TREATING FRIEDREICH’S ATAXIA WITH DIMETHYL FUMARATE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of US 62/665,438, filed May 1, 2018, incorporated by reference in its entirety for all purposes.

CROSS-REFERENCE TO SEQUENCE LISTING

[0002] Sequences disclosed in txt file named PCT513854ST25 of 4 kbytes created April 30, 2019 are incorporated by reference.

BACKGROUND

[0003] Friedriech's ataxia (FA) is caused by inheritance of GAA expansions and reduced expression the mitochondrial protein frataxin (FXN). FA is an ultimately lethal

neurodegenerative disease for which there is no current approved therapy (Albano et al , 2002). All pathophysiological consequences, severity and age of onset of FA are directly related to the extent of FXN deficiency, the greater the FXN deficiency, worse the outcome (Delatycki et al , 1999; Condo et al, 2006; Lu et al , 2007; Marmolino, 2011). Common symptoms associated with this disease include loss of muscle coordination, cardiomyopathy, hearing defect and diabetes (Rotig et al, 1997; Puccio et al, 2001).

[0004] FXN supports mitochondrial iron-sulfur cluster synthesis (Bridwell-Rabb et al, 2014; Cory et al, 2017). Recently we demonstrated that frataxin deficiency in FA patient cells, FA mice, and FA human patients causes a mitochondrial biogenesis defect (Jasoliya et al., 2017), so it is possible that a primary defect in iron-sulfur clusters (which are essential for several mitochondrial enzyme complexes) causes the mitochondrial biogenesis defect that ultimately triggers the disease.

SUMMARY OF THE CLAIMED INVENTION

[0005] The invention provides a method of treating a human subject having Friedreich’ s ataxia comprising administering to the subject an effective regime of dimethyl fumarate to increase the level of frataxin in the subject by at least 50%. Optionally, the dimethyl fumarate is administered orally. Optionally, the level of frataxin in the subject is increased by 50-100%. Optionally, the dimethyl fumarate is administered at 100-1000 mg per day. Optionally, the dimethyl fumarate is administered at 120 mg BID for a week and 240 mg BID thereafter. Optionally, the dimethyl fumarate is administered at 500-1000 mg per day for a period of at least six months. Optionally, the dimethyl fumarate is administered at 720 mg/day for a period of at least six months. Optionally, the dimethyl fumarate is administered at a dose of 700-1000 mg/day for a period of at least six months. Optionally, the dimethyl fumarate is administered at 100-400 mg per day for period of at least six months. Optionally, the method further comprises monitoring the level of frataxin in the subject. Optionally, the level of frataxin is measured by frataxin mRNA. Optionally, the level of frataxin is measured by frataxin protein. Optionally, the level of frataxin is measured in PBMCs. Optionally, the level of frataxin is measured in whole blood. Optionally, the effective regime is changed responsive to the measured level of frataxin. Optionally, the dose or frequency is increased if the level of frataxin is less than 50% of the pretreatment level. Optionally, the monitoring is performed quarterly. Optionally, the monitoring is performed responsive to a change in symptoms of the patient. Optionally, the subject is a fetus in utero. Optionally, the subject is asymptomatic. Optionally, the subject is symptomatic. Optionally, the administration at least partially reverses a deficit in at least one symptom of Friedreich’ s ataxia. Optionally, the administering is performed for the life of the patient.

DEFINITIONS

[0006] The term“Friedreich’ s ataxia” and“FRDA” interchangeably to an autosomal recessive congenital ataxia caused by a mutation in gene FXN (formerly known as X25) that codes for frataxin, located on chromosome 9. The genetic basis for FRDA involves GAA trinucleotide repeats in an intron region of the gene encoding frataxin. This segment is normally repeated 5 to 33 times within the FXN gene. In people with Friedreich ataxia, the GAA segment is repeated 66 to more than 1,000 times. People with GAA segments repeated fewer than 300 times tend to have a later appearance of symptoms (after age 25) than those with larger GAA trinucleotide repeats. The presence of these repeats results in reduced transcription and expression of the gene. Frataxin is involved in regulation of mitochondrial iron content. The mutation in the FXN gene causes progressive damage to the nervous system, resulting in symptoms ranging from gait disturbance to speech problems; it can also lead to heart disease and diabetes. The ataxia of Friedreich's ataxia results from the degeneration of nerve tissue in the spinal cord, in particular sensory neurons essential (through connections with the cerebellum) for directing muscle movement of the arms and legs. The spinal cord becomes thinner and nerve cells lose some of their myelin sheath (the insulating covering on some nerve cells that helps conduct nerve impulses). A subject with FRDA may exhibit one or more of the following symptoms: muscle weakness in the arms and legs, loss of coordination, vision impairment, hearing impairment, slurred speech, curvature of the spine (scoliosis), high plantar arches (pes cavus deformity of the foot), carbohydrate intolerance, diabetes mellitus, heart disorders (e.g., atrial fibrillation, tachycardia (fast heart rate) and hypertrophic cardiomyopathy). A subject with FRDA may further exhibit involuntary and/or rapid eye movements, loss of deep tendon reflexes, loss of extensor plantar responses, loss of vibratory and proprioceptive sensation, cardiomegaly, symmetrical hypertrophy, heart murmurs, and heart conduction defects. Pathological analysis may reveal sclerosis and degeneration of dorsal root ganglia, spinocerebellar tracts, lateral corticospinal tracts, and posterior columns.

[0007] Human frataxin is known to exist in three isoforms in humans, Accession numbers for nucleic acid forms are provided by GenBank Accession Nos. NM_000l44.4 (isoform 1); NM_181425.2 (isoform 2); NM_00l 161706.1 (isoform 3)). Accession numbers for protein forms are provided by GenBank Accession Nos. NP_000l35.2 (isoform 1); NP_852090.l (isoform 2); NP_00l 155178.1 (isoform 3)). Isoform 1 is the canonical form.

[0008] The term“alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e. unbranched) or branched chain, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. Ci-Cio means one to ten carbons). Examples of saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups

includevinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l ,4- pentadienyl), ethynyl, 1- and 3-prop ynyl, 3-butynyl, and the higher homologs and isomers.

An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-0-).

[0009] The term“alkylene” by itself or as part of another substituent means a divalent radical derived from an alkyl, as exemplified by -CH2CH2CH2CH2-, and further includes those groups described below as“heteroalkylene.” Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A“lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms and often 4 or fewer carbon atoms.

