Omaveloxolone for treating or preventing mitochondrial dysfunction caused by surf1 leigh syndrome

Abstract

Provided herein are compositions and methods of treating a subject in need thereof (e.g., a subject with Leigh Syndrome) comprising administering omaveloxolone to the subject.

Description

OMAVELOXOLONE FOR TREATING OR PREVENTING MITOCHONDRIAL DYSFUNCTION CAUSED BY SURF1 LEIGH SYNDROMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 517,866, filed August 4, 2023, which is incorporated herein in its entirety for all purposes.BACKGROUND

[0002] Leigh Syndrome can be caused by genetic variants in over 110 genes in both nuclear and mitochondrial DNA. It has a prevalence of 1 in 40,000 births and manifests as a complex neurological disorder with a panel of symptoms including developmental regression, seizures, muscle weakness, and loss of vision. Leigh Syndrome is most prevalent in infants, and typically leads to death within the first several years of life. Existing therapies are largely ineffective, and therefore, new therapeutic strategies are needed for patients suffering from Leigh Syndrome.SUMMARY

[0003] Disclosed herein, in various embodiments, is a method of treating Leigh Syndrome in a subject in need thereof, the method comprising administering an effective amount of omavel oxoIone or a pharmaceutically acceptable salt thereof to the subject.

[0004] In some embodiments, the subject carries one or more mutations in one or more genes associated with oxidative phosphorylation. In some embodiments, the subject has a dysfunction, deficiency, or reduction in oxidative phosphorylation. In some embodiments, the subject has reduced expression or activity of PP ARGCI A. In some embodiments, the subject has one or more mutations in a SURFI genomic sequence.

[0005] In some embodiments, the one or more mutations comprises a frameshift mutation, a missense mutation, a nonsense mutation, or a mutation introducing a splicing alteration. In some embodiments, the one or more mutations result in a mutant gene encoding a truncated protein.

[0006] In some embodiments, the subject is determined to have the one or more mutations by analysis of the subject's genomic sequence. In some embodiments, the subject is determined tohave the one or more mutations by a method selected from the group consisting of: targeted variant analysis, deletion analysis, mutation scanning of select exons mutation scanning of the entire coding region, sequence analysis of select exons, sequence analysis of the entire coding region, or RNA analysis of transcripts of a gene. In some embodiments, the subject has at least one SURF1 allele encoding a protein with residual function. In some embodiments, the method results in increased expression of SURF1 in the subject.

[0007] In some embodiments, the effective amount of omaveloxolone is administered to the subject orally or parenterally. In some embodiments, the effective amount of omaveloxolone is about 1 mg to about 1000 mg per day. In some embodiments, the effective amount of omaveloxolone is about 5 mg to about 150 mg per day. In some embodiments, the effective amount of omaveloxolone is about 10 mg, about 20 mg, about 30 mg, about 40 mg, 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg per day.

[0008] In some embodiments, the method results in plasma levels of omaveloxolone of at least 100 nM, at least 150 nM, at least 200 nM, at least 250 nM, at least 300 nM, at least 350 nM, at least 400 nM, at least 450 nM, or at least 500 nM.

[0009] In some embodiments, omaveloxolone is administered once or twice per day. In some embodiments, omaveloxolone is administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

[0010] In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, omaveloxolone is present in a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

[0011] In some embodiments, the method results in increased expression of one or more of GCLC, GCLM, HM0X1, NQO1, SRXN1, TXNRD1, FTL A FTHI. In some embodiments, the method results in increased expression of genes associated with cytochrome c oxidase IV (COX IV) and / or the oxidative phosphorylation (OXPHOS) system. In some embodiments, the method results in increased expression of PP ARGCI A.

[0012] In some embodiments, expression is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some embodiments, expression is increased within 1 month of beginning administration of omaveloxolone.

[0013] In some embodiments, the method results in reduction or improvement of one or more symptoms of Leigh Syndrome. In some embodiments, the method results in reduction of the subject’s NPMDS score.

[0014] In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[0015] Also disclosed herein, in various embodiments, is a method of increasing expression of one or more genes in the oxidative phosphorylation (OXPHOS) system. In some embodiments, the one or more genes in the OXPHOS system are part of one or more pathways comprising ATP Synthesis Coupled Electron Transport, Cytochrome Complex, Respiratory Chain Complex IV, and Electron Transport Chain OXPHOS System in Mitochondria. In some embodiments, the one or more genes are selected from NDUFA1, NDUFA2, NDUFA9, NDUFA10, NDUFA12, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NDUFV2; MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, MT-MD6, SDHA, UQCRQ, NDUFA4, COX8A; MT-CO3, MT-ATP6, NDUFAF2, NDUFAF4, NDUFAF5, NDUFAF6, C17ORF89, FOXRED1, NUBPL, SDHAF1, BCS1L, TTC19, SURF1, COX10, COX15, SCO2, PET100, PDHA1; PDHX, PDHB, DLAT, DLD, BLD, TPK1, SLC19A3, LIPT1, LIAS, BOLA3, HIBCH, ECHS1, PDSS2, and ('OO9.

[0016] In some embodiments, the method results in increased expression of genes associated with the oxidative phosphorylation (OXPHOS) system. In some embodiments, the method results in increased expression of PP ARGCI A.

[0017] Also disclosed herein, in various embodiments, is a method of treating a subject in need thereof, the method comprising administering an effective amount of omaveloxolone or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the subject carries one or more mutations in one or more genes associated with oxidative phosphorylation.

[0018] In some embodiments, the subject has a dysfunction, deficiency, or reduction in oxidative phosphorylation. In some embodiments, the subject comprises reduced expression oractivity of one or more genes associated with oxidative phosphorylation prior to the method. In some embodiments, the subject has reduced expression or activity of PP ARGCI A.

[0019] In some embodiments, the method results in increased expression of one or more genes associated with the oxidative phosphorylation (OXPHOS) system. In some embodiments, the method results in increased expression of PP ARGCI A.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0021] FIG. 1 depicts a volcano plot of primary compound screen data for SURF1 in healthy human glutamatergic neurons. The horizontal axis shows the fold change (log?) of SURF1 mRNA, while the vertical axis shows the statistical significance (q value, or p value corrected for multiple hypothesis testing by the Benjamini -Hochberg method). Dark gray data points represent compounds that are approved by the FDA and marketed for the treatment of other indications; light gray data points indicate compounds that were not FDA approved and marketed.

[0022] FIG. 2 depicts a process flow diagram showing the generation of cell models used in compound testing. Somatic (blood) cells were collected from a Leigh Syndrome patient having a SURF1 mutation and a related control, then reprogrammed into induced pluripotent stem cells (iPSCs).

[0023] FIG. 3A depicts a diagram showing an overview of the protocol used to differentiate neural progenitor cells from patient- and control-derived iPSC lines.

[0024] FIG. 3B depicts immunocytochemistry quality control images for NPCs generated from a SURF1 Leigh Syndrome patient iPSC line (PT3) or a related control iPSC line (PT2).

[0025] FIG. 4A depicts a diagram showing an overview of the protocol used to differentiate neurons from patient- and control-derived neural progenitor cell (NPC) lines.

[0026] FIG. 4B depicts immunocytochemistry quality control images for neurons produced from SURF1 Leigh Syndrome patient (PT3) and a related control (PT2) NPCs.

[0027] FIG. 5 depicts a bar graph (left panel) and line graph (right panel) of changes in SURF1 gene expression and cell viability for patient derived (PT3) and related control (PT2) NPCstreated with a range of concentrations of omaveloxolone (Oma) or vehicle (DMSO). *p<0.05, **p<0.01, ***p<0.001. ****p<0.0001; ns, not statistically significant.

[0028] FIG. 6 depicts a bar graph (left panel) and line graph (right panel) of changes in SURF1 gene expression and cell viability for patient derived (PT3) and related control (PT2) NPCs treated with a range of concentrations of niraparib tosylate (Nir) or vehicle (DMSO). *p<0.05, **p<0.01, ***p<0.001. ****p<0.0001; ns, not statistically significant.

[0029] FIG. 7 depicts graphs showing changes in SURF1 gene expression (left panel) and cell viability (right panel) for patient derived (PT3) and related control (PT2) neurons treated with a range of concentrations of omaveloxolone (Oma) or vehicle (DMSO). *p<0.05, **p<0.01, ***p<0.001. ****p<0.0001; ns, not statistically significant.

[0030] FIG. 8 depicts graphs showing changes in SURF1 gene expression and cell viability for patient derived (PT3) and related control (PT2) neurons treated with a range of concentrations of niraparib tosylate (Nir) or vehicle (DMSO). *p<0.05, **p<0.01, ***p<0.001. ****pO .0001; ns, not statistically significant.

[0031] FIG. 9 depicts plasma concentration (in nM) of omaveloxolone in patient plasma during omaveloxolone administration and dose reduction (arrow).

