WO2025007066A1 - Biomarkers and methods related to fragile x syndrome - Google Patents

Biomarkers and methods related to fragile x syndrome Download PDF

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WO2025007066A1
WO2025007066A1 PCT/US2024/036262 US2024036262W WO2025007066A1 WO 2025007066 A1 WO2025007066 A1 WO 2025007066A1 US 2024036262 W US2024036262 W US 2024036262W WO 2025007066 A1 WO2025007066 A1 WO 2025007066A1
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fxs
metabolite
subject
sample
level
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Joel D. Richter
Sneha Shah
Jessica SPINELLI
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University of Massachusetts Amherst
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • FXS is caused by a CGG triplet repeat expansion in a single gene, fragile X messenger ribonucleoprotein 1 (FMR1), which resides on the X chromosome.
  • FMR1 fragile X messenger ribonucleoprotein 1
  • FMRP protein fragile X messenger ribonucleoprotein
  • the present disclosure relates to methods and biomarkers to treat, and to supplement treatment of, fragile X-associated disorders such as fragile X syndrome (FXS).
  • FXS fragile X syndrome
  • the present disclosure relates to biomarkers, for example, metabolites that are differentially present in FXS subjects.
  • the present disclosure generally relates to biomarkers (e.g., metabolites) and methods that are useful for diagnosing, making prognoses, and/or treating a subject, e.g., a subject having, or at risk of having, a fragile X-associated disorder.
  • biomarkers e.g., metabolites
  • FXS fragile X syndrome
  • the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and administering to the subject a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product.
  • FMR1 fragile X messenger ribonucleoprotein 1
  • the disclosure also provides, among other things, methods of predicting a treatment outcome of, diagnosing, prognosing, or subclassifying fragile X syndrome (FXS) in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing.
  • FXS fragile X syndrome
  • FIGs.1A-1B Metabolic changes observed in FXS patient-derived post-mortem cortex (brain) samples.
  • FIG.1A Volcano plot depicting the changes in polar metabolite levels between post-mortem cortex tissue samples from six FXS males and six age-matched typically- developing (TD) males captured using liquid chromatography-mass spectrometry (LC-MS).
  • FIG.1B Ratios of glutathione (GSH) to glutathione disulfide (GSSG) levels compared between the FXS and TD samples used in FIG.1A, reflecting increased oxidative stress in the FXS cortex samples.
  • FIGs.2A-2B Metabolic changes observed in FXS patient-derived PBMC samples.
  • FIGs.3A-3C Common metabolic changes observed in FXS patient-derived PBMC and post-mortem cortex (brain) samples.
  • FIG.3A Venn diagram depicting the polar metabolite levels significantly altered in FXS males compared to TD males in PBMC samples, cortex samples, and both sample types.
  • the significance threshold of a p-value equal to 0.05 was used.
  • the left circle shows the number of polar metabolites with significantly changed levels in PBMC samples from FXS males compared to TD males; the right circle shows the equivalent comparison in cortex samples.
  • the overlapping region of the two circles and its accompanying list show the number and identity of polar metabolites with significantly changed levels in both PBMC and cortex samples from FXS males compared to TD males.
  • FIG.3B Plots depicting levels of amino acids from the overlapping region of FIG.3A that are reduced in cortex samples from FXS males compared to TD males.
  • FIG.3C The significance threshold of a p-value equal to 0.05 was used.
  • the left circle shows the number of polar metabolites with significantly changed levels in PBMC samples from FXS males compared to TD males; the right circle shows the equivalent comparison in cortex samples.
  • the overlapping region of the two circles and its accompanying list show the number and identity of
  • FIG.4 Metabolic changes observed in FXS patient-derived lymphoblastoid cell lines (LCLs). Volcano plot depicting the changes in polar metabolite levels in two human male FXS LCLs (GM05131 and GM07072) compared to two TD LCLs (GM07174 and GM06890) captured using LC-MS.
  • FIGs.5A-5D A. Schematic of FMR1 RNA isoforms in FXS. ASO against FMR1- 217 RNA used in FXS2 cells (160nM for 48hrs). B, C. qPCR data for FMR1-217 RNA and FMR1 RNA levels respectively (**** p ⁇ 0.0001, **p ⁇ 0.01). D. Western blot data for FMRP and GAPDH proteins in TD and FXS2 cells (with vehicle or ASO). [0017] FIGs.6A-6D.
  • FIG.7A Western blots to measure the FMRP levels in FXS1, FXS2 and TD lines compared to GAPDH. Below is a representation of FMRP levels in the cell lines.
  • FIG.7B PCA plot demonstrating the clustering of the FXS2 line (partial FMRP) with the TD as against the FXS1 cell line (no FMRP).
  • FIG.7, C, D and E Volcano plot depicting significantly altered metabolites (amino acids in green and their derivatives in red) in FXS2 vs TD (FIG.7C.), FXS1 vs TD (.
  • FIG.7D. and FXS2 vs FXS1 (FIG.7E., note the increase in amino acid levels).
  • FIG.7F. Examples of amino acid changes in the FXS lines compared to TD. (*p ⁇ 0.05, **p ⁇ 0.01).
  • FIGs.8A-8B iPSC derived forebrain neuron cultures.
  • FIG 8A The diagram depicts the conversion of LCLs from FXS patients into iPSCs, their subsequent differentiation into neuronal lineage, and the generation of mature forebrain neurons.
  • FIG.8B Representative images of cells in culture aligned to the different stages in FIG 8A. (panel A).
  • FXS LCLs will be treated with various inhibitors such as MG-132 or Bortezomib against proteosome, 3-methyladenine (3-MA) against autophagosome, Rapamycin to block mTORC1 activation and dexpramipexole (DEX) to block proton leak in the mitochondria will be used.
  • LC-MS assay will be used to analyze the effect of each inhibitor on the metabolome.
  • the i-TRAP qPCR method will be used to quantify ratios of individual uncharged to charged tRNAs. [0021] FIG.10.
  • FIGs.11A-C Lymphoblastoid cell lines (LCLs) from a typically developing (TD) male individual, a Fragile X Syndrome male (FXS) expressing some FMRP (FXS2), and a FXS male expressing no FMRP (FXS1) were analyzed for FMRP and GAPDH (as a loading control) on western blots in triplicate.
  • FIG.11B Polar metabolites from the cell lines noted in FIG.11A.
  • FIG.11C Relative amounts of leucine and methionine in the LCLs from the 3 genotypes noted in FIG.11A. - 5 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [0023] FIGs.12A-12B.
  • FIG.12A The culture media from the LCLs noted in FIG.11 were analyzed for metabolites by LC/MS. Shown are relative amounts of leucine and methionine.
  • FIG.12B PCA plot for metabolites in the culture media from the LCL lines denoted in FIG.11.
  • FIG.13 LC/MS analysis of the metabolome comparing FXS1 to TD LCLs.
  • the volcano plot at left is the cell pellets and at right is the culture media.
  • the metabolites above the dashed line are statistically different between the genotypes.
  • the vertical line separates metabolites that are decreased (to the left) or increased (to the right) when comparing FXS1 to TD. See also Table 5 (cell pellet) and Table 6 (culture media).
  • FIG.14 LC/MS analysis of the metabolome comparing FXS2 to TD LCLs.
  • the metabolites above the dashed line are statistically different between the genotypes.
  • FIG.15 LC/MS analysis of the metabolome comparing FXS2 to FXS1 LCLs. The volcano plot at left is the cell pellets and at right is the culture media. The metabolites above the dashed line are statistically different between the genotypes. The vertical line separates metabolites that are decreased (to the left) or increased (to the right) when comparing FXS2 to FXS1. See also Table 9 (cell pellet) and Table 10 (culture media). [0027] FIG.16A.
  • FIG.16B Amount of extracellular 13 C 5 , 15 N 2 glutamate with respect to the amount of extracellular 13 C 5 , 15 N 2 glutamine.
  • FIG.16C Amount of intracellular 13 C 5 , 15 N 2 glutamate with respect to the amount of intracellular 13 C 5 , 15 N 2 glutamine.
  • FIG.16D Relative amounts of extracellular 13 C 5 , 15 N 2 glutamate in wild-type and FXS2 cells.
  • FIG.16E Relative amounts of intracellular 13 C 5 , 15 N 2 glutamate in wild-type and FXS2 cells.
  • a first metabolite has a reduced level in a sample from a subject
  • a second metabolite has an increased level in the sample from a subject.
  • the term “comprising” can be substituted with the term “containing” or “including.”
  • the term “consisting of” excludes any element, step, or ingredient not specified in the claim element.
  • the term “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
  • Any of the terms “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment disclosed herein, can in some embodiments, be replaced with the term “consisting of,” or “consisting essentially of” to vary scopes disclosed herein.
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and/or” as used herein.
  • an acceptable error range for a particular value depends, at least in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of ⁇ 20%, e.g., ⁇ 10%, ⁇ 5% or ⁇ 1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular values used in the Exemplification.
  • the term “subject” refers to a mammalian subject, preferably human, diagnosed with or suspected of having a fragile X-associated disorder (e.g., FXS).
  • the term “metabolite” refers to an intermediate or product resulting from metabolism.
  • the term “metabolism” refers to chemical changes occurring within an organism. In some embodiments, a metabolite results from anabolism.
  • a metabolite results from catabolism.
  • Compounds described herein include those described generally, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated.
  • the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5 th Ed., Ed.: Smith, M.B.
  • a name of a compound may be generated using a chemical naming program (e.g., CHEMDRAW®, version 17.0.0.206, PerkinElmer Informatics, Inc.).
  • CHEMDRAW® version 17.0.0.206
  • PerkinElmer Informatics, Inc. a chemical naming program
  • the term “sample” refers to any sample that can be from or derived from a subject (e.g., a mammalian subject such as a human subject).
  • the methods disclosed herein can be performed using a variety of possible sample types. For example, a bodily fluid, a single cell or cell lysate, a population of cells, a cell culture, and/or a tissue.
  • phrases “pharmaceutically acceptable” means that the substance or composition the phrase modifies is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J.
  • salts of the agents/compounds described herein include salts derived from suitable inorganic and organic acids, and suitable inorganic and organic bases.
  • suitable acids include salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art, such as ion exchange.
  • salts derived from suitable acids include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxy-ethanesulfonate, hydroxymaleate, lactobionate, lactate, laurate, - 10 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 lauryl sulfate, malate, maleate, malate, mal
  • Salts derived from appropriate bases include salts derived from inorganic bases, such as alkali metal, alkaline earth metal, and ammonium bases, and salts derived from aliphatic, alicyclic or aromatic organic amines, such as methylamine, trimethylamine and picoline, or N + ((C 1 -C 4 )alkyl) 4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium and the like.
  • compositions include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • “Pharmaceutically acceptable carrier” refers to a non-toxic carrier or excipient that does not destroy the pharmacological activity of the agent with which it is formulated and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
  • Pharmaceutically acceptable carriers that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine,
  • Treating” or “treatment,” as used herein, refers to taking steps to deliver a therapy to a subject, such as a mammal, in need thereof (e.g., as by administering to a mammal one or more therapeutic agents). “Treating” or “treatment” includes inhibiting the disease or condition (e.g., - 11 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 as by slowing or stopping its progression or causing regression of the disease or condition) and relieving the symptoms resulting from the disease or condition.
  • the disease or condition e.g., - 11 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 as by slowing or stopping its progression or causing regression of the disease or condition
  • treating refers to the medical management of a subject with the intent to improve, ameliorate, stabilize (i.e., not worsen), prevent, or cure a disease, pathological condition, or disorder—such as the particular indications exemplified herein.
  • This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive treatment (treatment employed to supplement another therapy).
  • Treatment also includes diminishment of the extent of the disease or condition; preventing spread of the disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable.
  • “Ameliorating” or “palliating” a disease or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • treatment includes change or restoration of expression, e.g., endogenous FMRP expression.
  • treatment includes change or restoration of FMRP level(s), e.g., to ameliorate metabolic deficit(s) in FXS.
  • a “polynucleotide” is defined as a plurality of nucleotides and/or nucleotide analogs linked together in a single molecule.
  • a polynucleotide disclosed herein comprises deoxyribonucleotides.
  • the polynucleotide comprises ribonucleotides.
  • Non-limiting examples of polynucleotides include single-, double- or multi-stranded DNA or RNA, DNA-RNA hybrids (e.g., each “T” position may be independently substituted by a “U” or vice versa), or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide - 12 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 bases.
  • the backbone of the polynucleotide can comprise sugars and phosphate groups, modified or substituted sugar or phosphate groups, a polymer of synthetic subunits such as phosphoramidates, or a combination thereof.
  • nucleotide analog or “altered nucleotide” or “modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides.
  • a nucleotide analog may be modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability to perform its intended function.
  • positions of the nucleotide which may be derivatized include the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, and 5-propenyl uridine; the 6 position, e.g., 6-(2-amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, and 8- fluoroguanosine.
  • 5 position e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, and 5-propenyl uridine
  • the 6 position e.g., 6-(2-amino)propyl uridine
  • the 8-position for adenosine and/or guanosines e.g., 8-bro
  • Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated or N6-methyl adenosine) nucleotides.
  • the term “complementary” refers to sequence complementarity between two different polynucleotides or between two regions of the same polynucleotide.
  • a first region of a polynucleotide is complementary to a second region of the same or a different polynucleotide if, when the two regions are arranged in an anti-parallel fashion, at least one nucleotide residue of the first region is capable of base pairing (i.e., hydrogen bonding) with a residue of the second region, thus forming a hydrogen-bonded duplex.
  • sequence identity refers to the extent to which two nucleotide sequences have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as a reference sequence, to which test sequences are compared.
  • Sequence identity between reference and test sequences is expressed as a percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity.
  • two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide residue at 70% of the same positions over the entire length of the reference sequence.
  • alignment can include introduced gaps to provide for the maximal level of identity.
  • Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology).
  • test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • a commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through National Center for Biotechnology Information, National Library of Medicine, of the United States National Institutes of Health. (Altschul et al., 1990).
  • polypeptide refers to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
  • a polypeptide can comprise any suitable L-and/or D-amino acid, for example, common ⁇ -amino acids (e.g., alanine, glycine, valine), non- ⁇ -amino acids (e.g., ⁇ -alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine).
  • the amino, carboxyl, and/or other functional groups on a polypeptide can be free (e.g., unmodified) or protected with a suitable protecting group.
  • Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups are known in the art and are disclosed in, for example, Green and Wuts, “Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991.
  • the functional groups of a polypeptide can also be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label, such as a fluorogen or a hapten) using - 14 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 methods known in the art.
  • a polypeptide can comprise one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclizing modifications), N-methyl- ⁇ -amino group substitution), if desired.
  • a polypeptide can be an analog of a known and/or naturally-occurring peptide, for example, a peptide analog having conservative amino acid residue substitution(s).
  • antibody mimetic refers to polypeptides capable of mimicking an antibody’s ability to bind an antigen, but structurally differ from native antibody structures.
  • a “pharmaceutical composition” refers to a formulation of one or more therapeutic agents and a medium generally accepted in the art for delivery of a biologically active agent to subjects, e.g., humans.
