Fratzin-sensitive markers for determining the efficacy of fratzin replacement therapy

Abstract

The present disclosure is based, at least in part, on the provision of a set of markers, also referred to herein as FXN-sensitivity gene marker (or FSGM), the expression level of each of which is positively or negatively correlated with the level of Frataxin (FXN) in a cell. Thus, these FSGMs can be used to determine, assess and / or monitor the efficacy of FXN replacement therapy in a subject.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 840,878, filed April 30, 2019, the entire contents of which are expressly incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list of electronic submissions in ASCII format, which are incorporated herein by reference in their entirety. The ASCII copy created on April 30, 2020, is named 130197-00320_SL.txt and has a size of 8,701 bytes. Background Technology

[0005] Mitochondrial diseases are a group of conditions caused by dysfunctional 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 ataxia (FRDA) is the most common inherited ataxia in humans, caused by a deficiency of the mitochondrial protein frataxin (FXN) (specifically, human ataxia protein (hFXN)). FRDA is a rare disease with an estimated incidence of 1 in 29,000, a carrier frequency of approximately 1 in 85, and about 4,000–5,000 cases reported in the United States. FRDA is a progressive, multisystemic disease that typically begins in mid-childhood. Patients experience a variety of symptoms, including progressive neurological and cardiac dysfunction. Other clinical manifestations may include scoliosis, fatigue, diabetes, vision impairment, and hearing loss. The inheritance is autosomal recessive and is primarily caused by a hereditary GAA triplet amplification in the first intron of both alleles of the hFXN gene. This triplet amplification causes transcriptional repression of the FRDA gene, resulting in very low levels of hFXN in patients. hFXN heterozygotes typically have about 50% of the normal hFXN levels but are phenotypically normal. hFXN levels of ~45-70 pg / μl and ~5-25 pg / μl in whole blood of heterozygotes and patients with FRDA have been shown to be stable over time, respectively (Plasterer et al., 2013).

[0007] Currently, there are no FDA-approved treatments for FRDA. Antioxidants and iron chelation are not very effective, and despite treatment, patients often experience progressive loss of motor control and death, with cardiomyopathy being the leading cause of death.

[0008] Protein replacement therapy is a recognized approach for metabolic diseases such as diabetes, lysosomal storage disorders, and hemophilia. Work in patient-derived cell and animal models has demonstrated that functional FXN replacement can correct or improve the FRDA disease phenotype. However, reliable and effective trials are needed in the field to measure the clinical response and efficacy of FXN replacement. Summary of the Invention

[0009] In one aspect, this disclosure is (at least in part) based on providing a set of biomarkers, also referred to herein as FXN-sensitive genomic biomarkers (or FSGMs), the levels of which are positively or negatively correlated with the levels of the FXN protein in cells. In some embodiments, the FSGMs of this disclosure are inversely regulated by FXN gene ablation followed by FXN protein replacement. Thus, the FSGMs of this disclosure are all associated with FXN deficiency in subjects inversely correlated with FXN replacement. The FSGMs disclosed herein have been found to be sensitive to FXN and are considered biomarkers of FXN replacement.

[0010] Therefore, as described herein, these FSGMs can be used to determine, evaluate, and / or monitor the efficacy of FXN replacement therapy in subjects. In some embodiments, analysis of one or more FSGM expression profiles in subjects before and after the administration or initiation of FXN replacement therapy can determine, evaluate, and / or monitor the efficacy of FXN replacement therapy in subjects. Based on the results of FSGM expression profile analysis, FXN replacement therapy in subjects can be adjusted, for example, to initiate, increase, decrease, or terminate FXN replacement therapy in subjects.

[0011] This disclosure provides a method for evaluating FXN replacement therapy by measuring the expression profile of one or more FSGMs (“baseline FXN(-) profile”) in samples from patients with FXN deficiency prior to treatment with FXN replacement therapy; measuring the expression profile of one or more FSGMs (“FXN replacement profile”) in samples from patients with FXN deficiency after treatment with FXN replacement therapy; comparing the baseline FXN(-) profile and the FXN replacement profile; and using the comparison to determine the efficacy of the FXN replacement therapy.

[0012] In one aspect of this disclosure, determining the FXN expression profile of FSGM includes identifying an FXN feature vector that indicates the expression of FXN-sensitive genomic markers. Regardless of whether the sample is from an FXN-healthy subject, an FXN-deficient patient, or an FXN-deficient patient following FXN replacement therapy, the FXN feature vector reflects the FXN expression profile status of the sample.

[0013] In another aspect, the present invention provides a method for evaluating the efficacy of coagulin (FXN) replacement therapy, the method comprising: (a) determining the FXN substitution expression profile of one or more FSGMs in a sample from a patient with FXN deficiency treated with FXN replacement therapy; (b) comparing the patient's FXN substitution expression profile with a baseline FXN(-) expression profile; and (c) using the comparison to determine the efficacy of the FXN replacement therapy; wherein the one or more FSGMs are those listed in Tables 2, 4, and / or Figure 3 Any one or more markers defined in [the document / reference].

[0014] In one embodiment, the method further includes determining the baseline FXN(-) expression profile of one or more FXN-sensitive genomic markers (FSGMs) in samples from patients who exhibited FXN deficiency prior to FXN replacement therapy.

[0015] In one embodiment, one or more FSGMs include at least one or any combination of more than one of mitochondrial genes, EGR family genes, insulin-like genes, ribosome depletion response genes, mitochondrial energy production genes, proteasome regulatory genes, ribosome functional genes, respiratory chain genes, myocardial development genes, macromolecular catabolism genes, translation initiation genes, mitochondrial component genes, oxidative phosphorylation genes, negative regulatory genes of macromolecular catabolism processes, or regulatory genes of apoptosis processes.

[0016] In another embodiment, one or more FSGMs include a gene encoding a secretory protein or a secretory protein (e.g., a secretory protein as defined in Table 2). In one embodiment, one or more FSGMs include one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1. In another embodiment, one or more FSGMs include CYR61.

[0017] In another embodiment, one or more FSGMs include one or more of NR4A1, PTP4A1, ATF3, BTG2, EGR1, EGR2, EGR3, CYR61, and ABCE1.

[0018] In another embodiment, one or more FSGMs include one or more of EGR1, EGR2, EGR3, and IGF1.

[0019] In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, and CYCS.

[0020] In another embodiment, one or more FSGMs include one or more of OPS2, VBP1, PSMA3, SLIRP, CUL2, DCUN1D1, UBE2D3, ZNRF1, RNF2, and LAMP2.

[0021] In another embodiment, one or more FSGMs include one or more of RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, RPL38, RPS27L, and ABCE1.

[0022] In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, and CYCS.

[0023] In another embodiment, one or more FSGMs include one or more of NR4A1, EGR1, EGR3, ADAMTS1, THBS1, SERPINE1, IGF1, PTGS2, and CYR61.

[0024] In another embodiment, one or more FSGMs include one or more of PSMA3, CUL2, UBE2D3, ZNRF1, RPS15A, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38.

[0025] In another embodiment, one or more FSGMs include one or more of ABCE1, RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38.

[0026] In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, CYCS, TMEM-126A, MAOA, and ABCE1.

[0027] In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, and MT-ATP8.

[0028] In another embodiment, one or more FSGMs include one or more of ABCE1, RPL26, RPL38, RPL10, RPL32, RPS15A, RPL24, RPL39, SLIRP, COPS2, DCUN1D1, RNF2, EGR1, BTG2, ATF3, PTGS2, IGF1, SERPINE1, and THBS1.

[0029] In another embodiment, one or more FSGMs include one or more of RPL26, THBS1, SERPINE1, IGF1, PTGS2, RPL10, RPS27L, CYCS, ATF3, BTG2, EGR1, EGR3, and CYR61.

[0030] In one implementation, one or more FSGMs are upregulated after treatment with FXN replacement therapy.

[0031] In one implementation, one or more FSGMs that are upregulated after treatment with FXN replacement therapy are mt-RNR1, mt-RNR2, ADNP, AI480526, C230034O21RIK, CCDC85B, CCDC85C, CTCFL, NRTN, PDE4A, PHF1, RPL37RT, SLC26A10, SNORD17, SUV420H2, WNK2, YAM1, or ZNRF1.

[0032] In one implementation, one or more FSGMs are downregulated after treatment with FXN replacement therapy.

[0033] In one implementation, one or more FSGMs that are downregulated after treatment with FXN replacement therapy are CYR61, mt-ATP6, mt-ATP8, mt-CO2, mt-CO3, mt-ND1, mt-ND2, mt-ND3 and mt-ND4, EGR1, EGR2, EGR3, IGF1, LAMP2 or SLIRP.

[0034] In another embodiment, determining the FXN expression profile of FSGM includes determining FXN feature vectors indicating the value of FSGM expression. In one embodiment, the method includes determining the efficacy of FXN replacement therapy using comparisons, including determining first and second FXN feature vectors of the patient's FXN replacement expression profile and baseline FXN(-) expression profile, respectively, and determining the distance between the feature vectors.

[0035] In one implementation, determining the distance between eigenvectors includes determining the scalar product of the first and second eigenvectors.

[0036] In one implementation, the method further includes determining a third feature vector of the normal FXN expression profile of the FSGM in a healthy subject.

[0037] In one implementation, the method further includes determining the distance between the second and third feature vectors.

[0038] In one embodiment, the method further includes determining the distance between the first and third feature vectors, and normalizing the distance between the first and third feature vectors relative to the distance between the second and third feature vectors.

[0039] In one implementation, the method further includes using a standardized distance to determine the efficacy of the FXN alternative therapy.

[0040] In one implementation, the expression profile is determined by any one of sequencing, hybridization, or amplification of the sample RNA.

[0041] In one implementation, the expression profile is determined by HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, or any combination thereof.

[0042] In another embodiment, the method of the present invention further includes adjusting the treatment with FXN replacement therapy when the FXN replacement therapy is indicated to be ineffective.

[0043] In one implementation, the patient suffers from Friedreich ataxia (FRDA).

[0044] In one embodiment, the method further includes obtaining a biological sample from a patient exhibiting FXN deficiency.

[0045] In one aspect, the present invention provides a composition for determining FSGM expression profiles, the composition comprising the components used in Tables 2 and 4 and / or Figure 3 The reagents for detecting at least one or more FSGMs as described herein.

[0046] In another aspect, the present invention provides a treatment method for mitochondrial diseases, the method comprising providing a sample from a subject suffering from FXN deficiency, measuring the FXN expression profile of one or more FXN-sensitive genomic markers (FSGMs) in the sample, comparing the FXN expression profile of the sample with at least one other expression profile selected from the group consisting of normal FXN expression profiles of one or more FSGMs, baseline FXN(-) expression profiles of one or more FSGMs, and FXN alternative expression profiles of one or more FSGMs, classifying the sample FXN expression profile into corresponding to a normal FXN expression profile, a baseline FXN(-) expression profile, or an FXN alternative expression profile, and initiating, increasing, or decreasing the dose of FXN alternative therapy administered to the subject based on the classification of the sample FXN expression profile.

[0047] In another aspect, the present invention provides a treatment for mitochondrial diseases, the method comprising determining the expression of one or more FXN-sensitive genomic markers (FSGMs) in samples from individuals suffering from FXN deficiency, wherein the one or more FSGMs are those listed in Tables 2, 4, and / or Figure 3 The expression of any one or more biomarkers defined in the literature, and the initiation, increase or decrease of the dose of FXN replacement therapy administered to the subject based on the expression of one or more of the aforementioned FSGMs.

[0048] In one implementation, the method includes providing or obtaining a sample from a subject suffering from FXN deficiency.

[0049] In one implementation, the mitochondrial disease is Friedreich ataxia (FRDA).

[0050] In another embodiment, one or more FSGMs include secretory proteins, such as those defined in Table 2. In one embodiment, one or more FSGMs include one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1. In another embodiment, one or more FSGMs include CYR61.

[0051] In one embodiment, one or more FSGMs include one or more of NR4A1, PTP4A1, ATF3, BTG2, EGR1, EGR2, EGR3, CYR61, and ABCE1.

[0052] In another embodiment, one or more FSGMs include one or more of EGR1, EGR2, EGR3, and IGF1.

[0053] In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, and CYCS.

[0054] In another embodiment, one or more FSGMs include one or more of OPS2, VBP1, PSMA3, SLIRP, CUL2, DCUN1D1, UBE2D3, ZNRF1, RNF2, and LAMP2.

[0055] In another embodiment, one or more FSGMs include one or more of RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, RPL38, RPS27L, and ABCE1.

[0056] In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, and CYCS.

[0057] In another embodiment, one or more FSGMs include one or more of NR4A1, EGR1, EGR3, ADAMTS1, THBS1, SERPINE1, IGF1, PTGS2, and CYR61.

[0058] In another embodiment, one or more FSGMs include one or more of PSMA3, CUL2, UBE2D3, ZNRF1, RPS15A, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38.

[0059] In another embodiment, one or more FSGMs include one or more of ABCE1, RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38.

[0060] In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, CYCS, TMEM-126A, MAOA, and ABCE1.

[0061] In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, and MT-ATP8.

[0062] In another embodiment, one or more FSGMs include one or more of ABCE1, RPL26, RPL38, RPL10, RPL32, RPS15A, RPL24, RPL39, SLIRP, COPS2, DCUN1D1, RNF2, EGR1, BTG2, ATF3, PTGS2, IGF1, SERPINE1, and THBS1.

[0063] In another embodiment, one or more FSGMs include one or more of RPL26, THBS1, SERPINE1, IGF1, PTGS2, RPL10, RPS27L, CYCS, ATF3, BTG2, EGR1, EGR3, and CYR61.

[0064] In another aspect, the present invention provides a kit for detecting one or more FSGMs in biological samples from subjects exhibiting or receiving FXN deficiency treatment, comprising one or more reagents for measuring the levels of one or more FSGMs in the biological samples from said subjects, wherein said one or more FSGMs include those selected from Tables 2, 4 and / or Figure 3 One or more FSGMs, and a set of instructions for measuring the level of said FSGM.

[0065] In one embodiment, the reagent is an antibody that binds to one or more symbiotic protein-sensitive genomic markers (FSGMs) or an oligonucleotide complementary to the corresponding mRNA of one or more FSGMs.

[0066] In one embodiment, one or more FSGMs include secretory proteins, such as those defined in Table 2. In one embodiment, one or more FSGMs include one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1. In another embodiment, one or more FSGMs include CYR61.

[0067] In another aspect, the present invention provides a panel for monitoring or evaluating the efficacy of FXN (extra-acid protein) replacement therapy, the panel comprising one or more detection reagents, wherein each detection reagent is specific for the detection of one or more FXN-sensitive genomic markers (FSGMs), wherein the one or more FSGMs include those selected from Tables 2, 4 and / or Figure 3 One or more markers.

[0068] In one implementation, the coagulant-sensitive genomic marker (FSGM) includes at least two or more FSGMs.

[0069] In one embodiment, one or more FSGMs include secretory proteins, such as those defined in Table 2. In one embodiment, one or more FSGMs include one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1. In another embodiment, one or more FSGMs include CYR61.

[0070] In another aspect, the present invention provides a kit comprising the detection plate of the present invention and a set of instructions for obtaining information related to coagulant (FXN) replacement therapy based on the levels of one or more coagulant sensitivity genomic markers (FSGM).

[0071] In another aspect, the present invention provides a method for detecting one or more FSGM-sensitive genomic biomarkers in a biological sample by contacting a biological sample or a portion thereof from a patient suffering from FXN deficiency with one or more detection reagents specific for the detection of one or more FSGMs, optionally wherein the patient is receiving FXN replacement therapy, and wherein the one or more FSGMs include those selected from Tables 2, 4 and / or Figure 3 One or more FSGMs. In one embodiment, the sample is contacted with one or more detection reagents that are specific for the detection of one or more FSGMs. In another embodiment, a portion of the sample (e.g., isolated or purified nucleic acids or proteins) may be contacted with one or more detection reagents that are specific for the detection of one or more FSGMs.

[0072] In one embodiment, one or more FSGMs include secretory proteins, such as those defined in Table 2. In one embodiment, one or more FSGMs include one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1. In another embodiment, one or more FSGMs include CYR61.

[0073] In this discussion, unless otherwise stated, adjectives (e.g., “substantially” and “about”) modifying one or more features of an embodiment of this disclosure should be understood as limiting the condition or feature to the extent that it is operationally acceptable for the embodiment to which it is intended for use. Unless otherwise specified, the word “or” in the specification and claims is considered inclusive (having the meaning of and / or) rather than exclusive and indicates at least one or any combination of the matters it connects.

[0074] This summary is provided to introduce the selection of concepts in a simplified form, which will be further described in the detailed description below. This summary is not intended to indicate key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter. Attached Figure Description

[0075] Non-limiting examples of embodiments of this disclosure are described with reference to the accompanying drawings listed after this paragraph. Identical features appearing in more than one drawing are generally labeled with the same reference numerals as they appear in all the drawings in which they appear.

[0076] Figure 1 The diagram shows clusters generated by string analysis of predicted interactions of protein products from 85 FXN-sensitive genomic markers (FSGMs) from Table 2, according to embodiments of this disclosure.

[0077] Figure 2 These are photographs showing representative protein blots of FXN levels in normal dermal fibroblasts (Norm_#23971) and fibroblasts derived from FDRA patients (FA_#03816 and FA_#68).

[0078] Figure 3 This is a graph showing the baseline FXN(-) expression profiles in vector-treated FDRA-derived fibroblasts FA-GM03816, FA-GM04078, FA-4654 and FA-4675 according to embodiments of the present disclosure, and compared with normal fibroblast controls N-GM07522 and N-GM23971.

[0079] Figure 4A This is a chart showing gene expression analysis in fibroblasts from FRDA patients, which indicates that EGR1, EGR2, EGR3, and IGF1 are generally upregulated in fibroblasts from FRDA patients compared to normal fibroblasts. Figure 4BThis is a graph showing the effect of the FXN fusion protein described in Example 1 on the expression of hFXN, EGR1, EGR2, EGR3 and IGF1 in FDRA-derived fibroblasts FA-68, compared to vector-treated cells, according to embodiments of the present disclosure.

[0080] Figure 5 This is a schematic diagram of a process for evaluating FXN-induced signatures according to an embodiment of this disclosure.

[0081] Figure 6 These are images showing the amount of FXN protein in FXN knockdown (KD) HK293 clones A2 and A6, as well as in the disordered control clone. Figure 6 A table also shows the quantitative results of the amount of FXN protein in the protein blot.

[0082] Figure 7 This is a bar chart showing the amount of CYR61 protein in the culture medium from FXN-KD and disordered control HEK293 cells treated with the vector (black bar) or FXN fusion protein (gray bar).

[0083] Figure 8 This is a bar graph showing the amount of CYR61 protein in the culture medium of disordered control cells (KD-SRBL+V) transfected with empty vector; disordered control cells (SRBL5+hFXN) transfected with hFXN; hFXN-KD cells (KD-FXN+V) transfected with empty vector; and hFXN-KD cells (KD-FXN+hFXN) transfected with hFXN.

[0084] Figure 9 This is a bar chart showing the amount of FXN protein in total cellular proteins in WT mouse ES clones and homozygous mouse ES clone B9-46 treated with control or FXN knockout-inducing reagents (knockout agents).

[0085] Figure 10A This is a bar chart showing the amount of CYR61 expressed in mouse ES B9 cells treated with either a control reagent or a reagent that induces FXN gene knockdown. Figure 10B This is a bar graph showing the amount of CYR61 protein secreted from the culture medium of mouse ES B9 cells treated with either a control reagent or a reagent that induces FXN gene knockdown. Detailed Implementation

[0086] A. Overview

[0087] In one aspect, this disclosure is based (at least in part) on providing a set of biomarkers, also referred to herein as FXN-sensitive genomic biomarkers (or FSGMs), the levels of which are positively or negatively correlated with the levels of the FXN protein in cells. In some embodiments, the FSGMs of this disclosure are inversely regulated by FXN gene ablation followed by FXN protein replacement. Thus, the FSGMs of this disclosure are all associated with FXN deficiency in subjects and inversely correlated with FXN replacement. The FSGMs disclosed herein have been found to be sensitive to FXN and are considered biomarkers of FXN replacement. Therefore, as described herein, these FSGMs can be used to determine and / or monitor the efficacy of FXN replacement therapy in subjects. In one embodiment, the FSGMs include those selected from Tables 2, 4, and / or Figure 3 One or more biomarkers. In one embodiment, the FSGM includes secretory proteins, such as those defined in Table 2. In one embodiment, the FSGM includes one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1. In another embodiment, the FSGM includes CYR61.

[0088] In some implementations, analysis of one or more FSGM expression profiles before and after the administration or initiation of FXN replacement therapy in subjects can determine, evaluate, and / or monitor the efficacy of FXN replacement therapy in subjects. Based on the results of FSGM expression profile analysis, FXN replacement therapy in subjects can be adjusted, for example, to initiate, increase, decrease, or terminate FXN replacement therapy in subjects.

[0089] B. Definition

[0090] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. The following references (which are incorporated herein by reference in their entirety) provide general definitions of many terms used in this invention (unless otherwise defined herein): Singleton et al., Dictionary of Microbiology and Molecular Biology (2 nd ed.1994);The Cambridge Dictionary ofScience and Technology(Walker ed.,1988);The Glossary of Genetics,5 thEd., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, the Harper Collins Dictionary of Biology (1991). Generally, the steps and procedures described herein or inherent in molecular biology methods are common practices in the field. Such standard techniques can be found in reference manuals, such as Sambrook et al. (2000, Molecular Cloning—A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories); and Ausubel et al. (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0091] Unless otherwise defined, the following terms may have the meanings assigned to them below. However, it should be understood that other meanings known or understood by those skilled in the art are also possible and within the scope of this invention.

[0092] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “one,” and “the” include plural references. All technical and scientific terms used herein have the same meaning.

[0093] Unless specifically stated or obvious from the context, as used herein, the term "approximately" should be understood as falling within the normal tolerance range in the field, such as within 2 standard deviations of the mean. "Approximately" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context otherwise requires, all numerical values ​​provided herein may be modified by the term "approximately".

[0094] As used herein, the term "amplification" refers to any known in vitro process used to obtain multiple copies ("amplifiers") of a target nucleic acid sequence or its complementary sequence or fragment thereof. In vitro amplification refers to the production of nucleic acids that may contain fewer copies of the amplified nucleic acid than the complete target region sequence or its complementary sequence. 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 substitution amplification (SDA, including multi-strand substitution amplification methods (MSDA)). Replicase-mediated amplification uses self-replicating RNA molecules and replicases (e.g., Q-β-replicaase) (e.g., Kramer et al., U.S. Patent No. 4,786,600). PCR amplification is well-known, using DNA polymerase, primers, and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses at least four separate oligonucleotides to amplify the target and its complementary strand through multiple cycles of hybridization, ligation, and denaturation (e.g., EP Patent Application Publication No. 0320308). SDA is a method in which primers contain recognition sites for restriction endonucleases that allow the endonuclease to cleave one strand of a semi-modified DNA duplex containing a target sequence, followed by amplification in a series of primer extension and strand substitution steps (e.g., Walker et al., U.S. Patent No. 5,422,252). Two other known strand substitution amplification methods do not require endonuclease cleavage (Dattagupta et al., U.S. Patent No. 6,087,133 and U.S. Patent No. 6,124,120 (MSDA)). Those skilled in the art will understand that the oligonucleotide primer sequences of this invention can be readily used in any in vitro amplification method based on primer extension via polymerase. (See also Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25 and Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA) 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6: 1197-1202; Malek et al., 1994, Methods Mol. Biol., 28: 253-260; and Sambrook et al., 2000, Molecular Cloning A Laboratory Manual, Third Edition, CSH Laboratories). As is well known in the art, oligonucleotides are engineered to bind complementary sequences under selected conditions.

[0095] As used in this article, the term "marker" or "biomarker" refers to a biomolecule or biomolecule whose expression level is correlated with FXN level, for example, positively or negatively.

[0096] As used herein, biomarkers or markers of the present invention whose levels are positively or negatively correlated with FXN levels in cells are referred to as “FXN-sensitive genomic biomarkers” or “FSGMs”. In some embodiments, the FSGMs of the present disclosure are conversely regulated by FXN gene ablation followed by FXN protein replacement. Thus, in some embodiments, the FSGMs of the present disclosure are associated with FXN deficiency in subjects and inversely associated with FXN replacement. The FSGMs of the present invention can be used to detect and / or monitor FXN levels in samples (e.g., cell or tissue samples). In preferred embodiments, the FSGMs are selected from Tables 2, 4, or... Figure 3 The list includes those listed in Table 2, the human genes and proteins in Table 2, and the human homologs of the genes and proteins in Table 2. As used herein, refer to Tables 2, 4, and... Figure 3 References to FSGM in this context should be understood to include references to any of its mutants, variants, derivatives, or orthologs.