[0032] FIG. 10 shows NRF2 pathway activity in a subject administered omaveloxolone for 1 month, 2 months, 4 months, or 5 months (with dose reduction at 4 months).

[0033] FIG. 11 shows activation of NRF2 pathway genes in a subject administered omaveloxolone, compared to pre-treatment control. Points on the scatter plot each correspond to a gene. Points above the dashed line are statistically significant.

[0034] FIG. 12 shows expression of selected NRF2 pathway genes in a subject administered omaveloxolone, compared to pre-treatment control.

[0035] FIG. 13 shows the ratio of transcript counts of GOT1 over GPT encoding the liver enzymes aspartate and alanine aminotransferase, respectively.

[0036] FIG. 14 shows expression of the SURF1 gene in a subject administered omaveloxolone, compared to pre-treatment control.

[0037] FIG. 15 shows activity of oxidative phosphorylation (OXPHOS) pathways in a subject administered omaveloxolone, compared to pre-treatment control.

[0038] FIG. 16 shows activation of oxidative phosphorylation (OXPHOS) pathway genes in a subject administered omaveloxolone, compared to pre-treatment control. Points on the scatter plot each correspond to a gene. Points above the dashed line are statistically significant.

[0039] FIGs. 17A-17E. show SURF1 expression after omaveloxolone treatment in control and Leigh Syndrome fibroblasts.

[0040] FIGs. 18A and 18B show cytochrome C oxidase IV (COXIV) activity in PBMC samples from a Leigh Syndrome patient before and after omaveloxolone treatment (months 1 to 5).

[0041] FIGs. 19A and 19B show transcript levels of SURF1 (FIG. 19A) and normalized enrichment score for Leigh Syndrome deficient pathways (FIG. 19B) in a patient with Leigh Syndrome compared to a control population of healthy subjects.

[0042] FIG. 20 shows volcano plots for genes implicated in the pathways depicted in FIG. 19B.

[0043] FIGs. 21A and 21B show transcript levels of PPARGC1A in a neuronal line with a pathogenic SURF1 Leigh Syndrome (LS) mutation corrected compared to a neuronal line with the mutation intact (FIG. 21 A) and in the same SURF1 LS corrected and intact neurons comparing a more matured neuronal state at 8 weeks to an earlier less matured state at 4 weeks.(FIG. 21B)

[0044] FIG. 22 shows transcript levels of PP ARGCI A in a subject with Leigh Syndrome prior to omaveloxolone treatment compared to a population of healthy controls (top row) and in the subject with Leigh Syndrome administered omaveloxolone for 1 month, 2 months, 4 months, or 5 months (with dose reduction at 4 months) compared to pre-treatment.DETAILED DESCRIPTIONDefinitions

[0045] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0046] Unless defined otherwise, 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. The abbreviations used herein have their conventional meaning within the chemicaland biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0047] Throughout the description, where compositions and kits are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions and kits of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0048] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0049] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0050] The articles “a” and “an” are used in this disclosure to refer to one or more than one (z.e., to at least one) of the grammatical object of the article, unless the context is inappropriate. By way of example, “an element” means one element or more than one element.

[0051] The term “and / or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.

[0052] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0053] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0054] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred from the context.

[0055] At various places in the present specification, variable or parameters are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual sub-combination of the members of such groups and ranges. For example, an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, and 40, and an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0056] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0057] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.

[0058] As used herein, “administering” means oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration,intrathecal administration, intracranial administration, intranasal administration or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies (e.g., anti-cancer agent, chemotherapeutic, or immunotherapy). A compound of the present disclosure (e.g., omavel oxoIone or niraparib tosylate), or a pharmaceutically acceptable salt thereof, can be administered alone or can be co-administered to the patient. Co-administration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).

[0059] In general, an “effective amount” of a compound (e.g., omavel oxoIone or niraparib tosylate, or a pharmaceutically acceptable salt thereof) refers to an amount sufficient to elicit the desired biological response, e.g., to treat an advanced solid tumor and / or a blood cancer. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the disclosure may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject.

[0060] As used herein, “pharmaceutical composition” or “pharmaceutical formulation” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0061] “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.

[0062] As used herein, the term “pharmaceutically acceptable salt” refers to any salt of an acidic or a basic group that may be present in a compound of the present invention (e.g., omaveloxolone or niraparib tosylate), which salt is compatible with pharmaceutical administration.

[0063] As is known to those of skill in the art, “salts” of compounds may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acid. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0064] Examples of bases include, but are not limited to, alkali metal (e.g., sodium and potassium) hydroxides, alkaline earth metal (e.g., magnesium and calcium) hydroxides, ammonia, and compounds of formula NW4+, wherein W is Ci-4 alkyl, and the like.

[0065] Examples of salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy ethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, K+, Ca2+, NH4+, and NW4+(where W can be a Ci-4 alkyl group), and the like.

[0066] For therapeutic use, salts of the compounds of the present invention (e.g., omaveloxolone or niraparib tosylate) are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0067] As used herein, “pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to and / or absorption by a subject and can be included inthe compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, such as a phosphate buffered saline solution, emulsions (e.g., such as an oil / water or water / oil emulsions), lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates, fatty acid esters, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. For examples of excipients, see Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0068] As used herein, “solid dosage form” means a pharmaceutical dose(s) in solid form, e.g., tablets, capsules, granules, powders, sachets, reconstitutable powders, dry powder inhalers and chewables.

[0069] A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and / or a nonhuman animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a pediatric subject. In certain embodiments, the subject is an adult subject. In certain embodiments, the subject is a non-human animal.

[0070] As used herein, the phrase “subject in need thereof’ refers to a subject that exhibits and / or is diagnosed with one or more symptoms or signs of a disease or disorder as described herein.

[0071] As used herein, and unless otherwise specified, the terms “treat,” “treating” and “treatment” contemplate an action that occurs while a subject is suffering from the specified disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition (e.g., “therapeutic treatment”).Diseases and Conditions Associated with Oxidative Phosphorylation (OXPHOS) Deficiency or Dysfunction

[0072] In some aspects, the present disclosure relates to methods of treating a subject in need thereof, the method comprising administering an effective amount of omaveloxolone or a pharmaceutically acceptable salt thereof to the subject.

[0073] In some embodiments, the subject carries one or more mutations in one or more genes associated with oxidative phosphorylation. In some embodiments, the subject comprises a disease characterized by a dysfunction, deficiency, or reduction in mitochondrial energy production. In some embodiments, the subject comprises a disease featuring a dysfunction, deficiency, or reduction in oxidative phosphorylation (OXPHOS).

[0074] In some embodiments, the subject comprises reduced expression of one or more genes associated with oxidative phosphorylation prior to the method. Non-limiting examples of genes associated with the oxidative phosphorylation include NDUFA1, NDUFA2, NDUFA9, NDUFA10, NDUFA12, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NDUFV2; MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, MT-MD6, SDHA, UQCRQ, NDUFA4, COX8A; MT-C03, MT-ATP6, NDUFAF2, NDUFAF4, NDUFAF5, NDUFAF6, C17ORF89, F0XRED1, NUBPL, SDHAF1, BCS1L, TTC19, SURF1, COX10, COX15, SCO2, PET100, PDHA1; PDHX, PDHB, DLAT, DLD, BLD, TPK1, SLC19A3, LIPT1, LIAS, BOLA3, HIBCH, ECHS1, PDSS2, and COQ9. In some embodiments, the subject comprises one or more mutations in one or more of the genes in Table 1. In some embodiments, the subject has reduced expression or activity of PP ARGCI A.Table 1. Genes involved in oxidative phosphorylation (OXPHOS)

[0075] In some embodiments, the method results in increased expression of one or more genes associated with the oxidative phosphorylation (OXPHOS) system. In some embodiments, the method results in increased expression of one or more genes in Table 1. In some embodiments, the method results in increased expression of PPARGCIA. In some embodiments, the method results in increased activity of one or more components of the OXPHOS system in the subject. In some embodiments, the method results in increased Complex IV activity in the subject.Leigh Syndrome

[0076] In some aspects, the present disclosure relates to compositions and methods of treating a subject afflicted with Leigh Syndrome. Leigh Syndrome, otherwise known as subacute necrotizing encephalomyelopathy, is a mitochondrial disease characterized by impaired ATP synthesis. Leigh Syndrome (LS) (OMIM 256000) is a multifactorial neurological genetic disorder caused mainly by the damage in the mitochondrial energy production machinery. Leigh Syndrome can be caused by mutations in any of over 110 genes, many of which are associated with ATP synthesis. In some embodiments, the subject comprises one or more mutations in one or more genes associated with mitochondrial energy production.

[0077] Disorders of mitochondrial energy production of which LS is the most common childhood onset disorder, are mainly due to a dysfunction of the oxidative phosphorylation (OXPHOS) system. OXPHOS disorders mainly affect organ systems with high energy requirements, such as musculoskeletal and neurological. Consequently, in LS patients present with various neurological deficits. Although LS is highly specific to the central nervous system, there may in some cases be non-neurological symptoms.

