1963-1973 (1989). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993). The identification of Staphylococcus aureus (SaCas9) and other smaller Cas9 enzymes that can be packaged into adeno-associated viral (AAV) vectors that are highly stable and effective, easily produced, approved by FDA, and tested in multiple clinical trials, paves new avenues for therapeutic gene editing. Of high relevance to GBM, better tissue distribution of AAV provides an additional advantage for invasive and recurrent tumors. FDX2 -targeting AAV vectors of various serotypes, including AAV1, AAV2, AAV8, AAV9, and AAVrh.10, can be used, all of which were previously tested in clinical trials. Preferably, the CRISPR FDX2 editing or repression complex is specific, i.e., induces genomic alterations preferentially at the target site (FDX2), and does not induce alterations at other sites, or only rarely induces alterations at other sites. Making and Using Inhibitory and Other Nucleic Acids The nucleic acid sequences used to practice the methods described herein, whether RNA (e.g., inhibitory nucleic acid or guide RNA), cDNA (e.g., encoding a Attorney Docket No.29539-0796WO1 / MGH 2022-549 CRISPR protein), genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and / or expressed / generated recombinantly. Recombinant nucleic acid sequences can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including, e.g., in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems. Nucleic acid sequences can be inserted into delivery vectors and expressed from transcription units within the vectors. The recombinant vectors can be DNA plasmids or viral vectors. Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al. (Retroviruses. (1997)) and “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)). As will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring nucleic acids of the invention into cells. The selection of an appropriate vector to deliver nucleic acids and optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation. Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell. Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus. The recombinant vectors capable of expressing the nucleic acids of the invention can be delivered as described herein, and persist in target cells (e.g., stable transformants). Nucleic acid sequences can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066. Attorney Docket No.29539-0796WO1 / MGH 2022-549 Nucleic acid sequences can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. For example, the nucleic acid sequences can include a phosphorothioate at least at the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence. As another example, the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O- MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O- DMAEOE), or 2'-O--N-methylacetamido (2'-O--NMA). As another example, the nucleic acid sequence can include at least one 2'-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-O-methyl modification. In some embodiments, the nucleic acids are “locked,” i.e., comprise nucleic acid analogues in which the ribose ring is “locked” by a methylene bridge connecting the 2’-O atom and the 4’-C atom (see, e.g., Kaupinnen et al., Drug Disc. Today 2(3):287- 290 (2005); Koshkin et al., J. Am. Chem. Soc., 120(50):13252–13253 (1998)). For additional modifications see US 20100004320, US 20090298916, and US 20090143326. Techniques for the manipulation of nucleic acids used in the methods and compositions described herein, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook et al., Molecular Cloning; A Laboratory Manual 3d ed. (2001); Current Protocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons, Inc., New York 2010); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993). Pharmaceutical Compositions The methods described herein can include the administration of pharmaceutical compositions and formulations comprising inhibitory or CRISPR nucleic acid or protein sequences designed to target a FRDX RNA. In some embodiments, the compositions are formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions and Attorney Docket No.29539-0796WO1 / MGH 2022-549 formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The and Practice of Pharmacy, 21st ed., 2005. The inhibitory nucleic acids can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Formulations of the compositions can include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Pharmaceutical formulations can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc. Attorney Docket No.29539-0796WO1 / MGH 2022-549 Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers. Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences or RNPs as described herein) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n- Attorney Docket No.29539-0796WO1 / MGH 2022-549 propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity. In some embodiments, oil-based pharmaceuticals are used for administration of nucleic acid sequences or RNPs. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102. Pharmaceutical formulations can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, these injectable oil-in-water emulsions comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and / or an ethoxylated sorbitan trioleate. The pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107- 111). Suppositories formulations can be prepared by mixing the drug with a suitable Attorney Docket No.29539-0796WO1 / MGH 2022-549 non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols. In some embodiments, the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. In some embodiments, the pharmaceutical compounds can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674. In some embodiments, the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This Attorney Docket No.29539-0796WO1 / MGH 2022-549 suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time). In some embodiments, the pharmaceutical compounds and formulations can be lyophilized. Stable lyophilized formulations comprising an inhibitory nucleic acid or RNP can be made by lyophilizing a solution comprising an active agent and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg / mL protein, about 15 mg / mL sucrose, about 19 mg / mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670. The compositions and formulations can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH- sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells. Liposomes can also include "sterically stabilized" liposomes, i.e., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises Attorney Docket No.29539-0796WO1 / MGH 2022-549 one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860. The formulations can be administered for prophylactic and / or therapeutic treatments. In some embodiments, for therapeutic applications, compositions are administered to a subject who is known to have a mutation in the FXN gene and that have reduced expression of frataxin protein, but has not yet developed any of the symptoms of FRDA, in an amount sufficient to treat, alleviate, slow, delay development, or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount. For example, in some embodiments, pharmaceutical compositions of the invention are administered in an amount sufficient to decrease serum levels of triglycerides in the subject. The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use, i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents’ rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate. Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the Attorney Docket No.29539-0796WO1 / MGH 2022-549 degree and amount of therapeutic effect generated after each administration (e.g., effect on tumor size or growth), and the like. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms. Various studies have reported successful mammalian dosing using complementary nucleic acid sequences. For example, Esau C., et al., (2006) Cell Metabolism, 3(2):87-98 reported dosing of normal mice with intraperitoneal doses of miR-122 antisense oligonucleotide ranging from 12.5 to 75 mg / kg twice weekly for 4 weeks. The mice appeared healthy and normal at the end of treatment, with no loss of body weight or reduced food intake. Plasma transaminase levels were in the normal range (AST ¾ 45, ALT ¾ 35) for all doses with the exception of the 75 mg / kg dose of miR-122 ASO, which showed a very mild increase in ALT and AST levels. They concluded that 50mg / kg was an effective, non-toxic dose. Another study by Krützfeldt J., et al., (2005) Nature 438, 685-689, injected anatgomirs to silence miR- 122 in mice using a total dose of 80, 160 or 240 mg per kg body weight. The highest dose resulted in a complete loss of miR-122 signal. In yet another study, locked nucleic acids (“LNAs”) were successfully applied in primates to silence miR-122. Elmen J., et al., (2008) Nature 452, 896-899, report that efficient silencing of miR- 122 was achieved in primates by three doses of 10 mg kg-1 LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. In some embodiments, the methods described herein can include co- administration with other drugs or pharmaceuticals, e.g., compositions for providing cholesterol homeostasis. For example, the inhibitory nucleic acids can be co- administered with drugs for treating or reducing risk of a disorder described herein. Dosage An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic Attorney Docket No.29539-0796WO1 / MGH 2022-549 compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. Attorney Docket No.29539-0796WO1 / MGH 2022-549 EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Materials and Methods The following materials and methods were used in the Examples below. C. elegans were propagated on NGM plates seeded with E. coli strain OP5036. Frataxin frh-1(tm5913) animals were provided by the Mitani Lab through the National Bio-Resource Project of the MEXT, Japan. To generate mutants with CRISPR / Cas9, 30 pmol S. pyogenes Cas9 (IDT) was injected into C. elegans gonads along with 90 pmol tracrRNA (IDT), 95 pmol crRNA (IDT), ssODN repair template (when applicable), and 40 ng / µl PRF4::rol-6(su1006) plasmid was used as a marker of successful injections37. To measure C. elegans growth and development crowded plates of gravid animals were washed into tubes in M9 buffer [3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 liter] and incubated with 20% bleach and 10% 5M KOH for 5 minutes while vortexing. The resulting embryos were washed 3x in M9 buffer and allowed to hatch overnight while rocking in M9. The following day arrested L1 animals were dropped onto E. coli OP50 plates and incubated at 20˚C. For assays in hypoxia (1% oxygen), animals were incubated in a Hypoxic in vitro cabinet (Coy Laboratory Products, Inc) at room temperature. To measure animal length, images were acquired using a ZEISS Axio Zoom V16 microscope with ZEN PRO software and the midline of individual animals was quantified in FIJI software. To screen for genetic suppressor mutations of frh-1(tm5913), thousands of L4 animals were exposed to 47 mM EMS (ethyl methanesulfonate) (Sigma M0880) for four hours while rocking. Animals were then washed twice with M9 buffer and allowed to recover on standard NGM plates at 1% oxygen. F2 animals were produced by self-fertilization of the newly induced mutations (animals at the F2 are therefore m / m for about 100 new mutations per F2 strain tested). Animals were bleach prepped as described above to generate a synchronized L1 stage population of mutagenized F3 animals, which were then dropped onto standard NGM plates at 10% oxygen. Plates were checked daily and F3 individuals capable of growing to adulthood were transferred onto new plates at 1% oxgyen. Fertile isolates were retested using F4 or F5 progeny to confirm their phenotype and then genomic DNA for whole genome Attorney Docket No.29539-0796WO1 / MGH 2022-549 sequencing was isolated using Gentra Puregene Tissue Kit (Qiagen 158667). To identify candidate suppressor mutations screen isolates were whole genome sequenced as described38. Lists of protein-altering mutations from each suppressor strain were then compared to identify genes with multiple mutant alleles. These candidate genes were then verified using targeted CRISPR / Cas9-based editing. SDS-PAGE western blots were performed with whole worm lysate, prepared by snap-freezing worm pellets of equal volume in liquid nitrogen and then mixing with NuPAGE LDS sample buffer and boiling at 100˚C for 20 minutes, followed by centrifugation at maximum speed for 10 minutes. Samples were run in 4-12% NuPAGE Bis-Tris gels for 2 hours at 100 V. Gels were transferred to nitrocellulose membranes using the iBlot Dry Blotting system, and the blocked in 5% milk in TBST for 1 hour. Membranes were incubated in primary antibodies (Anti-NDUFS3 Abcam ab14711; Anti-ATP5A Abcam ab14748; Anti-actin Abcam ab179467; Anti-Lipoic acid Sigma 437695) at 1:1,000 dilution overnight at 4˚C in 5% milk / TBST. The following day membranes were washed for 1 hour in TBST, incubated with secondary antibodies at 1:10,000 dilution for 1 hour at room temperature in 5% milk / TBST, and then washed in TBST for another hour. Blots were developed using