WO2023240201A1 - Marqueurs sensibles à la frataxine pour surveiller la progression et le traitement du syndrome de leigh - Google Patents

Marqueurs sensibles à la frataxine pour surveiller la progression et le traitement du syndrome de leigh Download PDF

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WO2023240201A1
WO2023240201A1 PCT/US2023/068145 US2023068145W WO2023240201A1 WO 2023240201 A1 WO2023240201 A1 WO 2023240201A1 US 2023068145 W US2023068145 W US 2023068145W WO 2023240201 A1 WO2023240201 A1 WO 2023240201A1
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fxn
fsgms
therapy
subject
expression profile
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PCT/US2023/068145
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Joan David Bettoun
Matthew Garrett BAILE
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Larimar Therapeutics, Inc.
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Publication of WO2023240201A1 publication Critical patent/WO2023240201A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Mitochondrial diseases are a group of disorders caused by dysfunctional mitochondria, the cellular organelles that store potential energy in the form of adenosine triphosphate (ATP) molecules and are found in every cell of the human body except mature red blood cells.
  • ATP adenosine triphosphate
  • Leigh Syndrome or Leigh Disease is a progressive neurodegenerative disorder with a general onset in infancy or childhood that is characterized by the presence of lesions in the basal ganglia, brain stem and spinal cord. It is caused by a defect in oxidative phosphorylation (OXPHOS), with most mutations occurring in electron transport chain complex genes, while other mutations in genes that indirectly affect OXPHOS are also possible. Pathologic mutations in over 75 genes have been identified so far, with about 30% of Leigh Syndrome cases attributed to mutations in Complex I genes, and about 5% of Leigh Syndrome cases attributed to mutations in the NADH: ubiquinone oxidoreductase subunit S4 (NDUFS4) gene.
  • OXPHOS oxidative phosphorylation
  • Leigh Syndrome typically begins displaying symptoms within a few months to two years of age. Initial symptoms can include the loss of basic skills such as sucking, head control, walking and talking. These may be accompanied by other problems such as irritability, loss of appetite, vomiting and seizures. Eventually, the subject may also have heart, kidney, vision, and breathing complications. Leigh Syndrome is associated with neurodevelopmental delay and ataxia, and sometimes is also associated with epilepsy. Other characteristics of Leigh Syndrome include lactic acidosis, hypotonia, respiratory stress, visual impairment, and a possibility of additional cardiac, hepatologic, gastrointestinal and renal tubular symptoms.
  • the present disclosure is based, at least in part, on providing a set of markers, also referred to herein as FXN-sensitive genomic markers (or FSGMs), whose respective levels are positively or negatively correlated to frataxin (FXN) levels in a subject with a Leigh Syndrome or a cell from a subject with a Leigh Syndrome.
  • FXN-sensitive genomic markers or FSGMs
  • the FSGMs of the present disclosure are contrary regulated by a gene ablation or a genetic deficiency associated with Leigh Syndrome in a subject followed by FXN therapy.
  • said FSGMs of the present disclosure are both associated with a genetic deficiency associated with Leigh Syndrome in a subject and conversely associated with FXN in the subject with Leigh Syndrome.
  • the FSGMs disclosed herein were found to be sensitive to FXN levels in a model of Leigh Syndrome and are considered markers of FXN therapy in a subject with Leigh Syndrome.
  • any one or more of the FSGMs provided herein can serve as surrogate biomarker for FXN levels in a subject with Leigh Syndrome.
  • the FSGMs provided herein can be used to evaluate or to monitor efficacy of FXN therapy in a subject with Leigh Syndrome, as described herein.
  • the FSGMs provided herein can be used to evaluate and/or monitor an FXN therapy in a subject with Leigh Syndrome, e.g., determine, evaluate and/or monitor the efficacy of FXN therapy in a subject with Leigh Syndrome as described herein.
  • an FXN therapy e.g., efficacy of an FXN therapy
  • a subject with Leigh Syndrome can be determined, evaluated, and/or monitored based on the analysis of one or more FSGM expression profiles in samples obtained from a subject with Leigh Syndrome before, during and/or after administration or initiation of FXN therapy to the subject with Leigh Syndrome.
  • adjustments can be made to the FXN therapy in a subject with Leigh Syndrome, such as to, e.g., initiate an FXN therapy, increase a dose and/or administration frequency of an FXN therapy, decrease a dose and/or administration frequency of an FXN therapy, or cease FXN therapy in the subject.
  • subjects with Leigh Syndrome, French Canadian Type are excluded from the methods of the present disclosure.
  • the present disclosure provides a method for evaluating efficacy of a frataxin (FXN) therapy in a subject with Leigh Syndrome, the method comprising: (a) determining a baseline FXN expression profile for one or more FXN- sensitive genomic markers (FSGMs) in a sample obtained from the subject prior to administration of the FXN therapy; (b) determining an FXN therapy expression profile for the one or more FXN-sensitive genomic markers (FSGMs) in a sample obtained from the subject following administration of the FXN therapy; (c) comparing the FXN therapy expression profile determined in step (b) with the baseline FXN expression profile determined in step (a); and (d) determining efficacy of the FXN therapy based on the comparison in step (c); wherein the one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38,
  • the present disclosure provides a method for evaluating efficacy of a frataxin (FXN) therapy in a subject with Leigh Syndrome, the method comprising: (a) determining an FXN therapy expression profile for one or more FXN-sensitive genomic markers (FSGMs) in a sample obtained from the subject following administration of an FXN therapy; (b) comparing the subject FXN expression profile determined in step (a) with a reference FXN expression profile for the one or more FSGMs; and (c) determining efficacy of the FXN therapy based on the comparison in step (b); wherein the one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, m
  • the reference FXN expression profile is a baseline FXN expression profile for the one or more FSGMs.
  • the baseline FXN expression profile for the one or more FSGMs is determined in a sample obtained from a subject with Leigh Syndrome prior to administration of an FXN therapy.
  • the method further comprises determining a baseline FXN expression profile for the one or more FXN-sensitive genomic markers (FSGMs) in a sample obtained from the subject with Leigh Syndrome prior to administration of the FXN therapy.
  • FSGMs FXN-sensitive genomic markers
  • the one or more FSGMs comprise one or more of CYR61, THBS1, PTGS2, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or all 24 of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • At least one or more FSGMs are upregulated following treatment with FXN therapy.
  • the one or more FSGMs upregulated following treatment with FXN therapy comprise one or more of EGR1, EGR2, PTGS2, CUL2, ABCE1, EiFlAX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • At least one or more FSGMs are downregulated following treatment with FXN therapy.
  • the one or more FSGMs downregulated following treatment with FXN therapy comprise one or more of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A.
  • determining an FXN expression profile for one or more FSGMs comprises detecting the level of expression of the one or more FSGMs. In some embodiments, comparing the subject FXN therapy expression profile with the baseline FXN expression profile comprises comparing the level of expression of the one or more FSGMs in the FXN therapy expression profile with the level of expression of the corresponding one or more FSGMs in the baseline FXN expression profile.
  • the FXN therapy is determined to be effective.
  • EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP is increased in the FXN therapy expression profile as compared to the baseline FXN expression profile, the FXN therapy is determined to be effective.
  • the FXN therapy is determined to be effective when the expression level of CYR61, THBS1, UBE2D3, RPE26, RPE38, RPE32, RPE39 and RPS15A is decreased in the FXN therapy expression profile as compared to the baseline FXN expression profile.
  • determining an FXN therapy expression profile for one or more FSGMs comprises determining an FXN feature vector of values indicative of expression of the one or more FSGMs.
  • determining efficacy of the FXN therapy comprises determining a first FXN feature vector for the subject FXN therapy expression profile and a second FXN feature vector for the baseline FXN (-) expression profile and determining a distance between the first and second feature vectors.
  • determining the distance between the feature vectors comprises determining a scalar product of the first and second feature vectors.
  • the method further comprises determining a third feature vector for a normal FXN expression profile for the FSGMs for a healthy subject.
  • the method further comprises determining a distance between the second and third feature vectors. In some embodiments, the method further comprises determining a distance between the first and third feature vectors, and normalizing the distance between the first and third feature vectors to the distance between the second and third feature vectors. In some embodiments, the method further comprises using the normalized distance to determine effectiveness of the FXN therapy.
  • the FXN therapy expression profile is determined by one or more of sequencing, hybridization and amplification of RNA in the sample. In some embodiments, the FXN therapy expression profile is determined by HPEC/UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, EEISA, or any combination thereof.
  • the method further comprises recommending to a healthcare provider to modify the treatment with the FXN therapy based on the determination of efficacy for the FXN therapy. In some embodiments, the method further comprises obtaining a sample from the subject with Leigh Syndrome.
  • the sample is selected from the group consisting of a blood- derived sample, a buccal sample, a skin sample, a hair follicle and a muscle biopsy sample.
  • the blood-derived sample is a plasma sample, a serum sample, a whole blood sample or a platelet sample.
  • the sample from the subject with Leigh Syndrome is obtained at least 15 days following the last administration of the FXN therapy. In some embodiments, the sample from the subject with Leigh Syndrome is obtained 15 to 45 days following the last administraton of the FXN therapy.
  • the present disclosure provides a method of monitoring treatment of a subject with Leigh Syndrome with a frataxin (FXN) therapy, the method comprising: (a) determining a first FXN therapy expression profile for one or more FXN- sensitive genomic markers (FSGMs) in a first sample obtained from a subject with Leigh Syndrome at a first time point following administration of an FXN therapy to the subject, wherein the one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP; (b) determining a second FXN expression profile for the one or more FXN-sensitive FSGM
  • the method further comprises making a determination to maintain, increase or decrease the dose or administration frequency of the FXN therapy based on the comparison in step (c).
  • At least one dose of the FXN therapy is administered to the subject between obtaining the first time point and second time point.
  • the FXN therapy is not administered to the subject between obtaining the first time point and second time point.
  • the present disclosure provides a method for treating Leigh Syndrome, the method comprising: (a) determining an FXN therapy expression profile in a sample obtained from a subject with Leigh Syndrome for one or more FXN-sensitive genomic markers (FSGMs), (b) comparing the FXN expression profile of the sample with at least one other expression profile selected from the group consisting of normal FXN expression profile for the one or more FSGMs, baseline FXN expression profile for the one or more FSGMs, and FXN therapy expression profile for the one or more FSGMs, (c) classifying the FXN therapy expression profile determined in step (a) as corresponding to a normal FXN expression profile, baseline FXN expression profile or an FXN therapy expression profile, and (d) initiating or modulating an FXN therapy based on the classification of the FXN expression profile of the sample, wherein the one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE
  • modulating an FXN therapy comprises increasing the dosage, decreasing the dosage, increasing the administration frequency, or decreasing the administration frequence, of the FXN therapy.
  • the present disclosure provides a method of treating Leigh Syndrome in a subject, comprising: (a) determining an FXN expression profile for one or more FSGMs in a sample from a subject with Leigh Syndrome; and (b) recommending to a healthcare provider to administer an FXN therapy to the subject based on the subject FXN expression profile determined in step (a).
  • the pesent disclosure provides a method of treating Leigh Syndrome in a subject, comprising: (a) obtaining an FXN expression profile for one or more FSGMs in a sample obtained from a subject with Leigh Syndrome; and (b) administering an FXN therapy to the subject based on the subject FXN expression profile.
  • the method further comprises obtaining the sample from the subject for use in determing the FXN expression profile for the one or more FSGMs.
  • the present disclosure provides a method of detecting one or more frataxin- sensitive genomic markers (FSGMs) in a sample from a subject with Leigh Syndrome, comprising contacting the sample, or a portion thereof, with one or more reagents specific for detecting the level of each of one or more FSGMs, wherein the one or more FSGMs comprise one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, thereby detecting the FSGMs in the sample.
  • FSGMs frataxin- sensitive genomic markers
  • the subject is being treated or is scheduled to be treated with an FXN therapy.
  • the method further comprises obtaining the sample from the subject.
  • the FXN expression profile is determined by one or more of sequencing, hybridization and amplification of RNA in the sample. In some embodiments, the FXN expression profile is determined by HPLC/UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, or any combination thereof.
  • the method further comprises obtaining a sample from the subject.
  • the sample is selected from the group consisting of a blood- derived sample, a buccal sample, a skin sample, a hair follicle and a muscle biopsy sample.
  • the blood-derived sample is a plasma sample, a serum sample, a whole blood sample or a platelet sample.
  • the sample from the subject is obtained at least 15 days following the last administration of the FXN therapy. In some embodiments, the sample from the subject is obtained 15 to 45 days following the last administraton of the FXN therapy.
  • the FXN therapy comprises administration of an FXN fusion protein.
  • the FXN fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12.
  • the present disclosure provides a kit for detecting one or more frataxinsensitive genomic markers (FSGMs) in a sample obtained from a subject with Leigh Syndrome, comprising at least one reagent specific for detecting the level of each of the one or more FSGMs in the sample, wherein the one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, and a set of instructions for detecting the level of the one or more FSGMs in the sample from the subject.
  • FSGMs frataxinsensitive genomic markers
  • the reagent is an antibody that binds to the frataxin- sensitive genomic marker (FSGM) or an oligonucleotide that is complementary to the corresponding mRNA of the FSGM.
  • the present disclosure provides a panel of reagents for use in a method of monitoring or evaluating the efficacy of frataxin (FXN) therapy, the panel comprising at least two detection reagents, wherein each detection reagent is specific for the detection of at least one frataxin- sensitive genomic marker (FSGM) of a set of FSGMs, wherein the set of FSGMs comprises two or more markers selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt
  • the present disclosure provides a kit comprising the panel of the disclosure and a set of instructions for obtaining information relating to frataxin (FXN) therapy based on a level of the one or more frataxin- sensitive genomic markers (FSGMs).
  • FXN frataxin
  • FSGMs frataxin- sensitive genomic markers
  • panel A is a dot plot showing fold change in the levels of expression of selected gene markers in LRPPRC -deficient HEK293 cells vs. control cells (black dots) and in LRPPRC-deficient HEK293 cells treated with the exemplary FXN fusion protein vs. vehicle (white dots).
  • panel B is a dot plot showing fold change in the levels of expression of selected gene markers in LRPPRC-deficient Schwann cells vs. control cells (black dots) and in LRPPRC-deficient Schwann cells treated with the exemplary FXN fusion protein vs. vehicle (white dots).
  • Figure 2 is a dot plot of log2 mean gene expression levels vs. Iog2 fold change in gene expression in NDUFS4 KO mice vs. wild-type mice. DETAILED DESCRIPTION OF THE INVENTION
  • the present disclosure is based, at least in part, on providing a set of markers, also referred to herein as FXN-sensitive genomic markers (or FSGMs), whose respective levels are positively or negatively correlated to frataxin (FXN) levels in a subject with a Leigh Syndrome or a cell from a subject with a Leigh Syndrome.
  • FXN-sensitive genomic markers or FSGMs
  • the FSGMs of the present disclosure are contrary regulated by gene ablation or a genetic deficiency associated with Leigh Syndrome in a subject followed by FXN therapy.
  • said FSGMs of the present disclosure are both associated with a genetic deficiency associated with Leigh Syndrome in a subject and conversely associated with FXN in the subject with Leigh Syndrome.
  • the FSGMs disclosed herein were found to be sensitive to FXN in a model of Leigh Syndrome and are considered markers of FXN therapy in a subject with Leigh Syndrome. Therefore, these FSGMs can be used to determine and/or monitor efficacy of FXN therapy in a subject with Leigh Syndrome, as described herein.
  • the one or more FSGMs comprise one or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise two or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, , RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise three or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise four or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise five or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise six or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise seven or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eight or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise nine or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise ten or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eleven or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt- ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twelve or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise thirteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise fourteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise fifteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise sixteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise seventeen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eighteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise nineteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-one or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt- ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-two or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-three or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-four or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-five or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise one or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the efficacy of FXN therapy in a subject can be determined, evaluated, and/or monitored based on the analysis of one or more FSGM expression profiles before and after administration or initiation of FXN therapy in the subject. Based on the results of the FSGM expression profile analysis, adjustments can be made to the FXN therapy in a subject to, e.g., initiate, increase, decrease or cease FXN therapy in the subject.
  • the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.
  • amplification refers to any known in vitro procedure for obtaining multiple copies ("amplicons") of a target nucleic acid sequence or its complement or fragments thereof.
  • In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement.
  • Known in vitro amplification methods include, e.g., transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification and strand-displacement amplification (SDA including multiple stranddisplacement amplification method (MSDA)).
  • Replicase-mediated amplification uses selfreplicating RNA molecules, and a replicase such as Q-P-replicase (e.g., Kramer et al., U.S. Patent No. 4,786,600).
  • PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159).
  • LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., EP Pat.
  • SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., Walker et al., U.S. Patent. No. 5,422,252).
  • Two other known strand-displacement amplification methods do not require endonuclease nicking (Dattagupta et al., U.S. Patent. No. 6,087,133 and U.S. Patent. No. 6,124,120 (MS DA)).
  • oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase, (see generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8: 14-25 and (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6: 1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 2000, Molecular Cloning— A Laboratory Manual, Third Edition, CSH Laboratories).
  • the oligos are designed to bind to a complementary sequence under selected conditions.
  • the term "marker” or “biomarker” is a biological molecule, or a panel of biological molecules, whose expression level is correlated, e.g., either positively or negatively, with FXN levels in a subject with Leigh Syndrome or a cell from a subject with Leigh Syndrome.
  • a “marker” or a “biomarker” is an expressed gene, whose expression level may be measured by measuring levels of the corresponding mRNA or a protein.
  • a marker or biomarker of the invention whose respective levels are positively or negatively correlated to frataxin (FXN) levels in a cell or a sample obtained from a subject with Leigh Syndrome is referred to as a “Frataxin-sensitive genomic marker” or “FSGM”.
  • an FSGM is a marker or a biomarker, e.g., an expressed gene, that is differentially expressed in samples obtained from a healthy subject as compared to a sample obtained from a subject with Eeigh Syndrome.
  • an FSGM is contrary regulated by gene ablation or deficiency associated with Leigh Syndrome, followed by FXN therapy.
  • an FSGM may be a gene, e.g., PTGS2, that is expressed at a lower level in a sample obtained from a subject with Leigh Syndrome, as compared to a sample obtained from a healthy subject, and levels of which increase in a sample from a subject with Leigh Syndrome following FXN therapy.
  • an FSGM may be a gene, e.g., CYR61, that is expressed at a higher level in a sample obtained a subject with Leigh Syndrome, as compared to a sample obtained from a healthy subject, and levels of which decrease in a sample from a subject with Leigh Syndrome following FXN therapy.
