Tataxin-sensitive markers for monitoring ataxin replacement therapy

Abstract

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), each of which has an expression level that corresponds to a level of expression of an ataxin in a cell. FXN) levels are positively or negatively correlated. Thus, these FSGMs may be used to determine, assess, and / or monitor the effectiveness of FXN replacement therapy in a subject.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 328,238, filed on April 6, 2022, and U.S. Provisional Patent Application No. 63 / 393,968, filed on July 31, 2022. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on April 6, 2023, is named 130197-01520_SL.xml and is 12,288 bytes in size. Background Art

[0005] Mitochondrial diseases are a group of disorders caused by malfunctioning mitochondria, organelles that store potential energy in the form of adenosine triphosphate (ATP) molecules and are present in every cell of the human body except mature red blood cells.

[0006] Friedreich's Ataxia (FRDA) is the most common inherited ataxia in humans and is caused by a deficiency of the mitochondrial protein frataxin (FXN), and specifically human frataxin (hFXN). FRDA is a rare disease with an estimated incidence of 1:29,000, a carrier frequency of about 1:85, and about 4,000 to 5,000 cases reported in the United States. FRDA is a progressive multisystem disease that usually begins in middle childhood. Subjects suffer from a variety of symptoms, including progressive neurological and cardiac dysfunction. Other clinical findings may include scoliosis, fatigue, diabetes, visual impairment, and hearing loss. Inheritance is autosomal recessive and is primarily caused by an inherited GAA triplet expansion in the first intron of both alleles of the hFXN gene. This triplet expansion causes transcriptional repression of the FRDA gene, which in turn causes the subject to produce very small amounts of hFXN. hFXN heterozygotes typically have hFXN levels that are about 50% of normal values, but have a normal phenotype. hFXN levels in heterozygous whole blood and in whole blood of subjects with FRDA were approximately 45 to 70 pg / μl and approximately 5 to 25 pg / μl, respectively, and were shown to remain stable over time (Plasterer et al., 2013).

[0007] There is currently no FDA-approved treatment for FRDA. Antioxidants and iron chelation are less effective, and even with treatment, subjects typically experience a progressive loss of motor control and die, with cardiomyopathy being the primary cause of death.

[0008] Protein replacement therapy is an established approach for metabolic diseases such as diabetes, lysosomal storage diseases, and hemophilia. Subject-derived studies in cells and animal models have demonstrated that replacement of functional FXN can correct or ameliorate the FRDA disease phenotype. However, there remains a need in the art for reliable and efficient assays to measure the clinical response and effectiveness of FXN replacement. Summary of the invention

[0009] In one aspect, the present disclosure is based, at least in part, on providing a set of markers, also referred to herein as FXN sensitivity genomic markers (or FSGMs), each of which has a level that is positively or negatively correlated with the level of frataxin (FXN) in cells.

[0010] In some embodiments, the FSGM of the present disclosure is inversely regulated by FXN gene ablation or FXN deficiency in a subject, followed by FXN protein replacement. Thus, the FSGM of the present disclosure is associated with FXN deficiency in a subject and inversely correlated with FXN replacement. The FSGM disclosed herein was found to be sensitive to FXN levels and is considered a marker for FXN deficiency and FXN replacement therapy.

[0011] Thus, any one or more FSGMs provided herein can serve as surrogate biomarkers for FXN levels in a subject. For example, the FSGMs provided herein can be used to assess or monitor the progression of FXN deficiency in a subject, as described herein. In addition, the FSGMs provided herein can be used to assess and / or monitor FXN replacement therapy, e.g., to determine, assess and / or monitor the efficacy of FXN replacement therapy in a subject, as described herein.

[0012] In some embodiments, a FXN replacement therapy for a subject, e.g., the efficacy of a FXN replacement therapy, can be determined, evaluated, and / or monitored based on analyzing one or more FSGM expression profiles in a sample obtained from the subject before, during, and / or after administering or initiating a FXN replacement therapy to the subject. Based on the results of the FSGM expression profile analysis, adjustments can be made to the subject's FXN replacement therapy, e.g., to initiate FXN replacement therapy in the subject, increase the dose and / or frequency of administration of the FXN replacement therapy, decrease the dose and / or frequency of administration of the FXN replacement therapy, or discontinue FXN replacement therapy.

[0013] In some aspects, the present disclosure provides a method for evaluating the efficacy of a fraxin (FXN) replacement therapy, the method comprising: (a) determining a baseline FXN(-) expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a sample obtained from an FXN-deficient subject before administration of the FXN replacement therapy; (b) determining a FXN replacement expression profile of the one or more FXN sensitivity genomic markers (FSGMs) in a sample obtained from an FXN-deficient subject after administration of the FXN replacement therapy; (c) comparing the FXN replacement expression profile determined in step (b) with the baseline FXN(-) expression profile determined in step (a); and (d) determining the efficacy of the FXN replacement therapy based on the comparison in step (c); wherein the one or more FSGMs are selected from the markers defined in Table 1.

[0014] In some aspects, the present disclosure provides a method for evaluating the efficacy of a fraxin (FXN) replacement therapy, the method comprising: (a) determining a FXN replacement expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a sample obtained from a FXN-deficient subject after administration of a FXN replacement therapy; (b) comparing the subject FXN replacement expression profile determined in step (a) with a reference FXN expression profile of the one or more FSGMs; and (c) determining the efficacy of the FXN replacement therapy based on the comparison in step (b); wherein the one or more FSGMs are selected from the markers defined in Table 1.

[0015] In some embodiments, the reference FXN expression profile is a baseline FXN(-) expression profile of one or more FSGMs. In other embodiments, the reference FXN expression profile is a normal FXN expression profile of one or more FSGMs.

[0016] In some embodiments, a baseline FXN(-) expression profile of one or more FSGMs is determined in a sample obtained from a FXN-deficient subject prior to administration of FXN replacement therapy.

[0017] In some embodiments, the method further comprises determining a baseline FXN(-) expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a sample obtained from the FXN-deficient subject prior to administration of FXN replacement therapy.

[0018] In some embodiments, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In some embodiments, the one or more FSGMs comprise any 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In some embodiments, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP.

[0019] In some embodiments, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In some embodiments, the one or more FSGMs comprise any 2, 3, 4, 5, 6, 7, or all 8 of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In some embodiments, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9.

[0020] In some embodiments, the one or more FSGMs further comprise one or more FSGMs defined in Table 2 or Table 3. In some embodiments, the one or more FSGMs defined in Table 2 or Table 3 comprise 1, 2, 3, or all 4 of CTSS, BTG2, EGR1, and PTGS2. In some embodiments, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, TXNIP, CTSS, BTG2, EGR1, and PTGS2. In some embodiments, the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2. In some embodiments, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, CTSS, BTG2, EGR1, and PTGS2.

[0021] In some embodiments, the one or more FSGMs further comprise one or more FSGMs defined in Table 2 or Table 3. In some embodiments, the one or more FSGMs defined in Table 2 or Table 3 comprise 1, 2, 3, or all 4 of CTSS, BTG2, EGR1, and PTGS2. In some embodiments, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, CTSS, BTG2, EGR1, and PTGS2. In some embodiments, the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2. In some embodiments, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, CTSS, BTG2, EGR1, and PTGS2. In some embodiments, the one or more FSGMs comprise Xpo6, SOD2, FTL, TXNIP, CTSS, EGR1, BTG2, PTSG2, and LRKK2. In some embodiments, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, TXNIP, CTSS, BTG2, EGR1, and PTGS2.

[0022] In some embodiments, at least one or more FSGMs are upregulated following treatment with FXN replacement therapy. In some embodiments, the one or more FSGMs upregulated following treatment with FXN replacement therapy include one or more of LY96, LRRK2, Adgre1, Timp1, Xpo6, CD44, BASP1, PSMB8, C3ar1, TFRC, SOD2, PLCL2, S100a4, Mapre1, PSMB9, FTL, TXNIP, CFH, and Tmem70. In some embodiments, the one or more FSGMs upregulated following treatment with FXN replacement therapy include one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, CTSS, BTG2, EGR1, and PTGS2.

[0023] In some embodiments, at least one or more FSGMs are upregulated following treatment with FXN replacement therapy. In some embodiments, the one or more FSGMs upregulated following treatment with FXN replacement therapy include one or more of LY96, LRRK2, Adgre1, Timp1, Xpo6, Cd44, BASP1, PSMB8, C3ar1, TFRC, SOD2, PLCL2, S100a4, Mapre1, PSMB9, CFH, and Tmem70. In some embodiments, the one or more FSGMs upregulated following treatment with FXN replacement therapy include one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, CTSS, BTG2, EGR1, and PTGS2.

[0024] In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise LY96. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise LRRK2. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise Adgre1. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise Timp1. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise Xpo6. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise CD44. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise BASP1. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise PSMB8. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise C3ar1. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise TFRC. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise SOD2. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise PLCL2. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise S100a4. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise Mapre1. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise PSMB9. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise FTL. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise TXNIP. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise CFH. In some embodiments, one or more FSGMs upregulated after treatment with FXN replacement therapy comprise Tmem70.

[0025] In some embodiments, at least one or more FSGMs are downregulated following treatment with FXN replacement therapy. In some embodiments, the one or more FSGMs downregulated following treatment with FXN replacement therapy include one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and Ptms. In some embodiments, the one or more FSGMs downregulated following treatment with FXN replacement therapy include Lgals3.

[0026] In some embodiments, one or more FSGMs downregulated after treatment with FXN replacement therapy comprise Lgals3. In some embodiments, one or more FSGMs downregulated after treatment with FXN replacement therapy comprise Ube2v2. In some embodiments, one or more FSGMs downregulated after treatment with FXN replacement therapy comprise Sec61g. In some embodiments, one or more FSGMs downregulated after treatment with FXN replacement therapy comprise Zdhhc13. In some embodiments, one or more FSGMs downregulated after treatment with FXN replacement therapy comprise Yipf5. In some embodiments, one or more FSGMs downregulated after treatment with FXN replacement therapy comprise Tpp2. In some embodiments, one or more FSGMs downregulated after treatment with FXN replacement therapy comprise Ybx2. In some embodiments, one or more FSGMs downregulated after treatment with FXN replacement therapy comprise Ptms.

[0027] In some embodiments, determining the FXN expression profile of one or more FSGMs comprises detecting the expression level of the one or more FSGMs. In some embodiments, comparing the subject's FXN replacement expression profile with a baseline FXN(-) expression profile comprises comparing the expression level of one or more FSGMs in the FXN replacement expression profile with the expression level of the corresponding one or more FSGMs in the baseline FXN(-) expression profile.

[0028] In some embodiments, when the expression level of one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, CTSS, BTG2, EGR1, and PTGS2 is increased in the FXN replacement expression profile compared to the baseline FXN (-) expression profile, the FXN replacement therapy is determined to be effective. In some embodiments, when the expression level of Lgals3 is decreased in the FXN replacement expression profile compared to the baseline FXN (-) expression profile, the FXN replacement therapy is determined to be effective.

[0029] In some embodiments, determining the FXN expression profile of one or more FSGMs comprises determining an FXN feature vector indicating a value of expression of the one or more FSGMs. In some embodiments, determining the efficacy of the FXN replacement therapy comprises determining a first FXN feature vector of the subject's FXN replacement expression profile and a second FXN feature vector of a baseline FXN(-) expression profile, and determining a distance between the first feature vector and the second feature vector. In some embodiments, determining the distance between the feature vectors comprises determining a scalar product of the first feature vector and the second feature vector.

[0030] In some embodiments, determining the FXN expression profile of one or more FSGMs further comprises determining a third eigenvector of a normal FXN expression profile of FSGMs of a healthy subject. In some embodiments, determining the FXN expression profile of one or more FSGMs further comprises determining a distance between a second eigenvector and a third eigenvector. In some embodiments, determining the FXN expression profile of one or more FSGMs further comprises determining a distance between a first eigenvector and a third eigenvector, and normalizing the distance between the first eigenvector and the third eigenvector relative to the distance between the second eigenvector and the third eigenvector. In some embodiments, determining the FXN expression profile of one or more FSGMs further comprises determining the effectiveness of a FXN replacement therapy using the normalized distance.

[0031] In some embodiments, 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, enzyme analysis, mass spectrometry, NMR, immunoassay, ELISA, or any combination thereof. In some embodiments, the FXN expression profile is determined by determining the amount of expressed protein by one or more of HPLC / UV-Vis spectroscopy, enzyme analysis, mass spectrometry, NMR, immunoassay, ELISA, or any combination thereof.

[0032] In some embodiments, the methods of the present disclosure further comprise advising a healthcare provider to improve treatment with FXN replacement therapy based on the determination of the efficacy of the FXN replacement therapy.In some embodiments, the subject suffers from Friedreich's ataxia (FRDA).

[0033] In some embodiments, the methods of the present disclosure comprise obtaining a sample from a FXN-deficient subject. In some embodiments, the sample is selected from the group consisting of a cheek sample, a skin sample, a hair follicle, and a muscle biopsy sample. In some embodiments, the sample is a cheek sample. In some embodiments, the sample is a skin sample.

[0034] In some embodiments, the sample from the FXN-deficient subject is obtained at least 15 days after the last administration of FXN replacement therapy. In some embodiments, the sample from the FXN-deficient subject is obtained 15 to 45 days after the last administration of FXN replacement therapy.

[0035] In some aspects, the present disclosure provides a method for monitoring a subject treated with a fraxin (FXN) replacement therapy, the method comprising: (a) at a first time point after administering the FXN replacement therapy to a FXN-deficient subject, determining a first FXN replacement expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a first sample obtained from the subject, wherein the one or more FSGMs comprise one or more markers defined in Table 1; (b) at a second time point later than the first time point, determining a second FXN replacement expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a second sample obtained from the subject; (c) comparing the second FXN replacement expression profile to the first FXN replacement profile; thereby monitoring the subject treated with the FXN replacement therapy.

[0036] In some embodiments, the method further comprises determining to maintain, increase or decrease the dose or frequency of administration of the FXN replacement therapy based on the comparison in step (c). In some embodiments, at least one dose of the FXN replacement therapy is administered to the subject between obtaining the first time point and the second time point. In some embodiments, the FXN replacement therapy is not administered to the subject between obtaining the first time point and the second time point.

[0037] In some aspects, the present disclosure provides a method for treating FXN deficiency, the method comprising: (a) determining a FXN expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a sample obtained from a subject with FXN deficiency, (b) comparing the FXN expression profile of the sample to at least one other expression profile, the at least one other expression profile selected from the group consisting of: a normal FXN expression profile of one or more FSGMs, a baseline FXN(-) expression profile of one or more FSGMs, and a FXN replacement expression profile of one or more FSGMs, (c) classifying the FXN expression profile determined in step (a) as corresponding to a normal FXN expression profile, a baseline FXN(-) expression profile, or a FXN replacement expression profile, and (d) initiating or adjusting FXN replacement therapy based on the classification of the FXN expression profile of the sample, wherein the one or more FSGMs comprise one or more markers defined in Table 1.

[0038] In some embodiments, adjusting the FXN replacement therapy comprises increasing the dose of the FXN replacement therapy, decreasing its dose, increasing its frequency of administration, or decreasing its frequency of administration. In some embodiments, the FXN-deficient subject has Friedreich's ataxia (FRDA).

[0039] In some aspects, the present disclosure provides a method of treating FXN deficiency in a subject, comprising: (a) determining a FXN expression profile of one or more FSGMs in a sample from a subject with FXN deficiency; and (b) recommending to a healthcare provider that FXN replacement therapy be administered to the subject based on the subject's FXN expression profile determined in step (a).

[0040] In some aspects, the present disclosure provides a method of treating FXN deficiency in a subject, comprising: (a) obtaining a FXN expression profile of one or more FSGMs in a sample obtained from a subject with FXN deficiency; and (b) administering a FXN replacement therapy to the subject based on the subject's FXN expression profile.

[0041] In some embodiments, the methods of the present disclosure further comprise obtaining a sample from a FXN-deficient subject for determining the FXN expression profile of one or more FSGMs.

[0042] In some aspects, the present disclosure provides a method for detecting one or more fraxin-sensitive genomic markers (FSGMs) in a sample obtained from a subject deficient in fraxin (FXN), comprising: contacting the sample or a portion thereof with one or more reagents specifically for detecting the level of each of the one or more FSGMs, wherein the one or more FSGMs comprise one or more FSGMs selected from Table 1, thereby detecting the FSGMs in the sample.

[0043] In some embodiments, the subject is being treated with FXN replacement therapy or is scheduled to be treated with FXN replacement therapy.

[0044] In some embodiments, the method further comprises obtaining a sample from a FXN-deficient subject.

[0045] In some embodiments, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In some embodiments, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP.

[0046] In some embodiments, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In some embodiments, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, and PSMB9.

[0047] In some embodiments, the one or more FSGMs further comprise one of the plurality of FSGMs defined in Table 2 or Table 3. In some embodiments, the one or more FSGMs defined in Table 2 or Table 3 comprise one or more of CTSS, BTG2, EGR1, and PTGS2. In some embodiments, the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2. In some embodiments, the one or more FSGMs comprise Xpo6, SOD2, FTL, TXNIP, CTSS, EGR1, BTG2, PTSG2, and LRKK2.

[0048] In some embodiments, 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, enzyme analysis, mass spectrometry, NMR, immunoassay, ELISA, or any combination thereof. In some embodiments, the FXN expression profile is determined by determining the amount of expressed protein by HPLC / UV-Vis spectroscopy, enzyme analysis, mass spectrometry, NMR, immunoassay, ELISA, or any combination thereof.

[0049] In some embodiments, the subject has Friedreich's ataxia (FRDA).

[0050] In some embodiments, the methods of the present disclosure further comprise obtaining a sample from a FXN-deficient subject. In some embodiments, the sample is selected from the group consisting of a cheek sample, a skin sample, a hair follicle, and a muscle biopsy sample. In some embodiments, the sample is a cheek sample. In some embodiments, the sample is a skin sample.

[0051] In some embodiments, the sample from the FXN-deficient subject is obtained at least 15 days after the last administration of FXN replacement therapy. In some embodiments, the sample from the FXN-deficient subject is obtained 15 to 45 days after the last administration of FXN replacement therapy.

[0052] In some embodiments, FXN replacement therapy comprises administering a FXN fusion protein. In some embodiments, the FXN fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO:12.

[0053] In some aspects, the present disclosure also provides a kit for detecting one or more fraxin-sensitive genomic markers (FSGMs) in a sample obtained from a subject deficient in fraxin (FXN), comprising at least one reagent specifically for detecting the level of each of the one or more FSGMs in the sample, wherein the one or more FSGMs include one or more FSGMs defined in Table 1; and a set of instructions for detecting the level of the one or more FSGMs in the sample from the subject.

[0054] In some embodiments, the agent is an antibody that binds to a frataxin-sensitive genomic marker (FSGM) or an oligonucleotide that is complementary to the corresponding mRNA of FSGM.

[0055] In some embodiments, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP.

[0056] In some embodiments, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9.

[0057] In some embodiments, the kits of the present disclosure further comprise at least one reagent specifically for detecting the level of one of the plurality of FSGMs defined in Table 2 or Table 3. In some embodiments, the one or more FSGMs defined in Table 2 or Table 3 comprise one or more of CTSS, BTG2, EGR1, and PTGS2. In some embodiments, the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2. In some embodiments, the one or more FSGMs comprise Xpo6, SOD2, FTL, TXNIP, CTSS, EGR1, BTG2, PTSG2, and LRKK2.

[0058] In some aspects, the present disclosure provides a reagent detection combination in a method for monitoring or evaluating the efficacy of FXN replacement therapy, wherein the detection combination comprises at least two detection reagents, wherein each detection reagent is specifically used to detect at least one FSGM in a set of FXN sensitivity genomic markers (FSGMs), wherein the set of FSGMs comprises two or more markers defined in Table 1.

[0059] In some embodiments, the set of FSGMs comprises two or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP. In some embodiments, the set of FSGMs comprises two or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2 and PSMB9.

[0060] In some embodiments, the detection combination of the present disclosure further comprises at least one detection reagent specifically for detecting at least one of the FSGMs defined in Table 2 or Table 3. In some embodiments, at least one of the FSGMs defined in Table 2 or Table 3 comprises one or more of CTSS, BTG2, EGR1, and PTGS2. In some embodiments, at least one of the FSGMs comprises one or more of Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2. In some embodiments, at least one of the FSGMs comprises one or more of Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2. In some embodiments, at least one of the FSGMs comprises one or more of Xpo6, SOD2, FTL, TXNIP, CTSS, EGR1, BTG2, PTSG2, and LRKK2. In some embodiments, at least one of the FSGMs comprises Xpo6, SOD2, FTL, TXNIP, CTSS, EGR1, BTG2, PTSG2, and LRKK2.

[0061] In some aspects, the present disclosure provides a kit comprising a detection combination according to any one of technical solutions 76 to 80 and a set of instructions for obtaining information related to fraxin (FXN) replacement therapy based on the levels of one or more fraxin-sensitive genomic markers (FSGM).

[0062] In the discussion, unless otherwise indicated, adjectives such as "substantially" and "about" that modify conditions or relational characteristics of one or more features of an embodiment of the present disclosure should be understood to mean that the conditions or characteristics are defined within acceptable tolerances for the operation of the embodiment to which they are intended to be applied. Unless otherwise indicated, the word "or" in the specification and claims is considered to be an inclusive "or" (having and / or meaning) rather than an exclusive or, and indicates at least one and any combination of the items it connects.

[0063] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0064] BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Non-limiting examples of embodiments of the present disclosure are described below with reference to the drawings listed after this paragraph. Identical features appearing in more than one figure are generally labeled with the same reference numeral in all the figures in which they appear.

[0066] Figure 1 is a volcano plot resulting from principal component analysis of buccal samples obtained from healthy subjects versus buccal samples obtained from FRDA subjects who were not treated with FXN replacement therapy. Figure 1 The expression levels of various gene markers and their corresponding adjusted p values ​​are shown.

[0067] Figure 2 is a graphical representation showing the relative levels of selected gene markers in healthy subjects ("Normal") and FRDA subjects treated with placebo ("FA Placebo").

[0068] Figure 3 is a volcano plot generated from principal component analysis of samples from subjects treated with 100 mg of an exemplary FXN fusion protein obtained before dosing (Day -2) and on Day 22. Figure 3 The expression levels of various gene markers and their corresponding adjusted p values ​​are shown.

[0069] Figure 4 is a graphical representation showing the relative amounts of selected gene markers in cheek samples obtained from healthy subjects ("Normal") and FRDA subjects on day 22 following treatment with placebo ("FA Cohort"), 25 mg ("FA Cohort 1"), 50 mg (FA Cohort 2"), and 100 mg ("FA Cohort 3") of an exemplary FXN fusion protein.

[0070] Figure 5 is a graphical representation showing the ratios of the relative amounts of selected gene markers in FRDA subjects treated with placebo ("Placebo"), 25 mg ("Cohort 1"), 50 mg ("Cohort 2"), and 100 mg ("Cohort 3") of an exemplary FXN fusion protein at day 22 post-dosing compared to pre-dosing.

[0071] Figure 6is a graphical representation showing the relative amounts of selected FSGMs measured on Days 2, 15, 22, and 43 in buccal samples obtained following administration of placebo or 100 mg of an exemplary FXN fusion protein to FRDA subjects ("Cohort 3").

[0072] Figure 7 is a volcano plot resulting from principal component analysis of whole blood samples obtained from healthy subjects and FRDA subjects not treated with FXN replacement therapy.

[0073] Figure 8 is a graphical representation showing the relative amounts of FTL and TXNLP gene markers in cheek samples obtained from healthy subjects ("Normal") and FRDA subjects at day 22 following treatment with placebo ("FA Placebo") and 100 mg ("FA Cohort 3") of an exemplary FXN fusion protein.

[0074] Fig. 9 is a box plot showing the ratio of the relative amounts of gene markers in FRDA subjects treated with placebo ("Placebo") and 100 mg ("Cohort 3") of an exemplary FXN fusion protein at day 22 after dosing compared to pre-dose. DETAILED DESCRIPTION

[0075] A. Overview

[0076] In one aspect, 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), each of which has a level that is positively or negatively correlated with the level of frataxin (FXN) in cells. In some embodiments, the FSGMs of the present disclosure are reversely regulated by FXN gene ablation or deficiency, followed by FXN protein replacement. Therefore, the FSGMs of the present disclosure are associated with FXN deficiency in a subject and are reversely correlated with FXN replacement. The FSGMs disclosed herein are found to be sensitive to FXN and are considered markers of FXN replacement. Therefore, these FSGMs can be used to assess and / or monitor the progression of FXN deficiency in a subject, as described herein. These FSGMs can also be used to determine and / or monitor the efficacy of FXN replacement therapy in a subject, as described herein. In one embodiment, the FSGMs comprise one or more markers defined in Table 1 and / or Table 5.

[0077] In one embodiment, the one or more FSGMs include one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include two or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include three or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include four or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include five or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include six or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TxNIP. In another embodiment, the one or more FSGMs include seven or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include eight or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP.

[0078] In one embodiment, one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In another embodiment, one or more FSGMs comprise two or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In another embodiment, one or more FSGMs comprise three or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In another embodiment, one or more FSGMs comprise four or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In another embodiment, one or more FSGMs comprise five or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In another embodiment, the one or more FSGMs comprise six or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In another embodiment, the one or more FSGMs comprise seven or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In another embodiment, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9.

[0079] In one embodiment, the one or more FSGMs comprise one or more of LRRK2, SOD2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise two or more of LRRK2, SOD2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise three or more of LRRK2, SOD2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise LRRK2, SOD2, CD44, and Lgals3.

[0080] In one embodiment, one or more FSGMs include one or more of LRRK2, SOD2, CD44, Lgals3, FTL, and TXNIP. In another embodiment, one or more FSGMs include two or more of LRRK2, SOD2, CD44, Lgals3, FTL, and TXNIP. In another embodiment, one or more FSGMs include three or more of LRRK2, SOD2, CD44, Lgals3, FTL, and TXNIP. In another embodiment, one or more FSGMs include four or more of LRRK2, SOD2, CD44, Lgals3, FTL, and TXNIP. In another embodiment, one or more FSGMs include five or more of LRRK2, SOD2, CD44, Lgals3, FTL, and TXNIP. In another embodiment, one or more FSGMs include LRRK2, SOD2, CD44, Lgals3, FTL, and TXNIP.