[0010] The term“heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of at least one carbon atoms and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be

quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include-CPb-CPb-O-CPb, -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ - CH ₂ -N(CH ₃ )-CH ₃ , -CH ₂ -S-CH ₂ -CH ₃ , -CH ₂ -CH ₂ ,-S(0)-CH ₃ , -CH ₂ -CH ₂ -S(0) ₂ -CH ₃ , - CH=CH-0-CH ₃ , -Si(CH ₃ ) ₃ , -CH ₂ -CH=N-OCH ₃ , -CH=CH-N(CH ₃ )-CH ₃ , 0-CH ₃ , -0-CH ₂ - CH _3, and -CN. Up to two heteroatoms may be consecutive, such as, for example, -CH ₂ -NH- OCH ₃ . Similarly, the term“heteroalkylene” by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH ₂ -CH ₂ -S- CH ₂ -CH ₂ - and -CH ₂ -S-CH ₂ -CH ₂ -NH-CH ₂ -. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(0)2R’ - represents both -C(0)2R’ - and -R’C(0) ₂ -. AS described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(0)R’ , - C(0)NR’, -NR’R , -OR’, -SR , and/or -SO ₂ R’. Where“heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -NR’R or the like, it will be understood that the terms heteroalkyl and -NR’R’’ are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR’R or the like.

[0011] The terms“cycloalkyl” and“heterocycloalkyl,” by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of“alkyl” and “heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, l-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1 -(l,2,5,6-tetrahydropyridyl), l-piperidinyl, 2-piperidinyl, 3- piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0012] Certain agents of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. The agents of the present invention do not include those which are known to be too unstable to synthesize and/or isolate.

BRIEF DESCRIPTION OF THE FIGURES

[0013] Fig. 1. DMF dose dependently increases FXN expression in vitro in various patient derived lymphoblast cell models. Cells were treated with 0.01% DMSO vehicle, 1,3,10 or 30 mM DMF for 24hr. RNA was extracted and FXN expression was measured by qRT-PCR as FXN normalized to Actin. Vehicle, n=50; ImM, n=3; 3mM, n=20; IOmM, n=24; 30mM, n=42. Bars represent averages ± standard deviations (p<0.05*)

[0014] Fig. 2. DMF dose dependently increases FXN expression in vivo in cerebellar tissue of YG8 mice. Mice were IP dosed with 3, 5 and 10 mg/kg DMF for 7 day. Protein was extracted from Cerebellar tissue and measured using Western Blot analysis of FXN/Tubulin. (n=4; each group) Bars represent averages ± standard deviations (p<0.05*)

[0015] Fig. 3. DMF increases FXN expression in peripheral blood mononuclear cells (PBMC’s) when dosed at 480 mg/day in vivo in humans. 27 MS patients were recruited and 14 were treated with DMF and 13 with FTY for 3 months. Blood was drawn post treatment, PBMCs were isolated and FXN mRNA expression was measured pre and post treatment by qRT-PCR. Baseline values of FXN/mRNA expression were set at 1 and 3 months values are shown relative to baseline values. CNTRL = Controls (n=l2); DMF = Dimethylfumarate (n=l4); FTY = Fingolimod (n=l3);. Bars represent median ± error standard mean (p<0.05*)

[0016] Fig. 4. DMF induces both increased transcription initiation and procession at GAA repeat pause site of FXN. FXN pre-mRNA transcription was traced my qRT-PCR in healthy lymphoblast, patient derived lymphoblast and 24 hr treatment of 30uM DMF in patient lymphoblast cells. Bars represent averages ± standard deviations (n=3, each group; p<0.05*) [0017] Fig. 5. DMF reduces R-loop enrichment at GAA repeat pause site of FXN gene. R- loops were enriched using antibody specific for RNA-DNA hybrid and fold enrichment measured relative to input DNA by qPCR in healthy lymphoblast, patient derived lymphoblast and 24 hr treatment of 30uM DMF in patient lymphoblast cells. Bars represent averages ± standard deviations ( n=6, each group, p<0.05*)

[0018] Fig. 6. DMF increases mitochondrial biogenesis in patient derived fibroblast cells. Cells were treated with 0.01% DMSO vehicle, 1,3,10 or 30 mM DMF for 48 hr.

Mitochondrial DNA copy number was measured by q-PCR analysis as ratio of mitochondrial DNA over nuclear DNA; mt-TLl/B2M. Vehicle, n=9; 3mM, n=3; 10mM, n=5; 30mM, n=9; Healthy, n=3. Bars represent averages ± standard deviations ( p<0.05*)

DETAILED DESCRIPTION

[0019] The present application provides methods of treating Friedreich’ s ataxia using DMF, analogs or pharmaceutically acceptable salts thereof. The methods are based in part on human clinical data showing a rapid and substantial increase in frataxin levels in response to oral dimethyl fumarate.

I. Subjects amenable to treatment

[0020] Subjects amenable to treatment are humans homozygous for a mutation (a GAA expansion or point mutation) that inhibits or reduces the expression levels of frataxin. Such subjects can be treated in utero, after birth but before symptoms develop, and/or after symptoms develop. For subjects homozygous for a mutation in the frataxin gene that results in insufficient expression levels of the frataxin polypeptide, the risk of developing symptoms of Friedreich's ataxia generally increases with age. Accordingly, in asymptomatic subjects homozygous for a mutation in the frataxin gene that results in insufficient expression levels of the frataxin polypeptide, in certain embodiments, prophylactic application is contemplated for subjects over 5 years of age, for example, in subjects over about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years of age. Subjects with late or very late onset of disease, as described above can also be treated.

[0021] In some embodiments, the subject is asymptomatic but has familial and/or genetic risk factors for developing Friedreich's ataxia. In asymptomatic patients, treatment can begin at any age (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years of age, or older).

[0022] In some embodiments, the subject is exhibiting symptoms of Friedreich's ataxia, for example, muscle weakness in the arms and legs, loss of coordination, loss of deep tendon reflexes, loss of extensor plantar responses, loss of vibratory and proprioceptive sensation, vision impairment, involuntary and/or rapid eye movements, hearing impairment, slurred speech, curvature of the spine (scoliosis), high plantar arches (pes cavus deformity of the foot), carbohydrate intolerance, diabetes mellitus, and heart disorders (e.g., atrial fibrillation, tachycardia (fast heart rate), hypertrophic cardiomyopathy, cardiomegaly, symmetrical hypertrophy, heart murmurs, and heart conduction defects).

[0023] In some embodiments, the subject does not suffer from a disease condition other than Friedreich's ataxia. For example, the subject does not suffer from a disease condition other than Friedreich's ataxia that can be or is treated by the active agent. In some embodiments, the subject does not have or is not diagnosed with multiple sclerosis or psoriasis. In some embodiments, the subject does not have or is not diagnosed with a neurological disease other than Friedreich’ s ataxia.