  • an FSGM is a marker or a biomarker, e.g., an expressed gene, that is differentially expressed in a sample obtained from a healthy subject as compared to a sample obtained from a subject with Leigh Syndrome, as determined by principal component analysis, that shows an expression fold change of greater than 2 between groups, with Benjamini- Hochbert (BH) adjusted p-value ⁇ 0.05.
  • BH Benjamini- Hochbert
  • an FSGM is a marker or a biomarker, e.g., an expressed gene, that is differentially expressed in a sample obtained from a healthy subject as compared to a sample obtained from a subject with Leigh Syndrome, as determined by principal component analysis, that shows an expression fold change of less than 2, e.g., between 1 and 2, between groups, with Benjamini-Hochbert (BH) adjusted p-value ⁇ 0.05.
  • BH Benjamini-Hochbert
  • an FSGM is a marker or a biomarker, e.g., an expressed gene, that is differentially expressed in a sample obtained from a healthy subject as compared to a sample obtained from a subject with Leigh Syndrome, as determined by principal component analysis, that shows an expression fold change of greater than 2 between groups, with Benjamini- Hochbert (BH) adjusted p-value > 0.05.
  • BH Benjamini- Hochbert
  • the FSGMs of the present disclosure are contrary regulated by gene ablation or deficiency associated with Leigh Syndrome, followed by FXN therapy.
  • the FSGMs of the present disclosure are both associated with a gene ablation or deficiency associated with Leigh Syndrome in a subject and conversely associated with FXN.
  • An FSGM of the invention can be used to detect and/or monitor (e.g., serve as a surrogate for) FXN levels in a sample, e.g., a cell or tissue sample.
  • an FSGM is selected from the group consisting of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • an FSGM is selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • Reference to the FSGMs listed above is understood to include reference to any mutants, variants, derivatives, or orthologs thereof.
  • control sample refers to any clinically relevant comparative sample, or comparative FXN expression profile, including, for example, a sample from a healthy subject (z.e., a subject who does not have Leigh Syndrome), a normal FXN expression profile, a sample obtained from a subject with Leigh Syndrome, a baseline (FXN-) expression profile, or a sample obtained from a subject following administration of FXN therapy, or an FXN expression profile.
  • a control sample can also be a sample obtained from a subject with Leigh Syndrome from an earlier time point, e.g., prior to treatment with FXN therapy.
  • a control sample can be a purified sample, protein, and/or nucleic acid provided with a kit.
  • control samples can be diluted, for example, in a dilution series to allow for quantitative measurement of levels of analytes, e.g., markers, in test samples.
  • a control sample may include a sample derived from one or more subjects.
  • a control sample may also be a sample taken at an earlier time point from the subject to be assessed.
  • the control sample could be a sample taken from the subject to be assessed before treatment with FXN therapy.
  • the control sample may also be a sample from an animal model, or from a tissue or cell line derived from the animal model of Leigh Syndrome.
  • the level of activity or expression of one or more FSGMs consists of a group of measurements that may be determined, e.g., based on any appropriate statistical measurement, such as, for example, measures of central tendency including average, median, or modal values.
  • “different from a control” is preferably statistically significantly different from a control.
  • change, altered, increased or decreased is understood as having a level of the one or more FSGM to be detected at a level that is statistically different, e.g., increased or decreased, as compared to a control sample or to a predetermined threshold value, e.g., from a healthy subject (z.e., a subject who does not have Leigh Syndrome), or a sample from a subject with Leigh Syndrome.
  • Changed, altered, increased or decreased, as compared to control or threshold value can also include a difference in the rate of change of the level of one or more FSGMs obtained in a series of at least two subject samples obtained over time.
  • Determination of statistical significance is within the ability of those skilled in the art and can include any acceptable means for determining and/or measuring statistical significance, such as, for example, the number of standard deviations from the mean that constitute a positive or negative result, an increase in the detected level of an FSGM in a sample versus a control, wherein the increase is above some threshold value, or a decrease in the detected level of an FSGM in a sample versus a control, wherein the decrease is below some threshold value.
  • detecting”, “detection”, “determining”, and the like are understood to refer to identification of the presence and/or level of one or more FSGMs selected from the group consisting of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • DNA or "RNA” molecule or sequence (as well as sometimes the term “oligonucleotide”) refers to a molecule comprised generally of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C).
  • A deoxyribonucleotides
  • G guanine
  • T thymine
  • C cytosine
  • T is replaced by uracil (U).
  • the terms “subject with Leigh Syndrome” encompasses any subject who has been diagnosed with Leigh Syndrome.
  • a subject with Leigh Syndrome may have any one or more genetic abonormality or deficienty that is associated with Leigh Syndrome.
  • a subject with Leigh Syndrome has one or more deficiencies in the genes encoding pyruvate dehydrogenase (PDHC), Complex I, Complex II, Complex III, Complex IV, Complex V, SURF1 or MT-ATP6.
  • PDHC pyruvate dehydrogenase
  • a subject with Leigh Syndrome has not yet received FXN therapy and is, therefore, therapy naive.
  • a subject with Leigh Syndrome is scheduled to receive FXN therapy.
  • a subject with Eeigh Syndrome is currently undergoing FXN therapy.
  • a subject with Eeigh Syndrome has already undergone an FXN therapy.
  • the term “FXN therapy” refers to frataxin therapy in a subject which results in increased expression or activity of frataxin in the subject.
  • the FXN therapy may be carried out through delivery of any therapeutic agent capable of increasing levels of FXN in a subject with Leigh Syndrome or a cell obtained from a subject with Leigh Syndrome, or an in vitro or in vivo model of Leigh Syndrome.
  • the FXN therapy comprises FXN protein delivery or delivery of a nucleic acid encoding FXN to a subject.
  • FXN therapy comprises administration of a nucleic acid capable of increasing FXN levels in a subject with Leigh Syndrome.
  • the nucleic acid may be mRNA, siRNA or an antisense oligonucleotide.
  • the FXN therapy comprises FXN protein delivery to a subject with Eeigh Syndrome.
  • the FXN protein delivery can include delivery of FXN protein or delivery of a FXN fusion protein.
  • FXN fusion protein refers to full length FXN or a fragment of FXN fused to a full length or a fragment of a different protein, or to a peptide.
  • an FXN fusion protein comprises full-length hFXN (SEQ ID NO: 1) or mature hFXN (SEQ ID NO: 2), as described herein.
  • the FXN protein or fragment thereof is fused to a cell penetrating peptide (CPP).
  • the CPP is an HIV-TAT polypeptide.
  • FXN therapy comprises administering to a subject with Leigh Syndrome an FXN fusion protein comprising or consisting of SEQ ID NO: 12.
  • disorders disorders
  • diseases disorders
  • abnormal state refers to any deviation from the normal structure or function of any part, organ, or system of the body (or any combination thereof).
  • a specific disease is manifested by characteristic symptoms and signs, including biological, chemical, and physical changes, and is often associated with a variety of other factors including, but not limited to, demographic, environmental, employment, genetic, and medically historical factors.
  • An early stage disease state includes a state wherein one or more physical symptoms are not yet detectable. Certain characteristic signs, symptoms, and related factors can be quantitated through a variety of methods to yield important diagnostic information.
  • mitochondrial disease refers to a disease which is the result of either inherited or spontaneous mutations in mtDNA or nDNA which leads to altered functions of the proteins or RNA molecules that normally reside in mitochondria, which decreases the functions of the mitochondria to induce diseases of various types in, for example, the central nervous system, skeletal muscles, heart, eyes, liver, kidneys, large intestine (colon), small intestine, internal ear and pancreas; as well as blood, skin and endocrine glands.
  • the mitochondrial disease is Leigh Syndrome.
  • the methods of the present disclosure do not encompass Leigh Syndrome, French Canadian Type.
  • progression of Leigh Syndrome refers to worsening of a condition of a subject with Leigh Syndrome over time. This term encompasses an increase in severity and/or duration of existing symptoms of Leigh Syndrome in the subject and/or appearance of one or more new symptoms of Leigh Syndrome in the subject.
  • a sample obtained at an “earlier time point” is a sample that was obtained at a sufficient time in the past such that clinically relevant information could be obtained in the sample from the earlier time point as compared to the later time point.
  • an earlier time point is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or 1, 2, 3, 4, 5, 6, or 7 days earlier.
  • an earlier time point is at least one, two, three or four weeks earlier.
  • an earlier time point is at least six weeks earlier.
  • an earlier time point is at least two months earlier.
  • an earlier time point is at least three months earlier.
  • an earlier time point is at least six months earlier. In certain embodiments, an earlier time point is at least nine months earlier. In certain embodiments, an earlier time point is at least one year earlier.
  • Multiple subject samples e.g., 3, 4, 5, 6, 7, or more
  • Appropriate intervals for testing for a particular subject can be determined by one of skill in the art based on ordinary considerations.
  • expression is used herein to mean the process by which a polypeptide is produced from DNA. The process involves the transcription of the gene into mRNA and the translation of this mRNA into a polypeptide. Depending on the context in which used, “expression” may refer to the production of RNA, or protein, or both.
  • expression profile is used to include a genomic expression profile, meaning an expression profile of RNAs, or specifically of mRNAs or transcripts, or a protein expression profile.
  • expression profile may refer to a set of data obtained for mRNA expression. It may refer to the raw data in the readings of a PCR apparatus for example, or to the normalized expression values.
  • Expression profiles may be determined by any convenient means for measuring a level of a nucleic acid sequence such as quantitative hybridization of mRNA, labeled mRNA, amplified mRNA, cDNA, etc., quantitative PCR, and other techniques known to a person skilled in the art or described herein.
  • Expression profiles enable analysis of differential gene expression between two or more samples, between samples and control, as well as between samples and thresholds.
  • An expression profile can also be determined by any means known to a person skilled in the art or described herein for measuring the level of a protein or a polypeptide, e.g., mass spectrometry, immunodetection assays, e.g., ELISA, etc.
  • the term “FXN expression profile” includes any one of the following three FXN expression profiles: a normal FXN expression profile, a baseline FXN expression profile, or an FXN therapy expression profile.
  • the baseline FXN expression profile can be used as a control.
  • the normal FXN expression profile can be used as a control.
  • the term “FXN expression profile” for one or more FSGMs refers to the expression level (e.g., RNA level or protein level) of the one or more FSGMs or to a value or set of values indicative of the level of expression of the one or more FSGMs.
  • an FXN expression profile comprises a feature vector of values indicative of expression of the one or more FSGMs.
  • “determining an FXN expression profile” for one or more FSGMs comprises detecting the expression level (e.g., RNA level or protein level) of the one or more FSGMs.
  • “determining an FXN expression profile” for one or more FSGMs comprises determining a feature vector of values indicative of the expression level (e.g., RNA level or protein level) of the one or more FSGMs.
  • normal FXN expression profile refers to an expression profile of one or more FSGMs in a sample obtained from a normal, healthy subject or subjects (z.e., a subject or subjects that do not have Leigh Syndrome). In some embodiments, a “normal FXN expression profile” also encompasses an average of multiple, e.g., two or more, normal FXN expression profiles (e.g., from two or more subjects).
  • baseline FXN expression profile refers to the expression profile of one or more FSGMs in a sample from a subject with Leigh Syndrome prior to treatment with an FXN therapy. In some embodiments, the term “baseline FXN expression profile encompasses an average of multiple, e.g., two or more, baseline FXN expression profiles (e.g., from two more subjects).
  • An average FXN expression profile e.g., an average normal FXN expression profile or an average baseline FXN expression profile, may be determined by methods known in the art, e.g., by determining an expression level of one or more FSGMs in samples obtained from two or more subjects, e.g., normal subjects or subjects with Leigh Syndrome, and then calculating an average expression level of the one or more FSGMs.
  • the term "reference FXN expression profile” encompasses a “normal FXN expression profile” and a “baseline FXN expression profile”, i.e., can be either one.
  • the reference FXN expression profile e.g., reference normal FXN expression profile or reference baseline FXN expression profile can be used as a control, e.g., for comparing to an FXN expression profile to evaluate response of a subject to an FXN therapy.
  • FXN therapy expression profile refers to the expression profile for one or more FSGMs in a sample obtained from a subject with Leigh Syndrome subsequent to adminitration of at least one dose of an FXN therapy.
  • treatment with FXN therapy referes to administration to a subject with Leigh Syndrome of at least one dose of an FXN therapy.
  • following treatment with FXN therapy refers to at least 1 day, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or more days after administration of a dose of an FXN therapy.
  • the term “evaluate response of a subject to FXN therapy” encompasses evaluating efficacy of FXN therapy by (a) determining an FXN therapy expression profile in a sample from a subject with Leigh Syndrome following treatment with FXN therapy;
  • step (c) using the comparison in step (b) to evaluate or to determine efficacy of the FXN therapy.
  • the reference FXN expression profile is a baseline FXN expression profile, i.e., the expression profile of one or more FSGMs in a sample obtained from a subject with Leigh Syndrome prior to treatment with FXN therapy.
  • a difference between the FXN therapy expression profile and the baseline FXN expression profile is indicative of the efficacy of or response to the FXN therapy.
  • the reference FXN expression profile is a normal FXN expression profile, i.e., the expression profile of one or more FSGMs in a sample obtained from a normal subject, e.g., a subject who does not have Leigh Syndrome.
  • a comparison between the FXN therapy expression profile and the normal FXN expression profile e.g., a similarity between the two profiles, is indicative of the efficacy of or response to the FXN therapy.
  • a “higher level of expression”, “higher level”, “increased level,” and the like of an FSGM refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least 25% more, at least 50% more, at least 75% more, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten times the expression level of the FSGM in a control sample (e.g., a sample from a healthy subject, a sample from a subject with Leigh Syndrome, or a sample from a subject with Leigh Syndrome following FXN therapy) and preferably, the average expression level of the FSGM or FSGMs in several control samples.
  • a control sample e.g., a sample from a healthy subject, a sample from a subject with Leigh Syndrome, or a sample from a subject with Leigh Syndrome following FXN therapy
  • hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double- stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 2000, supra and Ausubel et al., 1994, supra, or further in Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach” IRL Press Oxford, Washington D.C., (1985)) and are commonly known in the art.
  • a nitrocellulose filter incubated overnight at a temperature representative of the desired stringency condition (60-65 °C for high stringency, 50-60 °C for moderate stringency and 40-45 °C for low stringency conditions) with a labeled probe in a solution containing high salt (6xSSC or 5xSSPE), 5xDenhardfs solution, 0.5% SDS, and 100 pg/ml denatured carrier DNA (e.g., salmon sperm DNA).
  • a temperature representative of the desired stringency condition 60-65 °C for high stringency, 50-60 °C for moderate stringency and 40-45 °C for low stringency conditions
  • 6xSSC or 5xSSPE high salt
  • 5xDenhardfs solution 0.5% SDS
  • 100 pg/ml denatured carrier DNA e.g., salmon sperm DNA
  • the non-specific ally binding probe can then be washed off the filter by several washes in 0.2xSSC/0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42 °C (moderate stringency) or 65 °C (high stringency).
  • the salt and SDS concentration of the washing solutions may also be adjusted to accommodate for the desired stringency.
  • the selected temperature and salt concentration is based on the melting temperature (Tm) of the DNA hybrid.
  • Tm melting temperature
  • RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well-known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al., 2000, supra).
  • hybridization kits e.g., ExpressHyb® from BD Biosciences Clonetech
  • the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • Hybridizing nucleic acid molecules also comprise fragments of the above described molecules.
  • nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules.
  • a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed).
  • a solid support e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed.
  • nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95% identity, more preferably at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection.
  • the term “identical” or “percent identity” refers to nucleic acid or amino acid sequences that do not comprise internal non-matching deletions or additions in the sequences relative to one another, i.e., the nucleic acid or amino acid sequences are of the same length and have a specified percentage of nucleotides or amino acid residues that are the same.
  • Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence.
  • the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length.
  • Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.
  • the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity.
  • CLUSTALW does take sequence gaps into account in its identity calculations.
  • the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10.
  • the present invention also relates to nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above-described hybridizing molecule. When used in accordance with the present invention the term "being degenerate as a result of the genetic code" means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.
  • the present invention also relates to nucleic acid molecules which comprise one or more mutations or deletions, and to nucleic acid molecules which hybridize to one of the herein described nucleic acid molecules, which show (a) mutation(s) or (a) deletion(s).
  • label refers to a molecular moiety or compound that can be detected or can lead to a detectable signal.
  • a label is joined, directly or indirectly, to a molecule, such as an antibody, a nucleic acid probe or the protein/antigen or nucleic acid to be detected (e.g., an amplified sequence).
  • Direct labeling can occur through bonds or interactions that link the label to the nucleic acid (e.g., covalent bonds or non-covalent interactions), whereas indirect labeling can occur through the use of a "linker” or bridging moiety, such as oligonucleotide(s) or small molecule carbon chains, which is either directly or indirectly labeled.
  • Bridging moieties may amplify a detectable signal.
  • Labels can include any detectable moiety (e.g., a radionuclide, ligand such as biotin or avidin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and fluorescent compound).
  • the label on a labeled probe is detectable in a homogeneous assay system, i.e., in a mixture, the bound label exhibits a detectable change compared to an unbound label.
  • level of expression of a gene refers to the level of mRNA, as well as pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s) and degradation products, or the level of protein, encoded by the gene in the cell.
  • level of one or more FSGMs means the absolute or relative amount or concentration of the FSGM in the sample.
  • a “lower level of expression” or “lower level” or “decreased level” and the like of an FSGM refers to an expression level in a test sample that is less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the expression level of the FSGM in a control sample (e.g., a sample from a healthy subject, a sample from a subject with Leigh Syndrome, or a sample from a subject following FXN therapy) and preferably, the average expression level of the FSGM in several control samples.
  • a control sample e.g., a sample from a healthy subject, a sample from a subject with Leigh Syndrome, or a sample from a subject following FXN therapy
  • nucleic acid molecule refers to a polymer of nucleotides, including an FSGM.
  • Non-limiting examples thereof include DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA) and chimeras thereof.
  • the nucleic acid molecule can be obtained by cloning techniques or synthesized.
  • DNA can be double- stranded or singlestranded (coding strand or non-coding strand [antisense]).
  • Conventional ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) are included in the term "nucleic acid” and polynucleotides as are analogs thereof.
  • a nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (referred to as "peptide nucleic acids” (PNA); Hydig-Hielsen et al., PCT Inti Pub. No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages or combinations thereof.
  • Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy substitutions (containing a 2'-O-methylribofuranosyl moiety; see PCT No.
  • Nitrogenous bases may be conventional bases (A, G, C, T, U), known analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992), or known derivatives of purine or pyrimidine bases (see, Cook, PCT Infl Pub. No. WO 93/13121) or "abasic" residues in which the backbone includes no nitrogenous base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481).
  • a nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs).
  • oligonucleotides or “oligos” define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill.
  • An oligonucleotide can be synthesized chemically or derived by cloning according to well-known methods. While they are usually in a single-stranded form, they can be in a double-stranded form and even contain a "regulatory region". They can contain natural rare or synthetic nucleotides. They can be designed to enhance a chosen criterion like stability for example. Chimeras of deoxyribonucleotides and ribonucleotides may also be within the scope of the present invention.
  • patient or “subject” can mean either a human or non-human animal, preferably a mammal.
  • subject is meant any animal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and birds.
  • a human subject may be referred to as a patient.
  • a "probe” is meant to include a nucleic acid oligomer or oligonucleotide that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (z.e., resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (z.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence).
  • a probe's "target” generally refers to a sequence within an amplified nucleic acid sequence (z.e., a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or "base pairing." Sequences that are "sufficiently complementary” allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely complementary.
  • a probe may be labeled or unlabeled.
  • a probe can be produced by molecular cloning of a specific DNA sequence or it can also be synthesized. Numerous primers and probes which can be designed and used in the context of the present invention can be readily determined by a person of ordinary skill in the art to which the present invention pertains.
  • a "reference level" of an FSGM may be an absolute or relative amount or concentration of the FSGM, a presence or absence of the FSGM, a range of amount or concentration of the FSGM, a minimum and/or maximum amount or concentration of the FSGM, a mean amount or concentration of the FSGM, and/or a median amount or concentration of the FSGM; and, in addition, “reference levels” of combinations of FSGMs may also be ratios of absolute or relative amounts or concentrations of two or more FSGMs with respect to each other.
  • Appropriate positive and negative reference levels of FSGMs for a particular disease state, phenotype, or lack thereof may be determined by measuring levels of desired FSGMs in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age-matched so that comparisons may be made between FSGM levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels may also be tailored to specific techniques that are used to measure levels of FSGMs in biological samples (e.g., LC-MS, GC-MS, etc.), where the levels of FSGMs may differ based on the specific technique that is used.
  • sample or “biological sample” includes a specimen or culture obtained from any source.
  • a sample includes any specimen or culture that comprises cells in which FXN therapy expression profile may be analyzed.
  • a sample includes any specimen or culture from a subject with Leigh Syndrome, e.g., a subject with Leigh Syndrome being treated with FXN therapy.
  • biological samples can be obtained from a solid tissue sample, preferably a buccal sample, alternatively a skin biopsy sample, skin strip, hair follicle, muscle biopsy sample, or a body fluid sample such as blood (including any blood product, such as whole blood, plasma, serum, or specific types of cells of the blood), urine, saliva, or seminal fluid.
  • the biological sample is a buccal sample.
  • the biological sample is a skin sample, e.g., a skin biopsy sample or a skin strip.
  • a sample can comprise exosomes which may be harvested in order to be tested for FSGMs transcripts.
  • the phrase "specific binding” or “specifically binding” when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (z.e., the antigenic determinant or epitope) on the protein; in other words the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A,” the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.
  • a “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or having a high percentage of identity (e.g., at least 80% identity) with all or a portion of a mature mRNA made by transcription of an FSGM of the invention and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.
  • a polynucleotide e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.
  • FXN-Sensitive Genomic Markers (FSGMs) of the Invention FXN-Sensitive Genomic Markers (FSGMs) of the Invention
  • the present disclosure provides a set of markers, also referred to herein as FXN-sensitive genomic markers (FSGMs), whose respective levels are positively or negatively correlated to frataxin (FXN) levels in a cell or a subject (e.g., a sample from a subject) with Leigh Syndrome.
  • FXN-sensitive genomic markers FXN-sensitive genomic markers
  • the FSGMs of the present disclosure are contrary regulated by in a subject with Leigh Syndrome before and after FXN therapy.
  • an FSGM of the present disclosure may be present at a modulated, i.e., lower or a higher, level in a subject with Leigh Syndrome as compared to the level of the FSGM present in a healthy subject, and the level of the FSGM may be altered towards, or similar to the level of the FSGM in a healthy subject following FXN therapy.
  • an FSGM of the present disclosure may be present at a lower level in a subject with Leigh Syndrome as compared to the level of the FSGM present in a healthy subject, and the level of the FSGM may be increased following FXN therapy.
  • an FSGM of the present disclosure may be present at a higher level in a subject with Leigh Syndrome as compared to the level of the FSGM present in a healthy subject, and the level of the FSGM may be decreased following FXN therapy.
  • the present invention provides a method for determining, evaluating, and/or monitoring the effectiveness of FXN therapy in a subject with Leigh Syndrome comprising determining: (i) a baseline FXN expression profile for one or more FSGMs in a sample from a subject with Leigh Syndrome prior to treatment with FXN therapy; and (ii) determining a subject FXN therapy expression profile for the one or more FSGMs in a sample from the subject undergoing FXN therapy or subsequent to treatment with FXN therapy; comparing the subject FXN therapy expression profile with the baseline FXN expression profile; and using the results of the comparison to determine, evaluate or monitor effectiveness of the FXN therapy.
  • adjustments can be made to the FXN therapy in the subject to, e.g., initiate, increase (e.g., increase dose and/or frequency of administration), decrease (e.g., decrease dose and/or frequency of administration) or cease FXN therapy in the subject.
  • increase e.g., increase dose and/or frequency of administration
  • decrease e.g., decrease dose and/or frequency of administration
  • Another aspect of the disclosure relates to providing a method for identifying one or more FSGMs, which are markers whose expression is sensitive to FXN levels in a cell from a subject with Leigh Syndrome.
  • the method comprises determining the expression profile of one or more genes in a sample obtained from a healthy subject, e.g., a subject who does not have Leigh Syndrome, referred to herein as a normal FXN expression profile; determining the expression profile of the one or more genes in a sample from a subject with Leigh Syndrome, referred to herein as a baseline FXN expression profile; and comparing the normal FXN expression profile with the baseline FXN expression profile; wherein the markers whose expression is altered in the baseline FXN expression profile compared to the normal FXN expression profile are identified as FSGMs.
  • the method for determining FSGMs may comprise a comparison between the expression profiles of one or more genes obtained from a sample from a subject with Leigh Syndrome before receiving FXN therapy (baseline FXN expression profile) and the expression profiles of the one or more genes obtained from a sample from a subject with Leigh Syndrome during or after receiving FXN therapy.
  • the gene expression profile from a sample obtained from a subject with Leigh Syndrome during or after FXN therapy is also referred to herein as an FXN therapy expression profile.
  • the markers whose expression is altered in the FXN expression profile as compared to the baseline FXN expression profile are identified as FSGMs.
  • the FSGMs that were determined by a method of an embodiment of the disclosure are selected from the group consisting of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • the FSGMs that were determined by a method of an embodiment of the disclosure are selected from the group consisting of ATF3, NR4al, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • the FSGMs that were determined by a method of an embodiment of the disclosure are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • Table 1 below lists the FSGMs of the present disclosure and their NCBI Accession Nos. for mRNA sequence.
  • the FSGMs of the invention include, but are not limited to any one or any combination of more than one of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • the FSGMs of the invention include any one or any combination of more than one of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • Methods, kits, and panels provided herein include one or any combination of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more FSGMs selected from ATF3, NR4al, EGR1, EGR2,
  • the one or more FSGMs of the invention comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) markers selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • markers selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3,
  • the one or more FSGMs comprise one or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise two or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise three or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise four or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise five or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise six or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise seven or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eight or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- ND1, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise nine or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise ten or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eleven or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt- ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twelve or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise thirteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise fourteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise fifteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise sixteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise seventeen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eighteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise nineteen or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-one or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt- ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-two or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-three or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-four or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-five or more of ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs of the invention comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) markers selected from EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • markers selected from EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt
  • the one or more FSGMs comprise one or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise two or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise three or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt- ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise four or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise five or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise six or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise seven or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eight or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise nine or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise ten or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eleven or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twelve or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise thirteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise fourteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise fifteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise sixteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise seventeen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eighteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise nineteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-one or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-two or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt- ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty-three or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise ATF3. In some embodiments, the one or more FSGMs comprise NR4al. In some embodiments, the one or more FSGMs comprise EGR1. In some embodiments, the one or more FSGMs comprise EGR2. In some embodiments, the one or more FSGMs comprise CYR61. In some embodiments, the one or more FSGMs comprise THBS1. In some embodiments, the one or more FSGMs comprise PTGS2. In some embodiments, the one or more FSGMs comprise UBE2D3. In some embodiments, the one or more FSGMs comprise CUL2. In some embodiments, the one or more FSGMs comprise ABCE1.
  • the one or more FSGMs comprise EiFlAX. In some embodiments, the one or more FSGMs comprise RPL26. In some embodiments, the one or more FSGMs comprise RPL38. In some embodiments, the one or more FSGMs comprise RPS27L. In some embodiments, the one or more FSGMs comprise RPL10. In some embodiments, the one or more FSGMs comprise RPL32. In some embodiments, the one or more FSGMs comprise RPL39. In some embodiments, the one or more FSGMs comprise , RPS15A. In some embodiments, the one or more FSGMs comprise mt- ATP8. In some embodiments, the one or more FSGMs comprise mt-ATP6.
  • the one or more FSGMs comprise mt-ND3. In some embodiments, the one or more FSGMs comprise mt- NDl. In some embodiments, the one or more FSGMs comprise mt-ND4. In some embodiments, the one or more FSGMs comprise mt-CO3. In some embodiments, the one or more FSGMs comprise CYCs. In some embodiments, the one or more FSGMs comprise SLIRP.
  • the one or more FSGMs comprise EGR1 and EGR2. In some embodiments, the one or more FSGMs comprise EGR1 and CYR61. In some embodiments, the one or more FSGMs comprise EGR1 and THBS1. In some embodiments, the one or more FSGMs comprise EGR1 and PTGS2. In some embodiments, the one or more FSGMs comprise EGR1 and UBE2D3. In some embodiments, the one or more FSGMs comprise EGR1 and CUL2. In some embodiments, the one or more FSGMs comprise EGR1 and ABCE1. In some embodiments, the one or more FSGMs comprise EGR1 and EiFlAX.
  • the one or more FSGMs comprise EGR1 and RPL26. In some embodiments, the one or more FSGMs comprise EGR1 and RPL38. In some embodiments, the one or more FSGMs comprise EGR1 and RPS27L. In some embodiments, the one or more FSGMs comprise EGR1 and RPL10. In some embodiments, the one or more FSGMs comprise EGR1 and RPL32. In some embodiments, the one or more FSGMs comprise EGR1 and RPL39. In some embodiments, the one or more FSGMs comprise EGR1 and RPS15A. In some embodiments, the one or more FSGMs comprise EGR1 and mt-ATP8.
  • the one or more FSGMs comprise EGR1 and mt-ATP6. In some embodiments, the one or more FSGMs comprise EGR1 and mt-ND3. In some embodiments, the one or more FSGMs comprise EGR1 and mt-NDl. In some embodiments, the one or more FSGMs comprise EGR1 and mt-ND4. In some embodiments, the one or more FSGMs comprise EGR1 and mt-CO3. In some embodiments, the one or more FSGMs comprise EGR1 and CYCs. In some embodiments, the one or more FSGMs comprise EGR1 and SLIRP.
  • the one or more FSGMs comprise EGR2 and CYR61. In some embodiments, the one or more FSGMs comprise EGR2 and THBS1. In some embodiments, the one or more FSGMs comprise EGR2 and PTGS2. In some embodiments, the one or more FSGMs comprise EGR2 and UBE2D3. In some embodiments, the one or more FSGMs comprise EGR2 and CUL2. In some embodiments, the one or more FSGMs comprise EGR2 and ABCE1. In some embodiments, the one or more FSGMs comprise EGR2 and EiFlAX. In some embodiments, the one or more FSGMs comprise EGR2 and RPL26.
  • the one or more FSGMs comprise EGR2 and RPL38. In some embodiments, the one or more FSGMs comprise EGR2 and RPS27L. In some embodiments, the one or more FSGMs comprise EGR2 and RPL10. In some embodiments, the one or more FSGMs comprise EGR2 and RPL32. In some embodiments, the one or more FSGMs comprise EGR2 and RPL39. In some embodiments, the one or more FSGMs comprise EGR2 and RPS15A. In some embodiments, the one or more FSGMs comprise EGR2 and mt-ATP8. In some embodiments, the one or more FSGMs comprise EGR2 and mt-ATP6.
  • the one or more FSGMs comprise EGR2 and mt-ND3. In some embodiments, the one or more FSGMs comprise EGR2 and mt-NDl. In some embodiments, the one or more FSGMs comprise EGR2 and mt- ND4. In some embodiments, the one or more FSGMs comprise EGR2 and mt-CO3. In some embodiments, the one or more FSGMs comprise EGR2 and CYCs. In some embodiments, the one or more FSGMs comprise EGR2 and SEIRP.
  • the one or more FSGMs comprise CYR61 and THBS1. In some embodiments, the one or more FSGMs comprise CYR61 and PTGS2. In some embodiments, the one or more FSGMs comprise CYR61 and UBE2D3. In some embodiments, the one or more FSGMs comprise CYR61 and CUL2. In some embodiments, the one or more FSGMs comprise CYR61 and ABCE1. In some embodiments, the one or more FSGMs comprise CYR61 and EiFlAX. In some embodiments, the one or more FSGMs comprise CYR61 and RPE26. In some embodiments, the one or more FSGMs comprise CYR61 and RPE38.
  • the one or more FSGMs comprise CYR61 and RPS27E. In some embodiments, the one or more FSGMs comprise CYR61 and RPE10. In some embodiments, the one or more FSGMs comprise CYR61 and RPE32. In some embodiments, the one or more FSGMs comprise CYR61 and RPE39. In some embodiments, the one or more FSGMs comprise CYR61 and RPS15A. In some embodiments, the one or more FSGMs comprise CYR61 and mt-ATP8. In some embodiments, the one or more FSGMs comprise CYR61 and mt-ATP6.
  • the one or more FSGMs comprise CYR61 and mt-ND3. In some embodiments, the one or more FSGMs comprise CYR61 and mt-NDl. In some embodiments, the one or more FSGMs comprise CYR61 and mt-ND4. In some embodiments, the one or more FSGMs comprise CYR61 and mt-CO3. In some embodiments, the one or more FSGMs comprise CYR61 and CYCs. In some embodiments, the one or more FSGMs comprise CYR61 and SLIRP.
  • the one or more FSGMs comprise CUL2 and THBS1. In some embodiments, the one or more FSGMs comprise CUL2 and UBE2D3. In some embodiments, the one or more FSGMs comprise CUL2 and ABCE1. In some embodiments, the one or more FSGMs comprise CUL2 and EiFlAX. In some embodiments, the one or more FSGMs comprise CUL2 and RPE26. In some embodiments, the one or more FSGMs comprise CUL2 and RPE38. In some embodiments, the one or more FSGMs comprise CUL2 and RPS27E. In some embodiments, the one or more FSGMs comprise CUL2 and RPL10.
  • the one or more FSGMs comprise CUL2 and RPL32. In some embodiments, the one or more FSGMs comprise CUL2 and RPL39. In some embodiments, the one or more FSGMs comprise CUL2 and RPS15A. In some embodiments, the one or more FSGMs comprise CUL2 and mt- ATP8. In some embodiments, the one or more FSGMs comprise CUL2 and mt-ATP6. In some embodiments, the one or more FSGMs comprise CUL2 and mt-ND3. In some embodiments, the one or more FSGMs comprise CUL2 and mt-NDl. In some embodiments, the one or more FSGMs comprise CUL2 and mt-ND4.
  • the one or more FSGMs comprise CUL2 and mt-CO3. In some embodiments, the one or more FSGMs comprise CUL2 and CYCs. In some embodiments, the one or more FSGMs comprise CUL2 and SLIRP.
  • the one or more FSGMs comprise EGR1, EGR2 and CYR61. In some embodiments, the one or more FSGMs comprise THBS1, PTGS2 and UBE2D3. In some embodiments, the one or more FSGMs comprise CUL2, ABCE1 and EiFlAX. In some embodiments, the one or more FSGMs comprise RPL26, RPL38 and RPS27L. In some embodiments, the one or more FSGMs comprise RPL10, RPL32 and RPL39. In some embodiments, the one or more FSGMs comprise RPS15A, mt-ATP8 and mt-ATP6. In some embodiments, the one or more FSGMs comprise mt-ND3, mt-NDl and mt-ND4. In some embodiments, the one or more FSGMs comprise mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise EGR1, EGR2 and THBS1. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and PTGS2. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and UBE2D3. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and CUL2. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and ABCE1. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and EiFlAX. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and RPL26.
  • the one or more FSGMs comprise EGR1, EGR2 and RPL38. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and RPS27L. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and RPL10. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and RPL32. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and RPL39. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and RPS15A. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and mt-ATP8.
  • the one or more FSGMs comprise EGR1, EGR2 and mt-ATP6. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and mt-ND3. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and mt-NDl. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and mt-ND4. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and CO3. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and CYCs. In some embodiments, the one or more FSGMs comprise EGR1, EGR2 and SLIRP.
  • the one or more FSGMs comprise PTGS2, THBS1 and UBE2D3. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and CUL2. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and ABCE1. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and EiFlAX. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and RPE26. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and RPE38. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and RPS27L.
  • the one or more FSGMs comprise PTGS2, THBS1 and RPE10. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and RPE32. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and RPE39. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and RPS15A. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and mt-ATP8. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and mt-ATP6.
  • the one or more FSGMs comprise PTGS2, THBS1 and mt-ND3. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and mt-NDl. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and mt-ND4. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and CO3. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and CYCs. In some embodiments, the one or more FSGMs comprise PTGS2, THBS1 and SEIRP.
  • the one or more FSGMs comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or all 24 of EGRl, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPE26, RPE38, RPS27E, RPE10, RPE32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, in any combination.
  • methods provided by the present disclosure comprise determining an FXN therapy expression profile for the one or more FSGMs described above.
  • an FXN therapy expression profile may be determined through the measurement of expression levels of at least one or any combination of more than one FSGM.