[0097] As used herein, the term "control sample" or "control" refers to any clinically relevant comparative sample, including, for example, samples from healthy FXN subjects (i.e., subjects with normal FXN levels), normal FXN expression profiles, samples from FXN-deficient subjects (i.e., subjects with complete or partial lack of FXN expression), baseline FXN(-) expression profiles, or samples or FXN replacement expression profiles from subjects following FXN replacement therapy. Control samples can also be samples from subjects at an earlier time point (e.g., before treatment with FXN replacement therapy). Control samples can be purified samples, proteins, and / or nucleic acids provided by a kit. Such control samples can be diluted, for example, serially to allow for quantitative measurement of the level of an analyte (e.g., a biomarker) in the test sample. Control samples can include samples derived from one or more subjects. Control samples can also be samples prepared from the subject to be evaluated at an earlier time point. For example, a control sample could be a sample obtained from the subject to be evaluated before treatment with FXN replacement therapy. Control samples may also be samples from animal models, or from tissues or cell lines derived from animal models of mitochondrial diseases (e.g., FRDA). The level of activity or expression 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 comprised of a set of measurable measurements, such as based on any suitable statistical measure, including measures of central tendency, such as the mean, median, or mode. In one embodiment, "different from the control" is preferably statistically significantly different from the control.

[0098] As used herein, “variable, altered, increased, or decreased” should be understood to mean a level of one or more detectable FSGMs that is statistically different (e.g., increased or decreased) compared to a control sample or threshold (e.g., from FXN-healthy subjects (i.e., subjects with normal FXN levels) or from FXN-deficient subjects (i.e., subjects lacking FXN expression). “Variable, altered, increased, or decreased” compared to a control or threshold may also include differences in the rate of change of levels of one or more FSGMs obtained from a series of at least two subject samples obtained over time. The determination of statistical significance is within the capabilities of those skilled in the art and may include any acceptable manner for determining and / or measuring statistical significance, such as the number of standard deviations of the means constituting positive or negative results, an increase in the detection level of FSGMs in the sample relative to the control, wherein said increase is above a certain threshold, or a decrease in the detection level of FSGMs in the sample relative to the control, wherein said decrease is below a certain threshold.

[0099] As used in this article, "detecting," "detection," and "measurement" should be understood as referring to the detection of samples selected from Tables 2, 4, and / or Tables 4. Figure 3 The identification of the presence and / or level of one or more FSGMs.

[0100] As used herein, the terms “DNA” or “RNA” molecules or sequences (and sometimes the term “oligonucleotide”) refer to molecules that are typically composed of the deoxyribonucleotides adenine (A), guanine (G), thymine (T), and / or cytosine (C). In “RNA”, T is replaced by uracil (U).

[0101] As used in this article, "FXN-deficient patients" and "FXN-deficient subjects" refer to subjects whose levels of FXN expression or activity are reduced compared to normal control subjects. Certain diseases cause FXN deficiency in patients, including mitochondrial diseases such as Friedreich ataxia (FRDA).

[0102] As used herein, the term "FXN replacement therapy" refers to the replacement of a coagulant protein in a subject, resulting in increased expression or activity of the coagulant protein in the subject. FXN replacement therapy can be performed by delivery of an FXN protein or by delivery of a nucleic acid encoding FXN to the subject. Delivery of an FXN protein to a subject can include delivery of an FXN protein or delivery of an FXN fusion protein. As used herein, the term "FXN fusion protein" refers to a full-length FXN or FXN fragment fused to a different protein or to a peptide. In some embodiments, as described herein, the FXN fusion protein comprises a full-length hFXN (SEQ ID NO: 1) or a mature hFXN (SEQ ID NO: 2). In some embodiments, the FXN protein or a fragment thereof is fused to a cell-penetrating peptide (CPP). In some embodiments, the CPP is an HIV-TAT peptide.

[0103] As used herein, the terms “symptom,” “disease,” and “abnormal state” are used inclusively and refer to any deviation from the normal structure or function of any part, organ, or system (or any combination thereof) of the body. Specific diseases are characterized 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, employment, genetic, and medical history factors. Early disease states include those where one or more physical symptoms have not yet been detected. Certain characteristic signs, symptoms, and associated factors can be quantified using various methods to produce important diagnostic information.

[0104] As used herein, the term "mitochondrial disease" refers to a disease caused by hereditary or spontaneous mutations in mtDNA or nDNA that result in functional alterations in proteins or RNA molecules normally present in mitochondria. These alterations impair mitochondrial function to induce various types of diseases in, for example, the central nervous system, skeletal muscle, heart, eyes, liver, kidneys, large intestine (colon), small intestine, inner ear, and pancreas, as well as the blood, skin, and endocrine glands. In a non-limiting embodiment, mitochondrial disease is Friedreich ataxia (FRDA).

[0105] As used herein, a sample obtained at an “earlier time point” is a sample obtained over a sufficient period of time in the past, from which clinically relevant information can be obtained compared to a later time point. In some embodiments, an earlier time point is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours earlier, or 1, 2, 3, 4, 5, 6, or 7 days earlier. In some embodiments, an earlier time point is at least one week, two weeks, three weeks, or four weeks earlier. In some embodiments, an earlier time point is at least six weeks earlier. In some embodiments, an earlier time point is at least two months earlier. In some embodiments, an earlier time point is at least three months earlier. In some embodiments, an earlier time point is at least six months earlier. In some embodiments, an earlier time point is at least nine months earlier. In some embodiments, an 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 intervals over time, and trends in FSGM levels can be analyzed. Those skilled in the art can determine the appropriate intervals for testing a particular subject based on conventional considerations.

[0106] As used in this article, “expression” refers to the process of producing polypeptides from DNA. This process involves the transcription of a gene into mRNA and the translation of that mRNA into a polypeptide. Depending on the context, “expression” may refer to the production of RNA or protein, or both.

[0107] The term "expression profile" is used to include genomic expression profiles, which refer to the expression profiles of RNAs, or particularly mRNAs or transcripts, or protein expression profiles. As used herein, an expression profile can refer to a set of data obtained relative to mRNA expression. It can refer to raw data from, for example, readings from a PCR device, or to normalized expression values. Expression profiles can be determined by any convenient means for measuring nucleic acid sequence levels, such as quantitative hybridization of mRNA, labeled mRNA, amplified mRNA, cDNA, etc., quantitative PCR, and other techniques known to those skilled in the art or described herein. Expression profiles enable the analysis of differential gene expression between two or more samples, between samples and controls, and between samples and thresholds. Expression profiles can also be determined by any means known to those skilled in the art or described herein for measuring protein or peptide levels, such as mass spectrometry, immunoassays (e.g., ELISA), etc.

[0108] As used herein, the term “FXN expression profile” includes any of the following three FXN expression profiles: normal FXN expression profile, baseline FXN(-) expression profile, or FXN alternative expression profile. As used herein, the baseline FXN(-) expression profile may also be referred to as the “threshold level” of FSGM expression. The baseline FXN(-) expression profile may also be used as a control.

[0109] As indicated in this article, the term “normal FXN profile” refers to the expression profile of one or more FSGMs in a sample from a normal patient (i.e., a patient who is not FXN deficient).

[0110] As indicated in this article, the term “baseline FXN(-) spectrum” refers to the expression profile of one or more FSGMs in a sample from a patient with FXN deficiency prior to treatment with FXN replacement therapy.

[0111] As indicated in this article, the term "FXN substitution profile" refers to the expression profile of one or more FSGMs in samples from FXN-deficient patients treated with FXN substitution therapy.

[0112] "Higher expression level", "higher level", "enhanced level", etc., of FSGM refers to the expression level of the test sample being higher than the standard error of the test used to assess the expression, and preferably at least 25%, at least 50%, at least 75%, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times higher than the FSGM expression level in control samples (e.g., samples from healthy subjects, samples from subjects lacking FXN, or samples from subjects after FXN replacement therapy) and the average expression level of FSGM or FSGMs in some preferred control samples.

[0113] As used herein, the term "hybridization," as in "nucleic acid hybridization," generally refers to the hybridization of two single-stranded nucleic acid molecules with complementary base sequences, which, under appropriate conditions, will form a thermodynamically favorable double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals mentioned above (Sambrook et al., 2000, ibid.; and Ausubel et al., 1994, ibid.; or further in Higgins and Hames (Eds.), "Nucleic acid hybridization, a practical approach," IRL Press, Oxford, Washington DC, (1985)), and are well known in the art. In hybridization with nitrocellulose membranes (or other such supports, such as nylon), as in well-known DNA blotting processes, the nitrocellulose membrane can be incubated overnight at the desired stringency conditions at typical temperatures (60-65°C for high stringency, 50-60°C for medium stringency, and 40-45°C for low stringency) with a probe labeled in a solution containing high salt (6xSSC or 5xSSPE), 5xDenhardt's solution, 0.5% SDS, and 100 μg / ml denatured vector DNA (e.g., salmon sperm DNA). The non-specifically bound probe is then washed off the membrane by multiple washes in 0.2xSSC / 0.1% SDS at a temperature chosen based on the desired stringency: room temperature (low stringency), 42°C (medium stringency), or 65°C (high stringency). The concentrations of salt and SDS in the washing solution can also be adjusted to suit the desired stringency. The chosen temperature and salt concentration are based on the denaturation temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In this case, the hybridization and washing conditions can be adjusted according to methods well known to those skilled in the art. Strict conditions are preferred (Sambrook et al., 2000, ibid.). Other methods using different annealing and washing solutions or commercially available hybridization kits (e.g., from BD BiosciencesClonetech) can also be used, as is well known in the art. As is well known, probe length and the composition of the nucleic acid to be measured constitute other parameters of the hybridization conditions. It is noteworthy that variations in these conditions can be achieved by including and / or replacing alternative blocking agents used to suppress background in the hybridization experiment. Typical blocking agents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available specialty formulations. Due to compatibility issues, including specific blocking agents may require changes to the hybridization conditions described above. Hybridized nucleic acid molecules also include fragments of the aforementioned molecules. Moreover, nucleic acid molecules hybridizing with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives, and allelic variants of these molecules. Furthermore, a hybridization complex refers to a complex formed between two nucleic acid sequences through the formation of hydrogen bonds 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 bound together by antiparallel conformational hydrogen bonds. Hybridization complexes can be formed in solution (e.g., in Cot or Rot analysis) or between a nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., a membrane, filter, chip, needle, or slide, on which cells have been immobilized).

[0114] As used herein, in the context of two or more nucleic acid or amino acid sequences, the term "identical" or "percentage identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of amino acid residues or nucleotides (e.g., 60% or 65% identity, preferably 70-95% identity, more preferably at least 95% identity) when compared and aligned over a comparison window or designated region to obtain maximum correspondence, as measured using sequence comparison algorithms known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or higher sequence identity are considered substantially identical. This definition also applies to complementary sequences of the test sequence. Preferably, the identity 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. As is well known in the art, those skilled in the art will know how to use algorithms, such as those based on the CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245)), to determine the percentage identity between / among sequences. Although the FASTDB algorithm generally does not consider internal mismatched deletions or additions (i.e., gaps) in the sequence in its calculations, this can be manually corrected to avoid overestimation of the % identity. However, CLUSTALW does consider sequence gaps in its identity calculations. Those skilled in the art can also use the BLAST and BLAST 2.0 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acid sequences uses a word length of 11 (W), an expected value of 10 (E), M=5, N=4, and a comparison of the two strands by default. For amino acid sequences, the BLASTP program defaults to a word length of 3 (W) and an expected value of 10 (E). The BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) uses an alignment of 50 (B), an expected value of 10 (E), M=5, N=4, and comparisons of the two strands. Furthermore, this invention also relates to nucleic acid molecules whose sequences are degenerate when compared with the sequences of the hybridized molecules described above. When the term "degenerate as a result of the genetic code" is used according to this invention, it means that different nucleotide sequences encode the same amino acid due to redundancy in the genetic code. This invention also relates to nucleic acid molecules comprising one or more mutations or deletions, and nucleic acid molecules hybridizing with one of the nucleic acid molecules described herein, said nucleic acid molecules exhibiting mutations or deletions.

[0115] The term "including" as used in this article refers to the phrase "including but not limited to" and may be used interchangeably with it.

[0116] As used herein, a “label” refers to a molecular part or compound that is detectable or can result in a detectable signal. Labels are directly or indirectly attached to molecules (e.g., antibodies), nucleic acid probes, or proteins / antigens or nucleic acids to be detected (e.g., amplified sequences). Direct labeling can occur by attaching the label to a bond or interaction (e.g., covalent or non-covalent) of a nucleic acid, while indirect labeling can occur by using a “connector” or bridging portion (e.g., an oligonucleotide or a small carbon chain) that is directly or indirectly labeled. Bridging portions can amplify the detectable signal. Labels can include any detectable part (e.g., radionuclides, ligands such as biotin or avidin, enzymes or enzyme substrates, reactive groups, chromophores such as stained or colored particles, luminescent compounds including bioluminescent, phosphorescent, or chemiluminescent compounds, and fluorescent compounds). Preferably, the label on the labeled probe is detectable in a homogeneous assay system (i.e., in a mixture), and the bound label exhibits a detectable change compared to an unbound label.

[0117] The terms "gene expression level," "gene expression level," and "FSGM level" refer to the levels of mRNA encoded by the gene, as well as pre-mRNA nascent transcripts, transcriptional intermediates, mature mRNA, and degradation products, or the levels of proteins in cells. The "level" of one or more FSGMs refers to the absolute or relative quantity or concentration of FSGMs in a sample.

[0118] "Lower expression level" or "lower level" or "reduced level" of FSGM refers to the expression level of FSGM in the test sample being lower than that in the control sample (e.g., a sample from a healthy subject, a sample from a subject lacking FXN, or a sample from a subject after FXN replacement therapy), and preferably 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the average expression level of FSGM in multiple control samples.

[0119] As used herein, “nucleic acid molecule” or “polynucleotide” refers to a polymer of nucleotides, including FSGM. Non-limiting examples include DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA), and their chimeras. Nucleic acid molecules can be obtained through cloning techniques or synthesis. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antsense]). Conventional ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) are included in the terms “nucleic acid” and “polynucleotide,” as are their analogues. The nucleic acid backbone may contain a variety of linkages known in the art, including sugar-phosphodiester linkages, peptide-nucleic acid bonds (referred to as “peptide nucleic acid” (PNA); Hydig-hielsen et al., PCT International Publication No. WO95 / 32305), phosphate thioester linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of nucleic acid can be ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-methoxy substitution (containing a 2'-O-methylfuranose moiety; see PCT No. WO98 / 02582) and / or 2'-halogen substitution. The nitrogenous base can be a conventional base (A, G, C, T, U), its known analogs (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992), or a known derivative of a purine or pyrimidine base (see Cook, PCT International Publication No. WO93 / 13121), or a "base-free" residue whose backbone does not contain one or more nitrogenous base residues (Arnold et al., U.S. Patent No. 5,585,481). Nucleic acids may consist only of the usual sugars, bases, and linkages, as found in RNA and DNA, or may include the usual components and substitutions (e.g., usual bases linked by a methoxy backbone, or nucleic acids containing a usual base and one or more base analogs). The term "isolated nucleic acid molecule" as commonly understood and used herein refers to a polymer of nucleotides, including but not limited to DNA and RNA. "Isolated" nucleic acid molecules are purified from their native in vivo state and obtained through cloning or chemical synthesis.

[0120] As used herein, "oligonucleotide" or "oligomer" defines a molecule having two or more nucleotides (ribose or deoxyribonucleotides). The size of an oligonucleotide will be determined by the specific circumstances and ultimately its particular use, and may be adapted accordingly by those skilled in the art. Oligonucleotides can be obtained by chemical synthesis or by clonal derivatization according to well-known methods. Although they are typically in single-stranded form, they can also be in double-stranded form, or even contain "regulatory regions." They can contain naturally rare or synthetic nucleotides. They can be engineered to enhance selected criteria, such as stability. Chimeras of deoxyribonucleotides and ribonucleotides are also within the scope of this invention.

[0121] As used in this article, “one or more” should be understood as covering each value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and any value greater than 10.

[0122] Unless the context clearly indicates otherwise, the term “or” as used herein is used inclusively to mean the term “and / or” and is used interchangeably with it.

[0123] As used herein, "patient" or "subject" may refer to a human or a non-human animal, preferably a mammal. "Subject" refers to any animal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and birds. Human subjects may be referred to as patients.

[0124] As used herein, a "probe" refers to a nucleic acid oligomer or oligonucleotide that specifically hybridizes with a target sequence in a nucleic acid or its complementary strand under conditions that promote hybridization, thereby allowing the detection of the target sequence or its amplified nucleic acid. Detection can be direct (i.e., generated by direct hybridization of the probe with the target or amplified sequence) or indirect (i.e., generated by hybridization of the probe with an intermediate molecular structure that links 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 the amplified sequence) that specifically hybridizes with at least a portion of the probe sequence via standard hydrogen bonds or "base pairing". "Sufficiently complementary" sequences allow the probe sequence to hybridize stably with the target sequence, even if the two sequences are not perfectly complementary. Probes can be labeled or unlabeled. Probes can be produced by molecular cloning of a specific DNA sequence, or they can be synthesized. A variety of primers and probes that can be designed and used in the context of this invention can be readily identified by those skilled in the art to which this invention pertains.

[0125] As used herein, a “reference level” for FSGM can be the absolute or relative amount or concentration of FSGM, the presence or absence of FSGM, the range of amounts or concentrations of FSGM, the minimum and / or maximum amount or concentration of FSGM, the average amount or concentration of FSGM, and / or the median amount or concentration of FSGM; furthermore, a “reference level” for a combination of FSGM can also be the ratio of the absolute or relative amounts or concentrations of two or more FSGMs relative to each other. Suitable positive and negative reference levels for FSGMs for a specific disease state, phenotype, or deficiency thereof can be determined by measuring the required levels of FSGM in one or more suitable subjects, and such reference levels can be tailored to a specific population of subjects (e.g., reference levels can be age-matched so that FSGM levels in samples from subjects of a specific age can be compared to reference levels for a specific disease state, phenotype, or deficiency thereof in a specific age group). Such reference levels can also be tailored to a specific technique (e.g., LC-MS, GC-MS, etc.) used to measure the levels of FSGM in biological samples, where the levels of FSGM can vary based on the specific technique used.

[0126] As used herein, “sample” or “biological sample” includes a sample or culture obtained from any source. In some embodiments, a sample includes any sample or culture containing cells capable of analyzing FXN expression profiles. In some embodiments, a sample includes any sample or culture from a subject lacking FXN or a subject receiving FXN replacement therapy. For example, a biological sample may be obtained from a bodily 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, or a solid tissue sample, such as a skin biopsy sample, skin band, hair follicle, or muscle biopsy sample, or an alternative sample may be an oral sample. Alternatively, a sample may contain exosomes harvested for testing FSGM transcripts.

[0127] As used herein, when the phrase “specifically binds” or “specifically binds” is used to refer to the interaction between an antibody and a protein or peptide, it means that the interaction depends on the presence of a specific structure on the protein (i.e., an antigenic determinant or epitope); in other words, the antibody recognizes and binds to a specific protein structure rather than the protein in general. For example, if an antibody is specific for epitope “A”, in a reaction involving labeled “A” and an antibody, the presence of a protein containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0128] The term “for example” is used in this document to mean the phrase “for example, but not limited to”, and can be used interchangeably with it.

[0129] “Transcribed polynucleotides” or “nucleotide transcripts” are polynucleotides (e.g., mRNA, hnRNA, cDNA, or analogs of such RNA or cDNA) that are wholly or partially complementary to or have a high percentage of identity (e.g., at least 80% identity) with mature mRNA, said mature mRNA being produced by transcription of the FSGM of the present invention and normal post-transcriptional processing (e.g., splicing) (if any) of the RNA transcripts and reverse transcription of the RNA transcripts.

[0130] Any composition or method provided herein may be combined with one or more of any other compositions and methods provided herein.

[0131] The ranges provided in this document should be understood as abbreviations of all values ​​within that range. For example, the range 1 to 50 should be understood as including any number, combination of numbers, or subrange of numbers in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0132] Exemplary embodiments of the invention will now be described in detail. While the invention will be described in conjunction with exemplary embodiments, it should be understood that this is not intended to limit the invention to these embodiments. Rather, it is intended to cover alternatives, modifications, and equivalents that may be included in the spirit and scope of the invention as set forth in the appended claims.

[0133] C. The FSGM of the present invention

[0134] In one aspect, the present invention provides a method for determining, evaluating, and / or monitoring the efficacy of FXN replacement therapy, comprising: (i) determining a baseline FXN(-) expression profile of one or more FSGMs in a sample from an FXN-deficient patient prior to treatment with FXN replacement therapy; and (ii) determining a patient FXN replacement expression profile of FSGMs in a sample from an FXN-deficient patient after treatment with FXN replacement therapy; comparing the patient FXN replacement expression profile with the baseline FXN(-) expression profile; and using said comparison to determine the efficacy of said FXN replacement therapy. Based on the results of the FSGM expression profile analysis, FXN replacement therapy in subjects can be adjusted to, for example, initiate, increase, decrease, or terminate FXN replacement therapy in subjects.

[0135] Another aspect of this disclosure relates to providing a method for identifying one or more FSGMs, wherein the FSGM is a biomarker whose expression is sensitive to FXN levels in cells. The method includes measuring an expression profile in samples from healthy subjects with normal FXN levels, referred herein as a normal FXN expression profile; measuring an expression profile in samples from subjects with deficient FXN levels, referred herein as a baseline FXN(-) expression profile; and comparing the normal FXN expression profile and the baseline FXN(-) expression profile; wherein the biomarker of altered expression in the baseline FXN(-) expression profile compared to the normal FXN expression profile is the FSGM. Additionally, or optionally, the method for measuring FSGM may include a comparison of expression profiles obtained from samples from subjects with FXN deficiency before and after FXN replacement therapy. Gene expression profiles of samples from FXN-deficient subjects after FXN replacement therapy are also referred herein as FXN replacement expression profiles. For example, Tables 2 and 4 of this document... Figure 3 The FSGM measured by the method of the embodiment of this disclosure is shown.

[0136] The FSGM of this invention includes, but is not limited to, Table 2, Table 4 and / or Figure 3 Any one or more of the FSGMs. In some embodiments of the invention, other biomarkers known in the art for measuring FXN expression or FXN replacement therapy may be used in conjunction with the methods of the invention.

[0137] As used herein, the term "one or more FSGMs" is intended to mean, for example, selected from Tables 2, 4 and / or Figure 3 One or more of the following (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) FSGM. The methods, kits and detection plates provided herein include, for example, those selected from Table 2, Table 4 and / or Figure 3 One or any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more FSGMs.

[0138] In some embodiments, one or more FSGMs of the present invention include those selected from Table 2, Table 4 and / or Figure 3One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) secretory proteins, namely the proteins listed in Table 2 that can be secreted by cells. For example, FSGM CYR61 is a secretory protein. Other FSGMs that are secretory proteins include, for example, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE, STC1, and THBS1. The expression level of FSGMs secreted from cells can be measured using, for example, any suitable method for detecting the peptide FSGMs of the present invention or any protein detection method described herein. In some embodiments, the detection method is an immunoassay method, such as ELISA, which involves antibodies that specifically bind to one or more secretory proteins (e.g., secretory proteins as defined in Table 2).

[0139] In one embodiment, one or more FSGMs include secreted proteins as defined in Table 2, alone or in combination with proteins selected from Table 2, Table 4 and / or Table 5. Figure 3 A combination of one or more other FSGMs. In another embodiment, one or more FSGMs include CYR61, which alone or in combination with those selected from Tables 2, 4 and / or Figure 3 A combination of one or more other FSGMs. In another embodiment, one or more FSGMs include one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and / or THBS1, alone or in combination with those selected from Tables 2, 4 and / or Figure 3 A combination of one or more other FSGMs.

[0140] CYR61, also known as Cell Communication Network Factor 1 (CCN1), Insulin-like Growth Factor Binding Protein 10, Cysteine-Rich Angiogenesis Inducer 61, IGF Binding Protein 10, CCN Family Member 1, Protein CYR61, IGFBP-10, IGFBP10, IBP-10, GIG1, Cysteine-Rich Heparin Binding Protein 61, Cysteine-Rich Angiogenesis Inducer 61, Cysteine-Rich Angiogenesis Inducer 61, and Protein GIG1, is a secreted protein encoded by the CYR61 gene. It is growth factor-induced and promotes endothelial cell adhesion. This protein interacts with several integrins and with heparan sulfate proteoglycans. It also functions as a linker between SERPINE1, EGR2, NR4A1, and THBS, and plays a role in pathways involving these genes. This protein also functions in cell proliferation, chemotaxis, angiogenesis, cell adhesion, differentiation, apoptosis, and extracellular matrix formation. Diseases associated with CYR61 include Wilms' tumor 5 and rhabdomyosarcoma. CYR61 can bind to α6 and β1 integrin heterodimers (which have been shown to regulate Schwann cell-axon interactions and promote axon regeneration after peripheral nerve injury), thus potentially inhibiting the process (Chang et al., Neuroscience 2018, 371:4-59).