II. Dimethyl fumarate and analogs

[0024] Dimethyl fumarate (CAS Number: 624-49-7) is a preferred compound for treatment. Analogs of dimethyl fumarate, which can also be used, are defined by formula I.

or a pharmaceutically acceptable salt thereof;

wherein Rl and R2 are independently selected from -CFF- _n E _n, where n is 1-3, OH, 0-, and (Cl-8) alkoxy (branched or unbranched), provided that at least one of Rl and R2 is (Cl-8) alkoxy. It is also to be understood that the present invention is considered to include cis and trans isomers, stereoisomers as well as optical isomers, e.g. mixtures of enantiomers as well as individual enantiomers and diastereomers, which arise as a consequence of structural asymmetry in selected compounds of the present series. Formula I compounds include trans (fumarate) and cis (maleate) isomers. E is an electron withdrawing group. Examples of electron withdrawing groups include N02, N(R2), N(R3)+, N(H3)+, S03H, -S03R’, - S(02)R’ (sulfone), -S(0)R’ (sulfoxide), -S(02)NH2 (sulfonamide), -S02NHR’, -S02NR’2, - PO(OR’)2, -P03H2, PO(NR’2)2, pyridinyl (2-, 3-, 4-), pyrazolyl, indazolyl, imidazolyl, thiazolyl, benzothiazolyl, oxazolyl, benzimidazolyl, benzoxazolyl, isoxazolyl,

benzisoxazolyl, triazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinazolinyl, pyrimidinyl, a 5 or 6-membered heteroaryl with a C-N double bond optionally fused to a 5 or 6 membered heteroaryl, pyridinyl N-oxide, -CºN, CX’3, C(0)X’, COOH, COOR’, C(0)R’,

C(0)NH2, C(0)NHR’ , C(0)NR’2, C(0)H, -P(0)(0R’)0R” and X’, wherein X’ is independently halogen (e.g. F, Cl, Br, I) and R, R’ and R’’ are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or similar Substituents (e.g. a substituent group, a size limited substituent group or a lower substituent group). Examples of dimethyl fumarate analogs include mo no methyl fumarate (MMF), monomethyl maleate, monoethyl fumarate, monoethyl maleate, monobutyl fumarate, monobutyl maleate, monooctyl fumarate, monoctyl maleate, mono (phenylmethyl) fumarate, mono

(phenylmethyl) maleate, mono (2-hydroxypropyl) fumarate, mono (2-hydroxypropyl) maleate, mono (2-ethylhexyl) fumarate, mono (2-ethylhexyl) maleate, dimethylfumarate, dimethyl maleate, diethyl fumarate, diethyl maleate, dipropyl fumarate, dipropyl maleate, diisopropyl fumarate, diisopropyl maleate, dibutyl fumarate, dibutyl maleate, diisobutyl fumarate, diisobutyl maleate, diheptyl fumarate, diheptyl maleate, bis (2-ethylhexyl) fumarate, bis (2-ethylhexyl) maleate, (-)-Dimenthyl fumarate, (-)-Bis ((S)-l- (ethoxycarbonyl)ethyl) fumarate, (-)-Bis ((S)-l-(ethoxycarbonyl)ethyl) maleate, Bis (2- trifluoroethyl) fumarate, Bis (2-trifluoroethyl) maleate.

III. Formulation and Administration of Active Agents A. Formulation

[0025] DMF and analogs described herein and/or pharmaceutically acceptable salts thereof can be administered orally, parenterally, (intravenously (IV), intramuscularly (IM), depo-IM, subcutaneously (SQ), and depo-SQ), sublingually, intranasally (e.g., inhalation, nasal mist or drops), intrathecally, topically, transmucosally, bucally, sublingually, ionophoretically or rectally.

[0026] Compositions are provided that contain therapeutically effective amounts of DMF or analogs. DMF or analogs are preferably formulated into suitable pharmaceutical preparations such as tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration.

[0027] DMF or analogs and/or pharmaceutically acceptable salts thereof can be administered in the“native” form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically, i.e., effective in the present method(s). Salts, esters, amides, prodrugs and other derivatives of the active agents can be prepared using standard procedures described, for example, by March (1992) Advanced Organic Chemistry; Reactions,

Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience. Prodrugs of the agents readily undergo chemical changes under physiological conditions to provide the agents of the present invention. Conversion usually occurs after administration to a patient.

[0028] Methods of formulating such derivatives are known. For example, the disulfide salts of a number of delivery agents are described in WO 2000/059863 which is incorporated herein by reference. Similarly, acid salts of agents can be prepared from the free base using conventional methodology that typically involves reaction with a suitable acid. Generally, the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto. The resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent. Suitable acids for preparing acid addition salts include, but are not limited to both organic acids, e.g., acetic acid, carboxylic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, suberic acid, lactic acid, benzene sulfonic acid, p-tolylsulfonic acid, arginine, glucuronic acid, galactunoric acid phthalic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid isobutyric, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like (see, e.g., Berge et a , J. Pharm. Sci. 66, 1-19 (1977). For the preparation of salt forms of basic drugs, the pKa of the counterion is preferably at least about 2 pH lower than the pKa of the drug. Similarly, for the preparation of salt forms of acidic drugs, the pKa of the counterion is preferably at least about 2 pH higher than the pKa of the drug. This permits the counterion to bring the solution's pH to a level lower than the pHmax to reach the salt plateau, at which the solubility of salt prevails over the solubility of free acid or base. The generalized rule of difference in pKa units of the ionizable group in the active pharmaceutical ingredient (API) and in the acid or base is meant to make the proton transfer energetically favorable. When the pKa of the API and counterion are not significantly different, a solid complex may form but may rapidly disproportionate (i.e., break down into the individual entities of drug and counterion) in an aqueous environment.

[0029] Preferably, the counterion is a pharmaceutically acceptable counterion. Suitable anionic salt forms include, but are not limited to acetate, benzoate, besylate, benzylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate (embonate), phosphate and diphosphate, salicylate and disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like. Suitable cationic salt forms include, but are not limited to aluminum, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, zinc, and the like.

[0030] Preparation of esters typically involves functionalization of hydroxyl and/or carboxyl groups that are present within the molecular structure of the active agent. In certain embodiments, the esters are typically acyl-substituted derivatives of free alcohol groups, i.e., moieties that are derived from carboxylic acids of the formula RCOOH where R is alky, and preferably is lower alkyl. Esters can be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.

[0031] Amides can also be prepared using techniques described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine.