[0078] In some embodiments, the subject comprises one or more mutations in one or more genes selected from NDUFA1, NDUFA2, NDUFA9, NDUFA10, NDUFA12, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NDUFV2 ; MT-ND 1 , MT-ND2, MT-ND3, MT-ND4, MT-ND5, MT-MD6, SDHA, UQCRQ, NDUFA4, COX8A; MT-CO3, MT- ATP6, NDUFAF2, NDUFAF4, NDUFAF5, NDUFAF6, C17ORF89, F0XRED1, NUBPL, SDHAF1, BCS1L, TTC19, SURF1, COXIO, COX15, SCO2, PET100, PDHA1; PDHX, PDHB, DLAT, DLD, BLD, TPK1, SLC19A3, LIPT1, LIAS, BOLA3, HIBCH, ECHS1, PDSS2, and COQ9. In some embodiments, the subject comprises one or more mutations in one or more of the genes in Table 1 above. In some embodiments, the subject has reduced expression or activity of PP ARGCI A.

[0079] In some embodiments, the method results in increased expression of one or more genes associated with the oxidative phosphorylation (OXPHOS) system disclosed herein. In some embodiments, the method results in increased expression of PPARGCIA. In some embodiments, the method results in increased activity of one or more components of the OXPHOS system in the subject. In some embodiments, the method results in increased Complex IV activity in the subject.

[0080] In various embodiments, the subject afflicted with Leigh Syndrome has one or more mutations in the SURF1 gene (i.e., SURF1 -mediated Leigh Syndrome). The SURF1 gene produces a protein product that is critical for the assembly of the cytochrome c oxidase complex in the mitochondrial membrane (also known as Complex IV). Without Complex IV, oxidative phosphorylation (a key driver of ATP generation in the cell) is severely impaired, leading to profound effects including neuronal cell death, which is a characteristic feature of the disorder. A7 / 7’7-mediated Leigh Syndrome is autosomal recessive; however, one of the two affected alleles can produce a protein variant (e.g., an allele harboring a missense mutation) having residual function (i.e., reduced, but not eliminated, function compared to a non-mutant SURF1 protein).

[0081] In some embodiments, the one or more mutations result in a SURF1 gene encoding a protein with reduced or substantially no function. In some embodiments, the one or more mutations result in a SURF1 gene encoding a truncated protein. In some embodiments, the one or more mutations comprise a frameshift mutation (z.e., a mutation that alters the reading frame of a gene by insertion or deletion of a number of nucleic acids that is not divisible by 3), a missense mutation (z.e., a mutation that results in a codon for a different amino acid compared to the original gene), a nonsense mutation (z.e., a mutation that introduces a premature stop codon), or a mutation that introduces a splice site. In some embodiments, the one or more mutations comprise a frameshift mutation. In some embodiments, the one or more mutations comprise a missense mutation. In some embodiments, the one or more mutations comprise a nonsense mutation, in some embodiments, the one or more mutations comprise a mutation that introduces a splice site. In some embodiments, the one or more mutations result in a gene encoding a truncated protein. In some embodiments, the subject has at least one SURF1 allele encoding a protein having residual function.

[0082] In various embodiments, the subject is determined to have the one or mutations by analysis of the subject’s genome sequence (e.g., SURF1 genomic sequence). In some embodiments, the subject is determined to have the one or more mutations by a method selected from the group consisting of targeted variant analysis, deletion analysis, mutation scanning of select exons, mutation scanning of the entire gene (e.g., SURF1) coding region, sequence analysis of select exons, sequence analysis of the entire gene (e.g., SURF1) coding region, or RNA analysis of transcripts of the gene (e.g., SURF1). In some embodiments, the subject is determined to have the one or more mutations by targeted variant analysis. In some embodiments, the subject is determined to have the one or more mutations by deletion analysis. In some embodiments, the subject is determined to have the one or more mutations by mutation scanning of select exons. In some embodiments, the subject is determined to have the one or more mutations by mutation scanning of the entire gene (e.g., SURF1) coding region. In some embodiments, the subject is determined to have the one or more mutations by sequence analysis of select exons. In some embodiments, the subject is determined to have the one or more mutations by sequence analysis of the entire gene (e.g., SURF1 coding region). In some embodiments, the subject is determined to have the one or more mutations by RNA analysis of transcripts of the gene (e.g., SURF1). Details of suitable methods to detect gene mutations insubjects afflicted with Leigh Syndrome are provided in the Genetic Testing Registry of the National Library of Medicine.

[0083] In some embodiments, the method results in increased expression of SURF1 in the subject compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the increased expression of SURF1 is increased expression of a SURF1 variant having residual function (e.g., having a point mutation).

[0084] In some embodiments, expression of SURF1 is increased by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 125% or more, 150% or more, 175% or more, 200% or more, 225% or more, 250% or more, 275% or more, 300% or more, 325% or more, 350% or more, 375% or more, 400% or more, 425% or more, 450% or more, 475% or more, or 500% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 10% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 20% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 30% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 40% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 50% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 60% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 70% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 80% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 90% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 100% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 125% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 150% or more compared to the expression of SURF1 in the subject prior to administration. In someembodiments, expression of SURF1 is increased by 175% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 200% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 225% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 250% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 275% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 300% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 325% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 350% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 375% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 400% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 425% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 450% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 475% or more compared to the expression of SURF1 in the subject prior to administration. In some embodiments, expression of SURF1 is increased by 500% or more compared to the expression of SURF1 in the subject prior to administration.

[0085] In some embodiments, the expression of SURF1 in the subject is increased by 10% to 500%, 20% to 500%, 30% to 500%, 40% to 500%, 50% to 500%, 60% to 500%, 70% to 500%, 80% to 500%, 90% to 500%, 100% to 500%, 125% to 500%, 150% to 500%, 175% to 500%, 200% to 500%, 225% to 500%, 250% to 500%, 275% to 500%, 300% to 500%, 325% to 500%, 350% to 500%, 375% to 500%, 400% to 500%, 425% to 500%, 450% to 500%, 475% to 500%, 20% to 475%, 20% to 450%, 20% to 425%, 20% to 400%, 20% to 375%, 20% to 350%, 20% to 325%, 20% to 300%, 20% to 275%, 20% to 250%, 20% to 225%, 20% to 200%, 20% to 175%, 20% to 150%, 20% to 125%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%,20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 50% to 400%, 50% to 350%, 50% to 300%, 50% to 250%, 50% to 200%, 50% to 150%, 50% to 100%, 100% to 400%, 100% to 350%, 100% to 300%, 100% to 250%, 100% to 200%, 100% to 150%, 150% to 400%, 150% to 350%, 150% to 300%, 150% to 250%, 150% to 200%, 200% to 400%, 200% to 350%, 200% to 300%, 200% to 250%, 250% to 400%, 250% to 350%, 250% to 300%, 300% to 400%, 300% to 350%, or 350% to 400% compared to the expression of SURF1 in the subject prior to administration.

[0086] In some embodiments, the expression of SURF1 in the subject is increased by 10% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 30% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 40% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 50% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 60% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 70% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 80% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 90% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 100% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 125% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 150% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 175% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 200% to 500%compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 225% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 250% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 275% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 300% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 325% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 350% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 375% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 400% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 425% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 450% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 475% to 500% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 475% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 450% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 425% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 400% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 375% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 350% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in thesubject is increased by 20% to 325% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 300% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 275% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 250% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 225% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 200% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 175% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 150% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 125% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 100% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 90% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 80% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 70% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 60% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 50% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 40% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 20% to 30% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 50% to 400% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject isincreased by 50% to 350% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 50% to 300% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 50% to 250% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 50% to 200% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 50% to 150% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 50% to 100% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 100% to 400% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 100% to 350% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 100% to 300% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 100% to 250% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 100% to 200% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 100% to 150% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 150% to 400% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 150% to 350% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 150% to 300% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 150% to 250% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 150% to 200% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 200% to 400% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, theexpression of SURF1 in the subject is increased by 200% to 350% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 200% to 300% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 200% to 250% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 250% to 400% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 250% to 350% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 250% to 300% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 300% to 400% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 300% to 350% compared to the expression of SURF1 in the subject prior to administration. In some embodiments, the expression of SURF1 in the subject is increased by 350% to 400% compared to the expression of SURF1 in the subject prior to administration.