[0141] Exemplary GenBank accession numbers for the nucleotide and amino acid sequences of each FSGM (or its human homolog) listed in Table 2 are listed in Table 4 below. These GenBank accession numbers are incorporated by reference to the version available on the earliest valid filing date of this application. AI480526, C230034O21Rik, D130020L05Rik, and Rp137rt are mouse genes without human homologs and therefore are not listed in Table 4.

[0142] It should be understood that the FSGM of the present invention includes human homologs of the genes and proteins listed in Table 2.

[0143]

[0144]

[0145] In one embodiment, one or more FSGMs include one or more of NR4A1, PTP4A1, ATF3, BTG2, EGR1, EGR2, EGR3, CYR61, and ABCE1. In another embodiment, one or more FSGMs include one or more of EGR1, EGR2, EGR3, and IGF1. In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, and CYCS. In another embodiment, one or more FSGMs include one or more of OPS2, VBP1, PSMA3, SLIRP, CUL2, DCUN1D1, UBE2D3, ZNRF1, RNF2, and LAMP2. In another embodiment, the one or more FSGMs include one or more of RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, RPL38, RPS27L, and ABCE1. In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, and CYCS. In another embodiment, one or more FSGMs include one or more of NR4A1, EGR1, EGR3, ADAMTS1, THBS1, SERPINE1, IGF1, PTGS2, and CYR61. In another embodiment, one or more FSGMs include one or more of PSMA3, CUL2, UBE2D3, ZNRF1, RPS15A, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38. In another embodiment, the one or more FSGMs include one or more of ABCE1, RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38. In another embodiment, the one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, CYCS, TMEM-126A, MAOA, and ABCE1. In yet another embodiment, the one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, and MT-ATP8.In another embodiment, the one or more FSGMs include one or more of ABCE1, RPL26, RPL38, RPL10, RPL32, RPS15A, RPL24, RPL39, SLIRP, COPS2, DCUN1D1, RNF2, EGR1, BTG2, ATF3, PTGS2, IGF1, SERPINE1, and THBS1. In another embodiment, the one or more FSGMs include one or more of RPL26, THBS1, SERPINE1, IGF1, PTGS2, RPL10, RPS27L, CYCS, ATF3, BTG2, EGR1, EGR3, and CYR61.

[0146] For example, FXN expression profiles can be determined by measuring the expression levels of at least one FSGM or any combination of more than one FSGM. As used herein, FSGMs include those in Tables 2 and 4 and / or Figure 3 Any one or more of the FSGMs listed in Table 2. FSGMs also include genes encoding secretory proteins (such as those defined in Table 2) (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, or THBS1), mitochondrial genes, EGR family genes, insulin-like genes, ribosome depletion response genes, mitochondrial energy production genes, proteasome regulatory genes, ribosome function genes, respiratory chain genes, cardiac development genes, macromolecular catabolism genes, translation initiation genes, mitochondrial component genes, oxidative phosphorylation genes, negative regulators of macromolecular metabolism, and regulators of apoptosis, or any one or more proteins encoded by any of these genes.

[0147] In the following text, the expression spectrum may also be referred to as the signature.

[0148] In one embodiment of this disclosure, the baseline FXN(-) expression profile may include, as shown in Table 2, fold adjustment via “KO (knockout) vs. WT (wild type)” and / or Figure 3 The expression pattern in.

[0149] In one embodiment of this disclosure, the baseline FXN(-) expression profile includes at least one or more FSGMs (e.g., Table 2, Table 4 and / or Figure 3Any combination of any one or more of the FSGMs listed in the table, or the expression of any one or more of the genes encoding secretory proteins (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, or THBS1) that encode secretory proteins (e.g., secretory proteins as defined in Table 2), mitochondrial genes, EGR family genes, insulin-like genes, ribosome depletion response genes, mitochondrial energy production genes, proteasome regulatory genes, ribosome function genes, respiratory chain genes, myocardial development genes, macromolecular catabolism genes, translation initiation genes, mitochondrial component genes, oxidative phosphorylation genes, negative regulatory genes of macromolecular metabolism processes, and regulatory genes of apoptosis processes, or altered expression of any one or more proteins encoded by any of these genes.

[0150] In another embodiment of this disclosure, the baseline FXN(-) expression profile may include downregulated expression levels of at least one of ADNP, AI480526, C230034O21RIK, CCDC85B, CCDC85C, CTCFL, D130020L05RIK, mt-RNR1, mt-RNR2, NRTN, PDE4A, PHF1, RPL37RT, SLC26A10, SNORD17, SUV420H2, WNK2, YAM1, and / or ZNRF1, or any combination thereof. The efficacy of FXN replacement therapy can be measured by upregulated patterns of any one or more of these FSGMs.

[0151] In another embodiment of this disclosure, the baseline FXN(-) expression profile may include upregulated expression levels of CYR61. The efficacy of FXN replacement therapy can be indicated by the downregulated pattern of CYR61.

[0152] In one implementation, the FXN alternative expression profile includes the reverse expression of the baseline FXN(-) expression profile.

[0153] In another embodiment, the FXN substitution expression profile used as an indicator of the efficacy of FXN substitution therapy may include Tables 2 and 4 and / or Figure 3 Any combination of one or two or more of the FSGMs shown, including secreted proteins (e.g., CYR61) detected in samples from patients treated with FXN replacement therapy, such as secreted proteins as defined in Table 2, or one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1.

[0154] In another implementation, the FXN alternative expression profile may include expression patterns as shown in Table 2 by fold adjustment of “drug vs. vector”.

[0155] In some embodiments, the FXN substitution expression profile is characterized by the inverse regulation of FSGMs, defined by any FSGM downregulated under FXN depletion conditions that becomes upregulated after FXN substitution therapy; conversely, it is also effective such that any FSGM upregulated under FXN depletion conditions becomes downregulated after FXN substitution therapy. Therefore, the detection of altered expression of one or more FSGMs in samples after FXN substitution therapy allows for monitoring the efficacy of FXN substitution therapy in subjects. For example, in one embodiment, the absence of altered expression of one or more FSGMs in samples after FXN substitution therapy indicates that FXN substitution therapy may be unsuccessful and / or may require enhancement. Similarly, in another embodiment, altered expression of one or more FSGMs in samples after FXN substitution therapy indicates that FXN substitution therapy is successful.

[0156] In some implementations, the altered expression is regulated or modified gene expression, which in the methods exemplified herein is itself presented as differential gene expression, also known as differential mRNA expression. Altered or regulated expression may include increased expression, also known as overexpression or upregulation, or decreased or suppressed expression, also known as downregulation.

[0157] As described in this article, an eigenvector is a set of values ​​that characterizes an expression profile. An eigenvector can include a set of n FSGMs, where n is the number of different genes whose expression levels are measured in the sample. For example, n could be from Tables 2, 4, and... Figure 3 All FSGMs provided in [the table]. Alternatively, n can be from Table 2, Table 4, and [other tables]. Figure 3 The FSGMs shown are at least 1, 2, 3, 4, 5, 6, or any combination of any number.

[0158] In one implementation, a set of FSGMs may include any combination of at least one or more FSGMs, such as those in Tables 2 and 4 and / or Figure 3Any one or more of the FSGMs listed herein, or any one or more genes encoding secretory proteins, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 or THBS1, mitochondrial genes, EGR family genes, insulin-like genes, ribosome depletion response genes, mitochondrial energy production genes, proteasome regulatory genes, ribosome function genes, respiratory chain genes, myocardial development genes, macromolecular catabolism genes, translation initiation genes, mitochondrial component genes, oxidative phosphorylation genes, negative regulatory genes of macromolecular metabolism processes and regulatory genes of apoptosis processes, or proteins encoded by any of these genes.

[0159] In one embodiment, one or more FSGMs include CYR61. In another embodiment, one or more FSGMs include one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1. In one embodiment, one or more FSGMs include one or more of NR4A1, PTP4A1, ATF3, BTG2, EGR1, EGR2, EGR3, CYR61, and ABCE1. In another embodiment, one or more FSGMs include one or more of EGR1, EGR2, EGR3, and IGF1. In yet another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO2, MT-CO3, MT-ATP6, MT-ATP8, and CYCS. In another embodiment, one or more FSGMs include one or more of OPS2, VBP1, PSMA3, SLIRP, CUL2, DCUN1D1, UBE2D3, ZNRF1, RNF2, and LAMP2. In another embodiment, the one or more FSGMs include one or more of RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, RPL38, RPS27L, and ABCE1. In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, and CYCS. In another embodiment, one or more FSGMs include one or more of NR4A1, EGR1, EGR3, ADAMTS1, THBS1, SERPINE1, IGF1, PTGS2, and CYR61. In another embodiment, one or more FSGMs include one or more of PSMA3, CUL2, UBE2D3, ZNRF1, RPS15A, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38. In another embodiment, the one or more FSGMs include one or more of ABCE1, RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38. In another embodiment, the one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, CYCS, TMEM-126A, MAOA, and ABCE1.In another embodiment, one or more FSGMs include one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, and MT-ATP8. In another embodiment, the one or more FSGMs include one or more of ABCE1, RPL26, RPL38, RPL10, RPL32, RPS15A, RPL24, RPL39, SLIRP, COPS2, DCUN1D1, RNF2, EGR1, BTG2, ATF3, PTGS2, IGF1, SERPINE1, and THBS1. In another embodiment, one or more FSGMs include one or more of RPL26, THBS1, SERPINE1, IGF1, PTGS2, RPL10, RPS27L, CYCS, ATF3, BTG2, EGR1, EGR3, and CYR61.

[0160] For example, a normal FXN expression profile obtained from a sample of a healthy subject may consist of a set of FSGM expression levels and may be represented by a normal FXN eigenvector, referred to as the normal FXN eigenvector. As described in the following embodiments, when measured in an FXN-deficient sample, FSGM may exhibit expression levels different from those in healthy subjects, and therefore may be represented by a lack-FXN eigenvector, referred to as the lack-FXN eigenvector. In one implementation, the difference between the lack-FXN eigenvector and the normal FXN eigenvector can be detected and quantified by the distance between the two eigenvectors. In another case, the FSGM expression levels in samples from FXN-deficient patients after FXN replacement therapy may exhibit different expression levels and may be represented by an FXN substitution eigenvector, referred to as the FXN substitution eigenvector. For the first two eigenvectors, the difference between the FXN substitution eigenvector and the normal FXN eigenvector or the lack-FXN eigenvector can be detected and quantified by the distance between the substitution FXN eigenvector and the normal FXN eigenvector or the lack-FXN eigenvector.

[0161] Therefore, using samples from patients with FXN deficiency obtained before treatment and samples obtained after FXN replacement therapy, a first FXN feature vector of the FXN replacement expression profile can be determined, and a second FXN feature vector of the baseline FXN(-) expression profile can be determined; wherein determining the distance or product between the first and second feature vectors can be used to determine the efficacy of the FXN replacement therapy. In one embodiment of this disclosure, a third feature vector of the normal FXN expression profile can be determined, establishing a normal expression profile 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 efficacy of the FXN replacement therapy. In one embodiment, the distance between the first and third feature vectors can be normalized relative to the distance between the second and third feature vectors, and the normalized distance can be used to determine the efficacy of the FXN replacement therapy. In one embodiment, the obtained normalized distance can be a value in the range of 0 (zero) to 1 (one), wherein the smaller the value (closest to zero), the more effective the treatment.

[0162] The markers of this invention, for example, are selected from Table 2, Table 4 and / or Figure 3 One or more FSGMs are associated with FXN levels in subjects. Therefore, in one aspect, the present invention provides Tables 2, 4, and / or Figure 3 Methods for the use, measurement, detection, quantification, etc. of one or more FSGMs (e.g., CYR61, or one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1) for determining and / or monitoring FXN status in subjects or for determining, evaluating, and / or monitoring FXN replacement therapy in subjects.

[0163] On the other hand, the present invention relates to Table 2, Table 4 and / or Figure 3 One or more FSGMs, such as CYR61, CYR61, or one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and THBS1, used, measured, detected, quantified, etc., alone or together with one or more other FSGMs for FXN expression levels.

[0164] Furthermore, in another implementation, FSGM can be used in combination with one or more other biomarkers for mitochondrial diseases (e.g., FRDA). It can be used in conjunction with Tables 2 and 4 and / or... Figure 3Other biomarkers used in combination with one or more FSGMs include any measurable characteristics described herein that can reflect an organism's physiological state quantitatively or qualitatively, such as whether the organism has a mitochondrial disease (e.g., FRDA). An organism's physiological state includes any disease or non-disease state, such as a subject with a mitochondrial disease (e.g., FRDA) or otherwise healthy subjects. This can be compared with Tables 2, 4, and / or... Figure 3 The FSGMs used in the present invention comprise characteristics that can be objectively measured and assessed as indicators of normal processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Such combined biomarkers can be clinical parameters (e.g., age, performance status), laboratory measurements (e.g., molecular biomarkers), or genetic or other molecular determinants. In other embodiments, the invention also relates to the analysis and consideration of any clinical and / or patient-related health data, such as data obtained from electronic medical records (e.g., collections of electronic health information about individual patients or groups associated with various types of data, such as demographics, medical history, medication and allergies, immune status, laboratory test results, radiographic images, vital signs, personal data such as age and weight, and billing information).

[0165] The present invention also considers Tables 2, 4 and / or Figure 3 The use of specific combinations of FSGMs, for example, combinations of FSGMs including CYR61 or combinations of FSGMs including CYR61 or CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1. In one embodiment, the invention contemplates a set of FSGMs having at least two (2) members, which may include Tables 2, 4, and / or Figure 3 Any two FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least three (3) members, which may include Table 2, Table 4 and / or Figure 3 Any three FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least four (4) members, which may include Table 2, Table 4 and / or Figure 3 Any four FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least five (5) members, which may include Table 2, Table 4 and / or Figure 3 Any five FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least six (6) members, which may include Table 2, Table 4 and / or Figure 3Any six FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least seven (7) members, which may include Table 2, Table 4 and / or Figure 3 Any seven FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least eight (8) members, which may include Table 2, Table 4 and / or Figure 3 Any eight FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least nine (9) members, which may include Table 2, Table 4 and / or Figure 3 Any nine FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least ten (10) members, which may include Table 2, Table 4 and / or Figure 3 Any ten FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least eleven (11) members, which may include Tables 2, 4 and / or Figure 3 Any ten FSGMs. In another embodiment, the invention contemplates a set of FSGMs having at least twelve (12) members, which may include Table 2, Table 4 and / or Figure 3 Any ten FSGMs. In other embodiments, the invention considers sets of FSGMs that include Tables 2, 4, and / or Figure 3 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, or 102 of the FSGMs listed. In one embodiment, the invention considers a set of FSGMs that includes Tables 2, 4, and / or Figure 3 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, or 102 of the FSGMs listed in Table 2. One or more of the FSGMs in this set are secreted proteins, such as those defined in Table 2, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1.

[0166] In another embodiment, the invention considers the FSGM set, which includes Table 2, Table 4 and / or Figure 3 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, or 102 of the FSGMs listed in the set, of which one of the FSGMs in this set is CYR61.

[0167] In some implementations, FSGM levels increase after FXN replacement therapy in subjects (e.g., subjects lacking FXN). In some implementations, FSGM is selected from a group consisting of mt-RNR1, mt-RNR2, ADNP, AI480526, C230034O21RIK, CCDC85B, CCDC85C, CTCFL, NRTN, PDE4A, PHF1, RPL37RT, SLC26A10, SNORD17, SUV420H2, WNK2, YAM1, and ZNRF1.

[0168] In other embodiments, FSGM levels decreased after treatment of subjects (e.g., subjects with FXN deficiency) with an FXN substitute. In some embodiments, FSGM was selected from a group consisting of CYR61, mt-ATP6, mt-ATP8, mt-CO2, mt-CO3, mt-ND1, mt-ND2, mt-ND3 and mt-ND4, EGR1, EGR2, EGR3, IGF1, LAMP2 and SLIRP.

[0169] In another aspect, the present invention provides the identification of a “diagnostic signature” or “diagnostic expression profile” based on the level of the FSGM of the present invention in a biological sample, which is correlated with FXN in the sample. “FSGM level” can refer to the protein level of FSGM in the biological sample. “FSGM level” can also refer to the expression level of the gene corresponding to the protein, for example, by measuring the expression level of the corresponding FSGM mRNAs. The set or totality of FSGM levels provides a diagnostic signature associated with FXN levels.

[0170] In some implementations, the diagnostic signature is obtained by: (1) detecting Tables 2 and 4 and / or in biological samples from subjects receiving FXN replacement therapy. Figure 3(2) The levels of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more FSGMs (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and / or THBS1), and (3) the levels of Tables 2 and 4 and / or Tables 4 and 5. Figure 3 The levels of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more FSGMs (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and / or THBS1) in the biological sample and the levels of the same FSGMs from the control sample, such as the baseline FXN(-) expression profile, and (3) the determination of the levels of the FSGMs detected in the biological sample as shown in Tables 2, 4 and / or Table 4. Figure 3 Whether at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more FSGMs (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and / or THBS1) are higher or lower than the levels of FSGMs in the control (e.g., baseline FXN(-) expression profile). If Table 2, Table 4 and / or Figure 3 If at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more FSGMs (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and / or THBS1) are higher or lower than the control (e.g., baseline FXN(-) expression profile), the diagnostic signature indicates the efficacy of FXN replacement therapy.

[0171] According to various implementations, the algorithm can be used to predict whether a biological sample from a subject contains FXN, or to assess or monitor whether a subject has effectively received FXN replacement therapy. Those skilled in the art will understand that the algorithm can 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 (n) detected at levels above a certain threshold level, or the number of markers (n) detected at levels below a certain threshold level) through multiple well-defined, sequential steps, ultimately producing a score or "output". This document considers any suitable algorithm—whether computer-based or human-based (e.g., lookup table).

[0172] In some embodiments, the FSGMs of the present invention (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) may include variant sequences. More specifically, certain binding agents / reagents used to detect specific FSGMs of the present invention may bind to and / or identify variants of these specific FSGMs of the present invention. As used herein, the term "variant" includes a nucleotide or amino acid sequence that differs from the specifically identified sequence, wherein one or more nucleotide or amino acid residues are deleted, substituted, or added. Variant sequences may be naturally occurring allelic variants or non-naturally occurring variants. Variant sequences (polynucleotides or polypeptides) preferably exhibit at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequences disclosed herein. Percentage identity is determined by comparing two sequences to be compared as described below, determining the number of identical residues in the compared portions, dividing that number by the total number of residues in the present invention (query) sequence, and multiplying the result by 100.

[0173] Variant sequences typically differ from the specifically identified sequence only in terms of conserved substitutions, deletions, or modifications. As used herein, a “conserved substitution” is the replacement of one amino acid with another amino acid of similar properties, such that those skilled in the art of peptide chemistry can expect the secondary structure and hydrophilicity of the polypeptide to remain substantially unchanged. Typically, the following groups of amino acids represent conserved 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. Variant sequences may also or optionally contain other modifications, including the deletion or addition of amino acids that have minimal impact on the antigenic properties, secondary structure, and hydrophilicity of the polypeptide. For example, a polypeptide may be conjugated to the N-terminus of a protein with a signal (or leader) sequence that directs co-translation or post-translational transfer of the protein. Peptides can also be conjugated to linkers or other sequences to facilitate peptide synthesis, purification, or identification (e.g., poly-His), or to enhance the binding of peptides to solid supports. For example, peptides can be conjugated to the Fc region of immunoglobulins.

[0174] Peptide and polynucleotide sequences can be aligned, and publicly available computer algorithms can be used to determine the percentage of identical amino acids or nucleotides in a specific region against another peptide or polynucleotide sequence. Percentage identity of polynucleotide or peptide sequences is determined by aligning polynucleotide and peptide sequences using an appropriate algorithm (e.g., BLASTN or BLASTP, respectively set to default parameters); identifying the number of identical nucleic acids or amino acids on the aligned portion; dividing the number of identical nucleic acids or amino acids by the total number of nucleic acids or amino acids in the polynucleotide or peptide of the present invention; and then multiplying by 100 to determine percentage identity.

[0175] Two exemplary algorithms for aligning and identifying the identity of polynucleotide sequences are the BLASTN and FASTA algorithms. The BLASTP algorithm can be used to check the alignment and identity of peptide sequences. The BLASTX and FASTX algorithms compare the translated nucleotide query sequence and peptide sequence across all reading frames. The FASTA and FASTX algorithms are described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988 and Pearson, Methods in Enzymol. 183:63-98, 1990. The FASTA software package is available from the University of Virginia, Charlottesville, Va. 22906-9025. The FASTA algorithm is set to the default parameters described in the documentation and is distributed with the algorithm; it can be used for the identification of polynucleotide variants. The readme files for FASTA and FASTX versions 2.0x, distributed with the algorithms, describe the use of the algorithms and the default parameters.

[0176] The BLASTN software is available on the NCBI anonymous FTP server and from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894. BLASTN algorithm versions 2.0.6 [September 10, 1998] and 2.0.11 [January 20, 2000] are set as the default parameters described in the documentation and distributed with the algorithm, which is preferably used for determining the 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 "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" by Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997.

[0177] In an alternative embodiment, the variant peptide is encoded by a polynucleotide sequence that hybridizes with a disclosed polynucleotide under stringent conditions. Stringent hybridization conditions used to determine complementarity include salt conditions of less than about 1 μM, more typically less than about 500 mM, and preferably less than about 200 mM. Hybridization temperatures can be as low as 5 °C, but are typically above about 22 °C, more preferably above about 30 °C, and most preferably above about 37 °C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency of hybridization can be affected by other factors such as probe composition, the presence of organic solvents, and the degree of base mismatch, the combination of parameters is more important than any single absolute measurement. An example of “stringent conditions” is pre-washing in a solution of 6X SSC, 0.2% SDS; hybridizing overnight at 65 °C in 6X SSC, 0.2% SDS; followed by washing twice for 30 minutes each in 1X SSC, 0.1% SDS at 65 °C, and then washing twice for 30 minutes each in 0.2X SSC, 0.1% SDS at 65 °C.

[0178] This invention provides for the use of various combinations and sub-combinations of FSGMs. For example, one or more secreted proteins, such as those defined in Table 2, may be used in the methods of this invention, including CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1. It should be understood that, unless otherwise expressly indicated, any single FSGM or combination of FSGMs provided herein may be used in this invention.

[0179] D. Tissue Samples

[0180] This invention can be implemented with any suitable biological sample that potentially contains, expresses, or includes detectable FSGM. For example, the biological sample can be obtained from a bodily 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, or a solid tissue sample, such as a skin biopsy sample, a muscle biopsy sample, or the sample can be a buccal sample. Alternatively, the sample can include exosomes harvested for testing FSGM transcripts.

[0181] 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 samples comprising a large number of cells, wherein the assay is an “averaged” expression across the entire collection of cells and tissues present in the sample. Preferably, there is a sufficient amount of tissue sample to accurately and reliably determine the target expression level.

[0182] Any commercially available equipment or system for separating and / or obtaining tissues and / or blood or other biological products, and / or for processing said materials prior to conducting a detection reaction, is considered.

[0183] In some embodiments, the present invention relates to the detection of FSGM nucleic acid molecules (e.g., those encoded in Tables 2, 4 and / or Tables 4 and 5) Figure 3 The mRNA of the protein FSGM, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1. In such an embodiment, RNA can be extracted from the biological sample prior to analysis. Methods for RNA extraction are well known in the art (see, for example, J. Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989, 2nd Ed., Cold Spring Harbour Laboratory Press: New York). Most methods for isolating RNA from body fluids or tissues are based on tissue destruction in the presence of protein denaturing agents to rapidly and effectively inactivate RNase. Typically, RNA isolation reagents include, among other components, guanidine thiocyanate and / or β-mercaptoethanol, which are known to act as RNase inhibitors. The isolated total RNA was then further purified from protein contaminants and concentrated by selective ethanol precipitation, phenol / chloroform extraction, followed by isopropanol precipitation (see, for example, P. Chomczynski and N. Sacchi, Anal. Biochem., 1987, 162: 156-159) or gradient centrifugation with cesium chloride, lithium chloride, or cesium trifluoroacetate.

[0184] Many different and universal kits are available for extracting RNA (i.e., total RNA or mRNA) from bodily fluids or tissues and are commercially available from companies such as Ambion, Inc. (Austin, Tex.), Amersham Biosciences (Piscataway, NJ), BDBiosciences Clontech (paloalto, Calif.), BioRad Laboratories (Hercules, Calif.), GIBCO BRL (Gaithersburg, Md.), and Giagen, Inc. (Valencia, Calif.). All of these kits typically include a user guide detailing the protocol to be followed. The sensitivity, processing time, and cost of each kit may vary. Those skilled in the art can easily select the kit best suited to their specific needs.