[0032] About 1 to 1000 mg of a compound or mixture of the one or more active agents or a physiologically acceptable salt or ester is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, and so forth, in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained. The compositions are preferably formulated in a unit dosage form, each dosage containing from about 1-1000 mg, 2-800 mg, 5-500 mg, 10-400 mg, 50-200 mg, e.g., about 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1000 mg of the active ingredient. The term "unit dosage from" refers to physically discrete units suitable as unitary (i.e., single) dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0033] To prepare compositions, the one or more active agents is mixed with a suitable pharmaceutically acceptable carrier. Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion, or the like. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and may be empirically determined.

[0034] Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to be suitable for the particular mode of administration. In addition, the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action. Dimethyl fumarate or analogs or pharmaceutically acceptable salts thererof may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

[0035] For oral administration, dimethyl fumarate, analogs and pharmaceutically acceptable salts thereof can be provided in a formulation that protects the active compound from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

[0036] Oral compositions generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.

[0037] Tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

[0038] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, medicated chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

[0039] The active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action.

[0040] Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required.

[0041] Suitable carriers for intravenous administration include physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known for example, as described in U.S. Pat. No. 4,522,811. [0042] Dimethyl fumarate, analogs and pharmaceutically acceptable salts thereof may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Controlled or delayed release is a mechanism of formulation to release a drug over an extended time. Use of controlled release formulation may reduce the frequency of administration, reduce fluctuations in blood concentration and protect the gastrointestinal tract from side effects. The active compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time-release formulations or coating. Such carriers include controlled release formulations (also known as modified, delayed, extended or sustained release or gastric retention dosage forms, such as the Depomed GR™ system in which agents are encapsulated by polymers that swell in the stomach and are retained for about eight hours, sufficient for daily dosing of many drugs). Controlled release systems include microencapsulated delivery systems, implants and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, matrix controlled release devices, osmotic controlled release devices, multiparticulate controlled release devices, ion- exchange resins, enteric coatings, multilayered coatings, microspheres, liposomes, and combinations thereof. The release rate of the active ingredient can also be modified by varying the particle size of the active ingredient(s). Examples of modified release include, e.g., those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123;

4,008,719; 5,674,533; 5,059,595; 5,591,767; 5, 120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891 ; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6, 113,943; 6, 197,350; 6,248,363; 6,264,970; 6,267,981 ; 6,376,461 ; 6,419,961 ; 6,589,548; 6,613,358; and 6,699,500.

[0043] Dimethyl fumarate is commercially available as a delayed-release oral formulation (Tecfidera®) that prevents release of the active ingredient in the gastric environment while allowing for rapid release of the active ingredient in the intestine region. The finished product is available as 2 mm enteric-coated microtablets in size 0 hard gelatin capsules, in strengths 120 mg and 240 mg, respectively. The 120 mg strength capsules have white body with green cap, for the 240 mg strength capsules with green body and green cap are used. The microtablets are composed of immediate release tablet core (dimethyl fumarate,

crosscarmellose sodium as disintegrant, microcrystalline cellulose as diluent and binder, magnesium stearate as lubricant and talc (for 120 mg strength only) and colloidal anhydrous silica as glidants), and two layers of coating. Dimethyl fumarate is also commercially available as Skilarence® in 30 mg or 120 mg gastro -resistance tables.

B. Route of Administration and Dosing

[0044] Dimethyl fumarate and analogs are administered in a regime effective to increase the baseline level of frataxin in a subject by at least 25%, 50%, 75%, 100%, 200 or 300%.

The baseline level is assessed before the First treatment. In some patients, the level is increased by 50-100% or 50-200% of pretreatment levels. In some patients, the level is increased to at least 25, 30, 40 or 50% of the level in control subjects not having Friedreich’s ataxia. A 200% increase relative to baseline level in a patient approximately corresponds to an increase to 50% of normal levels, which is known to be adequate for normal functioning because heterozygous carriers of a frataxin mutation are typically symptom free. However, lesser increases are still valuable in reducing symptoms due to frataxin deficiency because the severity of such symptoms approximately correspond with the extent of such deficiency. Levels of frataxin can be measured at the mRNA or protein level. Levels can be measured in PBMC’s or whole blood among other issue samples. Levels can be based on frataxin isoform 1 as a surrogate for all three known isoforms of frataxin or can measure iso form 1 with isoform 2 and/or isoform 3. Additionally or alternatively, the regime is effective to reduce, delay, or inhibit deterioration of at least one sign or symptom of Friedreich’ s ataxia.

Preferably, the regime is effective at least partially reverse at least one sign or symptom of Friedreich’ s ataxia. Preferably subjects receiving treatment live a normal life span.

[0045] An effective regime refers to a combination of dose, frequency and route of administration effective to achieve a stated purpose. Preferably the route of administration is oral. If by other route, the regime can be calculated to deliver the same, less or grater area under the curve as oral administration. Exemplary dosages for oral administration are between 100 and 2000 mg/day. Such dosages can be given at once or in divided form (e.g., a dose of 1000 mg split into two doses of 500 mg in the same). In some subjects the dose is 500-1000 mg per day. In some subjects, the dose is 500-1500 mg/day. In some subjects, the dose is 700-1500 mg/day. In some subjects, the dose is 700-1000 mg/day. In some subjects, the dose is 720 mg/day. In some subjects, the dose is 100-400 mg per day. An exemplary regime is oral administration of dimethyl fumarate at an induction dose of 120 mg BID for a week, and 240 mg BID thereafter. Higher or lower dosage can be administered. [0046] A dose regime is typically maintained for at least six months, 1 year, 5 years, 10 years or the life of a subject, or until the subject undergoes other therapy such as gene replacement or correction rendering further treatment unnecessary.

[0047] Subjects receiving treatment can be monitored to determine response to treatment. Monitoring is typically performed before commencing treatment (e.g., in the prior week), and at intervals after receiving treatment. These intervals can be regular intervals, such as monthly, quarterly, or annually, or can be responsive to changes in symptoms of a subject (e.g., noticeable improvement or deterioration). Monitoring can measure frataxin levels at the mRNA or protein level. Such levels can be measured in whole blood, serum, plasma or blood cells such as PBMC’s.

[0048] If monitoring in a subject indicates a subject’s response to treatment is within a desired range (e.g., an increase of 50-200% of baseline levels in that subject) or to within 25- 100% of an mean level in control subjects without Friedreich’s ataxia, then treatment can be continued as is, or increased in amount or frequency if toward the lower end of such ranges.

If a subject’s response is lower than such ranges, then the dose or frequency of treatment can be increased. If a subject shows no response after increasing dose and/or frequency to the maximum amount considered safe, then treatment can sometimes be discontinued.