  • a pharmaceutical composition may include one or more pharmaceutically acceptable excipients, diluents, or carriers.
  • a pharmaceutical composition suitable for use in methods disclosed herein further comprises one or more pharmaceutically acceptable carriers.
  • “Pharmaceutically acceptable carrier, diluent, or excipient” includes any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • the carrier may be a diluent, adjuvant, excipient, or vehicle with which the agent (e.g., polynucleotide) is administered.
  • agents e.g., polynucleotide
  • vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration).
  • compositions may - 15 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating, and coloring agents, etc.
  • concentration of the agent in such pharmaceutical formulation may vary widely, i.e., from less than about 0.5%, to at least about 1%, or to as much as 15% or 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight.
  • concentration will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the mode of administration.
  • Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing: 691-1092 (e.g., pages 958-89).
  • Non-limiting examples of pharmaceutically acceptable carriers are solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, such as salts, buffers, antioxidants, saccharides, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifying agents, or combinations thereof.
  • Non-limiting examples of buffers are acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffers, HEPPSO, and HEPES.
  • Non-limiting examples of salts are acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like.
  • Agents e.g., polynucleotides
  • described herein may be prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent, or eliminate, or to slow or halt progression of, a condition being treated (see, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, and Goodman and Gilman’s The Pharmaceutical Basis of Therapeutics, McGraw-Hill, New York, N.Y., the contents of which are incorporated herein by reference, for a general description of methods for administering various agents for human therapy).
  • administering refers to providing a compound, composition, or pharmaceutically acceptable salt thereof described herein to a subject in need of treatment or prevention. Administering can be performed, for example, once, a plurality of times, and/or over one or more extended periods. Administration includes both direct administration (including self-administration), and indirect administration (including an act of prescribing a drug or directing a subject to consume an agent).
  • the term “treat,” “treating” or “treatment” refers to therapeutic treatment wherein the objective is to slow down (lessen) an undesired physiological change or disease, such as the development or progression of the fragile X-associated disorder (e.g., FXS), or to provide a beneficial or desired clinical outcome during treatment.
  • Beneficial or desired clinical outcomes include alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, whether detectable or undetectable.
  • Non-limiting examples of - 18 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 symptoms include speech and motor development symptoms; cognitive disabilities, including learning and intellectual disabilities, hyperactivity, short attention span, anxiety, sensitivity to sensory stimulation, sleep problems, and seizures; recurrent ear infections, and mitral valve prolapse.
  • the term “poor” or “poorer” refers to greater degree of fragile X-associated disorder (e.g., FXS) symptoms, increased extent of disease, decreased (i.e., worsening) state of disease, increased or enhanced state of disease progression, deterioration or worsening of the disease state, whether detectable or undetectable.
  • LC-MS liquid chromatography-mass spectrometry- based metabolomics
  • a group of ions may originate from the same metabolite, and one metabolite can be represented by multiple peaks in LC-MS data with distinct m/z values but, at similar retention times, due to the presence of adducts (e.g., H + , Na + or K + ).
  • a “peak” is a point on a mass spectrum with a relatively high y-value.
  • methods of treating fragile X syndrome (FXS) in a subject disclosed herein comprise determining, in a sample from the subject: - 19 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing.
  • methods of treating fragile X syndrome (FXS) in a subject disclosed herein comprise determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and administering to the subject a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product.
  • FMR1 fragile X messenger ribonucleoprotein 1
  • the agent is an antisense oligonucleotide.
  • a therapeutically effective amount of the agent decreases an aberrant FMR1 transcript, a protein encoded by the aberrant FMR1 transcript, or both.
  • an aberrant FMR1 gene product comprises FMR1-217.
  • a method disclosed herein comprises determining a presence, absence, amount, or alteration of at least one FXS-associated metabolite (e.g., an FXS-associated metabolite), or an alteration of an FXS-associated ratio of two metabolites, in a sample from the - 20 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 subject.
  • FXS-associated metabolite e.g., an FXS-associated metabolite
  • a method further comprises treating FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, or the alteration of the FXS-associated ratio, in the sample.
  • Fragile X-associated disorders are caused by mutation of the fragile X messenger ribonucleoprotein 1 (FMR1, previously known as fragile X mental retardation 1) gene, located in the q27.3 locus of the X chromosome. The expansion of the trinucleotide CGG repeat above the normal range (greater than 54 repeats) in the non-coding region of the FMR1 gene has been associated with the development of fragile X-associated disorders.
  • FMR1 fragile X messenger ribonucleoprotein 1
  • the trinucleotide CGG repeat can range from 55-200 CGG repeats.
  • a fragile X-associated disorder is linked to greater than 77 CGG repeats in FMR1, e.g., greater than 98 CGG repeats in FMR1.
  • a fragile X-associated disorder is linked to at least 140 CGG repeats in FMR1.
  • a fragile X- associated disorder is linked to at least 201 CGG repeats in FMR1.
  • Non-limiting examples of fragile X-associated disorders include fragile X-associated tremor/ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), fragile X-associated neuropsychiatric disorders (FXAND), and fragile X syndrome (FXS).
  • a fragile X-associated disorder is fragile X syndrome (FXS), fragile X- associated primary ovarian insufficiency (FXPOI), or fragile X-associated tremor/ataxia syndrome (FXTAS), or a combination thereof.
  • a fragile X-associated disorder is FXS.
  • a subject is a mammal.
  • a subject includes humans, domestic animals, such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.), livestock (e.g., pigs, cattle, sheep, goats, horses, etc.), and non-domestic animals.
  • a subject is a human.
  • a subject e.g., human
  • a subject is male.
  • a subject e.g., human is female.
  • a subject e.g., a human
  • has, or is predisposed to have, a fragile X-associated disorder e.g., FXS.
  • a subject e.g., a human
  • has a fragile X-associated disorder e.g., FXS.
  • a subject e.g., a human
  • is predisposed to have a fragile X-associated disorder e.g., FXS.
  • a subject is diagnosed with a fragile X-associated disorder (e.g., FXS).
  • a subject is suspected of having a fragile X-associated disorder (e.g., FXS).
  • a subject has one or more of the physical and/or medical features associated with a fragile X-associated disorder (e.g., FXS).
  • FXS fragile X-associated disorder
  • Non-limiting examples of physical features associated with FXS include a long face, prominent ears and chin, arched palate, large testicles at puberty, low muscle tone, flat feet, and hyperextensible joints.
  • Non- limiting examples of medical or behavioral features associated with FXS include sleep problems, seizures, recurrent ear infections, mitral valve prolapse, behaviors of hyperactivity, short attention span, hand biting or hand flapping, poor eye contact and social skills, shyness, anxiety, autism, epilepsy, aggression, delayed speech and/or motor development, repetitive speech, sensitivity to sensory stimulation (including a hypersensitivity to being touched, to light or to sound), or any combination thereof.
  • a subject is a female with an IQ score of less than 115, 110, 105, 100, 95, or 90.
  • a subject is a male with an IQ score of less than 60, 55, 50, or 45.
  • a subject has one or more of the following: irregular menses, fertility problem, elevated FSH (follicle-stimulating hormone) level, premature ovarian failure, primary ovarian insufficiency, and vasomotor symptoms (e.g., “hot flash”).
  • a subject has one or more of the following: intention tremor, parkinsonism, ataxia, memory loss, white matter lesion involving middle cerebellar peduncles, and cognitive decline.
  • a subject has at least 55 CGG repeats in the 5’ untranslated region of an FMR1 gene, for example, having at least 77, at least 78, at least 98, at least 99, at least 140, or at least 201 CGG repeats in the 5’ untranslated region of the FMR1 gene. In some embodiments, a subject has between about 55-200 CGG repeats in the 5’ untranslated region of an FMR1 gene. In some embodiments, a subject has at least 200 CGG repeats in the 5’ - 22 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 untranslated region of an FMR1 gene.
  • a subject has at least 201 CGG repeats in the 5’ untranslated region of an FMR1 gene.
  • a subject e.g., a human
  • a subject e.g., a human
  • a subject has a CGG repeat expansion that is fully methylated.
  • a subject is a premutation carrier for FXS.
  • a subject has FXS.
  • a subject e.g., a human expresses or has an elevated expression of an aberrant FMR1 gene product.
  • a FMR1 gene encodes a fragile X messenger ribonucleoprotein (FMRP, previously known as fragile X mental retardation protein).
  • an FMR1 gene described herein is a human FMR1 gene (e.g., corresponding to GenBank reference number NC_000023.11), a mouse FMR1 gene (e.g., NC_000086.8), a rat FMR1 gene (e.g., NC_051356.1), a golden hamster FMR1 gene (e.g., NW_024429188.1), a Chinese hamster FMR1 gene (e.g., NW_003614110.1), a dog FMR1 gene (e.g., NC_051843.1), a pig FMR1 gene (e.g., NC_046383.1), or a monkey FMR1 gene (e.g., NC_041774.1).
  • a human FMR1 gene e.g., corresponding to GenBank reference number NC_000023.11
  • a mouse FMR1 gene e.g., NC_000086.8
  • a rat FMR1 gene e.g., NC_0513
  • the FMR1 gene is a human FMR1 gene.
  • the human FMR1 gene (Ensembl: ENSG00000102081.16) is located within chromosome band Xq27.3 between base pairs 147,911,919 and 147,951,125 (the numberings referring to Genome Reference Consortium Human Build 38 (GRCh38)).
  • an aberrant FMR1 gene product e.g., an aberrant FMR1 transcript and/or its protein product
  • contributes to pathology of a fragile X-associated disorder e.g., FXS
  • an aberrant FMR1 transcript contributes to pathology of a fragile X-associated disorder.
  • a protein encoded by an aberrant FMR1 transcript contributes to pathology of a fragile X-associated disorder.
  • an aberrant FMR1 transcript and its protein product contribute to pathology of a fragile X- associated disorder.
  • an aberrant FMR1 gene product is produced from a CGG expansion-dependent mis-splicing of an FMR1 transcript. - 23 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00105]
  • an aberrant FMR1 transcript is FMR1-217.
  • FMR1- 217 also referred to as “isoform 12” or “iso12,” is a transcript corresponding to A0A087X1M7 (ENST00000621447.1, 1,832 nucleotides).
  • FMR1-217 has 2 exons, and the splicing between Exon 1 of FMR1-217 (between base pairs 147,912,123 and 147,912,230, SEQ ID NO:1) and Exon 2 of FMR1-217 (between base pairs 147,912,728 and 147,914,451, SEQ ID NO:2) is considered aberrant FMR1 RNA splicing.
  • FMR1-217 is detected in a subpopulation of subjects with fragile X-associated disorder, including a subpopulation of FXS patients, and a subpopulation of premutation carriers for FXS.
  • CGCCCGCAGCCCACCTCTCGGGGGCGGGCTCCCGGCGCTAGCAGGGCTGA AGAGAAGATGGAGGAGCTGGTGGTGGAAGTGCGGGGCTCCAATGGCGCTTTCTACA AG (SEQ ID NO:1).
  • FMR1-217 encodes a 31-amino acid protein (SEQ ID NO:3).
  • MEELVVEVRGSNGAFYKHWDFGELHCSGRGL SEQ ID NO:3.
  • a subject e.g., a human
  • a subject does not express and/or does not have an elevated expression of an aberrant FMR1 gene product, for example, compared to typically developing subjects.
  • a subject does not express an aberrant FMR1 gene product.
  • a subject does not have an elevated expression of an aberrant FMR1 gene product.
  • Normal FMR1 Gene Products [00112]
  • a subject e.g., a human
  • expresses a normal FMR1 gene product i.e., a FMR1 gene product expressed in typically developing subjects.
  • Non-limiting examples of “normal” human FMR1 gene products include: a transcript corresponding to Q06787 (FMR1-205, ENST00000370475.9, 4,441 nucleotides), and its protein product (a 632-amino acid protein (NP_002015.1)), - 25 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 a transcript corresponding to NM_001185075.2 (4,170 nucleotides), and its protein product (a 537-amino acid protein (NP_001172004.1)), a transcript corresponding to NM_001185076.2 (4,378 nucleotides), and its protein product (a 611-amino acid protein (NP_001172005.1)), a transcript corresponding to NM_001185082.2 (4,303 nucleotides), and its protein product (a 586-amino acid protein (NP_001172011.1)), a transcript corresponding to NM_001185081.2 (4,
  • a normal FMR1 transcript is Q06787 (FMR1-205, ENST00000370475.9, 4,441 nucleotides, produces a 632-amino acid protein (NP_002015.1)).
  • FMR1-205 also referred to as “isoform 1” or “iso1,” is produced in typical developing subjects (subjects, e.g., humans who do not have FXS) and a subpopulation of FXS subjects.
  • a subject has a decreased level of isoform 1 of FMR1. In some embodiments, a subject has a decreased level of isoform 1 of FMR1 and an increased level of isoform 12 of FMR1.
  • Age a subject (e.g., a human) is a child (e.g., birth to 17 years of age). In some embodiments, a subject (e.g., a human) is an adult (18-64 years of age).
  • a subject e.g., a human
  • a subject is an older adult (65 years of age or older).
  • a subject e.g., a human
  • a subject is at least about 18 months of age.
  • a subject is at least about 24 months of age.
  • a subject is at least about 42 months of age.
  • a subject e.g., a human
  • a subject is at least about 1 year of age, for example, at least about: 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, or 50 years of age.
  • a subject is at least about: 2, 3, 5, 6, 7, 8, 10, 12, 13, - 27 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 14, 15, 18, 30, or 50 years of age.
  • a subject is at least 14 years of age.
  • a subject is at least 18 years of age.
  • a subject e.g., a human
  • a subject is about 90 years of age or younger, for example, about: 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 29, 25, 24, 23, 22, 20, 18, 17, 16, 15, 13, 11, 10, 7, 6 years of age or younger.
  • a subject is about 68 months of age or younger.
  • a subject e.g., a human
  • a subject is about 0-100 years of age, for example, about: 1-100, 1-85, 1-80, 1-65, 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-29, 1-24, 1- 23, 1-22, 1-20, 1-18, 1-17, 1-16, 1-13, 1-12, 1-11, 1-10, 1-8, 1-7, 1-6, 2-100, 2-85, 2-80, 2-65, 2- 60, 2-55, 2-50, 2-45, 2-40, 2-35, 2-30, 2-29, 2-24, 2-23, 2-22, 2-20, 2-18, 2-17, 2-16, 2-13, 2-12, 2-11, 2-10, 2-8, 2-7, 2-6, 3-100, 3-85, 3-80, 3-65, 3-60, 3-55, 3-50, 3-45, 3-40, 3-35, 3-30, 3-29, 3-24, 3-23, 3-22, 3-20, 3-18, 3-17, 3-16, 3-13, 3-12, 3-11, 3-11,
  • a subject is about: 1-7, 2-16, 3-10, 3-11, 3-17, 3-18, 3-23, 5- 11, 5-13, 5-23, 5-35, 5-50, 6-18, 6-40, 7-16, 8-18, 8-45, 10-17, 10-23, 10-45, 12-18, 12-29, 12- 40, 12-45, 12-50, 13-22, 13-29, 13-35, 13-40, 14-50, 14-100, 15-55, 18-24, 18-45, 18-50, 18-60, 18-65, 18-100, 30-80, or 50-85 years of age.