[0081] In another embodiment, the one or more FSGMs further include one or more FSGMs defined in Table 2 and / or Table 3. In one embodiment, the one or more FSGMs defined in Table 2 and / or Table 3 include one or more of CTSS, BTG2, EGR1, and PTGS2. In another embodiment, the one or more FSGMs defined in Table 2, 3, 4, and / or Table 6 include two or more of BTG2, EGR1, and PTGS2. In another embodiment, the one or more FSGMs defined in Table 2 and / or Table 3 include three or more of CTSS, BTG2, EGR1, and PTGS2. In another embodiment, the one or more FSGMs defined in Table 2, 3, 4, and / or Table 6 include CTSS, BTG2, EGR1, and PTGS2.

[0082] In some embodiments, the efficacy of FXN replacement therapy in a subject can be determined, evaluated and / or monitored based on analyzing one or more FSGM expression profiles before and after administration or initiation of FXN replacement therapy in a subject. Based on the results of the FSGM expression profile analysis, adjustments can be made to the subject's FXN replacement therapy, for example, to initiate, increase, decrease or stop the subject's FXN replacement therapy.

[0083] B. Definition

[0084] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention belongs. The following references (the entire disclosure of which is incorporated herein by reference) provide general definitions of many terms used in the present invention for those skilled in the art (unless otherwise defined in this article): Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); Cambridge Dictionary of Science and Technology (Walker, 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (Springer Verlag) (1991); and Hale and Marham, Harper Collins Dictionary of Biology (1991). In general, the procedures of molecular biological methods described or inherent herein, etc. are methods commonly used in the art. Such standard techniques can be found in reference manuals, such as Sambrook et al., (2000, Molecular Cloning--A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratories); and Ausubel et al., (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0085] Unless otherwise indicated, the following terms may have the following meanings assigned to them. However, it should be understood that other meanings known or understood by one of ordinary skill in the art are also possible and within the scope of the present invention.

[0086] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning.

[0087] Unless expressly stated or obvious from the context, as used herein, the term "about" should be understood as being within the general tolerance range in the art, such as within 2 standard deviations of the mean. About can be understood as being 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 the context, all numerical values ​​provided herein may be modified by the term about.

[0088] As used herein, the term "amplification" refers to any known in vitro procedure for obtaining multiple copies ("amplicons") of a target nucleic acid sequence, its complement, or a fragment thereof. In vitro amplification refers to the production of an amplified nucleic acid that may contain less than the entire target sequence or its complement. Known in vitro amplification methods include, for example, transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification, and strand displacement amplification (SDA, including multiple strand displacement amplification (MSDA)). Replicase-mediated amplification uses a self-replicating RNA molecule and a replicase, such as Q-β-replicase (e.g., Kramer et al., U.S. Pat. No. 4,786,600). PCR amplification is well known and uses a DNA polymerase, primers, and thermal cycling to synthesize multiple copies of two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat. 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 strands by using multiple cycles of hybridization, ligation, and denaturation (e.g., European Patent Application Publication No. 0 320 308). SDA is a method in which primers contain a recognition site for a restriction endonuclease that allows the endonuclease to cleave one strand of a semi-modified DNA duplex including the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., Walker et al., U.S. Pat. No. 5,422,252). Two other known strand displacement amplification methods do not require endonuclease cleavage (Dattagupta et al., U.S. Pat. No. 6,087,133 and U.S. Pat. No. 6,124,120 (MSDA)). One skilled in the art will appreciate that the oligonucleotide primer sequences of the present invention can 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, 3rd ed., Cold Spring Harbor Laboratory (CSH Laboratories)). As is generally known in the art, oligonucleotides are designed to bind to complementary sequences under selected conditions.

[0089] As used herein, the term "marker" or "biomarker" is a biological molecule or a group of biological molecules, the expression level of which is, for example, positively correlated or negatively correlated with the level of FXN. In some embodiments, a "marker" or "biomarker" is an expressed gene, the expression level of which can be measured by measuring the level of the corresponding mRNA or protein.

[0090] As used herein, markers or biomarkers of the invention whose respective levels are positively or negatively correlated with the level of frataxin (FXN) in cells or samples obtained from a subject are referred to as "frataxin sensitive genomic markers" or "FSGMs."

[0091] In some embodiments, FSGM is a marker or biomarker, such as an expressed gene, that is differentially expressed in a sample obtained from a healthy subject compared to a sample obtained from a FXN-deficient subject (e.g., a subject with FRDA). In other embodiments, FSGM is reversely regulated by FXN gene ablation or deficiency followed by FXN replacement therapy. For example, FSGM can be a gene that is expressed at a lower level in a sample obtained from a FXN-deficient subject compared to a sample obtained from a healthy subject, such as Timp1, and whose level is increased in a sample from a FXN-deficient subject following FXN replacement therapy. Or, for example, FSGM can be a gene that is expressed at a higher level in a sample obtained from a FXN-deficient subject compared to a sample obtained from a healthy subject, such as Lgals3, and whose level is decreased in a sample from a FXN-deficient subject following FXN replacement therapy.

[0092] In some embodiments, FSGM is a marker or biomarker, e.g., an expressed gene, that is differentially expressed in samples obtained from healthy subjects compared to samples obtained from FXN-deficient subjects, as determined by principal component analysis, exhibiting an expression fold change greater than 2 between the groups, with a Benjamini-Hochbert (BH) adjusted p value < 0.05.

[0093] In some embodiments, FSGM is a marker or biomarker, e.g., an expressed gene, differentially expressed in samples obtained from healthy subjects compared to samples obtained from FXN-deficient subjects, as determined by principal component analysis, exhibiting an expression fold change of less than 2, e.g., between 1 and 2, between the groups, with a Benjamini-Hochbert (BH) adjusted p-value < 0.05.

[0094] In some embodiments, FSGM is a marker or biomarker, e.g., an expressed gene, that is differentially expressed in samples obtained from healthy subjects compared to samples obtained from FXN-deficient subjects, as determined by principal component analysis, exhibiting an expression fold change of greater than 2 between the groups, with a Benjamini-Hochbert (BH) adjusted p value > 0.05.

[0095] In some embodiments, the FSGM of the present disclosure is reversely regulated by FXN gene ablation or deficiency followed by FXN protein replacement. Thus, in some embodiments, the FSGM of the present disclosure is associated with FXN deficiency in a subject and is reversely associated with FXN replacement. The FSGM of the present invention can be used to detect and / or monitor FXN levels in a sample, such as a cell or tissue sample (e.g., to serve as a surrogate thereof). In preferred embodiments, the FSGM is selected from those listed in Table 1 and / or Table 5, human genes and proteins in Table 1 and / or Table 5, and human homologs of genes and proteins in Table 1 and / or Table 5. In some embodiments, the FSGM is selected from those listed in Table 1 and / or Table 5 and one or more additional FSGMs in Table 2 and / or Table 3, human genes and proteins in Table 1 and / or Table 5, and human homologs of genes and proteins in Table 1 and / or Table 5. As used herein, reference to a FSGM in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs in Table 2 and / or Table 3 should be understood to include reference to any mutant, variant, derivative, or ortholog thereof.

[0096] As used herein, the term "control sample" or "control" refers to any clinically relevant comparative sample, or comparative expression profile, including, for example, a sample from a FXN healthy subject (i.e., a subject with normal FXN levels), a normal FXN expression profile, a sample from a FXN deficient subject (i.e., a subject completely or partially lacking FXN expression), a baseline FXN (-) expression profile, a sample obtained from a FXN deficient subject after administration of a FXN replacement therapy, or a FXN replacement expression profile. A control sample may also be a sample from a subject at an earlier time point (e.g., before treatment with a FXN replacement therapy). A control sample may be a purified sample, protein, and / or nucleic acid provided with a kit. Such a control sample may be diluted, for example, in a dilution series to allow for quantitative measurement of the level of an analyte (e.g., a marker) in a test sample. A control sample may include samples derived from one or more subjects. A control sample may also be a sample taken from a subject to be evaluated at an earlier time point. For example, a control sample may be a sample taken from a subject to be evaluated before treatment with a FXN replacement therapy. The control sample can also be a sample from an animal model, or a sample from a tissue or cell line derived from an animal model of a mitochondrial disease (such as FRDA). The activity or expression level of one or more FSGMs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more FSGMs) in the control sample is composed of a set of measurements that can be determined, for example, based on any appropriate statistical measurement (e.g., a measure of central tendency, including mean, median, or peak). In one embodiment, "different from the control" is preferably statistically significantly different from the control.

[0097] As used herein, "changed," "altered," "increased," or "decreased" is understood to mean that the level of one or more FSGM detected is a statistically different (e.g., increased or decreased) level compared to a control sample or a predetermined threshold (e.g., a sample from a FXN healthy subject (i.e., a subject with normal FXN levels)) or a sample from a FXN deficient subject (i.e., a subject completely or partially lacking FXN expression). A change, alteration, increase, or decrease compared to a control or threshold may also include a difference in the rate of change of the level of one or more FSGM obtained in a series of at least two subject samples obtained over time. Determination of statistical significance is within the capabilities of one skilled in the art and may include any acceptable means for determining and / or measuring statistical significance, such as the number of standard deviations constituting the mean for a positive or negative result, an increase in the detected level of FSGM in a sample compared to a control (where the increase is above a certain threshold), or a decrease in the detected level of FSGM in a sample compared to a control (where the decrease is below a certain threshold).

[0098] As used herein, "detecting," "detection," "determining," and the like are understood to refer to identifying the presence and / or level of one or more FSGMs selected from Table 1 and / or Table 5.

[0099] As used herein, the term "DNA" or "RNA" molecule or sequence (and sometimes the term "oligonucleotide") refers to a molecule typically composed of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and / or cytosine (C). In "RNA", T is replaced by uracil (U).

[0100] As used herein, the terms "FXN-deficient patient" or "FXN-deficient subject", which are used interchangeably herein, refer to a subject who has been determined to be FXN-deficient, e.g., has been diagnosed with Friedreich's ataxia (FRDA). In some embodiments, the FXN-deficient subject has a reduced level of FXN expression or activity, e.g., a partial or complete lack of FXN expression or activity (e.g., less than 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the FXN expression level or activity of a normal control subject) compared to a normal control subject. In some embodiments, the FXN-deficient subject has not yet received FXN replacement therapy and is therefore not undergoing therapy. In some embodiments, the FXN-deficient subject is scheduled to receive FXN replacement therapy. In some embodiments, the FXN-deficient subject is currently undergoing FXN replacement therapy. In some embodiments, the FXN-deficient subject has undergone FXN replacement therapy. In some embodiments, the FXN-deficient subject who is undergoing or has undergone FXN replacement therapy has had its FXN level partially or completely restored to that of a healthy subject.

[0101] Certain diseases cause FXN deficiency in subjects, including mitochondrial diseases, such as Friedreich's ataxia (FRDA). As used herein, the term "FXN replacement therapy" refers to the introduction or replacement of ataxia in a subject lacking FXN, which results in an increase in the level, expression and / or activity of ataxia in the subject. FXN replacement therapy can make the FXN level, expression or activity in the treated subject lower than (e.g., a certain proportion) the FXN level, expression or activity of a normal healthy subject (e.g., partial replacement) or can make the level, expression or activity of FXN in the treated subject close to or approximately equal to the level, expression or activity of a normal subject. FXN replacement therapy can be performed by delivering any therapeutic agent capable of increasing the level, expression or activity of FXN in a subject lacking FXN. In some embodiments, FXN replacement therapy can be performed by delivering FXN protein or a nucleic acid capable of increasing the level or expression of FXN in a subject lacking FXN (e.g., DNA or mRNA encoding FXN) to the subject. Delivery of FXN protein to the subject can include delivery of FXN protein or delivery of FXN fusion protein. In some embodiments, the nucleic acid capable of increasing the level or activity of FXN in a subject with FXN deficiency is an mRNA encoding FXN. In some embodiments, the nucleic acid capable of increasing the level or activity of FXN in a subject with FXN deficiency is an siRNA or an antisense oligonucleotide. In some embodiments, the nucleic acid capable of increasing the level or activity of FXN in a subject with FXN deficiency can be delivered to the subject via a viral vector.

[0102] As used herein, the term "FXN fusion protein" refers to full-length FXN or FXN fragments fused to the full length or fragments of different proteins or fused to peptides. In some embodiments, as described herein, the FXN fusion protein comprises full-length hFXN (SEQ ID NO: 1) or mature hFXN (SEQ ID NO: 2). In some embodiments, the FXN protein or its fragment is fused to a cell penetrating peptide (CPP). In some embodiments, the CPP is an HIV-TAT polypeptide. In some embodiments, FXN replacement therapy comprises administering a FXN fusion protein comprising SEQ ID NO: 12 or consisting thereof to a subject lacking FXN.

[0103] As used herein, the terms "disorder," "disease," and "abnormal state" are used inclusively and refer to any deviation from the normal structure or function of any part, organ, or system of the body (or any combination thereof). Specific diseases are manifested by characteristic symptoms and signs, including biological, chemical, and physical changes, and are often associated with a variety of other factors, including, but not limited to, demographic, environmental, occupational, genetic, and medical history factors. Early disease states include states in which one or more physical symptoms have not yet been detected. Certain characteristic signs, symptoms, and related factors can be quantified by a variety of methods to obtain important diagnostic information.

[0104] As used herein, the term "mitochondrial disease" refers to a disease caused by an inherited or spontaneous mutation of mtDNA or nDNA, which results in a change in the function of a protein or RNA molecule normally present in the mitochondria, thereby reducing mitochondrial function to induce, for example, various types of diseases in the central nervous system, skeletal muscle, heart, eyes, liver, kidneys, large intestine (colon), small intestine, inner ear, and pancreas; and blood, skin, and endocrine glands. In a non-limiting embodiment, the mitochondrial disease is Friedreich's ataxia (FRDA).

[0105] As used herein, the sample obtained at "early time point" is a sample obtained in the past sufficient time, so that compared with the later time point, clinically relevant information can be obtained in the sample at the earlier time point. In certain embodiments, the 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 earlier, or 1, 2, 3, 4, 5, 6 or 7 days. In certain embodiments, the earlier time point is at least one, two, three or four weeks earlier. In certain embodiments, the earlier time point is at least six weeks earlier. In certain embodiments, the earlier time point is at least two months earlier. In certain embodiments, the earlier time point is at least three months earlier. In certain embodiments, the earlier time point is at least six months earlier. In certain embodiments, the earlier time point is at least nine months earlier. In certain embodiments, the earlier time point is at least one year earlier. Multiple subject samples (e.g., 3, 4, 5, 6, 7 or more) can be obtained at regular or irregular time intervals over time and analyzed for trends in changes in FSGM levels. Appropriate time intervals for testing a particular subject can be determined by one skilled in the art based on typical considerations.

[0106] The term "expression" is used herein to refer to the process by which a polypeptide is produced from DNA. The process involves transcription of a gene into mRNA and translation of this mRNA into a polypeptide. Depending on the context in which it is used, "expression" may refer to the production of RNA, or protein, or both.

[0107] The term "expression profile" is used to include genomic expression profiles, meaning the expression profile of RNA, or specifically the expression profile of mRNA or transcripts, or protein expression profiles. As used herein, expression profiles can refer to a set of data obtained for mRNA expression. It can refer to the raw data in the reading of, for example, a PCR device, or a normalized expression value. Expression profiles can be determined by any suitable means of measuring the level of nucleic acid sequences (such as mRNA, labeled mRNA, quantitative hybridization of amplified mRNA, cDNA, etc., quantitative PCR and other technologies known to those skilled in the art or described herein). Expression profiles enable analysis of differential gene expression between two or more samples, between a sample and a control, and between a sample and a threshold value. Expression profiles can also be determined by any means known to those skilled in the art or described herein for measuring the level of a protein or polypeptide, such as mass spectrometry, immunodetection analysis, such as ELISA, etc.

[0108] As referred to herein, the term "FXN expression profile" includes any of the following three FXN expression profiles: a normal FXN expression profile, a baseline FXN (-) expression profile, or a FXN alternative expression profile. In some embodiments, a baseline FXN (-) expression profile can be used as a control. In some embodiments, a normal FXN expression profile can be used as a control.

[0109] In some embodiments, the term "FXN expression profile" of one or more FSGMs refers to the expression level (e.g., RNA level or protein level) of one or more FSGMs or a value or set of values ​​indicating the expression level of one or more FSGMs. In some embodiments, the FXN expression profile comprises a feature vector of values ​​indicating the expression of one or more FSGMs. In some embodiments, "determining the FXN expression profile of one or more FSGMs" comprises detecting the expression level (e.g., RNA level or protein level) of one or more FSGMs. In some embodiments, "determining the FXN expression profile of one or more FSGMs" comprises determining a feature vector of values ​​indicating the expression level (e.g., RNA level or protein level) of one or more FSGMs.

[0110] As referred to herein, the term "normal FXN expression profile" refers to the expression profile of one or more FSGMs in a sample obtained from one or more normal healthy subjects (i.e., subjects who are not deficient in FXN). In some embodiments, "normal FXN expression profile" also encompasses the average of multiple, e.g., two or more normal FXN expression profiles (e.g., from two or more subjects).

[0111] As referred to herein, the term "baseline FXN(-) expression profile" refers to the expression profile of one or more FSGMs in a sample from a FXN-deficient subject prior to treatment with FXN replacement therapy. In some embodiments, the term "baseline FXN(-) expression profile" encompasses an average of a plurality, such as two or more baseline FXN(-) expression profiles (e.g., from more than two subjects).

[0112] An average FXN expression profile, such as an average normal FXN expression profile or an average baseline FXN(-) expression profile, can be determined by methods known in the art, such as by determining the expression level of one or more FSGMs in samples obtained from two or more subjects (e.g., normal subjects or FXN-deficient subjects), and then calculating the average expression level of the one or more FSGMs.

[0113] As referred to herein, the term "reference FXN expression profile" encompasses "normal FXN expression profile" and "baseline FXN(-) expression profile", i.e., can be either. A reference expression profile, such as a reference normal FXN expression profile or a reference baseline FXN(-) expression profile can be used as a control, e.g., for comparison with a FXN replacement expression profile to assess a subject's response to FXN replacement therapy.

[0114] As referred to herein, the term "FXN replacement expression profile" refers to the expression profile of one or more FSGMs in a sample obtained from a FXN-deficient subject following administration of at least one dose of FXN replacement therapy.

[0115] As used herein, the term "treatment with FXN replacement therapy" refers to administering at least one dose of FXN replacement therapy to a subject. As used herein, the term "following treatment with FXN replacement therapy," "subsequent to treatment with FXN replacement therapy," "following administration of FXN replacement therapy," or "subsequent to administration of FXN replacement 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 days or more, after administration of a dose of FXN replacement therapy.

[0116] As referred to herein, the term "assessing a subject's response to a FXN replacement therapy" encompasses assessing the efficacy of a FXN replacement therapy by: (a) determining a FXN replacement expression profile in a sample from a FXN-deficient subject following treatment with a FXN replacement therapy; (b) comparing the FXN replacement expression profile to a reference FXN expression profile; and (c) using the comparison in step (b) to assess or determine the efficacy of the FXN replacement therapy.

[0117] In some embodiments, the reference FXN expression profile is a baseline FXN(-) expression profile, i.e., an expression profile of one or more FSGMs in a sample obtained from a FXN-deficient subject prior to treatment with a FXN replacement therapy. In some embodiments, a difference between the FXN replacement expression profile and the baseline FXN(-) expression profile indicates efficacy of or response to a FXN replacement therapy.

[0118] In some embodiments, the reference FXN expression profile is a normal FXN expression profile, i.e., an expression profile of one or more FSGMs in a sample obtained from a normal subject. In some embodiments, a comparison between the FXN replacement expression profile and the normal FXN expression profile, e.g., a similarity between the two profiles, indicates efficacy of or response to a FXN replacement therapy.

[0119] "Higher expression level", "higher level", "increased level" and the like of FSGM refers to an expression level in a test sample that is above the standard error of the analytical method used to assess expression, and preferably is at least greater than 25%, at least greater than 50%, at least greater than 75%, at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine-fold, or at least ten-fold the expression level of FSGM in a control sample (e.g., a sample from a healthy subject, a sample from a subject with FXN deficiency, or a sample from a subject after FXN replacement therapy), and preferably is the average expression level of one or more FSGM in several control samples.

[0120] As used herein, the term "hybridization" as in "nucleic acid hybridization" generally refers to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which will form a thermodynamically favorable double-stranded structure under appropriate conditions. Examples of hybridization conditions can be found in the two experimental manuals mentioned above (Sambrook et al., 2000, supra and Ausubel et al., 1994, supra, or in addition Higgins and Hames (eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985)) and are generally known in the art. In the case of hybridization to nitrocellulose filters (or other such supports, such as nylon), for example, in the well-known Southern blotting procedure, the nitrocellulose filter can be incubated overnight with the labeled probe in a solution containing high salt (6xSSC or 5xSSPE), 5xDenhardt's solution, 0.5% SDS, and 100 μg / ml denatured carrier DNA (e.g., salmon sperm DNA) at a temperature representing the desired stringency conditions (60 to 65°C for high stringency, 50 to 60°C for medium stringency, and 40 to 45°C for low stringency conditions). Nonspecifically bound probe can then be washed off the filter by several washes in 0.2xSSC / 0.1% SDS at a temperature selected taking into account the desired stringency: room temperature (low stringency), 42°C (medium stringency), or 65°C (high stringency). The salt and SDS concentrations of the wash solutions can also be adjusted to suit the desired stringency. The temperature and salt concentration selected are based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In this case, the conditions for hybridization and washing can be adjusted by the skilled artisan according to well-known methods. Stringent conditions will preferably be used (Sambrook et al., 2000, supra). Other protocols well known in the art using different annealing and washing solutions or commercially available hybridization kits (e.g., from BD Biosciences Clonetech) can also be used. ). As is well known, the length of the probe and the nucleic acid composition to be determined constitute other parameters of the hybridization conditions. It should be noted that changes in the above conditions can be achieved by including and / or replacing alternative blocking agents for suppressing background in hybridization experiments. Typical blocking agents include Denhart's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available special formulations. Including specific blocking agents may require modification of the hybridization conditions described above due to compatibility issues. The hybrid nucleic acid molecule also includes fragments of the molecules described above. In addition, the nucleic acid molecules hybridized with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules. In addition, a hybridization complex refers to a complex formed between two nucleic acid sequences by means of hydrogen bonds formed between complementary G and C bases and between complementary A and T bases; these hydrogen bonds can be further stabilized by base stacking interactions. Two complementary nucleic acid sequences are hydrogen bonded in an antiparallel configuration. Hybridization complexes can be formed in solution (e.g., Cot or Rot assays) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., a membrane, filter, chip, pin, or slide, such as a cell immobilized on).

[0121] As used herein, the term "identical" or "identity percentage" in the context of two or more nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are identical or have a specific percentage of identical amino acid residues or nucleotides (e.g., 60% or 65% identical, preferably 70% to 95% identical, more preferably at least 95% identical) when compared and aligned within a comparison window or within a specified region for maximum correspondence using a sequence comparison algorithm known in the art or measured by manual alignment and visual inspection. In some embodiments, the term "identical" or "identity percentage" refers to nucleic acid or amino acid sequences that do not include internal non-matching deletions or additions in the sequence relative to each other, that is, the nucleic acid or amino acid sequences have the same length and have a specific percentage of identical nucleotides or amino acid residues.

[0122] Sequences with, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. This definition also applies to the complement of the test sequence. Preferably, the described consistency is present in a region of at least about 15 to 25 amino acids or nucleotides in length, more preferably in a region of about 50 to 100 amino acids or nucleotides in length. Those skilled in the art will know how to use, for example, those algorithms known in the art based on CLUSTALW computer programs (Thompson, Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag, Comp. App. Biosci. 6 (1990), 237-245) to determine the percentage of consistency between / among sequences. Although the FASTDB algorithm does not generally consider internal non-matching deletions or additions in the sequence, that is, gaps in its calculations, the gaps can be manually corrected to avoid overestimating the consistency%. However, CLUSTALW does take sequence gaps into account in its consistency calculations. Those skilled in the art may also use BLAST and BLAST2.0 algorithms (Altschul, Nucleic Acids Research, 25 (1977), 3389-3402). The BLASTN program for nucleic acid sequences uses a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both chains by default. For amino acid sequences, the BLASTP program uses a word length (W) of 3 and an expectation (E) of 10 by default. The BLOSUM62 scoring matrix (Henikoff, Proceedings of the National Academy of Sciences of the United States of America, USA, 89, (1989), 10915) uses an alignment (B) of 50, an expectation (E) of 10, M=5, N=4, and a comparison of both chains. In addition, the present invention also relates to nucleic acid molecules whose sequences are degenerate compared to the sequences of the hybrid molecules described above. When used according to the present invention, the term "degenerate due to the genetic code" means that different nucleotide sequences encode the same amino acid due to the redundancy of the genetic code. The present invention also relates to nucleic acid molecules comprising one or more mutations or deletions and to nucleic acid molecules hybridizing to one of the nucleic acid molecules described herein, which exhibit (a) one or more mutations or (a) one or more deletions.

[0123] The term "including" is used herein to refer to, and is used interchangeably with, the phrase "including, but not limited to," "including ...

[0124] As used herein, "label" refers to a molecular part or compound that can detect or produce a detectable signal. The label is directly or indirectly attached to a molecule to be detected, such as an antibody, a nucleic acid probe or a protein / antigen or a nucleic acid (e.g., an amplified sequence). Direct labeling can be performed by connecting the label to the bond or interaction (e.g., a covalent bond or a non-covalent interaction) of the nucleic acid, while indirect labeling can be performed by using a "linker" or a bridging part of a direct or indirect label, such as one or more oligonucleotides or a small molecule carbon chain. The bridging part can amplify a detectable signal. The label can include any detectable part (e.g., a radionuclide, a ligand (e.g., biotin or avidin), an enzyme or an enzyme substrate, a reactive group, a chromophore (e.g., a dye or a colored particle), a luminescent compound (including bioluminescence, phosphorescence or a chemiluminescent compound) and a fluorescent compound). Preferably, the label on the labeled probe can be detected in a uniform analysis system (i.e., in a mixture), and the combined label exhibits a detectable change compared to the unbound label.

[0125] The terms "level of expression of a gene", "gene expression level", "level of an FSGM", etc. refer to the levels of mRNA and pre-mRNA nascent transcripts, transcriptional processing intermediates, mature mRNA and degradation products, or the level of protein encoded by the gene in a cell. The "level" of one or more FSGMs means the absolute or relative amount or concentration of the FSGM in a sample.

[0126] By "lower expression level" or "lower level" or "reduced level" etc. of FSGM is meant that the expression level in the test sample is lower than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% of the expression level of FSGM in a control sample (e.g., a sample from a healthy subject, a sample from a subject with FXN deficiency, or a sample from a subject after FXN replacement therapy) (and preferably, the average expression level of FSGM in several control samples).