  • an FSGM includes any one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2 UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • an FSGM includes any one or more of the FSGMs selected from EGR1, EGR2, CYR61, THBS1, PTGS2 UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • an expression profile may also be referred to as a signature.
  • a baseline FXN expression profile comprises altered expression of at least one or any combination of more than one FSGM, e.g., any one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • any one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, m
  • a baseline FXN expression profile may comprise the downregulated expression levels of at least one of EGR1, EGR2, PTGS2, UBE2D3, CUL2, ABCE1, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, or any combination thereof.
  • a measure of effectiveness of FXN therapy in a subject with Leigh Syndrome may be indicated by a pattern of upregulation of any one or more of these FSGMs.
  • a baseline FXN expression profile may comprise the upregulated expression level of ATF3, NR4al, CYR61, THBS1, RPL26, RPL38, RPL32, RPL39 and RPS 15A.
  • a measure of effectiveness of FXN therapy may be indicated by a pattern of downregulation of any one of these FSGMs.
  • an FXN therapy expression profile comprises the reversed expression of a baseline FXN expression profile.
  • an FXN therapy expression profile for use as an indicator of FXN treatment effectiveness may comprise one or any combination of two or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, detected in a sample from a subject with Leigh Syndrome treated with FXN therapy.
  • an FXN therapy expression profile may comprise an expression pattern exemplified in Figure 1, Panel A or Figure 1, Panel B.
  • an FXN therapy expression profile is characterized by the contrary regulation of FSGMs, which is defined by any FSGMs that were downregulated in a subject with Leigh Syndrome that become upregulated following FXN therapy; and the reverse is also valid, such that any FSGMs that were upregulated in a subject with Leigh Syndrome become downregulated following FXN therapy. Accordingly, detection of altered expression of one or more FSGMs in a sample following FXN therapy allows for monitoring of efficacy of the FXN therapy in a subject with Leigh Syndrome.
  • a lack of altered expression of one or more FSGMs in a sample from a subject with Leigh Syndrome following FXN therapy indicates that the FXN therapy may not have been successful and/or that increased FXN therapy may be needed.
  • altered expression of one or more FSGMs in a sample from a subject with Leigh Syndrome following FXN therapy indicates that FXN therapy was successful.
  • altered expression is modulated or altered gene expression, which in the method exemplified herein presents itself as differential gene expression, also known as differential mRNA expression.
  • Altered or modulated expression may comprise increased expression, also referred to as overexpression or upregulation, or decreased or inhibited expression, also referred to as downregulation.
  • feature vectors are a set of values that characterize an expression profile.
  • Feature vectors may comprise a set of n FSGMs, n being the number of different genes whose expression levels were measured in a sample.
  • n may be all the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • n may be at least one, two, or three, or four, or five, or six, or any number of FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • a set of FSGMs may comprise at least one or any combination of more than one FSGM, e.g., any one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, in any combination.
  • any one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A,
  • methods provided by the present disclosure comprise determining an FXN therapy expression profile for the one or more FSGMs described above, e.g. ,one or more FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • FSGMs described above e.g. ,one or more FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32,
  • a normal FXN expression profile obtained from samples of healthy subjects, e.g., subjects who do not have Leigh Syndrome, may be comprised of expression levels of a set of FSGMs, and may be represented by and referred to as a normal FXN feature vector.
  • FSGMs when measured in samples from subjects with Leigh Syndrome may present expression levels that are different from the levels of expression of FSGMs in healthy subjects, and thus may be represented by and referred to as a deficient FXN feature vector.
  • the difference between a deficient FXN feature vector and a normal FXN feature vector may be detected and quantified by the distance between the two feature vectors.
  • expression levels of FSGMs in a sample from a subject with Leigh Syndrome following FXN treatment may present yet different expression levels, and may be represented by and referred to as an FXN therapy feature vector.
  • an FXN therapy feature vector As for the previous two feature vectors, the difference between an FXN therapy feature vector and either a normal FXN feature vector or a deficient FXN feature vector may be detected and quantified by the distance between the FXN therapy feature vector and the normal FXN feature vector or the deficient FXN feature vector.
  • a first FXN feature vector may be determined for the FXN expression profile and a second FXN feature vector may be determined for the baseline FXN expression profile; wherein determining a distance, or scalar product, between the first and the second feature vectors may be used for determining effectiveness of the FXN therapy.
  • a third feature vector may be determined for the normal FXN expression profile, the normal expression profile being established for the FSGMs in a sample from a healthy subject.
  • the distance between the second (baseline FXN expression profile) and third (normal FXN expression profile) FXN feature vectors may be determined.
  • the distance between the first (FXN expression profile) and third (normal FXN expression profile) FXN feature vectors may be determined, and may be used for determining effectiveness of the FXN therapy.
  • the distance between the first and third feature vectors may be normalized to the distance between the second and third feature vectors, and the resulting normalized distance may be used to determine effectiveness of the FXN therapy.
  • the resulting normalized distance may be a value ranging from 0 (zero) to 1 (one), wherein the smaller the value (closest to zero) the more effective the therapy.
  • the markers of the disclosure e.g., one or more FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP, are correlated with FXN levels in a subject.
  • FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3,
  • the present disclosure provides methods for using, measuring, detecting, quantifying, and the like of one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, for determining and/or monitoring the FXN therapy status in a subject with Leigh Syndrome, or for determining, evaluating, and/or monitoring FXN therapy in a subject with Leigh Syndrome.
  • the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26
  • the FSGMs of the present disclosure may be used in combination with one or more additional markers for a mitochondrial disease, e.g., Leigh Syndrome.
  • the physiological state of an organism is inclusive of any disease or nondisease state, e.g., a subject having a mitochondrial disease, e.g., Leigh Syndrome, or a subject who is otherwise healthy.
  • the FSGMs of the disclosure include characteristics that can be objectively measured and evaluated as indicators of normal processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
  • Such combination markers can be clinical parameters (e.g., age, performance status), laboratory measures (e.g., molecular markers), or genetic or other molecular determinants.
  • the present invention also involves the analysis and consideration of any clinical and/or subject-related health data, for example, data obtained from an Electronic Medical Record (e.g., collection of electronic health information about individual subjects or populations relating to various types of data, such as, demographics, medical history, medication and allergies, immunization status, laboratory test results, radiology images, vital signs, personal statistics like age and weight, and billing information).
  • an Electronic Medical Record e.g., collection of electronic health information about individual subjects or populations relating to various types of data, such as, demographics, medical history, medication and allergies, immunization status, laboratory test results, radiology images, vital signs, personal statistics like age and weight, and billing information.
  • the present invention also contemplates the use of particular combinations of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least two (2) members, which may include any two of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least three (3) members, which may include any three of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS 15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least four (4) members, which may include any four of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least five (5) members, which may include any five of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least six (6) members, which may include any six of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least seven (7) members, which may include any seven of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least eight (8) members, which may include any eight of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least nine (9) members, which may include any nine of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt- ND4, mt-CO3, CYCs and SLIRP.
  • 9 members which may include any nine of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6,
  • the invention contemplates FSGM sets with at least ten (10) members, which may include any ten of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least eleven (11) members, which may include any eleven of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets with at least twelve (12) members, which may include any twelve of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates an FSGM set comprising at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 of the FSGMs listed in FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the invention contemplates FSGM sets comprising at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the level of the FSGM is increased following treatment of a subject with Leigh Syndrome.
  • the FSGM is selected from the group consisting of EGR1, EGR2, PTGS2, UBE2D3, CUL2, ABCE1, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the level of a FSGM is decreased following treatment of a subject with Leigh Syndrome.
  • the FSGM is ATF3, NR4al, CYR61, THBS1, RPL26, RPL38, RPL32, RPL39 and RPS15A.
  • the present invention provides for the identification of a “diagnostic signature” or “diagnostic expression profile” based on the levels of the FSGMs of the invention in a biological sample, that correlates with FXN in the sample.
  • the “levels of the FSGMs” can refer to the protein level of an FSGM in a biological sample.
  • the “levels of the FSGMs” can also refer to the expression level of the genes corresponding to the proteins, e.g., by measuring the expression levels of the corresponding FSGM mRNAs.
  • the collection or totality of levels of FSGMs provide a diagnostic signature that correlates with the level of FXN.
  • the diagnostic signature is obtained by (1) detecting the level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP in a biological sample from a subject with Leigh Syndrome receiving FXN therapy; (2) comparing the levels of the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, R
  • FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP are above or below the control (e.g., baseline FXN expression profile), then the diagnostic signature is indicative of the effectiveness of FXN therapy.
  • the control e.g., baseline FXN expression profile
  • an algorithm can be any computation, formula, statistical survey, nomogram, look-up Tables, decision tree method, or computer program which processes a set of input variables (e.g., number of markers (n) which have been detected at a level exceeding some threshold level, or number of markers (n) which have been detected at a level below some threshold level) through a number of well-defined successive steps to eventually produce a score or “output.”
  • Any suitable algorithm whether computer-based or manual-based (e.g., look-up Tables) — is contemplated herein.
  • the FSGMs of the disclosure can include variant sequences.
  • FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt- ND4, mt-CO3, CYCs and SLIRP, can include variant sequences.
  • certain binding agents/reagents used for detecting certain of the FSGMs of the invention can bind and/or identify variants of these certain FSGMs of the invention.
  • the term "variant” encompasses nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a sequence disclosed herein.
  • the percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.
  • Variant sequences generally differ from the specifically identified sequence only by conservative substitutions, deletions or modifications.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • Variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the antigenic properties, secondary structure and hydropathic nature of the polypeptide.
  • a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co- translationally or post-translationally directs transfer of the protein.
  • the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
  • a polypeptide may be conjugated to an immunoglobulin Fc region.
  • Polypeptide and polynucleotide sequences may be aligned, and percentages of identical amino acids or nucleotides in a specified region may be determined against another polypeptide or polynucleotide sequence, using computer algorithms that are publicly available.
  • the percentage identity of a polynucleotide or polypeptide sequence is determined by aligning polynucleotide and polypeptide sequences using appropriate algorithms, such as BLASTN or BLASTP, respectively, set to default parameters; identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percentage identity.
  • Two exemplary algorithms for aligning and identifying the identity of polynucleotide sequences are the BLASTN and FASTA algorithms.
  • the alignment and identity of polypeptide sequences may be examined using the BLASTP algorithm.
  • BLASTX and FASTX algorithms compare nucleotide query sequences translated in all reading frames against polypeptide sequences.
  • the FASTA and FASTX algorithms are described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and in Pearson, Methods in Enzymol. 183:63-98, 1990.
  • the FASTA software package is available from the University of Virginia, Charlottesville, Va. 22906-9025.
  • the FASTA algorithm set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of polynucleotide variants.
  • the readme files for FASTA and FASTX Version 2.0x that are distributed with the algorithms describe the use of the algorithms and describe the default parameters.
  • the BLASTN software is available on the NCBI anonymous FTP server and is available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894.
  • NCBI National Center for Biotechnology Information
  • the use of the BLAST family of algorithms, including BLASTN is described at NCBI's website and in the publication of Altschul, el al., "Gapped BLAST and PSLBLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, 1997.
  • variant polypeptides are encoded by polynucleotide sequences that hybridize to a disclosed polynucleotide under stringent conditions.
  • Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM, and preferably less than about 200 mM.
  • Hybridization temperatures can be as low as 5 °C, but are generally greater than about 22 °C, more preferably greater than about 30 °C, and most preferably greater than about 37 °C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization.
  • stringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone.
  • An example of "stringent conditions" is prewashing in a solution of 6XSSC, 0.2% SDS; hybridizing at 65°C, 6XSSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1XSSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2XSSC, 0.1% SDS at 65°C.
  • FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP. It is understood that any single FSGM or combination of the FSGMs provided herein can be used in the invention unless clearly indicated otherwise.
  • Tissue Samples e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-
  • the present invention may be practiced with any suitable biological sample that potentially contains, expresses, includes, a detectable FSGM.
  • the biological sample may be obtained from a solid tissue sample, such as a skin biopsy sample, muscle biopsy sample, preferably a buccal sample, or a body fluid sample such as blood (including any blood product, such as whole blood, plasma, serum, or specific types of cells of the blood), urine, saliva, or seminal fluid.
  • a sample can comprise exosomes which may be harvested in order to be tested for FSGM transcripts.
  • a sample which may be used for measuring an FXN expression profile in the context of the present disclosure may be selected from the group consisting of a buccal sample, a skin sample, a hair follicle and a muscle biopsy sample.
  • the sample is a buccal sample.
  • the sample is a skin sample.
  • a sample which may be used for measuring an FXN therapy expression profile may be obtained from subject with Leigh Syndrome prior to administration of FXN therapy, during administration of FXN therapy or after administration of FXN therapy.
  • a sample may be obtained from a subject with Leigh Syndrome at least 15 days following the last administration of the FXN therapy.
  • a sample may be obtained from a subject with Leigh Syndrome 15 to 45 days following the last administration of the FXN therapy, e.g., 15 to 20 days, 20 to 35 days or 25 to 45 days after the last administration of the FXN therapy.
  • a sample may be obtained from a subject with Leigh Syndrome 20 to 25 days, e.g., 20, 21, 22, 23 or 25 days, after the last administration of the FXN therapy.
  • a sample may be obtained from a subject with Leigh Syndrome at least 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 days after the last administration of the FXN therapy.
  • inventive methods may be performed at the single cell level. However, the inventive methods may also be performed using a sample comprising many cells, where the assay is "averaging" expression over the entire collection of cells and tissue present in the sample. Preferably, there is enough of the tissue sample to accurately and reliably determine the expression levels of interest. Any commercial device or system for isolating and/or obtaining tissue and/or blood or other biological products, and/or for processing said materials prior to conducting a detection reaction is contemplated.
  • the present invention relates to detecting FSGM nucleic acid molecules (e.g., mRNA encoding one or more FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP).
  • RNA can be extracted from a biological sample before analysis.
  • RNA isolation reagents comprise, among other components, guanidinium thiocyanate and/or beta-mercaptoethanol, which are known to act as RNase inhibitors.
  • Isolated total RNA is then further purified from the protein contaminants and concentrated by selective ethanol precipitations, phenol/chloroform extractions followed by isopropanol precipitation (see, for example, P. Chomczynski and N. Sacchi, Anal. Biochem., 1987, 162: 156-159) or cesium chloride, lithium chloride or cesium trifluoroacetate gradient centrifugations.
  • RNA z.e., total RNA or mRNA
  • kits can be used to extract RNA (z.e., total RNA or mRNA) from bodily fluids or tissues and are commercially available from, for example, Ambion, Inc. (Austin, Tex.), Amersham Biosciences (Piscataway, N.J.), BD Biosciences Clontech (Palo Alto, Calif.), BioRad Laboratories (Hercules, Calif.), GIBCO BRL (Gaithersburg, Md.), and Giagen, Inc. (Valencia, Calif.).
  • Sensitivity, processing time and cost may be different from one kit to another.
  • One of ordinary skill in the art can easily select the kit(s) most appropriate for a particular situation.
  • RNA is amplified, and transcribed into cDNA, which can then serve as template for multiple rounds of transcription by the appropriate RNA polymerase.
  • Amplification methods are well known in the art (see, for example, A. R. Kimmel and S. L. Berger, Methods Enzymol. 1987, 152: 307-316; J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2nd Ed., Cold Spring Harbour Laboratory Press: New York; “Short Protocols in Molecular Biology", F. M. Ausubel (Ed.), 2002, 5. sup. th Ed., John Wiley & Sons; U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,800,159).
  • Reverse transcription reactions may be carried out using non-specific primers, such as an anchored oligo- dT primer, or random sequence primers, or using a target- specific primer complementary to the RNA for each genetic probe being monitored, or using thermostable DNA polymerases (such as avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase).
  • non-specific primers such as an anchored oligo- dT primer, or random sequence primers
  • a target- specific primer complementary to the RNA for each genetic probe being monitored or using thermostable DNA polymerases (such as avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase).
  • the RNA isolated from the tissue sample (for example, after amplification and/or conversion to cDNA or cRNA) is labeled with a detectable agent before being analyzed.
  • a detectable agent is to facilitate detection of RNA or to allow visualization of hybridized nucleic acid fragments (e.g., nucleic acid fragments hybridized to genetic probes in an array-based assay).
  • the detectable agent is selected such that it generates a signal which can be measured and whose intensity is related to the amount of labeled nucleic acids present in the sample being analyzed.
  • the detectable agent is also preferably selected such that it generates a localized signal, thereby allowing spatial resolution of the signal from each spot on the array.
  • Standard nucleic acid labeling methods include: incorporation of radioactive agents, direct attachment of fluorescent dyes (see, for example, L. M. Smith et al., Nucl. Acids Res.
  • detectable agents include, but are not limited to: various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (such as, for example, quantum dots, nanocrystals, phosphors and the like), enzymes (such as, for example, those used horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, dioxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.
  • various ligands such as, for example, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (such as, for example, quantum dots, nanocrystals, phosphors and the like), enzymes (such as, for example, those used horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), color
  • the expression levels are determined by detecting the expression of a gene product (e.g., a protein, such as a secreted protein) thereby eliminating the need to obtain a genetic sample (e.g., RNA) from the sample.
  • a gene product e.g., a protein, such as a secreted protein
  • the distance may be achieved for example by calculating the mean squared error, which may be extracted from the difference in the expression pattern of each gene measured in two different profiles.
  • the distance may be achieved by calculating a correlation coefficient or applying a /-test.
  • said determining the expression profile of a sample of a subject comprises obtaining or provided a biological sample from a subject, extracting RNA from the sample, generating the corresponding cDNA, and detecting expression profile through any one of sequencing, hybridization or amplification.
  • the expression profile of a sample may be determined using qPCR.
  • the expression profile of a sample may be determined using Nanostring.
  • Detecting the FXN-sensitive expression profiles by sequencing may use, for example, next generation sequencing (NGS), RNASeq, and any sequencing techniques known to the man skilled in the art.
  • Detecting expression profile by hybridization comprises contacting a subject sample, or a portion thereof, with a probe or a set of probes that specifically hybridize with FSGMs (or their transcripts) selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • any one or more FSGMs of the disclosure or any combination thereof may be contacted with a subject sample.
  • determining the expression profile of a sample of a subject with Leigh Syndrome treated with FXN therapy by hybridization may comprise contacting the sample, or a portion thereof, with probes that hybridize with at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of its target nucleic acid, the target nucleic acid being the transcript, or the corresponding cDNA for any one of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • Detecting expression profile by amplification involves, by way of example, polymerase chain reaction techniques, such as real-time polymerase chain reaction (RT-PCR), which comprises contacting the sample with forward and reverse primers for each of the transcripts of interest as exemplified herein below in the examples and generating RT-PCR products.