  • a subject is about 24-68 months of age.
  • a subject is about 42 months to 16 years of age.
  • a subject is 12 years or older, and less than 18 years of age.
  • Samples [00123] In some embodiments, a sample is a sample from or derived from a human subject. In some embodiments, a sample comprises a single cell or cell lysate, a population of cells, a cell culture, a tissue, or a bodily fluid. [00124] In some embodiments, a sample comprises a bodily fluid sample, a hair sample (e.g., from hair follicles), a nasal sample (e.g., nasal swab), a buccal sample (e.g., buccal swab), or a skin sample. [00125] In some embodiments, a sample comprises a bodily fluid sample.
  • a bodily fluid comprises CSF.
  • a sample comprises a bodily fluid selected from cerebrospinal fluid (CSF), blood, serum, plasma, or urine.
  • CSF cerebrospinal fluid
  • PBMCs peripheral blood mononuclear cells
  • a sample comprises a tissue sample.
  • a sample comprises a brain sample.
  • a sample comprises a non-brain sample.
  • a sample comprises a non-neural sample.
  • a sample comprises a cell sample.
  • a - 29 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 sample comprises a blood cell (e.g., peripheral blood mononuclear cell (PBMC) or white blood cell), a fetal cell (e.g., circulating fetal cell), a blastomere, a trophectoderm cell, a stem cell (e.g., induced pluripotent stem cell (iPSC) or derived stem cell), a fibroblast (e.g., a dermal derived fibroblast cell or lung-derived fibroblast cell), a modified fibroblast, a leukocyte, a pluripotent cell, or a cultured cell, or a combination thereof.
  • PBMC peripheral blood mononuclear cell
  • a fetal cell e.g., circulating fetal cell
  • blastomere e.g., a blastomere, a trophectoderm cell
  • a stem cell
  • a sample comprises a brain sample, a PBMC sample, or a peripheral blood lymphocyte sample, or a combination thereof.
  • a method further comprises enriching (e.g., isolating) a cellular compartment or organelle from a sample.
  • a method further comprises enriching a cellular compartment (e.g., cytoplasm) from a sample.
  • a method further comprises enriching an organelle from a sample.
  • organelles include mitochondria, nuclei, Golgi apparatus, endoplasmic reticulum (ER), and ribosomes.
  • a method further comprises enriching mitochondria from a sample.
  • a sample is substantially free of macromolecules, e.g., molecules having molecular weights of greater than about 1,500 Daltons, for example, greater than about: 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Daltons.
  • FXS-Associated Metabolites [00132] In some embodiments, a method comprises determining, in a sample from a subject, a presence, an absence, or a level of an FXS-associated metabolite.
  • a method comprises determining, in a sample from a subject, an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite. - 30 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00134] In some embodiments, a method comprises determining, in a sample from a subject, a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites.
  • a method comprises determining a presence, absence, amount, and/or alteration of at least one FXS-associated metabolite (e.g., an FXS-associated metabolite).
  • an FXS-associated metabolite is present in aberrant amount in a FXS subject, is potentially involved in FXS initiation, progression, and/or prediction, or both.
  • an FXS-associated metabolite is present in aberrant amount in a FXS subject.
  • an FXS-associated metabolite is potentially involved in FXS initiation, progression, and/or prediction.
  • a method comprises determining a presence of at least one FXS-associated metabolite. In some embodiments, a method comprises determining an absence of at least one FXS-associated metabolite. In some embodiments, a method comprises determining an amount of at least one FXS-associated metabolite. In some embodiments, a method comprises determining an alteration of at least one FXS-associated metabolite.
  • an FXS-associated metabolite is absent in a sample (e.g., a CSF sample) from a subject (e.g., a human), but present in a control sample and/or a reference.
  • an FXS-associated metabolite is present in a sample (e.g., a CSF sample) from a subject (e.g., a human), but absent in a control sample and/or a reference.
  • a sample e.g., a CSF sample
  • at least one metabolite has an altered level in a sample (e.g., a CSF sample) from a subject (e.g., a human), relative to a control sample and/or a reference (e.g., reference level).
  • a control sample comprises a sample from one or more age- matched typically-developing individuals (e.g., two or more age-matched typically-developing individuals), a baseline sample from a subject (e.g., prior to the onset of the disease state), or a sample derived from healthy cells (e.g., not affected by the disease state and from a similar origin), or any combination thereof.
  • a control sample is a sample from one or more age-matched typically-developing individuals.
  • a control - 31 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 sample is a baseline sample from a subject.
  • a control sample is a sample derived from healthy cells.
  • the reference level is based on: a sample from an age-matched typically-developing individual, or a baseline sample from the subject.
  • a level of at least one metabolite is compared to a level of a reference (e.g., standard).
  • a reference is a theoretical value obtained from a population, e.g., a general population (e.g., from the same tissue, organ, bodily fluid, cell, organelle, and/or cellular compartment as a FXS sample). In some embodiments, a reference is a theoretical value obtained from one or more FXS subjects who were treated successfully.
  • the term “metabolite” e.g., an FXS-associated metabolite excludes molecules having molecular weights over 2,000 Daltons. In some embodiments, the term “metabolite” excludes molecules having molecular weights over 1,500 Daltons.
  • an FXS-associated metabolite excludes molecules having molecular weights over 1,000 Daltons.
  • an FXS-associated metabolite comprises a signaling molecule.
  • an FXS-associated metabolite comprises an amino acid, fatty acid, nucleotide, sugar, or an intermediate of a cellular process, or a combination thereof.
  • At least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference, comprises n-acetylneuraminate, D-alanine, L-alanine arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, L-carnitine, carnosine, citrulline, creatine, cystine, cytosine, fumarate, alpha-D-glucose, glucuronate, oxidized glutathione (GSSG), reduced glutathione (GSH), glycerol, glycine, guanine, 3-methyl-L-histidine, homoserine, hypoxanthine, indoleacetaldehyde, isoleucine, kynurenate, leucine, lysine, malate, methionine,
  • GSSG oxidized glutathione
  • GSH reduced glutathione
  • At least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference comprises n-acetylneuraminate, D-alanine, L-alanine arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, L-carnitine, carnosine, citrulline, creatine, cystine, cytosine, fumarate, alpha-D-glucose, glucuronate, oxidized glutathione (GSSG), reduced glutathione (GSH), glycerol, glycine, guanine, 3-methyl-L-histidine, homoserine, hypoxanthine, indoleacetaldehyde, isoleucine, kynurenate, leucine,
  • At least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference comprises: a) lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L- carnitine, arginine, cystine, urate, GSH, phosphoenolpyruvate, GSSG, orotate, or guanosine, or a combination thereof, b) argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl ace
  • At least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference comprises lysine, proline, glycine, - 33 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L-carnitine, arginine, cystine, urate, GSH, phosphoenolpyruvate, GSSG, orotate, or guanosine, or a combination thereof.
  • At least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference comprises argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-formyl-L-methionine, arginine, azelate, trehalose, or D-sedoheptulose, or a combination thereof.
  • At least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference comprises valine, norvaline, nicotinamide, D-alanine, creatine, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha-D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n-acetylneuraminate, asparagine, or L-alanine, or a combination thereof.
  • At least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference comprises arginine, leucine, lysine, or D-sedoheptulose, or a combination thereof.
  • at least one metabolite has a reduced level in a sample from a subject, relative to a control sample and/or a reference.
  • the FXS- associated metabolite has a reduced level in the sample from the subject, relative to a reference level of the same metabolite.
  • At least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference comprises: a) lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L- carnitine, arginine, cystine, urate, or GSH, or a combination thereof, b) argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-
  • a FXS-associated metabolite comprises n-acetylneuraminate, D-alanine, L-alanine arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, L-carnitine, carnosine, citrulline, creatine, cystine, cytosine, fumarate, alpha-D-glucose, glucuronate, oxidized glutathione (GSSG), reduced glutathione (GSH), glycerol, glycine, guanine, 3-methyl-L-histidine, homoserine, hypoxanthine, indoleacetaldehyde, isoleucine, kynurenate, leucine, lysine, malate, methionine, methyl acetoacetate, methylmalonate, mevalonate, n-formyl-L-methionine, nicotinamide
  • a FXS-associated metabolite comprises: a) lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L- carnitine, arginine, cystine, urate, or reduced glutathione (GSH), or a combination thereof, b) argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-
  • a FXS-associated metabolite comprises arginine, leucine, or lysine, or a combination thereof.
  • a FXS-associated metabolite comprises: a) phosphoenolpyruvate, oxidized glutathione disulfide (GSSG), orotate, or guanosine, or a combination thereof, or b) trehalose, or D-sedoheptulose, or both, or a combination thereof.
  • At least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference comprises lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L-carnitine, arginine, cystine, urate, or GSH, or a combination thereof.
  • At least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference comprises argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-formyl-L-methionine, arginine, or azelate, or a combination thereof.
  • At least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference comprises valine, norvaline, nicotinamide, D-alanine, creatine, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha-D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n-acetylneuraminate, asparagine, or L-alanine, or a combination thereof.
  • a polypeptide is an antibody mimetic. - 55 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00298]
  • Techniques, assays, and reagents for making and using therapeutic antibodies, or antigen-binding fragments thereof, against a target antigen e.g., an FMR1 transcript or protein product associated with a fragile X-associated disorder, such as FXS
  • a target antigen e.g., an FMR1 transcript or protein product associated with a fragile X-associated disorder, such as FXS
  • Therapeutic Monoclonal Antibodies From Bench to Clinic (Zhiqiang An eds., 1st ed. 2009); Antibodies: A Laboratory Manual (Edward A.
  • a gene editing system is used to decrease FMR1 CGG expansion in a subject prior to administering a polynucleotide.
  • a gene editing system is a CRISPR/Cas system, a transposon- based gene editing system, or a transcription activator-like effector nuclease (TALEN) system.
  • TALEN transcription activator-like effector nuclease
  • a gene editing system is a CRISPR/Cas system.
  • the gene editing system is a class II CRISPR/Cas system.
  • a gene editing system comprises a double nickase Cas9 (e.g., to achieve more accurate genome editing; see, e.g., Ran et al.., Cell 154: 1380-89 (2013).
  • Wild- type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA.
  • nickase Cas9 generates only a single-strand break.
  • dCas9 is catalytically inactive.
  • dCas9 is fused to a nuclease (e.g., a FokI to generate DSBs at target sequences homologous to two gRNAs).
  • compositions of the present disclosure include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations.
  • Pharmaceutical compositions and formulations of the present disclosure may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
  • penetration enhancers e.g., a coating agent
  • carriers e.g., a coating agent
  • excipients e.g., a coatings
  • Emulsions include but are not limited to heterogenous systems of one liquid dispersed in another in the form of droplets (e.g., droplets exceeding 0.1 ⁇ m in diameter).
  • Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.
  • Microemulsions are included as embodiments of the present disclosure. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.
  • Formulations of the present disclosure include but are not limited to liposomal formulations.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
  • Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered.
  • Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
  • Pharmaceutical formulations and compositions of the present disclosure may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat.
  • an agent or a composition of the present disclosure is administered separately.
  • an agent e.g., ASO
  • an agent e.g., ASO
  • an agent e.g., ASO
  • an agent e.g., ASO
  • a composition of the present disclosure is delivered locally to the central nervous system. This can include intrathecal or intraventricular injections, including the use of a catheter or Ommaya reservoir.
  • an agent or a composition of the present disclosure is delivered using an approach that enhances bioavailability in the central nervous system after systemic administration.
  • agents e.g., drugs
  • an agent or a composition of the present disclosure is delivered using an approach that enhances bioavailability in the central nervous system after systemic administration.
  • These approaches can include modification of the sugars or phosphate linkages, delivering as a duplex with a ligand-conjugated RNA molecule, formulation into an artificial exosome, liposome, polymer nanoparticle or lipid nanoparticle, or conjugation to lipids, antibodies, peptides, sugars, neuroactive molecules, or other moieties that enhance delivery to the central nervous system.
  • the agent is delivered after transiently - 61 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 disrupting the blood-brain barrier.
  • an agent e.g., ASO
  • a composition of the present disclosure is administered as intrathecal bolus injection.
  • an agent or a composition is delivered systemically, such as via intravenous or subcutaneous injection.
  • an agent e.g., ASO
  • a composition of the present disclosure is delivered using one or more controlled or sustained-release delivery systems (e.g., capsules, biodegradable matrices).
  • controlled or sustained-release delivery systems e.g., capsules, biodegradable matrices.
  • Non-limiting example delayed-release delivery systems for drug delivery that would be suitable for administration of a composition described herein are described in U.S. Patent Nos. US 5,990,092 (issued to Walsh); 5,039,660 (issued to Leonard); 4,452,775 (issued to Kent); and 3,854,480 (issued to Zaffaroni), the entire teachings of which are incorporated herein by reference.
  • a therapeutically effective amount of an agent is sufficient to: a) decrease splicing of an aberrant FMR1 transcript (e.g., between Exons 1 and 2 of FMR1-217), b) decrease level of an aberrant FMR1 gene product (e.g., an aberrant FMR1 transcript such as FMR1-217, and/or an aberrant FMR1-encoded protein), c) increase splicing of a normal FMR1 transcript (e.g., between Exons 1 and 2 of FMR1-205), d) increase level of a normal FMR1 gene product (e.g., a normal FMR1 transcript such as FMR1-205 and/or a normal FMR1-encoded protein), or e) increase level of FMRP, or any combination thereof, relative to a reference.
  • an aberrant FMR1 gene product e.g., an aberrant FMR1 transcript such as FMR1-217, and/or an aberrant FMR1-encoded protein
  • a therapeutically effective amount of an agent is sufficient to decrease splicing of an aberrant FMR1 transcript, relative to a reference, for example, by at least about 5% (e.g., by at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%).
  • a therapeutically effective amount of an agent is sufficient to decrease level of an aberrant FMR1 gene product, relative to a reference, for example, by at least - 62 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 about 5% (e.g., by at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%).
  • a therapeutically effective amount of an agent is sufficient to decrease level of an aberrant FMR1 gene product, relative to a reference, by at least about 25%.
  • splicing of an aberrant FMR1 transcript, level of an aberrant FMR1 gene product, splicing of a normal FMR1 transcript, level of a normal FMR1 gene product, level of FMRP, or any combination thereof is measured at least about 1 day after an agent is administered to a subject, e.g., for at least: about: 2 days, 3 days, 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 - 63 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 months, or 6 months, after administration.
  • splicing of an aberrant FMR1 transcript, level of an aberrant FMR1 gene product, splicing of a normal FMR1 transcript, level of a normal FMR1 gene product, level of FMRP, or any combination thereof is measured a tissue or a cell, for example, a white blood cell, a leukocyte, a fibroblast cell (e.g., a dermal derived fibroblast cell or a lung- derived fibroblast cell), a cortex tissue (e.g., a brain biopsy of superficial cortex), or a combination thereof.
  • a tissue or a cell for example, a white blood cell, a leukocyte, a fibroblast cell (e.g., a dermal derived fibroblast cell or a lung- derived fibroblast cell), a cortex tissue (e.g., a brain biopsy of superficial cortex), or a combination thereof.