[0127] As used herein, "nucleic acid molecule" or "polynucleotide" refers to a polymer of nucleotides, including FSGM. Non-limiting examples thereof include DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA), and chimeras thereof. Nucleic acid molecules can be obtained by cloning techniques or synthesis. DNA can be double-stranded or single-stranded (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 their analogs. The nucleic acid backbone may contain a variety of bonds known in the art, including one or more of a sugar-phosphodiester bond, a peptide-nucleic acid bond (referred to as "peptide nucleic acid" (PNA); Hydig-Hielsen et al., PCT International Publication No. WO 95 / 32305), a phosphorothioate bond, a methylphosphonate bond, or a combination thereof. The sugar moiety of the nucleic acid can be a ribose or deoxynucleoside, or similar compounds with known substitutions (e.g., 2' methoxy substitution (containing a 2'-O-methyl ribofuranosyl moiety; see PCT No. WO 98 / 02582) and / or 2' halide substitution). The nitrogenous base can be a conventional base (A, G, C, T, U), a known analog thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th edition, 1992), or a known derivative of a purine pyrimidine base (see Cook, PCT International Publication No. WO 93 / 13121), or an "abasic" residue, one or more residues of which the backbone does not include a nitrogenous base (Arnold et al., U.S. Pat. No. 5,585,481). Nucleic acids may contain 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 nucleic acids comprising conventional bases and one or more base analogs). "Isolated nucleic acid molecules" as generally understood and used herein refer to polymers of nucleotides, and include, but are not limited to, DNA and RNA. "Isolated" nucleic acid molecules are purified from their natural in vivo state obtained by cloning or chemical synthesis.

[0128] As used herein, "oligonucleotides" or "oligos" define molecules having two or more nucleotides (ribose or deoxyribonucleotides). The size of the oligonucleotide will be dictated by the particular situation and ultimately by its particular use and adapted accordingly by those skilled in the art. Oligonucleotides can be chemically synthesized or cloned according to well-known methods. Although they are usually in single-stranded form, they can be in double-stranded form and even contain "regulatory regions". They can contain naturally rare or synthetic nucleotides. They can be designed to enhance selected criteria, such as stability. Chimeras of deoxyribonucleotides and ribonucleotides can also be within the scope of the present invention.

[0129] As used herein, "one or more" is understood to encompass every value 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and any value greater than 10.

[0130] The term "or" is used herein inclusively to mean, and interchangeably with, the terms "and / or," unless the context clearly dictates otherwise.

[0131] As used herein, "patient" or "subject" may refer to a human or non-human animal, preferably a mammal. "Subject" means 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.

[0132] As used herein, the term "progression of FXN deficiency" refers to a worsening of the condition of a subject with FXN deficiency over time. This term encompasses an increase in severity and / or duration of existing symptoms of FXN deficiency and / or the development of one or more new symptoms of FXN deficiency in a subject with FXN deficiency. In some embodiments, the progression of FXN deficiency is associated with the level of FXN in a subject with FXN deficiency. For example, in some embodiments, the progression of FXN deficiency over time is associated with a decrease in FXN levels over time.

[0133] As used herein, "probe" means a nucleic acid oligomer or oligonucleotide that specifically hybridizes with a target sequence in a nucleic acid or its complement under conditions that promote hybridization, thereby allowing detection of a target sequence or its amplified nucleic acid. Detection can be direct (i.e., produced by direct hybridization of the probe with a target or amplified sequence) or indirect (i.e., produced by hybridization of the probe with an intermediate molecular structure that connects the probe to the target or amplified sequence). The "target" of a probe generally refers to a sequence within an amplified nucleic acid sequence (i.e., a subset of an amplified sequence) that specifically hybridizes with at least a portion of the probe sequence by standard hydrogen bonding or "base pairing". A "fully complementary" sequence allows stable hybridization of the probe sequence with the target sequence, even if the two sequences are not completely complementary. The probe may be labeled or unlabeled. The probe may be produced by molecular cloning of a specific DNA sequence or it may also be synthesized. Many primers and probes that can be designed and used in the context of the present invention can be easily determined by a person of ordinary skill in the art to which the present invention relates.

[0134] As used herein, a "reference level" of FSGM can be an absolute or relative amount or concentration of FSGM, the presence or absence of FSGM, a range of amounts or concentrations of FSGM, a minimum and / or maximum amount or concentration of FSGM, an average amount or concentration of FSGM, and / or a median amount or concentration of FSGM; and in addition, a "reference level" of a combination of FSGMs can also be a ratio of the absolute or relative amounts or concentrations of two or more FSGMs relative to one another. Appropriate positive and negative reference levels of FSGM for a particular disease state, phenotype, or deficiency can be determined by measuring the level of the desired FSGM in one or more appropriate subjects, and such reference levels can be customized for a particular subject population (e.g., a reference level can be matched to age so that the level of FSGM in a sample of a subject of a particular age can be compared to a reference level for a particular disease state, phenotype, or deficiency in a particular age group). Such reference levels can also be customized according to a particular technique (e.g., LC-MS, GC-MS, etc.) used to measure the level of FSGM in a biological sample, wherein the level of FSGM can differ based on the particular technique used.

[0135] As used herein, "sample" or "biological sample" includes a specimen or culture obtained from any source. In some embodiments, the sample includes any specimen or culture containing cells that can be analyzed for FXN expression profile. In some embodiments, the sample includes any specimen or culture from a FXN-deficient subject or a subject to be treated with FXN replacement therapy. For example, the biological sample can be obtained from a solid tissue sample, preferably a cheek sample, or a skin biopsy sample, skin epidermis, hair follicle, muscle biopsy sample, or a body fluid sample, such as blood (including any blood product, such as whole blood, plasma, serum or a specific type of blood cell), urine, saliva or semen. In some embodiments, the biological sample is a cheek sample. In another embodiment, the biological sample is a skin sample, for example, a skin biopsy sample or skin epidermis. Alternatively, the sample can include exosomes that can be collected to test for FSGM transcripts.

[0136] As used herein, the phrase "specific binding" or "specifically binds" when used in relation to the interaction of an antibody with a protein or peptide means that the interaction is dependent on the presence of a specific structure (i.e., an antigenic determinant or epitope) on the protein; in other words, the antibody recognizes and binds to a specific protein structure rather than to proteins in general. For example, if the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of a protein containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0137] The term "such as" is used herein to mean, and is used interchangeably with, the phrase "such as, but not limited to."

[0138] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or an analog of such RNA or cDNA) that is complementary to or has a high percentage of identity (e.g., at least 80% identity) to all or part of a mature mRNA prepared by transcription of the FSGM of the present invention and normal post-transcriptional processing (e.g., splicing) of the RNA transcript, if any, and reverse transcription of the RNA transcript.

[0139] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0140] 43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,

[0141] Reference will now be made in detail to exemplary embodiments of the present invention. Although the present invention will be described in conjunction with exemplary embodiments, it should be understood that it is not intended to limit the present invention to those embodiments. On the contrary, it is intended to cover alternatives, modifications and equivalents within the spirit and scope of the present invention as defined by the appended claims.

[0142] C. FXN sensitivity genomic markers (FSGM) of the present invention

[0143] The present disclosure provides a set of markers, also referred to herein as FXN-sensitive genomic markers (or FSGMs), each of which has a positive or negative correlation with the level of frataxin (FXN) in a cell or subject (e.g., a sample from a subject). In some embodiments, the FSGMs of the present disclosure are reversely regulated by FXN gene ablation or deficiency, followed by FXN protein replacement. Therefore, the FSGMs of the present disclosure are associated with FXN deficiency in a subject and are reversely correlated with FXN replacement. All FSGMs disclosed herein were found to be sensitive to FXN levels and are considered markers for FXN replacement therapy. Therefore, these FSGMs can be used to assess and / or monitor the progression of FXN deficiency in a subject, as described herein. These FSGMs can also be used to assess and / or monitor FXN replacement therapy, for example, to assess, monitor or determine the efficacy of FXN replacement therapy in a subject, as described herein.

[0144] For example, in one aspect, the present invention provides a method for determining, evaluating and / or monitoring the effectiveness of FXN replacement therapy, the method comprising determining: (i) a baseline FXN(-) expression profile of one or more FSGMs in a sample from a FXN-deficient subject prior to treatment with a FXN replacement therapy; and (ii) determining a subject FXN replacement expression profile of one or more FSGMs in a sample from a FXN-deficient subject undergoing FXN replacement therapy or after treatment with a FXN replacement therapy; comparing the subject FXN replacement expression profile with the baseline FXN(-) expression profile, and using the results of the comparison to determine, evaluate or monitor the effectiveness of the FXN replacement therapy. Based on the results of the FSGM expression profile analysis, adjustments can be made to the FXN replacement therapy in the subject to, for example, initiate, increase (e.g., increase dosage and / or frequency of administration), decrease (e.g., decrease dosage and / or frequency of administration) or stop the FXN replacement therapy in the subject.

[0145] Another aspect of the present disclosure relates to a method for identifying one or more FSGMs. The method comprises determining an expression profile of one or more genes in a sample from a healthy subject with normal FXN levels, referred to herein as a normal FXN expression profile; determining an expression profile of one or more genes in a sample from a subject with deficient FXN levels, referred to herein as a baseline FXN(-) expression profile; and comparing the normal FXN expression profile to the baseline FXN(-) expression profile; wherein markers whose expression is altered in the baseline FXN(-) expression profile compared to the normal FXN expression profile are identified as FSGMs. Additionally or alternatively, the method for determining FSGMs may comprise comparing an expression profile of one or more genes obtained from a sample of a FXN-deficient subject before receiving FXN replacement therapy (baseline FXN(-) expression profile) with an expression profile of one or more genes obtained from a sample of a FXN-deficient subject during or after receiving FXN replacement therapy. The gene expression profile of a sample obtained from a FXN-deficient subject during or after FXN replacement therapy is also referred to herein as a FXN replacement expression profile. In an embodiment, a marker whose expression is altered in a FXN alternative expression profile (e.g., an expression level change toward, or similar to, a normal FXN expression profile) compared to a baseline FXN expression profile is identified as a FSGM. For example, Table 1 herein presents FSGMs identified by a method of an embodiment of the present disclosure.

[0146] Table 1. FXN sensitivity genomic markers (FSGM) of the present disclosure

[0147]

[0148]

[0149] The FSGM of the present invention includes, but is not limited to, any one of the FSGMs listed in Table 1 and / or Table 5, or any combination of two or more. In some embodiments, the FSGM of the present invention includes any one of the FSMGs listed in Table 1, or any combination of two or more. In some embodiments, the FSGM of the present invention includes any one of the FSMGs listed in Table 5, or any combination of two or more. In some embodiments, the FSGM of the present invention includes any one of the FSMGs listed in Table 1 and / or Table 5, or any combination of two or more, in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In some embodiments, the FSGM of the present invention includes any one of the FSMGs listed in Table 1, or any combination of two or more, in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In some embodiments, the FSGM of the present invention includes any one of the FSMGs listed in Table 5, or any combination of two or more, in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. It will be appreciated that in some embodiments of the invention, other markers known in the art for measuring FXN expression levels or assessing the efficacy of FXN replacement therapy may be used in conjunction with the FSGM and / or methods of the invention.

[0150] As used herein, the term "one or more FSGMs" means one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) FSGMs selected from, for example, Table 1 and / or Table 5. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) FSGMs are selected from Table 1. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) FSGMs are selected from Table 5. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) FSGMs are selected from, for example, Table 1 and / or Table 5 alone or in combination with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) additional FSGMs listed in Tables 2, 3, 4, and / or 6. The methods, kits and detection combinations 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 Table 1 and / or Table 5. In some embodiments, the methods, kits and detection combinations 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 Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6.

[0151] In some embodiments, one or more FSGMs of the present invention comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) markers selected from Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs selected from Table 2, 3, 4 and / or 6.

[0152] In some embodiments, one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In some embodiments, one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP in combination with one or more additional FSGMs selected from Tables 2, 3, 4, and / or 6. In other embodiments, one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, and PSMB9. In other embodiments, one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, and PSMB9 in combination with one or more additional FSGMs selected from Tables 2, 3, 4, and / or 6. In some embodiments, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In other embodiments, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP in combination with one or more additional FSGMs selected from Tables 2, 3, 4, and / or 6.

[0153] In one embodiment, the one or more FSGMs comprise one or any combination of LRRK2, SOD2, CD44, and Lgals3 alone or in combination with one or more additional FSGMs selected from Table 4 and / or Table 5. In another embodiment, the one or more FSGMs comprise one or any combination of LRRK2, CD44, and Lgals3 alone or in combination with one or more additional FSGMs selected from Table 2, 3, 4, and / or 6. In one embodiment, the one or more FSGMs comprise one or any combination of LRRK2, SOD2, CD44, Lgals3, FTL, and TXNIP alone or in combination with one or more additional FSGMs selected from Table 4 and / or Table 5. In another embodiment, the one or more FSGMs comprise one or any combination of LRRK2, CD44, Lgals3, FTL, and TXNIP alone or in combination with one or more additional FSGMs selected from Table 2, 3, 4, and / or 6.

[0154] In one embodiment, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9 alone or in combination with one or any combination of CTSS, BTG2, EGR1, and PTGS2. In another embodiment, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, and PSMB9 alone or in combination with one or any combination of CTSS, BTG2, EGR1, and PTGS2.

[0155] In one embodiment, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, TXNIP alone or in combination with one or any combination of CTSS, BTG2, EGR1, and PTGS2. In another embodiment, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, TXNIP alone or in combination with one or any combination of CTSS, BTG2, EGR1, and PTGS2.

[0156] In one embodiment, the one or more FSGMs comprise one or any combination of LRRK2, SOD2, CD44 and Lgals3 alone or in combination with one or any combination of CTSS, BTG2, EGR1 and PTGS2. In another embodiment, the one or more FSGMs comprise one or any combination of LRRK2, CD44 and Lgals3 alone or in combination with one or any combination of CTSS, BTG2, EGR1 and PTGS2.

[0157] In one embodiment, the one or more FSGMs comprise one or any combination of LRRK2, SOD2, CD44, Lgals3, FTL, and TXNIP alone or in combination with one or any combination of CTSS, BTG2, EGR1, and PTGS2. In another embodiment, the one or more FSGMs comprise one or any combination of LRRK2, CD44, Lgals3, FTL, and TXNIP alone or in combination with one or any combination of CTSS, BTG2, EGR1, and PTGS2.

[0158] In one embodiment, the one or more FSGMs include Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2. In one embodiment, the one or more FSGMs include Xpo6, SOD2, FTL, TXNIP, CTSS, EGR1, BTG2, PTSG2, and LRKK2.

[0159] Additional FSGMs suitable for use in the context of the present disclosure are listed in Tables 2 and 3 below.

[0160] Table 2: Additional FSGMs of the present disclosure

[0161]

[0162]

[0163]

[0164] In one embodiment, the one or more FSGMs include one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include two or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include three or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include four or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include five or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include six or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include seven or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include eight or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise nine or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP.

[0165] In one embodiment, one or more FSGMs include one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, one or more FSGMs include two or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, one or more FSGMs include three or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, one or more FSGMs include four or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, one or more FSGMs include five or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise six or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise seven or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise eight or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP.

[0166] In some embodiments, one or more FSGMs comprise Timp 1. In some embodiments, one or more FSGMs comprise LRRK2. In some embodiments, one or more FSGMs comprise BASP1. In some embodiments, one or more FSGMs comprise CD44. In some embodiments, one or more FSGMs comprise Xpo6. In some embodiments, one or more FSGMs comprise Lgals3. In some embodiments, one or more FSGMs comprise SOD2. In some embodiments, one or more FSGMs comprise PSMB9. In some embodiments, one or more FSGMs comprise FTL. In some embodiments, one or more FSGMs comprise TXNIP.

[0167] In some embodiments, one or more FSGMs consist of Timp 1. In some embodiments, one or more FSGMs consist of LRRK2. In some embodiments, one or more FSGMs consist of BASP1. In some embodiments, one or more FSGMs consist of CD44. In some embodiments, one or more FSGMs consist of Xpo6. In some embodiments, one or more FSGMs consist of Lgals3. In some embodiments, one or more FSGMs consist of SOD2. In some embodiments, one or more FSGMs consist of PSMB9. In some embodiments, one or more FSGMs consist of FTL. In some embodiments, one or more FSGMs consist of TXNIP.

[0168] In some embodiments, one or more FSGMs comprise Timp 1 and LRRK2. In some embodiments, one or more FSGMs comprise Timp 1 and Basp1. In some embodiments, one or more FSGMs comprise Timp 1 and CD44. In some embodiments, one or more FSGMs comprise Timp 1 and Xpo6. In some embodiments, one or more FSGMs comprise Timp 1 and Lgals3. In some embodiments, one or more FSGMs comprise Timp 1 and SOD2. In some embodiments, one or more FSGMs comprise Timp 1 and PSMB9. In some embodiments, one or more FSGMs comprise Timp 1 and FTL. In some embodiments, one or more FSGMs comprise Timp 1 and TXNIP. In some embodiments, one or more FSGMs comprise LRRK2 and BASP1. In some embodiments, one or more FSGMs comprise LRRK2 and CD44. In some embodiments, one or more FSGMs comprise LRRK2 and Xpo6. In some embodiments, one or more FSGMs comprise LRRK2 and Lgals3. In some embodiments, one or more FSGMs comprise LRRK2 and SOD2. In some embodiments, one or more FSGMs comprise LRRK2 and PSMB9. In some embodiments, one or more FSGMs comprise LRRK2 and FTL. In some embodiments, one or more FSGMs comprise LRRK2 and TXNIP. In some embodiments, one or more FSGMs comprise BASP1 and CD44. In some embodiments, one or more FSGMs comprise BASP1 and Xpo6. In some embodiments, one or more FSGMs comprise BASP1 and Lgals3. In some embodiments, one or more FSGMs comprise BASP1 and SOD2. In some embodiments, one or more FSGMs comprise BASP1 and PSMB9. In some embodiments, one or more FSGMs comprise BASP1 and FTL. In some embodiments, one or more FSGMs comprise BASP1 and TXNIP. In some embodiments, one or more FSGMs comprise CD44 and Xpo6. In some embodiments, one or more FSGMs comprise CD44 and Lgals3. In some embodiments, one or more FSGMs comprise CD44 and SOD2. In some embodiments, one or more FSGMs comprise CD44 and PSMB9. In some embodiments, one or more FSGMs comprise CD44 and FTL. In some embodiments, one or more FSGMs comprise CD44 and TXNIP. In some embodiments, one or more FSGMs comprise Xpo6 and Lgals3. In some embodiments, one or more FSGMs comprise Xpo6 and SOD2.In some embodiments, one or more FSGMs comprise Lgals3 and PSMB9. In some embodiments, one or more FSGMs comprise Lgals3 and FTL. In some embodiments, one or more FSGMs comprise Lgals3 and TXNIP. In some embodiments, one or more FSGMs comprise Lgals3 and SOD2. In some embodiments, one or more FSGMs comprise Lgals3 and PSMB9. In some embodiments, one or more FSGMs comprise SOD2 and PSMB9. In some embodiments, one or more FSGMs comprise SOD2 and FTL. In some embodiments, one or more FSGMs comprise SOD2 and TXNIP.

[0169] In some embodiments, one or more FSGMs comprise Timp1, LRRK2, and BASP1. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, and CD44. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, and Xpo6. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, and Lgals3. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, and SOD2. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, and FTL. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, and FXNIP. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, and CD44. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, and Xpo6. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, and Lgals3. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, and SOD2. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, and PSMB9. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, and FTL. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, and TXNIP. In some embodiments, one or more FSGMs comprise BASP1, CD44, and Xpo6. In some embodiments, one or more FSGMs comprise BASP1, CD44, and Lgals3. In some embodiments, one or more FSGMs comprise BASP1, CD44, and SOD2. In some embodiments, one or more FSGMs comprise BASP1, CD44, and PSMB9. In some embodiments, one or more FSGMs comprise BASP1, CD44, and FTL. In some embodiments, one or more FSGMs comprise BASP1, CD44, and TXNIP. In some embodiments, one or more FSGMs comprise CD44, Xpo6, and Lgals3. In some embodiments, one or more FSGMs comprise CD44, Xpo6, and SOD2. In some embodiments, one or more FSGMs comprise CD44, Xpo6, and PSMB9. In some embodiments, one or more FSGMs comprise CD44, Xpo6, and FTL. In some embodiments, one or more FSGMs comprise CD44, Xpo6, and TXNIP. In some embodiments, one or more FSGMs comprise Xpo6, Lgals3, and SOD2.In some embodiments, one or more FSGMs comprise Xpo6, Lgals3, and PSMB9. In some embodiments, one or more FSGMs comprise Xpo6, Lgals3, and FTL. In some embodiments, one or more FSGMs comprise Xpo6, Lgals3, and TXNIP. In some embodiments, one or more FSGMs comprise Timp1, BASP1, and CD44. In some embodiments, one or more FSGMs comprise Timp1, BASP1, and Xpo6. In some embodiments, one or more FSGMs comprise Timp1, BASP1, and Lgals3. In some embodiments, one or more FSGMs comprise Timp1, BASP1, and SOD2. In some embodiments, one or more FSGMs comprise Timp1, BASP1, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, BASP1, and FTL. In some embodiments, one or more FSGMs comprise Timp1, BASP1, and TXNIP. In some embodiments, one or more FSGMs comprise LRRK2, CD44, and Xpo6. In some embodiments, one or more FSGMs comprise LRRK2, CD44, and Lgals3. In some embodiments, one or more FSGMs comprise LRRK2, CD44, and SOD2. In some embodiments, one or more FSGMs comprise LRRK2, CD44, and PSMB9. In some embodiments, one or more FSGMs comprise LRRK2, CD44, and FTL. In some embodiments, one or more FSGMs comprise LRRK2, CD44, and TXNIP. In some embodiments, one or more FSGMs comprise LRRK2, Xpo6, and Lgals3. In some embodiments, one or more FSGMs comprise LRRK2, Xpo6, and SOD2. In some embodiments, one or more FSGMs comprise LRRK2, Xpo6, and PSMB9. In some embodiments, one or more FSGMs comprise LRRK2, Xpo6, and FTL. In some embodiments, one or more FSGMs comprise LRRK2, Xpo6, and TXNIP. In some embodiments, one or more FSGMs comprise LRRK2, Lgals3, and SOD2. In some embodiments, one or more FSGMs comprise LRRK2, Lgals3, and PSMB9. In some embodiments, one or more FSGMs comprise LRRK2, Lgals3, and FTL. In some embodiments, one or more FSGMs comprise LRRK2, Lgals3, and TXNIP. In some embodiments, the one or more FSGMs comprise LRRK2, SOD2, and PSMB9. In some embodiments, the one or more FSGMs comprise LRRK2, SOD2, and FTL.In some embodiments, one or more FSGMs comprise LRRK2, SOD2, and TXNIP. In some embodiments, one or more FSGMs comprise BASP1, Xpo6, and Lgals3. In some embodiments, one or more FSGMs comprise BASP1, Xpo6, and SOD2. In some embodiments, one or more FSGMs comprise BASP1, Xpo6, and PSMB9. In some embodiments, one or more FSGMs comprise BASP1, Xpo6, and FTL. In some embodiments, one or more FSGMs comprise BASP1, Xpo6, and TXNIP. In some embodiments, one or more FSGMs comprise BASP1, Lgals 3, and SOD2. In some embodiments, one or more FSGMs comprise BASP1, Lgals3, and PSMB9. In some embodiments, one or more FSGMs comprise BASP1, Lgals3, and FTL. In some embodiments, one or more FSGMs comprise BASP1, Lgals3, and TXNIP. In some embodiments, one or more FSGMs comprise BASP1, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise BASP1, SOD2, and FTL. In some embodiments, one or more FSGMs comprise BASP1, SOD2, and TXNIP. In some embodiments, one or more FSGMs comprise Lgals3, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise Lgals3, SOD2, and FTL. In some embodiments, one or more FSGMs comprise Lgals3, SOD2, and TXNIP. In some embodiments, one or more FSGMs comprise Timp1, CD44, and Xpo6. In some embodiments, one or more FSGMs comprise Timp1, CD44, and Lgals3. In some embodiments, one or more FSGMs comprise Timp1, CD44, and SOD2. In some embodiments, one or more FSGMs comprise Timp1, CD44, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, CD44, and FTL. In some embodiments, one or more FSGMs comprise Timp1, CD44, and TXNIP. In some embodiments, one or more FSGMs comprise Timp1, Xpo6, and Lgals3. In some embodiments, one or more FSGMs comprise Timp1, Xpo6, and SOD2. In some embodiments, one or more FSGMs comprise Timp1, Xpo6, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, Xpo6, and FTL. In some embodiments, the one or more FSGMs include Timpl, Xpo6, and TXNIP.In some embodiments, one or more FSGMs include Timp1, Lgals3, and SOD2. In some embodiments, one or more FSGMs include Timp1, Lgals3, and PSMB9. In some embodiments, one or more FSGMs include Timp1, Lgals3, and FTL. In some embodiments, one or more FSGMs include Timp1, Lgals3, and TXNIP. In some embodiments, one or more FSGMs include Timp1, SOD2, and PSMB9. In some embodiments, one or more FSGMs include Timp1, SOD2, and FTL. In some embodiments, one or more FSGMs include Timp1, SOD2, and TXNIP. In some embodiments, one or more FSGMs include PSMB9, SOD2, and Lgals3. In some embodiments, one or more FSGMs include PSMB9, SOD2, and Xpo6.