[0185] In some implementations, after extraction, the mRNA is amplified and transcribed into cDNA, which can then be used as a template for multiple rounds of transcription via a suitable RNA polymerase. Amplification methods are well known in the art (see, for example, AR. Kimmel and S.L. Berger, Methods Enzymol. 1987, 152:307-316; J. Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989, 2nd Ed., Cold Spring Harbour Laboratory Press: New York; “Short Protocols in Molecular Biology”, F.M. U.S.A. Susubel (Ed.), 2002, 5th Ed., John Wiley & Sons; U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159). Reverse transcription can be performed using non-specific primers (e.g., anchored oligo-dT primers) or random sequence primers, or target-specific primers that are complementary to the RNA of each genetic probe being monitored, or using a thermostable DNA polymerase (e.g., avian myeloblastoma virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase).

[0186] In some embodiments, RNA isolated from tissue samples (e.g., after amplification and / or conversion to cDNA or cRNA) is labeled with a detectable reagent prior to analysis. The detectable reagent serves to facilitate RNA detection or allow visualization of hybridized nucleic acid fragments (e.g., nucleic acid fragments hybridized with genetic probes in array-based assays). Preferably, the detectable reagent is selected to produce a measurable signal whose intensity is correlated with the amount of labeled nucleic acid present in the sample being analyzed. In array-based analytical methods, the detectable reagent is also preferably selected to produce a localized signal, thereby allowing spatial resolution of the signal from each point on the array.

[0187] Methods for labeling nucleic acid molecules are well known in the art. For reviews of the latest advances in labeling schemes, labeling detection techniques, and related fields, see, for example, L.J. 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 ligation of fluorescent dyes (see, e.g., LMSmith et al., Nucl. Acids Res. 1985, 13:2399-2412) or enzymes (see, e.g., BA Connoly and P. Rider, Nucl. Acids Res. 1985, 13:4485-4502); and chemical modification of nucleic acid fragments to enable detection by immunochemistry or other affinity reactions (see, e.g., TR Broker et al., Nucl. Acids Res. 1978, 5:363-384; EA Bayer et al., Methods of Biochem. Analysis, 1980, 26:1-45; R. Langer et al., Proc. Natl. Acad. Sci. USA, 1981, 78:6633-6637; R. W Richardson et al., Nucl. Acids Res. 1985, 13:4485-4502). Res. 1983, 11:6167-6184; DJ Brigati et al., Virol. 1983, 126:32-50; P. Tchen et al., Proc. Natl Acad. Sci. USA, 1984, 81:3466-3470; JELandegent et al., Exp. Cell Res. 1984, 15:61-72; and AHHopman et al., Exp. Cell Res. 1987, 169:357-368); and enzyme-mediated labeling methods, such as random initiation, nick translation, PCR, and tailing with terminal transferases (for a review of enzyme labeling, see, for example, J. Temsamani and S. Agrawal, Mol. Biotechnol. 1996, 5:223-232).

[0188] In practice, any of a variety of detectable reagents may be used. Suitable detectable reagents include, but are not limited to: various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (e.g., quantum dots, nanocrystals, phosphorescent agents, etc.), enzymes (e.g., those used in ELISA, i.e., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and other haptens and proteins available from biotin, digoxin, or their antisera or monoclonal antibodies.

[0189] However, in some implementations, the expression level is determined by detecting the expression of gene products (e.g., proteins, such as secretory proteins), thereby eliminating the need to obtain genetic samples (e.g., RNA) from the sample.

[0190] Detection and / or measurement of E.FSGM

[0191] Various methods can be used to measure the distance between eigenvectors. Once the data is standardized, this distance can be obtained, for example, by calculating the mean squared error, which can be extracted from the difference in expression patterns of each gene measured in two different spectra (e.g., baseline FXN(-) and FXN substitution). Alternatively, the distance can be obtained by calculating the correlation coefficient or applying a t-test.

[0192] As described in detail herein, numerous methods for determining RNA expression profiles have been described, including sequencing, hybridization, or amplification of sample RNA. In a particular embodiment of this disclosure, determining the expression profile of the patient sample includes obtaining or providing a biological sample from a patient, extracting RNA from the sample, generating the corresponding cDNA, and detecting the expression profile by any of sequencing, hybridization, or amplification.

[0193] Detecting FXN-sensitive expression profiles through sequencing can be performed using next-generation sequencing (NGS), RNASeq, and any sequencing technology known to those skilled in the art.

[0194] Detection of expression profiles by hybridization involves contacting the patient sample or a portion thereof with Tables 2, 4 and / or Tables 4 and / or Tables 5 and 6. Figure 3The present disclosure discloses a probe or a set of probes that specifically hybridize to the FSGM (or its transcript). In one embodiment of the present disclosure, a specific probe of the transcript of at least one gene encoding at least one secretory protein (e.g., secretory proteins as defined in Table 2, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and / or THBS1, mitochondrial proteins, EGR family proteins, insulin-like proteins, ribosome depletion response proteins, mitochondrial energy production proteins, proteasome regulatory proteins, ribosome functional proteins, respiratory chain proteins, myocardial development proteins, macromolecular catabolism proteins, translation initiation proteins, mitochondrial component proteins, oxidative phosphorylation proteins, negative regulators of macromolecular catabolism processes, or regulators of apoptosis processes, or any combination thereof) may be contacted with a patient sample. For example, a specific probe of at least one or any combination of mt-ATP6, mt-ATP8, mt-CO2, mt-CO3, mt-ND1, mt-ND2, mt-ND3, mt-ND4, mt-RNR1, mt-RNR2, EGR1, EGR2, EGR3, IGF1, LAMP2, APOLD1, MAOA, PDE4A, YARS, RnF13 and RPL10, CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and / or THBS1 can be brought into contact with a patient sample. Therefore, determining the expression profile of a patient sample treated with FXN replacement therapy by hybridization may include contacting the sample or a portion thereof with a probe that hybridizes at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% with its target nucleic acid, which is listed in Tables 2 and 4 and / or Figure 3 Transcripts or corresponding cDNAs of any of the FSGMs provided (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1).

[0195] The amplification detection of expression profiles involves, for example, polymerase chain reaction (PCR) techniques, such as real-time polymerase chain reaction (RT-PCR), which involves contacting a sample with forward and reverse primers for each target transcript exemplified in the examples below, and generating RT-PCR products. Optionally, the RT-PCR products are detected with specific or universal probes or combinations thereof to facilitate their quantification. Thus, the FXN-induced signature can be determined by detecting FSGM transcripts in a sample. In one embodiment of this disclosure, forward and reverse primers are used to detect FSGM transcripts.

[0196] In an alternative implementation, the expression profile can be detected by analyzing the protein products and proteomic profiles of the FSGM. This can be done using protein detection methods, techniques involving specific antibodies, or protein quantification / characterization techniques, such as high-performance liquid chromatography (HPLC), mass spectrometry-based techniques, gel-based techniques such as differential gel electrophoresis, etc.

[0197] In another aspect, this disclosure provides compositions for the detection of FXN expression profiles, said compositions comprising at least one or more nucleotide sequences for the detection of FSGM. In one embodiment of this disclosure, the composition can be used for the detection of any of the following: FXN alternative expression profiles, baseline FXN(-) expression profiles, and / or normal FXN expression profiles. The composition may contain components for use in Tables 2 and 4 and / or Figure 3 The composition may contain at least one nucleotide sequence for the detection of transcripts of genes defined in Tables 2 and 4. Figure 3 The nucleotide sequences shown are 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 and / or up to all FSGMs. For example, a composition for detecting FXN signatures includes nucleotides for detecting at least one or any combination of mt-ATP6, mt-ATP8, mt-CO2, mt-CO3, mt-ND1, mt-ND2, mt-ND3, mt-ND4, mt-RnR1, mtRnR2, EGR1, EGR2, EGR3, IGF1, LAMP2, APOLD1, MAOA, PDE4A, YARS, RnF13, RPL10, SLIRP, CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and / or THBS1. The nucleotide sequences can be DNA or analogues thereof, or RNA or analogues thereof. The nucleotide sequence can be complementary to at least a portion of the FSGM. The binding of the nucleotide sequence used for the detection of the FSGM will depend on the level of stringency of the reaction. The nucleotide sequence can be an oligonucleotide, which can be used as a probe or primer, and therefore can contain modifications compatible with its function. For example, a short oligonucleotide used as a probe can carry a label (e.g., a fluorescent label) to enable detection and quantification.

[0198] This invention considers any suitable means, techniques and / or processes for detecting and / or measuring the FSGM of this invention. These methods are described in detail below.

[0199] 1. Detection of protein FSGM

[0200] This invention is intended for use in detecting the peptide FSGM of this invention (i.e., Tables 2, 4 and / or Figure 3The proteins, including secreted proteins CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1, can be detected using any suitable method. In some embodiments, the detection method is an immunoassay method that involves specific binding to the proteins listed in Tables 2 and 4 and / or Tables 4. Figure 3 Antibodies against one or more of the FSGMs (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1). The procedures for various useful immunoassay methods have been described in the scientific literature, such as Nakamura et al. (1987), which are incorporated herein by reference.

[0201] Generally, the immunobinding method involves obtaining a sample suspected of containing FSGM protein, peptide (e.g., FSGM secretory protein or peptide) or antibody, and contacting the sample or a portion thereof with the antibody or protein or peptide (as described in the invention) under conditions that allow for effective formation of immune complexes.

[0202] Immunobinding methods include those for detecting or quantifying the amount of a reactant component in a sample, requiring the detection or quantification of any immune complexes formed during the binding process. Here, a sample suspected of containing FSGM protein, peptide, or corresponding antibody may be obtained, and the sample may be contacted with the antibody or its encoded protein or peptide (as applicable), and then the amount of immune complexes formed under specific conditions may be detected or quantified.

[0203] The selected biological sample, or a portion thereof, is exposed to proteins under effective conditions for a period of time sufficient to allow the formation of immune complexes (primary immune complexes). Typically, complex formation simply involves adding the composition to the biological sample and incubating the mixture long enough for antibodies to form immune complexes with any present antigens (i.e., to bind to any present antigens). Subsequently, the sample-antibody composition is typically washed, for example with tissue sections, ELISA plates, dot blots, or protein blotting, to remove any non-specifically bound antibody species, allowing only those antibodies that specifically bind within the primary immune complexes to be detected.

[0204] Typically, the detection of immune complex formation is well known in the art and can be achieved through the application of a variety of methods. These methods are generally based on the detection of labels or FSGMs, such as any radioactive, fluorescent, biological, or enzyme-based tags or labels used standardly in the art. U.S. patents relating to the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference. Of course, as is known in the art, other advantages can be found in the use of a second binding ligand (e.g., a second antibody or biotin / antibiotin ligand binding configuration).

[0205] The protein used in the detection can itself be linked to a detectable label, which can then be easily detected, thereby allowing the amount of primary immune complexes in the composition to be determined.

[0206] Alternatively, the first added component bound within the primary immune complex can be detected by a second binding ligand having binding affinity for the encoded protein, peptide, or corresponding antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand itself is typically an antibody and may therefore be referred to as a "second" antibody. The primary immune complex is contacted with the labeled second binding ligand or antibody under effective conditions for a time sufficient to allow for the formation of secondary immune complexes. The secondary immune complex is then typically washed to remove any non-specifically bound labeled secondary antibody or ligand, and the remaining label in the secondary immune complex is then detected.

[0207] Other methods include a two-step approach for detecting primary immune complexes. As described above, a second binding ligand (e.g., an antibody) with binding affinity to the encoded protein, peptide, or corresponding antibody is used to form a secondary immune complex. After washing, the secondary immune complex is re-contaminated with a third binding ligand or antibody with binding affinity to the second antibody under effective conditions for a time sufficient to allow for the formation of an immune complex (the third immune complex). The third ligand or antibody is then linked to a detectable label, allowing for the detection of the resulting third immune complex. The system can provide signal amplification if desired.

[0208] The immunoassay method of this invention has significant utility in monitoring the efficacy of FXN replacement therapies (e.g., CTI-1601). In this document, biological or clinical samples suspected of containing encoded proteins or peptides or corresponding antibodies are used. However, these embodiments can also be applied to non-clinical samples, such as titration of antigen or antibody samples, selection of hybridomas, etc.

[0209] In particular, the present invention considers the use of ELISA as a type of immunoassay. It is contemplated that the FSGM proteins or peptides of the present invention (including secreted proteins or peptides, such as those defined in Table 2, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) may be used as immunogens in ELISA assays monitoring FXN replacement therapy. In its simplest and most direct sense, an immunoassay is a binding assay. Certain preferred immunoassays are various types of enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it is readily understood that the detection is not limited to these techniques; Western blotting, dot blots, FACS analysis, etc., may also be used.

[0210] In an exemplary ELISA, an antibody binding to the FSGM of the present invention (which includes secreted proteins, such as those defined in Table 2, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) is immobilized on a selected surface exhibiting protein affinity, such as wells in a polystyrene microtiter plate. A test composition, such as a clinical sample, suspected of containing the FSGM antigen, is then added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection is typically achieved by adding a second antibody specific to the target protein, linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection can also be achieved by adding a second antibody followed by a third antibody with binding affinity to the second antibody, wherein the third antibody is linked to a detectable label.

[0211] In another exemplary ELISA, a sample suspected of containing FSGM antigen is immobilized on 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 cases where the initial antibody is conjugated to a detectable label, the immune complex can be detected directly. Again, a second antibody with binding affinity to the first antibody can be used to detect the immune complex, wherein the second antibody is conjugated to a detectable label.

[0212] Regardless of the form used, ELISAs share certain common characteristics, such as coating, incubation or conjugation, washing to remove non-specific binding types, and detection of bound immune complexes. These are described below.

[0213] In antigen- or antibody-coated plates, the wells are typically incubated with the antigen or antibody solution overnight or for several hours. The wells are then washed to remove any incompletely adsorbed material. Any remaining usable surface of the wells is then “coated” with a nonspecific protein that is antigenically neutral to the test antiserum. These include bovine serum albumin (BSA), casein, and milk powder solutions. Coating allows for the blocking of nonspecific adsorption sites on the surface and thus reduces background caused by nonspecific binding of the antiserum to the surface.

[0214] In ELISA, secondary or tertiary detection methods are often more common than direct steps. Therefore, after protein or antibody binding to the wells, the wells are coated with a non-reactive material to reduce background and washed to remove unbound material. The immobilized surface is then brought into contact with the control and / or clinical or biological sample to be tested, under conditions that allow for effective formation of immune complexes (antigen / antibody). Detection of the immune complexes then requires the binding of a labeled second-level binding ligand or antibody, or a second-level binding ligand or antibody, to a labeled tertiary antibody or third-level binding ligand.

[0215] The phrase "under effective conditions that allow for the formation of immune complexes (antigen / antibody)" means that the conditions preferably include diluting the antigen and antibody with a solution (e.g., BSA, bovine gamma globulin (BGG), and phosphate-buffered saline (PBS) / Tween). These added reagents also help reduce nonspecific background.

[0216] "Suitable" conditions also refer to incubation at a temperature and duration sufficient to allow for effective binding. The incubation process typically lasts about 1 to 2 to 4 hours, preferably at a temperature of about 25 to 27°C, or overnight at a temperature of about 4°C.

[0217] After all incubation steps in ELISA, the contacted surfaces are washed to remove uncombined material. A preferred washing procedure includes washing with a solution (e.g., PBS / Tween or borate buffer). Specific immune complexes form between the test sample and the initially bound material, and even trace amounts of these immune complexes can be detected after subsequent washing.

[0218] To provide a detection method, the second or third antibody will have a relevant label to allow detection. Preferably, this will be an enzyme that produces colorimetric color upon incubation with a suitable chromogenic substrate. Thus, for example, it is desirable to contact the first or second immune complex with an antibody conjugated to urease, glucose oxidase, alkaline phosphatase, or catalase and incubate it for a period of time under conditions favorable to the development of further immune complex formation (e.g., 2 hours at room temperature in a solution containing PBS, such as PBS-Tween).

[0219] The amount of labeling is quantified, for example, by incubation with a chromogenic substrate (e.g., urea and bromocresol purple), after incubation with the labeled antibody and subsequent washing to remove unbound material. Quantification is then achieved by measuring the degree of color production (e.g., using a visible spectrophotometer).

[0220] The protein FSGMs of this invention (including secreted proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) can also be measured, quantified, detected, and analyzed using proteometry and instruments. Proteometry refers to the application of mass spectrometry in protein research. While not intended to be limiting, two methods are commonly used to characterize proteins using mass spectrometry. In the first method, the intact protein is ionized and then introduced into a mass spectrometer. This method is known as a “top-down” strategy for protein analysis. The two main methods for ionizing intact proteins are electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). In the second method, the protein is enzymatically digested into smaller peptides using a protease (e.g., trypsin). These peptides are then introduced into a mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. Thus, the latter approach (also known as “bottom-up” proteomics) uses the identification of peptide levels to infer the presence of proteins.

[0221] The total protein mass analysis of the FSGM of this invention (which includes secreted proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) can be performed using time-of-flight (TOF) MS or Fourier transform ion cyclotron resonance (FT-ICR). Both types of instruments are useful because of their wide mass range and, in the case of FT-ICR, high mass accuracy. The most widely used instruments for peptide mass analysis are MALDI time-of-flight instruments because they allow for high-speed acquisition of peptide mass fingerprints (PMFs) (1 PMF can be analyzed in approximately 10 seconds). Quadrupole-time-of-flight and quadrupole ion traps can also be used in this application.

[0222] The protein FSGM of this invention (including secreted proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) can also be determined in complex mixtures of proteins and molecules coexisting in biological media or samples; however, fractionation of the sample may be necessary and is considered herein. It should be understood that ionization of complex mixtures of proteins can lead to a tendency for more abundant proteins to be “flooded” or to suppress signals from less abundant proteins in the same sample. Furthermore, mass spectra from complex mixtures can be difficult to interpret due to the sheer number of components. Fractionation can be used to separate any complex protein mixture before mass spectrometry analysis. Two methods are widely used for fractionating proteins or peptide products derived from their enzymatic digestion. The first method fractionates whole proteins and is known as two-dimensional gel electrophoresis. The second method, high-performance liquid chromatography (LC or HPLC), is used for fractionating peptides after enzymatic digestion. In some cases, it may be desirable to combine these techniques. Any other suitable methods known in the art for fractionating protein mixtures are also considered herein.

[0223] Gel spots identified on 2D gels are typically attributed to a single protein. If protein identification is required, intragel digestion is usually employed, where the target protein spot is excised and digested via proteolytic hydrolysis. The mass of the peptides produced by digestion can be determined by mass spectrometry analysis using peptide mass fingerprinting. If this information does not allow for explicit protein identification, tandem mass spectrometry analysis of the peptides can be performed for de novo sequencing.

[0224] Characterizing protein mixtures using HPLC / MS is also known in the art as “shotgun proteomics” and MuDPIT (Multidimensional Protein Identification Technique). The peptide mixture produced by digesting the protein mixture is fractionated by one or two steps of liquid chromatography (LC). The eluent from the chromatographic stage can be directly introduced into the mass spectrometer via electrospray ionization or placed on a series of small spots for subsequent mass spectrometric analysis using MALDI.

[0225] The protein FSGMs of this invention (including secreted proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) can be identified using MS with a variety of techniques, all of which are considered herein. Peptide mass fingerprinting uses the mass of proteolytic peptides as input to a search of a predictive mass database generated by digestion of a known protein list. If a protein sequence in the reference list produces a large number of predictive masses matching experimental values, there is some evidence that the protein is present in the original sample. It can be further understood that the development of methods and instruments 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 identification of gel-separated proteins. Microcapillary LC-MS / MS has been successfully used for large-scale identification of single proteins directly from mixtures without gel electrophoretic separation (Link et al., 1999; Opitek et al., 1997).

[0226] Several recent methods have enabled the quantification of proteins via mass spectrometry. For example, carbon (… 13 C) or nitrogen ( 15 A stable (e.g., non-radioactive) heavier isotope of nitrogen can be incorporated into one sample, while another can be incorporated into a corresponding lighter isotope (e.g., nitrogen). 12 C and 14 N) labeling. Two samples are mixed before analysis. Peptides from different samples can be distinguished by their mass differences. The ratio of their peak intensities corresponds to the relative abundance ratio of the peptide (and protein). The most commonly used isotope labeling methods are SILAC (stable isotope labeling of amino acids in cell cultures) and trypsin-catalyzed... 18 O-labeling, ICAT (isotope-encoded affinity labeling), and iTRAQ (isoweight labeling for relative and absolute quantification) are all methods of "semi-quantitative" mass spectrometry, which can be performed without labeling the sample. This is typically done via MALDI analysis (in linear mode). The peak intensity or area from a single molecule (usually a protein) is correlated with the amount of protein in the sample. However, the individual signal depends on the protein's primary structure, sample complexity, and instrument settings. Other types of "label-free" quantitative mass spectrometry use spectral counting (or peptide counting) of digested proteins as a means of determining relative protein mass.

[0227] 2. Detection of nucleic acids corresponding to the protein FSGM

[0228] In some embodiments, the present invention relates to the detection of nucleic acid FSGMs, such as the corresponding gene or mRNA of the protein FSGM of the present invention, for example, Tables 2, 4 and / or Figure 3 These include CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1.

[0229] In various embodiments, the method of the present invention typically involves the determination of the expression levels of a set of genes in a biological sample. In practice, the determination of gene expression levels can be performed by any suitable method. For example, gene expression levels can be determined by detecting the expression of mRNA expressed by the target gene and / or by detecting the expression of a polypeptide encoded by the gene.

[0230] To detect nucleic acids encoding the FSGMs of this invention (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1), any suitable method may be used, including but not limited to DNA blotting, RNA blotting, polymerase chain reaction (PCR) (see, for example, U.S. Patent 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-PCT), anchored PCR, competitive PCR (see, for example, U.S. Patent No. 5,747,251), and rapid amplification of cDNA ends (RACE) (see, for example, Gene Cloning and Analysis: Current...). Innovations, 1997, pp. 99-115); ligase chain reaction (LCR) (see, for example, EP01320308), one-sided PCR (Ohara et al., Proc. Natl. Acad. Sci., 1989, 86: 5673-5677), in situ hybridization, Taqman-based detection (Holland et al., Proc. Natl. Acad. Sci., 1991, 88: 7276-7280), differential display (see, for example, Liang et al., Nucl. Acid. Res., 1993, 21: 3269-3275) and other RNA fingerprinting techniques, nucleic acid sequence-based amplification (NASBA) and other transcription-based amplification systems (see, for example, U.S. Patent Nos. 5,409,818 and 5,554,527), Qbeta replicase, strand displacement amplification (SDA), repair chain reaction (RCR), nuclease protection assay, subtraction-based methods, wait.

[0231] In other implementations, the gene expression levels of target FSGMs (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1) can be determined by amplifying complementary DNA (cDNA) or complementary RNA (cRNA) generated from mRNA and analyzing it using a microarray. Many different array configurations and their manufacturing methods are known to those skilled in the art (see, for example, U.S. Patent 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 for the simultaneous measurement of steady-state mRNA levels from a large number of genes. Currently widely used microarrays include cDNA arrays and oligonucleotide arrays. Analysis using microarrays is typically based on measurements of signal intensity received by self-labeled probes used to detect cDNA sequences from a sample that hybridize with nucleic acid probes immobilized at known locations on the microarray (see, for example, U.S. Patent 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 have been described in numerous scientific publications and patents (see, for example, M. Schena et al., Science, 1995, 270:467-470; M. Schena et al., Proc. Natl. Acad. Sci. USA 1996, 93:10614-10619; JJ Chen et al., Genomics, 1998, 51:313-324; U.S. Patent 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).

[0232] 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 fractionated or whole-cell RNA. When using RNA, it may be necessary to convert the RNA into complementary cDNA. In one embodiment, the RNA is whole-cell RNA and is used directly as a template for amplification.

[0233] Under conditions allowing selective hybridization, primer pairs that selectively hybridize to nucleic acids corresponding to any FSGM nucleotide sequence identified herein are contacted with isolated nucleic acids. Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that promote template-dependent nucleic acid synthesis. Multiple rounds of amplification (also known as “cycling”) are performed until a sufficient amount of amplification product is produced. The amplification product is then detected. In some applications, detection can be performed visually. Alternatively, detection may involve indirect identification of the product by chemiluminescence, radioactive imaging with incorporated radiolabels or fluorescent tags, or even by using a system using electrical or thermal pulse signals (Affymax technique; Bellus, 1994). After detection, the results observed in a given patient can be compared with, for example, a statistically significant control group of healthy patients. In this way, the amount of nucleic acid detected can be correlated with various clinical states.