  • a therapeutically effective amount of an agent is sufficient to raise an intelligence quotient (IQ) score, for example, to at least about 40, e.g., to at least about: 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130, to between about: 40-110, 40-100, 50-105, 60-80, 65-90, 70-80, 75-95, or 70-100, or to about: 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130.
  • IQ intelligence quotient
  • a therapeutically effective amount of an agent is sufficient to raise an IQ score by at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or - 64 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 20 points, by 1-10, 1-15, 2-20, 2-15, 2-10, 5-15, 5-10, 10-20, or 15-20 points, or by about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 points.
  • a therapeutically effective amount of an agent is sufficient to reduce (e.g., prevent) absent or irregular menses, fertility problems, elevated FSH (follicle- stimulating hormone) levels, premature ovarian failure, primary ovarian insufficiency, and/or hot flashes.
  • a therapeutically effective amount of an agent is sufficient to reduce (e.g., prevent) intention tremors, parkinsonism, ataxia, memory loss, white matter lesions involving middle cerebellar peduncles, and/or cognitive decline.
  • a therapeutically effective amount of an agent is sufficient to reduce (e.g., prevent) neuropathy of extremities, mood instability, irritability, explosive outbursts, personality changes, and/or autonomic function problems such as impotence or loss of bladder or bowel functions. In some embodiments, a therapeutically effective amount of an agent is sufficient to reduce (e.g., prevent) high blood pressure, thyroid disorders, and/or fibromyalgia. Additional Therapeutic Agents [00340] In some embodiments, one or more additional therapeutic agents are administered to a subject.
  • Non-limiting examples of additional therapeutic agents include a modulator of DNA methylation (e.g., by inhibiting DNA methylation and/or promoting DNA demethylation), a metabotropic glutamate receptor 5 (mGluR5) modulator (e.g., Basimglurant or Mavoglurant), a GABAB receptor activator (e.g., arbaclofen), a GABAA or GABAB receptor activator (e.g., acamprosate), an AMPAkine (e.g.,AX516), a CB1 inhibitor (e.g., rimonabant), a RAS signaling inhibitor (e.g., lovastatin), a STEP inhibitor, an S6K inhibitor, a PAK inhibitor (e.g., FRAX486), an MMP9 inhibitor (e.g., minocycline), a GSK3 ⁇ inhibitor (e.g., lithium), and combinations thereof.
  • mGluR5 metabotropic glutamate receptor 5
  • treating a subject comprises providing the subject with a ketogenic (“keto”) diet.
  • a therapeutically effective amount of a DNA-demethylating compound or DNA demethylase is administered to a subject, prior to, during, or after, administering an agent (e.g., ASO) or a composition described herein to the subject.
  • an agent e.g., ASO
  • a therapeutically effective amount of a DNA-demethylating compound or DNA - 65 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 demethylase is administered to a subject after administering an agent or a composition disclosed herein to the subject.
  • Non-limiting examples of DNA-demethylating compounds include 5-Azacytidine (5- Aza-CR) and 5-aza-2′-deoxycytidine (5-Aza-CdR), dihydro-5-azacytidine (DHAC), zebularine, 5-fluoro-2'-deoxycytidine, Hydralazine, RG108, procainamide, and SGI-1027.
  • a DNA-demethylating compound is a nucleoside analogue.
  • a DNA-demethylating compound is a non-nucleoside analogue.
  • a DNA demethylase e.g., DNA methylation modification enzymes Dnmt or Tet
  • Dnmt or Tet DNA methylation modification enzymes
  • dCas9-Dnmt/Tet a catalytically inactivate Cas9
  • sgRNA single guide RNA
  • a dCas9-Tet1 demethylates the FMR1 locus and promoter region when FMR1 has an expanded CGG repeat of 200 or more.
  • a therapeutically effective amount of a DNA-demethylating compound or DNA demethylase is sufficient to demethylate at least about 5% of an FMR1 gene, for example, at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, about: 10-100%, 10-90%, 15-90%, 15-80%, 15-75%, 20-75%, 20-70%, 25-60%, 25-55%, 25-50%, 30-40%, or 30-35%, or about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an FMR1 gene.
  • Such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times.
  • Therapeutic agents in a combination therapy can be administered via the same administration route or via different administration routes.
  • Powders and/or liquids may be - 66 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 reconstituted or diluted to a desired concentration (e.g., appropriate for dosage) prior to administration.
  • a treatment regimen will provide beneficial effects of a drug combination in treating diseases, conditions, or disorders described herein.
  • a method is used for monitoring (e.g., in a clinical trial and/or in a drug screen) effectiveness of an agent.
  • a method comprises obtaining level of a metabolite from a subject before administering an agent, and monitoring changes in the level as an indication of the effectiveness of the agent in the subject.
  • a level of a metabolite of a subject is compared to a predetermined reference (e.g., standard).
  • a method comprises altering (e.g., increasing or decreasing) the administration of an agent to a subject.
  • increased administration of an agent is desirable to increase effectiveness of an agent.
  • decreased administration of an agent is desirable to decrease effect(s) of an agent.
  • the disclosure provides, among other things, methods of predicting a treatment outcome of, diagnosing, prognosing, or subclassifying fragile X syndrome (FXS) in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing.
  • FXS fragile X syndrome
  • the disclosure also provides, among other things, methods of diagnosing FXS in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, - 67 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing.
  • the disclosure also provides, among other things, methods of prognosing FXS in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing.
  • the disclosure also provides, among other things, methods of subclassifying FXS in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing.
  • the disclosure also provides, among other things, methods of diagnosing, subclassifying or predicting a treatment outcome of FXS in a subject, the methods comprising: a) determining a presence, absence, amount, or alteration of at least one FXS- associated metabolite, or b) determining an alteration of an FXS-associated ratio of two metabolites, or both a) and b), in a sample from a subject, wherein the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS- associated ratio, in the sample is indicative of the presence, risk, progression, severity, or treatment outcome of FXS.
  • the disclosure also provides, among other things, methods of predicting a treatment outcome of FXS in a subject, the methods comprising: - 68 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 a) determining: i. a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii.
  • the disclosure also provides, among other things, methods of diagnosing FXS in a subject, the method comprising: a) determining: i. a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii.
  • the disclosure also provides, among other things, methods of prognosing FXS in a subject, the methods comprising: a) determining: i. a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii.
  • an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject and b) prognosing the subject as having a propensity to have a poorer prognosis of FXS based on the presence, absence, amount, or alteration of the at least one FXS- associated metabolite, or the alteration of the FXS-associated ratio, in the sample.
  • the subject may be prognosed as having a propensity to have a poorer prognosis of FXS, compared to another subject with a ratio of 1:4.
  • a subject is any one or more subjects described herein.
  • At least one FXS-associated metabolite is any one or more FXS-associated metabolites disclosed herein.
  • a sample is any one or more samples described herein.
  • an FXS-associated ratio is any one or more FXS-associated ratios disclosed herein.
  • a treatment outcome is any one or more treatment outcomes described herein.
  • an aberrant metabolite level e.g., an increased level or a reduced level of a metabolite, a presence of a metabolite not usually present, or an absence of a metabolite usually present
  • a method disclosed herein is used to identify a subject having or at risk for developing FXS.
  • a method is used to determine whether to administer an agent to a subject.
  • the subject is treated when the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS- associated ratio, in the subject’s sample, is determined or detected.
  • the subject’s treatment is altered when the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the subject’s sample, is determined or detected.
  • a method is used to determine whether a subject is effectively treated with an agent.
  • a method provides information useful in prognostication, staging and/or management of FXS. In some embodiments, a method provides information useful for designing a treatment regimen. [00368] In some embodiments, a method is used for monitoring (e.g., in a clinical trial and/or in a drug screen) effectiveness of an agent. In some embodiments, a method comprises obtaining level of a metabolite from a subject before administering an agent, and monitoring changes in the - 70 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 level as an indication of the effectiveness of the agent in the subject.
  • a level of a metabolite of a subject is compared to a predetermined reference (e.g., standard).
  • a predetermined reference e.g., standard.
  • the subject is treated when the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS- associated ratio, in the subject’s sample, is determined or detected, for example, in a method disclosed herein.
  • the subject’s treatment is altered when the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the subject’s sample, is determined or detected for example, in a method disclosed herein.
  • FXS is the most frequently inherited form of intellectual impairment and most common single-gene cause of autism. There are about 83,000 FXS cases in the United States; 1 in approximately 4,000 boys and 1 in approximately 7,000 girls are afflicted. FXS children present a range of symptoms beyond intellectual impairment, including speech and developmental delays, perseveration, hyperactivity, aggression, and epilepsy, among other maladies.
  • FXS is caused by a CGG triplet repeat expansion in a single gene, fragile X messenger ribonucleoprotein 1 (FMR1), which resides on the X chromosome.
  • FMR1 fragile X messenger ribonucleoprotein 1
  • FMRP protein fragile X messenger ribonucleoprotein protein
  • a therapeutic biomarker may entail measurement of brain tissue, or a brain-derived fluid – particularly cerebral spinal fluid (CSF), which is derived from the brain ventricles.
  • CSF cerebral spinal fluid
  • Metabolites may be determined with extreme accuracy and with strong statistical rigor using liquid chromatography-mass spectrometry (LC-MS).
  • LC-MS liquid chromatography-mass spectrometry
  • the samples were centrifuged at 22,000 x g for 10 minutes at 4°C, creating three layers.
  • the top layer contained polar metabolites
  • the middle layer was the protein interface
  • the bottom layer contained nonpolar metabolites.
  • the polar metabolites were transferred into a new tube and the fluid evaporated in a refrigerated CENTRIVAP® attached to a -105°C cold trap (Labconco Corporation, Kansas City, MO).
  • the bottom layer was discarded, and the protein interface was used to quantify protein.
  • RIPA buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 150mM NaCl) was added to the tube containing the protein interface and samples were vortexed 10 minutes at 4°C. The samples were centrifuged at 22,000 x g for 10 minutes at 4°C and protein was quantified with a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA).
  • BCA bicinchoninic acid
  • PBMCs were isolated from whole blood using a Cell Preparation (BD VACUTAINER® CPTTM) blood tube (BD Biosciences, Franklin Lakes, NJ). PBMCs were pelleted and washed twice with 1X PBS.
  • the PBMCs were cultured in RPMI-1640 medium (Sigma-Aldrich, Burlington, MA), supplemented with 15% fetal bovine serum (FBS) and 2.5% L-glutamine, at 37°C with 5% CO 2 in T25 flasks for 4 days. PBMCs were collected by centrifugation and pellets were frozen at -80°C. For LC-MS, the cells were pelleted and washed twice with 1X PBS. Samples were resuspended in 800 ⁇ L 80% LC-MS-grade methanol in LC- MS-grade water and vortexed for 10 minutes at 4°C.
  • Samples were centrifuged at 22,000 x g for 10 minutes at 4°C and supernatants were dried down in a refrigerated CENTRIVAP® attached to a -105°C cold trap (Labconco Corporation). Dried polar metabolite pellets were resuspended in 100 ⁇ L of LC-MS-grade water and vortexed for 10 minutes at 4°C. Samples were centrifuged at 22,000 x g for 10 minutes at 4°C and 20 ⁇ L was moved into LC-MS vials.
  • Lymphoblast cell lines were obtained from Coriell Institute (Camden, NJ) from two FXS individuals (GM07365 and GM06897) and two typically-developing control males (GM07174 and GM06890).
  • Cells were cultured in RPMI-1640 medium (Sigma-Aldrich), supplemented with 15% fetal bovine serum (FBS) and 2.5% L-glutamine, at 37°C with 5% CO 2 in T25 flasks.
  • LCLs were collected by centrifugation and pellets were frozen at -80°C.
  • Samples were resuspended in 800 ⁇ L 80% LC-MS-grade methanol in LC-MS-grade water and vortexed for 10 minutes at 4°C. Samples were centrifuged at 22,000 x g for 10 minutes at 4°C and supernatants were dried down in a refrigerated CENTRIVAP® attached to a -105°C cold trap (Labconco Corporation). Dried polar metabolite pellets were resuspended in 100 ⁇ L of LC-MS- grade water and vortexed for 10 minutes at 4°C. Samples were centrifuged at 22,000 x g for 10 minutes at 4°C and 20 ⁇ L was moved into LC-MS vials.
  • Cerebrospinal fluid was collected from non-Fragile X individuals at Rush University Medical Center (Chicago, IL). It was shipped to UMass Chan Medical School and used for LC- MS analysis.
  • a Q EXACTIVE® Plus Hybrid Quadrupole-ORBITRAP® mass spectrometer (Thermo Fisher Scientific) equipped with an Ion Max source and a HESI II probe coupled to a VANQUISH® Horizon UHPLC System (Thermo Fisher Scientific) was used to measure polar metabolites on post-mortem brain samples.
  • the instrument Prior to operation, the instrument underwent mass calibration for positive and negative ion mode using Calmix (Thermo Fisher Scientific). Two ⁇ L of resuspended polar metabolite samples were injected onto a SeQuant ZIC®-pHILIC 5 ⁇ m 150 x 2.1 mm analytical column equipped with a 2.1 x 20 mm guard column (MilliporeSigma, Burlington, MA). The column oven was held at 25°C and the autosampler tray was held at 4°C. Buffer A was comprised of 20 mM ammonium carbonate, 0.1% ammonium hydroxide. Buffer B was comprised of 100% acetonitrile.
  • the chromatographic gradient was run at a flow rate of 0.150 mL/min as follows: 0-20 min: linear gradient from 80-20% buffer B; 20-20.5 min: linear gradient from 20-80% buffer B; 20.5-28 min: hold at 80% buffer B.
  • the mass spectrometer was operated in full-scan, polarity switching mode, with the spray voltage set to 4.0 kV and the heated capillary at 350°C.
  • the sheath gas flow was 10 units, the auxiliary gas flow was 1 unit, and the sweep gas flow was 1 unit.
  • the resolution was set at 70,000, the automatic gain control (AGC) target at 1x10 6 , and the maximum injection time at 20 msec.
  • AGC automatic gain control
  • FIGs.6A-B a volcano plots display metabolites that exhibit statistical significance (-log(p>1.3)) in A. cerebellum tissue and B. cortex.
  • FIG.6C. depicts a bar graph displaying changes in leucine and lysine. Free amino acids are denoted in green, while their derivatives are highlighted in red, constituting most observed changes. The most significant reductions in brain tissues were observed in free amino acids, with 14 out of 20 reduced in the cerebellum and 13 out of 20 in the cortex.
  • FXS patient-derived cell lines with varying FMRP expression levels were utilized: the FXS2 line displayed 50% FMRP compared to TD cells, whereas the FXS1 line exhibited undetectable FMRP levels via western blot analysis (FIG.7A).
  • the PCA plot showed distinct clustering based on genotype, with FXS1 further apart from TD compared to FXS2, suggesting that partial FMRP expression in FXS2 cells influences the metabolite profile towards TD (FIG.7B).
  • the findings revealed a depletion of the free amino acid pool in both FXS LCL cultures (FIG.7C, D), mirroring the observations in FXS brain tissues (FIG.6). Indeed, upon comparing the FXS2 vs.