Monitoring can also check for potential side effects of treatment such as lymphopenia or PML infections as indications that treatment should be reduced in dose or frequency, or suspended.

[0049] Alternatively, dimethyl fumarate, analogs or pharmaceutically acceptable salts thereof can be administered parenterally, for example, by IV, IM, depo-IM, SC, depo-SC, sublingually, intranasally, intrathecally, transdermally

IV. Combination Therapies

[0050] Dimethyl fumarate, analogs and pharmaceutically acceptable salts thereof can be used in combination with each other or with other therapeutic agents or approaches used to treat, mitigate or prevent Friedreich’ s ataxia. For example, the one or more active agents described herein and/or analogs thereof can be co-administered with a histone deacetylase (HD AC) inhibitor. Preferably combinations act synergistically. EXAMPLES

[0051] To identify therapeutic strategies we screened 1,600 drugs of known safety profiles that are currently being used for other indications in humans (Sahdeo et al., 2014). Dimethyl fumarate (DMF) provided dose-dependent protection in cell-based screening. Chemically, DMF is methyl ester of fumaric acid and is currently used to treat Multiple Sclerosis (MS) (Venci et al., 2013). Here, we aim to analyze effect of DMF on FXN expression in patient derived lymphoblast cell model, mice model YG8 and in blood lymphocytes from humans dosed with DMF.

[0052] Here, we show that DMF dose-dependently increases FXN expression, both at mRNA and protein level, in FA patient derived lymphoblast cells and in mouse in vivo. At the molecular level, inherited GAA repeats are thought to repress FXN expression through the formation of thermodynamically stable R-loop structure composed of an RNA-DNA hybrid and a displaced DNA single strand (Ginno et al., 2013; Santos-Pereira et al., 2015). Presence of R-loop at expanded GAA site can result in stalling of RNA polymerase and premature transcription termination (Pandolfo, 2002; Krasilnikova et al., 2007; Sanz et al., 2016). Mechanistically, we confirm that there are increased R-loops at the GAA repeat regions in the FXN gene, and further demonstrate that DMF increases FXN expression in FA cells both by increasing transcription initiation, as well as reducing transcriptional stalling at GAA pause sites by decreasing R-loop enrichment. Because deficiency of mitochondrial FXN is the only cause of FA, we suggest that DMF could be considered as a potential therapy for FA.

METHODS

Cell Culture & Drug treatment

[0053] Human lymphoblast cells: Healthy- GM13068; FA-patient derived- GM14518, GM15850, GM16216, GM16214 and GM16220 were grown in RPMI-1640 supplemented with 15% Fetal Bovine Serum. Human Fibroblast cells: Healthy- GM3440; FA patient derived- GM04078 were grown in DMEM supplemented with 12% Fetal Bovine Serum. For both cell types, Media was changed every two days. These cells were obtained from Coriell Institute for Medical Research Repository. [0054] For in vitro drug treatment, 2x106 cells were treated with 0.01% DMSO as vehicle control or 1, 3, 10, 30 and 100mM of dimethyl fumarate for 24 or 48 hrs. Post treatment cells are harvested, washed with PBS and processed for downstream analysis.

ANIMAL PROCEDURE

[0055] 50 mg/ml of DMF was dissolved in DMSO to make stock solution of DMF. Prior to injection, 0.5 mg/ml of working DMF solution (1 :l00 dilution) was made by diluting the stock solution into phosphate-buffered saline with 5% Tween-20 and 5% polyethylene glycol (Sigma- Aldrich, St. Louis, MO, USA). The mice were injected intraperitoneally every day for 7 days with 0, 3, 5 and 10 mg/kg DMF. The mice were euthanized with C02 followed by cervical dislocation and tissues were immediately removed then flash frozen with liquid nitrogen. Samples were stored in -80 °C until utilized for experiments.

[0056] Mice were randomly allocated to 4 dose groups- 0 (vehicle), 3, 5 and 10 mg/kg DMF dose. Sample size of 4 animals per dose group was determined by power calculation (Mean l=vehicle= 1, Mean 2 =1.2 (20% Frataxin increase), Standard deviation

(SD)=0.l=l0%, from calculated SDs of many technical replicates of FXN measurements of mouse cerebellum, 2 sided test, alpha=0.05 and power of 0.80. Experimenter was not blinded to mice numbers during intra peritoneal injection, however after the western blot was performed, images were quantified objectively by Li-Cor Odyssey software.

PATIENTS

[0057] Patients were recruited from the Multiple Sclerosis Center of the Federico II

University of Naples. There were 12 controls, and 27 MS patients. Of these 14 were treated with DMF and 13 with Fingolimod (FTY). Controls demographics were: age, gender. For DMF treated MS patients: age 42.84+11.9, gender M/F=8/6, age at onset 29.2+9.8, disease duration 13.6+7.1, relapses in the previous year 0.6+0.7, EDSS 4.6+1.9. For FTY treated MS patients: age 45.9+23.3, gender M/F=3/l0, age at onset 34.2+20.9, disease duration 11.8+6.2, relapses in the previous year 0.5+0.7, EDSS 3.1+1.2.

[0058] Of the 14 DMF treated patients, 5 switched from interferon beta- la s.c., 3 were treatment naive, 2 switched from natalizumab, 2 from interferon beta-lb s.c., 1 from interferon beta- la i.m., and 1 from FTY. Of the 13 FTY treated patients, 3 were treatment naive, 3 switched from interferon beta- la s.c., 3 from interferon beta- la i.m., 2 from natalizumab, and 2 from interferon beta- lb s.c. [0059] Patients were included if a decision to start a therapy with Dimethylfumarate (DMF) or fingolimod (FTY) had already been taken as part of clinical practice. We obtained samples on the day before treatment was started, and after 3 months of continuous DMF or FTY therapy. Patients treated with DMF received 120 mg BID for one week and 240 mg BID for the remaining days until month 3. FTY patients were treated with 0.5 mg FTY for all 3 months. Healthy controls were recruited at our clinic through students and site personnel. Patients and controls were genotyped for the FXN gene expansions in order to exclude the presence of expanded alleles.

RNA extraction and qRT-PCR

[0060] In in vitro cultured cells, RNA was extracted using RNAesay kit (QIAGEN). cDNA was then synthesized from mRNA with iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) per manufacturer’s instruction in a C1000 Touch Thermal Cycler (Bio- Rad Laboratories, Hercules, CA, USA). A SensiFAST SYBR No-ROX Kit (Bioline,

Taunton, MA, USA) was used to perform qPCR on the synthesized cDNA in a Roche Lightcycler 480 (Roche Diagnostics, Indianapolis, IN, USA). The second derivative of the amplification curve was used to determine the cycle threshold, and the data were analyzed by a delta CT calculation. Primer sets used in qPCR are listed in Table S2.