[0234] As used herein, the term primer refers to any nucleic acid capable of initiating the synthesis of nascent nucleic acids in a template-dependent process. Typically, primers are oligonucleotides of 10 to 20 base pairs in length, but longer sequences may also be used. Primers can be provided in double-stranded or single-stranded form, although single-stranded form is preferred.

[0235] Many template-dependent methods can be used to amplify nucleic acid sequences present in a given template sample. One of the most well-known amplification methods is polymerase chain reaction (PCR), which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159 and Innis et al., 1990, each of which is incorporated herein by reference in its entirety.

[0236] In PCR, two primer sequences complementary to regions on the opposite complementary strands of the target nucleic acid sequence are prepared. An excess of deoxyribonucleoside triphosphates (DNPs) is added to the reaction mixture along with a DNA polymerase, such as Taq polymerase. If the target nucleic acid sequence is present in the sample, the primers will bind to the target nucleic acid, and the polymerase will extend the primers along the target nucleic acid sequence by adding nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers dissociate from the target nucleic acid to form the reaction product. Excess primers bind to both the target nucleic acid and the reaction product, and this process is repeated.

[0237] To quantify the amount of amplified mRNA, reverse transcriptase PCR amplification can be performed. Methods for reverse transcription of RNA into cDNA are well-known and described in Sambrook et al., 1989. Alternative methods of reverse transcription utilize thermostable DNA polymerases. These methods are described in WO90 / 07641, submitted December 21, 1990. Polymerase chain reaction methods are well-known in the art.

[0238] Another method for amplification is ligase chain reaction (“LCR”), disclosed in European Application No. 320308, 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 binds to the opposite complementary strand of the target, such that they are adjacent. In the presence of a ligase, the two probe pairs are ligated to form a single unit. By temperature cycling (as in PCR), the bound ligation unit dissociates from the target and then serves as the “target sequence” for ligating excess probe pairs. U.S. Patent No. 4,883,750 describes a similar method to LCR for binding probe pairs to a target sequence.

[0239] As described in PCT application number PCT / US87 / 00880, Qbeta replicase can also be used as another amplification method in this invention. In this method, an RNA replication sequence having a region complementary to the target is added to the sample in the presence of RNA polymerase. The polymerase replicates the subsequently detectable replication sequence.

[0240] Isothermal amplification can also be used for the amplification of nucleic acids in this invention, which uses restriction endonucleases and ligases to amplify target molecules containing nucleotides 5'-[α-thio]-triphosphate in one strand at the restriction site. Walker et al. (1992), which are incorporated herein by reference in their entirety.

[0241] Chain displacement amplification (SDA) is another method for isothermal amplification of nucleic acids, involving multiple rounds of chain displacement and synthesis, i.e., nick translation. A similar method, called repair chain reaction (RCR), involves annealing several probes over the entire region targeted for amplification, followed by a repair reaction where only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target-specific sequences can also be detected using cycling probe reaction (CPR). In CPR, a probe with the 3' and 5' sequences of non-specific DNA and the intermediate sequence of specific RNA hybridizes with DNA present in the sample. After hybridization, the reactants are treated with RNase H, and the probe products are identified as distinct products released after digestion. The original template is annealed with another cycling probe, and the reaction is repeated.

[0242] According to the present invention, other amplification methods can be used, which are described in GB application No. 2202328 and PCT application No. PCT / US89 / 01025, each of which is incorporated herein by reference in its entirety. In the former application, “modified” primers are used for 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., 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 is catalytically cleaved. After cleavage, the target sequence is fully released to be bound by the excess probe. Cleavage of the labeled probe indicates the presence of the target sequence.

[0243] Other nucleic acid amplification methods considered include transcription-based amplification systems (TAS), including nucleic acid sequence-based amplification (NASBA) and 3SR. Kwoh et al., (1989); Gingeras et al., PCT application WO88 / 10315, which is incorporated herein by reference in its entirety. In NASBA, nucleic acids for amplification can be prepared by standard phenol / chloroform extraction, thermal denaturation of clinical samples, treatment with lysis buffer, and extraction with guanidine hydrochloride for separating DNA and RNA using a microcentrifuge column or RNA. These amplification techniques involve annealing primers with target-specific sequences. After polymerization, the DNA / RNA hybrid is digested with RNase H, while the double-stranded DNA molecule is thermally denatured again. In either case, polymerization is followed by the addition of a second target-specific primer, resulting in complete double-stranding of the single-stranded DNA. The double-stranded DNA molecule is then subjected to multiple transcription by a polymerase (such as T7 or SP6). In an isothermal cyclic reaction, RNA is reverse transcribed into double-stranded DNA and then transcribed again by a polymerase (such as T7 or SP6). The resulting products, whether truncated or complete, all indicate target-specific sequences.

[0244] European Patent Application No. 329822 (incorporated herein by reference in its entirety) by Davey et al. discloses a method for nucleic acid amplification involving the cyclic synthesis of single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which can be used according to the present invention. The ssRNA is the first template of the first primer oligonucleotide, which is extended by reverse transcriptase (RNA-dependent DNA polymerase). DNA is then removed from the resulting RNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific to RNA in a DNA or RNA duplex). The resulting ssDNA is the second template of the second primer, which also includes the sequence of an RNA polymerase promoter (e.g., T7 RNA polymerase) homologous to its template at the 5' side. The primer is then extended by a DNA polymerase (e.g., the large “Klenow” fragment of E. coli DNA polymerase 1), thereby producing a double-stranded DNA (“dsDNA”) molecule having the same sequence as the original RNA between the primers and also having a promoter sequence at one end. This promoter sequence can be used by a suitable RNA polymerase to produce numerous RNA copies of the DNA. These copies can then be reintroduced into a cycle, resulting in very rapid amplification. With appropriate enzyme selection, this amplification can be completed isothermally in each cycle without the addition of enzymes. Due to the cyclic nature of the process, the starting sequence can be chosen in either DNA or RNA form.

[0245] Miller et al.'s PCT application WO89 / 06700 (the full text of which is incorporated herein by reference) discloses a nucleic acid sequence amplification protocol based on hybridization of a promoter / primer sequence with a target single-stranded DNA (“ssDNA”), followed by transcription of numerous RNA copies of that sequence. This protocol is non-cyclic, meaning it does not generate new templates from the resulting RNA transcripts. Other amplification methods include “race” and “one-sided PCR.” Frohman (1990) and Ohara (1989), each of whom is incorporated herein by reference in full.

[0246] The method of amplifying bi-oligonucleotides by linking two (or more) oligonucleotides in the presence of a nucleic acid having a generated bi-oligonucleotide sequence can also be used in the amplification step of this invention. Wu et al. (1989), the entire text of which is incorporated herein by reference.

[0247] The oligonucleotide probes or primers of this invention can be of any suitable length, depending on the specific assay format, specific needs, and the target sequence used. In a preferred embodiment, the oligonucleotide probes or primers are at least 10 nucleotides in length (preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32…), and they can be adapted to be particularly suitable for the selected nucleic acid amplification system and / or the hybridization system used. As is well known in the art, longer probes and primers are also within the scope of this invention. This invention also includes primers longer than 30, 40, and 50 nucleotides, and probes longer than 100, 200, 300, 500, 800, and 1000 nucleotides. Of course, longer primers have the disadvantage of being more expensive; therefore, primers with a length of 12 to 30 nucleotides are typically designed and used in the art. As is well known in the art, probes with lengths from 10 to greater than 2000 nucleotides can be used in the methods of the present invention. For the above-described identity percentage, probe and primer sizes (e.g., 16, 17, 31, 24, 39, 350, 450, 550, 900, 1240 nucleotides, ...) not specifically described are also within the scope of the present invention.

[0248] In other embodiments, detection methods may utilize hybridization techniques, such as the selection of specific primers or probes to anneal with the target FSGM followed by selective hybridization detection. As is well known in the art, oligonucleotide probes and primers can be designed by taking into account the melting point of hybridization with their target sequence (see below and Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1994, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., NY).

[0249] In order for hybridization to occur under the assay conditions of the present invention, the oligonucleotide primers and probes should contain an oligonucleotide sequence having 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 with a portion of the FSGM of the present invention. The probes and primers of the present invention are those that hybridize with the FSGM homologs of the present invention under stringent hybridization conditions, and those that hybridize with the FSGM homologs of the present invention under at least moderately stringent conditions. In some embodiments, the probes and primers of the present invention have complete sequence identity with the FSGM (gene sequence (e.g., cDNA or mRNA)) of the present invention. It should be understood that, based on the FSGM of the present invention disclosed herein, other probes and primers can be readily designed and used in the present invention using computer alignment and sequence analysis methods known in the art (see Molecular Cloning: A Laboratory Manual, Third Edition, edited by Cold Spring Harbor Laboratory, 2000).

[0250] 3. Antibodies and Labels

[0251] In some embodiments, the present invention provides methods and compositions for highly sensitive detection and quantification of FSGMs (e.g., CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1) of the present invention. Those skilled in the art will recognize that many strategies can be used to label target molecules to enable their detection or differentiation in a mixture of particles. Labels can be attached by any known method, including methods utilizing non-specific or specific interactions between the label and the target. Labels can provide a detectable signal or influence the migration rate of particles in an electric field. Furthermore, labeling can be performed directly or by binding a partner.

[0252] In some embodiments, the marking includes a binding partner that binds to the target FSGM, wherein the binding partner is attached to the fluorescent portion. The compositions and methods of the present invention can utilize highly fluorescent portions, for example, portions capable of emitting at least about 200 photons when stimulated by a laser emitting light of an excitation wavelength of said portion, wherein said laser is focused on a point containing said portion with a diameter not less than about 5 micrometers, and wherein the total energy directed by said laser to said point does not exceed about 3 microjoules. The portions suitable for the compositions and methods of the present invention are described in more detail below.

[0253] In some embodiments, the present invention provides a label for detecting biomolecules, the label comprising a binding partner of the biomolecule linked to a fluorescent portion, wherein the fluorescent portion is capable of emitting at least about 200 photons when stimulated by light of an excitation wavelength emitted by a laser, wherein the laser is focused on a point comprising the portion with a diameter not less than about 5 micrometers. And wherein the total energy directed by the laser to the point does not exceed about 3 microjoules. In some embodiments, the portion comprises a plurality of fluorescent entities, such as 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 portion 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 may be a fluorescent dye molecule. In some embodiments, the fluorescent dye molecule comprises at least one substituted indole ring system, wherein the substituent on the 3-carbon of the indole ring contains a chemically reactive group or a conjugated substance. In some embodiments, the dye molecule is an Alexa Flour molecule selected from Alexa Flour 488, Alexa Flour 532, Alexa Flour 647, Alexa Flour 680, or Alexa Flour 700. In some embodiments, the dye molecule is an Alexa Flour 647 dye molecule. In some embodiments, the dye molecule comprises a first type and a second type of dye molecule, such as two different Alexa Flour molecules, for example, wherein the first type and the second type of dye molecules have different emission spectra. The ratio of the first type of dye molecule to the second type of dye molecule can be, for example, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, or 1:4. The binding partner can be, for example, an antibody.

[0254] In some embodiments, the present invention provides a marker for the detection of biological FSGMs of the present invention, wherein the marker comprises a binding partner of the FSGM and a fluorescent portion, wherein the fluorescent portion is capable of emitting at least about 200 photons when stimulated by light of an excitation wavelength emitted by a laser, wherein the laser is focused on a point containing the portion with a diameter not less than about 5 micrometers. And wherein the total energy directed by the laser to the point does not exceed about 3 microjoules. In some embodiments, the fluorescent portion comprises fluorescent molecules. In some embodiments, the fluorescent portion comprises multiple 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 marker comprises about 2 to 4 fluorescent molecules. In some embodiments, the fluorescent dye molecule comprises at least one substituted indole ring system, wherein the substituent on the 3-carbon of the indole ring contains a chemically reactive group or a conjugated substance. In some embodiments, the fluorescent molecule is selected from Alexa Flour 488, Alexa Flour 532, Alexa Flour 647, Alexa Flour 680, or Alexa Flour 700. In some embodiments, the fluorescent molecule is an Alexa Flour 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.

[0255] As used herein, the term "antibody" is a broad term and is used in its general sense, including but not limited to naturally occurring antibodies and non-naturally occurring antibodies, including, for example, single-chain antibodies, chimeric antibodies, bifunctional antibodies, and humanized antibodies, as well as their antigen-binding fragments. An "antigen-binding fragment" of an antibody refers to the portion of the antibody involved in antigen binding. The antigen-binding site is formed by 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 choice of epitopes or regions of the molecules to which an antibody produces will determine its specificity, for example, for all forms of molecules, if present, or for all (e.g., all or substantially all) of the molecules.

[0256] Methods for preparing antibodies are well-established. Those skilled in the art will recognize that many methods can be used to produce antibodies, for example, as described in *Antibodies, A Laboratory Manual*, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, NY. Those skilled in the art will also understand that binding fragments or Fab fragments of analog antibodies can also be prepared from genetic information by various methods (*Antibody Engineering: A Practical Approach* (Borrebaeck, C., ed.), 1995, Oxford University Press, Oxford; *J. Immunol.* 149, 3914-3920 (1992)). Monoclonal and polyclonal antibodies targeting molecules (e.g., proteins) and biomarkers are also commercially available (Rand D Systems, Minneapolis, Minn.; HyTest, HyTest Ltd., Turku, Finland; Abeam Inc., Cambridge, Mass., USA; Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049, USA; Bios Pacific, Emeryville, Calif).

[0257] In some implementations, the antibody is a polyclonal antibody. In other implementations, the antibody is a monoclonal antibody.

[0258] Antibodies can be prepared using any of a variety of techniques known to those skilled in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Typically, antibodies can be produced using cell culture techniques, including the production of monoclonal antibodies as described herein, or by transfecting antibody genes into suitable bacterial or mammalian cell hosts to allow for the production of recombinant antibodies.

[0259] Monoclonal antibodies can be prepared using hybridoma methods, such as the techniques of Kohler and Milstein (Eur. J. Immunol. 6:511-519, 1976) and their modifications. These methods involve the preparation of immortalized cell lines capable of producing antibodies with desired specificity. Monoclonal antibodies can also be prepared using recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the antibody used in the disclosed methods can be isolated and sequenced using conventional methods. Recombinant antibodies, antibody fragments, and / or their fusions can be expressed in vitro or in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, or mammalian cells) and further purified as needed using known methods.

[0260] More specifically, monoclonal antibodies (MAbs) can be readily prepared using well-known techniques, such as those exemplified in U.S. Patent 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., purified or partially purified expressed protein, polypeptide, or peptide). The immunizing composition is administered in a manner that effectively stimulates antibody-producing cells. The methods used to produce monoclonal antibodies (MAbs) are generally the same as those used to prepare polyclonal antibodies. Rodents such as mice and rats are preferred animals; however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may offer certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, and most preferably BALB / c mice, as these are the most commonly used and generally yield a higher percentage of stable fusions.

[0261] Antibodies can also be derived from recombinant antibody libraries based on amino acid sequences encoded by polynucleotides that have been designed on a computer and synthesized. Methods for designing and obtaining sequences created on a computer 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. Patent No. 6,300,064).

[0262] Antibodies are digested using techniques known in the art to produce their antigen-binding fragments. For example, the proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (“F(ab)” fragments) each comprise a covalently heterodimer containing an intact antigen-binding site. Pepsin is capable of cleaving IgG molecules to provide several fragments, including the “F(ab’)2” fragment, which contains two antigen-binding sites. The “Fv” fragment can be produced by preferential proteolytic cleavage of IgM, IgG, or IgA immunoglobulin molecules, but is more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VL heterodimer containing antigen-binding sites that retain most of the antigen recognition and binding capabilities of the natural 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)).

[0263] 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 U.S. Patent No. 5,885,793).

[0264] Various expression systems exist in the art for the production of antibody fragments, including Fab fragments, scFv, VL, and VH. For example, expression systems derived from prokaryotes and eukaryotes can be used for the large-scale production of antibody fragments. Particularly advantageous are expression systems that allow for the secretion of large quantities of antibody fragments into culture media. Eukaryotic expression systems based on mammalian cells, insect cells, plants, transgenic animals, and lower eukaryotes have been described for the large-scale production of antibody fragments and antibody fusion proteins. For example, economical large-scale production of antibody fragments can be achieved in yeast fermentation systems. Large-scale fermentation of these organisms is well known in the art and is currently used for the large-scale production of several recombinant proteins.

[0265] In some cases, antibodies that bind to the protein FSGM used in the methods of this invention are commercially available or can be obtained without excessive experimentation.

[0266] In other embodiments, particularly when oligonucleotides are used as binding partners for detecting and hybridizing with mRNAN FSGM or other FSGM-based nucleic acids, the binding partner (e.g., oligonucleotide) may include a label, such as a fluorescent moiety or dye. Furthermore, any binding partner of the present invention (e.g., an antibody) may also be labeled with a fluorescent moiety. The fluorescence of this moiety will be sufficient to allow detection in a single-molecule detector (e.g., the single-molecule detector described herein). The term "fluorescent moiety" as used herein includes one or more fluorescent entities whose total fluorescence makes the moiety detectable in the single-molecule detector described herein. Thus, a 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 "molecular part" is used herein, it refers to a group of fluorescent entities, such as multiple fluorescent dye molecules, each individually ligated to a binding partner, or these entities may be ligated together, provided that the entities as a group provide sufficient fluorescence to be detected.

[0267] Typically, the fluorescence of the fraction involves a combination of quantum efficiency and photobleaching deficiency sufficient to make the fraction detectable above background levels in a single-molecule detector, exhibiting consistency with the desired detection limits, accuracy, and assay precision. For example, in some embodiments, the fluorescence of the fraction allows for detection limits of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.00001, or 0.000001 pg / ml and coefficients 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%) in the instruments described herein, and / or quantification of molecules (e.g., FSGM). In some implementations, the fluorescence of the fluorescent portion allows for the detection and / or quantification of molecules (e.g., FSGM) in the instruments described herein with limits of detection less than about 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, or 0.001 pg / ml and coefficients of variation less than about 10%. As used herein, the "limit of detection" or LoD includes the lowest concentration of a sample that can be identified as containing molecules of the target substance, such as a first non-zero value. It can be defined by zero variability and the slope of a standard curve. For example, the limit of detection for an experiment can be determined by running a standard curve, determining the zero value of the standard curve, and adding two standard deviations to that value. The concentration of the target substance that produces a signal equal to this value is the "lower limit of detection" concentration.

[0268] Furthermore, the portion possesses properties consistent with its use in the selected assay. In some embodiments, the assay is an immunoassay, wherein the fluorescent portion is linked to an antibody; the portion must have properties that prevent it from agglomerating with other antibodies or proteins, or from undergoing aggregation that does not exceed the accuracy and precision required for the assay. In some embodiments, a preferred fluorescent portion is a combination of 1) a high absorption coefficient; 2) a high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with the labeled target molecule (e.g., a protein), such as a dye molecule, so that it can be analyzed using the analyzers and systems of the present invention (e.g., without causing precipitation of the target protein, or of proteins already linked to the portion).

[0269] Any suitable fluorescent moiety can be used. Examples include, but are not limited to, Alexa Flour dyes (MolecularProbes, Eugene, Oreg.). Alexa Flour 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 use dyes selected from the following: Alexa Flour 647, Alexa Flour 488, Alexa Flour 532, Alexa Flour 555, Alexa Flour 610, Alexa Flour 680, Alexa Flour 700, and Alexa Flour 750. Some embodiments of the present invention use dyes selected from the following: Alexa Flour 488, Alexa Flour 532, Alexa Flour 647, Alexa Flour 700, and Alexa Flour 750. Some embodiments of the present invention use dyes selected from the following: Alexa Flour 488, Alexa Flour 532, Alexa Flour 555, Alexa Flour 610, Alexa Flour 680, Alexa Flour 700, and Alexa Flour 750. Some embodiments of the present invention utilize the Alexa Flour 647 molecule, which has maximum absorption between approximately 650 and 660 nm and maximum emission between approximately 660 and 670 nm. Alexa Flour 647 dye is used alone or in combination with other Alexa Flour dyes.

[0270] In some embodiments, the fluorescently labeled portion of the analyzer system of the present invention used to detect FSGM in a sample is a quantum dot. A quantum dot (QD), also known as a semiconductor nanocrystal or artificial atom, is a semiconductor crystal containing any number of electrons between 100 and 1000 and ranging from 2 to 10 nm. Some QDs can have diameters between 10 and 20 nm. QDs have high quantum yields, making them particularly suitable for optical applications. QDs are fluorophores that fluoresce by forming excitons, which are similar to the excited states of conventional fluorophores but have much longer lifetimes, up to 200 nanoseconds. This property provides QDs with low photobleaching. The energy levels of a QD can be controlled by changing its size and shape, as well as the depth of its potential. One optical characteristic of small exciton QDs is coloration, which is determined by the size of the dot. Larger dots produce redder fluorescence, or are more towards the red end of the spectrum. Smaller dots produce bluer fluorescence, or are more towards the blue end of the spectrum. The band gap energy that determines the energy and thus the color of the fluorescence is inversely proportional to the square of the QD size. Larger QDs have more closely spaced energy levels, thus allowing them to absorb photons with lower energy, i.e., those closer to the red end of the spectrum. Because the emission frequency of a point depends on the band gap, it is possible to control the output wavelength of the point with extremely high precision. In some embodiments, proteins detected by a single-molecule analyzer system are labeled with QDs. In some embodiments, the single-molecule analyzer is used to detect proteins labeled with a single QD and filters are used to allow the detection of different proteins at different wavelengths.

[0271] F. Separated FSGM

[0272] 1. Isolated polypeptide FSGM

[0273] One aspect of the present invention relates to isolated FSGM proteins and their biologically active portions, including secretory proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1, and polypeptide fragments suitable for use as immunogens against FSGM proteins or fragments thereof. In one embodiment, the native FSGM protein can be isolated using a suitable purification protocol employing standard protein purification techniques. In another embodiment, a protein or peptide comprising all or a fragment of the FSGM protein is generated using recombinant DNA technology. As an alternative to recombinant expression, such a protein or peptide can be chemically synthesized using standard peptide synthesis techniques.

[0274] "Isolated" or "purified" proteins, or their biologically active portions, are substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein originates, or, when chemically synthesized, substantially free of chemical precursors or other chemicals. The statement "substantially free of cellular material" includes protein formulations in which the protein is separated from the cellular components of the cells from which it is isolated or recombined. Therefore, proteins substantially free of cellular material include protein formulations having less than about 30%, 20%, 10%, or 5% (dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When the protein or its biologically active portion is recombined, it is also preferably substantially free of culture medium, i.e., the culture medium constitutes less than about 20%, 10%, or 5% of the volume of the protein formulation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from the chemical precursors or other chemicals involved in the protein synthesis. Therefore, such protein formulations, except for the target polypeptide, have less than about 30%, 20%, 10%, or 5% (dry weight) of chemical precursors or compounds.

[0275] The bioactive portion of the FSGM protein includes a polypeptide comprising an amino acid sequence sufficiently identical to or derived from the amino acid sequence of the FSGM protein, comprising fewer amino acids than the full-length protein, and exhibiting at least one activity of the corresponding full-length protein. Typically, the bioactive portion includes a domain or motif having at least one activity of the corresponding full-length protein. The bioactive portion of the FSGM protein of the present invention can be, for example, a polypeptide of length 10, 25, 50, 100, or more amino acids. Furthermore, other bioactive portions of the FSGM protein with missing regions can be prepared using recombinant techniques, and one or more functional activities against the native form of the FSGM protein can be evaluated.

[0276] Preferred FSGM proteins are listed in Tables 2 and 4 and / or Figure 3 Other useful 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 variations or mutagenesis.

[0277] To determine the percentage identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. Preferably, a global alignment is used to calculate the percentage identity between the two sequences. Alternatively, a local alignment is used to calculate the percentage identity between the two sequences. The percentage identity between two sequences is a function of the number of common positions shared by the sequences (i.e., % identity = # of common positions / total number of positions # (e.g., overlapping positions) × 100). In one embodiment, the two sequences are of the same length. In another embodiment, the two sequences are of different lengths.

[0278] Mathematical algorithms can be used to determine the percentage identity between two sequences. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) (Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm was incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the BLASTN program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of this invention. BLAST protein searches can be performed using the BLASTP program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of this invention. To obtain vacancy-bearing alignments for comparative purposes, as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402, a newer version of the BLAST algorithm called Gapped BLAST can be used. It is capable of performing vacancy-bearing local alignments for the programs BLASTN, BLASTP, and BLASTX. Alternatively, PSI-blast can be used for iterative searches to detect intermolecular distance relationships. When using the BLAST, Gapped BLAST, and PSI-blast programs, the default parameters for the respective programs (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 the algorithm of Myers and Miller (1988) CABIOS 4:11-17. This algorithm was incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residual table, a 12-fold vacancy length penalty, and a 4-fold vacancy penalty can be used. Another useful algorithm for identifying local sequence similarity and aligned regions is the FASTA algorithm, as described by 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 weighted residual table can be used, for example, with k-tuple values ​​of 2.