  • RT-PCR products are detected with specific or general probes, or a combination thereof, which facilitate their quantification.
  • an FXN-induced signature may be determined by detecting FSGMs transcripts in a sample.
  • forward and reverse primers are used for detecting FSGMs transcripts.
  • the expression profile may be detected through FSGMs protein products and analysis of protein profile, which may be performed through protein detection methodology, using techniques involving specific antibodies, or protein quantification/characterization techniques, such as high-performance liquid chromatography (HPLC), mass spectrometry-based techniques, gel-based techniques for example differential ingel electrophoresis, and the like.
  • HPLC high-performance liquid chromatography
  • mass spectrometry-based techniques mass spectrometry-based techniques
  • gel-based techniques for example differential ingel electrophoresis, and the like.
  • the present disclosure provides a composition for detection of an FXN expression profile, the composition comprising at least one or a plurality of nucleotide sequences for detection of FSGMs.
  • the composition may be for detection of any one of an FXN expression profile, a baseline FXN expression profile and/or a normal FXN expression profile.
  • composition may comprise at least one nucleotide sequence for the detection of transcripts of the genes selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the composition may comprise nucleotide sequences for the detection of two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, and/or up to all FSGMs FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • a nucleotide sequence may be DNA or an analog thereof, or RNA or an analog thereof.
  • the nucleotide sequence may be complementary to as least a portion of an FSGM. Binding of the nucleotide sequence for detection of FSGMs will depend on the level of stringency of the reaction.
  • the nucleotide sequence may be an oligonucleotide, which may function as a probe or a primer, and as such may comprise modifications compatible with their function.
  • short oligonucleotides used as probes may carry labels, for example fluorescent labels, that enable their detection and quantification.
  • the present invention contemplates any suitable means, techniques, and/or procedures for detecting and/or measuring the FSGMs of the invention. These methods are described in detail below.
  • the present invention contemplates any suitable method for detecting polypeptide FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • the detection method is an immunodetection method involving an antibody that specifically binds to one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura el al. (1987), which is incorporated herein by reference.
  • the immunobinding methods include obtaining a sample suspected of containing an FSGM protein, peptide, e.g., an FSGM secreted protein or peptide, or antibody, and contacting the sample, or a portion thereof, with an antibody or protein or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • an FSGM protein, peptide e.g., an FSGM secreted protein or peptide, or antibody
  • an antibody or protein or peptide in accordance with the present invention as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • the immunobinding methods include methods for detecting or quantifying the amount of a reactive component in a sample, which methods require the detection or quantitation of any immune complexes formed during the binding process.
  • a sample suspected of containing an FSGM protein, peptide or a corresponding antibody and contact the sample with an antibody or encoded protein or peptide, as the case may be, and then detect or quantify the amount of immune complexes formed under the specific conditions.
  • immune complexes primary immune complexes
  • complex formation is a matter of simply adding the composition to the biological sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any antigens present.
  • the sample-antibody composition such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non- specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or FSGM, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art.
  • U.S. patents concerning the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference.
  • a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
  • the protein employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first added component that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the encoded protein, peptide or corresponding antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two-step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the encoded protein, peptide or corresponding antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • the immunodetection methods of the present invention have evident utility in the monitoring of efficacy of FXN therapy, e.g., treatment with an FXN fusion protein.
  • FXN therapy e.g., treatment with an FXN fusion protein.
  • a biological or clinical sample suspected of containing either the encoded protein or peptide or corresponding antibody is used.
  • these embodiments also have applications to non- clinical samples, such as in the titering of antigen or antibody samples, in the selection of hybridomas, and the like.
  • the present invention contemplates the use of ELISAs as a type of immunodetection assay. It is contemplated that FSGMs of the invention will find utility as immunogens in ELISA assays in monitoring of FXN therapy.
  • Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like also may be used.
  • antibodies binding to the FSGMs of the invention including FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate.
  • a test composition suspected of containing the FSGM antigen such as a clinical sample, is added to the wells. After binding and washing to remove non- specifically bound immunecomplexes, the bound antigen may be detected. Detection is generally achieved by the addition of a second antibody specific for the target protein, that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA.” Detection also may be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the FSGM antigen are immobilized onto the well surface and then contacted with the anti-biomarker antibodies of the invention. After binding and washing to remove non-specific ally bound immunecomplexes, the bound antigen is detected. Where the initial antibodies are linked to a detectable label, the immunecomplexes may be detected directly. Again, the immunecomplexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non- specifically bound species, and detecting the bound immunecomplexes. These are described as follows.
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • the immobilizing surface is contacted with the control and/or clinical or biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
  • the phrase "under conditions effective to allow immunecomplex (antigen/antibody) formation” means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween.
  • suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25 °C to 27 °C, or may be overnight at about 4 °C or so.
  • the contacted surface is washed so as to remove non-complexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immunecomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immunecomplexes may be determined.
  • the second or third antibody will have an associated label to allow detection.
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immunecomplex formation (e.g., incubation for
  • the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • FSGMs of the invention e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, can also be measured, quantitated, detected, and otherwise analyzed using protein mass spectrometry methods and instrumentation.
  • FSGMs of the invention e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, R
  • Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins.
  • two approaches are typically used for characterizing proteins using mass spectrometry.
  • intact proteins are ionized and then introduced to a mass analyzer. This approach is referred to as "top-down" strategy of protein analysis.
  • the two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).
  • ESI electrospray ionization
  • MALDI matrix-assisted laser desorption/ionization
  • proteins are enzymatically digested into smaller peptides using a protease such as trypsin.
  • FSGMs of the invention e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP, can be conducted using time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance (FT- ICR).
  • TOF time-of-flight
  • FT- ICR Fourier transform ion cyclotron resonance
  • the most widely used instruments for peptide mass analysis are the MALDI time-of-flight instruments as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace (1 PMF can be analyzed in approx. 10 sec).
  • PMFs peptide mass fingerprints
  • Multiple stage quadrupole-time-of-flight and the quadrupole ion trap also find use in this application.
  • FSGMs of the invention e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, can also be measured in complex mixtures of proteins and molecules that co-exist in a biological medium or sample, however, fractionation of the sample may be required and is contemplated herein.
  • Fractionation can be used to first separate any complex mixture of proteins prior to mass spectrometry analysis.
  • Two methods are widely used to fractionate proteins, or their peptide products from an enzymatic digestion.
  • the first method fractionates whole proteins and is called two-dimensional gel electrophoresis.
  • the second method high performance liquid chromatography (LC or HPLC) is used to fractionate peptides after enzymatic digestion. In some situations, it may be desirable to combine both of these techniques. Any other suitable methods known in the art for fractionating protein mixtures are also contemplated herein.
  • Gel spots identified on a 2D Gel are usually attributable to one protein. If the identity of the protein is desired, usually the method of in-gel digestion is applied, where the protein spot of interest is excised, and digested proteolytically.
  • the peptide masses resulting from the digestion can be determined by mass spectrometry using peptide mass fingerprinting. If this information does not allow unequivocal identification of the protein, its peptides can be subject to tandem mass spectrometry for de novo sequencing.
  • Characterization of protein mixtures using HPLC/MS may also be referred to in the art as “shotgun proteomics” and MuDPIT (Multi-Dimensional Protein Identification Technology).
  • a peptide mixture that results from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography (LC).
  • the eluent from the chromatography stage can be either directly introduced to the mass spectrometer through electrospray ionization, or laid down on a series of small spots for later mass analysis using MALDI.
  • FSGMs of the invention e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, can be identified using MS using a variety of techniques, all of which are contemplated herein.
  • Peptide mass fingerprinting uses the masses of proteolytic peptides as input to a search of a database of predicted masses that would arise from digestion of a list of known proteins. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample. It will be further appreciated that the development of methods and instrumentation for automated, data-dependent electrospray ionization (ESI) tandem mass spectrometry (MS/MS) in conjunction with microcapillary liquid chromatography (LC) and database searching has significantly increased the sensitivity and speed of the identification of gel-separated proteins. Microcapillary LC-MS/MS has been used successfully for the large-scale identification of individual proteins directly from mixtures without gel electrophoretic separation (Link et al., 1999; Opitek et al., 1997).
  • ESI electrospray ionization
  • MS/MS microcapillary liquid chromatography
  • SILAC stable isotope labeling by amino acids in cell culture
  • ICAT isotope coded affinity tagging
  • iTRAQ isobaric tags for relative and absolute quantitation
  • MALDI mass spectrometry
  • the peak intensity, or the peak area, from individual molecules (typically proteins) is here correlated to the amount of protein in the sample.
  • the individual signal depends on the primary structure of the protein, on the complexity of the sample, and on the settings of the instrument.
  • Other types of "label-free" quantitative mass spectrometry uses the spectral counts (or peptide counts) of digested proteins as a means for determining relative protein amounts.
  • the invention involves the detection of nucleic acid FSGMs, e.g., the corresponding genes or mRNA of the FSGMs of the invention, e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A,
  • the methods of the present invention generally involve the determination of expression levels of a set of genes in a biological sample. Determination of gene expression levels in the practice of the inventive methods may be performed by any suitable method. For example, determination of gene expression levels may be performed by detecting the expression of mRNA expressed from the genes of interest and/or by detecting the expression of a polypeptide encoded by the genes.
  • FSGMs of the invention e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP
  • any suitable method can be used, including, but not limited to, Southern blot analysis, Northern blot analysis, polymerase chain reaction (PCR) (see, for example, U.S.
  • gene expression levels of FSGMs of interest may be determined by amplifying complementary DNA (cDNA) or complementary RNA (cRNA) produced from mRNA and analyzing it using a microarray.
  • FSGMs of interest e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, may be determined by amplifying complementary DNA (cDNA) or complementary RNA (cRNA) produced from mRNA and analyzing it using a microarray.
  • cDNA complementary DNA
  • cRNA complementary RNA
  • Microarray technology allows for the measurement of the steady-state mRNA level of a large number of genes simultaneously.
  • Microarrays currently in wide use include cDNA arrays and oligonucleotide arrays. Analyses using microarrays are generally based on measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid probe immobilized at a known location on the microarray (see, for example, U.S. Pat. Nos. 6,004,755; 6,218,114; 6,218,122; and 6,271,002).
  • Nucleic acid used as a template for amplification can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al., 1989).
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary cDNA.
  • the RNA is whole cell RNA and is used directly as the template for amplification.
  • Pairs of primers that selectively hybridize to nucleic acids corresponding to any of the FSGM nucleotide sequences identified herein are contacted with the isolated nucleic acid under conditions that permit selective hybridization.
  • the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis.
  • Multiple rounds of amplification, also referred to as "cycles,” are conducted until a sufficient amount of amplification product is produced.
  • the amplification product is detected. In certain applications, the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology; Bellus, 1994). Following detection, one may compare the results seen in a given subject with a statistically significant reference group of, for example, normal subjects. In this way, it is possible to correlate the amount of nucleic acid detected with various clinical states.
  • primer as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences may be employed.
  • Primers may be provided in double-stranded or single- stranded form, although the single- stranded form is preferred.
  • PCR polymerase chain reaction
  • two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target nucleic acid sequence.
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the target nucleic acid sequence is present in a sample, the primers will bind to the target nucleic acid and the polymerase will cause the primers to be extended along the target nucleic acid sequence by adding on nucleotides.
  • the extended primers will dissociate from the target nucleic acid to form reaction products, excess primers will bind to the target nucleic acid and to the reaction products and the process is repeated.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al., 1989.
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641 filed Dec. 21, 1990. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, also may be used as still another amplification method in the present invention.
  • a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence which may then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[a-thio]- triphosphates in one strand of a restriction site also may be useful in the amplification of nucleic acids in the present invention. Walker et al. (1992), incorporated herein by reference in its entirety.
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • Target specific sequences also may be detected using a cyclic probe reaction (CPR).
  • CPR a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA which is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products which are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • modified primers are used in a PCR like, template and enzyme dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR. Kwoh et al. (1989); Gingeras et al., PCT Application WO 88/10315, incorporated herein by reference in their entirety.
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • Kwoh et al. (1989); Gingeras et al., PCT Application WO 88/10315 incorporated herein by reference in their entirety.
  • the nucleic acids may be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6.
  • a polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA singlestranded RNA
  • dsDNA double- stranded DNA
  • the ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H(RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • the resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase 1), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence may be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies may then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification may be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence may be chosen to be in the form of either DNA or RNA.
  • Miller et al., PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence.
  • This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "race” and "one-sided PCR.” Frohman (1990) and Ohara et al. (1989), each herein incorporated by reference in their entirety.
  • Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted sequences employed.
  • the oligonucleotide probes or primers are at least 10 nucleotides in length (preferably, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or more nucleotides in length) and they may be adapted to be especially suited for a chosen nucleic acid amplification system and/or hybridization system used. Longer probes and primers are also within the scope of the present invention as well known in the art.
  • Primers having more than 30, more than 40, more than 50 nucleotides and probes having more than 100, more than 200, more than 300, more than 500 more than 800 and more than 1000 nucleotides in length are also covered by the present invention.
  • longer primers have the disadvantage of being more expensive and thus, primers having between 12 and 30 nucleotides in length are usually designed and used in the art.
  • probes ranging from 10 to more than 2000 nucleotides in length can be used in the methods of the present invention.
  • the detection means the use of a hybridization technique, e.g., where a specific primer or probe is selected to anneal to a target FSGM of interest, and thereafter detection of selective hybridization is made.
  • the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular Cloning— A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1994, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
  • oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least 70% (at least 71%, 72%, 73%, 74%), preferably at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) and more preferably at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to a portion of an FSGM of the invention.
  • Probes and primers of the present invention are those that hybridize under stringent hybridization conditions and those that hybridize to FSGM homologs of the invention under at least moderately stringent conditions.
  • probes and primers of the present invention have complete sequence identity to the FSGMs of the invention (gene sequences ⁇ e.g., cDNA or mRNA). It should be understood that other probes and primers could be easily designed and used in the present invention based on the FSGMs of the invention disclosed herein by using methods of computer alignment and sequence analysis known in the art (cf. Molecular Cloning: A Laboratory Manual, Third Edition, edited by Cold Spring Harbor Laboratory, 2000).
  • the invention provides methods and compositions that include labels for the highly sensitive detection and quantitation of the FSGMs of the invention, e.g., FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-
  • the labels may be attached by any known means, including methods that utilize non-specific or specific interactions of label and target. Labels may provide a detectable signal or affect the mobility of the particle in an electric field. In addition, labeling can be accomplished directly or through binding partners.
  • the label comprises a binding partner that binds to the FSGM of interest, where the binding partner is attached to a fluorescent moiety.
  • compositions and methods of the invention may utilize highly fluorescent moieties, e.g., a moiety capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules.
  • highly fluorescent moieties e.g., a moiety capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules.
  • the invention provides a label for detecting a biological molecule comprising a binding partner for the biological molecule that is attached to a fluorescent moiety, wherein the fluorescent moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules.
  • the moiety comprises a plurality of fluorescent entities, e.g., about 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, or about 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 fluorescent entities. In some embodiments, the moiety comprises about 2 to 4 fluorescent entities.
  • the biological molecule is a protein or a small molecule. In some embodiments, the biological molecule is a protein.
  • the fluorescent entities can be fluorescent dye molecules. In some embodiments, the fluorescent dye molecules comprise at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance.
  • the dye molecules are Alexa Fluor molecules selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the dye molecules are Alexa Fluor molecules selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the dye molecules are Alexa Fluor 647 dye molecules. In some embodiments, the dye molecules comprise a first type and a second type of dye molecules, e.g., two different Alexa Fluor molecules, e.g., where the first type and second type of dye molecules have different emission spectra.
  • the ratio of the number of first type to second type of dye molecule can be, e.g., 4 to 1, 3 to 1, 2 to 1, 1 to 1, 1 to 2, 1 to 3 or 1 to 4.
  • the binding partner can be, e.g., an antibody.
  • the invention provides a label for the detection of a biological FSGM of the invention, wherein the label comprises a binding partner for the FSGM and a fluorescent moiety, wherein the fluorescent moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules.
  • the fluorescent moiety comprises a fluorescent molecule. In some embodiments, the fluorescent moiety comprises a plurality of fluorescent molecules, e.g., about 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8, or 3 to 6 fluorescent molecules. In some embodiments, the label comprises about 2 to 4 fluorescent molecules. In some embodiments, the fluorescent dye molecules comprise at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. In some embodiments, the fluorescent molecules are selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or Alexa Fluor 700.
  • the fluorescent molecules are selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the fluorescent molecules are Alexa Fluor 647 molecules.
  • the binding partner comprises an antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.
  • antibody is a broad term and is used in its ordinary sense, including, without limitation, to refer to naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof.
  • An "antigen -binding fragment” of an antibody refers to the part of the antibody that participates in antigen binding.
  • the antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy (“H”) and light (“L”) chains.
  • epitope or region of the molecule to which the antibody is raised will determine its specificity, e.g., for various forms of the molecule, if present, or for total (e.g., all, or substantially all of the molecule).
  • Monoclonal and polyclonal antibodies to molecules e.g., proteins, and markers also commercially available (R and D Systems, Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abeam Inc., Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049 USA; BiosPacific, Emeryville, Calif.).
  • the antibody is a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as the technique of Kohler and Milstein (E r. J. Immunol. 6:511-519, 1976), and improvements thereto. These methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity. Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding antibodies employed in the disclosed methods may be isolated and sequenced using conventional procedures. Recombinant antibodies, antibody fragments, and/or fusions thereof, can be expressed in vitro or in prokaryotic cells (e.g. bacteria) or eukaryotic cells (e.g. yeast, insect or mammalian cells) and further purified as necessary using well known methods.
  • prokaryotic cells e.g. bacteria
  • eukaryotic cells e.g. yeast, insect or mammalian cells
  • monoclonal antibodies may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified expressed protein, polypeptide or peptide.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • the methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • Antibodies may also be derived from a recombinant antibody library that is based on amino acid sequences that have been designed in silico and encoded by polynucleotides that are synthetically generated. Methods for designing and obtaining in silico-created sequences are known in the art (Knappik et al., J. Mol. Biol. 296:254:57-86, 2000; Krebs et al., J. Immunol. Methods 254:67-84, 2001; U.S. Pat. No. 6,300,064).
  • Digestion of antibodies to produce antigen-binding fragments thereof can be performed using techniques well known in the art.