[0170] In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, and CD44. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, and Xpo6. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, and Lgals3. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, and SOD2. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, and FTL. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, and TXNIP. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, and Xpo6. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, and Lgals3. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, and SOD2. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, and PSMB9. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, and FTL. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, and TXNIP. In some embodiments, one or more FSGMs comprise BASP1, CD44, Xpo6, and Lgals3. In some embodiments, one or more FSGMs comprise BASP1, CD44, Xpo6, and Lgals3. In some embodiments, one or more FSGMs comprise BASP1, CD44, Xpo6, and SOD2. In some embodiments, one or more FSGMs comprise BASP1, CD44, Xpo6, and PSMB9. In some embodiments, one or more FSGMs comprise BASP1, CD44, Xpo6, and FTL. In some embodiments, one or more FSGMs comprise BASP1, CD44, Xpo6, and TXNIP. In some embodiments, one or more FSGMs comprise CD44, Xpo6, Lgals3, and SOD2. In some embodiments, one or more FSGMs comprise CD44, Xpo6, Lgals3, and SOD2. In some embodiments, one or more FSGMs comprise CD44, Xpo6, Lgals3, and PSMB9. In some embodiments, one or more FSGMs comprise CD44, Xpo6, Lgals3, and FTL. In some embodiments, one or more FSGMs comprise CD44, Xpo6, Lgals3, and TXNIP.In some embodiments, one or more FSGMs comprise Xpo6, Lgals3, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise Xpo6, Lgals3, SOD2, and FTL. In some embodiments, one or more FSGMs comprise Xpo6, Lgals3, SOD2, and TXNIP. In some embodiments, one or more FSGMs comprise PSMB9, SOD2, Lgals3, and Xpo6. In some embodiments, one or more FSGMs comprise PSMB9, SOD2, Lgals3, and CD44. In some embodiments, one or more FSGMs comprise PSMB9, SOD2, Lgals3, and BASP1. In some embodiments, one or more FSGMs comprise PSMB9, SOD2, Lgals3, and LRRK2. In some embodiments, one or more FSGMs comprise PSMB9, SOD2, Lgals3, and Timp1. In some embodiments, one or more FSGMs comprise FTL, SOD2, Lgals3, and Xpo6. In some embodiments, one or more FSGMs comprise FTL, SOD2, Lgals3, and CD44. In some embodiments, one or more FSGMs comprise FTL, SOD2, Lgals3, and BASP1. In some embodiments, one or more FSGMs comprise FTL, SOD2, Lgals3, and LRRK2. In some embodiments, one or more FSGMs comprise FTL, SOD2, Lgals3, and Timp1. In some embodiments, one or more FSGMs comprise TXNIP, SOD2, Lgals3, and Xpo6. In some embodiments, one or more FSGMs comprise TXNIP, SOD2, Lgals3, and CD44. In some embodiments, one or more FSGMs comprise TXNIP, SOD2, Lgals3, and BASP1. In some embodiments, one or more FSGMs comprise TXNIP, SOD2, Lgals3, and LRRK2. In some embodiments, one or more FSGMs comprise TXNIP, SOD2, Lgals3, and Timp1.

[0171] In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, and Xpo6. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, and Lgals3. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, and SOD2. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, and PSMB9. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, Xpo6, and Lgals3. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, Xpo6, and SOD2. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, Xpo6, and PSMB9. In some embodiments, one or more FSGMs comprise BASP1, CD44, Xpo6, Lgals3, and SOD2. In some embodiments, one or more FSGMs comprise BASP1, CD44, Lgals3, and PSMB9. In some embodiments, the one or more FSGMs comprise CD44, Xpo6, Lgals3, SOD2, and PSMB9.

[0172] In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, and Lgals3. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, and SOD2. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, and FTL. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, and TXNIP. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, Xpo6, Lgals3, and SOD2. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, Xpo6, Lgals3, and PSMB9. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, Xpo6, Lgals3, and FTL. In some embodiments, one or more FSGMs comprise LRRK2, BASP1, CD44, Xpo6, Lgals3, and TXNIP. In some embodiments, one or more FSGMs comprise Timp1, BASP1, CD44, Xpo6, Lgals3, and SOD2. In some embodiments, one or more FSGMs comprise Timp1, BASP1, CD44, Xpo6, Lgals3, and PSMB9. In some embodiments, one or more FSGMs comprise PSMB9, SOD2, Lgals3, Xpo6, CD44, and BASP1. In some embodiments, one or more FSGMs comprise FTL, SOD2, Lgals3 Xpo6, CD44, and BASP1. In some embodiments, one or more FSGMs comprise TXNIP, SOD2, Lgals3, Xpo6, CD44, and BASP1.

[0173] In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, and SOD2. In some embodiments, one or more FSGMs comprise Timp1, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, LRKK2, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, LRKK2, BASP1, Xpo6, Lgals3, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Lgals3, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, and PSMB9. In some embodiments, one or more FSGMs comprise LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and FTL. In some embodiments, one or more FSGMs comprise LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and TXNIP.

[0174] In some embodiments, one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and PSMB9. In some embodiments, one or more FSGMs comprise Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and FTL. In some embodiments, one or more FSGMs comprise Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, and TXNIP. In some embodiments, one or more FSGMs comprise Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, and FTL. In some embodiments, one or more FSGMs comprise Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, and TXNIP.

[0175] In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with one or more additional gene markers described in Table 6 (i.e., EGR1, CTSS, BTG2, and / or PTGS2).

[0176] In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with EGR1. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with CTSS. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with BTG2. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with PTGS2.

[0177] In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with EGR1 and CTSS. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with EGR1 and BTG2. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with EGR1 and PTGS2. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with CTSS and BTG2. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with CTSS and PTGS2. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with BTG2 and PTGS2.

[0178] In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with EGR1, CTSS, and BTG2. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with EGR1, CTSS, and PTGS2. In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with CTSS, BTG2, and PTGS2.

[0179] In some embodiments, any combination of one or more of Timp1, LRKK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP as described above may be further combined with EGR1, CTSS, BTG2, and PTGS2.

[0180] In one embodiment, the method provided by the present disclosure comprises determining the FXN expression profile of one or more FSGMs described above. In another embodiment, the method provided by the present disclosure comprises determining the FXN expression profile of one or more FSGMs described above and one or more FSGMs defined in Tables 2, 3, 4 and / or 6. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise one or more of CTSS, BTG2, EGR1 and PTGS2. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise two or more of CTSS, BTG2, EGR1 and PTGS2. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise three or more of CTSS, BTG2, EGR1 and PTGS2. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise CTSS, BTG2, EGR1 and PTGS2.

[0181] In one aspect, the present disclosure provides a method for detecting one or more frataxin-sensitive genomic markers (FSGMs) in a sample from a subject with frataxin (FXN) deficiency, comprising contacting the sample or a portion thereof with one or more reagents specifically for detecting the level of each of the one or more FSGMs disclosed herein, thereby detecting the FSGMs in the sample. In one embodiment, the sample is a cheek sample. In one embodiment, the sample is a skin sample. In some embodiments, the subject suffers from FRDA. In some embodiments, the FXN-deficient subject is undergoing treatment or is planned to be treated with FXN replacement therapy. In some embodiments, the one or more FSGMs comprise one or more FSGMs selected from Table 1 and / or Table 5. In some embodiments, the one or more FSGMs comprise one or more FSGMs selected from Table 1. In some embodiments, the one or more FSGMs comprise one or more FSGMs selected from Table 5. In some embodiments, the one or more FSGMs comprise a combination of one or more FSGMs selected from Table 1 and / or Table 5 and one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In some embodiments, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44CD44, Xpo6, Lgals3, SOD2, and PSMB9, FTL, and TXNIP. In some embodiments, one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44CD44, Xpo6, Lgals3, SOD2, and PSMB9, FTL, and TXNIP in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2). In some embodiments, one or more FSGMs comprise or consist of Timp1, LRRK2, BASP1, CD44CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, TXNIP, CTSS, BTG2, EGR1, and PTGS2. In some embodiments, one or more FSGMs comprise or consist of LRRK2 and Xpo6. In some embodiments, one or more FSGMs comprise or consist of LRRK2, Xpo6, CTSS, BTG2, EGR1, and PTGS2.

[0182] In one embodiment, the one or more FSGMs comprise one or more of LRRK2, SOD2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise two or more of LRRK2, SOD2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise three or more of LRRK2, SOD2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise LRRK2, SOD2, CD44, and Lgals3.

[0183] In one embodiment, the one or more FSGMs comprise one or more of LRRK2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise two or more of LRRK2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise LRRK2, CD44, and Lgals3.

[0184] In one embodiment, the present disclosure provides a method for detecting one or more frataxin-sensitive genomic markers (FSGMs) in a buccal sample obtained from a frataxin (FXN)-deficient subject (e.g., a subject with FRDA), comprising contacting the sample or a portion thereof with one or more reagents specifically for detecting the level of each of the one or more FSGMs disclosed herein, wherein the FSGMs comprise one or more FSGMs selected from Table 5, thereby detecting the FSGMs in the sample.

[0185] In one embodiment, the present disclosure provides a method for detecting one or more frataxin-sensitive genomic markers (FSGMs) in a buccal sample from a frataxin (FXN)-deficient subject (e.g., a subject with FRDA) who is undergoing FXN replacement therapy, comprising contacting the sample or a portion thereof with one or more reagents specifically for detecting the level of each of the one or more FSGMs disclosed herein, wherein the FSGMs comprise one or more FSGMs selected from Table 5, thereby detecting the FSGMs in the sample.

[0186] In one embodiment, the method provided by the present disclosure comprises determining the FXN expression profile of one or more FSGMs described above. In another embodiment, the method provided by the present disclosure comprises determining the FXN expression profile of one or more FSGMs described above and one or more FSGMs defined in Tables 2, 3, 4 and / or 6. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise one or more of CTSS, BTG2, EGR1 and PTGS2. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise two or more of CTSS, BTG2, EGR1 and PTGS2. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise three or more of CTSS, BTG2, EGR1 and PTGS2. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise CTSS, BTG2, EGR1 and PTGS2.

[0187] For example, the FXN expression profile can be determined by measuring the expression level of at least one FSGM or any combination of more than one FSGM. As used herein, FSGM includes any one or more FSGM listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGM listed in Table 2, 3, 4 and / or 6.

[0188] Hereinafter, expression profiles may also be referred to as signatures.

[0189] In one embodiment of the present disclosure, the baseline FXN(-) expression profile comprises altered expression of at least one FSGM or any combination of more than one FSGM (e.g., any one or more FSGMs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6).

[0190] In another embodiment of the present disclosure, the baseline FXN(-) expression profile may include downregulated expression levels of at least one of Timp1, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, and TXNIP, or any combination thereof. The effectiveness of FXN replacement therapy may be indicated by an upregulation pattern of any one or more of these FSGMs.

[0191] In another embodiment of the present disclosure, the baseline FXN(-) expression profile may comprise an upregulated expression level of Lgals3. A measure of the effectiveness of FXN replacement therapy may be indicated by a downregulation pattern of Lgals3.

[0192] In one embodiment, the FXN replacement expression profile comprises the inverse expression of a baseline FXN (-) expression profile.

[0193] In another embodiment, the FXN replacement expression profile used as an indicator of the effectiveness of FXN replacement therapy may comprise one of the FSGMs presented in Table 1 and / or Table 5 alone, or in combination of any two or more, including, for example, Timp1, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL and one or more of TXNIP, CTSS, BTG2, EGR1 and PTGS2 detected in a sample of a subject treated with FXN replacement therapy.

[0194] In another embodiment, the FXN alternative expression profile may comprise Table 2 and Figure 2 , 3 and / or the expression pattern exemplified in 4 by fold regulation of the FXN replacement expression profile in a subject treated with FXN replacement therapy.

[0195] In some embodiments, the FXN replacement expression profile is characterized by directional regulation of FSGMs, which are defined as any FSGM that is downregulated under FXN depletion conditions (e.g., in subjects with FRDA) and becomes upregulated following FXN replacement therapy; and vice versa, any FSGM that is upregulated under FXN depletion conditions (in subjects with FRDA) and becomes downregulated following FXN replacement therapy. Thus, detecting altered expression of one or more FSGMs in a sample following FXN replacement therapy allows monitoring the efficacy of FXN replacement therapy in a subject. For example, in one embodiment, no change in expression of one or more FSGMs in a sample following FXN replacement therapy indicates that FXN replacement therapy may not be effective and / or that an increase in FXN replacement therapy may be required. Likewise, in another embodiment, a change in expression of one or more FSGMs in a sample following FXN replacement therapy indicates that FXN replacement therapy is effective.

[0196] Altered or modulated expression may comprise increasing expression (also referred to as overexpression or upregulation) or decreasing or inhibiting expression (also referred to as downregulation).

[0197] As referred to herein, a feature vector is a set of values ​​that characterize an expression profile. A feature vector may comprise a set of n FSGMs, where n is the number of different genes whose expression levels are measured in a sample. For example, n may be the total number of FSGMs provided in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. Alternatively, n may be at least one, two, or three, or four, or five, or six, or any number of FSGMs presented in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6, in any combination.

[0198] In some embodiments, a set of FSGMs may include at least one FSGM or any combination of more than one FSGM, such as any one or more FSGMs listed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. For example, the one or more FSGMs include one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, BTG2, EGR1, and PTGS2, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2), in any combination.

[0199] In one embodiment, the one or more FSGMs include one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include two or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include three or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include four or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include five or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include six or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include seven or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs include eight or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise nine or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP.

[0200] In one embodiment, one or more FSGMs include one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, one or more FSGMs include two or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, one or more FSGMs include three or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, one or more FSGMs include four or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, one or more FSGMs include five or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise six or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise seven or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP.

[0201] In one embodiment, the method provided by the present disclosure comprises determining the FXN expression profile of one or more FSGMs described above. In another embodiment, the method provided by the present disclosure comprises determining the FXN expression profile of one or more FSGMs described above and one or more FSGMs defined in Tables 2, 3, 4 and / or 6. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise one or more of CTSS, BTG2, EGR1 and PTGS2. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise two or more of CTSS, BTG2, EGR1 and PTGS2. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise three or more of CTSS, BTG2, EGR1 and PTGS2. In one embodiment, the one or more FSGMs defined in Tables 2, 3, 4 and / or 6 comprise CTSS, BTG2, EGR1 and PTGS2.

[0202] In one embodiment, the one or more FSGMs comprise one or more of LRRK2, SOD2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise two or more of LRRK2, SOD2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise three or more of LRRK2, SOD2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise LRRK2, SOD2, CD44, and Lgals3.

[0203] In one embodiment, the one or more FSGMs comprise one or more of LRRK2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise two or more of LRRK2, CD44, and Lgals3. In another embodiment, the one or more FSGMs comprise LRRK2, CD44, and Lgals3.

[0204] For example, a normal FXN expression profile obtained from a sample of a healthy subject may be composed of a set of expression levels of FSGM, and may be represented by a normal FXN feature vector and referred to as a normal FXN feature vector. As described in the following example, when measured in a FXN-deficient sample (e.g., a sample from a subject with FRDA), FSGM may exhibit expression levels different from those of FSGM in a healthy subject, and may therefore be represented by a lack of FXN feature vector and referred to as a lack of FXN feature vector. In one embodiment, the difference between the lack of FXN feature vector and the normal FXN feature vector may be detected and quantified by the distance between the two feature vectors. In an alternative scenario, the expression levels of FSGM of a sample from a FXN-deficient subject (e.g., a subject with FRDA) after FXN replacement therapy may also exhibit different expression levels, and may be represented by a FXN replacement feature vector and referred to as a FXN replacement feature vector. For the aforementioned two feature vectors, the difference between the FXN replacement feature vector and the normal FXN feature vector or the lack of FXN feature vector may be detected and quantified by the distance between the replacement FXN feature vector and the normal FXN feature vector or the lack of FXN feature vector.

[0205] Thus, samples from FXN-deficient subjects obtained before treatment and samples obtained after FXN replacement therapy are prepared, a first FXN feature vector for a FXN replacement expression profile can be determined and a second FXN feature vector for a baseline FXN (-) expression profile can be determined; wherein determining the distance or scalar product between the first feature vector and the second feature vector can be used to determine the effectiveness of FXN replacement therapy. In one embodiment of the present disclosure, a third feature vector for a normal FXN expression profile can be determined, the normal expression profile being established for FSGM in samples from healthy subjects. In one embodiment, the distance between the second (baseline FXN (-) expression profile) and the third (normal FXN expression profile) FXN feature vectors can be determined. In another embodiment, the distance between the first (FXN replacement expression profile) and the third (normal FXN expression profile) FXN feature vectors can be determined and used to determine the effectiveness of FXN replacement therapy. In one embodiment, the distance between the first feature vector and the third feature vector can be normalized relative to the distance between the second feature vector and the third feature vector, and the resulting normalized distance can be used to determine the effectiveness of FXN replacement therapy. In one embodiment, the resulting normalized distance may be a value in the range of 0 (zero) to 1 (one), where the smaller the value (closest to zero), the more effective the therapy.

[0206] The markers disclosed herein (e.g., one or more FSGMs selected from Table 1 and / or Table 5, alone or in combination with one or more FSGMs selected from Tables 2, 3, 4, and / or 6) are associated with FXN levels in a subject. Thus, in one aspect, the present invention provides methods of using, measuring, detecting, quantifying, etc., one or more FSGMs in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP, alone or in combination with one or more FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2), methods for determining and / or monitoring FXN status in a subject, or methods for determining, evaluating, and / or monitoring FXN replacement therapy in a subject.

[0207] In another aspect, the invention relates to using, measuring, detecting, quantifying, etc. one or more FSGMs in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6 (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP alone or in combination with one or more FSGMs listed in Tables 2, 3, 4 and / or 6 (e.g., one or more of CTSS, BTG2, EGR1 and PTGS2)) as an alternative for measuring FXN expression levels.

[0208] Additionally, in another embodiment, the FSGMs may be used in combination with one or more additional markers of a mitochondrial disease (e.g., FRDA). Other markers that may be used in combination with one or more FSGMs listed in Tables 1 and / or 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 include any measurable feature described herein that reflects the physiological state of an organism (e.g., whether the organism suffers from a mitochondrial disease, e.g., FRDA) in a quantitative or qualitative manner. The physiological state of an organism includes any disease or non-disease state, e.g., a subject suffering from a mitochondrial disease (e.g., FRDA) or an otherwise healthy subject. The FSGMs of the present invention that may be used in combination with one or more additional FSGMs in Tables 2, 3, 4, and / or 6 include features that can be objectively measured and assessed as indicators of normal processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Such combination markers may be clinical parameters (e.g., age, performance status), laboratory measurements (e.g., molecular markers), or genetic or other molecular determinants. In other embodiments, the present invention also relates to the analysis and consideration of any clinical and / or subject-related health data, such as, for example, data obtained from electronic medical records (e.g., a collection of electronic health information about an individual subject or a population involving a variety of data types such as demographic data, medical history, medications and allergies, immune status, laboratory test results, radiological images, vital signs, personal statistics such as age and weight, and billing information).

[0209] The present invention also encompasses the use of specific combinations of FSGMs listed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., a combination of FSGMs comprising one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP, alone or in combination with one or more FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2), in any combination. In one embodiment, the present invention encompasses a panel of FSGMs having at least two (2) members, which may include any two FSGMs listed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In another embodiment, the present invention encompasses a panel of FSGMs having at least three (3) members, which may include any three FSGMs listed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In one embodiment, the present invention encompasses a FSGM group having at least four (4) members, which may include any four FSMGs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In another embodiment, the present invention encompasses a FSGM group having at least five (5) members, which may include any five FSMGs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In another embodiment, the present invention encompasses a FSGM group having at least six (6) members, which may include any six FSMGs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In another embodiment, the present invention encompasses a FSGM group having at least seven (7) members, which may include any seven FSMGs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In another embodiment, the present invention encompasses a FSGM group having at least eight (8) members, which may include any eight FSMGs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In another embodiment, the present invention encompasses a FSGM group having at least nine (9) members, which may include any nine FSMGs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6.In another embodiment, the present invention encompasses a FSGM group having at least ten (10) members, which may include any ten FSMGs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In another embodiment, the present invention encompasses a FSGM group having at least eleven (11) members, which may include any eleven FSMGs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In another embodiment, the present invention encompasses a FSGM group having at least twelve (12) members, which may include any twelve FSMGs listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In other embodiments, the present invention encompasses a FSGM group comprising at least 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 , 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 or 138 FSGMs or a combination thereof with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6.In one embodiment, the present invention encompasses a FSGM group comprising at least 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 11, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 or 138 FSGMs or a combination thereof with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6, wherein the one or more FSGMs in the group are FSGMs defined in Tables 1 and / or 5, e.g., Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP. In some embodiments, the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2 and LRKK2. In some embodiments, the one or more FSGMs include Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2.

[0210] In another embodiment, the present invention encompasses a FSGM group comprising at least 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 4, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100 or 102 FSGMs or a combination thereof with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6, wherein one FSGM in the group is Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP and CTSS, BTG2, EGR1 and PTGS2. In some embodiments, the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2 and LRKK2.

[0211] In certain embodiments, the level of FSGM is increased following treatment of a subject (e.g., a subject deficient in FXN) with FXN replacement. In some embodiments, FSGM is selected from the group consisting of Timp1, Timp1, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, and TXNIP.

[0212] In other embodiments, the level of FSGM is reduced following treatment of a subject (eg, a FXN-deficient subject) with FXN replacement. In some embodiments, the FSGM is Lgals3.

[0213] In another aspect, the invention provides for identifying a "diagnostic signature" or "diagnostic expression profile" based on the level of FSGM of the invention in a biological sample, which is associated with FXN in the sample. "Level of FSGM" can refer to the protein level of FSGM in a biological sample. "Level of FSGM" can also refer to the expression level of a gene corresponding to the protein, for example by measuring the expression level of the corresponding FSGM mRNA. The collection or population of levels of FSGM provides a diagnostic signature associated with the level of FXN.

[0214] In certain embodiments, 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 listed in Tables 1 and / or 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6 (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP, alone or in combination with one or more of CTSS, BTG2, EGR1, and PTGS2) in a biological sample of a subject receiving FXN replacement therapy; (2) detecting the level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more FSGMs listed in Tables 1 and / or 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6 (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP, alone or in combination with one or more of CTSS, BTG2, EGR1, and PTGS2) in a biological sample of a subject receiving FXN replacement therapy; 4, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP or one or more thereof together with CTSS, BTG2, EGR1 and PTGS2) detected in the biological sample is compared to the level of the same FSGM in a control sample (e.g., a baseline FXN(-) expression profile); and (3) determining whether at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more FSGMs listed in Tables 1 and / or 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6 (e.g., Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP, alone or in combination with CTSS, BTG2, EGR1 and PTGS2) detected in the biological sample is higher or lower than the level of the FSGM in the control (e.g., a baseline FXN(-) expression profile). If at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more FSGMs listed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6 (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP alone or in combination with one or more of CTSS, BTG2, EGR1 and PTGS2) are higher or lower than a control (e.g., a baseline FXN(-) expression profile), then the diagnostic signature indicates the effectiveness of FXN replacement therapy. In some embodiments, the one or more FSGMs include Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2 and LRKK2.

[0215] According to various embodiments, an algorithm may be employed to predict whether a biological sample from a subject contains FXN, or to assess or monitor whether a subject is effectively receiving FXN replacement therapy. Those skilled in the art will appreciate that an algorithm may be any calculation, formula, statistical survey, nomogram, lookup table, decision tree method, or computer program that processes a set of input variables (e.g., the number of markers detected at a level above a certain threshold level (n), or the number of markers detected at a level below a certain threshold level (n)) through a number of well-defined sequential steps to ultimately produce a score or "output". Any suitable algorithm, whether computer-based or manual-based (e.g., lookup table) is contemplated herein.

[0216] In certain embodiments, the FSGM of the present invention (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP alone or together with one or more of CTSS, BTG2, EGR1, and PTGS2) may include variant sequences. More specifically, certain binding agents / reagents for detecting certain FSGM of the present invention can bind to and / or recognize variants of these certain FSGM of the present invention. As used herein, the term "variant" encompasses nucleotide or amino acid sequences that are different from the specifically identified sequence, in which one or more nucleotides or amino acid residues are deleted, substituted, or added. The variant may be a naturally occurring allelic variant or a non-naturally occurring variant. The variant sequence (polynucleotide or polypeptide) preferably exhibits at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence disclosed herein. The percent identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portions, dividing that number by the total number of residues in the subject (query) sequence and multiplying the result by 100.

[0217] Variant sequences generally differ from a specifically identified sequence only by conservative substitutions, deletions or modifications. As used herein, a "conservative substitution" is a substitution in which one amino acid is replaced by another amino acid of similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydrophilicity of the polypeptide to remain essentially unchanged. In general, the following amino acid groups represent conservative changes: (1) ala, pro, gly, glu, asp, gln, 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 deletions or additions of amino acids that have minimal effect on the antigenic properties, secondary structure and hydrophilicity of the polypeptide. For example, a polypeptide may be bound to a signal (or leader) sequence at the N-terminus of a protein that co-translationally or post-translationally directs the transfer of the protein. The polypeptide can also be conjugated to a linker or other sequence to facilitate synthesis, purification or identification of the polypeptide (eg, poly-His), or to enhance binding of the polypeptide to a solid support. For example, the polypeptide can be conjugated to an immunoglobulin Fc region.

[0218] Polypeptide and polynucleotide sequences can be compared, and publicly available computer algorithms can be used to determine the percentage of identical amino acids or nucleotides in a specified region for another polypeptide or polynucleotide sequence. The percent identity of a polynucleotide or polypeptide sequence is determined by: comparing the polynucleotide and polypeptide sequences using an appropriate algorithm (such as BLASTN or BLASTP set to default parameters, respectively); identifying the number of identical nucleic acids or amino acids on the compared portions; dividing the number of identical nucleic acids or amino acids by the total number of nucleic acids or amino acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percent identity.

[0219] Two exemplary algorithms for comparing and identifying the consistency of polynucleotide sequences are BLASTN and FASTA algorithms. The BLASTP algorithm can be used to check the alignment and consistency of polypeptide sequences. BLASTX and FASTX algorithms compare the nucleotide query sequence translated in all reading frames with the polypeptide sequence. FASTA and FASTX algorithms are described in Pearson and Lipman, Proceedings of the National Academy of Sciences, 85:2444-2448, 1988; and Pearson, Methods in Enzymol. 183:63-98, 1990. The FASTA software package is obtained from the University of Virginia, Charlottesville, Va. 22906-9025. The FASTA algorithm set to the default parameters described in the document and assigned with the algorithm can be used to determine polynucleotide variants. The self-reading files of FASTA and FASTX versions 2.0x distributed with the algorithm describe the use of the algorithm and describe the default parameters.

[0220] 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. The BLASTN algorithm version 2.0.6 [September 10, 1998] and version 2.0.11 [January 20, 2000] set to the default parameters described in the document and distributed with the algorithm are preferably used to determine variants according to the present invention. The use of the BLAST algorithm family (including BLASTN) is described on the NCBI website and in the publication Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Research, 25: 3389-3402, 1997.

[0221] In an alternative embodiment, the variant polypeptide is encoded by a polynucleotide sequence that hybridizes to the disclosed polynucleotide under stringent conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more typically less than about 500 mM, and preferably less than about 200 mM. The hybridization temperature can be as low as 5°C, but is 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 hybridizations. Since other factors (such as probe composition, the presence of organic solvents, and the degree of base mismatching) can affect hybridization stringency, the combination of parameters is more important than the absolute measurement of any one parameter alone. An example of "stringent conditions" is a prewash in a solution of 6XSSC, 0.2% SDS; hybridization overnight at 65°C, 6XSSC, 0.2% SDS; followed by two washes in 1XSSC, 0.1% SDS at 65°C for 30 minutes each, and two washes in 0.2XSSC, 0.1% SDS at 65°C for 30 minutes each.