  • FXS1 metabolite profiles a few amino acids exhibit higher expression levels, which may correlate with the increased FMRP levels in FXS2 line (FIG.7E). Examples of amino acids changes are depicted in FIG.7F. These findings suggest that restoring FMRP levels could potentially ameliorate metabolic deficits in FXS. - 93 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00397] Research Strategy: [00398] Methods: [00399] a) Cell culture- LCL lines will be grown in RPMI media with 15% FBS as per instructions from Coriell Institute, NJ for all experiments pertaining to 2.1 and 2.2.
  • iPSC derived forebrain neurons via reprogramming of the LCL lines (FIG.8) have been generated.
  • the forebrain neurons will be cultured in maturation media (STEMCELL Technologies) for a period of 9 days. ASOs resuspended in water will be applied to cultures at 5uM concentration for a minimum of 7 days before testing.
  • Drug treatments- FXS and TD LCLs will be treated with inhibitors such as Rapamycin, MG-132 or Bortezomib, 3-methyladenine (3-MA) and dexpramipexole (DEX) or vehicle control.
  • LC-MS will be performed on the samples.
  • c) Protein assays- Western blots will be performed using antibodies against FMRP, GAPDH, phospho-mTOR, mTOR, anti–LC3-I/II, ubiquitin, Hexokinase I.
  • LC-MS- Metabolites will be extracted using an acetonitrile:methanol:water based solution and subjected to a quadrupole orbitrap mass spectrometer coupled to a Vanquish UHPLC system with electrospray ionization.
  • f) Data analysis- The LC-MS data will be analyzed using the MAVEN software and Compound Discoverer software.
  • Power and statistical analysis- All experiments will be performed using at least three biological replicates. Controls will include cells from typically developing individuals cultured parallel to FXS cell lines and treated with the inhibitors or ASOs as well as their respective vehicle treatments. All samples within an experiment will be run simultaneously for LC-MS.
  • Reduced amino acids could also alter charged tRNA: uncharged tRNA ratios and impact protein synthesis.
  • a qPCR-based method, i-trap (individual tRNA acetylation PCR), will be used to measure tRNA charging (Y. Tsukamoto et al.., i-tRAP (individual tRNA acylation PCR): a convenient method for selective quantification of tRNA charging. Rna 29, 111–112 (2023)).
  • This method includes a demethylation step which makes uncharged tRNAs’ 3′-end CC while charged tRNAs’ 3′-end CCA, and using TaqMan-MGB probes designed to recognize tRNAs of interest as well as the CC and CCA polymorphisms the charged tRNA fraction will be quantified.
  • 2.1B Effect of protein degradation deficits on amino acid levels in FXS [00411] Increased activity of the ubiquitin proteasome system (UPS) in FXS model mice, elevates protein degradation. Normalizing proteasome activity with bortezomib or MG-132 inhibitors has been shown to correct FXS phenotypes such as excessive hippocampal neuronal firing (S. R.
  • UPS ubiquitin proteasome system
  • Inhibitors such as 3-methyladenine (3-MA) may be used to alter autophagy and its effect tested using LC-MS. - 95 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00412] 2.1C: Study of whether restoring mitochondrial deficits can influence amino acid levels in FXS cells [00413] Mitochondrial deficits, including inner membrane proton leaks, may exacerbate amino acid imbalances by altering their synthesis via the increased flux in the TCA cycle that can be ameliorated by inhibitors such as dexpramipexole (DEX) (P.
  • DEX dexpramipexole
  • FMRP may be restored in FXS cells and metabolome changes measured using LC-MS (FIG.10).
  • 2.2A Study of whether FMRP rescue can restore amino acid levels in FXS LCLs
  • ASOs antisense oligonucleotides
  • FXS is a neurodevelopmental disorder.
  • FXS LCLs have been re-programmed (FIGs.7 and 8) to derive forebrain excitatory mature neurons.
  • LC-MS will be used to determine whether the metabolome changes observed in FXS patient derived brain tissues and LCLs (FIGs. 6 and 7) are mirrored in the iPSC derived neurons in culture.
  • Tables 5-10 show the raw data of all the metabolites in the LCL cell lines (TD, FXS1, FXS2). Table 5. Metabolic changes comparing FXS1 to TD LCLs (cell pellet) Metabolite log 2 (fold change) -log 10 (p-value) HISTIDINOL -44532700 35617310 - 97 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Metabolite log 2 (fold change) -log 10 (p-value) TYROSINE -0.9744300 2.3462150 Table 6.
  • FIG.16A Increased glutamine incorporation into citrate in FXS is indicative of increased reverse carboxylation of glutamine in the citric acid cycle.
  • FXS cells increased percent of glutamine incorporation into glutamate was observed which accumulated more in the extracellular media than intracellularly (FIGs.16B-E).
  • Increased ion count of glutamine-derived glutamate was observed in media in FXS cultures (FIGs.16B and 16D) but a smaller increase was observed intracellularly (FIGs.16C and 16E).
  • a method of diagnosing, subclassifying, or predicting a treatment outcome of fragile X syndrome (FXS) in a subject comprising: a) determining a presence, absence, amount, or alteration of at least one FXS- associated metabolite, or b) determining an alteration of an FXS-associated ratio of two metabolites, - 103 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 or both a) and b), in a sample from the subject, wherein the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS- associated ratio, in the sample is indicative of the presence, risk, progression, severity, or treatment outcome of FXS.
  • FXS fragile X syndrome
  • a method of predicting a treatment outcome of fragile X syndrome (FXS) in a subject comprising: a) determining: i) a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii) an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject, and b) predicting the treatment outcome of FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the sample.
  • a method of diagnosing fragile X syndrome (FXS) in a subject comprising: a) determining: i) a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii) an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject, and b) diagnosing the subject as having or having a propensity to develop FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the sample. 4.
  • FXS fragile X syndrome
  • a method of prognosing fragile X syndrome (FXS) in a subject comprising: a) determining: i) a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii) an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject, and - 104 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 b) prognosing the subject as having a propensity to have a poorer prognosis of FXS based on the presence, absence, amount, or alteration of the at least one FXS- associated metabolite, and/or the alteration of the FXS-associated ratio, in the sample.
  • FXS fragile X syndrome
  • treating FXS comprises administering to the subject a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product.
  • FMR1 fragile X messenger ribonucleoprotein 1
  • ASO is chemically modified to comprise: a) a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2’-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2’-O,4’-C-aminomethylene bridged nucleic acid (2’,4’-BNA(NC)), an alpha-L-locked nucleic acid, or a tricyclo-DNA, or a combination thereof, - 106 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 b) a ribose group comprising 2’-O-methyl, 2’-fluoro, 2’-deoxy, 2’-O-(2- methoxyethyl) (MOE), 2’-O-alkyl, 2’-O-alkoxy, 2’-O
  • LNA locked nucleic acid
  • CSF cerebrospinal fluid
  • PBMCs peripheral blood mononuclear cells
  • the at least one metabolite comprises n- acetylneuraminate, D-alanine, L-alanine arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, L-carnitine, carnosine, citrulline, creatine, cystine, cytosine, fumarate, alpha-D-glucose, glucuronate, oxidized glutathione (GSSG), reduced glutathione (GSH), glycerol, glycine, guanine, 3-methyl-L-histidine, homoserine, hypoxanthine, indoleacetaldehyde, isoleucine, kynurenate, leucine, lysine, malate, methionine, methyl acetoacetate, methylmalonate, mevalonate, n-formyl-L-methionine, nicotinamide,
  • the at least one metabolite comprises: a) lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L- carnitine, arginine, cystine, urate, or reduced glutathione (GSH), or a combination thereof, b) argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuron
  • Embodiment 31 or 32 wherein the control sample: a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject.
  • the at least one metabolite has an increased level in the sample from the subject, relative to a control sample and/or reference level.
  • a level of the at least one FXS-associated metabolite has a log 2 fold increase of ⁇ 0.50 in the sample, relative to a control sample and/or reference level, optionally, wherein the log 2 fold increase is ⁇ 0.90. 36.
  • Embodiment 34 or 35 wherein the control sample: a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject.
  • the at least one metabolite comprises: a) phosphoenolpyruvate, oxidized glutathione disulfide (GSSG), orotate, or guanosine, or a combination thereof, or - 109 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 b) trehalose, or D-sedoheptulose, or both, or a combination thereof. 38.
  • Embodiment 37 wherein the at least one metabolite has an increased level in the sample from the subject, relative to a control sample and/or reference level.
  • 39. The method of Embodiment 38, wherein a level of at least one FXS-associated metabolite has a log 2 fold increase of ⁇ 0.50 in the sample, relative to a control sample and/or reference level, optionally, wherein the log 2 fold increase is ⁇ 0.90.
  • the control sample a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject. 41.
  • Embodiment 37 wherein the at least one metabolite has a reduced level in the sample from the subject, relative to a control sample and/or reference level.
  • 42. The method of Embodiment 41, wherein a level of the at least one FXS-associated metabolite has a log 2 fold reduction of ⁇ (-0.50) in the sample, relative to a control sample and/or reference level, optionally, wherein the log 2 fold increase is ⁇ (-0.90).
  • the control sample a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject. 44.
  • the method of Embodiment 46, wherein the FXS-associated ratio is a ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP).
  • 48. The method of any one of Embodiments 44-47, wherein the FXS-associated ratio is reduced in the sample from the subject, relative to a control sample and/or reference level.
  • 49. The method of Embodiment 48, wherein the control sample: a) is a sample from an age-matched typically developing individual, or b) is a baseline sample from the subject.
  • 50 The method of any one of Embodiments 44-47, wherein the FXS-associated ratio is increased in the sample from the subject, relative to a control sample and/or reference level. 51.
  • Embodiment 50 wherein the control sample: a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject.
  • the control sample a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject.
  • 52. The method of any one of Embodiments 1-51, wherein the subject is a human male.
  • 53. The method of any one of Embodiments 1-51, wherein the subject is a human female.
  • determining the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, or the alteration of the FXS-associated ratio comprises performing liquid chromatography-mass spectrometry (LC-MS). 55.
  • LC-MS liquid chromatography-mass spectrometry
  • a method of diagnosing, subclassifying, or predicting a treatment outcome of fragile X syndrome (FXS) in a subject comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing, wherein a), b), or c), or a combination of the foregoing is indicative of the presence, risk, progression, severity, or treatment outcome of FXS.
  • FXS fragile X syndrome
  • a method of predicting a treatment outcome of fragile X syndrome (FXS) in a subject comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and predicting the treatment outcome of FXS based on a), b), or c), or a combination of the foregoing.
  • FXS fragile X syndrome
  • a method of diagnosing fragile X syndrome (FXS) in a subject comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and - 112 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 diagnosing the subject as having or having a propensity to develop FXS based on a), b), or c), or a combination of the foregoing.
  • FXS fragile X syndrome
  • a method of prognosing fragile X syndrome (FXS) in a subject comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and prognosing the subject as having a propensity to have a poorer prognosis of FXS based on a), b), or c), or a combination of the foregoing.
  • FXS fragile X syndrome
  • a method of treating fragile X syndrome (FXS) in a subject comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and treating FXS based on a), b), or c), or a combination of the foregoing.
  • FXS fragile X syndrome

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Abstract

Provided herein, in various embodiments, are methods of treating fragile X syndrome (FXS), comprising determining in a sample from the subject: a presence, an absence, or a level of an FXS-associated metabolite; an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite; a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS- associated metabolites, or a combination of the foregoing. Also provided herein, in various embodiments, are methods of diagnosing, prognosing, subclassifying and/or predicting a treatment outcome of FXS in a subject.