  • the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the "F(ab)" fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site.
  • the enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the "F(ab')2" fragment, which comprises both antigen-binding sites.
  • "Fv" fragments can be produced by preferential proteolytic cleavage of an IgM, IgG or IgA immunoglobulin molecule, but are more commonly derived using recombinant techniques known in the art.
  • the Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule (Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem. 15:2706-2710 (1976); and Ehrlich et al., Biochem. 19:4091-4096 (1980)).
  • Antibody fragments that specifically bind to the protein FSGMs disclosed herein can also be isolated from a library of scFvs using known techniques, such as those described in U.S. Pat. No. 5,885,793.
  • a wide variety of expression systems are available in the art for the production of antibody fragments, including Fab fragments, scFv, VL and VHs.
  • expression systems of both prokaryotic and eukaryotic origin may be used for the large-scale production of antibody fragments.
  • Particularly advantageous are expression systems that permit the secretion of large amounts of antibody fragments into the culture medium.
  • Eukaryotic expression systems for large-scale production of antibody fragments and antibody fusion proteins have been described that are based on mammalian cells, insect cells, plants, transgenic animals, and lower eukaryotes.
  • the cost-effective, large-scale production of antibody fragments can be achieved in yeast fermentation systems. Large-scale fermentation of these organisms is well known in the art and is currently used for bulk production of several recombinant proteins.
  • Antibodies that bind to the protein FSGMs employed in the present methods are, in some cases, available commercially or can be obtained without undue experimentation.
  • the binding partners can comprise a label, e.g., a fluorescent moiety or dye.
  • a label e.g., a fluorescent moiety or dye.
  • any binding partner of the invention e.g., an antibody, can also be labeled with a fluorescent moiety. The fluorescence of the moiety will be sufficient to allow detection in a single molecule detector, such as the single molecule detectors described herein.
  • a “fluorescent moiety,” as that term is used herein, includes one or more fluorescent entities whose total fluorescence is such that the moiety may be detected in the single molecule detectors described herein.
  • a fluorescent moiety may comprise a single entity (e.g., a Quantum Dot or fluorescent molecule) or a plurality of entities (e.g., a plurality of fluorescent molecules). It will be appreciated that when “moiety,” as that term is used herein, refers to a group of fluorescent entities, e.g., a plurality of fluorescent dye molecules, each individual entity may be attached to the binding partner separately or the entities may be attached together, as long as the entities as a group provide sufficient fluorescence to be detected.
  • the fluorescence of the moiety involves a combination of quantum efficiency and lack of photobleaching sufficient that the moiety is detectable above background levels in a single molecule detector, with the consistency necessary for the desired limit of detection, accuracy, and precision of the assay.
  • the fluorescence of the fluorescent moiety is such that it allows detection and/or quantitation of a molecule, e.g., an FSGM, at a limit of detection of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.00001, or 0.000001 pg/ml and with a coefficient of variation of less than about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less, e.g., about 10% or less, in the instruments described herein.
  • the fluorescence of the fluorescent moiety is such that it allows detection and/or quantitation of a molecule, e.g., an FSGM, at a limit of detection of less than about 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 pg/ml and with a coefficient of variation of less than about 10%, in the instruments described herein.
  • "Limit of detection,” or LoD, as those terms are used herein, includes the lowest concentration at which one can identify a sample as containing a molecule of the substance of interest, e.g., the first non-zero value. It can be defined by the variability of zeros and the slope of the standard curve.
  • the limit of detection of an assay may be determined by running a standard curve, determining the standard curve zero value, and adding 2 standard deviations to that value.
  • a concentration of the substance of interest that produces a signal equal to this value is the "lower limit of detection" concentration.
  • the moiety has properties that are consistent with its use in the assay of choice.
  • the assay is an immunoassay, where the fluorescent moiety is attached to an antibody; the moiety must have properties such that it does not aggregate with other antibodies or proteins, or experiences no more aggregation than is consistent with the required accuracy and precision of the assay.
  • fluorescent moieties that are preferred are fluorescent moieties, e.g., dye molecules that have a combination of 1) high absorption coefficient; 2) high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with labeling the molecule of interest (e.g., protein) so that it may be analyzed using the analyzers and systems of the invention (e.g., does not cause precipitation of the protein of interest, or precipitation of a protein to which the moiety has been attached).
  • fluorescent moieties e.g., dye molecules that have a combination of 1) high absorption coefficient; 2) high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with labeling the molecule of interest (e.g., protein) so that it may be analyzed using the analyzers and systems of the invention (e.g., does not cause precipitation of the protein of interest, or precipitation of a protein to which the moiety has been attached).
  • Alexa Fluor dyes Molecular Probes, Eugene, Oreg.
  • the Alexa Fluor dyes are disclosed in U.S. Pat. Nos. 6,977,305; 6,974,874; 6,130,101; and 6,974,305 which are herein incorporated by reference in their entirety.
  • Some embodiments of the invention utilize a dye chosen from the group consisting of Alexa Fluor 647, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750.
  • Some embodiments of the invention utilize a dye chosen from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 700 and Alexa Fluor 750. Some embodiments of the invention utilize a dye chosen from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. Some embodiments of the invention utilize the Alexa Fluor 647 molecule, which has an absorption maximum between about 650 and 660 nm and an emission maximum between about 660 and 670 nm. The Alexa Fluor 647 dye is used alone or in combination with other Alexa Fluor dyes.
  • the fluorescent label moiety that is used to detect an FSGM in a sample using the analyzer systems of the invention is a quantum dot.
  • Quantum dots also known as semiconductor nanocrystals or artificial atoms, are semiconductor crystals that contain anywhere between 100 to 1,000 electrons and range from 2-10 nm. Some QDs can be between 10-20 nm in diameter. QDs have high quantum yields, which makes them particularly useful for optical applications.
  • QDs are fluorophores that fluoresce by forming excitons, which are similar to the excited state of traditional fluorophores, but have much longer lifetimes of up to 200 nanoseconds. This property provides QDs with low photobleaching.
  • the energy level of QDs can be controlled by changing the size and shape of the QD, and the depth of the QDs' potential.
  • One optical feature of small excitonic QDs is coloration, which is determined by the size of the dot. The larger the dot, the redder, or more towards the red end of the spectrum the fluorescence. The smaller the dot, the bluer or more towards the blue end it is.
  • the bandgap energy that determines the energy and hence the color of the fluoresced light is inversely proportional to the square of the size of the QD. Larger QDs have more energy levels which are more closely spaced, thus allowing the QD to absorb photons containing less energy, i.e., those closer to the red end of the spectrum.
  • the protein that is detected with the single molecule analyzer system is labeled with a QD.
  • the single molecule analyzer is used to detect a protein labeled with one QD and using a filter to allow for the detection of different proteins at different wavelengths.
  • One aspect of the invention pertains to isolated FSGM proteins and biologically active portions thereof, including ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against an FSGM protein or a fragment thereof.
  • the native FSGM protein can be isolated by an appropriate purification scheme using standard protein purification techniques.
  • a protein or peptide comprising the whole or a segment of the FSGM protein is produced by recombinant DNA techniques.
  • Alternative to recombinant expression such protein or peptide can be synthesized chemically using standard peptide synthesis techniques.
  • An "isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • heterologous protein also referred to herein as a "contaminating protein”
  • the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the protein When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
  • Biologically active portions of an FSGM protein include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the FSGM protein, which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein.
  • biologically active portions comprise a domain or motif with at least one activity of the corresponding full-length protein.
  • a biologically active portion of an FSGM protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • other biologically active portions, in which other regions of the FSGM protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of the FSGM protein.
  • Preferred FSGM proteins are listed in Table 1.
  • Other useful proteins are substantially identical (e.g., at least about 40%, preferably 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to one of these sequences and retain the functional activity of the corresponding naturally-occurring FSGM protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is calculated using a global alignment.
  • the percent identity between the two sequences is calculated using a local alignment.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, which is able to perform gapped local alignments for the programs BLASTN, BLASTP and BLASTX.
  • PSLBlast can be used to perform an iterated search which detects distant relationships between molecules.
  • the default parameters of the respective programs e.g., BLASTX and BLASTN
  • BLASTX and BLASTN the default parameters of the respective programs. See the NCBI website.
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • a PAM120 weight residue table can, for example, be used with a k-tuple value of 2.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
  • nucleic acid molecules which encode an FSGM protein or a portion thereof.
  • isolated nucleic acids of the invention also include nucleic acid molecules sufficient for use as hybridization probes to identify FSGM nucleic acid molecules, and fragments of FSGM nucleic acid molecules, e.g., those suitable for use as PCR primers for the amplification of a specific product or mutation of FSGM nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule preferably a protein-encoding sequences
  • is free of sequences which naturally flank the nucleic acid i.e., sequences located at the 5' and 3' ends of the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule that is substantially free of cellular material includes preparations having less than about 30%, 20%, 10%, or 5% of heterologous nucleic acid (also referred to herein as a "contaminating nucleic acid").
  • a nucleic acid molecule of the present invention can be isolated using standard molecular biology techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • a nucleic acid molecule of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • nucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which has a nucleotide sequence complementary to the nucleotide sequence of an FSGM nucleic acid or to the nucleotide sequence of a nucleic acid encoding an FSGM protein.
  • a nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.
  • a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence, wherein the full length nucleic acid sequence comprises an FSGM nucleic acid or which encodes an FSGM protein.
  • Such nucleic acids can be used, for example, as a probe or primer.
  • the probe/primer typically is used as one or more substantially purified oligonucleotides.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 15, more preferably at least about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a nucleic acid of the invention.
  • Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences corresponding to one or more FSGMs of the invention.
  • the probes hybridize to nucleic acid sequences that traverse splice junctions.
  • the probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as part of a diagnostic test kit or panel for identifying cells or tissues which express or mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein or its translational control sequences have been mutated or deleted.
  • the invention further encompasses nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding an FSGM protein (e.g., protein having the sequence provided in the sequence listing), and thus encode the same protein.
  • an FSGM protein e.g., protein having the sequence provided in the sequence listing
  • DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. In addition, it will be appreciated that DNA polymorphisms that affect RNA expression levels can also exist that may affect the overall expression level of that gene (e.g., by affecting regulation or degradation).
  • allelic variant refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide corresponding to an FSGM of the invention.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene.
  • Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
  • an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or more nucleotides in length and hybridizes under stringent conditions to an FSGM nucleic acid or to a nucleic acid encoding a marker protein.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%) identical to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C.
  • FXN therapy involves the administration of an FXN therapeutic to a subject with Leigh Syndrome.
  • FXN therapeutic may be provided by administration of any agent capable of increasing FXN levels in a subject, e.g., a subject with Leigh Syndrome.
  • FXN protein delivery may be delivery of full length FXN or delivery of a FXN fusion protein.
  • FXN fusion protein refers to FXN or a fragment of FXN fused to a full length or a fragment of a different protein, or to a peptide.
  • an FXN fusion protein comprises a polypeptide that comprises FXN, e.g., full-length hFXN (SEQ ID NO: 1) or mature hFXN (SEQ ID NO: 2).
  • the FXN fusion protein also comprises a cell penetrating peptide (CPP).
  • cell penetrating peptide refers to a short peptide sequence, typically between 5 and 30 amino acids long, that can facilitate cellular intake of various molecular cargo, such as proteins.
  • a CPP present in an FXN fusion protein facilitates the delivery of the FXN fusion protein into a cell, e.g., a recipient cell.
  • CPPs may be polycationic, i.e., have an amino acid composition that either contains a high relative abundance of positively charged amino acids, such as lysine or arginine.
  • CCPs may also be amphipathic, i.e., have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
  • CPPs may also be hydrophobic, i.e., contain only apolar residues with low net charge, or have hydrophobic amino acid groups that are crucial for cellular uptake.
  • a CPP that may be comprised in the FXN fusion protein may be any CPP known to a person skilled in the art.
  • the CPP may be any CPP listed in the Database of Cell- Penetrating Peptides CPPsite 2.0, the entire contents of which are hereby incorporated herein by reference.
  • CPPs useful in the context of the present invention may a cell penetrating peptide derived from a protein selected from the group consisting of HIV TransActivator of Transcription peptide (HIV-TAT), galanin, mastoparan, transportan, penetratin, polyarginine, VP22, transportan, amphipathic peptides such as MAP, KALA, ppTG20, prolinerich peptides, MPG-derived peptides, Pep-1, and also loligomers, arginine-rich peptides and calcitonin-derived peptides.
  • HIV-TAT HIV TransActivator of Transcription peptide
  • the CPP comprises a TAT protein domain comprising amino acids 47-57 of the 86 amino acid full length HIV-TAT protein (which 11 amino acid peptide may also be referred to herein as “HIV-TAT”; SEQ ID NO:4).
  • the CPP consists of HIV-TAT (SEQ ID NO:4).
  • the CPP comprises amino acids 47-57 of the 86 amino acid full length HIV-TAT protein with a methionine added at the amino terminus for initiation (12 AA; “HIV-TAT+M”): MYGRKKRRQRRR (SEQ ID NO: 5). Table 2 below lists amino acid sequences of exemplary CPPs.
  • the CPP comprised in the FXN fusion protein is HIV-TAT (SEQ ID NO: 4).
  • the FXN fusion protein comprises full-length FXN, e.g., SEQ ID NO: 1, and HIV-TAT, e.g., SEQ ID NO: 4, as CPP.
  • the CPP may be fused together with the FXN, e.g., full-length FXN, via a linker to form a single polypeptide chain.
  • FXN fusion proteins include TAT-FXN fusion proteins, where TAT or a fragment of TAT may be directly or indirectly (through a linker) linked to either the N- or the C- terminus of FXN.
  • the linker may comprise the amino acid sequence GG.
  • the CPP e.g., HIV-TAT
  • an FXN fusion protein of the present disclosure facilitates delivery of the FXN fusion protein into a cell, e.g., a cell that may be present in vitro, ex vivo, or in a subject with Eeigh Syndrome.
  • the FXN fusion protein may be processed by cellular machinery to remove the CPP, e.g., HIV-TAT, from the FXN.
  • the exemplary FXN fusion protein is a 24.9 kDa fusion protein currently under investigation as an FXN therapy to restore functional levels of FXN in the mitochondria of FRDA subjects.
  • the exemplary FXN fusion protein includes the HIV-TAT peptide linked to the N-terminus of the full-length hFXN protein.
  • the mechanism of action of the exemplary FXN fusion protein relies on the cell-penetrating ability of the HIV-TAT peptide to deliver the exemplary FXN fusion protein into cells and the subsequent processing into mature hFXN after translocation into the mitochondria.
  • the exemplary FXN fusion protein is described in US 2021/0047378, the entire contents of which are hereby incorporated herein by reference.
  • the exemplary FXN fusion protein comprises the following amino acid sequence (224 amino acids): MYGRKKRRQRRRGGMWTEGRRAVAGEEASPSPAQAQTETRVPRPAEEAPECGRRGER TDIDATCTPRRASSNQRGENQIWNVKKQSVYEMNERKSGTEGHPGSEDETTYEREAEET EDSEAEFFEDEADKPYTFEDYDVSFGSGVETVKEGGDEGTYVINKQTPNKQIWESSPSS GPKRYDWTGKNWVYSHDGVSEHEEEAAEETKAEKTKEDESSEAYSGKDA (SEQ ID NO: 12).
  • FXN may also be delivered by viral gene delivery, which may utilize retroviral, lentiviral, and adeno-associated viral vectors, as well as adenoviruses.
  • FXN therapy may be achieved by upregulation of endogenous mutant FXN gene, which depending on the number of GAA repeats is expressed in varying levels in carriers of the mutant FXN allele.
  • the present disclosure provides methods for evaluating and/or monitoring efficacy of FXN therapy in a subject with Leigh Syndrome.
  • the disclosure further provides methods for determining whether a subject is in need of FXN therapy or a change in FXN therapy, e.g., determining whether FXN therapy should be initiated, increased, decreased or ceased in a subject in Leigh Syndrome.
  • the methods are carried out by the subject using a sample obtained from the same subject or as a point of care test, and results can be assessed by the subject or by a physician.
  • the present disclosure provides methods for evaluating efficacy of a frataxin (FXN) therapy in a subject with Leigh Syndrome, the method comprising: (a) determining a baseline FXN expression profile for one or more FXN- sensitive genomic markers (FSGMs) in a sample obtained from the subject prior to administration of the FXN therapy; (b) determining an FXN therapy expression profile for the one or more FXN-sensitive genomic markers (FSGMs) in a sample obtained from the subject following administration of the FXN therapy; (c) comparing the FXN therapy expression profile determined in step (b) with the baseline FXN expression profile determined in step (a); and (d) determining efficacy of the FXN therapy based on the comparison in step (c); wherein the one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, R
  • comparing the FXN therapy expression profile determined in step (b) with the baseline FXN expression profile comprises comparing the expression level of one or more FSGMs in the FXN therapy expression profile to the expression level of the corresponding one or more FSGMs in the baseline FXN expression profile.
  • the present disclosure also provides a method for evaluating efficacy of a frataxin (FXN) therapy in a subject with Leigh Syndrome, the method comprising: (a) determining an FXN therapy expression profile for one or more FXN-sensitive genomic markers (FSGMs) in a sample obtained from the subject following administration of an FXN therapy; (b) comparing the subject FXN expression profile determined in step (a) with a reference FXN expression profile for the one or more FSGMs; and (c) determining efficacy of the FXN therapy based on the comparison in step (b); wherein the one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3,
  • the baseline FXN expression profile for the one or more FSGMs is determined in a sample obtained from a subject prior with Leigh Syndrome prior to administration of an FXN therapy.
  • comparing the FXN therapy expression profile determined in step (a) with a reference FXN expression profile comprises comparing the expression level of one or more FSGMs in the FXN therapy expression profile to the expression level of the corresponding one or more FSGMs in the reference FXN expression profile.
  • a reference FXN expression profile e.g., a baseline FXN expression profile
  • an increase in the expression level of EGR1 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of EGR2 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of PTGS2 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of CUL2 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of ABCE1 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of EiFlAX in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of RPS27L in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of RPL10 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-ATP8 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-ATP6 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-ND3 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-NDl in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-ND4 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-C03 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of CYCs in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of SLIRP in a FXN therapy expression profile is an indication that the expression level of SLIRP in a reference FXN expression profile, e.g., a baseline FXN expression profile, is an indication that the FXN therapy is effective.
  • a decrease in the expression level of CYR61 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • a decrease in the expression level of THBS1 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • a decrease in the expression level of UBE2D3 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPL26 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPL38 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPL32 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPL39 in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPS 15A in a FXN therapy expression profile is an indication that the FXN therapy is effective.