[0222] The present invention provides for the use of various combinations and subcombinations of FSGMs. For example, one or more FSGMs listed in Table 1 and / or Table 5 (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP) can be used alone or in combination (in any combination) with one or more FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2). It should be understood that any single FSGM or combination of FSGMs provided herein can be used in the present invention unless otherwise expressly indicated.

[0223] D. Biological samples

[0224] The present invention can be practiced with any suitable biological sample that potentially contains, expresses, includes detectable FSGM. For example, the biological sample can be obtained from a solid tissue sample, such as a skin biopsy sample, a muscle biopsy sample, preferably a cheek sample, or a body fluid sample, such as blood (including any blood product, such as whole blood, plasma, serum, or specific types of blood cells), urine, saliva, or semen. Alternatively, the sample can include exosomes that can be collected to test for FSGM transcripts.

[0225] In some embodiments, samples useful for measuring FXN expression profiles in the context of the present disclosure may be selected from the group consisting of cheek samples, skin samples, hair follicles, and muscle biopsy samples. In one embodiment, the sample is a cheek sample. In another embodiment, the sample is a skin sample. In some embodiments, the sample is a serum sample.

[0226] In some embodiments, samples that can be used to measure the FXN expression profile can be obtained from FXN-deficient subjects before, during, or after the administration of FXN replacement therapy. In some embodiments, the sample can be obtained from a FXN-deficient subject at least 15 days after the last administration of FXN replacement therapy. In some embodiments, the sample can be obtained from a FXN-deficient subject 15 to 45 days after the last administration of FXN replacement therapy (e.g., 15 to 20 days, 20 to 35 days, or 25 to 45 days after the last administration of FXN replacement therapy). In some embodiments, the sample can be obtained from a FXN-deficient subject 20 to 25 days (e.g., 20, 21, 22, 23, or 25 days) after the last administration of FXN replacement therapy. In some embodiments, the sample may be obtained from a FXN-deficient subject 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 FXN replacement therapy.

[0227] The method of the present invention can be performed at the single cell level. However, the method of the present invention can also be performed using a sample comprising many cells, wherein the analysis is an "average" representation over the entire collection of cells and tissues present in the sample. Preferably, there is enough tissue sample to accurately and reliably determine the expression level of interest.

[0228] Any commercial device or system for separating 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.

[0229] In certain embodiments, the invention relates to detecting FSGM nucleic acid molecules (e.g., mRNA encoding one or more FSGMs listed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of Timpl, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2)). In such embodiments, RNA can be extracted from the biological sample prior to analysis. Methods of RNA extraction are well known in the art (see, e.g., J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2nd edition, Cold Spring Harbor Laboratory Press, New York). Most methods of isolating RNA from body fluids or tissues are based on disrupting the tissue in the presence of a protein denaturant to quickly and effectively inactivate RNases. In general, RNA isolation reagents contain components such as guanidine thiocyanate and / or beta-mercaptoethanol, which are known to act as RNase inhibitors. The isolated total RNA is then further purified from protein contaminants and concentrated by selective ethanol precipitation, phenol / chloroform extraction followed by isopropanol precipitation (see, e.g., P. Chomczynski and N. Sacchi, Anal. Biochem., 1987, 162: 156-159), or cesium chloride, lithium chloride, or cesium trifluoroacetate gradient centrifugation.

[0230] A variety of different and common kits are available for extracting RNA (i.e., total RNA or mRNA) from body fluids or tissues and are commercially available from, for example, Ambion, Inc. (Austin, Tex.), Amersham Biosciences (Piscataway, NJ), Bo Leide Biotech Cloning Technology (Palo Alto, Calif.), BioRad Laboratories (Hercules, Calif.), GIBCO BRL (Gaithersburg, Md.), and Giagen, Inc. (Valencia, Calif.). User guides describing the protocols to be followed in great detail are typically included in all of these kits. The sensitivity, processing time, and cost may vary from one kit to another. One of ordinary skill in the art can readily select the most appropriate kit for a particular situation.

[0231] In certain embodiments, after extraction, the mRNA is amplified and transcribed into cDNA, which can then be used as a template for multiple rounds of transcription by an appropriate RNA polymerase. Amplification methods are well known in the art (see, e.g., AR Kimmel and SL Berger, Methods in Enzymology, 1987, 152: 307-316; J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2nd edition, Cold Spring Harbor Laboratory Press, New York; "Short Protocols in Molecular Biology", FM Ausubel (ed.), 2002, 5th edition supplement, John Wiley & Sons; U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,800,159). The reverse transcription reaction can be performed using nonspecific primers (such as anchored oligo-dT primers or random sequence primers) or, using target-specific primers complementary to the RNA of each genetic probe being monitored, or using a thermostable DNA polymerase (such as avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase).

[0232] In certain embodiments, RNA isolated from a tissue sample (e.g., after amplification and / or conversion to cDNA or cRNA) is labeled with a detectable agent prior to analysis. The role of the detectable agent is to facilitate detection of RNA or to allow observation of hybridized nucleic acid fragments (e.g., nucleic acid fragments hybridized to genetic probes in array-based analysis). Preferably, the detectable agent is selected so that it produces a measurable signal and the intensity of the signal is related to the amount of labeled nucleic acid present in the sample to be analyzed. In array-based analysis methods, the detectable agent is also preferably selected so that it produces a localization signal, thereby generating a spatial resolution of the signal at each point on the array.

[0233] Methods for labeling nucleic acid molecules are well known in the art. For a review of labeling protocols, label detection techniques, and the latest developments in the field, see, e.g., LJ Kricka, Ann. Clin. Biochem. 2002, 39: 114-129; RP van Gijlswijk et al., Expert Rev. Mol. Diagn. 2001, 1: 81-91; and S. Joos et al., J. Biotechnol. 1994, 35: 135-153. Standard nucleic acid labeling methods include: incorporation of radioactive reagents, direct attachment of fluorescent dyes (see, e.g., LM Smith et al., Nucleic Acids Research, 1985, 13:2399-2412) or enzymes (see, e.g., BA Connoly and P. Rider, Nucleic Acids Research, 1985, 13:4485-4502); chemical modification of nucleic acid fragments so that they can be detected immunochemically or by other affinity reactions (see, e.g., TR Broker et al., Nucleic Acids Research, 1978, 5:363-384; EA Bayer et al., Methods of Biochemical Analysis (see, e.g., BA Connoly and P. Rider, Nucleic Acids Research, 1985, 13:4485-4502); Biochem. Analysis), 1980, 26: 1-45; R. Langer et al., Proceedings of the National Academy of Sciences of the United States of America, 1981, 78: 6633-6637; R. W. Richardson et al., Nucleic Acids Research, 1983, 11: 6167-6184; DJ Brigati et al., Virology (Virol.) 1983, 126: 32-50; P. Tchen et al., Proceedings of the National Academy of Sciences of the United States of America, 1984, 81: 3466-3470; J. E. Landegent et al., Exp. Cell Res. Res. 1984, 15: 61-72; and AH Hopman et al., Experimental Cell Res. 1987, 169: 357-368); and enzyme-mediated labeling methods such as random priming, nick translation, PCR, and terminal transferase tailing (for a review of enzyme labeling, see, e.g., J. Temsamani and S. Agrawal, Mol. Biotechnol. 1996, 5: 223-232).

[0234] Any of a wide variety of detectable agents may be used in the practice of the present invention. Suitable detectable agents include, but are not limited to, various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (e.g., quantum dots, nanocrystals, phosphors, etc.), enzymes (e.g., those used in ELISA, i.e., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, digoxigenin, or other haptens and proteins for which antisera or monoclonal antibodies are available.

[0235] However, in some embodiments, expression levels are determined by detecting expression of a gene product (eg, a protein, such as a secreted protein), thereby eliminating the need to obtain a genetic sample (eg, RNA) from the sample.

[0236] E. Detection and / or Measurement of FSGM

[0237] Various methods can be used to measure the distance between feature vectors. Once the data is normalized, the distance can be obtained, for example, by calculating the mean square error, which can be obtained from the difference in the expression pattern of each gene measured in two different spectra (e.g., baseline FXN (-) and FXN replacement). Alternatively, the distance can be obtained by calculating the correlation coefficient or applying a t test.

[0238] As described in detail herein, many methods for determining RNA expression profiles have been described, including sequencing, hybridization, or amplification of sample RNA. In a specific embodiment of the present disclosure, the expression profile of the sample of the subject is determined, including obtaining or providing a biological sample from the subject, extracting RNA from the sample, generating corresponding cDNA, and detecting the expression profile by any one of sequencing, hybridization, or amplification. In some embodiments, the expression profile of the sample can be determined using qPCR. In some embodiments, the expression profile of the sample can be determined using Nanostring.

[0239] Detection of FXN sensitivity expression profile by sequencing can be performed using, for example, next generation sequencing (NGS), RNASeq, and any sequencing technology known to those skilled in the art.

[0240] Detecting the expression profile by hybridization comprises contacting the subject sample or a portion thereof with a probe or a set of probes that specifically hybridizes to the FSGMs (or transcripts thereof) disclosed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In one embodiment of the present disclosure, any one or more FSGMs of the present disclosure, or any combination thereof, may be contacted with the subject sample. For example, a specific probe encoding at least one of the gene transcripts of one or more FSGMs listed in Table 1 and / or Table 5, alone (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP), or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2), or any combination thereof, may be contacted with the subject sample. Thus, determining the expression profile of a sample from a subject treated with FXN replacement therapy by hybridization can comprise contacting the sample, or a portion thereof, with a probe that hybridizes to at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of a target nucleic acid thereof, the target nucleic acid being a transcript or corresponding cDNA of any of the FSGMs listed in Tables 1 and / or 5 (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP) alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2).

[0241] Detection of expression profiles by amplification involves, for example, polymerase chain reaction techniques, such as real-time polymerase chain reaction (RT-PCR), which comprises contacting a sample with forward and reverse primers for each of the transcripts of interest as exemplified in the examples below, and generating RT-PCR products. Optionally, the RT-PCR products are detected with specific or general probes, or a combination thereof, which facilitates their quantification. Thus, a signature of FXN induction can be determined by detecting FSGM transcripts in a sample. In one embodiment of the present disclosure, forward and reverse primers are used to detect FSGM transcripts.

[0242] In an alternative embodiment, the expression profile can be detected by FSGM protein products and analysis of the protein profile, which can be performed by protein detection methods using techniques involving specific antibodies or protein quantification / characterization techniques such as high performance liquid chromatography (HPLC), mass spectrometry-based techniques, gel-based techniques (e.g., differential gel electrophoresis), etc.

[0243] In another aspect, the present disclosure provides a composition for detecting an expression profile of FXN, the composition comprising at least one or more nucleotide sequences for detecting FSGM. In one embodiment of the present disclosure, the composition can be used to detect any of an alternative expression profile of FXN, a baseline FXN (-) expression profile, and / or a normal FXN expression profile. The composition may comprise at least one nucleotide sequence for detecting a gene transcript defined in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. The composition may comprise a nucleotide sequence for detecting two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, and / or up to all FSGMs presented in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. For example, the composition for detecting the FXN signature includes nucleotides for detecting one or more of Timpl, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6 (e.g., one or more of CTSS, BTG2, EGR1 and PTGS2), or any combination thereof. The nucleotide sequence may be DNA or an analog thereof, or RNA or an analog thereof. The nucleotide sequence may be complementary to at least a portion of the FSGM. The binding of the nucleotide sequence for detecting the FSGM will depend on the stringency of the reaction. The nucleotide sequence may be an oligonucleotide that may act as a probe or primer, and thus may include modifications compatible with its function. For example, a short oligonucleotide used as a probe may carry a label that enables its detection and quantification, such as a fluorescent label.

[0244] The present invention encompasses any suitable means, techniques and / or procedures for detecting and / or measuring the FSGM of the present invention. These methods are described in detail below.

[0245] 1. Detection of protein FSGM

[0246] The present invention encompasses any suitable method for detecting the polypeptide FSGM listed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. In certain embodiments, the detection method is an immunodetection method involving an antibody that specifically binds to one or more FSGMs listed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of Timpl, LRRK2, BASPl, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2). The steps of various suitable immunodetection methods are described in the scientific literature, such as Nakamura et al. (1987), which is incorporated herein by reference.

[0247] Generally, the immunological binding method comprises obtaining a sample suspected of containing a FSGM protein, peptide (e.g., a FSGM secreted protein or peptide) or antibody, and contacting the sample or a portion thereof with an antibody or protein or peptide according to the invention (as the case may be) under conditions effective to allow the formation of an immune complex.

[0248] Immunobinding methods include methods for detecting or quantifying the amount of reactive components in a sample, which methods require detecting or quantifying any immune complexes formed during the binding process. Here, the method will obtain a sample suspected of containing FSGM protein, peptide or corresponding antibody, and contact the sample with the antibody or encoded protein or peptide (as the case may be), and then detect or quantify the amount of immune complexes formed under specific conditions.

[0249] The selected biological sample or part thereof is contacted with the protein under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes). Generally, complex formation requires only the addition of the composition to the biological sample and the incubation of the mixture for a period of time long enough for the antibody to form an immune complex with any antigen present (i.e., bind to any antigen present). After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, is typically washed to remove any non-specifically bound antibody species, thereby detecting only those antibodies that are specifically bound within the primary immune complex.

[0250] In general, detection of immune complex formation is well known in the art and can be achieved by applying many methods. These methods are generally based on detection of markers or FSGMs (any radioactive, fluorescent, biological and enzyme tags or labels used as standard in the art). U.S. patents on the use of such markers include U.S. Patents No. 3,817,837; No. 3,850,752; No. 3,939,350; No. 3,996,345; No. 4,277,437; No. 4,275,149 and No. 4,366,241, each of which is incorporated herein by reference. Of course, as is known in the art, additional advantages can be found by using secondary binding ligands such as second antibodies and / or biotin / avidin ligand binding arrangements.

[0251] The protein used in the assay may itself be linked to a detectable label, wherein this label can then be simply detected, thereby allowing the amount of primary immune complexes in the composition to be determined.

[0252] Alternatively, the first added component bound to the primary immune complex can be detected by means of a second binding ligand with binding affinity to the encoded protein, peptide or corresponding antibody. In these cases, the second binding ligand can be connected to a detectable label. The second binding ligand itself is usually an antibody, so it can be referred to as a "secondary" antibody. The primary immune complex is contacted with the labeled secondary binding ligand or antibody under effective conditions and continues for a period of time sufficient to allow the formation of a secondary immune complex. The secondary immune complex is then usually washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complex is then detected.

[0253] Other methods include detecting a primary immune complex by a two-step approach. As described above, a second binding ligand (such as an antibody) having binding affinity to a coded protein, peptide or corresponding antibody is used to form a secondary immune complex. After washing, the secondary immune complex is contacted with a third binding ligand or antibody having binding affinity to the second antibody again under effective conditions and continues for a period of time sufficient to allow the formation of an immune complex (tertiary immune complex). The third ligand or antibody is connected to a detectable label, thereby allowing the detection of the tertiary immune complex thus formed. This system can provide signal amplification if this is desired.

[0254] The immunoassay methods of the present invention have obvious utility in monitoring the efficacy of FXN replacement therapy (e.g., treatment with FXN fusion proteins). Here, biological or clinical samples suspected of containing the encoded protein or peptide or the corresponding antibody are used. However, these embodiments also have application to non-clinical samples, such as in the titration of antigen or antibody samples, in the selection of fusion tumors, etc.

[0255] Specifically, the present invention encompasses the use of ELISA as a class of immunoassays. It is contemplated that the FSGMs of the present invention (including one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2)) will be used as immunogens in ELISA assays for monitoring FXN replacement therapy. In the simplest and most direct sense of an immunoassay, an immunoassay is a binding assay. Certain preferred immunoassays are various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical assays using tissue sections are also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blots, dot blots, FACS assays, and the like may also be used.

[0256] In an exemplary ELISA, antibodies that bind to FSGMs of the invention (including one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2)) are immobilized on a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. A test composition suspected of containing FSGM antigens (e.g., a clinical sample) is then added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can generally be achieved by adding a second antibody that is specific (i.e., specific for the target protein), the second antibody being linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by adding a second antibody and then adding a third antibody that has binding affinity for the second antibody, wherein the third antibody is linked to a detectable label.

[0257] In another exemplary ELISA, a sample suspected of containing FSGM antigen is fixed to the well surface and then contacted with the anti-biomarker antibody of the present invention. After binding and washing to remove non-specifically bound immune complexes, the bound antigen is detected. In the case where the initial antibody is linked to a detectable label, the immune complex can be detected directly. Similarly, the immune complex can be detected using a second antibody that has binding affinity for the first antibody, wherein the second antibody is linked to a detectable label.

[0258] Regardless of the format used, ELISAs have certain common features such as coating, incubation or binding, washing to remove non-specifically bound species, and detection of bound immune complexes. These are described below.

[0259] When coating the plate with an antigen or antibody, the wells of the plate are usually incubated with a solution of the antigen or antibody 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 surface of the wells is then "coated" with nonspecific proteins that are antigenically neutral to the test antiserum. These nonspecific proteins include bovine serum albumin (BSA), casein, or milk powder solutions. The coating allows the nonspecific adsorption sites on the fixed surface to be blocked, and therefore reduces the background caused by the nonspecific binding of the antiserum to the surface.

[0260] In ELISA, it may be more customary to use secondary or tertiary detection means rather than direct procedures. Thus, after the protein or antibody is bound to the wells, coated with a non-reactive material to reduce background, and washed to remove unbound material, the fixed surface is contacted with a 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 bound to a labeled tertiary antibody or third binding ligand.

[0261] The phrase "under conditions effective to allow immune complex (antigen / antibody) formation" means that the conditions preferably include diluting the antigen and antibody with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween. These added agents also help reduce nonspecific background.

[0262] "Suitable" conditions also mean that the incubation is at a temperature sufficient to allow effective binding and for a period of time sufficient to allow effective binding. The incubation step is generally about 1 hour to 2 hours to 4 hours at a temperature preferably about 25°C to 27°C, or can be overnight at about 4°C.

[0263] After all incubation steps in ELISA, the contacted surfaces are washed to remove uncomplexed material. Preferred washing procedures include washing with solutions such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the initially bound material and subsequent washing, the presence of even trace amounts of immune complexes can be determined.

[0264] In order to provide detection means, the second or third antibody will have a related label that allows detection. Preferably, the label will be an enzyme that produces color development when cultivated with an appropriate chromogenic substrate. Therefore, for example, it will be necessary to contact the first or second immune complex with an antibody bound to urease, glucose oxidase, alkaline phosphatase or catalase and cultivate together, the time period and conditions of the contact and cultivation are conducive to further forming immune complexes (e.g., at room temperature in a solution containing PBS such as PBS-Tween, cultivate 2 hours).

[0265] After incubation with the labeled antibody, and washing to remove unbound material, the amount of label is quantified by, for example, incubation with a chromogenic substrate such as urea and bromocresol purple. Quantification is then achieved by measuring the degree of color produced, for example, using a visible spectrum spectrophotometer.

[0266] Protein mass spectrometry methods and instruments can also be used to measure, quantify, detect and otherwise analyze proteins encoded by one or more FSGMs of the present invention (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP). Protein mass spectrometry refers to the application of mass spectrometry to study proteins. Although not intended to be limiting, two methods are generally used to characterize proteins using mass spectrometry. In the first method, the intact protein is ionized and then introduced into a mass analyzer. This method is referred to as the "top-down" strategy for protein analysis. The two main methods for ionizing whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). In the second method, a protease such as trypsin is used to enzymatically digest the protein into smaller peptides. These peptides are then introduced into a mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. The latter approach (also called "bottom-up" proteomics) therefore uses down-regulation of recognition at the peptide level to infer the presence of proteins.

[0267] The whole protein quality analysis of proteins encoded by one or more FSGMs of the present invention (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, TXNIP) can be performed using time-of-flight (TOF) MS or Fourier transform ion cyclotron resonance (FT-ICR). These two types of instruments are applicable due to their wide mass range and high mass accuracy in the case of FT-ICR. The most widely used instrument for peptide mass analysis is the MALDI time-of-flight instrument because it allows the acquisition of peptide mass fingerprints (peptide mass fingerprint; PMF) at a high step rate (about 10 seconds to analyze 1 PMF). Multistage quadrupole time-of-flight and quadrupole ion traps can also be used in this application.

[0268] Proteins encoded by one or more FSGMs of the present invention (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP) can also be measured in complex mixtures of proteins and molecules coexisting in biological culture media or samples, however, fractionation of the samples may be required and considered herein. It should be understood that the ionization of complex mixtures of proteins may result in the following situation, where more abundant proteins tend to "swamp" or suppress the signals of less abundant proteins in the same sample. In addition, due to the large number of mixture components, it may be difficult to interpret the mass spectrum of complex mixtures. Prior to mass spectrometry analysis, fractionation can be used first to separate any complex protein mixture. Two methods are widely used to fractionate proteins or their peptide products from enzymatic digestion. The first method fractionates the whole protein 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 cases, it may be necessary to combine two of these techniques. Any other suitable method known in the art for fractionating protein mixtures is also contemplated herein.

[0269] The gel spots identified on the 2D gel are usually attributable to a protein. If the identity of the protein is needed, then the in-gel digestion method is usually applied, wherein the protein spots of interest are excised and digested in a proteolytic manner. The peptide masses produced by the digestion can be determined by mass spectrometry using peptide mass fingerprinting. If this information does not allow clear identification of the protein, then a tandem mass spectrometry can be performed on its peptides for de novo sequencing.

[0270] Characterizing protein mixtures using HPLC / MS may also be referred to in the art as "shotgun proteomics" and MuDPIT (Multi-Dimensional Protein Identification Technology). The peptide mixture resulting from the digestion of the protein mixture is fractionated by one or two steps of liquid chromatography (LC). The eluate from the chromatography stage can be introduced directly into a mass spectrometer by electrospray ionization, or placed on a series of small spots for later mass analysis using MALDI.

[0271] MS can be used to identify proteins encoded by one or more FSGMs of the present invention (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP) using a variety of techniques (all of which are contemplated herein). Peptide mass fingerprinting uses the masses of proteolytic peptides as input to search a database containing predicted masses that would be generated by digestion of a series of known proteins. If a protein sequence in a reference list produces a large number of predicted masses that match the experimental values, there is some evidence that the protein was present in the original sample. It should be further appreciated that the development of methods and instrumentation for automated data-dependent electrospray ionization (ESI) tandem mass spectrometry (MS / MS) combined with microcapillary liquid chromatography (LC) and database searching has significantly increased the sensitivity and speed of identifying gel-separated proteins. Microcapillary LC-MS / MS has been successfully used to identify individual proteins on a large scale directly from a mixture without gel electrophoresis (Link et al., 1999; Opitek et al., 1997).

[0272] Several recent methods allow the quantification of proteins by mass spectrometry. For example, carbon ( 13 C) or nitrogen ( 15 A stable (e.g., non-radioactive) heavy isotope of N) can be incorporated into one sample, while another sample can be treated with the corresponding light isotope (e.g., 12 C and 14 N) labeling. The two samples are mixed before analysis. Peptides from different samples can be distinguished due to their mass differences. The ratio of their peak intensities corresponds to the relative abundance ratio of peptides (and proteins). The most popular isotope labeling methods are SILAC (stable isotope labeling with amino acids in cell culture), trypsin-catalyzed 18O labeling, ICAT (isotope-coded affinity tag), iTRAQ (isobaric tags for relative and absolute quantification). "Semi-quantitative" mass spectrometry can be performed without sample labeling. Typically, it is performed by MALDI analysis (in linear mode). Here, the peak intensity or peak area of ​​individual molecules (usually proteins) is related to the amount of protein in the sample. However, individual signals depend on the primary structure of the protein, the complexity of the sample, and the settings of the instrument. Other types of "label-free" quantitative mass spectrometry use spectrum counting (or peptide counting) of digested proteins as a means of determining relative protein amounts.

[0273] 2. Detection of nucleic acids corresponding to the FSGM protein

[0274] In certain embodiments, the present invention relates to detecting a nucleic acid FSGM, for example, a corresponding gene or mRNA of a FSGM of the present invention (e.g., a FSGM listed in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6, including one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP).

[0275] In various embodiments, the method of the present invention generally relates to determining the expression level of a group of genes in a biological sample. The determination of gene expression level in the practice of the method of the present invention can be carried out by any suitable method. For example, the determination of gene expression level can be carried out by detecting the expression of mRNA expressed by the gene of interest and / or by detecting the expression of a polypeptide encoded by the gene.

[0276] To detect nucleic acids encoding FSGM (e.g., Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP) of the present invention, any suitable method can be used, including but not limited to Southern blot analysis, Northern blot analysis, polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202 and 6,040,166; "PCR Protocols: A Guide to Methods and Applications", Innis et al. (eds.), 1990, Academic Press: New York), reverse transcriptase PCR (RT-PCR), quantitative PCR (qPCR), anchored PCR, competitive PCR (see, e.g., U.S. Pat. No. 5,747,251), rapid amplification of cDNA ends (RACE) (see, e.g., "Gene Cloning and Analysis: Current Innovations"). Innovations", 1997, pp. 99-115); ligase chain reaction (LCR) (see, e.g., EP 01 320 308), one-sided PCR (Ohara et al., Proc. Natl. Acad. Sci. USA, 1989, 86:5673-5677), in situ hybridization, Taqman-based analysis (Holland et al., Proc. Natl. Acad. Sci. USA, 1991, 88:7276-7280), differential display (see, e.g., Liang et al., Nucleic Acids Res., 1993, 21:3269-3275) and other RNA fingerprinting techniques, nucleic acid sequence-based amplification (NASBA) and other transcription-based amplification systems (see, e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527), Qβ replicase, strand displacement amplification (SDA), repair chain reaction (RCR), nuclease protection assays, subtraction-based methods, wait.

[0277] In other embodiments, the gene expression level of the FSGM of interest (e.g., Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP) can be determined by amplifying complementary DNA (cDNA) or complementary RNA (cRNA) generated from the mRNA and analyzing it using a microarray. Many different array configurations and methods for their production are known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436 ,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734 and 5,700,637). Microarray technology allows the simultaneous measurement of steady-state mRNA levels of a large number of genes. Currently widely used microarrays include cDNA arrays and oligonucleotide arrays. Analysis using microarrays is generally based on measuring the intensity of the signal received from labeled probes used to detect cDNA sequences of a sample that hybridize to nucleic acid probes immobilized at known locations on the microarray (see, e.g., U.S. Pat. Nos. 6,004,755; 6,218,114; 6,218,122; and 6,271,002). Array-based gene expression methods are known in the art and are described in numerous scientific publications as well as patents (see, e.g., M. Schena et al., Science, 1995, 270:467-470; M. Schena et al., Proc. Natl. Acad. Sci. USA, 1996, 93:10614-10619; J. J. Chen et al., Genomics, 1998, 51:313-324; U.S. Pat. Nos. 5,143,854; 5,445,934; 5,807,522; 5,837,832; 6,040,138; 6,045,996; 6,284,460 and 6,607,885).