[0279] Whether gaps are allowed or not, a technique similar to the one described above can be used to determine the percentage identity between two sequences. When calculating the percentage identity, only exact matches are considered.

[0280] 2. Isolated nucleic acid FSGM

[0281] One aspect of this invention relates to isolated nucleic acid molecules encoding FSGM proteins or portions thereof (e.g., secretory proteins or portions thereof). The isolated nucleic acids of this invention also include nucleic acid molecules sufficient to be used as hybridization probes for identifying FSGM nucleic acid molecules, and fragments of FSGM nucleic acid molecules, such as those suitable for use as PCR primers for amplifying 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), as well as analogs of DNA or RNA produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred.

[0282] "Isolated" nucleic acid molecules are nucleic acid molecules separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. In one embodiment, the "isolated" nucleic acid molecule (preferably a protein-encoding sequence) does not contain sequences naturally flanking the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid). For example, in various embodiments, the isolated nucleic acid molecule may contain fewer than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences naturally flanking the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. In another embodiment, when "isolated" nucleic acid molecules (such as cDNA molecules) are produced by recombinant technology, they may be substantially free of other cellular material or culture medium, or substantially free of chemical precursors or other chemicals when chemically synthesized. Nucleic acid molecules substantially free of cellular material include formulations having fewer than about 30%, 20%, 10%, or 5% heterologous nucleic acid (also referred to herein as "contaminating nucleic acid").

[0283] The nucleic acid molecules of the present invention can be isolated using standard molecular biology techniques and sequence information from database records described herein. Using all or part of these nucleic acid sequences, the nucleic acid molecules of the present invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0284] The nucleic acid molecules of this invention can be amplified using standard PCR amplification techniques, employing cDNA, mRNA, or genomic DNA as templates and suitable oligonucleotide primers. The amplified nucleic acids can then be cloned into suitable vectors and characterized by DNA sequence analysis. Furthermore, all or part of the nucleotides corresponding to the nucleic acid molecules of this invention can be prepared using standard synthetic techniques, such as automated DNA synthesizers.

[0285] 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 the FSGM nucleic acid or the nucleotide sequence of the nucleic acid encoding the FSGM protein. The nucleic acid molecule complementary to the given nucleotide sequence is a nucleic acid molecule sufficiently complementary to the given nucleotide sequence, which can hybridize with the given nucleotide sequence to form a stable double strand.

[0286] Furthermore, the nucleic acid molecules of the present invention may comprise only a portion of a nucleic acid sequence, wherein the full-length nucleic acid sequence comprises FSGM nucleic acid or encoding an 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 nucleotide sequence region 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 the nucleic acid of the present invention.

[0287] Probes based on the nucleic acid molecular sequences of this invention can be used to detect transcripts or genomic sequences corresponding to one or more FSGMs of this invention. In some embodiments, the probe hybridizes to a nucleic acid sequence that crosses splice junctions. The probe contains a labeling group attached thereto, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used as part of a diagnostic test kit or test strip for identifying cells or tissues that express or misexpress the protein, for example by measuring the level of nucleic acid molecules encoding the protein in a cell sample from a subject, such as detecting mRNA levels or determining whether the gene encoding the protein or its translation control sequence has been mutated or deleted.

[0288] The invention further includes nucleic acid molecules that, due to the degeneracy of the genetic code, have nucleotide sequences different from those encoding FSGM proteins (e.g., proteins having sequences provided in the sequence listing) and thus encode the same protein.

[0289] Those skilled in the art will understand that DNA sequence polymorphisms leading to changes in amino acid sequences can exist in populations (e.g., human populations). Such genetic polymorphisms can also exist in individuals within a population due to natural allele variations. An allele is one of a set of genes that alternate at a given gene locus. Furthermore, it should be understood that DNA polymorphisms affecting RNA expression levels can also exist, which can influence the overall expression level of that gene (e.g., by affecting regulation or degradation).

[0290] As used herein, the phrase “allelic variant” refers to a nucleotide sequence present at a given gene locus or a polypeptide encoded by said nucleotide sequence.

[0291] As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide corresponding to the FSGM of this invention. Such natural allelic variations typically result in 1-5% variation in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the target gene in many different individuals. This can be readily performed using hybridization probes to identify the same gene loci in multiple individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that result from natural allelic variations and do not alter functional activity are within the scope of this invention.

[0292] In another embodiment, the isolated nucleic acid molecules of the present invention are 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 long, and hybridize with FSGM nucleic acid or nucleic acid encoding marker proteins under stringent conditions. As used herein, the term “hybridization under stringent conditions” is intended to describe the conditions used for hybridization and washing, under which nucleotide sequences having at least 60% (65%, 70%, preferably 75%) identity with each other generally 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 Protocols in Molecular Biology, John Wiley & Sons, NY (1989). A preferred, non-limiting example of stringent hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing once or multiple times in 0.2X SSC, 0.1% SDS at 50–65°C.

[0293] G. Coagulant Replacement Therapy

[0294] The methods provided in this disclosure refer to the determination of gene expression profiles related to FXN replacement therapy. FXN replacement therapy involves administering FXN replacement therapy to a subject in need. Various alternatives for delivering exogenous FXN are conceivable. FXN replacement therapy can be provided by delivery of FXN protein or by delivery of nucleic acid encoding FXN. FXN protein delivery can be the delivery of full-length FXN or the delivery of an FXN fusion protein.

[0295] As used herein, the term "FXN fusion protein" refers to an FXN or FXN fragment fused to a full-length or fragment thereof or to a peptide of a different protein. In some embodiments, the FXN fusion protein comprises a polypeptide containing FXN, such as a full-length hFXN (SEQ ID NO:1) or a mature hFXN (SEQ ID NO:2). In some embodiments, the FXN fusion protein further comprises a cell-penetrating peptide (CPP).

[0296] As used herein, the term "cell-penetrating peptide" or "CPP" refers to a short peptide sequence, typically 5-30 amino acids in length, that facilitates cellular uptake of various molecular cargoes (e.g., proteins). In the context of this invention, CPPs present in FXN fusion proteins facilitate the delivery of FXN fusion proteins to cells (e.g., recipient cells). CPPs can be polycationic, i.e., having an amino acid composition containing a high relative abundance of positively charged amino acids, such as lysine or arginine. CPPs can also be amphiphilic, i.e., having a sequence containing alternating patterns of polar / charged amino acids and nonpolar, hydrophobic amino acids. CPPs can also be hydrophobic, i.e., containing only nonpolar residues with a low net charge, or containing hydrophobic amino acid groups essential for cellular uptake.

[0297] The CPP that can be included in the FXN fusion protein can be any CPP known to those skilled in the art. For example, the CPP can be any CPP listed in the Database of Cell Penetrating Peptides CPPsite 2.0, the entire text of which is incorporated herein by reference. For example, a CPP useful in the context of this invention can be a cell-penetrating peptide derived from a protein selected from the following: transactivator of HIV transcription peptides (HIV-TAT), glycopeptide, wasp venom, transportan, permein, polyarginine, VP22, transportan, amphiphilic peptides such as MAP, KALA, ppTG20, proline-rich peptides, MPG-derived peptides, pep-1, and oligomers, arginine-rich peptides, and calcitonin-derived peptides.

[0298] In some embodiments, the CPP includes a TAT protein domain comprising amino acids 47-57 of the full-length HIV-TAT protein, which is 86 amino acids long (this 11-amino acid peptide may also be referred to herein as "HIV-TAT"; SEQ ID NO:4). In one embodiment, the CPP consists of HIV-TAT (SEQ ID NO:4). In some embodiments, the CPP comprises amino acids 47-57 of the full-length HIV-TAT protein, wherein a methionine is added at the amino terminus for initiation (12AA; "HIV-TAT+M"): MYGRKKRRQRRR (SEQ ID NO:5). Table 5 below lists the amino acid sequences of exemplary CPPs.

[0299] Table 5. Exemplary CPPs and Corresponding Sequences

[0300]

[0301]

[0302] 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 comprises full-length FXN (e.g., SEQ ID NO:1) and HIV-TAT (e.g., SEQ ID NO:4) as CPP.

[0303] In some embodiments, in the FXN fusion proteins of this disclosure, the CPP can be fused to an FXN (e.g., a full-length FXN) via a linker to form a single polypeptide chain. Examples of FXN fusion proteins include TAT-FXN fusion proteins, wherein a TAT or a TAT fragment can be directly or indirectly (via a linker) attached to the N- or C-terminus of the FXN. In one specific instance, the linker may comprise the amino acid sequence GG.

[0304] In some respects, CPP (e.g., HIV-TAT) present in the FXN fusion protein of this disclosure facilitates the delivery of the FXN fusion protein to cells (e.g., cells that may be present in vitro, outside the body, or in a subject). Once inside the cell, the FXN fusion protein can be processed by cellular mechanisms to remove CPP (e.g., HIV-TAT) from the FXN.

[0305] A specific example of a TAT-FXN fusion protein is called CTI-1601. CTI-1601 is a 24.9 kDa fusion protein that is currently being investigated as an FXN replacement therapy to restore functional levels of FXN in the mitochondria of patients with FRDA. CTI-1601 comprises an HIV-TAT peptide linked to the N-terminus of a full-length hFXN protein. The mechanism of action of CTI-1601 depends on the cell-penetrating ability of the HIV-TAT peptide to deliver CTI-1601 into cells, where it is subsequently translocated to the mitochondria and post-processed into mature hFXN. CTI-1601 is described in U.S. Provisional Patent Applications Nos. 62 / 880,073 and 62 / 891,029, each of which is incorporated herein by reference in its entirety. CTI-1601 includes the following amino acid sequence (amino acid 224): MYGRKKRRQRRRGGMWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGH PGSLDETTYERLAEETLDSLAFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA(SEQ ID NO:12).

[0306] FXN replacement can also be delivered via viral gene replacement, utilizing retroviruses, lentiviruses, adeno-associated virus vectors, and adenoviruses. Alternatively, FXN replacement therapy can be achieved through the upregulation of endogenous mutated FXN genes, depending on the number of GAA repeats, which are expressed at varying levels in carriers of mutated FXN alleles.

[0307] H.FSGM Application

[0308] In some aspects, the present invention provides methods for evaluating and / or monitoring the efficacy of FXN replacement therapy in subjects. The invention further provides methods for determining whether a subject requires FXN replacement therapy or for adjusting FXN replacement therapy, for example, determining whether FXN replacement therapy should be started, increased, decreased, or terminated in the subject. In some embodiments, the methods are performed by the subject using samples obtained from the same subject or as a point-of-care test, and the results can be evaluated by the subject or a physician. In one aspect, the invention constitutes the ability to obtain, analyze, detect, and / or measure one or more FSGMs of the invention (i.e., Tables 2, 4, and / or) through the methods of the invention. Figure 3The application of information related to FSGMs. In one embodiment, one or more FSGMs include secretory proteins, such as those defined in Table 2. For example, in one embodiment, one or more FSGMs include CYR61. In another embodiment, one or more FSGMs include one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1.

[0309] For example, when performing the detection and / or measurement of one or more protein FSGMs of the present invention (as described herein, i.e., Tables 2, 4 and / or Figure 3 When using the method of the present invention for FSGMs such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1, biological samples can be contacted with a detection reagent (e.g., a monoclonal antibody) that selectively binds to the target FSGM to form a protein-protein complex, which can then be further detected directly (if the antibody contains a label) or indirectly (if a secondary detection reagent, such as a second antibody, is used, which is also labeled). Therefore, the method of the present invention can detect the polypeptide FSGMs of the present invention (i.e., those listed in Tables 2, 4, and / or...). Figure 3 One or more FSGMs, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1, are converted into a protein-protein complex containing a detectable first antibody or a first and further second antibody. To identify the presence of a target FSGM, it is necessary to form such a protein-protein complex, and it is necessary to modify the physical properties and characteristics of the target FSGM by implementing the method of the present invention.

[0310] When the method of the present invention is used to detect one or more FSGMs corresponding to the present invention (i.e., Tables 2, 4 and / or Figure 3 The same principle applies when dealing with FSGMs, such as nucleic acids of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1. Specifically, when using amplification methods, this results in the formation of a new population of amplicones—newly synthesized molecules not present in the original biological sample—thus physically transforming the biological sample. Similarly, when hybridization probes are used to detect target FSGMs, physically new molecular species are efficiently generated through hybridization of the probe (optionally including a label) with the target biomarker mRNA (or other nucleic acids), which are then detected. These polynucleotide products are efficiently newly generated or formed due to the implementation of the methods of the present invention.

[0311] Methods for monitoring or evaluating the efficacy of FXN replacement therapy over time in subjects are also provided. In these methods, one or more FSGMs (i.e., Tables 2, 4, and / or Tables 4) are evaluated in a pair of samples (a first sample obtained from the subject at an earlier time point or prior to the treatment regimen and a second sample obtained from the subject at a later time point, such as when the subject has undergone at least a portion of the treatment regimen) are evaluated. Figure 3 The method of the present invention involves obtaining and analyzing more than two samples (e.g., 3, 4, 5, 6, 7, 8, 9 or more samples) at regular or irregular intervals to assess FSGM levels. Paired comparisons can be made between consecutive or non-consecutive subject samples. Trends in FSGM levels and rates of change in FSGM levels can be analyzed for any two or more consecutive or non-consecutive subject samples.

[0312] Exemplary methods for detecting the presence, absence, or alteration of expression levels of FSGM protein or its corresponding nucleic acid in a biological sample include obtaining a biological sample from a subject and contacting the biological sample with a compound or reagent capable of detecting peptides or nucleic acids (e.g., mRNA, genomic DNA, or cDNA). In some embodiments, the detection method of the present invention can therefore be used to detect mRNA, protein, cDNA, or genomic DNA in biological samples, for example, in vitro and in vivo.

[0313] The method provided herein for detecting the presence, absence, or variation of expression levels of FSGM proteins (e.g., secreted proteins, such as those defined in Table 2, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) or corresponding nucleic acids in biological samples includes obtaining a biological sample from a subject who may or may not contain the FSGM protein or nucleic acid to be detected, contacting the sample with an FSGM-specific binder (i.e., one or more FSGM-specific binders) capable of forming a complex with the FSGM protein or nucleic acid to be detected, and contacting the sample with a detection reagent for detecting the FSGM-FSGM-specific binder complex (if formed). It should be understood that the method provided herein for detecting FSGM expression levels in biological samples includes the step of performing a assay. In some embodiments of the detection method, the level of the FSGM protein or nucleic acid in the sample is absent or below a detection threshold.

[0314] The method involves forming a transient or stable complex between FSGM and an FSGM-specific binder. The method requires the complex (if formed) to form for a sufficient period of time to allow the detection reagent to bind to the complex and generate a detectable signal (e.g., a fluorescent signal, a signal from the product of an enzymatic reaction (e.g., a peroxidase reaction, a phosphatase reaction, a β-galactosidase reaction, or a polymerase reaction)).

[0315] In some embodiments, the same method is used to detect all FSGMs. In some embodiments, the same biological sample (e.g., the same body fluid or tissue) is used to detect all FSGMs. In some embodiments, various methods are used to detect different FSGMs. In some embodiments, FSGMs are detected in different biological samples. In some embodiments, the biological sample is 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, or a solid tissue sample, such as a skin biopsy sample, skin band, hair follicle, muscle biopsy sample, or oral sample.

[0316] FSGM levels can be detected based on absolute expression levels, or normalized or relative expression levels. Absolute FSGM levels are preferred when monitoring a subject's treatment or determining if there are changes in the subject's FXN status. For example, the expression levels of one or more FSGMs can be monitored in subjects undergoing FXN replacement therapy, e.g., at regular intervals, such as monthly intervals. Regulation of one or more FSGM levels over time can be monitored to observe trends in FSGM level changes. The expression levels of the FSGMs of this invention in subjects can be higher than those in normal samples, but lower than previous expression levels, thus indicating a lack of efficacy of the subject's FXN replacement therapy. Changes in FSGM levels, or no change at all, may be more relevant to a subject's treatment decision than the FSGM levels present in the population. Rapid changes in FSGM levels in subjects can be an indication of abnormal FXN levels, even if FSGM levels are within the normal range for the population.

[0317] As an alternative to determining the absolute expression level of FSGM, it can be determined based on the normalized expression level of FSGM. The expression level is normalized by comparing the absolute expression level of FSGM with that of genes that are not FSGM (e.g., constitutively expressed housekeeping genes). Suitable genes for normalization include housekeeping genes such as actin genes or epithelial cell-specific genes. This normalization allows for comparisons of expression levels between one sample (e.g., a sample from FXN-deficient subjects) and another sample (e.g., a normal sample) or samples from different sources.

[0318] This disclosure describes a method for assessing and / or monitoring the efficacy of FXN replacement therapy in patients in need, wherein a sample from the patient is analyzed. As used herein, the sample may be a bodily fluid sample, such as a blood sample, or a solid tissue sample, such as a skin biopsy sample, a muscle biopsy sample, or the sample may be a buccal sample. Essentially, any tissue or bodily fluid sample containing cells from which the FXN expression profile can be analyzed can be used in any of the methods disclosed herein. Alternatively, exosomes may be harvested to detect FSGM transcripts.

[0319] As described in the examples, a cell-based system was used to validate baseline FXN(-) expression profiles where FXN was downregulated, and treatment with FXN replacement therapies (e.g., FXN fusion proteins) demonstrated the opposite regulation of FSGM.

[0320] Any of the FXN expression profiles described herein may be part of one or more algorithms that can be used to analyze the FXN expression profile of a sample and determine whether the sample represents a sample from a normal subject, a sample from a patient before FXN replacement therapy, or a sample from a patient after FXN replacement therapy. One or more algorithms can be used to analyze samples from patients treated with FXN replacement drugs and determine whether the patient responded effectively to the treatment and, therefore, whether expressed a characteristic profile of the FXN replacement expression profile. Thus, the algorithm used to analyze the expression profile of a sample may use any one of a baseline FXN(-) expression profile, an FXN replacement expression profile, or a normal FXN expression profile or a combination thereof. Samples having an FXN signature expression pattern consistent with the baseline FXN(-) expression profile represent a lack of efficacy of FXN replacement therapy; samples having an FXN expression profile consistent with the FXN replacement expression profile and / or the normal FXN expression profile represent efficacy of FXN replacement therapy. In one embodiment, a classifier may be applied to the FXN expression profile obtained from a patient sample to obtain information about the sample, such as characterizing the state of the FXN expression profile or determining whether the patient has received FXN replacement therapy. Alternatively, a classifier can be applied to assess whether the FXN expression profile of a patient sample reaches a specific threshold required for FXN replacement therapy to be considered effective.

[0321] This disclosure also provides a treatment method for patients with mitochondrial diseases exhibiting FXN deficiency, the method comprising measuring an FXN expression profile in a sample from the patient and comparing the FXN expression profile obtained from the sample with at least one of a normal FXN expression profile, a baseline FXN(-) expression profile, or an FXN replacement expression profile. The sample may be further classified as having a normal FXN, a baseline FXN(-), or an FXN replacement profile. Using the comparison results of the sample FXN profile and the FXN profile described herein, a treatment regimen using FXN replacement therapy may be started, paused, or terminated. Alternatively, the FXN replacement therapy dosage regimen may be adjusted, for example, by increasing or decreasing it. In one embodiment, the method further comprises obtaining or providing the sample from a subject (e.g., a subject with FXN deficiency).

[0322] In some embodiments of the methods provided herein, the levels of one or more FSGMs selected from Tables 2, 4 and / or in biological samples are compared to the levels in control samples (e.g., samples from subjects lacking FXN). Figure 3 Elevated or decreased levels of one or more FSGMs (e.g., secreted proteins, such as those defined in Table 2, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1 and / or THBS1) indicate that FXN replacement therapy is effective.

[0323] In some embodiments of the methods provided herein, the expression levels of one or more FSGMs detected in biological samples, selected from Tables 2, 4 and / or [other data], are compared with those in control samples (e.g., samples from subjects lacking FXN). Figure 3 The absence of an increase or decrease in the expression levels of one or more FSGMs (e.g., secreted proteins, such as those defined in Table 2, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) is an indication that FXN alternative therapy (e.g., at the current dose) is ineffective and should be adjusted.

[0324] In some embodiments, the method may further include monitoring a subject receiving FXN replacement therapy. In some embodiments, the levels of one or more FSGMs detected in a second sample obtained from the subject after receiving FXN replacement therapy are compared to the levels in a first sample obtained from the subject before FXN replacement therapy is administered, with the levels selected from Tables 2, 4, and / or Tables 4 being compared to the levels in a first sample obtained from the subject before FXN replacement therapy is administered. Figure 3The absence or absence of elevated or decreased expression levels of one or more FSGMs (e.g., secreted proteins, such as those defined in Table 2, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) indicates ineffectiveness of FXN replacement therapy and / or lack of response in the subject to FXN replacement therapy. The method may further include the step of adjusting the FXN replacement therapy to a higher dose.

[0325] In other embodiments, the expression levels of one or more FSGMs (e.g., secreted proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) in a second sample obtained from the subject after administration of FXN replacement therapy are compared with those in a first sample obtained from the subject before administration of FXN replacement therapy. Figure 3 Elevated or decreased expression levels of FSGM (e.g., secreted proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1) indicate effectiveness of FXN replacement therapy and / or a response in the subject to FXN replacement therapy. The method may further include steps of adjusting FXN replacement therapy to a lower dose or discontinuing treatment.

[0326] In some implementations, FSGM levels increase after FXN replacement therapy in subjects (e.g., subjects lacking FXN). In some implementations, the FSGM is selected from a group consisting of mt-RNR1, mt-RNR2, ADNP, AI480526, C230034O21RIK, CCDC85B, CCDC85C, CTCFL, NRTN, PDE4A, PHF1, RPL37RT, SLC26A10, SNORD17, SUV420H2, WNK2, YAM1, or ZNRF1.

[0327] In other embodiments, FSGM levels decrease after the subject is treated with FXN replacement therapy (e.g., a subject lacking FXN). In some embodiments, the FSGM is selected from a group consisting of CYR61, mt-ATP6, mt-ATP8, mt-CO2, mt-CO3, mt-ND1, mt-ND2, mt-ND3 and mt-ND4, EGR1, EGR2, EGR3, IGF1, LAMP2 or SLIRP.

[0328] In other embodiments, the invention also relates to the analysis and consideration of any clinical and / or patient-related health data, such as data obtained from electronic medical records (e.g., the collection of electronic health information about individual patients or groups associated with various types of data, such as demographics, medical history, medication and allergies, immune status, laboratory test results, radiographic images, vital signs, personal statistics such as age and weight, and billing information).

[0329] In some implementations, the methods provided herein further include obtaining biological samples from subjects suspected of having mitochondrial diseases (such as FRDA).

[0330] In some implementations, the methods provided herein further include methods based on a selection from Table 2, Table 4, and / or Figure 3 The level of one or more FSGMs is used to select a treatment regimen for the subject. In some embodiments, the treatment method is initiated, modified, revised, or maintained based on the results of the method of the present invention, for example, when it is determined that the subject has responded to the treatment regimen, or when it is determined that the subject has not responded to the treatment regimen, or when it is determined that the subject has not responded to the treatment regimen adequately. In some embodiments, the treatment method is modified based on the results of the method.

[0331] In some embodiments of the diagnostic and monitoring methods provided herein, the methods further include separating components of a biological sample.

[0332] In some embodiments of the diagnostic and monitoring methods provided herein, the methods further include labeling components of biological samples.

[0333] In some embodiments of the diagnostic and monitoring methods provided herein, the methods further include amplifying components of a biological sample.

[0334] In some embodiments of the methods provided herein, the methods include forming a complex with a probe and components of a biological sample. In some embodiments, forming a complex with a probe includes forming a complex with at least one non-naturally occurring reagent. In some embodiments of the methods provided herein, the methods include treating a biological sample. In some embodiments of the methods provided herein, methods for detecting levels of at least two FSGMs include groups of FSGMs. In some embodiments of the methods provided herein, methods for detecting levels include attaching the FSGM to be detected to a solid surface.

[0335] I. Reagent Kit / Test Plate

[0336] The present invention also provides compositions and kits for evaluating and monitoring the efficacy of FXN replacement therapy. In some embodiments, the kits disclosed herein may be used by subjects for self-assessment or may be administered by subjects for assessment by a physician, or may be used as point-of-care kits.

[0337] These kits may contain one or more of the following: reagents that specifically bind to the FSGM of the present invention, and a set of instructions for measuring FSGM levels. In one embodiment, the FSGM includes secreted proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, or THBS1. In another embodiment, the FSGM includes CYR61.