Pierce ECL Western Blotting Substrate (Fisher 32209) and imaged with a GE Amersham Imager. Quantitative tandem mass tag proteomics was performed by the Thermo Fisher Center for Multiplexed Proteomics in the Department of Cell Biology at Harvard Medical School. From frozen worm pellets total protein quantification was performed using the micro-BCA assay by Pierce. Samples were reduced with DTT and alkylated with iodoacetimide. Proteins were precipitated using methanol / chloroform and the pellet resuspended in 200 mM EPPS, pH 8.0. Digestion was performed sequentially using LysC (1:50) and Trypsin (1:100) based on protease to protein ratio. Peptides were detected (MS1) in the Orbitrap; sequenced (MS2) in the ion trap; and quantified (MS3) in the Orbitrap. ~2 µl of each TMT-labelled sample was mixed to verify labelling success. Peptides were separated using a gradient of 3 to 27% 90% Acetonitrile in 0.1% formic acid over 180 minutes. MS2 spectra were searched using the Comet algorithm against a custom C. elegans + E. coli database containing its reversed complement and known contaminants. Peptide spectral matches were filtered to a 1% false discovery rate (FDR) using the target-decoy Attorney Docket No.29539-0796WO1 / MGH 2022-549 strategy combined with linear discriminant analysis. Proteins were quantified only from peptides with a summed SN threshold of >100. Protein Expression and Purification. SDAec, ISCU2, and FXN were expressed and purified as previously described (Cory et. al., 2017). FDX2 (aa 66-183, Q6P4F2) was synthesized and inserted into a pet29b+ vector from Twist Bioscience after codon optimization using Vector Builder software. The pet29b+ plasmid encoding FDX2 was transformed into BL21(DE3) cells. The cells were grown in Terrific Broth (Research Products International) at 37 °C and induced with 0.4 mM of IPTG. Following IPTG induction, the temperature was dropped to 16 °C, and the cells were harvested 16 hr later. Cells were resuspended in lysis buffer (50 mM HEPES, 500 mM NaCl, 50 mM imidazole, 10 mM MgCl2, 10 mM CaCl2, pH 8.0). Lysozyme (10 mg·L, Sigma-Aldrich), protease inhibitor cocktail (10 mg·L, Sigma-Aldrich), and 1 µg / mL DNase was added to resuspension before lysing by sonication (Branson Sonifier 450), and the soluble fraction was loaded onto 2 x 5 mL HisTrap columns (Cytiva). The protein was eluted using a linear gradient of Buffer A (50 mM HEPES, 500 mM NaCl, 50 mM imidazole pH 8.0) and Buffer B (50 mM HEPES, 500 mM NaCl, 500 mM imidazole pH 8.0) over 6 column volumes (CV). The eluted protein was digested with TEV protease (prepared in house; 1:50, protease: protein) overnight at 4 °C and the digested product was loaded onto a 5 mL HisTrap column (Cytiva) to remove TEV protease and His-tag. The flow-through containing cleaved product was concentrated to 20 mL, diluted to 150 mL with Anion A buffer [50 mM HEPES, 10 % glycerol (vol / vol) pH 7.5] and loaded onto an anion exchange column (26 / 20 POROS 50HQ, Applied Biosystems). FDX2 was eluted with Anion Buffer B [50 mM HEPES, 1 M NaCl, 10 % glycerol (vol / vol) pH 7.5] using a linear gradient over 5 CV. Brown fractions containing FDX2 were pooled and concentrated to 5 mL and loaded onto a HiLoad 16 / 100 Superdex 75 pg (Cytiva). The fractions corresponding to FDX2 were concentrated, frozen in liquid nitrogen, and stored at -80 °C. The concentration of FDX2 was determined using the extinction coefficient 11,000 M-1 cm-1 at 414 nm. Cysteine Desulfurase SDAec Complex Activity Measurements. Velocities of each complex were determined using a modified methylene blue assay (Das et. al., 2019). Protein complexes were prepared using 0.5 µM SDAec, 1.5 µM ISCU2 and when included, variable concentrations of FXN and FDX2. Each reaction was mixed with 4 mM D,L-DTT and 5 µM Fe2+, incubated for 15 min at 37 °C, initiated with 1 Attorney Docket No.29539-0796WO1 / MGH 2022-549 mM L-cysteine and quench with 20 mM N,N-diphenyl-p-phenylenediamine and 30 mM Fe3+. After quenching, samples were incubated for 20 min at 37 °C and centrifuged at 13,000 rpm for 5 min to remove precipitated protein. Absorbance was measured at 670 nm. Sulfide produced during reaction was determined using standard curve. Activity measurements were conducted in an anaerobic glovebox (mBraun, 22 °C, <1 ppm O2; monitored by Teledyne Model 311- Pxygen Gas analyzer). Cell Lines and Culturing: K562(female) and HEK293T (female) cells were obtained from ATCC and cultured in DMEM (Gibco) with 25 mM Glucose, 10% FBS (non-dialyzed, Invitrogen), 1 mM Pyruvate, 50 ug / mL Uridine, and 4mM Glutamine along with 100 U / mL Penicillin / Streptomycin under 5% CO2 at 37 C. Cell lines were checked by ATCC profiling before purchase, and were tested to ensure absence of mycoplasma every three months. Cells were passaged every two to three days. 293T cells were washed with PBS (Invitrogen) and dissociated using TrypLE (Gibco). For experiments with hypoxia, cells were placed in a Coy O2 Control Dual Hypoxia Chamber maintained at 37 C, 1% O2 and 5% CO2 with appropriate humidity control. Cloning: For overexpression assays, cDNA’s were either purchased from ORIGENE or custom synthesized from IDT. Point mutations were introduced using Gibson Assembly and custom synthesized primers from IDT. Constructs were cloned into pLYS6 bearing a Geneticin selection cassette, using the NheI and EcoRI sites. All plasmids were verified by sequencing. pMD2.G (Addgene 12259) and psPAX2 (Addgene 12260) were used for lentiviral packaging. Proliferation Assays: Cell proliferation assays were performed between 8-10 days following lentiviral infection. Cells were seeded at an initial density between 1- 2x10^5 cells / mL (K562) or 1x10^5 cells per well in a 6 well plate (293T) and cultured for 3 days in either 21% or 1% oxygen tensions. Viable cell numbers were then determined using a Vi-Cell Counter (Beckman). Lentivirus production: 2.5x10^6293T cells were seeded in 5 mL in a T25cm^2 flask (one lentivirus per flask). The following day the cells were transfected with 1 mL of transfection mixture, which contained 25 uL Lipofectamine 2000 (Thermo Fisher Scientific), 3.75 ug psPAX2, 2.5 ug pMD2.G, 5 ug of lentiviral vector of interest and Opti-MEM medium (Gibco) up to 1 mL. The mixture was incubated at room temperature for 20 min before adding it to cells and then incubating for six Attorney Docket No.29539-0796WO1 / MGH 2022-549 hours. Following the incubation, the media was replaced with fresh DMEM. Two days after transfection, media was collected, filtered through a 0.45 uM filter, and stored at -80. Infection : Cells were seeded at 5x10^6 cells / mL (K562) or 1x10^5 (293T) in 2 mL per well in a 6 well plate the day of infection. Cells were infected with virus and polybrene was added at a concentration of 1:1000 final volume (Invitrogen). Cells were incubated for 48 hours before being selected with Geneticin (500 ug / mL) (Gibco) for 48 hours. Polyacrylamide gel electrophoresis and protein immunoblotting: 2-5x10^6 K562 or 293T cells were harvested, washed in cold PBS and lysed for 15-30 minutes on ice in RIPA lysis buffer (Thermofisher) containing 1X HALT protease and phosphatase and Pierce Universal Nuclease for Cell Lysis (Thermo Fisher). Lysates were further clarified by centrifugation for 10 min at 10000xg at 4C. Supernatant was collected into fresh tubes, and protein concentration measured with Pier 660nm protein assay (Thermo Fisher). 