[0061] For FXN gene expression in DMF treated patients, PBMCs were extracted from 30 mL of EDTA anticoagulated whole blood using Leucosep® tubes (Greiner bio-one,

Frickenhausen, Germany) and frozen at -80°C until analysis. Total mRNA was extracted from PBMCs using RIboPure RNA Purification Kit (ThermoFisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. 500 ng mRNA was reversely transcribed using the one-step High Capacity RNA-to-cDNA Master Mix (ThermoFisher Scientific, Waltham, MA, USA) following manufacturer’s instructions in a total volume of 20pL. mRNA was quantified using a Gene Expression Assay for frataxin (Life Sciences, catalog n.

Hs00l75940_ml) and standardized by quantification of hypoxanthine phosphoribosyl- transferase 1 as a reference gene. Relative expression was calculated with the efficiency- calibrated model as previously described (Sacca et ak, 2013)

Western blot

[0062] Cells/ mice tissues were lysed using lysis buffer (Cell Signalling) supplemented with a complete protease inhibitor cocktail (Roche Applied Science) and

phenylmethylsulfonyl fluoride (Sigma- Aldrich Corp.). Tissues were further homogenized using 0.5 mm glass beads in a Bullet Blender high-throughput homogenizer (Next Advance, Inc.). After pelleting cellular debris by spinning at 16000 rpm at 4°C for 15 min, protein was quantified by Bradford assay. For western blotting, 40-50 pg protein was added per lane of 4-12% Bis-Tris gels (Invitrogen Corp.). Primary antibodies were diluted in Odyssey blocking buffer (LI-COR Biosciences). Antibodies used included: anti-FXN (provided by Franco Taroni M.D., Istituto Besta) and anti-actin (#A2668, Sigma). Direct conjugated secondary antibodies (anti-rabbit IRdye800Cw and anti-mouse IRdye680 from LI-COR) were used to detect and quantify the signal of primary antibodies and imaged using a LI-COR Odyssey.

Harvesting Nucleic Acids for DRIP

[0063] Cell pellets were washed with DPBS (Life Technologies) and resuspended in 4 mL of 10 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl pH 8, lysed with 0.5% SDS, and digested with 400 units of Proteinase K (Thermo Fisher Scientific, Waltham, MA) at 37°C overnight. Cell lysates were then extracted once with 1 volume of equilibrated phenol pH 8 (USB, Cleveland, OH) and twice with 1 volume of chloroform (Sigma- Aldrich). DNA was precipitated with 1 volume of isopropanol and 300 mM sodium acetate, and was swirled out of solution with a glass shepherd's hook. The DNA pellet was washed twice by rinsing the hook with 400 pL of 70% ethanol, and was rehydrated in 10 mM Tris-HCl pH 8.

DRIP-PCR

[0064] Harvested nucleic acids (~50 pg) were digested using a restriction enzyme cocktail (20 units each of EcoRI, Hindlll, BsrGI, Puvl, Sspl) (New England Biolabs, Ipswich, MA; NEB) overnight at 37°C in lx NEBuffer 2. Digests were cleaned by phenol and chloroform extraction followed by precipitation in isopropanol. The resulting fragmented DNA was pelleted at full speed (16,100c g) at 4°C and washed twice with 70% ethanol. Air-dried pellets were rehydrated in 10 mM Tris-HCl pH 7.5, 1 mM EDTA (TE).

[0065] We adapted the previously described DRIP protocol (Ginno et a , 2013). Six to eight pg of digested nucleic acids were diluted in 450 pL of TE, and 10 pL was reserved as input for qPCR. Fifty-two pL of lOx IP buffer was added for a final buffer concentration of 10 mM sodium phosphate, 140 mM sodium chloride, 0.05% Triton X-100, and 20 pL of S9.6 antibody (1 mg/ml; KeraFAST) The samples were incubated with the antibody at 4°C for 2 hours. This incubation and all wash steps were performed on a rotisserie mixer. 40 pL of Protein A/G Agarose beads (Pierce, Rockford, IL) was washed twice with 800 pL of lx IP buffer for 5 minutes at room temperature. After adding agarose beads to each sample, they were incubated for 2 hours at 4°C. Each DRIP was then washed three times with 700 pL lx IP buffer for 10 minutes per wash at room temperature. After the final wash, the agarose beads were resuspended in 250 pL of lx IP buffer and incubated with 60 units of Proteinase K for 30 minutes at 50 °C. Digested DRIP samples were then cleaned with phenol/chloroform extraction and isopropanol precipitation. Air-dried DRIP pellets were resuspended in 80 pL of 10 mM Tris-HCl pH 8.

[0066] We used 10 pL reactions with Sensi-FAST Lo-Rox 2x qPCR mix (Bioline, London, UK) to assay for genomic loci on FXN gene using Primer listed in the table S2. For each DRIP sample, 5 pL of the output and 5 pL of diluted input (DlOO) were assayed in triplicate. Fold enrichment for a given locus was calculated using the comparative Ct method.

Materials

[0067] Dimethyl Fumarate was purchased from (Sigma- Aldrich, St. Louis, MO, USA) and stock solution was prepared in DMSO. Media for cell culture was obtained from (Corning, Inc., NY, USA) and Fetal Bovine Serum from Fetal Bovine Serum (JR Scientific, Woodland, CA).

Statistics

[0068] Cell and Mice data are expressed as mean ± standard deviation. Relative comparison of data were performed with a t-test or one way repeated measures analysis of Variance (ANOVA) using a host hoc Bonferroni correction. All statistical analysis was performed with Prism (GraphPad Software, La Jolla, USA) Differences were considered significant when p<0.05

[0069] For Human dosing data, normality was tested with the Kolmogorov-Smirnov test. P values less than 0.05 were considered significant. The effect of DMF and FTY on frataxin expression was analyzed with a General Linear Model for repeated measures with FXN expression as the dependent variable at baseline and 3 months after treatment, and drug as a factor. Statistical analysis was performed with SPSS 23.0.0.2 running on MacOS 10.11.6.

[0070] The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). RESULTS:

[0071] 1) DMF dose dependently increases FXN expression in vitro in cell models

[0072] To study the effect of DMF on FXN expression we treated patient-derived FA lymphoblast cells GM14518, GM15850, GM16214, GM16216 and GM16220 that has different number of GAA repeats (Table Sl) with 1, 3, 10, 30 and 100 mM DMF for 24 hr. FXN mRNA expression was measured by qRT-PCR. We observed dose dependent increase in FXN mRNA expression with significant increase at 10 pM and 30 pM DMF concentration by 25% and 93% respectively, compared to their respective vehicle treated control [Fig. 1]. The 100 pM dose was toxic and triggered significant cell death and very low RNA yields (data not shown). Overall, DMF significantly increased FXN expression in various patient derived lymphoblast cell models.