Description

Docket No.5439.1032-002; UMMS 23-42 BIOMARKERS AND METHODS RELATED TO FRAGILE X SYNDROME RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/554,913, filed on February 16, 2024, which claims the benefit of U.S. Provisional Patent Application No. 63/511,638, filed on June 30, 2023, each of which is incorporated by reference herein in its entirety. INCORPORATION BY REFERENCE OF MATERIAL IN XML [0002] This application incorporates by reference the Sequence Listing contained in the following eXtensible Markup Language (XML) file being submitted concurrently herewith: File name: 54391032-002_SL.xml; created April 1, 2024, 41,224 Bytes in size. GOVERNMENT SUPPORT [0003] This invention was made with government support under GM135087 and GM046779 from National Institutes of Health. The government has certain rights in the invention. BACKGROUND [0004] Fragile X syndrome (FXS) is an autism spectrum disorder that is the most frequently inherited form of intellectual impairment. FXS afflicts 1 in approximately 4,000 boys and 1 in approximately 7,000 girls. In addition to intellectual impairment, children with FXS present a range of symptoms including speech and developmental delays, perseveration, hyperactivity, aggression, and epilepsy, among other maladies. FXS is caused by a CGG triplet repeat expansion in a single gene, fragile X messenger ribonucleoprotein 1 (FMR1), which resides on the X chromosome. When the CGG triplet expands to 200 repeats or more, the FMR1 gene is methylated and thereby transcriptionally inactivated. The loss of the FMR1 gene product, the protein fragile X messenger ribonucleoprotein (FMRP), is the cause of the disorder. [0005] There is a critical need to develop methods and biomarkers to supplement treatment of fragile X-associated disorders such as fragile X syndrome (FXS). - 1 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 SUMMARY [0006] The present disclosure relates to methods and biomarkers to treat, and to supplement treatment of, fragile X-associated disorders such as fragile X syndrome (FXS). [0007] The present disclosure relates to biomarkers, for example, metabolites that are differentially present in FXS subjects. Experiments conducted during the course of development of embodiments of the present disclosure identified a series of metabolites as being differentially present in samples of FXS subjects (e.g., versus one or more control samples and/or reference levels). Accordingly, the present disclosure generally relates to biomarkers (e.g., metabolites) and methods that are useful for diagnosing, making prognoses, and/or treating a subject, e.g., a subject having, or at risk of having, a fragile X-associated disorder. [0008] Provided herein, among other things, are methods of treating fragile X syndrome (FXS) in a subject, the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and administering to the subject a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product. [0009] The disclosure also provides, among other things, methods of predicting a treatment outcome of, diagnosing, prognosing, or subclassifying fragile X syndrome (FXS) in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing. - 2 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 BRIEF DESCRIPTION OF THE DRAWINGS [0010] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [0011] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments. [0012] FIGs.1A-1B. Metabolic changes observed in FXS patient-derived post-mortem cortex (brain) samples. FIG.1A. Volcano plot depicting the changes in polar metabolite levels between post-mortem cortex tissue samples from six FXS males and six age-matched typically- developing (TD) males captured using liquid chromatography-mass spectrometry (LC-MS). The horizontal dotted line represents a significance threshold of a p-value equal to 0.05 (-log10(0.05) = 1.3). The left arm of the plot, where log2 of the fold change is less than zero, represents a decrease in levels of metabolites (18 metabolites reached statistical significance) in the FXS samples compared to TD samples. The right arm of the plot, where log2 of the fold change is greater than zero, represents an increase in levels of metabolites (four metabolites reached statistical significance) in the FXS samples compared to TD samples. See also Table 1. FIG.1B. Ratios of glutathione (GSH) to glutathione disulfide (GSSG) levels compared between the FXS and TD samples used in FIG.1A, reflecting increased oxidative stress in the FXS cortex samples. [0013] FIGs.2A-2B. Metabolic changes observed in FXS patient-derived PBMC samples. FIG.2A. Volcano plot depicting the changes in polar metabolite levels between PBMC samples from six FXS males and five age-matched TD males captured using LC-MS. The horizontal dotted line represents a significance threshold of a p-value equal to 0.05 (-log10(0.05) = 1.3). The left arm of the plot, where log2 of the fold change is less than zero, represents a decrease in levels of metabolites (24 metabolites reached statistical significance) in the FXS samples compared to TD samples. See also Table 2. FIG.2B. Ratios of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) levels compared between the FXS and TD samples used in FIG.2A, reflecting altered mitochondrial metabolism in the FXS PBMC samples. - 3 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [0014] FIGs.3A-3C. Common metabolic changes observed in FXS patient-derived PBMC and post-mortem cortex (brain) samples. FIG.3A. Venn diagram depicting the polar metabolite levels significantly altered in FXS males compared to TD males in PBMC samples, cortex samples, and both sample types. The significance threshold of a p-value equal to 0.05 was used. The left circle shows the number of polar metabolites with significantly changed levels in PBMC samples from FXS males compared to TD males; the right circle shows the equivalent comparison in cortex samples. The overlapping region of the two circles and its accompanying list show the number and identity of polar metabolites with significantly changed levels in both PBMC and cortex samples from FXS males compared to TD males. FIG.3B. Plots depicting levels of amino acids from the overlapping region of FIG.3A that are reduced in cortex samples from FXS males compared to TD males. FIG.3C. Plots depicting the levels of amino acids from the overlapping region of FIG.3A that are reduced in PBMC samples from FXS males compared to TD males. [0015] FIG.4. Metabolic changes observed in FXS patient-derived lymphoblastoid cell lines (LCLs). Volcano plot depicting the changes in polar metabolite levels in two human male FXS LCLs (GM05131 and GM07072) compared to two TD LCLs (GM07174 and GM06890) captured using LC-MS. The horizontal dotted line represents a significance threshold of a p- value equal to 0.05 (-log10(0.05) = 1.3). The left arm of the plot, where log2 of the fold change is less than zero, represents a decrease in levels of metabolites (21 metabolites reached statistical significance) in the FXS samples compared to TD samples. See also Table 4. [0016] FIGs.5A-5D. A. Schematic of FMR1 RNA isoforms in FXS. ASO against FMR1- 217 RNA used in FXS2 cells (160nM for 48hrs). B, C. qPCR data for FMR1-217 RNA and FMR1 RNA levels respectively (**** p<0.0001, **p<0.01). D. Western blot data for FMRP and GAPDH proteins in TD and FXS2 cells (with vehicle or ASO). [0017] FIGs.6A-6D. Volcano plot illustrating log2FC in FXS compared to TD samples (N=6 each). Dotted lines indicate cutoffs at log2FC=0 and –log(p>1.3). Amino acids and their derivatives are represented by green and red dots respectively in cerebellum (A) and cortex tissues (B). C. Box plots display the levels of leucine and lysine in TD and FXS samples (*p<0.05, *p<0.01). D. Venn diagram shows the overlapping differential metabolites (amino acids and their derivatives highlighted in green and red respectively). - 4 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [0018] FIGs.7A-E. Changes in metabolites may correlate with FMRP expression. FIG.7A. Western blots to measure the FMRP levels in FXS1, FXS2 and TD lines compared to GAPDH. Below is a representation of FMRP levels in the cell lines. FIG.7B. PCA plot demonstrating the clustering of the FXS2 line (partial FMRP) with the TD as against the FXS1 cell line (no FMRP). FIG.7, C, D and E. Volcano plot depicting significantly altered metabolites (amino acids in green and their derivatives in red) in FXS2 vs TD (FIG.7C.), FXS1 vs TD (. FIG.7D.) and FXS2 vs FXS1 (FIG.7E., note the increase in amino acid levels). FIG.7F. Examples of amino acid changes in the FXS lines compared to TD. (*p<0.05, **p<0.01). [0019] FIGs.8A-8B. iPSC derived forebrain neuron cultures. FIG 8A. The diagram depicts the conversion of LCLs from FXS patients into iPSCs, their subsequent differentiation into neuronal lineage, and the generation of mature forebrain neurons. FIG.8B. Representative images of cells in culture aligned to the different stages in FIG 8A. (panel A). [0020] FIG.9. Model proposing the influence of dysregulated proteostasis and/or mitochondrial function on free amino acids levels in FXS. FXS LCLs will be treated with various inhibitors such as MG-132 or Bortezomib against proteosome, 3-methyladenine (3-MA) against autophagosome, Rapamycin to block mTORC1 activation and dexpramipexole (DEX) to block proton leak in the mitochondria will be used. LC-MS assay will be used to analyze the effect of each inhibitor on the metabolome. To assess the effect of reduced free amino acids on tRNA charging, the i-TRAP qPCR method will be used to quantify ratios of individual uncharged to charged tRNAs. [0021] FIG.10. Model proposing restoration of FMRP based rescue of metabolic deficits in FXS. Using ASOs to restore FMRP in FXS cells, whether the metabolite expression specifically amino acids are restored to those in TD cells will be tested. [0022] FIGs.11A-C. FIG.11A. Lymphoblastoid cell lines (LCLs) from a typically developing (TD) male individual, a Fragile X Syndrome male (FXS) expressing some FMRP (FXS2), and a FXS male expressing no FMRP (FXS1) were analyzed for FMRP and GAPDH (as a loading control) on western blots in triplicate. FIG.11B. Polar metabolites from the cell lines noted in FIG.11A. (panel A) were assessed by LC/MS. A Principal Component Analysis (PCA) shows tight clustering of the metabolites based on the genotype. FIG.11C. Relative amounts of leucine and methionine in the LCLs from the 3 genotypes noted in FIG.11A. - 5 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [0023] FIGs.12A-12B. FIG.12A. The culture media from the LCLs noted in FIG.11 were analyzed for metabolites by LC/MS. Shown are relative amounts of leucine and methionine. FIG.12B. PCA plot for metabolites in the culture media from the LCL lines denoted in FIG.11. [0024] FIG.13. LC/MS analysis of the metabolome comparing FXS1 to TD LCLs. The volcano plot at left is the cell pellets and at right is the culture media. The metabolites above the dashed line are statistically different between the genotypes. The vertical line separates metabolites that are decreased (to the left) or increased (to the right) when comparing FXS1 to TD. See also Table 5 (cell pellet) and Table 6 (culture media). [0025] FIG.14. LC/MS analysis of the metabolome comparing FXS2 to TD LCLs. The volcano plot at left is the cell pellets and at right is the culture media. The metabolites above the dashed line are statistically different between the genotypes. The vertical line separates metabolites that are decreased (to the left) or increased (to the right) when comparing FXS2 to TD. See also Table 7 (cell pellet) and Table 8 (culture media). [0026] FIG.15. LC/MS analysis of the metabolome comparing FXS2 to FXS1 LCLs. The volcano plot at left is the cell pellets and at right is the culture media. The metabolites above the dashed line are statistically different between the genotypes. The vertical line separates metabolites that are decreased (to the left) or increased (to the right) when comparing FXS2 to FXS1. See also Table 9 (cell pellet) and Table 10 (culture media). [0027] FIG.16A. Relative amounts of 13C5 citrate in wild-type and FXS2 cells. [0028] FIG.16B. Amount of extracellular 13C5,15N2 glutamate with respect to the amount of extracellular 13C5,15N2 glutamine. [0029] FIG.16C. Amount of intracellular 13C5,15N2 glutamate with respect to the amount of intracellular 13C5,15N2 glutamine. [0030] FIG.16D. Relative amounts of extracellular 13C5,15N2 glutamate in wild-type and FXS2 cells. [0031] FIG.16E. Relative amounts of intracellular 13C5,15N2 glutamate in wild-type and FXS2 cells. DETAILED DESCRIPTION [0032] A description of example embodiments follows. - 6 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [0033] Several aspects of the invention are described below, with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or practiced with other methods, protocols, reagents, cell lines and animals. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts, steps or events are required to implement a methodology in accordance with the present invention. Many of the techniques and procedures described, or referenced herein, are well understood and commonly employed using conventional methodology by those skilled in the art. Definitions [0034] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly-used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and/or as otherwise defined herein. [0035] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. [0036] When introducing elements disclosed herein, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. Further, the one or more elements may be the same or different. For example, unless the context clearly indicates otherwise, “an FXS-associated metabolite” includes a single metabolite, and two or more metabolites. In some embodiments, a first metabolite has a reduced level in a sample from a subject, and a second metabolite has an increased level in the sample from a subject. - 7 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [0037] Throughout this specification and the claims which follow, unless the context requires otherwise, the term “comprise,” and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of, e.g., a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein, the term “comprising” can be substituted with the term “containing” or “including.” [0038] As used herein, the term “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, the term “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. [0039] Any of the terms “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment disclosed herein, can in some embodiments, be replaced with the term “consisting of,” or “consisting essentially of” to vary scopes disclosed herein. [0040] As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and/or.” [0041] It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” “fewer than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2, 1- 3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera. [0042] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For - 8 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.” [0043] As used herein, the term “about” means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art. Typically, an acceptable error range for a particular value depends, at least in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of ± 20%, e.g., ± 10%, ± 5% or ± 1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular values used in the Exemplification. When “about” precedes a range, as in “about 55-200 CGG repeats,” the term “about” should be read as applying to both given values of the range, such that “about 55-200 CGG repeats” means about 55 CGG repeats to about 200 CGG repeats. [0044] The term “subject” refers to a mammalian subject, preferably human, diagnosed with or suspected of having a fragile X-associated disorder (e.g., FXS). [0045] As used herein, the term “metabolite” refers to an intermediate or product resulting from metabolism. As used herein, the term “metabolism” refers to chemical changes occurring within an organism. In some embodiments, a metabolite results from anabolism. In some embodiments, a metabolite results from catabolism. [0046] Compounds described herein include those described generally, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the relevant contents of which are incorporated herein by reference. [0047] Unless specified otherwise within this specification, the nomenclature used in this specification generally follows the examples and rules stated in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979, which is - 9 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 incorporated by reference herein for its chemical structure names and rules on naming chemical structures. Optionally, a name of a compound may be generated using a chemical naming program (e.g., CHEMDRAW®, version 17.0.0.206, PerkinElmer Informatics, Inc.). [0048] As used herein, the term “sample” refers to any sample that can be from or derived from a subject (e.g., a mammalian subject such as a human subject). The methods disclosed herein can be performed using a variety of possible sample types. For example, a bodily fluid, a single cell or cell lysate, a population of cells, a cell culture, and/or a tissue. [0049] The phrase “pharmaceutically acceptable” means that the substance or composition the phrase modifies is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. [0050] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the agents/compounds described herein include salts derived from suitable inorganic and organic acids, and suitable inorganic and organic bases. [0051] Examples of salts derived from suitable acids include salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts derived from suitable