30 ug was loaded per well in Novex Tris-Glycine 4- 20% gels (Life Technologies). Gels were run for 55 min at 180 V and subsequently transferred onto a nitrocellulose membrane, 0.45 uM (BioRad). Membranes were stained with Ponceau S to check for adequate loading. Membranes were then blocked for 1-2 hours with Odyssey Blocking Buffer (LI-COR Biosciences) at room temperature (RT). Afterwards, membranes were incubated overnight at 4 C with a solution of primary antibody diluted in Odyssey Blocking Buffer + 0.1% Tween-20. The next day, membranes were washed at RT 3 times for 3 minutes in TBS-T. The membrane was incubated with goat anti-rabbit or anti-mouse conjugated to IRDye800 or IRDye680 (LI-COR Biosciences) in a 1:1 solution of Odyssey Blocking Buffer (LI-COR Biosciences) and TBS-T. Membranes were incubated for 1 hours at room temperature, and then washed three times in TBS-T for 10 min each. Membranes were then scanned for infrared signal using the Odyssey Imaging System (LI-COR Biosciences). Band Intensities were analyzed with Image Studio LITE (LI- COR Biosciences). Mice: C57BL / 6J-shFxn mice (Chandran et al. eLife 2017;6:e30054) were generously provided by the Geshwind Laboratory at the University of California, Los Angeles. C57BL / 6NJ-Fdx2em1Murr / Murr mice were bought from the Jackson laboratory (strain No: 030192). C57BL / 6J-shFxn mice and C57BL / 6J-shFxn mice Attorney Docket No.29539-0796WO1 / MGH 2022-549 were bred, pups were weaned and genotyped at !25 days after birth. Mouse genotypes from tail biopsies were determined using real time PCR with specific probes designed for each gene (Transnetyx, Cordova, TN). All cages were provided with food and water ad-libitum. Food and water were monitored daily and replenished as needed, and cages were changed weekly. A standard light–dark cycle of !12 h light exposure was used. Two to five animals were housed per cage. Body weights were recorded regularly, and mice were humanely euthanized when they had lost 20% of peak body weight, in accordance with the American Veterinary Medical Association guidelines. For all experiments, animals were randomized on a 1:1 basis, balanced by age and sex. All animal studies were approved by the Subcommittee on Research Animal Care and the Institutional Animal Care and Use Committee of Massachusetts General Hospital. Doxycycline knockdown in Fdx / shFxn mice: The average age of the animals at the start of experiments was 2–3 months. Doxycycline treatment followed the established optimal dosing protocol; 2 mg / ml Doxycycline (Sigma) was added to the drinking water of all animals which was changed weekly. In addition, animals were injected intraperitoneally with doxycycline twice a week, starting with 5 mg / kg body weight for 10 weeks followed by 10 mg Dox / kg body at later timepoints. Accelerating rotarod test: The amount of time mice were able to maintain their position on an accelerating rotating rod (Ugo Basile) was recorded. Mice were acclimated to the experimental room for at least 30 minutes before the start of the measurements. Rotarod parameters were as follows: acceleration of 5 rpm / m and a maximum speed of 40 rpm. If mice used their body to grasp the rod (rather than walking on it) for more than 10s, this time was recorded as time of fall. Each mouse was tested three times. Grip strength test: fore and hind limbs grip strength was measured using a commercial dynamometer (Bioseb) over 3 consecutive efforts with 30 seconds of rest in between. The maximal effort (g) was used as absolute force (g) to assess whole- body strength. Mice were excluded if they refused the test (hanging < 10 seconds on 3 repeated attempts). Each mouse was tested three times. Attorney Docket No.29539-0796WO1 / MGH 2022-549 Example 1. Mutations in NFS1 / NFS-1 or FDX2 / FDX-2 partially rescue the growth defect caused by Frataxin loss in C. elegans. With the ability to cultivate Frataxin / frh-1 null C. elegans using hypoxia, we performed a forward genetic selection for rare suppressor mutations among hundreds of thousands of random EMS-induced mutations that allow frh-1 mutants to grow at a non-permissive oxygen tension (Fig. 1A). frh-1(tm5913) animals carrying a deletion in the Frataxin gene were randomly mutagenized at 1% oxygen, generating hundreds of new mutations throughout the genome in each animal. Two generations later tens of thousands of F2 progeny were transferred to 10% oxygen, and while the vast majority of animals arrested development as larvae, like the frh-1(tm5913) strain, rare animals carrying newly induced suppressor mutations reached adulthood and were isolated. These mutants still carried a deletion in the Frataxin gene. Following whole genome sequencing, we identified that suppressor mutant strains also carried missense mutations in one of two genes: the cysteine desulfurase NFS1 / nfs-1 (one allele corresponding to R244K) or the mitochondrial ferredoxin Y73F8A.27 (three independent alleles corresponding to E117K, A126V, and P127S) (Fig. 1B & Table 1). Most animals encode two ferredoxin paralogs, FDX1 and FDX2, both of which harbor 2Fe-2S clusters and are localized to mitochondria. FDX2 is required for de novo 2Fe-2S cluster biosynthesis, donating electrons to a persulfide on the scaffold protein ISCU4,12,13. On the other hand, FDX1 has long been known to be required for steroidogenesis, and more recently, been shown to be key for synthesis of heme a (for Attorney Docket No.29539-0796WO1 / MGH 2022-549 cytochrome c oxidase) and lipoyl synthase14–16. These ferredoxins cannot functionally complement each other due to sequence differences that lend specificity to target substrates13–16. Although basal metazoa such as sponges and anemones encode both paralogs, C. elegans contain only one mitochondrial ferredoxin, Y73F8A.27, and through phylogenetic