  • an increase in the expression level of one or more FSGMs selected from the group consisting of EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX,RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy, as compared to the expression level of the corresponding one or more FSGMs in a sample obtained from the subject with Leigh Syndrome prior to administration of FXN therapy is an indication that the FXN therapy is effective.
  • a decrease in the expression level of one or more FSGMs selected from the group consisting of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy, as compared to the expression level of the corresponding one or more FSGMs in a sample obtained from the subject with Leigh Syndrome prior to administration of FXN therapy is an indication that the FXN therapy is effective.
  • lack of a decrease in the expression level of one or more FSGMs selected from the group consisting of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy, as compared to the expression level of the corresponding one or more FSGMs in a sample obtained from the subject with Leigh Syndrome prior to administration of FXN therapy is an indication that the FXN therapy is not effective.
  • an increase in the expression level of EGR1 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of EGR2 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of PTGS2 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of CUL2 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of ABCE1 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of EiFlAX in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of RPS27L in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of RPL10 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-ATP8 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-ATP6 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-ND3 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-NDl in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-ND4 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of mt-CO3 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of CYCs in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • an increase in the expression level of SLIRP in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • a decrease in the expression level of CYR61 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • a decrease in the expression level of THBS1 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • a decrease in the expression level of UBE2D3 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPL26 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPL38 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPL32 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPL39 in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • a decrease in the expression level of RPS15A in a sample obtained from a subject with Leigh Syndrome following administration of FXN therapy is an indication that the FXN therapy is effective.
  • lack of an increase or decrease in the expression level of one or more FSGMs e.g., one or any combination of one or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, in a sample obtained from a subject with Leigh Syndrome following administration of an FXN therapy, as compared to the expression level of the corresponding one or more FSLMs in a control sample, e.g., a sample obtained from the subject with Leigh Syndrome prior to administration of the FXN therapy, is an indication that the FXN therapy is ineffective, e.g., at the current dose, and
  • the methods provided herein may also include monitoring a subject with Leigh Syndrome being administered FXN therapy.
  • lack of an increase or decrease in the detected expression level of one or more FSGMs e.g., one or any combination of one or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, in a second sample obtained from a subject with Leigh Syndrome after administration of FXN therapy, as compared to the expression level of the corresponding one or more FSGMs in a first sample obtained from the subject with Leigh Syndrome before administration of FXN therapy, is an indication that the FXN therapy is not effic
  • an increased or decreased expression level of one or more FSGMs lipid amount of one or more FSLMs e.g., one or any combination of one or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, in a second sample obtained from a subject with Leigh Syndrome after administration of FXN therapy, as compared to the amount of the one or more FSGMs in a first sample obtained from the subject with Leigh Syndrome before administration of FXN therapy, is an indication that the FXN therapy is efficacious and/or the subject with Leigh Syndrome is responsive to the FXN therapy.
  • the method may further include the step of
  • the expression level of one or more of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A is decreased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of CYR61 is decreased in a subject with Leigh Syndrome following treatment with FXN therapy.
  • the expression level of THBS1 is decreased in a subject with Leigh Syndrome following treatment with FXN therapy.
  • the expression level of UBE2D3 is decreased in a subject with Leigh Syndrome following treatment with FXN therapy.
  • the expression level of RPL26 is decreased in a subject with Leigh Syndrome following treatment with FXN therapy.
  • the expression level of RPL38 is decreased in a subject with Leigh Syndrome following treatment with FXN therapy.
  • the expression level of RPL32 is decreased in a subject with Leigh Syndrome following treatment with FXN therapy.
  • the expression level of RPL39 is decreased in a subject with Leigh Syndrome following treatment with FXN therapy.
  • the expression level of RPS15A is decreased in a subject with Leigh Syndrome following treatment with FXN therapy.
  • the expression level of one or more of EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of EGR1 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of EGR2 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of PTGS2 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of CUL2 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of ABCE1 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of EiFl AX is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of RPS27L is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of RPL10 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of mt- ATP8 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of mt-ATP6 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of mt-ND3 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of mt-NDl is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of mt-ND4 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of mt-CO3 is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of CYCs is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the expression level of SLIRP is increased in a subject with Leigh Syndrome following treatment with an FXN therapy.
  • the present disclosure also provides a method of monitoring treatment of a subject with Leigh Syndrome with a frataxin (FXN) therapy, the method comprising: (a) determining a first FXN therapy expression profile for one or more FXN- sensitive genomic markers (FSGMs) in a first sample obtained from a subject with Leigh Syndrome at a first time point following administration of an FXN therapy to the subject, wherein the one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP; (b) determining a second FXN expression profile for the one or more FXN-
  • comparing the second FXN therapy expression profile with the first FXN therapy expression profile comprises comparing the expression level of one or more FSGMs in the second FXN therapy expression profile with the amount of the corresponding one or more FSGMs in the first FXN therapy expression profile.
  • a decrease in the amount of one or more of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A in the second FXN therapy expression profile, as compared to the first FXN therapy expression profile is an indication that the FXN therapy is effective.
  • lack of a decrease in the amount of one or more of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A in the second FXN therapy expression profile, as compared to the first FXN therapy expression profile is an indication that the FXN therapy is not effective.
  • an increase in the amount of one or more of EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP in the second FXN therapy expression profile, as compared to the first FXN therapy expression profile, is an indication that the FXN therapy is effective.
  • lack of an increase in the amount of one or more of EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP in the second FXN therapy expression profile, as compared to the first FXN therapy expression profile, is an indication that the FXN therapy is not effective.
  • the method for monitoring treatment of a subject with Leigh Syndrome with FXN therapy further comprises making a determination, or making a recommendation to a healthcare provider, to maintain the FXN therapy regimen based on the comparison in step (c). In some embodiments, the method further comprises making a determination, or making a recommendation to a healthcare provider, to alter the FXN therapy regimen based on the comparison in step (c). In some embodiments, the method further comprises making a determination to maintain the dose and/or administration frequency, increase the dose and/or administration frequency, or decrease the dose and/or administration frequency of the FXN therapy based on the comparison in step (c).
  • the method further comprises making a recommendation, e.g., to a healthcare provider, to maintain the dose and/or administration frequency, increase the dose and/or administration frequency, or decrease the dose and/or administration frequency of the FXN replacement therapy based on the comparison in step (c).
  • a recommendation e.g., to a healthcare provider, to maintain the dose and/or administration frequency, increase the dose and/or administration frequency, or decrease the dose and/or administration frequency of the FXN replacement therapy based on the comparison in step (c).
  • the method comprises continuing to administer the FXN therapy regimen (e.g., without changing the regimen, e.g., maintaining the dose and/or administration frequency) to the subject with Leigh Syndrome based on the comparison in step (c).
  • the method further comprises altering the FXN replacement therapy regimen based on the comparison in step (c).
  • the method further comprises administering an altered FXN therapy regimen based on the comparison in step (c).
  • administering an altered FXN therapy regimen comprises administering an increased dose and/or administration frequency, or administering a decreased dose and/or administration frequency of the FXN therapy.
  • the method for monitoring treatment of a subject with Leigh Syndrome with FXN therapy may further comprise the step of continuing administering the FXN therapy to the subject without adjustments, or decreasing the dose and/or administration frequency of the FXN therapy, if the FXN therapy is determined to be effective.
  • the method for monitoring treatment of a subject with FXN therapy may further comprise a step of adjusting FXN therapy by increasing the dose and/or administration frequency of the FXN therapy if the FXN therapy is determined to be not effective.
  • a decrease in the expression level of one or more FSGMs selected from the group consisting of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A in the second FXN therapy expression profile, as compared to the first FXN therapy expression profile, is an indication that the dose and/or administration schedule of the FXN therapy should be maintained or decreased.
  • lack of a decrease in the expression level of one or more FSGMs selected from the group consisting of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A in the second FXN therapy expression profile, as compared to the first FXN therapy expression profile, is an indication that dose and/or administration schedule of the FXN therapy should be maintained or increased.
  • a biological sample is a solid tissue sample, preferably a buccal sample, alternatively a skin biopsy sample, skin strip, hair follicle, muscle biopsy sample, or a body fluid sample such as blood (including any blood product, such as whole blood, plasma, serum, or specific types of cells of the blood), urine, saliva, or seminal fluid.
  • the biological sample is a skin sample.
  • the biological sample is a buccal sample.
  • the biological sample is a blood-derived sample, e.g., a plasma sample or a platelet sample.
  • FSGM levels can be detected based on the absolute level or a normalized or relative level. Detection of absolute FSGM levels may be preferable when monitoring the treatment of a subject with Eeigh Syndrome. For example, the levels of one or more FSGMs can be monitored in a subject undergoing treatment with an FXN therapy, e.g., at regular intervals, such as a monthly intervals. A modulation in the levels of one or more FSGMs can be monitored over time to observe trends in changes of the FSGM levels.
  • Eevels of the FSGMs of the disclosure in the subject may be higher than the levels of those FSLMs in a normal sample, but may be lower than the prior levels, thus indicating a lack of efficacy of the FXN therapy in the subject. Changes, or not, in FSGM levels may be more relevant to treatment decisions for the subject than FSGM levels present in the population.
  • determinations may be based on the normalized level of the FSGM.
  • FSGM levels are normalized by correcting the absolute level of an FSGM by comparing its level to the expression level of a gene markers that is not FSGM, e.g., a gene marker that is not sensitive to FXN levels. This normalization allows the comparison of the FSGM level in one sample, e.g., a sample from a subject with Leigh Syndrome, to another sample, e.g., a normal sample, or between samples from different sources.
  • Also provided by the disclosure is a method for treating Leigh Syndrome, the method comprising: (a) determining an FXN therapy expression profile in a sample obtained from a subject with Leigh Syndrome for one or more FXN-sensitive genomic markers (FSGMs), (b) comparing the FXN expression profile of the sample with at least one other expression profile selected from the group consisting of normal FXN expression profile for the one or more FSGMs, baseline FXN expression profile for the one or more FSGMs, and FXN therapy expression profile for the one or more FSGMs, (c) classifying the FXN therapy expression profile determined in step (a) as corresponding to a normal FXN expression profile, baseline FXN expression profile or an FXN therapy expression profile, and (d) initiating or modulating an FXN therapy based on the classification of the FXN expression profile of the sample, wherein the one or more FSGMs are selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2
  • a therapy regime using FXN therapy may be initiated, paused or ceased.
  • an FXN therapy dosage regime may be modified, e.g., increased or decreased.
  • the method further comprises obtaining or providing a sample from a subject with Leigh Syndrome.
  • modulating an FXN therapy comprises increasing the dosage, decreasing the dosage, increasing the administration frequency, or decreasing the administration frequence, of the FXN therapy.
  • the FXN expression profile determined in step (a) is classified as corresponding to a baseline FXN expression profile.
  • the FXN expression profile determined in step (a) is classified as corresponding to a baseline FXN expression profile.
  • the FXN expression profile in the sample is classified as a baseline FXN expression profile
  • administration of an FXN therapy is initiated in the subject with Leigh Syndrome.
  • the FXN therapy regiment is altered, e.g., the dose and/or administration frequency of the FXN therapy is increased.
  • the FXN expression profile determined in step (a) is classified as corresponding to a normal FXN expression profile.
  • the FXN expression profile in the sample when the FXN expression profile in the sample is classified as a normal FXN expression profile, administration of an FXN therapy is not initiated in the subject with Leigh Syndrome. In some embodiments, when the FXN expression profile in the sample is classified as a normal FXN expression profile, and the subject with Leigh Syndrome is already undergoing FXN therapy, the FXN therapy regimen is maintained (z.e., not changed), e.g., the dose and/or administration frequency of the FXN therapy is maintained.
  • the present disclosure also provides a method of treating Leigh Syndrome in a subject, comprising: (a) determining an FXN expression profile for one or more FSGMs in a sample from a subject with Leigh Syndrome; and (b) recommending to a healthcare provider to administer an FXN therapy to the subject based on the subject FXN expression profile determined in step (a).
  • the present disclosure also provides a method of treating Leigh Syndrome in a subject, comprising: (a) obtaining an FXN expression profile for one or more FSGMs in a sample obtained from a subject with Leigh Syndrome; and (b) administering an FXN therapy to the subject based on the subject FXN expression profile.
  • modulating an FXN therapy comprises increasing the dosage, decreasing the dosage, increasing the administration frequency, decreasing the administration frequency, or any combination thereof, of the FXN therapy.
  • FSGMs selected from the group consisting of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A in the sample obtained from the subject with Leigh Syndrome as compared to the expression level of the corresponding one or more FSGMs in a normal FXN expression profile
  • an FXN therapy is administered to the subject with Leigh Syndrome, or a recommendation to a healthcare provider is made to administer an FXN replacement therapy to the subject with Leigh Syndrome.
  • the FXN therapy regimen is altered (e.g., the dosage and/or administration frequency is increased), or a recommendation to a healthcare provider is made to alter the FXN replacement therapy regimen.
  • FSGMs selected from the group consisting of CYR61, THBS1, UBE2D3, RPL26, RPL38, RPL32, RPL39 and RPS15A in the sample obtained from the subject with Leigh Syndrome as compared to the expression level of the corresponding one or more FSGMs in a normal FXN expression profile
  • an FXN therapy is not administered to the subject with Leigh Syndrome, or a recommendation to a healthcare provider is made to not administer an FXN replacement therapy to the subject.
  • the FXN replacement therapy regimen is maintained (z.e., not changed), or a recommendation to a healthcare provider is made to maintain the FXN replacement therapy regimen.
  • FSGMs selected from the group consisting of EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP in the sample obtained from the subject with Leigh Syndrome as compared to the expression level of the corresponding one or more FSGMs in a normal FXN expression profile, then an FXN therapy is administered to the subject with Leigh Syndrome, or a recommendation to a healthcare provider is made to administer an FXN replacement therapy to the subject with Leigh Syndrome.
  • FSGMs selected from the group consisting of EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs
  • the FXN therapy regimen is altered (e.g., the dosage and/or administration frequency is increased), or a recommendation to a healthcare provider is made to alter the FXN replacement therapy regimen.
  • FSGMs selected from the group consisting of EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP in the sample obtained from the subject with Leigh Syndrome as compared to the expression level of the corresponding one or more FSGMs in a normal FXN expression profile, then an FXN therapy is not administered to the subject with Leigh Syndrome, or a recommendation to a healthcare provider is made to not administer an FXN replacement therapy to the subject.
  • FSGMs selected from the group consisting of EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and S
  • FSGMs selected from the group consisting of EGR1, EGR2, PTGS2, CUL2, ABCE1, EIF1AX, RPS27L, RPL10, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP in the sample obtained from the subject with Leigh Synrome as compared to the expression level of the corresponding one or more FSGMs in a normal FXN expression profile, and the subject with Leigh Syndrome is undergoing an FXN therapy, then the FXN replacement therapy regimen is maintained (z.e., not changed), or a recommendation to a healthcare provider is made to maintain the FXN replacement therapy regimen.
  • the present disclosure also involves the analysis and consideration of any clinical and/or subject-related health data, for example, data obtained from an Electronic Medical Record (e.g., collection of electronic health information about individual subjects or populations relating to various types of data, such as, demographics, medical history, medication and allergies, immunization status, laboratory test results, radiology images, vital signs, personal statistics like age and weight, and billing information).
  • an Electronic Medical Record e.g., collection of electronic health information about individual subjects or populations relating to various types of data, such as, demographics, medical history, medication and allergies, immunization status, laboratory test results, radiology images, vital signs, personal statistics like age and weight, and billing information.
  • the methods provided herein further comprise obtaining a biological sample from a subject suspected of having Leigh Syndrome.
  • the methods provided herein further comprise selecting a treatment regimen for the subject based on the expression level of the one or more FSGMs selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the treatment method is started, changed, revised, or maintained based on the results from the methods of the disclosure, e.g., when it is determined that the subject with Leigh Syndrome is responding to the treatment regimen, or when it is determined that the subject with Leigh Syndrome is not responding to the treatment regimen, or when it is determined that the subject with Leigh Syndrome is insufficiently responding to the treatment regimen.
  • the treatment method is changed based on the results from the methods.
  • the method further comprises isolating a component of the biological sample.
  • the method further comprises concentrating a component of the biological sample.
  • the present disclosure also provides a method of detecting one or more frataxin- sensitive genomic markers (FSGMs) in a sample from a subject with Leigh Syndrome, comprising contacting the sample, or a portion thereof, with one or more reagents specific for detecting the level of each of one or more FSGMs, wherein the one or more FSGMs comprise one or more FSGMs selected from the group consisting of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, thereby detecting the FSGMs in the sample.
  • FSGMs frataxin- sensitive genomic markers
  • the sample is selected from the group consisting of a buccal sample, a skin sample, a hair follicle or a blood-derived sample.
  • the sample is a blood-derived sample, e.g., a plasma sample or a serum sample.
  • the same is a urine sample.
  • the sample is obtained from the subject with Leigh Syndrome before the subject is administered FXN therapy. In some embodiments, the sample is obtained from the subject with Leigh Syndrome after the subject is administered FXN replacement therapy. In some embodiments, the sample is obtained from the subject with Leigh Syndrome at different time points, e.g., 1, 2, 3, 4 or more time points, while the subject is being administered FXN therapy. In some embodiments, the method further comprises obtaining a sample from the subject with Leigh Syndrome. In some embodiments, the sample is selected from the group consisting of a buccal sample, a skin sample, a hair follicle or a blood-derived sample. In some embodiments, the sample is a blood-derived sample, e.g., a plasma sample or a serum sample. In some embodiments, the same is a urine sample.
  • the one or more FSGMs comprise one or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise two or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise three or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt- ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise four or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise five or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise six or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise seven or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eight or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise nine or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise ten or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eleven or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twelve or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise thirteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise fourteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise fifteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise sixteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise seventeen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise eighteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise nineteen or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise twenty or more of EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt- ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the one or more FSGMs comprise EGR1. In one embodiment, the one or more FSGMs comprise EGR2. In one embodiment, the one or more FSGMs comprise CYR61. In one embodiment, the one or more FSGMs comprise THBS1. In one embodiment, the one or more FSGMs comprise PTGS2. In one embodiment, the one or more FSGMs comprise UBE2D3. In one embodiment, the one or more FSGMs comprise CUE2. In one embodiment, the one or more FSGMs comprise ABCE1. In one embodiment, the one or more FSGMs comprise EiFlAX. In one embodiment, the one or more FSGMs comprise RPE26. In one embodiment, the one or more FSGMs comprise RPE38.