Table SI: Cell line

[0073] 2) DMF increases FXN expression in vivo in YG8 mice model of FA

[0074] To further determine if in vitro effects of DMF on FXN expression were translated in vivo in FA YG8 mice model. Mice were Intraperitonieally (IP) dosed with DMF at 0, 3, 5 and lOmg/kg concentration for 7 days. Cerebellum, which is a primary tissue affected in FA was collected and FXN protein level was measured by western blot. Consistently, we observed significant dose-dependent increase in FXN protein level by 23% and 52% respectively in cerebellum of animal dosed with 5 and 10 mg/kg DMF compared to vehicle treated control animals [Fig. 2]. [0075] 3) DMF increases FXN expression in vivo in MS patients

[0076] We recruited 12 controls, and 27 MS patients from Multiple Sclerosis Center of the Federico II University of Naples. Of these 14 were treated with DMF and 13 with Fingolimod (FTY). Blood was collected before and after 3 months of treatment and PBMCs were isolated to determine FXN mRNA expression.

[0077] Baseline levels of FXN mRNA were similar between controls and MS patients (Fig. 3).

[0078] We observed significant 85.1% increase in FXN expression in 3 months DMF treated MS patient. However, FXN expression was not much affected in FTY treated patients. Overall, consistent with cell and mice model, we observed a significant increase in FXN mRNA expression in peripheral blood lymphocytes of DMF treated MS patients.

[0079] 4) DMF induces transcription initiation and reverses transcriptional 'pausing' in mutant frataxin gene

[0080] The GAA repeats causing decreased FXN expression appear to do so by multiple mechanisms. In particular, GAA repeats can cause RNA polymerase 'pausing', which may be the result of co-transcriptional R-loop structures (Punga et al., 2010; Sandi et al., 2013). We studied the consequences of DMF dosing on FXN pre-mRNA transcription by qRT-PCR in patient derived lymphoblast cell line GM-14518 with -900 GAA repeats and healthy individual derived lymphoblast cell line GM-13068 by using primers (Table S2) that are designed to follow RNA synthesis along the of FXN pre-mRNA (Fig.4).

Table $2. qPCR P imer list

[0081] Consistent with a prior study (Groh et al., 2014), we observed significant 50% decrease in RNA transcript level in patient cells, just downstream of the GAA repeats, consistent with the idea that GAA repeats cause a 'stalling' of RNA polymerase in this region, resulting in decreased frataxin expression. We observed additional transcriptional reduction along the transcript such that RNA levels were 60 % lower than controls near Intron2-Exon3 and further reduced by 85% near exon 5, which is representative of mature FXN transcript.

[0082] Interestingly, when we treated the same GM14518 patient cells with 30 mM DMF treatment for 24 hour, we measured a significant 48% increase in transcript levels at the 5’UTR (Fig. 4). In addition, transcript level were significantly higher at all the loci downstream of GAA pause site, increasing the level of mature transcript by -146% as measured by Ex3-Ex4 primers and Ex5 primers, compared to untreated patient cells. Thus, DMF increases FXN expression by at least two mechanisms: increased transcriptional initiation (Fig. 4, UTR) and reversal of pausing (Fig. 4, Down-GAA to Ex5).

[0083] 5) DMF reduces R-loop concentrations at mutant frataxin pause sites

[0084] One of the mechanism proposed for RNA polymerase transcriptional pausing in mutant frataxin is through the formation of R- loops structure (Groh et al., 2014). R- loops formed over GAA repeats are thought to silence FXN expression through the recruitment of repressive histone marks and impairment of transcription caused by chromatin condensation. We tested whether DMF has any effect on R-loop formation using DNA-RNA hybrid immuno-precipitation (DRIP) to selectively enrich DNA-RNA hybrid containing R-loops in healthy lymphoblast cells GM-13068, patient derived lymphoblast cells GM-14518, and in patient cells treated with 30 mM DMF for 24 hr. DRIP was followed by qRT-PCR to detect R-loop enrichment at desired locations along the FXN gene. We observed a trend of increased R-loop enrichment at all loci near GAA repeats in FA patient cells, with significant enrichment at Up GAA and down GAA loci of FXN gene by 5 fold and 3.6 fold respectively (Fig. 5) compared to healthy cells. Positive control genes, FBLX17 and TFPT showed higher R-loop enrichments both in healthy cells and patient cells as expected. DMF treated patient cells showed an overall trend of decrease in R-loop enrichment compared to untreated patient cells with significant decrease at UTR-Exl and UP GAA loci by 3.6 and 3.4 fold

respectively. R-loop enrichment at TFPT and FBLX17 were not affected by DMF treatment. As a negative control for this experiment, DNA from patient derived lymphoblast cells was treated with RNase-H before DRIP. RNase-H treatment decreased R-loop enrichment in patient cells at loci near GAA repeats in FXN gene as well as decreased R-loop enrichment at positive control genes.

[0085] Overall, DMF treatment reduced R-loop loads specifically around the GAA repeat region of FXN, suggesting that the increased transcription observed in DMF treated patient cells might be linked to the reduction of R-loop induced pausing.

6) DMF increases mitochondrial biogenesis in patient derived fibroblast cells.

[0086] We recently demonstrated a mitochondrial biogenesis defect in FA patient cells, FA mice, and FA patient blood lymphocyte tissue (Jasoliya et al., 2017). We also showed that the mitochondrial biogenesis defect was dependent on frataxin concentration through siRNA knockdown and over-expression experiments. Furthermore, we showed that the

mitochondrial biogenesis defect in FA patient blood was directly and significantly correlated with their blood frataxin expression (Jasoliya et al., 2017). Given the highly energetic nature of neurons, this frataxin-dependent mitochondrial biogenesis defect could be a significant contributor to the neuropathomechanism of Friedreich's ataxia. We treated FA fibroblasts GM-4078 with DMF for 48 hr. Total DNA was extracted and mitochondrial copy number per cell was measured as mtDNA/nDNA ratio. Dose-dependent increases in mitochondrial copy number were observed, with significant increases at 10 mM and 30 pM concentration by 20% and 76% respectively compared to vehicle treated control. Overall, DMF significantly increased mitochondrial copy number in FA patient derived primary cells. We recently demonstrated that DMF also increases mitochondrial biogenesis and gene expression in normal human fibroblasts, control mice, and in humans with Multiple Sclerosis (Hayashi Jasoliya). Since frataxin deficiency triggers the mitobiogenic defect in cells, and DMF dose dependently increases frataxin expression (Fig.l, 2), the increase in mitochondrial biogenesis we observed could potentially be the direct consequence of frataxin induction.