[0338] This invention also includes kits for detecting the presence of FSGM protein or nucleic acid in biological samples. These kits can be used to assess and / or monitor the efficacy of FXN replacement therapy. For example, the kits may contain labeling compounds or reagents capable of detecting FSGM protein or nucleic acid in biological samples and methods for determining the amount of protein or mRNA in the sample (e.g., antibodies binding to the protein or fragments thereof, or oligonucleotide probes binding to DNA or mRNA encoding the protein). The kits may also include instructions for use, for practicing any of the methods provided herein or interpreting results obtained using the kit based on the teachings provided herein. The kits may also contain reagents for detecting control proteins in the sample (e.g., actin for tissue samples, albumin in blood or blood-derived samples) to standardize the amount of FSGM present in the sample. The kits may also contain purified FSGM for use as a control in detection or for quantification in assays performed with the kit. In some embodiments, the biological sample evaluated by the kits or test strips of this disclosure is a bodily 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, or a solid tissue sample, such as a skin biopsy sample, skin band, hair follicle, muscle biopsy sample, or oral sample.

[0339] The kit comprises a reagent assay strip used in methods for assessing and / or monitoring the efficacy of FXN replacement therapy. The assay strip includes at least two assay reagents, each specific for a single FSGM selected from the FSGM protein set provided herein. In one embodiment, the FSGM includes secreted proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, or THBS1. In another embodiment, the FSGM includes CYR61.

[0340] For antibody-based kits, the kit may comprise, for example: (1) a first antibody that binds to a first FSGM protein (e.g., attached to a solid support); and optionally, (2) a second, different antibody that binds to the first FSGM protein or the first antibody and is conjugated to a detectable label. In some embodiments, the kit comprises (1) a second antibody that binds to a second FSGM protein (e.g., attached to a solid support); and optionally, (2) a third, different antibody that binds to the second FSGM protein or the second antibody and is conjugated to a detectable label. The first and second FSGM proteins are different. In one embodiment, the first and second FSGMs are the FSGMs of the present invention, for example selected from Tables 2, 4 and / or Figure 3 One or more FSGMs. In some embodiments, the kit comprises a third antibody that binds to a third FSGM protein that is different from the first and second FSGM proteins, and a fourth different antibody that binds to the third FSGM protein or an antibody that binds to the third FSGM protein, wherein the third FSGM protein is different from the first and second FSGM proteins.

[0341] For oligonucleotide-based kits, the kit may contain, for example: (1) an oligonucleotide, such as a detectable labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding an FSGM protein, or (2) a pair of primers for amplifying FSGM nucleic acid molecules. In some embodiments, the kit may further contain, for example: (1) an oligonucleotide, such as a second detectable labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a second FSGM protein, or (2) a pair of primers for amplifying the second FSGM nucleic acid molecule. The first and second FSGMs are different. In one embodiment, the first and second FSGMs are the FSGMs of the present invention, for example selected from Tables 2, 4 and / or Figure 3 One or more FSGMs. In some embodiments, the kit may further comprise, for example: (1) an oligonucleotide, such as a third detectable labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a third FSGM protein, or (2) a pair of primers for amplifying the third FSGM nucleic acid molecule, wherein the third FSGM is different from the first and second FSGMs. In some embodiments, the kit contains a third primer specific to each nucleic acid FSGM to allow detection using quantitative PCR methods.

[0342] For chromatographic methods, the kit may contain FSGM, including labeled FSGM, to allow for the detection and identification of one or more FSGMs of the present invention by chromatography, for example selected from Tables 2, 4 and / or Figure 3One or more FSGMs. In some embodiments, the kit for the chromatographic method comprises a derivatized compound of one or more FSGMs used in the present invention. In some embodiments, the kit for the chromatographic method comprises a column for resolving the FSGMs used in the method.

[0343] For detecting the FSGM of the present invention (e.g. selected from Table 2, Table 4 and / or Figure 3 This invention relates to one or more FSGM-specific reagents that allow for the detection and quantification of FSGM in complex mixtures, such as cell or tissue samples. In some embodiments, the reagent is species-specific. In some embodiments, the reagent is not species-specific. In some embodiments, the reagent is isotype-specific. In some embodiments, the reagent is not isotype-specific.

[0344] In some implementations, the kit for assessing and / or monitoring the efficacy of FXN replacement therapy contains assays selected from Tables 2, 4, and / or Figure 3 The kit contains at least one reagent that is specific for detecting the levels of one or more FSGMs. In some embodiments, the kit further comprises a reagent for detecting the levels of one or more FSGMs selected from Table 2, Table 4, and / or Figure 3 Instructions for use in detecting, evaluating, and / or monitoring the level of at least one FSGM for the efficacy of FXN replacement therapy.

[0345] This invention provides a method for selecting from Table 2, Table 4 and / or Figure 3 A kit for detecting the level of at least one FSGM using at least one specific reagent. In one embodiment, the FSGM comprises secreted proteins such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, or THBS1. In another embodiment, the FSGM comprises CYR61.

[0346] In some embodiments, the kit may also contain, for example, buffers, preservatives, protein stabilizers, and reaction buffers. The kit may further contain components (e.g., enzymes or substrates) required for detecting the detectable label. The kit may also contain a control sample or a series of control samples that can be measured and compared with the test sample. Where appropriate, the control may be a control serum sample or a control sample of purified protein or nucleic acid with a known target FSGM level. Each component of the kit may be packaged in a separate container, and all the different containers may be in a single package, along with instructions for interpreting the results of assays performed using the kit.

[0347] The kit of the present invention may optionally contain other components for carrying out the methods of the present invention.

[0348] The present invention further provides a reagent test strip and at least one control reagent for detecting one or more FSGMs in a subject sample. In some embodiments, the FSGMs include at least two or more FSGMs, wherein each of the two or more FSGMs is selected from Table 2, Table 4 and / or Figure 3 In one embodiment, the one or more FSGMs include secreted proteins, such as those defined in Table 2, such as CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and / or THBS1. In another embodiment, the one or more FSGMs include CYR61.

[0349] In some embodiments, a control reagent is used in the detection of FSGM in biological samples, wherein the detection plate provides a control sample containing FSGM as a positive control and optionally for quantifying the amount of FSGM present in the biological sample. The detection plate may provide reagents for detecting control proteins (e.g., actin in tissue samples, albumin in blood or blood-derived samples) to standardize the amount of FSGM present in the sample. The detection plate may provide purified FSGM for detection, used as a control, or for quantification of the assay performed with the detection plate.

[0350] In some implementations, FSGM levels in the test strip are increased when compared to a control or subjects treated with an FXN substitute (e.g., subjects lacking FXN). In some implementations, FSGM is selected from a group consisting of mt-RNR1, mt-RNR2, ADNP, AI480526, C230034O21RIK, CCDC85B, CCDC85C, CTCFL, NRTN, PDE4A, PHF1, RPL37RT, SLC26A10, SNORD17, SUV420H2, WNK2, YAM1, or ZNRF1.

[0351] In some implementations, FSGM levels are reduced in the group when compared to a control or subjects receiving FXN replacement (e.g., subjects lacking FXN). In some implementations, FSGM is selected from groups consisting of CYR61, mt-ATP6, mt-ATP8, mt-CO2, mt-CO3, mt-ND1, mt-ND2, mt-ND3 and mt-ND4, EGR1, EGR2, EGR3, IGF1, LAMP2 or SLIRP.

[0352] In some embodiments, the detection plate includes one or more FSGMs that have elevated levels compared to controls after treatment of subjects (e.g., subjects lacking FXN) with FXN substitutes, and / or one or more FSGMs that have decreased levels compared to controls after treatment of subjects (e.g., subjects lacking FXN) with FXN substitutes.

[0353] In a preferred embodiment, the detection plate includes a method for detecting two or more FSGMs of the present invention (e.g., Tables 2, 4, and / or...). Figure 3The reagents listed in section 2, 3, 4, 5, 6, 7, 8, 9, or up to all FSGMs are preferably combined with a control reagent. In some embodiments, the detection plate contains a reagent for detecting CYR61; in some embodiments, the detection plate contains a reagent for detecting one or more of CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1; in some embodiments, the detection plate contains a reagent for detecting one or more of NR4A1, PTP4A1, ATF3, BTG2, EGR1, EGR2, EGR3, CYR61, and ABCE1; one or more of EGR1, EGR2, EGR3, and IGF1; MT-ND1, MT- One or more of ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, and CYCS; one or more of OPS2, VBP1, PSMA3, SLIRP, CUL2, DCUN1D1, UBE2D3, ZNRF1, RNF2, and LAMP2; one or more of RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, RPL38, RPS27L, and ABCE1; one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, and CYCS. Multiple; one or more of NR4A1, EGR1, EGR3, ADAMTS1, THBS1, SERPINE1, IGF1, PTGS2, and CYR61; one or more of PSMA3, CUL2, UBE2D3, ZNRF1, RPS15A, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38; one or more of ABCE1, RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, and RPL38; MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT One or more of -CO3, MT-ATP6, MT-ATP8, CYCS, TMEM-126A, MAOA and ABCE1; one or more of MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6 and MT-ATP8; one or more of ABCE1, RPL26, RPL38, RPL10, RPL32, RPS15A, RPL24, RPL39, SLIRP, COPS2, DCUN1D1, RNF2, EGR1, BTG2, ATF3, PTGS2, IGF1, SERPINE1 and THBS1;Or one or more of the following reagents: RPL26, THBS1, SERPINE1, IGF1, PTGS2, RPL10, RPS27L, CYCS, ATF3, BTG2, EGR1, EGR3, and CYR61.

[0354] In this detection plate, each FSGM is detected by a reagent specific to that FSGM. In some embodiments, the detection plate includes repeat wells, spots, or portions to allow analysis of various dilutions (e.g., serial dilutions) of biological samples and control samples. In a preferred embodiment, the detection plate allows for the quantitative detection of one or more FSGMs of the present invention.

[0355] In some embodiments, the detection plate is a protein chip for detecting one or more FSGMs. In some embodiments, the detection plate is an ELISA plate for detecting one or more FSGMs. In some embodiments, the detection plate is a detection plate for quantitative PCR to detect one or more FSGMs.

[0356] In some embodiments, the test kit is provided on a single device, comprising a test kit for one or more FSGMs of the present invention and at least one control sample. In some embodiments, the test kit is provided on a single device comprising a test kit for two or more FSGMs of the present invention and at least one control sample. In some embodiments, multiple test kits for detecting different FSGMs of the present invention have at least one homogeneous control sample to facilitate comparison of results between test kits.

[0357] Example

[0358] Example 1: Generation of FXN-induced signatures

[0359] FXN fusion protein

[0360] The FXN fusion protein used in this embodiment is a fusion protein comprising TAT-cpp and hFXN linked by a linker at the N-terminus of hFXN (Vyas et al., (2012) Hum. Mol. Genet. 21, 1230-1247), referred to herein as CTI-1601. The hFXN in the fusion protein is a full-length 210aa consortium precursor form containing an 80aa mitochondrial targeting sequence (MTS) at the N-terminus. The full-length hFXN protein (amino acids 1-210) comprises the amino acid sequence of SEQ ID NO:1.

[0361]

[0362]

[0363] When the protein is introduced into the mitochondrial matrix, it is cleaved at amino acid 81, producing the mature form of FXN, resulting in a mature 130-amino acid active FXN with a predicted molecular weight of 14.2 kDa (SEQ ID NO:1).

[0364]

[0365] The full-length hFXN (SEQ ID NO:1) comprises mature hFXN (SEQ ID NO:2) and a mitochondrial targeting sequence (MTS) having the following amino acid sequence: MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRK (SEQ ID NO:3)

[0366] The fusion protein comprises an HIV-TAT peptide (YGRKKRRQRRR) linked to the N-terminus of the full-length hFXN protein via a linker. The mechanism of action of the fusion protein relies on the cell-penetrating ability of the HIV-TAT peptide to deliver the fusion protein into the cell, where it is subsequently translocated to the mitochondria and post-processed into mature hFXN. Specific fusion proteins, such as CTI-1601, are described in U.S. Provisional Patent Applications Nos. 62 / 880,073 and 62 / 891,029, both of which are incorporated herein by reference in their entirety.

[0367] CTI-1601 includes the following amino acid sequence (224 amino acids):

[0368] MYGRKKRRQRRRGGMWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAE ETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA(SEQ ID NO:12).

[0369] FXN conditional knockout (KO) animals

[0370] The FRDA mouse model (FXN-KO:MCK-Cre) established by the Jackson laboratory was used. In this model, FXN... flox / null::MCK-Cre mice carry the Cre-conditional coagulant allele, the coagulant global knockout allele, and the cardiac / skeletal muscle-specific Cre recombinase transgene. Fxn flox / null ::MCK-Cre mice (cargo No. 029720) develop progressive cardiomyopathy due to a deficiency of coagulants in the heart and skeletal muscle. The mutants exhibit peak body weight at 9 weeks of age and have a mean survival of 86 ± 5 days. The cardiomyopathy phenotype is characterized by decreased heart rate and ejection fraction, as well as a shortening fraction distinguishable from non-mutant littermates at approximately 7 weeks of age. At 9 weeks of age, left ventricular mass is significantly increased compared to non-mutant littermates.

[0371] In vivo administration of FXN fusion protein

[0372] Three groups of animals (n=8 per group) were used in this study: one control and two knockout FXN-KO:MCK-Cre groups. At 5 weeks of age, animals in each group were administered 10 mg / kg of the FXN fusion protein or vector (50 mM NaOAc, 0.1 PEG). The drug was administered subcutaneously at a dose of 10 mL / kg. Animals received the test drug or vector every 48 hours until they reached 77 days of age. Twenty-four hours after the last dose (at 11 weeks), all animals were sacrificed, and tissues were cleaned by perfusion with PBS. The hearts were removed and preserved in an RNase-free reagent compatible with tissue preservation for further RNA analysis. One such reagent inactivates RNase and stabilizes RNA within the tissue, such as RNA Later. TM .

[0373] Cardiac performance

[0374] Since conditional knockout mice lose FXN in the heart, cardiac performance was assessed by conscious ECG and anesthetized echocardiography in all eight animals from each group before administration of the FXN fusion protein at 4 weeks of age and after administration of the FXN fusion protein at 8 and 10 weeks of age.

[0375] RNA sequencing (RNASeq)

[0376] RNA was isolated and prepared for sequencing from representatives of all groups (one control animal treated with the vector, two knockout animals treated with the vector, and two knockout animals treated with the FXN fusion protein). The KAPA Stranded RNA-seq kit was used with RiboErase (HMR). RNA sequencing was performed using the KR1151-v4.16 platform. Approximately 100 million paired-end Illumina reads, 151 nt in length (before trimming), were sequenced from each sample. Adapter sequences were trimmed from the FastQ files using cutadapt v1.2.1. Low-quality bases (Q<30) were removed from the 3' ends of the reads, and reads with more than 30% low-quality bases (Q<30) were filtered out. The remaining reads were aligned to the April 2018 Ensembl release (GRCm38 v92 primary set) of the mouse reference genome using RSEM v1.3.0 with STAR v2.5.3 specified as the aligner. RSEM was used to generate *.genes.results files for each sample.

[0377] Fibroblasts from patients with Friedreich ataxia (FDRA)

[0378] Fibroblasts from FDRA patients are designated FA GM03816 and FA 68 in the results and figures. Cells were maintained in high-glucose DMEM medium supplemented with 10% FBS and grown to confluence. Once confluence was achieved, cells were held for 24 hours without changing the medium before RNA isolation.

[0379] RNA extraction

[0380] Upon reaching confluence, cells were rinsed with PBS buffer. Total RNA was extracted using the RNeasy Mini Kit (Qiagen catalog number 74104), which includes an optional genomic DNA removal step, according to the manufacturer's protocol. The total RNA concentration in solution was measured using a Beckman Coulter DU730 UV / Vis spectrophotometer.

[0381] Reverse transcription (RT)-cDNA synthesis

[0382] According to the manufacturer's instructions, reverse transcription was performed using 4 μg of total RNA in a 30 μL reaction with the Superscript IV VILO Master Mix with ezDNase kit (Invitrogen catalog number 766500).

[0383] Quantitative real-time polymerase chain reaction (PCR)

[0384] This paper describes the use of interchangeable quantitative PCR or real-time (RT) PCR, performed using the Quant Studio 5 automated system (Applied Biosystems). The master mix was TaqMan Fast Advanced Master mix (ThermoFisher 4444557), and the plates were MicroAmp rapid 96-well plates (ThermoFisher 4346907). Typically, each reaction (per well) consisted of the following: 10 μL Master Mix (20X) + 0.33 μL housekeeping gene primers / probes (60X) + 1 μL target gene primers / probes (20X) + 6.67 μL nuclease-free H2O + 2 μL cDNA (approximately 25 ng). PCR cycles consisted of 40 cycles of incubation at 50°C for 2 min with UNG (from the uracil-DNA glycosylase family, used to remove uracil), incubation at 95°C for 2 min for polymerase activation, and incubation at 95°C for 1 sec and 60°C for 20 sec.

[0385] PCR reactions involve forward and reverse primers. For example, the forward primer is 18 to 22 nucleotides in length and may include 15, 16, 17, 18, 19, 20, or 21 nucleotides identical to the target nucleic acid, which is listed in Tables 2 and 4 and / or... Figure 3 The sequence of any of the FSGMs shown. The reverse primer can be complementary to the target nucleic acid. The reverse primer may also include a sequence complementary to the adapter sequence.

[0386] Quantitative PCR (qPCR) of housekeeping genes

[0387] β-actin transcripts were used as housekeeping genes because their expression levels were constant in fibroblasts derived from FA patients (Disease Models & Mechanisms (2017) 10, 1353-1369 doi:10.1242 / dmm.030536). Primer-probe kits (Hs01060665_g1) were purchased from Thermo Fisher. Probe oligonucleotides were labeled with fluorescent dyes (e.g., VIC dyes with a maximum absorption of 538 nm and a maximum emission of 554 nm, thus emitting in the green-yellow portion of the visible spectrum, or HEX dyes) and non-fluorescent quenchers (NFQ-MGB quencher).

[0388] Quantitative PCR of FXN-sensitive genomic biomarkers (FXN signature)

[0389] To develop the methods disclosed herein, target genes were selected based on RNASeq analysis from the hearts of FXN conditionally knockout mice treated with or without FXN fusion proteins; these are referred to herein as FXN-sensitive genomic biomarkers (FSGMs). The ThermoFisherq PCR primer-probe sets for the selected targets are shown in Table 1. Target gene probes were labeled with a fluorescent dye (Fluorescein amidite, FAM) along with a quencher (NFQ-MGB).

[0390] Table 1. Primer-probe set

[0391] ABCE1-Hs00759267_s1 Lars2-Hs01118920_m1 RAP2c-Hs00221801_m1 ADAMTS1-Hs00199608_m1 MAOA-Hs00165140_m1 RnF13-Hs00961508_g1 ALAS1-Hs00167441_m1 MKI67-Hs00606991_m1 RPL10-Hs01095478_g1 APOLD1-Hs00707371_S1 MPC1-Hs00211484_m1 RPL24-Hs02338570_gH ATF3-Hs00231069_m1 mt-ATP6-Hs02596862_g1 RPL26-Hs00864008_m1 CH25H-Hs02379634_s1 mt-ATP8-H202596863_g1 RPL32-Hs00851655_g1 CYR61-Hs00155479_m1 mt-CO2-Hs02596865_g1 RPL38-Hs01019601_g1 CUL2-Hs00180203_m1 mt-CO3-Hs02596866_g1 RPL39-Hs04194816_g1 CYCS-Hs01588974_g1 mt-ND1-Hs02596873_s1 RPS15A-Hs03043791_m1 EGR1-Hs00152928_m1 mt-ND2-Hs02596874_g1 RPS23-Hs01922548_s1 EGR2-Hs00166165_M1 mt-ND3-Hs02596875_s1 RPS27L-Hs00955038_g1 EGR3-Hs00231780_m1 mt-ND4-Hs02596876_g1 SLC25A25-Hs01595834_g1 EIF1AX-Hs00796778_s1 mt-RnR1-Hs02596859_g1 SLIRP-Hs00364015_m1 hFXN-Hs00175940_m1 mt-RnR2-Hs02596860_s1 SMTN-Hs01022255_g1 HIF1a-Hs00153153_m1 NR4A1-Hs00374226_m1 UBE2D3-Hs00704312_s1 IGF1-Hs01547656_m1 PDE4a-Hs00183479_m1 YARS-Hs00169373_m1 LAMP2-Hs00903587_m1 PICALM-Hs00200318_m1 ZNRF1-Hs00936381_m1

[0392] To verify the validity of the PCR results, two tests were used as quality control: (1) linearity of the signal, which was established for each primer / probe by titrating normal HEK293 RNA as a function of cDNA concentration with dCT (differential cycle threshold); and (2) CT in reverse transcriptase (RT) control samples that were not quantifiable, to confirm that the signal was not due to contamination of genomic DNA (gDNA) in the RNA preparation.

[0393] Cycle threshold (CT) values ​​are generated using a PCR apparatus. Two CT values ​​are assigned to each well, one for β-actin and one for the target gene. The ΔCT value (target CT - β-actin CT) is calculated by subtracting the β-actin CT from the target gene CT in each well. The mean ΔCT of the baseline sample (i.e., untreated) is calculated and used as the baseline sample for either "normal" or "untreated". The baseline sample ΔCT is subtracted from the baseline sample ΔCT of the "treated" sample. (Treatment sample ΔCT [subtract] baseline sample ΔCT) yields the ΔΔCT for each "treated" sample. The fold change for each individual sample is calculated using Equation 2ΔΔCT. The mean of the replicates is then taken, and the standard deviation is calculated as the error.

[0394] In vivo processed genome expression

[0395] Hearts were collected from knockout (KO) mice or control mice treated with the FXN fusion protein, and RNA was extracted for analysis. RNA sequencing, as described above, was used to obtain transcriptional expression profiles triggered by or without FXN fusion protein treatment. Differential gene expression analysis after FXN fusion protein treatment was performed as described below.

[0396] Differential expression (DE) analysis of RNASeq results was performed using Bioconductor libraries in versions R3.44 and 3.7. Unadjusted “Expected Counts” columns from rsem were entered as tximport and used as input for DEseq2. Tximport and DESeq2 were used with all default settings except that genes with an epigenetic length of 0 were reclassified as having a length of 0.1 before running DESeq2. Two initial reports (data not shown) were compiled based on the DESeq2 analysis: “All conaffin knockout samples vs. all wild-type samples”, and “All drug-treated samples vs. all vector control samples”. Data in the “Drug-treated samples vs. all vector control samples” report were categorized according to adjusted p-values ​​(padj). Genes with padj < 0.005 were considered for further evaluation.

[0397] In the “Coagulant Knockout (KO) Samples vs. All Wild-Type (WT) Samples” report, a cutoff value of 320 (readout from RNASeq analysis) was applied, and genes below this threshold were not further considered if they were downregulated in the “Drug-Treatment Samples vs. All Vector-Treatment Control Samples” report.

[0398] Genes meeting these criteria, namely those whose expression is (i) greater than 320 in “coagulant knockout vs. WT samples” and (ii) upregulated or downregulated in “drug-treated vs. vector-treated knockout animals”; or those whose expression is (i) less than 320 in “coagulant knockout vs. WT samples” and (ii) upregulated only in “drug-treated vs. vector-treated knockout animals”, are further restricted to genes with a log2 fold change greater than 0.584 or less than -0.584, corresponding to approximately 2-fold induction or inhibition, respectively.

[0399] Genes meeting all the above criteria were used as coagulant-sensitive genomic markers (FXN-induced signatures) to generate FXN expression profiles and to examine the opposite regulation between different treatments. Other genes slightly below the above criteria, but with strong plausibility after further careful examination, were included in the potential FSGM list as genes to be tested in other models. For example, mt-CO2 was included, which was upregulated 3.21-fold in FXN KO compared to WT animals and downregulated 0.57-fold in CTI-1601-treated KO compared to vector-treated KO, as it just missed the significance cutoff. Other mt-DNA encoding complex IV subunits did show being affected (mt-Co2 was expected to be similarly regulated since the gene is polycistronic), and one of the major protein levels regulated by LRPPRC (significant hit) was mt-Co2. This progressive selection approach allowed for the identification of genes oppositely regulated by FXN protein substitution after FXN gene ablation, defining alternative FXN expression profiles. These genes respond to FXN and are likely target genes of FXN, and are considered true markers of FXN substitution, unlike other genes that are not regulated in the opposite way and may simply be markers of tissue remodeling or inflammatory changes (data not shown).

[0400] In vivo treatment of mice with the FXN fusion protein resulted in significant differential expression of 102 (102) genes, either upregulated or downregulated compared to control (fold regulation in “KO vs. WT” = baseline coagulant (-) signature), which are detailed in Table 2. Most importantly, these genes were found to be conversely regulated in coagulant-deficient mouse models when treated with the FXN fusion protein (fold regulation in “drug (FXN fusion protein) vs. vector (Veh)” = alternative coagulant signature). In other words, some genes that were upregulated in the absence of coagulant were downregulated after treatment with the FXN fusion protein. Conversely, some genes that were downregulated in the absence of coagulant were upregulated after treatment with the FXN fusion protein. This result is particularly surprising because coagulant has never been described as a transcriptional regulator, and therefore regulation of downstream genes was not expected.