  • the one or more FSGMs comprise RPS27E. In one embodiment, the one or more FSGMs comprise RPE10. In one embodiment, the one or more FSGMs comprise RPE32. In one embodiment, the one or more FSGMs comprise RPE39. In one embodiment, the one or more FSGMs comprise RPS15A. In one embodiment, the one or more FSGMs comprise mt- ATP8. In one embodiment, the one or more FSGMs comprise mt-ATP6. In one embodiment, the one or more FSGMs comprise mt-ND3. In one embodiment, the one or more FSGMs comprise mt-NDl. In one embodiment, the one or more FSGMs comprise mt-ND4. In one embodiment, the one or more FSGMs comprise mt-CO3. In one embodiment, the one or more FSGMs comprise CYCs. In one embodiment, the one or more FSGMs comprise SEIRP.
  • kits for evaluating and monitoring effectiveness of FXN therapy in a subject with Eeigh Syndrome may be used by a subject for self-evaluation or may be carried out by a subject for evaluation by a physician, or as point of care kits.
  • kits may include one or more of the following: a reagent that specifically binds to an FSGM of the invention, and a set of instructions for measuring the level of the FSGM.
  • the FSGM comprises any one of FSGMs selected from ATF3, NR4al, EGR1, EGR2,CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPE26, RPE38, RPS27E, RPE10, RPE32, RPE39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP. Ill
  • kits for detecting the presence of an FSGM protein or nucleic acid in a biological sample can be used to evaluate and/or monitor effectiveness of FXN therapy in a subject with Leigh Syndrome.
  • the kit can comprise a labeled compound or agent capable of detecting an FSGM protein or nucleic acid in a biological sample and means for determining the amount of the protein or mRNA in the sample (e.g., an antibody which binds the protein or a fragment thereof, or an oligonucleotide probe which binds to DNA or mRNA encoding the protein).
  • Kits can also include instructions for use of the kit for practicing any of the methods provided herein or interpreting the results obtained using the kit based on the teachings provided herein.
  • kits can also include reagents for detection of a control protein in the sample, e.g., actin for tissue samples, albumin in blood or blood derived samples for normalization of the amount of the FSGM present in the sample.
  • the kit can also include the purified FSGM for detection for use as a control or for quantitation of the assay performed with the kit.
  • a biological sample which is evaluated by a kit or panel of the disclosure is a solid tissue sample, preferably a buccal sample, alternatively, a skin biopsy sample, skin strip, hair follicle, muscle biopsy sample, or a body fluid sample such as blood (including any blood product, such as whole blood, plasma, serum, or specific types of cells of the blood), urine, saliva, or seminal fluid.
  • Kits include a panel of reagents for use in a method to evaluate and/or monitor effectiveness of FXN therapy, the panel comprising at least two detection reagents, wherein each detection reagent is specific for one FSGM, wherein said FSGMs are selected from the FSGM protein sets provided herein.
  • At least one of the FSGMs comprises a protein encoded by any one of FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a first FSGM protein; and, optionally, (2) a second, different antibody which binds to either the first FSGM protein or the first antibody and is conjugated to a detectable label.
  • the kit includes (1) a second antibody (e.g., attached to a solid support) which binds to a second FSGM protein; and, optionally, (2) a third, different antibody which binds to either the second FSGM protein or the second antibody and is conjugated to a detectable label.
  • the first and second FSGM proteins are different.
  • the first and second FSGMs are FSGMs of the invention, e.g., one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt
  • the kit comprises a third antibody which binds to a third FSGM protein which is different from the first and second FSGM proteins, and a fourth different antibody that binds to either the third FSGM protein or the antibody that binds the third FSGM protein wherein the third FSGM protein is different from the first and second FSGM proteins.
  • the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding an FSGM protein or (2) a pair of primers useful for amplifying an FSGM nucleic acid molecule.
  • an oligonucleotide e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding an FSGM protein
  • a pair of primers useful for amplifying an FSGM nucleic acid molecule.
  • the kit can further include, for example: (1) an oligonucleotide, e.g., a second detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a second FSGM protein or (2) a pair of primers useful for amplifying the second FSGM nucleic acid molecule.
  • the first and second FSGMs are different.
  • the first and second FSGMs are FSGMs of the invention, e.g., one or more of the FSGMs selected from Table 1.
  • the kit can further include, for example: (1) an oligonucleotide, e.g., a third detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a third FSGM protein or (2) a pair of primers useful for amplifying the third FSGM nucleic acid molecule wherein the third FSGM is different from the first and second FSGMs.
  • the kit includes a third primer specific for each nucleic acid FSGM to allow for detection using quantitative PCR methods.
  • the kit can include FSGMs, including labeled FSGMs, to permit detection and identification of one or more FSGMs of the invention, e.g., one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, by chromatography.
  • kits for chromatography methods include compounds for derivatization of one or more FSGMs of the invention.
  • kits for chromatography methods include columns for resolving the FSGMs of the method.
  • Reagents specific for detection of an FSGM of the invention e.g., one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP, allow for detection and quantitation of the FSGM in a complex mixture, e.g., cell or tissue sample.
  • the reagents are species specific.
  • the reagents are not species specific.
  • the reagents are isoform specific. In certain embodiments, the reagents are not isoform specific.
  • kits for evaluation and/or monitoring of the effectiveness of FXN therapy comprise at least one reagent specific for the detection of the level of one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt- ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the kits for evaluation and/or monitoring of the effectiveness of FXN therapy comprise at least one reagent specific for the detection of the level of one or more of the FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26,
  • kits further comprise instructions for the detection, evaluation and/or monitoring of the effectiveness of FXN therapy based on the level of at least one FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt- ATP6, m
  • kits comprising at least one reagent specific for the detection of the level of at least one FSGMs selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the reagent detects a protein.
  • the reagent detects an mRNA.
  • kits can also comprise, e.g., a buffering agents, a preservative, a protein stabilizing agent, reaction buffers.
  • the kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate).
  • the kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample.
  • the controls can be control serum samples or control samples of purified proteins or nucleic acids, as appropriate, with known levels of target FSGMs.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
  • the kits of the invention may optionally comprise additional components useful for performing the methods of the invention.
  • the invention further provides panels of reagents for detection of one or more FSGM in a subject sample and at least one control reagent.
  • the FSGM comprises at least two or more FSGMs, wherein at least one of the FSGMs is selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-CO3, CYCs and SLIRP.
  • the control reagent is to detect the FSGM for detection in the biological sample
  • the panel is provided with a control sample containing the FSGM for use as a positive control and optionally to quantitate the amount of FSGM present in the biological sample.
  • the panel can be provided with reagents for detection of a control protein, e.g., actin for tissue samples, albumin in blood or blood derived samples for normalization of the amount of the FSGM present in the sample.
  • the panel can be provided with a purified FSGM for detection for use as a control or for quantitation of the assay performed with the panel.
  • the level of the FSGM in the panel is increased when compared to a control or in a subject with Leigh Syndrome following administration of an FXN therapy.
  • the FSGM is selected from the group consisting of EGR1, EGR2, PTGS2, CUL2, ABCE1, mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • the level of the FSGM in the panel is decreased when compared to a control or in a subject with Leigh Syndrome following administration of an FXN therapy.
  • the FSGM is selected from the group consisting of ATF3, NR4al, CYR61, THBS1, RPL26, RPL38, RPL32, RPL39 and RPS15A.
  • the panel comprises one or more FSGMs with an increased level when compared to a control following treatment of a subject with Leigh Syndrome with FXN therapy, and/or one or more FSGMs with a decreased level when compared to a control or following treatment of a subject with Leigh Syndrome with FXN therapy.
  • the panel includes reagents for detection of two or more
  • FSGMs of the invention (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or up to all of the FSGMs is selected from ATF3, NR4al, EGR1, EGR2, CYR61, THBS1, PTGS2, UBE2D3, CUL2, ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A, mt-ATP8, mt-ATP6, mt-ND3, mt- ND1, mt-ND4, mt-CO3, CYCs and SLIRP, preferably in conjunction with a control reagent.
  • each FSGM is detected by a reagent specific for that FSGM.
  • the panel includes replicate wells, spots, or portions to allow for analysis of various dilutions (e.g., serial dilutions) of biological samples and control samples.
  • the panel allows for quantitative detection of one or more FSGMs of the invention.
  • the panel is a protein chip for detection of one or more FSGMs. In certain embodiments, the panel is an EEISA plate for detection of one or more FSGMs. In certain embodiments, the panel is a plate for quantitative PCR for detection of one or more FSGMs.
  • the panel of detection reagents is provided on a single device including a detection reagent for one or more FSGMs of the invention and at least one control sample. In certain embodiments, the panel of detection reagents is provided on a single device including a detection reagent for two or more FSGMs of the invention and at least one control sample. In certain embodiments, multiple panels for the detection of different FSGMs of the invention are provided with at least one uniform control sample to facilitate comparison of results between panels.
  • the goal of this experiment was to identify gene markers that are differentially expressed in an in vitro cell model of Leigh Syndrome as compared to a control cell line, and that are modulated by treatment with an exemplary FXN fusion protein.
  • LRPPRC deficient human embryonic kidney 293 HEK293 cells and LRPPRC deficient Schwann cells as an in vitro model of Leigh Syndrome.
  • LRPPRC is known to be involved in mitochondrial energy production, and LRPPRC deficient cells were expected to have mitochondrial impairment.
  • HEK293 cells and Schwann cells were stably transfected with LRPPRC short hairpin (sh)RNA, resulting in targeted gene silencing of LRPPRC and production of LRPPRC knock down cells and Schwann LRPPRC knock down cells.
  • Corresponding control cell lines transfected with a scramble sequence were generated in parallel, resulting in the scramble control clones.
  • the FXN therapy used in this example involved treatment of LRPPRC deficient HEK293 cells and LRPPRC deficient cells with an exemplary FXN fusion protein.
  • the exemplary FXN fusion protein is a fusion protein comprising TAT-cpp and human FXN (hFXN) linked through a linker at the N-terminus of hFXN.
  • the hFXN in the exemplary FXN fusion protein is the full-length 210 aa frataxin long precursor form, which contains an 80 aa mitochondrial targeting sequence (MTS) at the N-terminus.
  • the full-length hFXN protein (amino acids 1-210) has the amino acid sequence of SEQ ID NO: 1.
  • hFXN protein As the hFXN protein is imported into the mitochondrial matrix, it is cleaved at amino acid 81, resulting in the mature form of FXN, having 130 aa and a predicted molecular weight of 14.2 kDa (SEQ ID NO: 2).
  • the full-length hFXN (SEQ ID NO: 1) comprises mature hFXN (SEQ ID NO: 2) and a mitochondrial targeting sequence (MTS) having the amino acid sequence MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQ RGLNQIWNVKKQSVYLMNLRK (SEQ ID NO: 3).
  • the exemplary FXN fusion protein also includes the HIV-TAT peptide YGRKKRRQRRR (SEQ ID NO: 4) linked via a linker to the N-terminus of the full-length hFXN protein.
  • the mechanism of action of the fusion protein relies on the cell-penetrating ability of the HIV-TAT peptide to deliver the fusion protein into cells and the subsequent processing into mature hFXN after translocation into the mitochondria.
  • the exemplary FXN fusion protein used in this Example is described in US 2021/0047378, the entire contents of which are hereby incorporated herein by reference, and has the following amino acid sequence (224 amino acids): MYGRKKRRQRRRGGMWTEGRRAVAGEEASPSPAQAQTETRVPRPAEEAPECGRRGER TDIDATCTPRRASSNQRGENQIWNVKKQSVYEMNERKSGTEGHPGSEDETTYEREAEET EDSEAEFFEDEADKPYTFEDYDVSFGSGVETVKEGGDEGTYVINKQTPNKQIWESSPSS GPKRYDWTGKNWVYSHDGVSEHEEEAAEETKAEKTKEDESSEAYSGKDA (SEQ ID NO: 12).
  • the LRPPRC-deficient HEK293 cells and Schwann cells were grown in growth media (DMEM containing 10% FBS) until -50% confluency. Cells were then treated with 20pM of the exemplary FXN fusion protein or vehicle in transduction media (DMEM containing 5% heat-inactivated FBS, 20mM glycerol) for 3 hours, then cells were supplemented with an equal volume of growth media. Treatment was repeated on 3 consecutive days.
  • Figure 1 is a dot plot showing fold change in the levels of expression of selected gene markers in LRPPRC -deficient HEK293 cells vs. control cells (black dots) and in LRPPRC-deficient HEK293 cells treated with the exemplary FXN fusion protein vs. vehicle (white dots).
  • the selected gene markers include genes involved in regulation of neuronal development and survival, such as ATF3, NR4al, EGR1, CYR61, THBS1 and PTGS2; genes involved in protein degradation, such as CUL2; genes involved in transation, such as ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A; and genes involved electron transport chain, such as mt-ATP8, mt- ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • genes involved in regulation of neuronal development and survival such as ATF3, NR4al, EGR1, CYR61, THBS1 and PTGS2
  • genes involved in protein degradation such as CUL2
  • genes involved in transation such as ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL
  • panel B is a dot plot showing fold change in the levels of expression of selected gene markers in LRPPRC-deficient Schwann cells vs. control cells (black dots) and in LRPPRC-deficient Schwann cells treated with the exemplary FXN fusion protein vs. vehicle (white dots).
  • the selected gene markers include genes involved in regulation of neuronal development and survival, such as ATF3, NR4al, EGR2, CYR61, THBS1 and PTGS2; genes involved in protein degradation, such as CUL2; genes involved in transation, such as ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39, RPS15A; and genes involved electron transport chain, such as mt-ATP8, mt-ATP6, mt-ND3, mt-NDl, mt-ND4, mt-C03, CYCs and SLIRP.
  • genes involved in regulation of neuronal development and survival such as ATF3, NR4al, EGR2, CYR61, THBS1 and PTGS2
  • genes involved in protein degradation such as CUL2
  • genes involved in transation such as ABCE1, EiFlAX, RPL26, RPL38, RPS27L, RPL10, RPL32, RPL39
  • the results presented in Figure 1 indicate that selected gene markers are modulated in LRPPRC-deficient cells, which represent an in vitro model of Leigh Syndrome, as compared to control cells.
  • the results presented in Figure 1 also indicate that these gene markers are also modulated, e.g., contrary regulated, by treatment with an exemplary FXN fusion protein.
  • the gene markers identified in Example 1 are frataxin-sensitive gene markers (FSGMs).
  • Example 1 demonstrate that the gene markers identified as being modulated in an in vitro model of Leigh Syndrome and also regulated by treatment with an exemplary FXN fusion protein may be used a biomarkers for evaluating efficacy of FXN therapy in subjects with Leigh Syndrome.
  • the goal of this experiment is study differential expression of gene markers in a mouse model of Leigh Syndrome as compared to a WT mouse.
  • the mouse model of Leigh Syndrome used in this experiment was the Ndufs KO mouse model harboring a mutation in the NDUFS4 gene encoding a small 18 kD protein of mitochondrial Complex I. Mutations in the NDUFS4 gene lead to mitochondrial dysfunction and demyelination.
  • the NDUFS4 KO mice are characterized by smaller body size as compared to WT mice, are blind, develop severa ataxia by day 35, and die by day 60. These mice also develop progressive neuronal deterioration, as evidenced by bilateral lesions in the brain, gliosis and immune activation.
  • Ndufs KO mouse model is described, e.g., in Kruse el al., “Mice with mitochondrial complex I deficiency develop a fatal encephalomyopathy”, Cell Metab. 2008 Apr; 7(4):312-20, the entire contents of which are hereby incorporated herein by reference.
  • mice at the Jackson laboratory were treated with 30 mg/kg of the exemplary FXN fusion protein or vehicle by daily subcutaneous injections for 6 weeks beginning at 1 week of age.
  • mice were sacrificed and perfused with PBS. Brains were excised and preserved in an RNase-free reagent compatible with preservation of tissues for further RNA analysis, such as RNA Later. RNA was extracted from whole brain, and RNA sequencing (RNA seq) was performed.
  • Transcript counts for each sample were used to build a DESeq2 object including a design of the formula ⁇ SampleGroup, which was used downstream to perform differential expression of genes (DEGs).
  • the samples were subject to quality control (QC) measures, including performing PCA on all samples using the plot PCA function from DESeq2 in R to determine if there were any visual outliers.
  • the count matrix was transformed using vst (variance stabilizing transformation) prior to plotting. None of the samples were outliers and the WT and KO samples clustered well together separated in PCI. Targeted pairwise comparisons using DESeq2 for use in conjunction with the reversal analysis was performed.
  • DESeq2 utilizes raw counts, metadata and a formula design ( ⁇ SampleGroup or a combination of genotype + drug treatment) as input in a generalized linear model.
  • Performing differential gene expression (DEG) using DESeq2 involved three general steps: 1. Estimation of size factors (which control for differences in the library size of the sequencing experiments);
  • the count matrix was normalized using the median of ratios whereby the counts are divided by sample- specific size factors determined by the median ratio of gene counts relative to the geometric mean per gene.
  • DESeq2 also filters out genes with low row counts (z.e., less than 10 counts across samples).
  • Figure 2 is a dot plot of log2 mean gene expression levels vs. Iog2 fold change in gene expression in NDUFS4 KO mice vs. wild-type mice.
  • the results presented in Figure 2 indicate that there are 752 genes that are upregulated and 234 genes that are downregulated in NDUFS4 KO mice vs. wild-type mice.
  • these results show that there are 986 genes that are differentially regulated in NDUFS4 KO mice vs. wild-type mice.
  • the expression of 94 genes was fully or partially reversed by treatment with an exemplary FXN fusion protein.
  • Example 2 The results presented in Example 2 indicate that there are gene markers that are modulated in a mouse model of Eeigh Syndrome and are also modulated, e.g., contrary modulated, by treatment with an exemplary FXN fusion protein. These gene markers may be used as biomarkers for evaluating efficacy of FXN therapy in subjects with Eeigh Syndrome.

Abstract

La présente invention est basée, au moins en partie, sur la fourniture d'un ensemble de marqueurs, également appelés ici marqueurs génomiques sensibles à la FXN (ou FSGM), dont les niveaux d'expression respectifs sont corrélés positivement ou négativement aux niveaux de frataxine (FXN) dans une cellule d'un sujet atteint du Syndrome de Leigh. Par conséquent, ces FSGM peuvent être utilisés pour déterminer, évaluer et/ou surveiller l'efficacité d'une thérapie FXN chez un sujet atteint du syndrome de Leigh.
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