[0278] The nucleic acid used as a template for amplification can be isolated from cells contained in a biological sample according to standard methods (Sambrook et al., 1989). The nucleic acid can be genomic DNA or partially isolated or whole cell RNA. In the case of using RNA, it may be necessary to convert the RNA into complementary cDNA. In one embodiment, the RNA is whole cell RNA and is directly used as a template for amplification.

[0279] Primer pairs 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 allow selective hybridization. After hybridization, the nucleic acid: primer complex is contacted with one or more enzymes that promote template-dependent nucleic acid synthesis. Multiple rounds of amplification (also referred to as "cycling") are performed until a sufficient amount of amplified product is produced. Then, the amplified product is detected. In certain applications, detection can be performed by visual inspection. Alternatively, detection can involve indirect identification of the product via chemiluminescence, radioactive scintigraphy incorporating radioactive or fluorescent labels, or even via a system using electrical or thermal pulse signals (Affymax technology; Bellus, 1994). After detection, the results seen in a given subject can be compared 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 conditions.

[0280] As defined herein, the term primer is intended to encompass any nucleic acid capable of initiating the synthesis of a nascent nucleic acid in a template-dependent process. Typically, a primer is an oligonucleotide of ten to twenty base pairs in length, but longer sequences may be employed. Primers may be provided in double-stranded or single-stranded form, but the single-stranded form is preferred.

[0281] Many template-dependent processes can be used to amplify nucleic acid sequences present in a given template sample. One of the most well-known amplification methods is the polymerase chain reaction (referred to as PCR), which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, and Innes et al., 1990, each of which is incorporated herein by reference in its entirety.

[0282] In PCR, two primer sequences complementary to the region on the relative complementary strand of the target nucleic acid sequence are prepared. Excessive deoxynucleoside triphosphates are added to the reaction mixture together with an archaeal dna polymerase (e.g., Taq polymerase). If the target nucleic acid sequence is present in the sample, primers will be bonded to the target nucleic acid and polymerase will cause the primers to extend along the target nucleic acid sequence by being added on the nucleotides. By raising and lowering the temperature of the reaction mixture, extension primers will dissociate from the target nucleic acid to form a reaction product, and excess primers will be bonded to the target nucleic acid and the reaction product, and the process is repeated.

[0283] Reverse transcriptase PCR amplification procedures can be performed to quantify the amount of mRNA amplified. Methods for reverse transcription of 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 December 21, 1990. Polymerase chain reaction methods are well known in the art.

[0284] Another amplification method is the ligase chain reaction ("LCR"), which is disclosed in European Application No. 320 308, which is incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of a target sequence, each pair will bind to the opposite complementary strands of the target so that they are adjacent. In the presence of a ligase, the two probe pairs will be connected to form a single unit. By temperature cycling, as in PCR, the bound connection unit dissociates from the target and then serves as a "target sequence" for connecting excess probe pairs. U.S. Patent No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

[0285] The Qβ replicase described in PCT Application No. PCT / US87 / 00880 can also be used as another amplification method in the present invention. In this method, an RNA replicating sequence having a region complementary to the target region is added to the sample in the presence of an RNA polymerase. The polymerase will replicate the replicating sequence which can then be detected.

[0286] Isothermal amplification methods using restriction endonucleases and ligases to achieve amplification of target molecules containing nucleotide 5'-[a-thio]-triphosphates in one strand of the restriction site can also be used to amplify nucleic acids in the present invention. Walker et al. (1992) is incorporated herein by reference in its entirety.

[0287] Chain displacement amplification (SDA) is another method for isothermal amplification of nucleic acids, which involves multiple rounds of chain displacement and synthesis (i.e., nick translation). A similar method called repair chain reaction (RCR) involves annealing several probes throughout the region for amplification, followed by a repair reaction, in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for ease of detection. A similar method is used in SDA. Target-specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, probes with 3' and 5' sequences of nonspecific DNA and an intermediate sequence of specific RNA are hybridized with the DNA present in the sample. After hybridization, the reactant is treated with RNase H, and the product of the probe is identified as a unique product released after digestion. The initial template is annealed to another cyclic probe and the reaction is repeated.

[0288] Other amplification methods described in GB application No. 2 202 328 and PCT application No. PCT / US89 / 01025 (each of which is incorporated herein by reference in its entirety) can be used according to the present invention. In the former application, "modified" primers are used in PCR (such as template and enzyme-dependent synthesis). Primers can be modified by labeling with a capture portion (e.g., biotin) and / or a detection portion (e.g., an enzyme). In the latter application, an excess of labeled probe is added to the sample. In the presence of the target sequence, the probe binds and cleaves in a catalytic manner. After cleavage, the target sequence is released intact to be bound by the excess probe. The cleavage of the labeled probe signals the presence of the target sequence.

[0289] Other nucleic acid amplification procedures covered include transcription-based amplification systems (TAS), including nucleic acid sequence-based amplification (NASBA) and 3SR. Guo et al. (1989); Gingeras et al., PCT application WO 88 / 10315 is incorporated herein by reference in its entirety. In NASBA, nucleic acids can be prepared for amplification by standard phenol / chloroform extraction, thermal denaturation of clinical samples, treatment with lysis buffer and separation of DNA and RNA from small spin columns, or extraction of RNA with guanidine chloride. These amplification techniques involve annealing primers with target-specific sequences. After polymerization, the DNA / RNA hybrid is digested with RNase H, and the double-stranded DNA molecules are thermally denatured again. In either case, single-stranded DNA is completely converted to double-stranded by adding a second target-specific primer, followed by polymerization. The double-stranded DNA molecule is then transcribed multiple times by a polymerase such as T7 or SP6. In an isothermal cycle reaction, RNA is reverse transcribed into double-stranded DNA and transcribed once more with a polymerase such as T7 or SP6. The resulting products (whether truncated or intact) are indicative of the target-specific sequence.

[0290] Davey et al., European Application No. 329 822 (incorporated herein by reference in its entirety) discloses a nucleic acid amplification process involving cyclic synthesis of single-stranded RNA ("ssRNA"), ssDNA and double-stranded DNA (dsDNA), which can be used according to the present invention. ssRNA is the first template of the first primer oligonucleotide, which is extended by reverse transcriptase (RNA-dependent DNA polymerase). RNA is then removed from the resulting DNA: RNA double helix by the action of ribonuclease H (RNase H, an RNase specific for RNA in a double helix with DNA or RNA). The resulting ssDNA is the second template of the second primer, which also includes a sequence homologous to the template at the 5' end of an RNA polymerase promoter (exemplified by T7 RNA polymerase). The primer is then extended by a DNA polymerase (exemplified by the larger "Klenow" fragment of Escherichia coli DNA polymerase 1) to produce a double-stranded DNA ("dsDNA") molecule having a sequence consistent with the sequence of the initial RNA between the primers and additionally having a promoter sequence at one end. This promoter sequence can be used by an appropriate RNA polymerase to prepare many RNA copies of the DNA. These copies can then be re-entered into the cycle, thereby causing very rapid amplification. In the case of a suitable selection enzyme, this amplification can be performed isothermally without adding enzyme in each cycle. Due to the cyclic nature of this process, the starting sequence can be selected to be in the form of DNA or RNA.

[0291] Miller et al., PCT Application WO 89 / 06700 (incorporated herein by reference in its entirety) discloses a nucleic acid sequence amplification scheme based on 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 generated from the resulting RNA transcripts. Other amplification methods include "race" and "single-sided PCR". Frohman (1990) and Obara et al. (1989) are each incorporated herein by reference in their entirety.

[0292] Methods based on the ligation of two (or more) oligonucleotides in the presence of a nucleic acid having the sequence of the resulting "di-oligonucleotide," thereby amplifying di-oligonucleotides, can also be used in the amplification step of the present invention. Wu et al. (1989) is incorporated herein by reference in its entirety.

[0293] The oligonucleotide probe or primer of the present invention can have any suitable length, depending on the specific analysis format and specific needs and the target sequence used. In a preferred embodiment, the length of the oligonucleotide probe or primer is at least 10 nucleotides (preferably, a length of 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) and it can be adapted to be particularly suitable for the selected nucleic acid amplification system and / or the hybridization system used. As well known in the art, longer probes and primers are also within the scope of the present invention. The present invention also encompasses primers with a length greater than 30, greater than 40, greater than 50 nucleotides and probes with a length greater than 100, greater than 200, greater than 300, greater than 500, greater than 800 and greater than 1000 nucleotides. Of course, longer primers have the disadvantage of being more expensive, and therefore primers with lengths between 12 and 30 nucleotides are generally designed and used in the art. As is well known in the art, probes ranging from 10 to greater than 2000 nucleotides in length can be used in the methods of the present invention. With respect to the percent consistency described above, non-specifically described sizes of probes and primers (e.g., 16, 17, 31, 24, 39, 350, 450, 550, 900, 1240 nucleotides, etc.) are also within the scope of the present invention.

[0294] In other embodiments, detection means using hybridization techniques, for example, where specific primers or probes are selected to anneal to the target FSGM of interest, and then selective hybridization is performed. As is generally known in the art, oligonucleotide probes and primers can be designed by considering their melting points for hybridization to the target sequence (see below and Sambrook et al., 1989, Molecular Cloning--A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory; Ausubel et al., 1994, A Laboratory Manual of Modern Molecular Biology, John Wiley & Sons, New York).

[0295] To be able to hybridize under the assay conditions of the present invention, 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 the FSGM of the present invention. The probes and primers of the present invention are those that hybridize to the FSGM homologs of the present invention under stringent hybridization conditions and those that hybridize to the FSGM homologs of the present invention under at least moderate stringency conditions. In certain embodiments, the probes and primers of the present invention have complete sequence identity to the FSGM of the present invention (gene sequence (e.g., cDNA or mRNA)). It is understood that other probes and primers can be easily designed and used in the present invention based on the present FSGM disclosed herein by using computer alignment and sequence analysis methods known in the art (see Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, ed., 2000).

[0296] 3. Antibodies and Labeling

[0297] In some embodiments, the present invention provides methods and compositions comprising labels for highly sensitive detection and quantification of FSGMs (e.g., Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP) of the present invention. Those skilled in the art will recognize that many strategies can be used to label target molecules to achieve their detection or identification in a particle mixture. The labels can be attached by any known means, including methods that utilize nonspecific or specific interactions of the labels with the target. The labels can provide a detectable signal or affect the mobility of the particles in an electric field. In addition, labeling can be achieved directly or through a binding partner.

[0298] In some embodiments, the label comprises a binding partner that binds to the FSGM of interest, wherein the binding partner is linked to a fluorescent moiety. The compositions and methods of the present invention can utilize highly fluorescent moieties, for example, moieties 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 having a diameter of no less than about 5 microns containing the moiety, and wherein the total energy of the laser directed at the spot is no greater than about 3 microjoules. Moieties suitable for use in the compositions and methods of the present invention are described in more detail below.

[0299] In some embodiments, the present invention provides a label for detecting a biomolecule, the label comprising a binding partner for the biomolecule attached to a fluorescent moiety, wherein the moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the fluorescent moiety, wherein the laser is focused on a point having a diameter of not less than about 5 microns containing the moiety, and wherein the total energy of the laser directed at the point is not greater than about 3 microjoules. In some embodiments, the moiety comprises a plurality of fluorescent entities, for example, 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. In some embodiments, the biomolecule is a protein or a small molecule. In some embodiments, the biomolecule is a protein. The fluorescent entity can be a fluorescent dye molecule. In some embodiments, the fluorescent dye molecule comprises at least one substituted indolium ring system, wherein the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. In some embodiments, the dye molecule is an Alexa Fluor molecule 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 molecule is an Alexa Fluor molecule selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680, or Alexa Fluor 700. In some embodiments, the dye molecule is an Alexa Fluor 647 dye molecule. In some embodiments, the dye molecule comprises a first type and a second type of dye molecule, for example, two different Alexa Fluor molecules, for example, wherein the first type and the second type of dye molecule have different emission spectra. The ratio of the number of dye molecules of the first type to the number of dye molecules of the second type can be, for example, 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, for example, an antibody.

[0300] In some embodiments, the present invention provides a label for detecting FSGM of an organism of the present invention, wherein the label comprises a binding partner for FSGM and a fluorescent moiety, wherein the moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the fluorescent moiety, wherein the laser is focused on a point having a diameter of not less than about 5 microns containing the moiety, and wherein the total energy of the laser directed at the point is not greater than about 3 microjoules. In some embodiments, the fluorescent moiety comprises a fluorescent molecule. In some embodiments, the fluorescent moiety comprises a plurality of fluorescent molecules, for example, 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 molecule comprises at least one substituted indolium ring system, wherein the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. In some embodiments, the fluorescent molecule is 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 fluorescent molecule is selected from the group consisting of: Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680, or Alexa Fluor 700. In some embodiments, the fluorescent molecule is an Alexa Fluor 647 molecule. In some embodiments, the binding partner comprises an antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0301] As used herein, the term "antibody" is a broad term and is used in its usual sense, including but not limited to referring to naturally occurring antibodies and non-naturally occurring antibodies, including, for example, single-chain antibodies, chimeric antibodies, bifunctional antibodies and humanized antibodies, and antigen-binding fragments thereof. An "antigen-binding fragment" of an antibody refers to the portion of an antibody that participates in antigen binding. The antigen binding site is formed by the amino acid residues of the N-terminal variable region ("V") of the heavy chain ("H") and the light chain ("L"). It should be understood that the epitope or region of the molecule selected to produce the antibody will determine its specificity for various forms of the molecule (if present) or the entirety (e.g., all or substantially all of the molecule).

[0302] The method of producing antibodies is recognized. Those skilled in the art will recognize that many procedures can be used to produce antibodies, for example, as described in Antibodies, A Laboratory Manual, Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, New York. Those skilled in the art will also understand that the binding fragments or Fab fragments of the simulated antibodies can also be prepared by various procedures from genetic information (Antibody Engineering: A Practical Approach (Borrebaeck, C., ed.), 1995, Oxford University Press, Oxford; J. Immunol., 149, 3914-3920 (1992)). Monoclonal and polyclonal antibodies and markers against molecules (e.g., proteins) are also commercially available (R and D Systems, Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abcam Inc., Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049 USA; BiosPacific, Emeryville, Calif.).

[0303] In some embodiments, the antibody is a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody.

[0304] Antibodies can be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be produced by cell culture techniques including production of monoclonal antibodies as described herein or by transfecting antibody genes into suitable bacterial or mammalian cell hosts to allow production of recombinant antibodies.

[0305] Monoclonal antibodies can be prepared using hybridoma methods, such as the technology of Kohler and Milstein (Eur. J. Immunol., 6:511-519, 1976) and modifications thereof. These methods involve the preparation of immortalized cell lines capable of producing antibodies with the desired specificity. Monoclonal antibodies can be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the antibody used in the disclosed method can be separated 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 using well-known methods as needed.

[0306] More specifically, monoclonal antibodies (MAbs) can be easily prepared using well-known techniques (such as those exemplified in U.S. Pat. No. 4,196,265, which is incorporated herein by reference). Typically, this technique involves immunizing a suitable animal with a selected immunogenic composition (e.g., a purified or partially purified expressed protein, polypeptide, or peptide). The immune composition is administered in a manner that effectively stimulates antibody-producing cells. The method for producing monoclonal antibodies (MAbs) generally begins along the same route as the method for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, it is also possible to use rabbits, sheep, or frog cells. The use of rats can provide certain advantages (Goding, 1986, pages 60-61), but mice are preferred, with BALB / c mice being the most preferred because this is the most commonly used and generally provides a higher percentage of stable fusions.

[0307] Antibodies can also be derived from recombinant antibody libraries that are based on amino acid sequences designed in silico and encoded by synthetically produced polynucleotides. Methods for designing and obtaining in silico produced 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).

[0308] Digestion of antibodies to produce antigen-binding fragments thereof can be performed using techniques well known in the art. For example, the proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer including a complete antigen-binding site. Pepsin is capable of cleaving IgG molecules to provide several fragments, including a "F(ab')2" fragment containing two antigen-binding sites. "Fv" fragments can be produced by preferential proteolytic cleavage of IgM, IgG or IgA immunoglobulin molecules, but are more commonly produced using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VL heterodimer that includes an antigen binding site that retains most 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)).

[0309] Antibody fragments that specifically bind to the protein FSGM disclosed herein can also be isolated from scFv libraries using known techniques, such as those described in US Pat. No. 5,885,793.

[0310] A wide variety of expression systems can be used in the art to produce antibody fragments, including Fab fragments, scFv, VL and VH. For example, expression systems of protokaryotic and eukaryotic origin can be used for large-scale production of antibody fragments. Particularly advantageous is the expression system that allows a large amount of antibody fragments to be secreted into the culture medium. Eukaryotic expression systems for large-scale production of antibody fragments and antibody fusion proteins have been described, which are based on mammalian cells, insect cells, plants, transgenic animals and lower eukaryotic organisms. For example, the cost-saving large-scale production of antibody fragments can be realized in yeast fermentation systems. The large-scale fermentation of these organisms is well-known in the art and is currently used for the large-scale production of some recombinant proteins.

[0311] In some cases, antibodies that bind to the protein FSGM employed in the methods of the invention are commercially available or can be obtained without undue experimentation.

[0312] In yet other embodiments, specifically, when an oligonucleotide is used as a binding partner to detect and hybridize with an mRNA FSGM or other nucleic acid-based FSGM, the binding partner (e.g., an oligonucleotide) may include a label, such as a fluorescent moiety or a dye. In addition, any binding partner (e.g., an antibody) of the present invention may 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 detector described herein). The term "fluorescent moiety" as used herein includes one or more fluorescent entities whose total fluorescence allows the moiety to be detected in a single molecule detector described herein. Therefore, the fluorescent moiety may include a single entity (e.g., a quantum dot or a fluorescent molecule) or multiple entities (e.g., multiple fluorescent molecules). It should be understood that when the term "moiety" as used herein refers to a group of fluorescent entities (e.g., multiple fluorescent dye molecules), each individual entity can be individually attached to a binding partner or the entities can be attached together, as long as the entities in the form of a group provide sufficient fluorescence to be detected.

[0313] Typically, the fluorescence of the moiety involves a combination of quantum efficiency and lack of photobleaching sufficient to allow the moiety to be detected above background levels in a single molecule detector with the consistency, accuracy, and analytical precision necessary for the desired detection limit. For example, in some embodiments, the fluorescence of the fluorescent moiety is such that it allows detection and / or quantification of a molecule (e.g., FSGM) with a detection limit of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.00001, or 0.000001 pg / mL in an instrument described herein and 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 some embodiments, the fluorescence of the fluorescent moiety is such that it allows detection and / or quantification of a molecule (e.g., FSGM) with 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 a coefficient of variation of less than about 10% in the instruments described herein. "Limit of detection" or LoD as those terms used herein include the lowest concentration at which a sample containing a molecule of the substance of interest can be identified, e.g., the first non-zero value. It can be defined by the zero rate of change and the slope of the standard curve. For example, the limit of detection of an assay can be determined by running a standard curve, determining the standard curve zero value, and increasing the value by 2 standard deviations. The concentration of the substance of interest that produces a signal equal to the value is the "lower limit of detection" concentration.

[0314] In addition, the moiety has properties consistent with its use in the selected analysis. In some embodiments, the analysis is an immunoassay in which a fluorescent moiety is attached to an antibody; the moiety must have properties such that it does not aggregate with other antibodies or proteins, or does not undergo aggregation consistent with the accuracy and precision required for the analysis. In some embodiments, a preferred fluorescent moiety is a fluorescent moiety (e.g., a dye molecule) having the following combination: 1) a high absorption coefficient; 2) a high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with labeling a molecule of interest (e.g., a protein) so that it can be analyzed using the analyzer and system of the present invention (e.g., without causing precipitation of the protein of interest, or precipitation of the protein attached to the moiety).

[0315] Any suitable fluorescent moiety may be used. Examples include, but are not limited to, Alexa Fluor dyes (Molecular Probes, Eugene, Oreg.). Alexa Fluor dyes are disclosed in U.S. Patent Nos. 6,977,305; 6,974,874; 6,130,101 and 6,974,305, which are incorporated herein by reference in their entirety. Some embodiments of the present invention utilize dyes selected 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 present invention utilize dyes selected 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 present invention utilize dyes selected 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 present 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.

[0316] In some embodiments, the fluorescent marker moiety used to detect FSGM in a sample using the analyzer system of the present invention is a quantum dot. Quantum dots (QDs), also known as semiconductor nanocrystals or artificial atoms, are semiconductor crystals that contain 100 to 1,000 electrons at any position and are in the range of 2 to 10 nm. The diameter of some QDs can be between 10 and 20 nm. QDs have a high quantum yield, which makes them particularly suitable for optical applications. QDs are fluorophores that fluoresce by forming excitons, which are similar to the excited state of traditional fluorophores, but have a much longer lifetime of up to 200 nanoseconds. This property provides QDs with low photobleaching. The energy level of the QD can be controlled by changing the size and shape of the quantum dots and the depth of the QD potential. One optical feature of small exciton QDs is coloration, which is determined by the size of the dots. The larger the dots, the redder the fluorescence or the closer it is to the red end of the spectrum. The smaller the dots, the bluer the fluorescence or the closer it is to the blue end. The energy of the fluorescence and therefore the band gap energy of the color is inversely proportional to the square of the size of the QD. Larger QDs have more energy levels that are more closely spaced, thus allowing the QDs to absorb photons containing less energy, i.e., photons closer to the red end of the spectrum. Because the emission frequency of the dots depends on the band gap, it is possible to control the output wavelength of the dots very precisely. In some embodiments, proteins detected with a single molecule analyzer system are labeled with QDs. In some embodiments, a single molecule analyzer is used to detect proteins labeled with one QD and filters are used to allow detection of different proteins at different wavelengths.

[0317] F. Isolated FSGM

[0318] 1. Isolation of FSGM polypeptide

[0319] One aspect of the invention relates to isolated FSGM proteins and biologically active portions thereof, including Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP, as well as polypeptide fragments suitable for use as immunogens to generate antibodies against FSGM proteins or fragments thereof. In one embodiment, native FSGM proteins can be isolated using standard protein purification techniques by an appropriate purification scheme. In another embodiment, proteins or peptides comprising intact FSGM proteins or fragments thereof are produced by recombinant DNA technology. As an alternative to recombinant expression, such proteins or peptides can be chemically synthesized using standard peptide synthesis techniques.

[0320] 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 was produced, or substantially free of chemical precursors or other chemicals when chemically synthesized. The phrase "substantially free of cellular material" includes protein preparations in which the protein is separated from cellular components of the cells from which the protein was isolated or recombinantly produced. Thus, proteins substantially free of cellular material include protein preparations having less than about 30%, 20%, 10% or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When a protein or biologically active portion thereof is produced recombinantly, it is also preferably substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10% or 5% of the volume of the protein preparation. When a 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 that participate in protein synthesis. Thus, such a protein preparation has less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

[0321] Biologically active portions of FSGM proteins include polypeptides comprising an amino acid sequence that is substantially identical to or derived from the amino acid sequence of FSGM proteins, comprising fewer amino acids than the full-length protein, and exhibiting at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif having at least one activity of the corresponding full-length protein. Biologically active portions of FSGM proteins of the present invention can be polypeptides of, for example, 10, 25, 50, 100 or more amino acids in length. In addition, 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 functional activities of the native form of the FSGM protein.

[0322] Preferred FSGM proteins are listed in Table 1 and / or Table 5 alone, or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6. Other suitable proteins are substantially identical to one of these sequences (e.g., at least about 40%, preferably 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) and retain the functional activity of the corresponding naturally occurring FSGM protein, but differ in amino acid sequence due to natural allelic variation or mutational induction.

[0323] In order to determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the sequence of the first amino acid or nucleic acid sequence to optimally align with the second amino or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at the position. Preferably, a global alignment is used to calculate the percent identity between the two sequences. Alternatively, a local arrangement is used to calculate the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / positions (e.g., overlapping positions) total × 100). In one embodiment, the lengths of the two sequences are the same. In another embodiment, the lengths of the two sequences are different.

[0324] Mathematical algorithms can be used to determine the percent identity between two sequences. A preferred non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altshull (1990) Proc. Nat. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altshull (1993) Proc. Nat. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into Altshull et al. (1990) J. Molecular Biol., 215:403-410 BLASTN and BLASTX programs. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the BLASTP program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. In order to obtain a gapped alignment for comparison purposes, a newer version of the BLAST algorithm called gapped BLAST can be used as described in Ortschul et al. (1997) Nucleic Acids Research, 25:3389-3402, which can perform gapped local alignments of programs BLASTN, BLASTP and BLASTX. Alternatively, PSI-Blast can be used to perform an iterative search that detects long-range relationships between molecules. When using BLAST, gapped BLAST and PSI-Blast programs, the default parameters of each program (e.g., BLASTX and BLASTN) can be used. See the NCBI website. Another preferred non-limiting example of a mathematical algorithm for sequence comparison is Myers and Miller, (1988) CABIOS 4:11-17 algorithm. This algorithm is incorporated into the ALIGN program (version 2.0), which is a part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, PAM120 weight residue tables, gap length penalties of 12 and gap penalties of 4 can be used. Another useful algorithm for identifying local sequence similarity and alignment regions is the FASTA algorithm as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA, 85:2444-2448. When using the FASTA algorithm to compare nucleotide or amino acid sequences, the PAM120 weight residue table can be used, for example, with a k-tuple value of 2.

[0325] The percent identity between two sequences can be determined, with or without gaps, using techniques similar to those described above. In calculating the percent identity, only exact matches are counted.

[0326] 2. Isolation of FSGM Nucleic Acids

[0327] One aspect of the invention relates to isolated nucleic acid molecules encoding FSGM proteins or portions 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, such as those nucleic acid molecules suitable for use as PCR primers to amplify specific products or mutations of FSGM nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of DNA or RNA generated using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0328] An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. In one embodiment, an "isolated" nucleic acid molecule (preferably a protein coding sequence) does not contain sequences that 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 that produces the nucleic acid. For example, in various embodiments, an isolated nucleic acid molecule may contain less than about 5kB, 4kB, 3kB, 2kB, 1kB, 0.5kB, or 0.1kB of nucleotide sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell that produces the nucleic acid. In another embodiment, an "isolated" nucleic acid molecule (e.g., a cDNA molecule) may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Nucleic acid molecules that are substantially free of cellular material include preparations with less than about 30%, 20%, 10%, or 5% heterologous nucleic acids (also referred to herein as "contaminating nucleic acids").