DISCUSSION:

Effect of R-loops on FXN gene expression

[0087] GAA repeat expansions in the first intron of the FXN gene result in decreased FXN protein expression. Understanding the mechanistic relationship between increased GAA repeats and reduced FXN expression is important to therapeutic approaches for FA.

Expanded GAA repeat generates a region with high G versus C strand asymmetry or GC skew, which thermodynamically favors re-invasion of the GA-rich transcript on the template DNA strand behind the advancing RNA polymerase, thus forming R-loops. R-loop formation has been shown to play a vital role in transcription termination in humans and could thus lead to pausing and/ or termination in the FXN intronl as observed here (Fig. 4)

[0088] An alternative, non-exclusive model for the effect of R-loop is the recruitment of repressive histone marks, leading to the compaction of chromatin (Al-Mahdawi et a , 2007; Greene et ak, 2007; Skourti-Stathaki et ak, 2014). Spreading of such repressive modifications to the promoter region could lead to the epigenetic silencing and reduced transcription initiation. Alternatively, chromatin compaction could lead to reduced elongation through the GAA repeats, as has been observed (Li et ak, 2015). Thus R-loops could decrease FXN expression by causing premature transcription termination and/or decreasing transcription initiation or elongation because of chromatin compaction. Recent evidence also hints at a possible mechanism by which repressive histone marks can be recruited by R-loops

[0089] Formation of R-loops at expanded GAA repeats leaves the GA-rich non-template DNA strand in a single stranded state that is accessible to the transcription machinery. This has been shown to trigger antisense RNA transcription (De Biase et ak, 2009). The sense and antisense FXN transcripts may lead to formation of dsRNA thereby recruiting the RNA interference machinery and further decreasing the formation of mature FXN transcript (Skourti-Stathaki et ak, 2014).

DMF induces frataxin expression by increased transcriptional initiation.

[0090] We show that DMF induces frataxin expression through increased transcriptional initiation, and also through decreasing transcriptional pausing in mutant frataxin genes. One possible cause of increased FXN initiation events is increased activation through the Nrf2 mediated mechanism, as described previously for dyclonine. In this view, DMF which is a known Keapl/Nrf2 inducer, triggers Nrf2 binding to 3 Antioxidant Response Elements (AREs) upstream of the frataxin gene, triggering increased transcriptional initiation (Sahdeo et al., 2014). Another possibility is that DMF, that drives the expression of mitochondrial biogenesis factors NRF1 and TFAM (Hayashi et al., 2017) drives the expression of all mitochondrial proteins including frataxin as a consequence of increased mitochondrial biogenesis overall (Fig. 6).

DMF increases frataxin expression by reversal of R-loops and transcriptional pausing.

[0091] We show that DMF reverses transcriptional pausing at multiple sites post-GAA repeat, to increase RNA polymerase procession through the GAA-laden frataxin gene, resulting in increased fxn expression in the mutant gene. We also show that simultaneously DMF is decreasing R-loop enrichment, which has been proposed by others to inhibit RNA polymerase procession through the frataxin gene by the two mechanisms described above, repressive histone marks and antisense activity. Thus we demonstrate a novel activity of DMF on mutant frataxin expression, i.e. DMF-dependent reversal of transcriptional pausing that may be driven by DMF-dependent reversal of transcriptional-repressive R-loops.

However, DMF's activity appears to be not general to all R-loops, but rather only to those R- loops internal to mutant Frataxin (Fig. 5).

DMF induces FXN and mitochondrial biogenesis at the concentrations it reaches in human dosing.

[0092] Friedreich’s ataxia is a rare, autosomal recessive disease caused by depletion of mitochondrial protein FXN whose likely role is in Fe-S cluster biogenesis. Loss of FXN results in decrease in mitochondrial copy number (Jasoliya et al., 2017), which primarily effects high energy demanding tissues, including dorsal root ganglia, dentate nuclei cerebellum, skeletal muscle and heart. Primary clinical features involve progressive loss of muscle coordination and balance, leading to gait and limb ataxia resulting in being wheel chair bound as the disease progresses. Cardiomyopathy is the leading cause of death in FA (Strawser et al., 2017; Indelicato et al., 2018). Currently there is no approved therapy for FA (Tai et al., 2018). Here we show that DMF, approved for use in humans for multiple sclerosis and psoriasis, dose-dependently induces both FXN expression whose deficiency is the only cause of FA, and also induces mitochondrial biogenesis. Both the FXN induction and the mitochondrial biogenesis induction are happening at -10 mM, which is close to the ~7 mM concentration reached by DMF's major bioactive metabolite MMF in 480mg dosing in humans. We show that DMF significantly increases FXN expression by 93% in FA derived lymphoblast cell model at 30mM dose, by 52% in FA mice Yg8 at lOmg/kg dose and by 85% in MS patient treated for 3 month. FXN mRNA expression is reduced to 19.4% in FA patients, and to 53% in carriers, as compared to healthy controls (Sacca et al., 2011). Carriers are healthy individuals that discover their status only during genetic counseling, and their condition has never been linked to any neurological or cardiological abnormality. Therefore, the increase in FXN mRNA after DMF treatment can counteract the pathological process of reduced FXN at the increase found after in vivo administration.

[0093] Although there have been more than 50 clinical trials in Friedreich's ataxia, and more than 190 clinical trials of mitochondrial disease (clintrials.gov), no drug dosed in humans has ever been shown to steadily increase FXN expression with an acceptable safety and tolerability profile. Similarly, no drug dosed in humans has ever been demonstrated to increase mitochondrial biogenesis in humans except DMF. Thus, DMF is unique, it is safe and well tolerated, it has more than 200,000 patient treated worldwide for Multiple Sclerosis and Psoriasis. This provides a basis for Friedreich's ataxia and mitochondrial disease therapy.

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[0094] The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof are to be included within the spirit and purview of this application and scope of the appended claims. Each embodiment, aspect, element, feature, step or the like can be used in combination with any other unless the context requires otherwise. For example, although the invention is sometimes described with reference to Friedreich’s ataxia, the disclosure also apply to other neurodegenerative diseases mentioned. All publications (including accession numbers, websites and the like), patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if so individually denoted. To the extend a reference, such as an accession number is associated with different content at different times, the version in effect at the effective filing date of the application is meant. Effective filing date means the actual filing date or earlier filing date in which such reference was cited.