[0087] In various embodiments, a subject afflicted with Leigh Syndrome (e.g., SURF1- mediated Leigh Syndrome) is administered an effective amount of one or more small molecule compounds that upregulate the expression of SURF1. In some embodiments, administration of the one or more small molecule compounds increases expression of SURF1 variants having residual function. In some embodiments, increased expression of SURF1 variants having residual function ameliorates the mechanistic root cause of the disorder.Compounds and Methods of Treatment

[0088] In some embodiments, the subject is administered an effective amount of omaveloxolone or a pharmaceutically acceptable salt thereof. Omaveloxolone, also known as N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-l l-cyano-2,2,6a,6b,9,9, 12a-heptamethyl-10,14- dioxo-l,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2- difluoropropanamide or RTA-408, has the chemical structure of formula (I), as d epicted below:

[0089] In some embodiments, the subject is administered an effective amount of niraparib tosylate or a pharmaceutically acceptable salt thereof. Niraparib tosylate, also known as (S)-2- (4-(piperi din-3 -yl)phenyl)-2H-indazole-7-carboxamide 4-methylbenzenesulfonate, MK-4827 tosylate, or MK-4827 4-methylbenzenesulfonate, has the chemical structure of formula (II), as depicted below:

[0090] In some embodiments, the subject is administered at least one of omavel oxoIone and niraparib tosylate. In some embodiments, the subject is administered omavel oxoIone and niraparib tosylate. In some embodiments, the subject is administered an effective amount of omaveloxolone and an effective amount of niraparib tosylate. In some embodiments, the subject is administered an effective amount of omaveloxolone.

[0091] As noted above, the present disclosure provides methods of treatment comprising administering an effective amount of omaveloxolone or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the method comprises administering omaveloxolone to the subject orally or parenterally. In some embodiments, comprises administering omaveloxolone to the subject orally. In some embodiments, comprises administering omaveloxolone to the subject parenterally, e.g., intravenously, rectally, or by injection. In some embodiments, the method comprises administering omaveloxolone to the subject locally, e.g., topically or intramuscularly. In some embodiments, the method comprises administering omaveloxolone to target tissues. The skilled artisan can determine an appropriatesite and route of administration based on factors including, but not limited to, the disease or condition being treated.

[0092] The dose and dosage regimen may depend upon a variety of factors readily determined by a physician, such as the nature of the disease or condition, the characteristics of the subject, and the subject's history. In some embodiments, the effective amount of omavel oxoIone is about 1 mg to about 1000 mg per day. In some embodiments, the effective amount of omaveloxolone is about 2 mg to about 500 mg per day. In some embodiments, the effective amount of omaveloxolone is about 4 mg to about 200 mg per day. In some embodiments, the effective amount of omaveloxolone is about 5 mg to about 150 mg per day. In some embodiments, the effective amount of omaveloxolone is about 10 mg, 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, or about 200 mg per day. In some embodiments, the effective amount of omaveloxolone is about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg per day.

[0093] In some embodiments, omaveloxolone is administered once or twice per day. The skilled artisan can determine an appropriate dosage regimen based on factors including, but not limited to, the nature of the disease or condition, the characteristics of the subject, and the subject's history.

[0094] In some embodiments, omaveloxolone is administered for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years. The skilled artisan can determine an appropriate duration of treatment based on factors including, but not limited to, the nature of the disease or condition, the characteristics of the subject, and the subject's history.

[0095] In some embodiments, the method results in plasma levels of omaveloxolone of at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 150 nM, at least 200 nM, at least 250 nM, at least 300 nM, at least 350 nM, at least 400 nM, at least 450 nM, or at least 500nM. In some embodiments, the method results in plasma levels of omaveloxolone of at least 100 nM, at least 150 nM, at least 200 nM, at least 250 nM, at least 300 nM, at least 350 nM, at least 400 nM, at least 450 nM, or at least 500 nM. In some embodiments, the method results in plasma levels of omaveloxolone of at least 100 nM, In some embodiments, the method results in plasma levels of omaveloxolone of at least 200 nM, In some embodiments, the method results in plasma levels of omaveloxolone of at least 300 nM. In some embodiments, the method results in plasma levels of omaveloxolone of at least 400 nM, In some embodiments, the method results in plasma levels of omaveloxolone of at least 500 nM.

[0096] In some embodiments, the method further comprises administering an additional therapeutic agent.Pharmaceutical Compositions

[0097] In various embodiments, a compound of the present invention (e.g., omaveloxolone or niraparib tosylate) or a pharmaceutically acceptable salt thereof is administered to the subject in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, diluents, or reagents. In some embodiments, omaveloxolone is combined with one or more pharmaceutically acceptable carriers, diluents, excipients, and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use.

[0098] The pharmaceutical compositions described herein can be administered by a variety of routes including, but not limited to, oral (enteral) administration, parenteral (by injection) administration, rectal administration, transdermal administration, intradermal administration, intrathecal administration, subcutaneous (SC) administration, intravenous (IV) administration, intramuscular (IM) administration, and intranasal administration. In certain embodiments, the pharmaceutical compositions described herein are administered orally.

[0099] The pharmaceutical compositions described herein may also be administered chronically (“chronic administration”). Chronic administration refers to administration of a compound or pharmaceutical composition thereof over an extended period of time, e.g., for example, over 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may be continued indefinitely, for example, for the rest of the subject’s life. In certain embodiments, the chronic administration is intended to provide a constant level of the compound in the blood, e.g., within the therapeutic window over the extended period of time.

[0100] The pharmaceutical compositions described herein may be presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary 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. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.

[0101] In certain embodiments, the pharmaceutical compositions provided herein are administered to the patient as a solid dosage form. In certain embodiments, the solid dosage form is a capsule.

[0102] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with ordinary experimentation. General considerations in the formulation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21sted., Lippincott Williams & Wilkins, 2005.

[0103] Examples of carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile.

[0104] Pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some embodiments, the composition is sterile and may be fluid such that it can be drawn into a syringe or delivered to a subject from a syringe. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In certain embodiments, the pharmaceutical composition is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0105] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent disclosed herein.Effects Following Treatment

[0106] As noted above, the present disclosure provides methods of treating a subject in need thereof, comprising administering an effective amount of omaveloxolone or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the subject, having received the method of treatment, will have outcomes including, but not limited to, altered gene expression in blood and / or target tissues. In some embodiments, the subject, having received the method of treatment, will have an improvement or reduction in one or more symptoms of a disease or condition.

[0107] As noted above, the present disclosure provides methods of treating Leigh Syndrome comprising administering an effective amount of omaveloxolone or a pharmaceuticallyacceptable salt thereof to the subject. In some embodiments, the subject, having received the method of treatment, will have outcomes including, but not limited to, altered gene expression in blood and / or target tissues. In some embodiments, the subject, having received the method of treatment, will have an improvement or reduction in one or more symptoms of Leigh Syndrome.

[0108] In some embodiments, the method results in altered gene expression in the blood and / or one or more target tissues of the subject. In some embodiments, the method results in increased expression of one or more of GCLC, GCLM, HM0X1, NQO1, SRXN1, TXNRD1, FTL and FTH1. In some embodiments, the method results in increased expression of GCLM. In some embodiments, the method results in increased expression of SRXNL In some embodiments, the method results in increased expression of FTL. In some embodiments, the method results in increased expression of GCLM and SRXNL In some embodiments, the method results in increased expression of GCLM, SRXN1, and FTL.

[0109] In some embodiments, the method results in increased expression of one or more pathways from the group consisting of ATP Synthesis Coupled Electron Transport, Cytochrome Complex, Respiratory Chain Complex IV, and Electron Transport Chain OXPHOS System in Mitochondria. In some embodiments, the method results in increased expression of one or more pathways from the group consisting of Complex IV (COX), mitochondrial, NADELubiquinone oxidoreductase (NDUF) (Complex I), succinate dehydrogenase complex genes (SDHA and SDHB), ubiquinol-cytochrome C reductase (UQCRs) (Complex III), and ATP synthase genes (Complex V).

[0110] In some embodiments, the method results in increased expression of genes associated with COX IV and / or the OXPHOS system. In some embodiments, the method results in increased expression of genes in the COX IV pathway. In some embodiments, the method results in increased expression of genes in the OXPHOS pathway. In some embodiments, the method results in increased expression of one or more genes encoding a component of Complex IV (COX), mitochondria, NADH:Ubiquinone Oxidoreductase (NDUF) (Complex I), Succinate Dehydrogenase Complex (SDHA and SDHB) (Complex II), and Ubiquinol-Cytochrome C Reductase (UQCRs) (Complex III). In some embodiments, the method results in increased expression of COX, mitochondrial, and / or Ubiquinol-Cytochrome C Reductase genes (UQCRs). In some embodiments, the method results in increased expression of COX andmitochondrial genes involved in the formation of complex IV. In some embodiments, the method results in increased expression of ATP Synthase genes which are a part of Complex V.[OHl] In some embodiments, the method results in increased expression of one or more genes selected from NDUFA1, NDUFA2, NDUFA9, NDUFA10, NDUFA12, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NDUFV2; MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, MT-MD6, SDHA, UQCRQ, NDUFA4, COX8A; MT-CO3, MT-ATP6, NDUFAF2, NDUFAF4, NDUFAF5, NDUFAF6, C17ORF89, FOXRED1, NUBPL, SDHAF1, BCS1L, TTC19, SURF1, COXIO, COX15, SCO2, PET100, PDHA; PDHX, PDHB, DLAT, DLD, BLD, TPK1, SLC19A3, LIPT1, LIAS, BOLA3, HIBCH, ECHS1, PDSS2, and COQ9. In some embodiments, the method results in increased expression of one or more of the genes in Table 1 above. In some embodiments, the method results in increased expression of PPARGC1A.