[0401] The coagulant-sensitive genomic markers (FSGMs) shown in Table 2 can be grouped, for example, according to homology and / or function. For instance, several mitochondrial genes that are induced or repressed in knockout animals show a reversal of their expression patterns after treatment with the FXN fusion protein. The mitochondrial gene transcripts CYR61, mt-ATP6, mt-ATP8, mt-CO2, mt-CO3, mt-ND1, mt-ND2, mt-ND3, and mt-ND4 are downregulated after treatment with the FXN fusion protein, while the mitochondrial gene transcripts mt-RNR1 and mt-RNR2 are upregulated, as shown in Table 2 under “FXN fusion protein vs. Veh”. After treatment with the FXN fusion protein, the expression of transcripts from the EGR family, EGR1, EGR2, and EGR3, or transcripts from the insulin-like growth factor family, IGF1 and LAMP2, is also downregulated. Similarly, SLIRP expression is downregulated upon treatment with the FXN fusion protein. Another set of biomarkers showing altered expression included ADNP, AI480526, C230034O21RIK, CCDC85B, CCDC85C, CTCFL, NRTN, PDE4A, PHF1, RPL37RT, SLC26A10, SNORD17, SUV420H2, WNK2, YAM1, and ZNRF1; these biomarkers were upregulated after treatment with the FXN fusion protein.

[0402] The FSGMs shown in Table 2 can also be grouped, for example, according to whether they are secreted proteins. For example, as shown in Table 2, CYR61, ADAMTS1, ASPN, FAM177A, IGF1, LOX, NRTN, SERPINE1, STC1, and THBS1 are all secreted proteins.

[0403] Table 2. Differential gene expression after treatment with FXN fusion protein (FXN-induced signature)

[0404]

[0405]

[0406] In Table 2, the values ​​contained in the columns labeled “Knockout (KO) vs. Wild Type (WT)” (Column 1) and “FXN-Fusion vs. Vector” (Column 2) indicate whether FSGM increases or decreases with effective FXN replacement therapy.

[0407] More specifically, for a given FSGM, if the value in column 2 (FXN-fusion vs. carrier) is less than 1.0 and the value in column 1 (KO vs. WT) is greater than 1.0, the FSGM level increases (compared to wild type) under FXN depletion conditions and decreases when effective FXN replacement therapy is administered, and is thus modulated inversely (e.g., by CYR61).

[0408] Conversely, for a given FSGM, if the value in column 2 (FXN-fusion vs. vector) is greater than 1.0 and the value in column 1 (KO vs. WT) is less than 1.0, the FSGM level is reduced under FXN depletion conditions (compared to wild type) and increased when effective FXN replacement therapy is administered, and is thus modulated inversely (e.g., YAM1).

[0409] Example 2: String Analysis of FSGM

[0410] This example describes a string analysis of the FSGMs shown in Table 2, demonstrating that the protein products of the FSGMs are at least partially biologically linked as a group.

[0411] String analysis was performed using the string database (string-db.org; Szklarczyk et al. (2015) DOI:10.1093 / nar / gkv1277 and its references) on 85 protein products of FXN-sensitive genomic markers described in Table 2. String analysis is as follows: Figure 1 As shown. String analysis represents instances of known and / or predicted protein-protein interactions based on their functions. Parameters used to generate clusters in string analysis are: nodes = 85; edges = 97; average node degree = 2.28; average local clustering coefficient = 0.345; expected number of edges = 35; PPI enrichment p-value < 1.0e-16. The minimum required interaction score is 0.700 (high confidence). Disconnected nodes in the network are hidden. The following parameters are used as active interaction sources: text mining, experiments, databases, co-expression, neighborhood, gene fusion, and co-occurrence. The partitioning used allows a marker to be part of more than one cluster. For simplification, from Figure 1 Only some clusters are clearly visible in the image, while other clusters are not visible in the image, but these are also listed below in this article.

[0412] Under the parameters mentioned above, the following are examples of clusters and their respective markers obtained through string analysis:

[0413] - Response to ribosome depletion or endoplasmic reticulum (ER) stress - NR4A1, PTP4A1, ATF3, BTG2, EGR1, EGR2, EGR3, CYR61, ABCE1;

[0414] - Mitochondrial energy production - MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, CYCS;

[0415] - Regulation of proteasome and unfolded protein responses - COPS2, VBP1, PSMA3, SLIRP, CUL2, DCUN1D1, UBE2D3, ZNRF1, RNF2, LAMP2;

[0416] - Ribosome functions - RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, RPL38, RPS27L; ABCE1,

[0417] - Respiratory chain - MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, CYCS;

[0418] - Cardiac development - NR4A1, EGR1, EGR3, ADAMTS1, THBS1, SERPINE1, IGF1, PTGS2, CYR61;

[0419] - Macromolecular catabolic metabolism - PSMA3, CUL2, UBE2D3, ZNRF1, RPS15A, RPL24, RPL32, RPL26, RPL10, RPL39, RPL38;

[0420] - Translation Start- ABCE1, RPS15A, EIF1AX, RPL24, RPL32, RPL26, RPL10, RPL39, RPL38;

[0421] - Mitochondrial components - MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8, CYCS, TMEM-126A, MAOA, ABCE1;

[0422] -Oxidative phosphorylation-MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-CO3, MT-ATP6, MT-ATP8;

[0423] - Negative regulation of macromolecular catabolic processes - ABCE1, RPL26, RPL38, RPL10, RPL32, RPS15A, RPL24, RPL39, SLIRP, COPS2, DCUN1D1, RNF2, EGR1, BTG2, ATF3, PTGS2, IGF1, SERPINE1, THBS1;

[0424] - Regulation of apoptosis - RPL26, THBS1, SERPINE1, IGF1, PTGS2, RPL10, RPS27L, CYCS, ATF3, BTG2, EGR1, EGR3, CYR61.

[0425] Other clusters with the highest false discovery rate of 0.003 derived from string analysis include: membrane-targeting proteins, membrane-targeting SRP-dependent co-translational proteins, translation, nuclear transcription mRNA catabolism, primary metabolism, cellular metabolism, protein targeting, peptide metabolism, negative regulation of cellular processes, cellular macromolecular metabolism, organic matter metabolism, regulation of cellular protein metabolism, regulation of protein metabolism, skeletal muscle cell differentiation, respiratory electron transport chain, metabolic processes, cytoplasmic translation, cell cycle regulation, cellular component organization of biogenesis, mitochondrial electron transport, NADH to ubiquinone, angiogenesis, regulation of macromolecular metabolism, catabolism of nucleobase-containing compounds, establishment of protein localization in organelles, cellular processes, cellular macromolecular catabolism, purine ribonucleoside monophosphate metabolism, macromolecular catabolism, responses to stress, and responses to oxygen.

[0426] The protein clusters based on string analysis revealed that FSGM protein products have more potential interactions among themselves than would be expected from a random set of proteins of similar size taken from the genome. This enrichment suggests that the protein products of FSGM are at least partially biologically linked as groups.

[0427] Example 3: Selection of potential FXN target genes after in vitro treatment

[0428] Identification of genes conversely regulated by FXN gene ablation and subsequent in vivo FXN protein replacement suggests that gene expression changes induced by FXN replacement therapy can serve as an indicator of treatment efficacy in patients treated with FXN replacement therapy. Based on this premise, baseline FXN-induced signatures were tested in two in vitro human cell models: fibroblasts derived from Friedreich ataxia (FDRA) and...

[0429] Expression of conamin protein and mRNA in human cell models

[0430] Detection of conamin in FDRA-derived fibroblasts protein Conamin protein expression and mRNA expression were visualized and quantified by Western blotting gel electrophoresis, while conamin mRNA expression was quantified by qRT-PCR. Results are shown in... Figure 2 And in Table 3.

[0431] Figure 2The assay shows the presence of coagulants in control GM23971 cells and FDRA-derived fibroblasts FAGM03816 and FA 68. β-actin signaling was used for coagulant signaling normalization during protein expression quantification. Coagulant levels in control GM23971 cells were considered 100%, compared to 64% and 31% of the control levels in FDRA-derived fibroblasts FAGM03816 and FA 68, respectively. Coagulant mRNA quantification showed similar results when compared to control cells, with approximately 66% and 32% mRNA expression in FAGM03816 and FA 68, respectively (Table 3).

[0432] Table 3. Expression of co-electrolyte proteins and mRNAs in FRDA-derived fibroblasts (FA) compared to normal fibroblasts.

[0433]

[0434] Development of conamin-induced genetic signature in cell models

[0435] Development of baseline FXN(-) expression profile In a comparison between normal cells (N-GM07522 and N-GM23971) and coagulant-depleted cells derived from FDRA-derived fibroblasts (FA-GM 03816, FA-GM 04078, FA-4654, FA-68 (not shown), FA-4675, and FA-4194 (not shown)), an instance of baseline FXN deficiency (FXN(-)) expression profile was identified and as follows: Figure 3 As shown. The expression of altered ABCE1, APOLD1, ATF3, CYR61, CUL2, CYCs, EGR1, EGR2, EGR3, EiFIAX, IGF1, LAMP2, MAOA, NR4a1, PDE4A, RnF13, RPL10, RPL24, RPL26, RPL32, RPL38, RPL39, RPS15A, RPS23, RPS27L, SLIRP, UBE2D3, YARS, ZNRF1, and mitochondrial transcripts mt-ATP6, mt-ATP8, mt-CO2, mt-CO3, mt-ND1, mt-ND2, mt-ND3, mt-ND4, mt-RNR1, and mt-RNR2 was identified.

[0436] Effects of administering coagulants to FDRA-derived fibroblasts:

[0437] Gene expression analysis of fibroblasts derived from FRDA patients showed that, compared with normal fibroblasts, several transcription factors and secreted proteins were generally upregulated in patient-derived fibroblasts. Figure 3 and Figure 4ATo assess the role of contraprotein substitution in FDRA, FA-derived fibroblasts (lineage FA-68) were treated with the FXN fusion protein or vector, and RNA was collected and processed for PCR analysis. Results are shown in... Figure 4B In the figure, represents the fold increase in gene expression in cells treated with the FXN fusion protein relative to cells treated with the vector. hFXN expression is shown as an internal control. Figure 4B An exemplary FXN alternative expression profile is provided, represented as the downregulation of EGR1, EGR2, EGR3, and IGF1 expression detected in coagulant-depleted cell lines after treatment with the FXN fusion protein. Figure 5 A schematic diagram of the process is shown.

[0438] Example 4: Detection of FXN signature in patient samples treated with FXN fusion protein

[0439] Blood, oral, or muscle cell samples were collected from FDRA patients before and after treatment with a contraceptive replacement therapy (e.g., FXN fusion protein). Both samples (pre-treatment and post-treatment) were processed for RNA extraction and compared with Tables 2 and 4 and / or... Figure 3 RT-PCR was performed on the FSGMs shown. Analysis of the RT-PCR results from both samples will show which transcripts were altered, upregulated, or downregulated after treatment, and will provide an indication of the efficacy of FXN replacement therapy. The presence of inverse regulation of FSGMs when comparing FSGM expression before and after FXN replacement therapy will be an indication of effective treatment. For example, the detection of downregulation of at least one of CYR61, EGR1, EGR2, EGR3, and / or IGF1 after treatment indicates that FXN replacement therapy is effective. Conversely, if no inverse regulation is detected in at least one FSGM when comparing before and after treatment, it indicates treatment failure. Similarly, obtaining the eigenvectors of the FXN expression profiles in the samples before and after treatment and comparing them with the lack-of-FXN eigenvectors and FXN replacement eigenvectors described above will provide an indication of the efficacy of FXN replacement therapy. As a result of the FXN signature obtained from the patient samples, a new FXN replacement therapy dosing regimen can be adopted by increasing or decreasing the dose of FXN replacement therapy given to the patient.

[0440] Example 5: In vitro cell model of generating conamin (FXN) knockdown (KD)

[0441] HEK293 cells were transfected with the KD-hFXN shRNA construct to inhibit the expression of conamin mRNA and protein in the cells. A non-FXN-specific disordered control shRNA construct was used as a control. Figure 6 As shown, the expression of FXN protein was significantly reduced in KD-FXN clones A2 and A6 compared to the disordered control. Figure 6 The table shows the results of protein quantification in Western blot, expressed as the amount of FXN in KD cells relative to the amount of FXN in disordered control cells. Figure 6 The results shown in the table indicate that, compared with the disordered control, the amount of FXN protein in KD-FXN clones A2 and A6 was reduced by 82% and 72%, respectively.

[0442] Example 6: Effect of FXN fusion protein treatment on CYR61 protein expression in hFXN-KD cells

[0443] The aim of this experiment was to determine changes in CYR61 levels in response to treatment with the FXN fusion protein CTI-1601 in the disordered control and hFXN-KD cell lines produced as described in Example 5. For this purpose, disordered control and hFXN-KD (clone A6) cells were seeded at a density of 150,000 cells / well in 1 mL of treatment medium (DMEM, 5% heat-inactivated FBS, 20 mM glycerol, and 20 mM HEPES) in 6-well tissue culture plates pre-coated with 1% fibronectin solution. After 1 hour, the cells in each well were treated with different concentrations of CTI-1601. Specifically, 50 μL of serially diluted CTI-1601 (20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, and 0 μM control) in formulation buffer (20 mM histidine, 250 mM sucrose, 0.05% polysorbate 20, pH 5.8) was added to each well, and the plates were incubated in an incubator for 3 hours. Subsequently, 1 mL of complete culture medium (10% FBS, DMEM containing antibiotics) was added to each well, and the plates were incubated for 21 hours. This cycle was repeated three times on days 1, 2, and 3, followed by incubation of the plates for another day. On day 5, the plates were photographed, 1 mL of culture medium was harvested, 10 μL of HALT protease inhibitor was added, and the plates were frozen at -80°C for further analysis.

[0444] The amount of CYR61 protein secreted into the cell culture medium was measured using the CYR61 ELISA (R&D Biosystems-CDYR10) according to the manufacturer's protocol. The culture medium from the randomized control and hFXN-KD cells was diluted 1:2 prior to analysis.

[0445] ELISA analysis results of hFXN-KD cells are as follows: Figure 7As shown. The results indicated a relatively low level of CYR61 protein (approximately 63.3 pg / mL) in the culture medium from the disordered control cells, and this level was unaffected by treatment with 10 μM CTI-1601. Conversely, consistent with the mRNA data, the level of CYR61 protein secreted from the culture medium from hFXN-KD cells was significantly higher (approximately 1,198.5 pg / mL) compared to the CYR61 protein level in the culture medium from the disordered control cells. Furthermore, Figure 7 It was also shown that treatment with 10 μM CTI-1601 significantly reduced the level of CYR61 protein secreted by hFXN-KD cells to the control level (approximately 87.6 pg / mL).

[0446] These results again demonstrate that CYR61 is inversely regulated by FXN protein substitution following FXN knockdown, and that, in addition to changes in gene expression levels, the detection of secreted CYR61 protein can serve as a biomarker for FXN protein substitution.

[0447] Example 7: Transfection of hFXN into hFXN-KD cells resulted in a decrease in the amount of secreted CYR61 protein.

[0448] The aim of this experiment was to determine whether transfection of hFXN-KD cells with hFXN could reverse mitochondrial damage in these cells, as measured by the amount of secreted CYR61 protein. For this purpose, hFXN-KD and disordered control HEK293 cells, as described in Example 5, were transfected with Fugene-6 reagent using either an empty pCDNA3 vector (+V) or a full-length hFXN expression vector: pCDNA3-hFXN (+hFXN), and incubated for 48 hours, according to the manufacturer's instructions. The transfected cells were then incubated for another 48 hours. After the second 48-hour incubation, 1 mL of culture medium was removed, and 10 μL of HALT protease inhibitor was added to each aliquot. Following the manufacturer's protocol, HALT was administered using a protease inhibitor derived from ABCAM. TM The Simple Step CYR61 ELISA (ab238267) measured the amount of CYR61 protein in the culture medium. For the measurement, the culture medium from the randomized control and hFXN-KD cells was diluted 1 / 10. Data were plotted using Graphpad Prism Bar plots with standard deviation as error bars.

[0449] The results of the experiment are as follows: Figure 8As shown, this figure is a bar chart displaying the amount of CYR61 protein in the culture medium of cells transfected with empty vector (KD-SRBL+V); cells transfected with hFXN (SRBL5+hFXN); hFXN-KD cells transfected with empty vector (KD-FXN+V); and hFXN-KD cells transfected with hFXN (KD-FXN+hFXN). Figure 8 This indicates that, in the presence or absence of exogenously expressed hFXN, disordered control cells do not secrete detectable levels of CYR61 protein. hFXN-KD cells transfected with the empty vector secrete large amounts of CYR61 protein, and transient expression of hFXN in these cells reduces the amount of secreted CYR61 protein.

[0450] These results demonstrate that CYR61 is inversely regulated by the expression of an alternative FXN protein driven by FXN knockdown followed by nucleic acid-mediated expression, and further confirm that the detection of secreted CYR61 protein can be used as a biomarker for FXN protein substitution.

[0451] Example 8: Increased CYR61 levels in embryonic stem cells of FXN knockout mice

[0452] The purpose of this experiment was to determine whether the level of CYR61 protein secreted in mouse embryonic stem (ES)B9 cells with FXN gene deletion (knockout) was altered.

[0453] Generation of FNX-knockout mouse cell lines

[0454] A mouse embryonic stem cell line with FXN deficiency was generated. Specifically, as a result of this experiment, a homozygous mouse ES clone B9-46 was generated, which could be induced to knock out two alleles of the FXN gene. Figure 9 This is a bar chart showing the amount of FXN protein in total cellular protein in WT mouse ES clones and homozygous mouse ES clone B9-46 treated with control or FXN knockout-inducing reagents (knockout agents). Mouse FXN protein levels were measured using the Mouse FXN Elisa Kit (Abcam ab199078) according to the manufacturer's protocol. Figure 9 This indicates that treatment with the reagent to induce FXN knockout resulted in the elimination of FXN protein in B9-46 cells. No decrease in FXN protein levels was observed in WT cells or control-treated B9-46 cells.

[0455] Measurement of CYR61 gene expression (mRNA and protein)

[0456] Mouse B9 cells were treated with either a control reagent or a reagent that induced FXN gene knockdown. To measure the level of CYR61 gene expression, RNA was extracted from B9 mouse cells, and the amount of CYR61 mRNA was measured using qPCR as described previously. TaqMan Primer was used for qPCR analysis. TM Purchased from Thermo Fisher, β-actin was used as the housekeeping gene (β-actin VIC PL: Hs01060665_g1; CYR61: Hs00155479_m1). Two biological replicates were analyzed for both reagent and control treatments. To measure the amount of secreted CYR61 in cell culture medium, 1 mL of cell culture medium was harvested, 10 μL of HALT protease inhibitor was added, and the medium was frozen at -80°C for further analysis. As previously described, the amount of secreted CYR61 protein was determined by ELISA.

[0457] The results of CYR61 gene expression analysis are shown in Figure 10, Figure A. The results indicate that knockout of the FXN gene in B9 cells led to an approximately two-fold increase in CYR61 mRNA expression. The results of secreted CYR61 protein level measurement are shown in Figure 10, Figure B. The results indicate that knockout of the FXN gene in B9 cells led to an approximately two-fold increase in the amount of CYR61 protein in the cell culture medium.

[0458] The description of embodiments of this disclosure in this application is provided by way of example and is not intended to limit the scope of this disclosure. The described embodiments include different features, and not all of these features are required in all embodiments. Some embodiments utilize only some features or possible combinations of features. Variations of the embodiments of this disclosure described, as well as embodiments including different combinations of the features mentioned in the described embodiments, will occur to those skilled in the art. The scope of embodiments of this disclosure is defined only by the claims.

[0459] All references or documents cited or referenced herein, and all manufacturer's instructions for use, specifications, and product manuals for any product mentioned herein or in any other document, are incorporated herein by reference and may be used in the practice of this invention.

Claims

1. Use of a coagulant-sensitive genomic biomarker (FSGM) in the preparation of a pharmaceutical agent for evaluating the efficacy of coagulant (FXN) replacement therapy, said method comprising: (a) Determine the FXN substitution expression profile of one or more FSGMs in samples from FXN-deficient patients treated with FXN substitution therapy; (b) Compare the patients’ FXN alternative expression profiles with the baseline FXN(-) expression profiles; as well as (c) Use the comparison to determine the efficacy of the FXN replacement therapy; The one or more FSGMs mentioned above include EGR1, and The patient in question is receiving FXN replacement therapy.

2. The use according to claim 1, wherein the one or more FSGMs further comprises one or more of BTG2, PTGS2 and CTSS.

3. The use according to claim 1 or 2, further comprising determining the baseline FXN(-) expression profile of one or more FXN-sensitive genomic markers (FSGMs) in samples from patients exhibiting FXN deficiency prior to FXN replacement therapy.

4. The use according to claim 1, wherein the one or more FSGMs further comprises BTG2.

5. The use according to claim 1, wherein the one or more FSGMs further comprises one or more of MPEG1, RNF13, EGR2, CALM2, DCUN1D1, NRTN, RAP2C, MKI67, EIF1AX, and Kctd12b.

6. The use according to claim 1, wherein the expression profile is determined by any one of sequencing, hybridization, or amplification of the sample RNA.

7. The use according to claim 1, wherein the expression profile is determined by HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, or any combination thereof.

8. The use according to claim 1, wherein the patient suffers from Friedreich ataxia (FRDA).

9. A method for detecting one or more FSGMs in a biological sample by contacting a biological sample or a portion thereof from a patient with a deficiency of FXN with one or more detection reagents that specifically detect one or more FSGMs, wherein the one or more FSGMs include EGR1, and wherein the patient is receiving FXN replacement therapy.

10. The method of claim 9, wherein the one or more FSGMs further comprises one or more of BTG2, PTGS2 and CTSS.

11. The method of claim 9, wherein the FXN replacement therapy comprises treatment with an FXN fusion protein.

12. The method of claim 9, wherein the FXN replacement therapy comprises treatment with CTI-1601.

13. The method of claim 9, wherein the one or more FSGMs further comprises BTG2.

14. The method of claim 9, wherein the one or more FSGMs further comprises one or more of MPEG1, RNF13, EGR2, CALM2, DCUN1D1, NRTN, RAP2C, MKI67, EIF1AX, and Kctd12b.

15. Use of a coagulant-sensitive genomic marker (FSGM) in the preparation of a pharmaceutical preparation for a method of assessing whether the dose of FXN replacement therapy should be initiated, increased, or decreased in a subject during treatment for mitochondrial disease, said use including: Samples were provided from subjects suffering from FXN deficiency; The FXN expression profiles of one or more FSGMs in the sample were determined. The FXN expression profile of the sample was compared with at least one other expression profile selected from the group consisting of normal FXN expression profiles of one or more FSGMs, baseline FXN(-) expression profiles of one or more FSGMs, and FXN alternative expression profiles of one or more FSGMs. The FXN expression profiles of the samples were classified into those corresponding to normal FXN expression profiles, baseline FXN(-) expression profiles, or FXN substitution expression profiles. Based on the classification of the FXN expression profile of the sample, the dose of FXN replacement therapy administered to the subject can be started, increased, or decreased. The one or more FSGMs mentioned above include EGR1; and The mitochondrial disease mentioned is Friedreich ataxia (FRDA).

16. The use according to claim 15, wherein the one or more FSGMs further comprises one or more of BTG2, PTGS2 and CTSS.

17. The use according to claim 15, wherein the one or more FSGMs include BTG2.

18. The use according to claim 15, wherein the one or more FSGMs further comprises one or more of MPEG1, RNF13, EGR2, CALM2, DCUN1D1, NRTN, RAP2C, MKI67, EIF1AX, and Kctd12b.

19. A kit for detecting two or more FSGMs in biological samples from subjects exhibiting FXN deficiency and receiving FXN replacement therapy, comprising: Multiple reagents for measuring the levels of the two or more FSGMs in the biological sample from the subject, wherein the two or more FSGMs include EGR1, and one or more FSGMs selected from the group consisting of BTG2, PTGS2, and CTSS. A set of instructions for measuring the levels of the two or more FSGMs.

20. The kit of claim 19, wherein the reagent for measuring the levels of the two or more FSGMs is an antibody that binds to the two or more FSGMs or an oligonucleotide complementary to the corresponding mRNA of the two or more FSGMs.

21. The kit according to claim 19 or 20, wherein the two or more FSGMs further include one or more of MPEG1, RNF13, EGR2, CALM2, DCUN1D1, NRTN, RAP2C, MKI67, EIF1AX, and Kctd12b.

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