[0329] Can use standard molecular biology technology and the sequence information in the database record described herein to separate nucleic acid molecules of the present invention.Use all or part of this type of nucleotide sequence, can use standard hybridization and cloning technique to separate nucleic acid molecules of the present invention (for example, such as people such as Sambrook, Molecular Cloning: Experimental Guide, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, described in 1989).

[0330] Can use cDNA, mRNA or genomic DNA as template and suitable oligonucleotide primer to increase nucleic acid molecules of the present invention according to standard PCR amplification technique.The nucleic acid molecules amplified in this way can be cloned in a suitable vector and characterized by DNA sequence analysis.In addition, corresponding to all or part of Nucleotide of nucleic acid molecules of the present invention can be prepared by standard synthesis technology, for example, using automated DNA synthesizer.

[0331] In another preferred embodiment, the isolated nucleic acid molecule of the present invention comprises a nucleic acid molecule having a nucleotide sequence complementary to the nucleotide sequence of a FSGM nucleic acid or a nucleotide sequence of a nucleic acid encoding a FSGM protein. A nucleic acid molecule complementary to a given nucleotide sequence is a nucleic acid molecule sufficiently complementary to a given nucleotide sequence that it can hybridize with the given nucleotide sequence to form a stable double helix.

[0332] In addition, the nucleic acid molecules of the invention may comprise only a portion of a nucleic acid sequence, wherein the full-length nucleic acid sequence comprises a FSGM nucleic acid or encodes a FSGM protein. Such nucleic acids can be used, for example, as probes or primers. Probes / primers are typically used as one or more substantially purified oligonucleotides. Oligonucleotides typically comprise 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.

[0333] Probes based on the sequences of nucleic acid molecules of the present invention can be used to detect transcripts or genomic sequences corresponding to one or more FSGMs of the present invention. In certain embodiments, the probe hybridizes to a nucleic acid sequence spanning a splice junction. The probe comprises a labeling group, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor, attached thereto. Such probes can be used as part of a diagnostic test kit or a detection combination, which is used to identify cells or tissues that express or do not express proteins by measuring the level of nucleic acid molecules encoding proteins in a cell sample from a subject, such as detecting mRNA levels or determining whether a gene encoding a protein or its translation control sequence has mutated or deleted.

[0334] The present invention further encompasses nucleic acid molecules that differ in nucleotide sequence from nucleic acids encoding FSGM proteins (eg, proteins having a sequence provided in the sequence listing) due to the degeneracy of the genetic code and that therefore encode the same protein.

[0335] Those skilled in the art will appreciate that DNA sequence polymorphisms that cause amino acid sequence changes can exist within a population (e.g., a human population). Due to natural allele variation, this genetic polymorphism can exist between individuals within a population. An allele is one of a group of genes that alternately appear on a given genetic locus. In addition, it should be understood that there may also be DNA polymorphisms that affect RNA expression levels, which can affect the overall expression level of the gene (e.g., by affecting regulation or degradation).

[0336] As used herein, the phrase "allelic variant" refers to a nucleotide sequence or a polypeptide encoded by a nucleotide sequence occurring at a specified locus.

[0337] As used herein, the terms "gene" and "recombinant gene" refer to a nucleic acid molecule comprising an open reading frame encoding a polypeptide corresponding to the FSGM of the present invention. Such natural allelic variation can typically result in 1-5% variation 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 performed 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 result from natural allelic variation and do not alter functional activity are within the scope of the present invention.

[0338] In another embodiment, the 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 to a FSGM nucleic acid or a nucleic acid encoding a marker protein under stringent conditions. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences that are at least 60% (65%, 70%, preferably 75%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in sections 6.3.1-6.3.6 of Current Molecular Biology, John Wiley & Sons, New York (1989). A preferred, non-limiting example of stringent hybridization conditions is hybridization in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.

[0339] G. Fraxin replacement therapy

[0340] In some aspects, the methods provided by the present disclosure refer to determining a gene expression profile associated with a FXN replacement therapy. FXN replacement therapy involves administering a therapeutic compound capable of increasing FXN levels, e.g., a FXN replacement therapeutic agent, to a subject in need thereof. The present disclosure can contemplate and encompass many alternatives for delivering exogenous FXN or otherwise increasing FXN levels in a subject (e.g., a FXN-deficient subject). FXN therapy, e.g., a FXN replacement therapeutic agent can increase FXN protein delivery or be provided by delivering a nucleic acid (e.g., a nucleic acid encoding FXN (e.g., DNA or mRNA)) capable of increasing FXN levels, expression, or activity in a subject. FXN protein delivery can be delivery of full-length FXN or delivery of FXN fusion protein.

[0341] In some embodiments, FXN replacement therapy comprises administering to a subject in need thereof a nucleic acid capable of increasing the level, expression and / or activity of FXN in the subject. In some embodiments, the nucleic acid may be a nucleic acid encoding FXN, e.g., FXN mRNA. In some embodiments, the nucleic acid may be an antisense oligonucleotide, e.g., an antisense oligonucleotide for activating the expression of FXN protein as described in Li et al., Nucleic Acid Ther., 2018, 28(1):23-33 or Mikaeili et al., Scientific Reports 2018, 8:17217, each of which is hereby incorporated by reference herein in its entirety. In some embodiments, the nucleic acid may be an siRNA capable of activating the expression of FXN protein as described in, e.g., Shen et al., Bioorganic & Medicinal Chemistry Letters 2018, 28(17):2850-2855, the entirety of which is hereby incorporated by reference herein.

[0342] In some embodiments, a nucleic acid capable of increasing the level, expression, and / or activity of FXN in a subject (eg, a FXN-deficient subject) can be delivered to a subject via a viral vector.

[0343] In some embodiments, FXN replacement therapy comprises administering FXN fusion protein. As used herein, the term "FXN fusion protein" refers to FXN or FXN fragments fused to the full length or fragments of different proteins or fused to peptides. In some embodiments, the FXN fusion protein comprises a polypeptide comprising FXN, e.g., full-length hFXN (SEQ ID NO: 1) or mature hFXN (SEQ ID NO: 2). In some embodiments, the FXN fusion protein further comprises a cell penetrating peptide (CPP). Exemplary FXN fusion proteins are described, e.g., in US2021 / 0047378, the entire contents of which are incorporated herein by reference.

[0344] As used herein, the term "cell penetrating peptide" or "CPP" refers to a short peptide sequence, typically 5 to 30 amino acids long, that can facilitate the uptake of various molecular cargoes (such as proteins) into cells. In the context of the present invention, the CPP present in the FXN fusion protein facilitates the delivery of the FXN fusion protein into cells (e.g., recipient cells). The CPP can be polycationic, i.e., its amino acid composition contains a higher relative abundance of positively charged amino acids (such as lysine or arginine). The CCP can also be amphipathic, i.e., its sequence contains an alternating pattern of polar / charged amino acids and non-polar hydrophobic amino acids. The CPP can also be hydrophobic, i.e., contain only non-polar residues with low net charge or have hydrophobic amino acid groups that are critical for cellular uptake.

[0345] The CPP that may be included in the FXN fusion protein may be any CPP known to a person skilled in the art. For example, the CPP may be any CPP listed in the cell penetrating peptide CPPsite 2.0 database, the entire contents of which are hereby incorporated herein by reference. For example, a CPP suitable for use in the context of the present invention may be a cell penetrating peptide derived from a protein selected from the group consisting of HIV transcript peptide transactivator (HIV-TAT), galanin, melittin, transporter, penetratin, polyarginine, VP22, transporter, amphipathic peptides (such as MAP, KALA, ppTG20), proline-rich peptides, MPG-derived peptides, Pep-1 and oligomers, arginine-rich peptides, and calcitonin-derived peptides.

[0346] In some embodiments, CPP comprises a TAT protein domain, and the TAT protein domain comprises amino acids 47-57 of 86 amino acids of full-length HIV-TAT protein (the 11 amino acid peptides may also be referred to herein as "HIV-TAT"; SEQ ID NO: 4). In one embodiment, CPP consists of HIV-TAT (SEQ ID NO: 4). In some embodiments, CPP comprises amino acids 47-57 of 86 amino acids of full-length HIV-TAT protein, wherein methionine (12AA; "HIV-TAT+M") is added to the starting amino terminus: MYGRKKRRQRRR (SEQ ID NO: 5). Table 8 below lists the amino acid sequences of exemplary CPPs.

[0347] Table 8. Exemplary CPPs and corresponding sequences

[0348] SEQ ID NO. CPP Amino acid sequence 4 HIV-TAT YGRKKRRQRRR 5 HIV-TAT+M MYGRKKRRQRRR 6 Galanin GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS 7 Melittin <![CDATA[INLKALAALAKKIL-NH2]]> 8 Transporter GWTLNSAGYLLGKINLKALAALAKKIL 9 Cell-penetrating peptides RQIKIWFQNRRMKWKK 10 Polyarginine RRRRRRRRR 11 VP22 DAATATRGRSAASRPTERPRAPARSASRPRRPVE

[0349] In some embodiments, the CPP contained in the FXN fusion protein is HIV-TAT (SEQ ID NO: 4). In some embodiments, the FXN fusion protein contains full-length FXN, e.g., SEQ ID NO: 1, and HIV-TAT, e.g., SEQ ID NO: 4, as CPP.

[0350] In some embodiments, in the FXN fusion protein of the present disclosure, CPP can be fused to FXN, for example, full-length FXN via a linker to form a single polypeptide chain. Examples of FXN fusion proteins include TAT-FXN fusion proteins, in which TAT or a fragment of TAT can be directly or indirectly (via a linker) connected to the N-terminus or C-terminus of FXN. In a specific example, the linker can include the amino acid sequence GG.

[0351] In some aspects, the CPP (e.g., HIV-TAT) present in the FXN fusion protein of the present disclosure facilitates delivery of the FXN fusion protein to a cell (e.g., a cell that may be present in vitro, ex vivo, or in a subject). Once inside the cell, the FXN fusion protein may be processed by the cell machinery to remove the CPP (e.g., HIV-TAT) from FXN.

[0352] A specific example of a TAT-FXN fusion protein is referred to as an exemplary FXN fusion protein. An exemplary FXN fusion protein is a 24.9 kDa fusion protein that is currently being studied as an FXN replacement therapy to restore the functional level of FXN in the mitochondria of FRDA subjects. An exemplary FXN fusion protein includes an HIV-TAT peptide connected to the N-terminus of a full-length hFXN protein. The mechanism of action of an exemplary FXN fusion protein relies on the cell penetration ability of the HIV-TAT peptide to deliver the exemplary FXN fusion protein into cells, and is subsequently processed into mature hFXN after translocation into mitochondria. An exemplary FXN fusion protein is described in US 2021 / 0047378, the entire contents of which are hereby incorporated herein by reference. An exemplary FXN fusion protein comprises the following amino acid sequence (224 amino acids): MYGRKKRRQRRRGGMWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA (SEQ ID NO: 12).

[0353] FXN replacement can also be delivered by viral gene replacement, which can utilize retroviral, lentiviral and adeno-associated viral vectors, as well as adenovirus. Alternatively, FXN replacement therapy can be achieved by upregulating the endogenous mutant FXN gene, which depends on the number of GAA repeats expressed at different levels in carriers of the mutant FXN allele. Upregulation of the endogenous mutant FXN gene can be achieved by using, for example, antisense oligonucleotides or siRNA.

[0354] How to use H.FSGM

[0355] In some aspects, the disclosure provides methods for assessing FXN deficiency or monitoring or assessing the progression of FXN deficiency in a subject over time. In these methods, the amount of one or more FSGMs described herein (i.e., one or more FSGMs listed in Table 1 and / or Table 5, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP, alone or in combination with one or more of CTSS, BTG2, EGR1, and PTGS2) is assessed in a pair of samples obtained from a subject. The pair of samples may comprise a first sample obtained from the subject at an earlier time point and a second sample obtained from the subject at a later time point. It is understood that the methods of the present disclosure include obtaining and analyzing more than two samples (e.g., 3, 4, 5, 6, 7, 8, 9 or more samples) at regular or irregular intervals for assessing FSGM levels. Pairwise comparisons may be made between consecutive or non-consecutive subject samples. Trends in FSGM levels and rates of change in FSGM levels may be analyzed for any two or more consecutive or non-consecutive subject samples.

[0356] Thus, the present disclosure provides a method for assessing FXN deficiency in a subject, comprising (a) determining the FXN expression profile of one or more FSGMs in a sample obtained from a subject (e.g., a FXN-deficient subject or a subject suspected of FXN deficiency), wherein the one or more FSGMs comprise one or more markers defined in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6; and (b) comparing the FXN alternative expression profile determined in step (a) with a reference FXN expression profile of the one or more FSGMs; and making a determination regarding FXN deficiency in the subject based on the comparison in step (b). In one embodiment, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP. In another embodiment, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2). In one embodiment, a determination is made regarding the severity of FXN deficiency. In one embodiment, the one or more FSGMs comprise or consist of LRRK2 and Xpo6. In one embodiment, the one or more FSGMs comprise or consist of LRRK2, Xpo6, CTSS, BTG2, EGR1, and PTGS2.

[0357] i. Methods for monitoring the progression of FXN deficiency

[0358] The present disclosure further provides a method for monitoring the progression of FXN deficiency in a subject, the method comprising: (a) at a first time point, determining a first FXN expression profile of one or more FSGMs in a first sample obtained from a subject (e.g., a FXN-deficient subject or a subject suspected of FXN deficiency), wherein the one or more FSGMs comprise one or more markers defined in Table 1 and / or Table 5 alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6; (b) at a second time point later than the first time point, determining a second FXN expression profile of one or more FSGMs in a second sample obtained from the subject; and (c) comparing the second FXN expression profile to the first FXN profile; thereby monitoring FXN deficiency in the subject. In one embodiment, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP. In another embodiment, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP, alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6 (e.g., one or more of CTSS, BTG2, EGR1, and PTGS2). In one embodiment, the one or more FSGMs comprise or consist of LRRK2, Xpo6, CTSS, BTG2, EGR1, and PTGS2.

[0359] In some embodiments, a decrease in the amount of any one or more of Timp, LRRK2, BASP1, CD44, XPO6, Lgals3, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH, and / or Tmem70 in a sample obtained from a subject with FXN deficiency over time indicates that FXN deficiency is progressing.

[0360] In some embodiments, a decrease in the amount of Timp1 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of LRRK2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of BASP1 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of CD44 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of Xpo6 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, an increase in the amount of Lgals3 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of SOD2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of PSMB9 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of FTL in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of TXNIP in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of EGR1 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of CTSS in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of BTG2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of PTGS2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of LY96 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of Adgre1 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of PSMB8 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of C3ar1 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of TFRC in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of PLCL2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed.In some embodiments, a decrease in the amount of S100a4 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of Mapre1 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of CFH in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of Tmem70 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, a decrease in the amount of any two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more) of the aforementioned FSMGs in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed.

[0361] In some embodiments, an increase in the amount of Ube2v2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, an increase in the amount of Sec61g in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, an increase in the amount of Zdhhc13 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, an increase in the amount of Yipf5 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, an increase in the amount of Tpp2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, an increase in the amount of Ybx2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, an increase in the amount of Ptms in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has progressed. In some embodiments, an increase in the amount of any two or more (eg, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the foregoing FSMGs in the second FXN expression profile compared to the first FXN expression profile indicates progression of FXN deficiency.

[0362] In some embodiments, the amount of Timp1 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of LRRK2 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of BASP1 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of CD44 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of Xpo6 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of Lgals3 in the second FXN expression profile is not increased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of SOD2 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of PSMB9 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of FTL in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of TXNIP in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of EGR1 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of CTSS in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of BTG2 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of PTGS2 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of LY96 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of Adgre1 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of PSMB8 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of C3ar1 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of TFRC in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed.In some embodiments, the amount of PLCL2 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of S100a4 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of Mapre1 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of CFH in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of Tmem70 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the amount of Ube2v2 in the second FXN expression profile is not decreased compared to the first FXN expression profile, indicating that FXN deficiency has not progressed. In some embodiments, the absence of a decrease in the amounts of any two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) of the foregoing FSMGs in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has not progressed.

[0363] In some embodiments, the lack of an increase in the amount of Sec61g in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has not progressed. In some embodiments, the lack of an increase in the amount of Zdhhc13 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has not progressed. In some embodiments, the lack of an increase in the amount of Yipf5 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has not progressed. In some embodiments, the lack of an increase in the amount of Tpp2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has not progressed. In some embodiments, the lack of an increase in the amount of Ybx2 in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has not progressed. In some embodiments, the lack of an increase in the amount of Ptms in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has not progressed. In some embodiments, the absence of an increase in the amounts of any two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) of the foregoing FSMGs in the second FXN expression profile compared to the first FXN expression profile indicates that FXN deficiency has not progressed.

[0364] ii. Methods for evaluating the efficacy of FXN replacement therapy

[0365] The present disclosure provides methods for evaluating and / or monitoring the efficacy of FXN replacement therapy in a subject. The present invention further provides methods for determining whether a subject needs FXN replacement therapy or changing FXN replacement therapy, for example, determining whether FXN replacement therapy should be started, changed (e.g., increased or decreased dosage and / or frequency of administration) or stopped in a subject.

[0366] In some aspects, the present disclosure provides a method for evaluating the efficacy of FXN replacement therapy, the method comprising: (a) determining a baseline FXN(-) expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a sample obtained from an FXN-deficient subject before administration of the FXN replacement therapy; (b) determining a FXN replacement expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a sample obtained from an FXN-deficient subject after administration of the FXN replacement therapy; (c) comparing the FXN replacement expression profile determined in step (b) with the baseline FXN(-) expression profile determined in step (a); and (d) determining the efficacy of the FXN replacement therapy based on the comparison in step (c); wherein the one or more FSGMs are selected from the markers defined in Table 1 alone; or the markers defined in Table 1 and / or Table 5 or a combination thereof with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6. In one embodiment, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44CD44, Xpo6, Lgals3, SOD2 and PSMB9, FTL and TXNIP. In another embodiment, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44CD44, Xpo6, Lgals3, SOD2 and PSMB9, FTL and TXNIP alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6 (e.g., one or more of CTSS, BTG2, EGR1 and PTGS2). In one embodiment, the one or more FSGMs comprise or consist of LRRK2 and Xpo6. In one embodiment, the one or more FSGMs comprise or consist of LRRK2, Xpo6, CTSS, BTG2, EGR1 and PTGS2.

[0367] In some embodiments, an increase in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject after administration of a FXN replacement therapy as compared to the amount in a sample obtained from a FXN-deficient subject before administration of the FXN replacement therapy indicates that the FXN replacement therapy is effective.

[0368] In some embodiments, a decrease in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in a sample obtained from a FXN-deficient subject after administration of FXN replacement therapy as compared to the amount in a sample obtained from a FXN-deficient subject before administration of FXN replacement therapy indicates that the FXN replacement therapy is effective.

[0369] In some embodiments, the absence of an increase in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject after administration of a FXN replacement therapy as compared to the amount in a sample obtained from a FXN-deficient subject before administration of a FXN replacement therapy indicates that the FXN replacement therapy is ineffective.

[0370] In some embodiments, the absence of a decrease in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in a sample obtained from a FXN-deficient subject after administration of FXN replacement therapy as compared to the amount in a sample obtained from a FXN-deficient subject before administration of FXN replacement therapy indicates that the FXN replacement therapy is ineffective.

[0371] In some aspects, the present disclosure also provides a method for evaluating the efficacy of a fraxin (FXN) replacement therapy, comprising: (a) determining a FXN replacement expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a sample obtained from a FXN-deficient subject after administration of a FXN replacement therapy; (b) comparing the subject FXN replacement expression profile determined in step (a) with a reference FXN expression profile of one or more FSGMs; and (c) determining the efficacy of the FXN replacement therapy based on the comparison in step (b); wherein the one or more FSGMs are selected from the markers defined in Table 1 alone; or the markers defined in Table 1 and / or Table 5 or a combination thereof with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6. In one embodiment, the one or more FSGMs comprise one or any combination of Timp1, LRRK2, BASP1, CD44CD44, Xpo6, Lgals3, SOD2 and PSMB9, FTL and TXNIP. In another embodiment, the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44CD44, Xpo6, Lgals3, SOD2 and PSMB9, FTL and TXNIP alone or in combination with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6 (e.g., one or more of CTSS, BTG2, EGR1 and PTGS2). In one embodiment, the one or more FSGMs comprise or consist of LRRK2, Xpo6, CTSS, BTG2, EGR1 and PTGS2.

[0372] In some embodiments, the reference FXN expression profile is a baseline FXN(-) expression profile of one or more FSGMs. In some embodiments, wherein the reference FXN expression profile is a baseline FXN(-) expression profile of one or more FSGMs, an increase in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject after administration of FXN replacement therapy compared to the amount in the baseline FXN(-) expression profile (e.g., a FXN(-) expression profile of a sample obtained from a FXN-deficient subject before administration of FXN replacement therapy) indicates that the FXN replacement therapy is effective.

[0373] In some embodiments, wherein the reference FXN expression profile is a baseline FXN(-) expression profile of one or more FSGMs, a decrease in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2 and / or Ptms in a sample obtained from a FXN-deficient subject after administration of a FXN replacement therapy as compared to the amount in the baseline FXN(-) expression profile (e.g., a FXN(-) expression profile of a sample obtained from a FXN-deficient subject before administration of the replacement therapy) indicates that the FXN replacement therapy is effective.

[0374] In some embodiments, wherein the reference FXN expression profile is a baseline FXN(-) expression profile of one or more FSGMs, the absence of an increase in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject after administration of a FXN replacement therapy as compared to the amount in the baseline FXN(-) expression profile (e.g., a FXN(-) expression profile of a sample obtained from a FXN-deficient subject before administration of a FXN replacement therapy) indicates that the FXN replacement therapy is ineffective.

[0375] In some embodiments, wherein the reference FXN expression profile is a baseline FXN(-) expression profile of one or more FSGMs, the absence of a decrease in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2 and / or Ptms in a sample obtained from a FXN-deficient subject after administration of FXN replacement therapy as compared to the amount in the baseline FXN(-) expression profile (e.g., a FXN(-) expression profile of a sample obtained from a FXN-deficient subject before administration of replacement therapy) indicates that the FXN replacement therapy is ineffective.

[0376] In embodiments where the reference FXN expression profile FXN(-) is a baseline FXN(-) expression profile, the baseline FXN(-) expression profile of one or more FSGMs may be determined in a sample obtained from the same FXN-deficient subject administered FXN replacement therapy. Alternatively, the baseline FXN(-) expression profile of one or more FSGMs may be determined in a sample obtained from a different FXN-deficient subject (i.e., a different FXN-deficient subject not administered FXN replacement therapy). In some embodiments, the baseline FXN(-) expression profile is determined from samples obtained from a plurality of FXN-deficient subjects and may comprise, for example, one or an average of FSGMs based on measurements from a plurality of subjects.

[0377] In other embodiments, the reference FXN expression profile is a normal FXN expression profile.

[0378] In some embodiments, wherein the reference FXN expression profile is a normal FXN expression profile of one or more FSGMs, an amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject following administration of a FXN replacement therapy that is similar to the amount in the normal FXN expression profile indicates that the FXN replacement therapy is effective, and / or the dosage and / or administration frequency of the FXN replacement therapy should be maintained.

[0379] In some embodiments, wherein the reference FXN expression profile is a normal FXN expression profile of one or more FSGMs, an amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in a sample obtained from a FXN-deficient subject following administration of a FXN replacement therapy that is similar to the amount in the normal FXN expression profile indicates that the FXN replacement therapy is effective, and / or the dosage and / or frequency of administration of the FXN replacement therapy should be maintained. In some embodiments, wherein the reference FXN expression profile is a normal FXN expression profile of one or more FSGMs, a decrease in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject following administration of a FXN replacement therapy as compared to the amount in the normal FXN expression profile indicates that the FXN replacement therapy is ineffective, and / or the dose and / or frequency of administration of the FXN replacement therapy should be increased.

[0380] In some embodiments, wherein the reference FXN expression profile is a normal FXN expression profile of one or more FSGMs, an increase in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in a sample obtained from a FXN-deficient subject following administration of a FXN replacement therapy compared to the amount in the normal FXN expression profile indicates that the FXN replacement therapy is ineffective, and / or the dosage and / or frequency of administration of the FXN replacement therapy should be increased.

[0381] In the aforementioned methods, when it is determined that FXN replacement therapy is ineffective, changes can be made to the FXN replacement therapy regimen to improve the efficacy of the therapy or ultimately achieve efficacy. For example, the administration of a higher dose, the administration of the same or similar dose at a higher frequency, or the administration of a higher dose at a higher frequency can improve the results of FXN replacement therapy. Therefore, in some embodiments of the methods provided herein, the method further comprises determining to change the FXN replacement therapy regimen based on the efficacy obtained. In some embodiments of the methods provided herein, the method further comprises a step of recommending (e.g., to a healthcare provider) to change the FXN replacement therapy regimen based on the efficacy obtained. Changing the FXN replacement therapy regimen may include adjusting (e.g., increasing or decreasing) the dose of the FXN replacement therapy, adjusting (e.g., increasing or decreasing) the frequency of administration of the FXN replacement therapy, or both.

[0382] iii. Methods for monitoring treatment with FXN replacement therapy

[0383] In some aspects, the present disclosure provides a method for monitoring a subject treated with a FXN replacement therapy, the method comprising: (a) determining a first FXN replacement expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a first sample obtained from a FXN-deficient subject at a first time point after administering a FXN replacement therapy to the subject, wherein the one or more FSGMs comprise one or more markers defined in Table 1 alone; or markers defined in Table 1 and / or Table 5 or a combination thereof with one or more additional FSGMs listed in Tables 2, 3, 4 and / or 6; (b) determining a second FXN replacement expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a second sample obtained from the subject at a second time point later than the first time point; and (c) comparing the second FXN replacement expression profile to the first FXN replacement profile; thereby monitoring the subject treated with the FXN replacement therapy. In one embodiment, the one or more FSGMs comprise or consist of LRRK2 and Xpo6. In one embodiment, the one or more FSGMs comprise or consist of LRRK2, Xpo6, CTSS, BTG2, EGR1 and PTGS2.

[0384] In some embodiments, the method further comprises deciding or recommending to a healthcare provider to maintain the FXN replacement therapy regimen based on the comparison in step (c). In some embodiments, the method further comprises deciding or recommending to a healthcare provider to change the FXN replacement therapy regimen based on the comparison in step (c). In some embodiments, the method further comprises deciding to maintain, increase, or decrease the dosage and / or frequency of administration of the FXN replacement therapy based on the comparison in step (c). In some embodiments, the method further comprises recommending, for example, to a healthcare provider to maintain, increase, or decrease the dosage and / or frequency of administration of the FXN replacement therapy based on the comparison in step (c).