[0112] In some embodiments, expression of the one or more genes is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%.

[0113] In some embodiments, expression of the one or more genes and / or pathways is increased within 1 month of beginning administration of omaveloxolone. In some embodiments, expression of the one or more genes and / or pathways is increased within 2 months of beginning administration of omaveloxolone.

[0114] Subjects with Leigh Syndrome often have one or more associated symptoms including, but not limited to, impaired mobility, impaired communication, impaired feeding, impaired self-care, and extrapy rami dal symptoms. Severity of individual symptoms or overall disease burden (z.e., the cumulative effect of the symptoms affecting a subject with Leigh Syndrome) may be measured by any method known in the art. A non-limiting example of a method to measure disease burden in a subject with Leigh Syndrome is the Newcastle Paediatric Mitochondrial Disease Scale (NPMDS, Phoenix et al. Neuromusc. Disord. 2006; 16:814-820). The scale has sections focusing on the current function (Section I), the system-specific involvement (Section II), and the current clinical assessment (Section III). Each item has 4 responses: normal (0), and mild (1), moderate (2), and severe (3) impairment. The total scorefrom all 3 sections, which reflects disease burden, can be categorized into mild (0-14), moderate (15-25), and severe (>25).

[0115] In some embodiments, the method results in improvement or reduction of one or more symptoms of Leigh Syndrome. In some embodiments, the method results in improvement of one or more of mobility, communication, feeding, self-care, or extrapy rami dal symptoms. In some embodiments, the method results in increased food intake, improved gastrointestinal symptoms, increased stamina, and / or reduced tremors. In some embodiments, the method results in reduction of the subject’s NPMDS score by at least 1 point, at least 2 points, at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, at least 10 points, at least 11 points, at least 12 points, at least 13 points, at least 14 points, at least 15 points, at least 16 points, at least 17 points, at least 18 points, at least 19 points, or at least 20 points.EXAMPLESExample 1: High-Throughput Screening of Small Molecule Modulators of SURF1 Expression

[0116] A library of 3,884 small molecules was screened using human neurons derived from the induced pluripotent stem cells (iPSCs) of a presumably healthy donor. The neurons, which were primarily glutamatergic, were directly differentiated from the iPSCs plated in 384-well plates by overexpression of the transcription factor NGN2 as described by Wang and Ward Stem Cell Reports. 2017 Oct 10; 9(4): 1221-1233). At day 15 of differentiation, triplicate wells of the neurons were treated with a single concentration (1 pM) of each compound in the library or with vehicle for 3 days. The effect of each compound on gene expression was then assessed for each well using a massively parallel 3 ’-end RNA-seq protocol.

[0117] Candidate molecules were identified based on increased expression of the SURF1 gene relative to vehicle-treated cells (log2 fold change greater than 0) with statistically significant at the level of q less than or equal to 0.1. These candidates are shown in the volcano plot depicted in FIG. 1 (green points indicate compounds that are approved by a regulatory agency, and pink points indicate compounds that are in development).

[0118] Applying cutoff criteria for fold change, q value, and cell viability (> 70% of vehicle- treated cells) yielded 22 overall candidate molecules, 6 of which were selected for follow-up testing in patient-derived NPCs at multiple concentrations.Example 2: Preparation of a Cell Model of SURFl-mediated Leigh Syndrome

[0119] ,S7 / A7’7-mediated Leigh Syndrome patient-derived primary cell models were prepared alongside control cells prepared from an immediate family member. The patient was identified as having one allele with a truncating mutation (c.312_321dell0insAT, p.Leul05X) and the other allele with a missense mutation (c.574C>T, p.Argl92Trp). An overview of the preparation of the cell models is outlined in FIG. 2. Somatic cells were collected from the peripheral blood of both individuals and reprogrammed to iPSCs using the CytoTuneTM-iPS Reprogramming Kit (Gibco, Life Technologies, Publication Part Number MAN0006712) and banked in cryogenic storage.

[0120] The patient-derived and control iPSCs were then differentiated into neural progenitor cells (NPCs) according to the manufacturer’s protocol (StemCell Technology, Generation and Culture of Neural Progenitor Cells Using the STEMdiff™ Neural System, Document Number 10000005588) using the embryoid body protocol for neural induction as shown in FIG. 3A. Immunocytochemistry on these cells revealed the robust expression of key markers of the early neuronal differentiation lineage (Nestin, PAX6, and SOX2), as shown in FIG. 3B.

[0121] Banked NPCs were then differentiated into neurons according to the manufacturer’s protocol (StemCell Technology, BrainPhys™ Neuronal Medium, Document #10000000225). Briefly, NPCs were expanded in STEMdiff™ Neuron Differentiation Media for 7 days. Neuron maturation was achieved by changing from Neuron Differentiation to Brainphys media (BrainPhys™ hPSC Neuron Kit, StemCell Technology) and a defined set of soluble factors for 2 weeks. (FIG. 4A). Immunocytochemistry revealed robust expression of the early neuron marker Tuj 1 and showed the extensive network of dendrites that are characteristic of neuron morphology (FIG. 4B).Example 3: Candidate Testing in Patient-Derived NPCs and NeuronsPatient-Derived NPCs

[0122] A range of concentrations of omaveloxolone and niraparib tosylate were tested on patient-derived and related control -derived NPCs (produced as described in Example 2). NPCswere plated on 96-well plates at different densities and cultured for 4 days in NPC media. Compounds were added at days 1 and 3 after plating, and cells were harvested for gene expression analysis by qPCR at day 4 after plating. FIGs. 5 and 6 show the results for omaveloxolone and niraparib tosylate, respectively, with mean ± SEM. Statistically significant differences were calculated with 1-way ANOVA and Dunnett’s multiple comparison tests. Both compounds showed a dose-dependent, statistically significant increase in SURF1 gene expression in both patient-derived and control -derived NPCs. For niraparib tosylate, statistically significant increases in gene expression were only observed at the two highest concentrations tested.Patient-Derived Neurons

[0123] Compounds were added to patient-derived neurons (produced as described in Example 2) at neuron differentiation day 7, and cells were exposed for three additional days before assessing gene expression changes by qPCR. A range of concentrations of omaveloxolone and niraparib tosylate were tested on patient-derived and related control -derived neurons. NPCs were plated onto 96-well plates coated with PDL and laminin. The plating density for the 96- well plates was 40000 cells per well for the control cells and 60,000 cells per well for the patient-derived cells. NPCs were differentiated in complete BrainPhys media for seven days before being subjected to compound treatment. On day 7, media was replaced with fresh BrainPhys media, and cells were treated with three compounds at four different drug concentrations. Before takedown on day 10, the plates were imaged with the neuronal live staining marker NeuO and the nuclei marker DAPI.

[0124] Cell viability after drug treatment was determined by the quantification of the live cell fraction in comparison to DMSO-treated wells (fraction of live DAPI-positive nuclei / total number of DAPI-positive nuclei).

[0125] Gene expression for SURF1 and a housekeeping gene (B2M) were measured for each condition using qPCR. Briefly, rRNA-depleted, polyadenylated mRNA was isolated using a KingFisher magnetic bead RNA isolation kit and reverse transcribed to cDNA. Gene expression levels were measured using custom multiplexed primer-probe sets for each target gene and a QuantStudio 7 qPCR instrument.

[0126] FIGs. 7 and 8 show the results for omaveloxolone and niraparib tosylate, respectively. Results of 2 independent experiments are presented with Mean ± SEM. Statistically significantdifferences were calculated with 1-way ANOVA and Dunnett’s multiple comparison tests. Both compounds showed a dose-dependent, statistically significant increase in SURF1 gene expression in both patient- and control -derived neurons, while preserving cell viability at certain concentrations.Example 4: Administration of Omaveloxolone to a SubjectPlasma omaveloxolone concentration

[0127] A patient with Leigh Syndrome was administered omaveloxolone (100 mg / day, orally) for four months, then administered a lower dose of omaveloxolone (50 mg / day, orally) for two additional months. The patient was diagnosed with Leigh Syndrome associated with heterozygous NM_003172.4(SURFl):c.312_321delinsAT (p.Prol04_Leul05insTer); NM_003172.4(SURFl):c.-l l_13del (p.Metl_Ala5del) mutations in the SURF1 gene. Both mutations are pathogenic and are known to cause Leigh Syndrome (Ribeiro et al. Mitchondrion. 2016; 31 :84-88; Zhu et al. Nature Genetics. 1998; 20:337-343; and Lee and Chiang. Antioxidants (Basel). 2021. 10(12: 1950)).