analysis we inferred that C. elegans retain FDX2 and have lost FDX1. Thus, Y73F8A.27 has been renamed fdx-2. Interestingly, alignment of C. elegans FDX-2 with human FDX2 and FDX1 revealed that C. elegans FDX-2 contains key sequence features14of both FDX2 and FDX1 (Fig. 1G), raising the possibility that it has acquired new FDX1-like functionality. Notably, all identified nfs-1 and fdx-2 suppressor mutations occur in highly conserved residues also present in human NFS1 and FDX2, respectively (Fig. 1G). We used CRISPR / Cas9 to generate nfs-1 and fdx-2 mutant alleles in a clean genetic background and confirmed that nfs-1(R244K), fdx-2(E117K), or fdx-2(A126V) mutations improved the growth of frh-1 null animals at non-permissive 7-10% oxygen (Fig. 1C,D & H), which is the natural environmental oxygen tension for C. elegans nematodes17. Analyzing Frataxin null animals carrying heterozygous mutations in nfs- 1(R244K) or fdx-2(A126V) revealed that these suppressor mutations dominantly rescue a Frataxin loss of function (Fig. 1E). The point mutations in nfs-1 and fdx-2 also significantly improved the growth of frh-1 mutants at the more severe 21% oxygen (Fig. 1H). In a frh-1(wt) background the nfs-1(R244K) and fdx-2(A126V) point mutations caused no growth defect at 21% oxygen (Fig. 1I). At 1% oxygen, a permissive condition that supports the growth and fertility of Frataxin mutants11, the nfs-1 and fdx-2 suppressor mutations further rescued the frh-1 growth rate to wild- type levels (Figs. 1F & H). Taken together, these results identify suppressor mutations within the ISC assembly complex that partially rescue the growth and developmental defects of Frataxin loss. Example 2. Frataxin suppressor mutations restore levels of ISC- containing complex I. To understand how the nfs-1 and fdx-2 suppressor mutations suppress the growth defects of Frataxin null animals, we first analyzed transcriptional reporters for C. elegans stress responses. Both the hsp-6::gfp reporter for mitochondrial stress and the gst-4::gfp reporter for oxidative stress were elevated in the frh-1 mutant at 21% oxygen but not induced at 1% oxygen11. The striking rescue by hypoxia may result Attorney Docket No.29539-0796WO1 / MGH 2022-549 from increased ISC production and stability11,18, as well as rescue of downstream pathways dependent on ISCs such as the electron transport chain19,20and lipoylation15. In contrast, the fdx-2(A126V) suppressor mutation did not change the induction of either stress reporter, and the nfs-1(R244K) partially decreased hsp-6::gfp fluorescence while increasing gst-4::gfp fluorescence, arguing against a global restoration of iron sulfur clusters to wild-type levels. To identify compromised cellular processes in the Frataxin mutant that are rescued by nfs-1 or fdx-2 mutation, we performed quantitative TMT proteomics in animals incubated continuously at 1% oxygen or animals shifted from 1% to 21% oxygen for two days. Analysis of Fe-S cluster-containing proteins revealed that C. elegans Frataxin mutants shifted to 21% oxygen were depleted for 4Fe-4S proteins while 2Fe-2S proteins were largely unaffected. Following prolonged (10 day) loss of Frataxin in cell culture virtually all ISC proteins are depleted21. The C. elegans proteomics may reflect differences in half-life between 4Fe-4S- and 2Fe-2S- containing proteins after two days of ISC biogenesis interruption. Of the 23 ISC- containing proteins whose levels were depleted by at least 1.5-fold, hypoxia partially increased the levels of 21 / 23 proteins, while the fdx-2(A126V) and nfs-1(R244K) suppressor mutations restored levels of only 10 Fe-S containing proteins, which included subunits of ETC complex I and complex II. In C. elegans mutations in core subunits of complex I can destabilize the entire complex and result in loss of other individual proteins22. Indeed, we found that all complex I subunits (with the exception of the acyl carrier protein NDUFAB1) were depleted in frh-1 mutants and rescued by fdx-2 or nfs-1 suppressor mutations (Fig. 2A). We confirmed these proteomics results with western blots against complex I subunit NDUFS3 (Figs. 2B & D), which showed that at 1% or 21% oxygen Frataxin loss caused low levels of complex I that were rescued by fdx-2 or nfs-1 mutation. In fact, bypass of complex I and complex II through expression of the yeast NADH dehydrogenase NDI1, a single polypeptide which donates electrons directly to ubiquinone without proton pumping, was sufficient to partially rescue the growth of frh-1 mutants (Fig. 2C). At 21% oxygen we also observed that lipoylation – a post- translational modification dependent on Fe-S clusters – was partially restored by fdx-2 or nfs-1 suppressors (Fig. 2D). Taken together, these results suggest fdx-2 and nfs-1 point mutations partially restore Fe-S biosynthesis in the absence of Frataxin, and that Attorney Docket No.29539-0796WO1 / MGH 2022-549 the resulting increased flux through the ETC may be paramount to the rescue of animal growth and development. Example 3. Excess FDX2 is detrimental to NFS1 activity and ISC biosynthesis. To understand the mechanism by which point mutations in nfs-1 or fdx-2 partially bypass the need for Frataxin in Fe-S biosynthesis, we mapped our mutations to recent cryo-EM structures of the ISC assembly complex. In structures containing Frataxin23we observed that NFS-1(R244K) lies at the interface with Frataxin, however our suppressor mutations were isolated in a Frataxin null background. Biochemical work has revealed that both Frataxin and the ferredoxin FDX2 interact with NFS1 at overlapping binding sites. Hence, they can compete for binding to NFS16. Cryo-EM of the ISC assembly complex containing FDX2 confirmed that NFS1 binds FDX2 at the same interface as Frataxin7. Indeed, the three suppressor mutations we isolated in FDX-2 (C. elegans E117K, A126V, and P127S) lie at the NFS1-FDX2 interaction surface along with NFS-1(R244K), and corresponding human residues NFS1(R289) and FDX2(E131) form salt-bridges that stabilize the NFS1-FDX2 interaction7. We therefore hypothesized that (1) all of our suppressor mutations function similarly to perturb the NFS1-FDX2 interface, and (2) in the absence of Frataxin, ISC synthesis is partially compromised due to constitutive binding of FDX2 to NFS1, and our suppressor mutations function to increase levels of free NFS1. Our model is further supported by the following experiments employing C. elegans genetics, in vitro biochemistry, and mammalian cell culture. First, a double mutant of the C. elegans suppressor mutations nfs-1(R244K); fdx-2(A126V) in a wild- type background was