acids include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxy-ethanesulfonate, hydroxymaleate, lactobionate, lactate, laurate, - 10 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 2-phenoxybenzoate, phenylacetate, 3-phenylpropionate, phosphate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. [0052] Either the mono-, di- or tri-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form. [0053] Salts derived from appropriate bases include salts derived from inorganic bases, such as alkali metal, alkaline earth metal, and ammonium bases, and salts derived from aliphatic, alicyclic or aromatic organic amines, such as methylamine, trimethylamine and picoline, or N+((C1-C4)alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. [0054] “Pharmaceutically acceptable carrier” refers to a non-toxic carrier or excipient that does not destroy the pharmacological activity of the agent with which it is formulated and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent. Pharmaceutically acceptable carriers that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. [0055] “Treating” or “treatment,” as used herein, refers to taking steps to deliver a therapy to a subject, such as a mammal, in need thereof (e.g., as by administering to a mammal one or more therapeutic agents). “Treating” or “treatment” includes inhibiting the disease or condition (e.g., - 11 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 as by slowing or stopping its progression or causing regression of the disease or condition) and relieving the symptoms resulting from the disease or condition. The term “treating,” or “treatment” refers to the medical management of a subject with the intent to improve, ameliorate, stabilize (i.e., not worsen), prevent, or cure a disease, pathological condition, or disorder—such as the particular indications exemplified herein. This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive treatment (treatment employed to supplement another therapy). Treatment also includes diminishment of the extent of the disease or condition; preventing spread of the disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. In some embodiments, treatment includes change or restoration of expression, e.g., endogenous FMRP expression. In some embodiments, treatment includes change or restoration of FMRP level(s), e.g., to ameliorate metabolic deficit(s) in FXS. [0056] As used herein, a “polynucleotide” is defined as a plurality of nucleotides and/or nucleotide analogs linked together in a single molecule. In some embodiments, a polynucleotide disclosed herein comprises deoxyribonucleotides. In some embodiments, the polynucleotide comprises ribonucleotides. Non-limiting examples of polynucleotides include single-, double- or multi-stranded DNA or RNA, DNA-RNA hybrids (e.g., each “T” position may be independently substituted by a “U” or vice versa), or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide - 12 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, modified or substituted sugar or phosphate groups, a polymer of synthetic subunits such as phosphoramidates, or a combination thereof. [0057] As used herein, the term “nucleotide analog” or “altered nucleotide” or “modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. A nucleotide analog may be modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability to perform its intended function. Non-limiting examples of positions of the nucleotide which may be derivatized include the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, and 5-propenyl uridine; the 6 position, e.g., 6-(2-amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, and 8- fluoroguanosine. Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated or N6-methyl adenosine) nucleotides. [0058] As used herein, the term “complementary” refers to sequence complementarity between two different polynucleotides or between two regions of the same polynucleotide. A first region of a polynucleotide is complementary to a second region of the same or a different polynucleotide if, when the two regions are arranged in an anti-parallel fashion, at least one nucleotide residue of the first region is capable of base pairing (i.e., hydrogen bonding) with a residue of the second region, thus forming a hydrogen-bonded duplex. [0059] As used herein, the term “sequence identity” refers to the extent to which two nucleotide sequences have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as a reference sequence, to which test sequences are compared. Sequence identity between reference and test sequences is expressed as a percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity. As an example, two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide residue at 70% of the same positions over the entire length of the reference sequence. - 13 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [0060] Alignment of sequences for comparison to achieve maximal levels of identity can be readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm. In some instances, alignment can include introduced gaps to provide for the maximal level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology). [0061] When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. A commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through National Center for Biotechnology Information, National Library of Medicine, of the United States National Institutes of Health. (Altschul et al., 1990). [0062] As used herein, the term “polypeptide” refers to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). A polypeptide can comprise any suitable L-and/or D-amino acid, for example, common ^-amino acids (e.g., alanine, glycine, valine), non- ^-amino acids (e.g., ^-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine). The amino, carboxyl, and/or other functional groups on a polypeptide can be free (e.g., unmodified) or protected with a suitable protecting group. Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups are known in the art and are disclosed in, for example, Green and Wuts, “Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991. The functional groups of a polypeptide can also be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label, such as a fluorogen or a hapten) using - 14 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 methods known in the art. A polypeptide can comprise one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclizing modifications), N-methyl- ^-amino group substitution), if desired. In addition, a polypeptide can be an analog of a known and/or naturally-occurring peptide, for example, a peptide analog having conservative amino acid residue substitution(s). [0063] As used herein, the term “antibody mimetic” refers to polypeptides capable of mimicking an antibody’s ability to bind an antigen, but structurally differ from native antibody structures. Examples of antibody mimetics include, but not limited to, Adnectins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies, nanobodies, nanoCLAMPs, and Versabodies. [0064] A “pharmaceutical composition” refers to a formulation of one or more therapeutic agents and a medium generally accepted in the art for delivery of a biologically active agent to subjects, e.g., humans. In some embodiments, a pharmaceutical composition may include one or more pharmaceutically acceptable excipients, diluents, or carriers. In some embodiments, a pharmaceutical composition suitable for use in methods disclosed herein further comprises one or more pharmaceutically acceptable carriers. [0065] “Pharmaceutically acceptable carrier, diluent, or excipient” includes any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. [0066] “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some embodiments, the carrier may be a diluent, adjuvant, excipient, or vehicle with which the agent (e.g., polynucleotide) is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may - 15 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating, and coloring agents, etc. The concentration of the agent in such pharmaceutical formulation may vary widely, i.e., from less than about 0.5%, to at least about 1%, or to as much as 15% or 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight. The concentration will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the mode of administration. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing: 691-1092 (e.g., pages 958-89). [0067] Non-limiting examples of pharmaceutically acceptable carriers are solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, such as salts, buffers, antioxidants, saccharides, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifying agents, or combinations thereof. [0068] Non-limiting examples of buffers are acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffers, HEPPSO, and HEPES. [0069] Non-limiting examples of antioxidants are ascorbic acid, methionine, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, lecithin, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, and tartaric acid. [0070] Non-limiting examples of amino acids are histidine, isoleucine, methionine, glycine, arginine, lysine, L-leucine, tri-leucine, alanine, glutamic acid, L-threonine, and 2-phenylamine. [0071] Non-limiting examples of surfactants are polysorbates (e.g., polysorbate-20 or polysorbate-80); polyoxamers (e.g., poloxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUA™ series (Mona Industries, Inc., - 16 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.). [0072] Non-limiting examples of preservatives are phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkylparaben (methyl, ethyl, propyl, butyl, and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. [0073] Non-limiting examples of saccharides are monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, nonreducing sugars such as glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, or iso-maltulose. [0074] Non-limiting examples of salts are acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as from nontoxic organic amines, such as N,N’-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like. In some embodiments, the salt is sodium chloride (NaCl). [0075] Agents (e.g., polynucleotides) described herein may be prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent, or eliminate, or to slow or halt progression of, a condition being treated (see, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, and Goodman and Gilman’s The Pharmaceutical Basis of Therapeutics, McGraw-Hill, New York, N.Y., the contents of which are incorporated herein by reference, for a general description of methods for administering various agents for human therapy). - 17 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [0076] “Administering” or “administration,” as used herein, refers to providing a compound, composition, or pharmaceutically acceptable salt thereof described herein to a subject in need of treatment or prevention. Administering can be performed, for example, once, a plurality of times, and/or over one or more extended periods. Administration includes both direct administration (including self-administration), and indirect administration (including an act of prescribing a drug or directing a subject to consume an agent). For example, as used herein, one (e.g., a physician) who instructs a subject (e.g., a human patient) to self-administer an agent (e.g., a drug), or to have an agent administered by another and/or who provides a patient with a prescription for a drug is administering an agent to a subject. [0077] “A therapeutically effective amount” or “an effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or biological result (e.g., treatment, healing, inhibition or amelioration of physiological response or condition, etc.). A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual. [0078] Non-limiting examples of desired therapeutic or biological results include improvement of behavior and/or cognition (e.g., clinical global impression-improvement (CGI-I) scale, pediatric anxiety rating scale (PARS) total score, visual analogue scale (VAS), anxiety, depression, and mood scale (ADAMS), aberrant behavior checklist (ABC), receptive language, attention, vigilance), improvement of mitochondrial function (e.g., mitochondrial membrane potential, mass, ATP production, and/or Ca2+ transfer), mitigation of mitochondrial dysregulation (e.g., mitochondrial ER stress (e.g., GRP78 level and/or CHOP level) and/or Bax/Bcl-2 ratio), or any combination of the foregoing. [0079] As used herein, the term “treat,” “treating” or “treatment” refers to therapeutic treatment wherein the objective is to slow down (lessen) an undesired physiological change or disease, such as the development or progression of the fragile X-associated disorder (e.g., FXS), or to provide a beneficial or desired clinical outcome during treatment. Beneficial or desired clinical outcomes include alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, whether detectable or undetectable. Non-limiting examples of - 18 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 symptoms include speech and motor development symptoms; cognitive disabilities, including learning and intellectual disabilities, hyperactivity, short attention span, anxiety, sensitivity to sensory stimulation, sleep problems, and seizures; recurrent ear infections, and mitral valve prolapse. [0080] The term “poor” or “poorer” refers to greater degree of fragile X-associated disorder (e.g., FXS) symptoms, increased extent of disease, decreased (i.e., worsening) state of disease, increased or enhanced state of disease progression, deterioration or worsening of the disease state, whether detectable or undetectable. [0081] “Mass Spectrometry” (MS) is a technique for measuring and analyzing molecules to produce a mass spectrum that serves as a “molecular fingerprint.” MS involves fragmenting a target molecule (e.g., a metabolite), then analyzing the fragments, based on their mass/charge ratios. A “mass spectrum” is a plot of data produced by a mass spectrometer. In some embodiments, a mass spectrum contains m/z values on x-axis and intensity values on y-axis. The term “m/z” or “mass-to- charge” ratio refers to the dimensionless quantity formed by dividing the mass number of an ion by its charge number. In liquid chromatography-mass spectrometry- based (LC-MS) metabolomics, a group of ions may originate from the same metabolite, and one metabolite can be represented by multiple peaks in LC-MS data with distinct m/z values but, at similar retention times, due to the presence of adducts (e.g., H+, Na+ or K+). A “peak” is a point on a mass spectrum with a relatively high y-value. Methods of Treating Fragile X-Associated Disorders [0082] Treatments for fragile X syndrome (and other autism spectrum disorders), which are mostly based on animal models, have met with very limited success in human clinical trials (Hagerman et al., Nature Rev Disease Primers 3:17065 (2017); Berry-Kravis et al.., Nature Rev Drug Disc.17:280-299 (2018)). There is no widely available therapy that shows even modest efficacy for FXS. Moreover, there are no established molecular readouts with statistical rigor of any FXS treatment. [0083] Provided herein, among other methods, are methods of treating fragile X-associated disorders. [0084] In some embodiments, methods of treating fragile X syndrome (FXS) in a subject disclosed herein comprise determining, in a sample from the subject: - 19 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing. [0085] In some embodiments, methods of treating fragile X syndrome (FXS) in a subject disclosed herein comprise determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and administering to the subject a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product. [0086] In some embodiments, the agent is an antisense oligonucleotide. [0087] In some embodiments, a therapeutically effective amount of the agent decreases an aberrant FMR1 transcript, a protein encoded by the aberrant FMR1 transcript, or both. [0088] In some embodiments, an aberrant FMR1 gene product comprises FMR1-217. [0089] For example, if the ratio between levels of two FXS-associated metabolites in a sample from the subject is determined to be 1:3 and suppose the reference ratio of the two FXS- associated metabolites is 1:5, the subject may be administered a therapeutically effective amount of an agent that that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product. In another example, after administering a therapeutically effective amount of an agent, if the ratio between levels of two FXS-associated metabolites in a sample from the subject is determined to be 1:2 a month later, the treatment regimen for the subject may be modified. [0090] In some embodiments, a method disclosed herein comprises determining a presence, absence, amount, or alteration of at least one FXS-associated metabolite (e.g., an FXS-associated metabolite), or an alteration of an FXS-associated ratio of two metabolites, in a sample from the - 20 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 subject. In some embodiments, a method further comprises treating FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, or the alteration of the FXS-associated ratio, in the sample. Fragile X-Associated Disorders [0091] Fragile X-associated disorders are caused by mutation of the fragile X messenger ribonucleoprotein 1 (FMR1, previously known as fragile X mental retardation 1) gene, located in the q27.3 locus of the X chromosome. The expansion of the trinucleotide CGG repeat above the normal range (greater than 54 repeats) in the non-coding region of the FMR1 gene has been associated with the development of fragile X-associated disorders. For example, in those carrying the premutation, the trinucleotide CGG repeat can