[0128] Blood was collected from the patient at approximately monthly intervals, including one pre-treatment sample. Dose was reduced three days prior to the month four blood collection.

[0129] Plasma omaveloxolone levels were quantified using mass spectrometry. FIG. 9 depicts plasma concentration (in nM) of omaveloxolone in the patient as a function of elapsed time of omaveloxolone treatment (in months). The arrow indicates the time point when the dose reduction occurred. Prior to the reduction in omaveloxolone dose at four months, the plasma concentration of Omaveloxolone was 350.2 ± 51.9 nM (months 1 and 2). Plasma levels of omaveloxolone were not significantly reduced within days after dose reduction, but decreased over the following months to 258.8 and 108.6 nM at months 5 and 6, respectively. Omaveloxolone concentration measured in the patient's blood agreed with previously published results (Lynch et al. Ann Clin Transl Neurol. 2018;6(l): 15-26) for the respective dosage.Analysis of Biomarkers of Omaveloxolone in Patient Blood

[0130] RNA was extracted, barcoded libraries for RNA-seq were prepared with 100 ng of RNA, and paired-end sequencing (150 bp x 2) was performed. RNA-seq reads were aligned using the pseudo-alignment software Kallisto (https: / / pachterlab.github.io / kallisto / ) v 0.46.1 with bootstrapping (number of bootstraps=100) (Bray et al. Nature BiotechnoL 2016. 34:525-527). Reads were aligned to the human genome assembly GRCh38 to generate transcript-level counts. Transcript counts were converted to gene counts using the human transcript to gene map version 107.

[0131] Differential expression was performed at the gene-level using Kallisto’s sister package Sleuth (https: / / pachterlab.github.io / sleuth / about.html) v0.30.0 using median-of-ratios normalization followed by shrinkage in R (Pimentel et al. Nat Methods. 2017. 14:687-690). Differential expression was performed between a control and a test group. The output yielded a gene-level log2(Fold Change) of the test group compared to the control group. For all comparisons the control group was the pre-treatment sample unless mentioned otherwise.

[0132] Gene set enrichment analysis (GSEA) was performed using the fGSEA package vl.22.0 (https: / / bioconductor.org / packages / release / bioc / html / fgsea.html) based on the genelevel fold change values from two-condition DE analysis (Korotkevich et al. bioRxiv. 2021.). Enrichment was calculated for pathways downloaded from the Molecular Signatures Database (https: / / www.gsea-msigdb.org / gsea / msigdb) MSigDB.

[0133] NRF2 pathway activity was generated from the canonical NRF2 gene set in the WikiPathways database (https: / / www.wikipathways.org / pathways / WP2884.html). The effect of omaveloxolone treatment on NRF2 pathway activation was calculated using Gene Set Enrichment Analysis from RNA-seq gene counts. Briefly, the genes were ranked based on the respective fold change from the largest to the smallest. The enrichment was then calculated based on this ranking, pathways with genes with the highest ranks were positively enriched while those with genes with the lowest ranks were negatively enriched. The magnitude of enrichment corresponds to the size of the enrichment. The significance of each enrichment was calculated based on an adaptive Monte Carlo sampling algorithm.

[0134] Analysis of change in enrichment of the pathway post-treatment showed an increase in NRF2 pathway activity (FIG. 10). The length of each bar in the graph corresponds to the magnitude of the enrichment at each month post-treatment start. Solid bars show statistically significant changes, and hatched bars show statistically insignificant changes. Error bars represent the log2(errors) natively generated from the GSEA analysis of fold changes. The NRF2 pathway activation effect compounded over the duration of omaveloxolone treatment, reaching peak value after four months of treatment. The consecutive reduction inomaveloxolone dosage prior to month four preceded a reduction in NRF2 pathway activation at month five.

[0135] In-depth analysis of the genes that form the NRF2 pathway showed a similar trend (FIG 11). From left to right, panels represent one, two, four, and five months of treatment with omaveloxolone, respectively, compared to the pre-treatment control. Each point on the scatter plot corresponds to a gene, where the x-axis represents the log2(Fold Change) of that gene at the respective treatment time point compared to the pre-treatment control and the y-axis represents the -logio(False discovery rate adjusted significance). Gene regulations above the dashed black line are significant. An increase in effect on NRF2 pathway genes during omaveloxolone treatment was evident from the increase in both the spread in Log2(Fold Change) on the x-axis as well as the increase in significance of these changes on the y-axis, up to month four. This increase is followed by a significant decrease in effect size at month five, following the reduction in omaveloxolone dose.

[0136] While the NRF2 pathway consists of > 1000s of genes, a small panel of 6 genes have been characterized as robust markers of NRF2 pathway activity (Morgenstern et. al, Redox Biol. 2024; 72: 103134.). These genes are GCLC, GCLM, HM0X1, NQO1, SRXN1 and TXNRD1. In addition, FTL (ferritin light chain) and F / T / / (ferritin heavy chain) were identified as NRF2 target genes in the trial of omaveloxolone for Friedreich’s Ataxia (https: / / www.accessdata.fda.gov / drugsatfda_docs / nda / 2023 / 216718Origls000MedR.pdf). FTL and FTH1, involved in iron metabolism, together encode the protein ferritin. In this trial, ferritin levels were up-regulated during omaveloxolone treatment providing additional supportive pharmacodynamic evidence of NRF2 pathway activation.

[0137] The GCLC, GCLM, HM0X1, NQO1, SRXN1, TXNRD1, FTL, and FTH1 genes were quantified in patient blood using RNA-seq and showed activity that roughly tracked with the dosing schedule (FIG. 12), i.e. increased expression followed by a decrease post dose reduction. The shading of each bar corresponds to the elapsed time after treatment start date in months. Error bars show the standard error. Solid and hatched bars show significant and nonsignificant fold change values, respectively.

[0138] While the up-regulation of FTH1 was non-significant across all timepoints (not shown), significant up-regulation of FEZ was observed. FTL levels did not drop significantly post dosereduction.

[0139] SRXN1 expression was significantly increased over the first four months, with a decrease in SRXN1 expression at month five following the omaveloxolone dose reduction. GCLM expression was consistently increased through month four, then returned to approximately baseline levels at month five.

[0140] GCLC and HM0X1 expression were significantly increased through month two, but the increase was not significant and the dose reduction abolished any increase at month four. NQO1 did not show any significant increase in expression, possibly due to high variation as shown by the error bars. This observation is further evidence of the limited dynamic range of NQO1 increase as a marker of NRF2 (Yagishita et al. Antioxidants (Basel). 2020; 9(8):716). TXNRD1 expression was increased through month four, followed by a small decrease at month five; however, these changes were not significant.

[0141] The RNA-seq analyses also illustrated that genes responded at different velocities to the omaveloxolone dose reduction. The dose reduction occurred only three days before the month four blood collection. GCLC and HM0X1 expression were reduced at month four blood collection, while expression of GCLM, NQO1, SRXN1 and TXNRD1 was not decreased until month five.Effect of Omaveloxolone on Liver Enzymes

[0142] Omaveloxolone treatment of Friedreich’s Ataxia had been associated with increased levels of liver enzymes alanine and aspartate aminotransferase (ALT and AST, respectively) previously, albeit without increase in other signs of liver injury (Lynch et al. Ann Clin Transl Neurol. 2018;6(1): 15-26). AST and ALT are encoded by genes GOT1 and GPT, respectively.

[0143] RNA-seq analysis of blood during the present study of omaveloxolone treatment of Leigh Syndrome showed an increase in AST / ALT ratio through month two, followed by a decrease to baseline, pre-treatment, levels at months four and five, following the omaveloxolone dose reduction (FIG. 13). Error bars represent standard error.Effect of Omaveloxolone on SURF1 in Patient Blood

[0144] RNA-seq analysis of patient blood during omaveloxolone treatment did not show a significant increase in SURF1 transcripts. Changes in SURF1 were variable across the four timepoints (FIG. 14). Due to the high variability, no direct conclusions can be drawn on the effect of omaveloxolone on SURF1 based on the RNA-seq profile of patient blood.Effect of Omaveloxolone on Leigh Syndrome (LS) Pathways

[0145] Mutations in the SURF1 gene are associated with LS due to cytochrome C oxidase (COX) deficiency. The SURF1 gene encodes the SURF1 protein essential for the correct assembly of the COX complex (Pecina etal. Biochimica et Biophysica Acta (BB A)- Molecular Basis of Disease, 2003; 1639(l):53-63; Wedatilake et al. Orphanet J. Rare Dis. 2013. 8:96; Yao and Shoubridge. Human Molecular Genetics. 1999; 8(13):2541-2549).