synthetic sterile and grew slowly (Fig. 3A), despite each suppressor mutation on their own having no growth defect (Fig. 1I). The nfs- 1(R244K); fdx-2(A126V) double mutant also displayed reduced lipoic acid staining, consistent with an additive weakening of the NFS1-FDX2 interaction and reduced ISC biosynthesis. Interestingly, both the slow growth, sterility, and low lipoic acid staining of the nfs-1(R244K); fdx-2(A126V) double mutant was rescued by hypoxia (Fig 3A), similar to other ISC-deficient mutants and further supporting a loss of ISC biosynthesis. Together, these results are consistent with the nfs-1 and fdx-2 suppressor mutations partially weakening NFS-1 / FDX-2 binding. Attorney Docket No.29539-0796WO1 / MGH 2022-549 To test whether FDX2 would also inhibit ISC synthesis in vivo, we over- expressed wild-type FDX2 in K562 and 293T human cells lines. We observed that overexpression of wild-type FDX2 caused a growth defect in both cell lines in normoxia (21% O2) that was rescued under continuous hypoxia (1% O2) (Fig. 3B). In contrast, other members of iron sulfur cluster machinery, such as NFS1 and ISCU, did not produce adverse effects when over-expressed11. Immunoblotting of K562 and 293T lysates from cells over-expressing GFP or FDX2 revealed that cells over- expressing FDX2 exhibited biochemical phenotypes of iron sulfur cluster deficiency: we observed reduction in lipoate synthesis due to loss of the ISC containing LIAS protein (Fig. 3C), and loss of ISC containing respiratory chain complexes I, II, and in the case of K562 cells, IV (Fig. 3C). Many of these defects were partially or fully restored by growth under low oxygen tension (Figs. 3B&C). These results validate our in vitro experiments and suggest that Frataxin loss in mammalian systems may also cause ISC defects through stoichiometric excess of FDX2. To confirm that FDX2 inhibits ISC production through excess NFS1 binding, we over-expressed mutant versions of FDX2. First, we found that over-expression of an FDX2 variant with a mutated iron sulfur cluster binding site (C117A) did not cause a reduction in growth in K562 cells nor a reduction in ISC production (Supplementary Figs. 3A&B). This is consistent with apo-FDX2 being unable to bind the NFS1- ISD11-ACPeccomplex24. Importantly, the C117A variant was expressed to levels equivalent to wild type over-expression. These results indicated that FDX2 must be functional in its capacity as an electron donor to cause a growth defect when over- expressed. We also tested whether over-expression of the homologous mammalian FDX2 mutants to those identified by our C. elegans suppression genetics (Table 1) also conferred a mammalian cell culture growth phenotype. We found that over- expression of the E131K mutant did not cause a growth defect in K562 cells nor a depletion of iron sulfur cluster levels as seen through immunoblotting of lipoic acid. Over-expression of the A140V mutant also failed to deplete iron sulfur cluster levels, but did cause a growth defect possibly through alternative mechanisms. These results support our hypothesis that the FDX2 mutants isolated in our screen weaken binding to the ISC assembly complex. Attorney Docket No.29539-0796WO1 / MGH 2022-549 Example 4. Partial loss of wild type FDX2 activity suppresses Frataxin mutants and rescues neurological defects associated with Frataxin loss. Inspired by these findings, we reasoned that simply lowering the amount of FDX2 by two-fold may boost ISC production in a Frataxin mutant, and so we attempted to rescue Frataxin loss by removing one copy of wild-type fdx-2 in C. elegans. We used CRISPR / Cas9 to generate a null allele of fdx-2 that carried an 8 bp deletion resulting in a frameshift (Fig. 4A). These animals were homozygous lethal (Fig. 4E), but the heterozygotes of fdx-2(null) / fdx-2(+) were viable and had a wild- type growth rate at all oxygen tensions (Fig. 4B). Similarly, humans heterozygous for disease-causing mutations in FDX2 are healthy25,26and the gnomAD database reveals that humans are not intolerant of carrying one FDX2 loss-of-function allele27. Introduction of the heterozygous fdx-2(null) / fdx-2(+) into frh-1(tm5913) partially rescued the growth and developmental defects of Frataxin mutants at all oxygen tensions, and was not significantly different from the homozygous fdx-2(A126V) suppressor mutations isolated in our screen (Fig. 4C). These results confirm our fdx-2 suppressors were dominant due to haploinsufficiency and strongly support our model that in an animal bearing a Frataxin mutation, lowering the amount of NFS1 bound by FDX2 can restore ISC biosynthesis. To test whether a 50% decrease in Fdx2 gene dosage could also suppress the movement disorders of a Frataxin mouse mutant, we asked whether lowering Fdx2 levels would improve any disease phenotypes in a mouse model of Friedreich’s ataxia. We obtained mice from The Jackson Laboratory carrying a loss-of-function Fdx2em1Murrallele generated by CRISPR / Cas9. As in C. elegans and humans, murine Fdx2 homozygotes are embryonic lethal, but heterozygotes are viable and appear healthy. We confirmed through western blotting that Fdx2em1Murrheterozygous mice have approximately 50% wild-type levels of Fdx2 protein in the brain (Fig. 4F), validating the use of this mutant. Fdx2 heterozygotes were crossed with mice heterozygous for doxycycline- inducible shRNA against Frataxin (shFxn, GGATGGCGTGCTCACCATTAA (SEQ ID NO:9), a genetic model that recapitulates the cardinal triad of Friedreich’s ataxia, namely, ataxia, cardiomyopathy, and diabetes28. From this single genetic cross we generated four genotypes of F1 hybrid mice: wild type, Fdx2 / +, shFxn, and shFxn; Fdx2 / +. We began Dox treatment at 3 months of age to knockdown Fxn and assessed Attorney Docket No.29539-0796WO1 / MGH 2022-549 body weight and survival at weekly intervals and motor defects using grip strength and rotarod tests following 12- and 15-weeks of Dox treatment. At both time points, we observed improvement of ataxia phenotypes in the shFxn; Fdx2 / + double mutants (Fig. 4D & G). Depletion of Fdx2 had no effect on body weight or life span in the shFxn background (Figs. 4G & H), reminiscent of the effects of hypoxia in this model11. We conclude that lowering Fdx2 levels partially rescues the neurological defects associated with Frataxin loss. Example 5. FRDA Knockdown in human cells We have reported previously that when FXN is knocked out in human cells, there is a loss of protein lipoylation (a pathway dependent on Fe-S clusters) and a growth defect in normoxia (21%) (Ast Meisel Cell 2019). ASOs are used to knockdown partially the expression of FDX2 and then assays are performed to determine whether CRISPR knockout of FXN still leads to lipoylation and growth defects or whether they are alleviated. Alternatively, lentiviral-based delivery of shRNA targeting FXN is also used. Table 1. List of mutations reported in this study isolated from Frataxin suppressor screens * NP_001384335.1, SEQ ID NO:2 References 1. Fox, N. G., Das, D., Chakrabarti, M., Lindahl, P. A. & Barondeau, D. P. Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe–S Assembly Complex. Biochemistry 54, 3880–3889 (2015). 2. Tsai, C.-L. & Barondeau, D. P. Human frataxin is an allosteric switch that activates the Fe-S cluster biosynthetic complex. Biochemistry 49, 9132–9139 (2010). 3. Schulz, V. et al. Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2. Nat. Commun. 15, 3269 (2024). Attorney Docket No.29539-0796WO1 / MGH 2022-549 4. Gervason, S. et al. Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin. Nat. Commun. 10, 3566 (2019). 5. Campuzano, V. et al. Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271, 1423–1427 (1996). 6. Uzarska, M. A. et al. During FeS cluster biogenesis, ferredoxin and frataxin use overlapping binding sites on yeast cysteine desulfurase Nfs1. J Biological Chem 298, 101570 (2021). 7. Steinhilper, R., Freibert, S.-A., Kaltwasser, S., Lill, R. & Murphy, B. J. Structural evidence for two-stage binding of mitochondrial ferredoxin 2 to the core iron-sulfur cluster assembly complex. bioRxiv 2024.02.19.580858 (2024) doi:10.1101 / 2024.02.19.580858. 8. Andreini, C., Banci, L. & Rosato, A. Exploiting Bacterial Operons To Illuminate Human Iron-Sulfur Proteins. J. Proteome Res. 15, 1308–1322 (2016). 9. Lill, R. & Freibert, S.-A. Mechanisms of Mitochondrial Iron-Sulfur Protein Biogenesis. Annu. Rev. Biochem. 89, 1–29 (2020). 10. Maio, N. & Rouault, T. A. Outlining the Complex Pathway of Mammalian Fe-S Cluster Biogenesis. Trends Biochem. Sci. 45, 411–426 (2020). 11. Ast, T. et al. Hypoxia Rescues Frataxin Loss by Restoring Iron Sulfur Cluster Biogenesis. Cell 177, 1507-1521.e16 (2019). 12. Kim, J. H., Frederick, R. O., Reinen, N. M., Troupis, A. T. & Markley, J. L. [2Fe-2S]-Ferredoxin Binds Directly to Cysteine Desulfurase and Supplies an Electron for Iron–Sulfur Cluster Assembly but Is Displaced by the Scaffold Protein or Bacterial Frataxin. J. Am. Chem. Soc. 135, 8117–8120 (2013). 13. Webert, H. et al. Functional reconstitution of mitochondrial Fe / S cluster synthesis on Isu1 reveals the involvement of ferredoxin. Nat. Commun. 5, 5013 (2014). 14. Schulz, V. et al. Functional spectrum and specificity of mitochondrial ferredoxins FDX1 and FDX2. Nat. Chem. Biol. 19, 206–217 (2023). 15. Joshi, P. R., Sadre, S., Guo, X. A., McCoy, J. G. & Mootha, V. K. Lipoylation is dependent on the ferredoxin FDX1 and dispensable under hypoxia in human cells. J. Biol. Chem. 299, 105075 (2023). Attorney Docket No.29539-0796WO1 / MGH 2022-549 16. Dreishpoon, M. B. et al. FDX1 regulates cellular protein lipoylation through direct binding to LIAS. J. Biol. Chem. 299, 105046 (2023). 17. Gray, J. M. et al. Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue. Nature 430, 317–322 (2004). 18. Fernandez, S. et al. Yeast cells depleted of the frataxin homolog Yfh1 redistribute cellular iron: Studies using Mössbauer spectroscopy and mathematical modeling. J. Biol. Chem. 298, 101921 (2022). 19. Jain, I. H. et al. Hypoxia as a therapy for mitochondrial disease. Science 352, 54–61 (2016). 20. Jain, I. H. et al. Genetic Screen for Cell Fitness in High or Low Oxygen Highlights Mitochondrial and Lipid Metabolism. Cell 181, 716-727.e11 (2019). 21. Ast, T. et al. METTL17 is an Fe-S cluster checkpoint for mitochondrial translation. Mol. Cell 84, 359-374.e8 (2024). 22. Meisel, J. D. et al. Hypoxia and intra-complex genetic suppressors rescue complex I mutants by a shared mechanism. Cell (2024) doi:10.1016 / j.cell.2023.12.010. 23. Fox, N. G. et al. Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism. Nat Commun 10, 2210–8 (2019). 24. Cai, K., Tonelli, M., Frederick, R. O. & Markley, J. L. Human Mitochondrial Ferredoxin 1 (FDX1) and Ferredoxin 2 (FDX2) Both Bind Cysteine Desulfurase and Donate Electrons for Iron–Sulfur Cluster Biosynthesis. Biochemistry 56, 487–499 (2017). 25. Gurgel-Giannetti, J. et al. A novel complex neurological phenotype due to a homozygous mutation in FDX2. Brain 141, 2289–2298 (2018). 26. Spiegel, R. et al. Deleterious mutation in FDX1L gene is associated with a novel mitochondrial muscle myopathy. Eur. J. Hum. Genet. 22, 902–906 (2014). 27. Chen, S. et al. A genomic mutational constraint map using variation in 76,156 human genomes. Nature 625, 92–100 (2024). 28. Chandran, V. et al. Inducible and reversible phenotypes in a novel mouse model of Friedreich’s Ataxia. eLife 6, e30054 (2017). 29. Yoon, H. et al. Frataxin-bypassing Isu1: characterization of the bypass activity in cells and mitochondria. Biochem. J. 459, 71–81 (2014). Attorney Docket No.29539-0796WO1 / MGH 2022-549 30. Das, D., Patra, S., Bridwell-Rabb, J. & Barondeau, D. P. Mechanism of frataxin “bypass” in human iron-sulfur cluster biosynthesis with implications for Friedreich’s ataxia. Journal of Biological Chemistry 294, 9276–9284 (2019). 31. Belbellaa, B., Reutenauer, L., Messaddeq, N., Monassier, L. & Puccio, H. High Levels of Frataxin Overexpression Lead to Mitochondrial and Cardiac Toxicity in Mouse Models. Mol. Ther. Methods Clin. Dev. 19, 120–138 (2020). 32. Huichalaf, C. et al. In vivo overexpression of frataxin causes toxicity mediated by iron-sulfur cluster deficiency. Mol. Ther. Methods Clin. Dev. 24, 367– 378 (2022). 33. Boniecki, M. T., Freibert, S. A., Mühlenhoff, U., Lill, R. & Cygler, M. Structure and functional dynamics of the mitochondrial Fe / S cluster synthesis complex. Nat. Commun. 8, 1287 (2017). 34. Baik, A. H. et al. Oxygen toxicity causes cyclic damage by destabilizing specific Fe-S cluster-containing protein complexes. Mol. Cell 83, 942-960.e9 (2023). 35. Itoh, Y. et al. Structure of the mitoribosomal small subunit with streptomycin reveals Fe-S clusters and physiological molecules. eLife 11, e77460 (2022). 36. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974). 37. Ghanta, K. S. & Mello, C. C. Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans. Genetics 216, 643– 650 (2020). 38. Lehrbach, N. J., Ji, F. & Sadreyev, R. Next‐Generation Sequencing for Identification of EMS‐Induced Mutations in Caenorhabditis elegans. Curr Protoc Mol Biology 117, 7.29.1-7.29.12 (2017). OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.29539-0796WO1 / MGH 2022-549 WHAT IS CLAIMED IS:

1. A method of treating a subject having a disorder associated with a mutation in the frataxin (FXN) gene and / or having reduced expression of frataxin protein, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of human ferredoxin 2 (FDX2).

2. The method of claim 1, wherein the inhibitor of FDX2 comprises an inhibitory nucleic acid or a CRISPR / Cas9 complex targeting the FDX2 gene.

3. The method of claim 2, wherein the inhibitory nucleic acid comprises a small hairpin RNA (shRNA), antisense oligonucleotide, small nuclear RNA (snRNA), small interfering RNA (siRNA), or a single guide RNA (sgRNA) targeting a FDX2 nucleic acid.

4. The method of claim 3, wherein the inhibitory nucleic acid comprises a small hairpin RNA (shRNA) targeting the FDX2 nucleic acid.

5. The method of claim 3, wherein the inhibitory nucleic acid comprises an antisense oligonucleotide targeting the FDX2 nucleic acid.

6. The method of claim 2, wherein the inhibitor comprises a CRISPR / Cas9 complex targeting the FDX2 gene.

7. The method of any of claims 1-6, wherein the subject has Friedreich ataxia (FRDA).

8. An inhibitor of human ferredoxin 2 (FDX2) for use in a method of treating a subject having a disorder associated with a mutation in the frataxin (FXN) gene and / or having reduced expression of frataxin protein, the method comprising administering to the subject a therapeutically effective amount of the inhibitor of human ferredoxin 2 (FDX2).

9. The inhibitor for the use of claim 8, wherein the inhibitor of FDX2 comprises an inhibitory nucleic acid or a CRISPR / Cas9 complex targeting the FDX2 gene.

10. The inhibitor for the use of claim 9, wherein the inhibitory nucleic acid comprises a small hairpin RNA (shRNA), antisense oligonucleotide, small nuclear RNAAttorney Docket No.29539-0796WO1 / MGH 2022-549 (snRNA), small interfering RNA (siRNA), or a single guide RNA (sgRNA) targeting a FDX2 nucleic acid.

11. The inhibitor for the use of claim 10, wherein the inhibitory nucleic acid comprises a small hairpin RNA (shRNA) targeting the FDX2 nucleic acid.

12. The inhibitor for the use of claim 10, wherein the inhibitory nucleic acid comprises an antisense oligonucleotide targeting the FDX2 nucleic acid.

13. The inhibitor for the use of claim 9, wherein the inhibitor comprises a CRISPR / Cas9 complex targeting the FDX2 gene.

14. The inhibitor for the use of any of claims 8-13, wherein the subject has Friedreich ataxia (FRDA).

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