[0385] In some embodiments, the method further comprises continuing to administer the FXN replacement therapy regimen to the subject based on the comparison in step (c) (e.g., without changing the regimen, e.g., maintaining the dose and / or frequency of administration). In some embodiments, the method further comprises changing the FXN replacement therapy regimen based on the comparison in step (c). In some embodiments, the method further comprises administering an altered FXN replacement therapy regimen based on the comparison in step (c). In some embodiments, administering an altered FXN replacement therapy regimen comprises administering an increased dose and / or frequency of administration of a FXN replacement therapy, or administering a decreased dose and / or frequency of administration thereof.

[0386] In some embodiments, an increase in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in the second sample compared to the amount in the first sample indicates that the level of FXN in the subject is increased, and / or FXN replacement therapy is effective.

[0387] In some embodiments, a decrease in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in the second sample compared to the amount in the first sample indicates that the level of FXN in the subject is increased, and / or FXN replacement therapy is effective.

[0388] In some embodiments, an increase in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in the second sample compared to the amount in the first sample indicates that the level of FXN in the subject is increased, and / or FXN replacement therapy is effective.

[0389] In some embodiments, a decrease in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in the second sample compared to the amount in the first sample indicates that the level of FXN in the subject is increased, and / or FXN replacement therapy is effective.

[0390] In some embodiments, the absence of an increase in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in the second sample compared to the amount in the first sample indicates that the level of FXN in the subject remains constant, and / or FXN replacement therapy is ineffective.

[0391] In some embodiments, the lack of a decrease in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in the second sample compared to the amount in the first sample indicates that the level of FXN in the subject remains constant, and / or FXN replacement therapy is ineffective.

[0392] It will be appreciated that after administering FXN replacement therapy to a FXN-deficient subject, the FXN level achieved in the subject may reach a desired or target level of FXN, e.g., similar to the level of FXN present in a normal healthy subject, or similar to the level of FXN present in a hFXN heterozygote. In this case, it is advantageous to maintain the dosage and / or frequency of administration of the FXN replacement therapy. In addition, it is advantageous to maintain the dosage and / or frequency of administration of the FXN replacement therapy when the desired level of FXN in the subject is achieved and maintained constant over time. It will be further appreciated that after administering FXN replacement therapy to a FXN-deficient subject, the FXN level achieved in the subject may be higher than the desired or target level of FXN, e.g., higher than the level of FXN present in a normal healthy subject, or higher than the level of FXN present in a hFXN heterozygote. In this case, it is advantageous to reduce the dosage and / or frequency of administration of the FXN replacement therapy. Alternatively, the FXN level achieved in the subject may be lower than the desired or target level of FXN, e.g., lower than the level of FXN present in a normal healthy subject, or lower than the level of FXN present in a hFXN heterozygote. In such cases, it may be advantageous to increase the dose and / or frequency of administration of FXN replacement therapy.

[0393] Thus, in some embodiments, an increase in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH, and / or Tmem70 in the second sample compared to the amount in the first sample indicates that the dose and / or frequency of administration of FXN replacement therapy should be maintained or reduced.

[0394] In some embodiments, a decrease in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in the second sample compared to the amount in the first sample indicates that the dose and / or frequency of administration of FXN replacement therapy should be maintained or reduced.

[0395] In some embodiments, the absence of an increase in the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH, and / or Tmem70 in the second sample compared to the amount in the first sample indicates that the dose and / or frequency of administration of FXN replacement therapy should be maintained or increased.

[0396] In some embodiments, the absence of a decrease in the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in the second sample compared to the amount in the first sample indicates that the dose and / or frequency of administration of FXN replacement therapy should be maintained or increased.

[0397] iv. Methods for treating FXN deficiency

[0398] In some aspects, the present disclosure also provides a method for treating FXN deficiency in a subject, the method comprising: (a) determining a FXN expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a sample obtained from a FXN-deficient subject; (b) comparing the FXN expression profile of the sample to at least one other expression profile, the at least one other expression profile selected from the group consisting of: a normal FXN expression profile of one or more FSGMs, a baseline FXN(-) expression profile of one or more FSGMs, and a FXN replacement expression profile of one or more FSGMs; (c) classifying the FXN expression profile determined in step (a) as corresponding to a normal FXN expression profile, a baseline FXN(-) expression profile, or a FXN replacement expression profile; and (d) maintaining, initiating, or adjusting FXN replacement therapy based on the classification of the FXN expression profile of the sample; wherein the one or more FSGMs comprise one or more markers defined in Table 1 alone; or markers defined in Table 1 and / or Table 5, or a combination thereof with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6.

[0399] In one embodiment, the one or more FSGMs comprise or consist of LRRK2 and Xpo6. In one embodiment, the one or more FSGMs comprise or consist of LRRK2, Xpo6, CTSS, BTG2, EGR1, and PTGS2.

[0400] In some embodiments, adjusting the FXN replacement therapy comprises increasing the dosage of the FXN replacement therapy, decreasing its dosage, increasing its frequency of administration, or decreasing its frequency of administration, or any combination thereof.

[0401] In some embodiments, the FXN expression profile determined in step (a) is classified as corresponding to a baseline FXN (-) expression profile when the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject is decreased compared to said amount in a normal FXN expression profile.

[0402] In some embodiments, when the FXN expression profile in the sample is classified as a baseline FXN (-) expression profile, administration of FXN replacement therapy is initiated in the FXN-deficient subject. In some embodiments, when the FXN expression profile in the sample is classified as a baseline FXN (-) expression profile and the FXN-deficient subject has undergone FXN replacement therapy, the FXN replacement therapy regimen is changed, for example, the dose and / or frequency of administration of the FXN replacement therapy is increased.

[0403] In some embodiments, the FXN expression profile determined in step (a) is classified as corresponding to a normal FXN expression profile when the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject is not changed (e.g., not decreased) compared to the amount in a normal FXN expression profile.

[0404] In some embodiments, when the FXN expression profile in the sample is classified as a normal FXN expression profile, administration of FXN replacement therapy is not initiated in the FXN-deficient subject. In some embodiments, when the FXN expression profile in the sample is classified as a normal FXN expression profile and the FXN-deficient subject has undergone FXN replacement therapy, the FXN replacement therapy regimen is maintained (i.e., not changed), e.g., the dosage and / or frequency of administration of the FXN replacement therapy is maintained.

[0405] In some embodiments, the FXN expression profile determined in step (a) is classified as corresponding to a baseline FXN (-) expression profile when the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2 and / or Ptms in a sample obtained from a FXN-deficient subject is increased compared to said amount in a normal FXN expression profile.

[0406] In some embodiments, when the FXN expression profile in the sample is classified as a baseline FXN (-) expression profile, administration of FXN replacement therapy is initiated in the FXN-deficient subject. In some embodiments, when the FXN expression profile in the sample is classified as a baseline FXN (-) expression profile and the FXN-deficient subject has undergone FXN replacement therapy, the FXN replacement therapy regimen is changed, for example, the dose and / or frequency of administration of the FXN replacement therapy is increased.

[0407] In some embodiments, the FXN expression profile determined in step (a) is classified as corresponding to a normal FXN expression profile when the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2 and / or Ptms in a sample obtained from a FXN-deficient subject is not altered (e.g., not increased) compared to the amount in a normal FXN expression profile.

[0408] In some embodiments, when the FXN expression profile in the sample is classified as corresponding to a normal FXN expression profile, administration of FXN replacement therapy is not initiated in the FXN-deficient subject. In some embodiments, when the FXN expression profile in the sample is classified as a normal FXN expression profile and the FXN-deficient subject has undergone FXN replacement therapy, the FXN replacement therapy regimen is maintained (i.e., not changed), e.g., the dosage and / or frequency of administration of the FXN replacement therapy is maintained.

[0409] In some embodiments, adjusting the FXN replacement therapy comprises increasing the dosage of the FXN replacement therapy, decreasing its dosage, increasing its frequency of administration, decreasing its frequency of administration, or any combination thereof.

[0410] In some aspects, the present disclosure provides a method of treating FXN deficiency in a subject, comprising: (a) determining the FXN expression profile of one or more FSGMs in a sample from a subject with FXN deficiency; and (b) based on the FXN expression profile of the subject determined in step (a), recommending to a healthcare provider to administer FXN replacement therapy to the subject. In some aspects, the present disclosure also provides a method of treating FXN deficiency in a subject, comprising: (a) obtaining the FXN expression profile of one or more FSGMs in a sample obtained from a subject with FXN deficiency; and (b) administering FXN replacement therapy to the subject based on the FXN expression profile of the subject. In some embodiments, the one or more FSGMs comprise one or more markers defined in Table 1 alone; or markers defined in Table 1 and / or Table 5 or a combination thereof with one or more additional FSGMs listed in Tables 2, 3, 4, and / or 6.

[0411] In one embodiment, the one or more FSGMs comprise or consist of LRRK2 and Xpo6. In one embodiment, the one or more FSGMs comprise or consist of LRRK2, Xpo6, CTSS, BTG2, EGR1, and PTGS2.

[0412] In some embodiments, the method further comprises comparing the FXN expression profile of one or more FSGMs in the sample to at least one other expression profile, the at least one other expression profile selected from the group consisting of: a normal FXN expression profile of the one or more FSGMs, a baseline FXN(-) expression profile of the one or more FSGMs, and a FXN replacement expression profile of the one or more FSGMs. In some embodiments, the method further comprises classifying the FXN expression profile of the one or more FSGMs in the sample as corresponding to a normal FXN expression profile of the one or more FSGMs, a baseline FXN(-) expression profile of the one or more FSGMs, or a FXN replacement expression profile of the one or more FSGMs.

[0413] In some embodiments, when the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject is reduced compared to the amount in a normal FXN expression profile, a FXN replacement therapy is administered to the FXN-deficient subject, or a healthcare provider is advised to administer a FXN replacement therapy to the FXN-deficient subject. In some embodiments, when the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject is decreased compared to the amount in a normal FXN expression profile and the FXN-deficient subject is undergoing FXN replacement therapy, the FXN replacement therapy regimen is changed (e.g., the dosage and / or frequency of administration is increased), or a healthcare provider is advised to change the FXN replacement therapy regimen.

[0414] In some embodiments, when the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject is not reduced (e.g., no difference) compared to the amount in a normal FXN expression profile, then FXN replacement therapy is not administered to the FXN-deficient subject, or a healthcare provider is advised not to administer FXN replacement therapy to the subject. In some embodiments, when the amount of any one or more of Timp, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, EGR1, CTSS, BTG2, PTGS2, LY96, Adgre1, PSMB8, C3ar1, TFRC, PLCL2, S100a4, Maprel, CFH and / or Tmem70 in a sample obtained from a FXN-deficient subject is not reduced (e.g., no difference) compared to the amount in a normal FXN expression profile and wherein the FXN-deficient subject is undergoing FXN replacement therapy, then the FXN replacement therapy regimen is maintained (i.e., not changed), or a healthcare provider is advised to maintain the FXN replacement therapy regimen.

[0415] In some embodiments, when the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in a sample obtained from a FXN-deficient subject is increased compared to the amount in a normal FXN expression profile, a FXN replacement therapy is administered to the FXN-deficient subject, or a healthcare provider is advised to administer a FXN replacement therapy to the FXN-deficient subject. In some embodiments, when the amount of any one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and / or Ptms in a sample obtained from a FXN-deficient subject is increased compared to the amount in a normal FXN expression profile and the FXN-deficient subject is undergoing FXN replacement therapy, the FXN replacement therapy regimen is changed (e.g., the dosage and or frequency of administration is increased), or a healthcare provider is advised to change the FXN replacement therapy regimen.

[0416] In some emb...

Claims

1. A method for evaluating the efficacy of frataxin (FXN) replacement therapy, the method comprising: (a) determining a baseline FXN(-) expression profile of one or more FXN sensitivity genomic markers (FSGM) in a sample obtained from a FXN-deficient subject prior to administration of said FXN replacement therapy; (b) determining a FXN replacement expression profile of said one or more FXN sensitivity genomic markers (FSGM) in a sample obtained from said FXN-deficient subject following administration of said FXN replacement therapy; (c) comparing the FXN replacement expression profile determined in step (b) to the baseline FXN(-) expression profile determined in step (a); and (d) determining the efficacy of the FXN replacement therapy based on the comparison in step (c); Wherein the one or more FSGMs are selected from the markers defined in Table 1.

2. A method for evaluating the efficacy of FXN replacement therapy, the method comprising: (a) determining a FXN replacement expression profile of one or more FXN sensitivity genomic markers (FSGM) in a sample obtained from a FXN-deficient subject following administration of FXN replacement therapy; (b) comparing the FXN alternative expression profile determined in step (a) to a reference FXN expression profile of the one or more FSGMs; and (c) determining the efficacy of the FXN replacement therapy based on the comparison in step (b); Wherein the one or more FSGMs are selected from the markers defined in Table 1. 3 . The method of claim 2 , wherein the reference FXN expression profile is a baseline FXN(-) expression profile of the one or more FSGMs.

4. The method of claim 3, further comprising determining a baseline FXN(-) expression profile of the one or more FXN sensitivity genomic markers (FSGM) in a sample obtained from the FXN-deficient subject prior to administering the FXN replacement therapy. 5 . The method of claim 2 , wherein the reference FXN expression profile is a normal FXN expression profile of the one or more FSGMs.

6. The method of any one of claims 1 to 5, wherein the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP.

7. The method of claim 6, wherein the one or more FSGMs comprise any 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP.

8. The method of any one of claims 1 to 7, wherein the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP.

9. The method according to any one of claims 1 to 8, wherein the one or more FSGMs further comprise one or more FSGMs defined in Table 2 or Table 3.

10. The method of claim 9, wherein the one or more FSGMs defined in Table 2 or Table 3 include 1, 2, 3 or all 4 of CTSS, BTG2, EGR1 and PTGS2.

11. The method of claim 9, wherein the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, TXNIP, CTSS, BTG2, EGR1, and PTGS2.

12. The method of claim 9, wherein the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2.

13. The method of claim 9, wherein the one or more FSGMs comprise LRRK2, Xpo6, EGR1, BTG2, PTSG2, and LRKK2.

14. The method of claim 9, wherein the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, CTSS, BTG2, EGR1, and PTGS2.

15. The method of any one of claims 1 to 14, wherein at least one or more FSGMs are upregulated following treatment with FXN replacement therapy.

16. The method of claim 15, wherein the one or more FSGMs upregulated following treatment with FXN replacement therapy comprise one or more of LY96, LRRK2, Adgre1, Timp1, Xpo6, CD44, BASP1, PSMB8, C3ar1, TFRC, SOD2, FTL, TXNIP, PLCL2, S100a4, Maprel, PSMB9, CFH, and Tmem70.

17. The method of claim 16, wherein the one or more FSGMs upregulated following treatment with FXN replacement therapy comprise one or more of Timpl, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, CTSS, BTG2, EGR1, and PTGS2.

18. The method of any one of claims 1 to 14, wherein at least one or more FSGMs are downregulated following treatment with FXN replacement therapy.

19. The method of claim 18, wherein the one or more FSGMs that are downregulated following treatment with FXN replacement therapy comprise one or more of Lgals3, Ube2v2, Sec61g, Zdhhc13, Yipf5, Tpp2, Ybx2, and Ptms.

20. The method of claim 19, wherein the one or more FSGMs that are downregulated following treatment with FXN replacement therapy comprises Lgals3.

21. The method of any one of claims 1 to 20, wherein determining the FXN expression profile of one or more FSGMs comprises detecting the expression level of the one or more FSGMs.

22. The method of claim 21, wherein comparing the subject's FXN replacement expression profile to the baseline FXN(-) expression profile comprises comparing the expression level of the one or more FSGMs in the FXN replacement expression profile to the expression level of the corresponding one or more FSGMs in the baseline FXN(-) expression profile.

23. The method of any one of claims 1 to 5 and 21 to 22, wherein the FXN replacement therapy is determined to be effective when the expression level of one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, SOD2, PSMB9, FTL, TXNIP, CTSS, BTG2, EGR1 and PTGS2 is increased in the FXN replacement expression profile compared to the baseline FXN(-) expression profile.

24. The method of any one of claims 1 to 14 and 18 to 22, wherein the FXN replacement therapy is determined to be effective when the expression level of Lgals3 is reduced in the FXN replacement expression profile compared to the baseline FXN(-) expression profile.

25. The method of any one of claims 1 to 24, wherein determining the FXN expression profile of one or more FSGMs comprises determining a FXN feature vector of values ​​indicative of expression of the one or more FSGMs.

26. The method of claim 25, wherein determining the efficacy of the FXN replacement therapy comprises determining a first FXN feature vector of the subject's FXN replacement expression profile and a second FXN feature vector of the baseline FXN(-) expression profile, and determining a distance between the first feature vector and the second feature vector.

27. The method of claim 26, wherein determining the distance between the eigenvectors comprises determining a scalar product of the first eigenvector and the second eigenvector.

28. The method of any one of claims 25 to 27, further comprising determining a third eigenvector of a normal FXN expression profile of the FSGM of a healthy subject.

29. The method of claim 28, further comprising determining a distance between the second eigenvector and the third eigenvector.

30. The method of claim 29, further comprising determining a distance between the first eigenvector and the third eigenvector, and normalizing the distance between the first eigenvector and the third eigenvector relative to the distance between the second eigenvector and the third eigenvector.

31. The method of claim 30, further comprising using the normalized distance to determine the effectiveness of the FXN replacement therapy.

32. The method of any one of claims 1 to 31, wherein the FXN expression profile is determined by one or more of sequencing, hybridization, and amplification of RNA in the sample.

33. The method of any one of claims 1 to 31, wherein the FXN expression profile is determined by HPLC / UV-Vis spectroscopy, enzyme assay, mass spectrometry, NMR, immunoassay, ELISA, or any combination thereof.

34. The method of any one of claims 1 to 33, further comprising advising a healthcare provider to improve treatment of the FXN-deficient subject with the FXN replacement therapy based on the determination of the efficacy of the FXN replacement therapy.

35. The method of any one of claims 1 to 34, wherein the FXN-deficient subject suffers from Freidrich's Ataxia (FRDA).

36. The method of any one of claims 1 to 35, further comprising obtaining a sample from the FXN-deficient subject.

37. The method of claim 36, wherein the sample is selected from the group consisting of a cheek sample, a skin sample, a hair follicle, and a muscle biopsy sample.

38. The method of claim 37, wherein the sample is a buccal sample.

39. The method of claim 37, wherein the sample is a skin sample.

40. The method of any one of claims 36 to 39, wherein the sample from the FXN-deficient subject is obtained at least 15 days after the last administration of the FXN replacement therapy.

41. The method of claim 40, wherein the sample from the FXN-deficient subject is obtained 15 to 45 days after the last administration of the FXN replacement therapy.

42. A method of monitoring a subject being treated with FXN replacement therapy, the method comprising: (a) determining a first FXN replacement expression profile of one or more FXN sensitivity genomic markers (FSGMs) in a first sample obtained from a FXN-deficient subject at a first time point after administration of a FXN replacement therapy to the subject, wherein the one or more FSGMs comprise one or more markers defined in Table 1; (b) determining a second FXN alternative expression profile of the one or more FXN sensitivity genomic markers (FSGM) in a second sample obtained from the subject at a second time point later than the first time point; (c) comparing the second FXN replacement expression profile to the first FXN replacement profile; thereby monitoring treatment of the subject with the FXN replacement therapy.

43. The method of claim 42, further comprising deciding to maintain, increase, or decrease the dosage and / or administration frequency of the FXN replacement therapy based on the comparison in step (c).

44. The method of claim 42, wherein at least one dose of the FXN replacement therapy is administered to the subject between obtaining the first time point and the second time point.

45. The method of claim 42, wherein the FXN replacement therapy is not administered to the subject between obtaining the first time point and the second time point.

46. ​​A method for treating FXN deficiency in a subject, the method comprising: (a) determining the FXN expression profile of one or more FXN sensitivity genomic markers (FSGM) in a sample obtained from a subject with FXN deficiency; (b) comparing the FXN expression profile of the sample to at least one other expression profile, the at least one other expression profile selected from the group consisting of: a normal FXN expression profile of the one or more FSGMs, a baseline FXN(-) expression profile of the one or more FSGMs, and a FXN replacement expression profile of the one or more FSGMs; (c) classifying the FXN expression profile determined in step (a) as corresponding to a normal FXN expression profile, a baseline FXN(-) expression profile, or a FXN replacement expression profile; and (d) initiating or adjusting FXN replacement therapy based on said classification of said FXN expression profile of said sample; Wherein the one or more FSGMs comprise one or more markers defined in Table 1.

47. The method of claim 46, wherein adjusting the FXN replacement therapy comprises increasing the dosage of the FXN replacement therapy, decreasing its dosage, increasing its frequency of administration, or decreasing its frequency of administration.

48. The method of claim 46, wherein the FXN-deficient subject suffers from Friedreich's ataxia (FRDA).

49. A method of treating FXN deficiency in a subject, comprising: (a) determining the FXN expression profile of one or more FSGMs in a sample from a subject with FXN deficiency; and (b) based on the subject's FXN expression profile determined in step (a), recommending to a healthcare provider that FXN replacement therapy be administered to the subject.

50. A method of treating FXN deficiency in a subject, comprising: (a) obtaining a FXN expression profile of one or more FSGMs in a sample obtained from a subject with FXN deficiency; and (b) administering a FXN replacement therapy to the subject based on the FXN expression profile of the subject.

51. The method of claim 49 or 50, further comprising obtaining the sample from the FXN-deficient subject for determining the FXN expression profile of the one or more FSGMs.

52. A method for detecting one or more fraxin-sensitive genomic markers (FSGMs) in a sample from a subject deficient in fraxin (FXN), comprising The sample or a portion thereof is contacted with one or more reagents specifically for detecting the level of each of one or more FSGMs, wherein the one or more FSGMs comprise one or more FSGMs selected from Table 1, thereby detecting the FSGMs in the sample.

53. The method of claim 52, wherein the FXN-deficient subject is being treated or is planned to be treated with FXN replacement therapy.

54. The method of claim 52, further comprising obtaining the sample from the FXN-deficient subject.

55. The method of any one of claims 42 to 54, wherein the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP.

56. The method of any one of claims 42 to 54, wherein the one or more FSGMs comprise Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, PSMB9, FTL, and TXNIP.

57. The method of any one of claims 42 to 54, wherein the one or more FSGMs further comprises one of the plurality of FSGMs defined in Table 2 or Table 3.

58. The method of claim 57, wherein the one or more FSGMs defined in Table 2 or Table 3 comprise one or more of CTSS, BTG2, EGR1 and PTGS2.

59. The method of claim 58, wherein the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2.

60. The method of claim 59, wherein the one or more FSGMs comprise LRRK2, Xpo6, EGR1, BTG2, PTSG2, and LRKK2.

61. The method of any one of claims 42 to 60, wherein the FXN expression profile is determined by one or more of sequencing, hybridization, and amplification of RNA in the sample.

62. The method of any one of claims 42 to 60, wherein the FXN expression profile is determined by HPLC / UV-Vis spectroscopy, enzyme assay, mass spectrometry, NMR, immunoassay, ELISA, or any combination thereof.

63. The method of any one of claims 42 to 62, wherein the subject suffers from Friedreich's ataxia (FRDA).

64. The method of any one of claims 42 to 63, further comprising obtaining a sample from the FXN-deficient subject.

65. The method of claim 64, wherein the sample is selected from the group consisting of a cheek sample, a skin sample, a hair follicle, and a muscle biopsy sample.

66. The method of claim 64, wherein the sample is a cheek sample.

67. The method of claim 64, wherein the sample is a skin sample.

68. The method of any one of claims 64 to 67, wherein the sample from the FXN-deficient subject is obtained at least 15 days after the last administration of the FXN replacement therapy.

69. The method of claim 68, wherein the sample from the FXN-deficient subject is obtained 15 to 45 days after the last administration of the FXN replacement therapy.

70. The method of any one of claims 1 to 51, 53, and 55 to 69, wherein the FXN replacement therapy comprises administering a FXN fusion protein.

71. The method of claim 70, wherein the FXN fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO:

12.

72. A kit for detecting one or more fraxin-sensitive genomic markers (FSGMs) in a sample obtained from a subject with fraxin (FXN) deficiency, comprising at least one reagent specifically for detecting the level of each of the one or more FSGMs in the sample, wherein the one or more FSGMs include one or more FSGMs defined in Table 1; and a set of instructions for detecting the level of the one or more FSGMs in the sample from the subject.

73. The kit of claim 72, wherein the reagent is an antibody that binds to a frataxin-sensitive genomic marker (FSGM) or an oligonucleotide that is complementary to the corresponding mRNA of FSGM.

74. The kit of claim 72 or 73, wherein the one or more FSGMs comprise one or more of Timp1, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL, and TXNIP.

75. The kit according to any one of claims 72 to 74, further comprising at least one reagent specifically for detecting the level of one of the plurality of FSGMs defined in Table 2 or Table 3.

76. The kit of claim 75, wherein the one or more FSGMs defined in Table 2 or Table 3 comprise one or more of CTSS, BTG2, EGR1 and PTGS2.

77. The kit of claim 76, wherein the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2.

78. The method of claim 76, wherein the one or more FSGMs comprise LRRK2, Xpo6, EGR1, BTG2, PTSG2, and LRKK2.

79. A reagent detection combination for use in a method for monitoring or evaluating the efficacy of FXN replacement therapy, wherein the detection combination comprises at least two detection reagents, each of which is specifically used to detect at least one FSGM in a set of FXN-sensitive genomic markers (FSGMs), wherein the set of FSGMs comprises two or more markers defined in Table 1.

80. The detection combination of claim 79, wherein the panel FSGM comprises two or more of Timpl, LRRK2, BASP1, CD44, Xpo6, Lgals3, SOD2, PSMB9, FTL and TXNIP.

81. The detection combination according to claim 79 or 80, further comprising at least one detection reagent specifically used to detect at least one FSGM among the FSGMs defined in Table 2 or Table 3.

82. The detection combination of claim 81, wherein at least one of the FSGMs defined in Table 2 or Table 3 comprises one or more of CTSS, BTG2, EGR1 and PTGS2.

83. The detection combination of claim 82, wherein the one or more FSGMs comprise Xpo6, SOD2, CTSS, EGR1, BTG2, PTSG2, and LRKK2.

84. The method of claim 82, wherein the one or more FSGMs comprise LRRK2, Xpo6, EGR1, BTG2, PTSG2, and LRKK2.

85. A kit comprising a detection combination according to any one of claims 79 to 83 and a set of instructions for obtaining information related to fraxin (FXN) replacement therapy based on the level of one or more fraxin sensitivity genomic markers (FSGM).

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