[0146] COX IV is the fourth complex of the mitochondrial oxidative phosphorylation (OXPHOS) system. In the OXPHOS system complexes I-IV generate an electrochemical gradient via electron transfer coupled to proton pumping. This gradient is then utilized by complex V (ATP synthase) to generate ATP from ADP and inorganic phosphate. COX deficiency in SURF1 associated LS leads to a reduction in OXPHOS output (Wedatilake et al. Orphanet J. Rare Dis. 2013. 8:96) and consecutive reduction in the ATP synthesis ability of cells.

[0147] RNA-seq of patient blood post treatment with Omaveloxolone treatment revealed an increase in activity of pathways associated with COX IV and the OXPHOS system (FIG. 15). Error bars represent the log2(errors) natively generated from the GSEA analysis of fold changes. Solid and hatched bars show significant and non-significant enrichment values, respectively. The magnitude of enrichment of each pathway showed a compounding effect with time similar to the NRF2 pathway activity described above. Expression was increased across all timepoints, though not all timepoints were significant. Further, expression correlated with the omaveloxolone dosing schedule; a reduction in pathway enrichment was observed at month five, following the dose reduction. Pathways analyzed are summarized below.

[0148] The ATP Synthesis Coupled Electron Transport pathway is a part of the GO (Gene Ontology) Biological Process database and is a large collection of genes involved in the transfer of electrons across a series of electron donors and acceptors, generating an electrochemical potential ultimately used for the synthesis of ATP. The biological relevance of this collection of genes is associated closely with the OXPHOS system. The ATP Synthesis Coupled Electron Transport pathway prominently features the following gene groups: COX (Complex IV), mitochondrial, NADH:Ubiquinone Oxidoreductase (NDUF) (Complex I), Succinate Dehydrogenase Complex genes (SDHA and SDHB) (Complex II) and Ubiquinol-Cytochrome C Reductase (UQCRs) (Complex III).

[0149] The Cytochrome Complex pathway is a part of the GO (Gene Ontology) Cellular Components database and consists of genes involved in the formation of various subunits of the cytochrome complex including respiratory chain complex III and IV. This includes COX, mitochondrial, and Ubiquinol-Cytochrome C Reductase genes (UQCRs).

[0150] The Respiratory Chain Complex IV pathway is a subset of the cytochrome complex pathway, and mainly includes COX and mitochondrial genes involved in the formation of complex IV.

[0151] The Electron Transport Chain OXPHOS System in Mitochondria pathway annotated in WikiPathways database measures the activity of the OXPHOS system using a set of genes mainly involved in the transport of electrons. This pathway shares many similarities with the GO pathway “ATP Synthesis Coupled Electron Transport”. In addition to the gene sets mentioned above for “ATP Synthesis Coupled Electron Transport” it also includes ATP Synthase genes which are a part of Complex V.

[0152] RNA-seq of patient blood post treatment with Omaveloxolone treatment revealed an increase in individual genes involved in complexes I-V that are a part of the oxidative phosphorylation (OXPHOS) pathway. Peak OXPHOS pathway enrichment was observed at month four post-treatment with Omaveloxolone followed by a decrease at month five due to the reduction in dose. Key OXPHOS pathway genes up-regulated at month four post Omaveloxolone treatment are summarized in the table below (Table 2). All fold changes are statistically significant. Genes are grouped by their function in the respective OXPHOS complex subunit.

[0153] Without wishing to be bound by theory, the up-regulation of genes across all major subcomplexes (complexes I-V) of the OXPHOS system involved in mitochondrial oxidative phosphorylation with Omaveloxolone indicates that OXPHOS enrichment is not due to a single gene but ubiquitous across all major OXPHOS related gene groups. This universality of Omaveloxolone’ s effect on all major gene groups involved in oxidative phosphorylation vs. a single gene could indicate that it could be therapeutic in other OXPHOS deficiencies.Table 2. OXPHOS pathway genes with increased expression in a LS subject following omaveloxolone treatment.Effect of omaveloxolone on COXIV activity

[0154] Blood was collected from a patient with Leigh Syndrome (as previously described) prior to treatment with omaveloxolone and at two, three, four, and five months after beginning omaveloxolone administration. PBMCs were prepared by Ficoll-Paque PLUS and cryopreserved until use. For the assessment of CoxIV activity, PBMCs were thawed, suspended in mitochondrial extraction buffer containing 225 mM D-Mannitol, 75 mM sucrose, two mM K2HPO4, and 20 mM HEPES, and lysed. A cytochrome c oxidase assay was performed according to the manufacturer's manual (Abeam, ab23971). Briefly, cell lysates were added to each well of a 96-well clear plate, ferrocytochrome c substrate solution was added to initiate the reaction, and absorbance at 550 nm was measured over 40 minutes at 30-second intervals. Cytochrome c oxidase activity, measured as absorbance at 550 nm caused by the oxidation of reduced cytochrome c, is expressed as micromoles of cytochrome c oxidized per minute per mg of protein, using an extinction coefficient (a) of 7.04 mM1FIG. 18A shows representative curves of Complex IV activity before and after omaveloxolone treatment, and FIG. 18B shows a comparison of Complex IV activity I pooled post-treatment samples versus pre-treatment samples. CoxIV activity significantly increases with omaveloxolone treatment.Effect of Omaveloxolone on PPARGC1A Expression

[0155] Oxidative phosphorylation is regulated by an extensive network of genes of which <1% are encoded by mitochondrial DNA and the rest (>1000) by nuclear DNA. Expression of these nuclear encoded genes is under control of a network of transcription factors, transcriptional coactivators, and transcriptional corepressors. Among the most well characterized is the transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGClu, gene - PPARGC1A). The expression of PGClu is tightly regulated both at the transcript and protein level with its main function being the recruitment of other factors that ultimately modulate the genes involved in mitochondrial energy production and oxidative phosphorylation. An increase in PPARGC1A could therefore be both an upstream factor viawhich Omaveloxolone up-regulates OXPHOS genes and a biomarker of increased OXPHOS activity.

[0156] Analysis of a publicly available dataset (GEO: GSE126360) from a study comparing a neuronal line with a pathogenic SURF1 mutation, and a neuronal line with the mutation corrected, showed up-regulation of PPARGC1A in the mutation-corrected cell line (FIG. 21 A). The analysis showed that the correction of the pathogenic SURF1 mutation led to an increase in PP ARGCI A transcripts; a ~2-fold increase was observed in 4-week-old neurons and ~4-fold in 8-week-old neurons (FIG. 21A). Additionally, PPARGC1A did not increase in Leigh Syndrome (LS, FIG. 21B, hatched bar) neurons during maturation, which showed low OXPHOS activity and consequently reduction of neurogenesis potential (which relies on energy production via oxidative phosphorylation). These analyses demonstrate that PPARGC1A is down-regulated in SURF1 neurons with reduced OXPHOS activity and that increase in PPARGC1A is correlated with neuronal maturation which involves increased energy production via increased OXPHOS activity.

[0157] PPARGC1A could therefore be a key biomarker of increased OXPHOS activity in a subject after administration of omaveloxolone. PPARGC1A was significantly lower in the plasma of a subject with Leigh Syndrome, prior to omaveloxolone administration, compared to the cohort of healthy age-matched adult controls (FIG. 22). PPARGC1A expression consistently increased during omaveloxolone administration through month 4, consistent with it being a biomarker for increased OXPHOS activity (FIG. 22). PPARGC1A expression dropped significantly at month 5, following reduction of omaveloxolone dose, which is consistent with the observed decreased OXPHOS activity at month 5. PPARGC1A could therefore be a key biomarker and the transcriptional coactivator via which Omaveloxolone rescues OXPHOS activity.Effect of Omaveloxolone on Leigh Syndrome (LS) Pathways

[0158] In-depth analysis of leading genes in LS pathways showed the effect of omaveloxolone treatment on OXPHOS pathways (FIG. 16). From left to right, panels represent one, two, four, and five months of treatment with omaveloxolone, respectively, compared to the pre-treatment control. Each point on the scatter plot corresponds to a gene, where the x-axis represents the log2(Fold Change) of that gene at the respective treatment time point compared to the pretreatment control and the y-axis represents the -logio(False discovery rate adjusted significance). Gene regulations above the dashed black line are significant.

[0159] The effect of omaveloxolone compounded up to month four, as demonstrated by the overall increase in significance and fold change magnitude of genes in the third panel. This was followed by a dramatic decrease in overall significance of fold change for these genes in the fourth panel at month 5 after the reduction in dosage. These results are consistent with data showing that omaveloxolone treatment of COX-deficient cells (which have an impaired OXPHOS system) showed a small but significant improvement in growth rate in glucose-free medium, in which OXPHOS system-impaired cells are restricted (Zighan et al. Mol. Biosci. 2022; 9: 890653). These data provide support for improvement in OXPHOS pathways during omaveloxolone treatment.Example 5: Omaveloxolone Effects on Control and Leigh Syndrome Fibroblasts

[0160] One healthy control cell line, one heterozygous healthy control cell line, and three patient-derived fibroblast cell lines were administered a range of concentrations of omaveloxolone. Mutations in the SURF1 gene in each cell line are listed in Table 3.Table 3. SURF1 mutations in fibroblast cell lines

[0161] Fibroblasts were plated onto 96-well plates in DMEM with 15% fetal bovine serum and incubated for one day, then omaveloxolone was administered at three concentrations (125 nM, 250 nM, and 500 nM). On Day 2 (24 hours after omaveloxolone treatment) cells were imaged using brightfield microscopy and then lysed for RNA extraction. Briefly, rRNA-depleted, polyadenylated mRNA was isolated with a KingFisher magnetic bead RNA isolation kit and reverse transcribed to cDNA.

[0162] Gene expression levels of SURF1 and the housekeeping gene beta-2-microglobin (B2M) were measured for each condition using qPCR (FIGs. 17A-17E). Omaveloxolone treatment demonstrated a dose-dependent, statistically significant increase in SURF1 gene expression in both patient- and control-derived fibroblasts while maintaining cell viability at all concentrations.Example 6: Expression of OXPHOS genes in Leigh Syndrome

[0163] RNA-seq of PBMCs collected from 13 healthy young adult donors aged 29 ± 1 years, was downloaded from project GEO: GSE263013 (Smith et al. GeroScience . 2024. https: / / doi.org / 10.1007 / sl l357-024-01126-y). The young adult subjects in the study were tightly controlled for lifestyle and age and shared a characteristic sedentary lifestyle.

[0164] The RNA-seq data from this control group was compared to that of a patient with Leigh Syndrome (described above; a 19-year-old female) prior to omaveloxolone administration, using two-condition differential expression analysis. The results from the differential expression were used to generate enrichment of biological pathways using Gene Set Enrichment Analysis (GSEA).

[0165] The patient with Leigh Syndrome comprised heterozygous mutations in the SURF1 gene. One of these mutations is a deletion of a start codon (c.312_321delinsAT (p.L105X); surfl c.-13_l ldel24). SURF1 transcripts in the Leigh Syndrome patient were present at only 20% of the amount present in the control subjects; this difference was statistically significant (FIG. 19A) Error bars show standard error in measurement. The Log2(Fold Change) value was significant with a q-value of 0.000004.

[0166] Pathways associated with biological functions impaired in Leigh Syndrome were significantly down-regulated in the patient with Leigh Syndrome, compared to the control subjects (FIG. 19B). All bars show statistically significant normalized enrichment scores. Pathways reported in the figure have q-value < 0.0005. These pathways are associated withdeficient electron transport and mitochondrial OXPHOS (oxidative phosphorylation) which lead to a reduction in the ATP synthesis ability of cells. Genes in these pathways were mostly down-regulated, indicating that these pathways are indeed deficient in Leigh Syndrome (FIG. 20). Crosses show significant and dots show insignificant fold changes, respectively. Each panel consists of genes comprising a pathway of interest.

[0167] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0168] All references, issued patents, and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entireties, for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of treating Leigh Syndrome in a subject in need thereof, the method comprising administering an effective amount of omaveloxolone or a pharmaceutically acceptable salt thereof to the subject.

2. The method of claim 1, wherein the subject carries one or more mutations in one or more genes associated with oxidative phosphorylation.

3. The method of claim 1 or claim 2, wherein the subject has a dysfunction, deficiency, or reduction in oxidative phosphorylation.

4. The method of any one of claims 1-3, wherein the subject has reduced expression or activity of PP ARGCI A.

5. The method of any one of claims 1-4 wherein the subject has one or more mutations in a SURF1 genomic sequence.

6. The method of any one of claims 2-5, wherein the one or more mutations comprises a frameshift mutation, a missense mutation, a nonsense mutation, or a mutation introducing a splicing alteration.

7. The method of any one of claims 2-6, wherein the one or more mutations result in a mutant gene encoding a truncated protein.

8. The method of any one of claims 2-7, wherein the subject is determined to have the one or more mutations by analysis of the subject’s genomic sequence.

9. The method of claim 8, wherein the subject is determined to have the one or more mutations by a method selected from the group consisting of: targeted variant analysis, deletion analysis, mutation scanning of select exons, mutation scanning of the entire coding region, sequence analysis of select exons, sequence analysis of the entire coding region, or RNA analysis of transcripts of a gene.

10. The method of any one of claims 1-9, wherein the subject has at least one SURF1 allele encoding a protein with residual function.

11. The method of any one of claims 1-10, wherein the method results in increased expression of SURF1 in the subject.

12. The method of any of claims 1-11, wherein omaveloxolone is administered to the subject orally or parenterally.

13. The method of any of claims 1-12, wherein the effective amount of omaveloxolone is about 1 mg to about 1000 mg per day.

14. The method of any of claims 1-13, wherein the effective amount of omaveloxolone is about 5 mg to about 150 mg per day.

15. The method of claim 14, wherein the effective amount of omaveloxolone is about 10 mg, about 20 mg, about 30 mg, about 40 mg, 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg per day.

16. The method of any one of claims 1-15, wherein the method results in plasma levels of omaveloxolone of at least 100 nM, at least 150 nM, at least 200 nM, at least 250 nM, at least 300 nM, at least 350 nM, at least 400 nM, at least 450 nM, or at least 500 nM.

17. The method of any of claims 1-16, wherein omaveloxolone is administered once or twice per day.

18. The method of any of claims 1-17, wherein omaveloxolone is administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

19. The method of any of claims 1-18, wherein the method further comprises administering an additional therapeutic agent.

20. The method of any of claims 1-19, wherein omaveloxolone is present in a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

21. The method of any of claims 1-20, wherein the method results in increased expression of one or more of GCLC, GCLM, HM0X1, NQO1, SRXN1, TXNRD1, FTL A FTHI.

22. The method of any of claims 1-21, wherein the method results in increased expression of one or more genes associated with cytochrome c oxidase IV (COX IV) and / or the oxidative phosphorylation (OXPHOS) system.

23. The method of any one of claims 1-22, wherein the method results in increased expression of PP ARGCI A.

24. The method of any one of claims 21-23, wherein expression is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%.

25. The method of claim 24, wherein expression is increased within 1 month of beginning administration of omaveloxolone.

26. The method of any one of claims 1-25, wherein the method results in reduction or improvement of one or more symptoms of Leigh Syndrome.

27. The method of any one of claims 1-26, wherein the method results in reduction of the subject’s NPMDS score.

28. The method of any of claims 1-27, wherein the subject is a mammal.

29. The method of claim 28, wherein the subject is human.

30. A method of increasing expression of one or more genes in the oxidative phosphorylation (OXPHOS) system.

31. The method of claim 30, wherein the one or more genes in the OXPHOS system are part of one or more pathways comprising ATP Synthesis Coupled Electron Transport, Cytochrome Complex, Respiratory Chain Complex IV, and Electron Transport Chain OXPHOS System in Mitochondria.

32. The method of claim 30 or claim 31, wherein the one or more genes are selected from NDUFA1, NDUFA2, NDUFA9, NDUFA10, NDUFA12, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NDUFV2; MT-ND1, MT-ND2, MT-ND3, MT- ND4, MT-ND5, MT-MD6, SDHA, UQCRQ, NDUFA4, COX8A; MT-CO3, MT-ATP6, NDUFAF2, NDUFAF4, NDUFAF5, NDUFAF6, C17ORF89, FOXRED1, NUBPL, SDHAF1, BCS1L, TTC19, SURF1, COXIO, COX15, SCO2, PET100, PDHA1; PDHX, PDHB, DLAT, DLD, BLD, TPK1, SLC19A3, LIPT1, LIAS, BOLA3, HIBCH, ECHS1, PDSS2, and ('009.

33. The method of any one of claims 30-32, wherein the method results in increased expression of one or more genes associated with the oxidative phosphorylation (OXPHOS) system.

34. The method of any one of claims 30-33, wherein the method results in increased expression of PP ARGCI A.

35. A method of treating a subject in need thereof, the method comprising administering an effective amount of omaveloxolone or a pharmaceutically acceptable salt thereof to the subject.

36. The method of claim 35, wherein the subject carries one or more mutations in one or more genes associated with oxidative phosphorylation.

37. The method of claim 35 or claim 36, wherein the subject has a dysfunction, deficiency, or reduction in oxidative phosphorylation.

38. The method of any one of claims 35-37, wherein the subject comprises reduced expression or activity of one or more genes associated with oxidative phosphorylation prior to the method.

39. The method of any one of claims 35-38, wherein the subject has reduced expression or activity of PP ARGCI A prior to the method.

40. The method of any one of claims 35-39, wherein the method results in increased expression of one or more genes associated with the oxidative phosphorylation (OXPHOS) system.

41. The method of any one of claims 35-40, wherein the method results in increased expression of PP ARGCI A.

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