range from 55-200 CGG repeats. In some embodiments, a fragile X-associated disorder is linked to greater than 77 CGG repeats in FMR1, e.g., greater than 98 CGG repeats in FMR1. In some embodiments, a fragile X-associated disorder is linked to at least 140 CGG repeats in FMR1. In some embodiments, a fragile X- associated disorder is linked to at least 201 CGG repeats in FMR1. [0092] Non-limiting examples of fragile X-associated disorders include fragile X-associated tremor/ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), fragile X-associated neuropsychiatric disorders (FXAND), and fragile X syndrome (FXS). In some embodiments, a fragile X-associated disorder is fragile X syndrome (FXS), fragile X- associated primary ovarian insufficiency (FXPOI), or fragile X-associated tremor/ataxia syndrome (FXTAS), or a combination thereof. In some embodiments, a fragile X-associated disorder is FXS. Subjects [0093] In some embodiments, a subject is a mammal. As used herein, the term “subject” includes humans, domestic animals, such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.), livestock (e.g., pigs, cattle, sheep, goats, horses, etc.), and non-domestic animals. In some embodiments, a subject is a human. In some embodiments, a subject (e.g., human) is male. In some embodiments a subject (e.g., human) is female. - 21 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [0094] In some embodiments, a subject (e.g., a human) has, or is predisposed to have, a fragile X-associated disorder (e.g., FXS). In some embodiments, a subject (e.g., a human) has a fragile X-associated disorder (e.g., FXS). In some embodiments, a subject (e.g., a human) is predisposed to have a fragile X-associated disorder (e.g., FXS). [0095] In some embodiments, a subject is diagnosed with a fragile X-associated disorder (e.g., FXS). In some embodiments, a subject is suspected of having a fragile X-associated disorder (e.g., FXS). [0096] In some embodiments, a subject has one or more of the physical and/or medical features associated with a fragile X-associated disorder (e.g., FXS). Non-limiting examples of physical features associated with FXS include a long face, prominent ears and chin, arched palate, large testicles at puberty, low muscle tone, flat feet, and hyperextensible joints. Non- limiting examples of medical or behavioral features associated with FXS include sleep problems, seizures, recurrent ear infections, mitral valve prolapse, behaviors of hyperactivity, short attention span, hand biting or hand flapping, poor eye contact and social skills, shyness, anxiety, autism, epilepsy, aggression, delayed speech and/or motor development, repetitive speech, sensitivity to sensory stimulation (including a hypersensitivity to being touched, to light or to sound), or any combination thereof. In some embodiments, a subject is a female with an IQ score of less than 115, 110, 105, 100, 95, or 90. In some embodiments, a subject is a male with an IQ score of less than 60, 55, 50, or 45. [0097] In some embodiments, a subject has one or more of the following: irregular menses, fertility problem, elevated FSH (follicle-stimulating hormone) level, premature ovarian failure, primary ovarian insufficiency, and vasomotor symptoms (e.g., “hot flash”). In some embodiments, a subject has one or more of the following: intention tremor, parkinsonism, ataxia, memory loss, white matter lesion involving middle cerebellar peduncles, and cognitive decline. [0098] In some embodiments, a subject has at least 55 CGG repeats in the 5’ untranslated region of an FMR1 gene, for example, having at least 77, at least 78, at least 98, at least 99, at least 140, or at least 201 CGG repeats in the 5’ untranslated region of the FMR1 gene. In some embodiments, a subject has between about 55-200 CGG repeats in the 5’ untranslated region of an FMR1 gene. In some embodiments, a subject has at least 200 CGG repeats in the 5’ - 22 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 untranslated region of an FMR1 gene. In some embodiments, a subject has at least 201 CGG repeats in the 5’ untranslated region of an FMR1 gene. [0099] In some embodiments, a subject (e.g., a human) has a CGG repeat expansion that is at least partially methylated. In some embodiments, a subject (e.g., a human) has a CGG repeat expansion that is partially methylated. In some embodiments, a subject has a CGG repeat expansion that is fully methylated. [00100] In some embodiments, a subject is a premutation carrier for FXS. In some embodiments, a subject has FXS. Aberrant FMR1 Gene Products [00101] In some embodiments, a subject (e.g., a human) expresses or has an elevated expression of an aberrant FMR1 gene product. [00102] A FMR1 gene encodes a fragile X messenger ribonucleoprotein (FMRP, previously known as fragile X mental retardation protein). In some embodiments, an FMR1 gene described herein is a human FMR1 gene (e.g., corresponding to GenBank reference number NC_000023.11), a mouse FMR1 gene (e.g., NC_000086.8), a rat FMR1 gene (e.g., NC_051356.1), a golden hamster FMR1 gene (e.g., NW_024429188.1), a Chinese hamster FMR1 gene (e.g., NW_003614110.1), a dog FMR1 gene (e.g., NC_051843.1), a pig FMR1 gene (e.g., NC_046383.1), or a monkey FMR1 gene (e.g., NC_041774.1). In some embodiments, the FMR1 gene is a human FMR1 gene. The human FMR1 gene (Ensembl: ENSG00000102081.16) is located within chromosome band Xq27.3 between base pairs 147,911,919 and 147,951,125 (the numberings referring to Genome Reference Consortium Human Build 38 (GRCh38)). [00103] In some embodiments, an aberrant FMR1 gene product (e.g., an aberrant FMR1 transcript and/or its protein product) contributes to pathology of a fragile X-associated disorder (e.g., FXS). In some embodiments, an aberrant FMR1 transcript contributes to pathology of a fragile X-associated disorder. In some embodiments, a protein encoded by an aberrant FMR1 transcript contributes to pathology of a fragile X-associated disorder. In some embodiments, an aberrant FMR1 transcript and its protein product contribute to pathology of a fragile X- associated disorder. [00104] In some embodiments, an aberrant FMR1 gene product is produced from a CGG expansion-dependent mis-splicing of an FMR1 transcript. - 23 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00105] In some embodiments, an aberrant FMR1 transcript is FMR1-217. In humans, FMR1- 217, also referred to as “isoform 12” or “iso12,” is a transcript corresponding to A0A087X1M7 (ENST00000621447.1, 1,832 nucleotides). FMR1-217 has 2 exons, and the splicing between Exon 1 of FMR1-217 (between base pairs 147,912,123 and 147,912,230, SEQ ID NO:1) and Exon 2 of FMR1-217 (between base pairs 147,912,728 and 147,914,451, SEQ ID NO:2) is considered aberrant FMR1 RNA splicing. FMR1-217 is detected in a subpopulation of subjects with fragile X-associated disorder, including a subpopulation of FXS patients, and a subpopulation of premutation carriers for FXS. [00106] CGCCCGCAGCCCACCTCTCGGGGGCGGGCTCCCGGCGCTAGCAGGGCTGA AGAGAAGATGGAGGAGCTGGTGGTGGAAGTGCGGGGCTCCAATGGCGCTTTCTACA AG (SEQ ID NO:1). [00107] CATTGGGACTTCGGAGAGCTCCACTGTTCTGGGCGAGGGCTGTGAAGAAA GAGTAGTAAGAAGCGGTAGTCGGCACCAAATCACAATGGCAACTGATTTTTAGTGG CTTCTCTTTGTGGATTTCGGAGGAGATTTTAGATCCAAAAGTTTCAGGAAGACCCTA ACATGGCCCAGCAGTGCATTGAAGAAGTTGATCATCGTGAATATTCGCGTCCCCCTT TTTGTTAAACGGGGTAAATTCAGGAATGCACATGCTTCAGCGTCTAAAACCATTAGC AGCGCTGCTACTTAAAAATTGTGTGTGTGTGTTTAAGTTTCCAAAGACCTAAATATA TGCCATGAAACTTCAGGTAATTAACTGAGAGTATATTATTACTAGGGCATTTTTTTTT TAACTGAGCGAAAATATTTTTGTGCCCCTAAGAACTTGACCACATTTCCTTTGAATTT GTGGTGTTGCAGTGGACTGAATTGTTGAGGCTTTATATAGGCATTCATGGGTTTACT GTGCTTTTTAAAGTTACACCATTGCAGATCAACTAACACCTTTCAGTTTTAAAAGGA AGATTTACAAATTTGATGTAGCAGTAGTGCGTTTGTTGGTATGTAGGTGCTGTATAA ATTCATCTATAAATTCTCATTTCCTTTTGAATGTCTATAACCTCTTTCAATAATATCCC ACCTTACTACAGTATTTTGGCAATAGAAGGTGCGTGTGGAAGGAAGGCTGGAAAAT AGCTATTAGCAGTGTCCAACACAATTCTTAAATGTATTGTAGAATGGCTTGAATGTT TCAGACAGGACACGTTTGGCTATAGGAAAATAAACAATTGACTTTATTCTGTGTTTA CCAATTTTATGAAGACATTTGGAGATCAGTATATTTCATAAATGAGTAAAGTATGTA AACTGTTCCATACTTTGAGCACAAAGATAAAGCCTTTTGCTGTAAAAGGAGGCAAA AGGTAACCCCGCGTTTATGTTCTTAACAGTCTCATGAATATGAAATTGTTTCAGTTGA CTCTGCAGTCAAAATTTTAATTTCATTGATTTTATTGATCCATAATTTCTTCTGGTGA - 24 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 GTTTGCGTAGAATCGTTCACGGTCCTAGATTAGTGGTTTTGGTCACTAGATTTCTGGC ACTAATAACTATAATACATATACATATATATGTGTGAGTAACGGCTAATGGTTAGGC AAGATTTTGATTGACCTGTGATATAAACTTAGATTGGATGCCACTAAAGTTTGCTTA TCACAGAGGGCAAGTAGCACATTATGGCCTTGAAGTACTTATTGTTCTCTTCCAGCA ACTTATGATTTGCTCCAGTGATTTTGCTTGCACACTGACTGGAATATAAGAAATGCC TTCTATTTTTGCTATTAATTCCCTCCTTTTTTGTTTTGTTTTGTAACGAAGTTGTTTAA CTTGAAGGTGAATGAAGAATAGGTTGGTTGCCCCTTAGTTCCCTGAGGAGAAATGTT AATACTTGAACAAGTGTGTGTCAGACAAATTGCTGTTATGTTTATTTAATTAAGTTTG ATTTCTAAGAAAATCTCAAATGGTCTGCACTGATGGAAGAACAGTTTCTGTAACAAA AAAGCTTGAAATTTTTATATGACTTATAATACTGCTGTGAGTTTTAAAAGTAAAGCA AAAGTAAACTGAGTTGCTTGTCCAGTGGGATGGACAGGAAAGATGTGAAATAAAAA CCAATGAAAAATGAA (SEQ ID NO:2). [00108] FMR1-217 encodes a 31-amino acid protein (SEQ ID NO:3). [00109] MEELVVEVRGSNGAFYKHWDFGELHCSGRGL (SEQ ID NO:3). [00110] Additional information on FMR1-217 and its protein product can be found at the web address below, the contents of which are incorporated herein by reference in their entirety: useast.ensembl.org/Homo_sapiens/Transcript/Summary?db=core;g=ENSG00000102081;r=X:14 7911951-147951125;t=ENST00000621447. [00111] In some embodiments, a subject (e.g., a human) does not express and/or does not have an elevated expression of an aberrant FMR1 gene product, for example, compared to typically developing subjects. In some embodiments, a subject does not express an aberrant FMR1 gene product. In some embodiments, a subject does not have an elevated expression of an aberrant FMR1 gene product. Normal FMR1 Gene Products [00112] In some embodiments, a subject (e.g., a human) expresses a normal FMR1 gene product (i.e., a FMR1 gene product expressed in typically developing subjects). Non-limiting examples of “normal” human FMR1 gene products include: a transcript corresponding to Q06787 (FMR1-205, ENST00000370475.9, 4,441 nucleotides), and its protein product (a 632-amino acid protein (NP_002015.1)), - 25 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 a transcript corresponding to NM_001185075.2 (4,170 nucleotides), and its protein product (a 537-amino acid protein (NP_001172004.1)), a transcript corresponding to NM_001185076.2 (4,378 nucleotides), and its protein product (a 611-amino acid protein (NP_001172005.1)), a transcript corresponding to NM_001185082.2 (4,303 nucleotides), and its protein product (a 586-amino acid protein (NP_001172011.1)), a transcript corresponding to NM_001185081.2 (4,107 nucleotides), and its protein product (a 516-amino acid protein (NP_001172010.1)), a transcript corresponding to Q06787-9 (FMR1-201, ENST00000218200.12, 4,333 nucleotides), and its protein product (a 611-amino acid protein), a transcript corresponding to Q06787-8 (FMR1-208, ENST00000440235.6, 4,271 nucleotides), and its protein product (a 586-amino acid protein), a transcript corresponding to X5D907 (FMR1-223, ENST00000687593.1, 4,159 nucleotides), and its protein product (a 594-amino acid protein), a transcript corresponding to Q06787-10 (FMR1-204, ENST00000370471.7, 4,125 nucleotides), and its protein product (a 537-amino acid protein), a transcript corresponding to G3V0J0 (FMR1-207, ENST00000439526.6, 3,699 nucleotides), and its protein product (a 592-amino acid protein), a transcript corresponding to A8MQB8 (FMR1-206, ENST00000370477.5, 3,437 nucleotides), and its protein product (a 582-amino acid protein), a transcript corresponding to A0A087WY29 (FMR1-212, ENST00000495717.6, 2,874 nucleotides), and its protein product (a 561-amino acid protein), a transcript corresponding to A0A087WXI3 (FMR1-214, ENST00000616382.5, 2,799 nucleotides), and its protein product (a 536-amino acid protein), and a transcript corresponding to R9WNI0 (“FMR1-218”, ENST00000621453.5, 1,827 nucleotides), and its protein product (a 548-amino acid protein). [00113] In some embodiments, a normal FMR1 transcript is Q06787 (FMR1-205, ENST00000370475.9, 4,441 nucleotides, produces a 632-amino acid protein (NP_002015.1)). FMR1-205, also referred to as “isoform 1” or “iso1,” is produced in typical developing subjects (subjects, e.g., humans who do not have FXS) and a subpopulation of FXS subjects. FMR1-205 - 26 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 has 17 exons, and the splicing between Exon 1 of FMR1-205 (between base pairs 147,911,919 and 147,912,230, SEQ ID NO:4) and Exon 2 of FMR1-205 (between base pairs 147,921,933 and 147,921,985, SEQ ID NO:5) is considered normal FMR1 RNA splicing. Additional information on FMR1-205 and its protein product can be found at the web address below, the contents of which are incorporated herein by reference in their entirety: useast.ensembl.org/Homo_sapiens/Transcript/Summary?db=core;g=ENSG00000102081;r=X:14 7911951-147951125;t=ENST00000370475. [00114] CTCAGTCAGGCGCTCAGCTCCGTTTCGGTTTCACTTCCGGTGGAGGGCCGC CTCTGAGCGGGCGGCGGGCCGACGGCGAGCGCGGGCGGCGGCGGTGACGGAGGCG CCGCTGCCAGGGGGCGTGCGGCAGCGCGGCGGCGGCGGCGGCGGCGGCGGCGGCG GAGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCTGGGCCTCGAGCGCCCGCAGCCC ACCTCTCGGGGGCGGGCTCCCGGCGCTAGCAGGGCTGAAGAGAAGATGGAGGAGCT GGTGGTGGAAGTGCGGGGCTCCAATGGCGCTTTCTACAAG (SEQ ID NO:4). [00115] GCATTTGTAAAGGATGTTCATGAAGATTCAATAACAGTTGCATTTGAAAA CAA (SEQ ID NO:5). [00116] In some embodiments, a subject has a decreased level of isoform 1 of FMR1. In some embodiments, a subject has a decreased level of isoform 1 of FMR1 and an increased level of isoform 12 of FMR1. Age [00117] In some embodiments, a subject (e.g., a human) is a child (e.g., birth to 17 years of age). In some embodiments, a subject (e.g., a human) is an adult (18-64 years of age). In some embodiments, a subject (e.g., a human) is an older adult (65 years of age or older). [00118] In some embodiments, a subject (e.g., a human) is at least about 1 month of age, for example, at least about: 2, 5, 10, 12, 18, 24, 30, 36, 42, or 48 months of age. In some embodiments, a subject is at least about 18 months of age. In some embodiments, a subject is at least about 24 months of age. In some embodiments, a subject is at least about 42 months of age. [00119] In some embodiments, a subject (e.g., a human) is at least about 1 year of age, for example, at least about: 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, or 50 years of age. In some embodiments, a subject is at least about: 2, 3, 5, 6, 7, 8, 10, 12, 13, - 27 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 14, 15, 18, 30, or 50 years of age. In some embodiments, a subject is at least 14 years of age. In some embodiments, a subject is at least 18 years of age. [00120] In some embodiments, a subject (e.g., a human) is about 90 years of age or younger, for example, about: 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 29, 25, 24, 23, 22, 20, 18, 17, 16, 15, 13, 11, 10, 7, 6 years of age or younger. In some embodiments, a subject is about 68 months of age or younger. [00121] In some embodiments, a subject (e.g., a human) is about 0-100 years of age, for example, about: 1-100, 1-85, 1-80, 1-65, 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-29, 1-24, 1- 23, 1-22, 1-20, 1-18, 1-17, 1-16, 1-13, 1-12, 1-11, 1-10, 1-8, 1-7, 1-6, 2-100, 2-85, 2-80, 2-65, 2- 60, 2-55, 2-50, 2-45, 2-40, 2-35, 2-30, 2-29, 2-24, 2-23, 2-22, 2-20, 2-18, 2-17, 2-16, 2-13, 2-12, 2-11, 2-10, 2-8, 2-7, 2-6, 3-100, 3-85, 3-80, 3-65, 3-60, 3-55, 3-50, 3-45, 3-40, 3-35, 3-30, 3-29, 3-24, 3-23, 3-22, 3-20, 3-18, 3-17, 3-16, 3-13, 3-12, 3-11, 3-10, 3-8, 3-7, 3-6, 4-100, 4-85, 4-80, 4-65, 4-60, 4-55, 4-50, 4-45, 4-40, 4-35, 4-30, 4-29, 4-24, 4-23, 4-22, 4-20, 4-18, 4-17, 4-16, 4- 13, 4-12, 4-11, 4-10, 4-8, 4-7, 4-6, 5-100, 5-85, 5-80, 5-65, 5-60, 5-55, 5-50, 5-45, 5-40, 5-35, 5- 30, 5-29, 5-24, 5-23, 5-22, 5-20, 5-18, 5-17, 5-16, 5-13, 5-12, 5-11, 5-10, 5-8, 5-7, 5-6, 6-100, 6- 85, 6-80, 6-65, 6-60, 6-55, 6-50, 6-45, 6-40, 6-35, 6-30, 6-29, 6-24, 6-23, 6-22, 6-20, 6-18, 6-17, 6-16, 6-13, 6-12, 6-11, 6-10, 6-8, 6-7, 7-100, 7-85, 7-80, 7-65, 7-60, 7-55, 7-50, 7-45, 7-40, 7-35, 7-30, 7-29, 7-24, 7-23, 7-22, 7-20, 7-18, 7-17, 7-16, 7-13, 7-12, 7-11, 7-10, 7-8, 8-100, 8-85, 8- 80, 8-65, 8-60, 8-55, 8-50, 8-45, 8-40, 8-35, 8-30, 8-29, 8-24, 8-23, 8-22, 8-20, 8-18, 8-17, 8-16, 8-13, 8-12, 8-11, 8-10, 10-100, 10-85, 10-80, 10-65, 10-60, 10-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10-29, 10-24, 10-23, 10-22, 10-20, 10-18, 10-17, 10-16, 10-13, 10-12, 10-11, 12-100, 12- 85, 12-80, 12-65, 12-60, 12-55, 12-50, 12-45, 12-40, 12-35, 12-30, 12-29, 12-24, 12-23, 12-22, 12-20, 12-18, 12-17, 12-16, 12-13, 13-100, 13-85, 13-80, 13-65, 13-60, 13-55, 13-50, 13-45, 13- 40, 13-35, 13-30, 13-29, 13-24, 13-23, 13-22, 13-20, 13-18, 13-17, 13-16, 14-100, 14-85, 14-80, 14-65, 14-60, 14-55, 14-50, 14-45, 14-40, 14-35, 14-30, 14-29, 14-24, 14-23, 14-22, 14-20, 14- 18, 14-17, 14-16, 15-100, 15-85, 15-80, 15-65, 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, 15-29, 15-24, 15-23, 15-22, 15-20, 15-18, 15-17, 15-16, 18-100, 18-85, 18-80, 18-65, 18-60, 18- 55, 18-50, 18-45, 18-40, 18-35, 18-30, 18-29, 18-24, 18-23, 18-22, 18-20, 20-100, 20-85, 20-80, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 20-29, 20-24, 20-23, 20-22, 30-100, 30- 85, 30-80, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 40-100, 40-85, 40-80, 40-65, 40-60, - 28 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 40-55, 40-50, 40-45, 50-100, 50-85, 50-80, 50-65, 50-60, or 50-55 years of age. [00122] In some embodiments, a subject is about: 1-7, 2-16, 3-10, 3-11, 3-17, 3-18, 3-23, 5- 11, 5-13, 5-23, 5-35, 5-50, 6-18, 6-40, 7-16, 8-18, 8-45, 10-17, 10-23, 10-45, 12-18, 12-29, 12- 40, 12-45, 12-50, 13-22, 13-29, 13-35, 13-40, 14-50, 14-100, 15-55, 18-24, 18-45, 18-50, 18-60, 18-65, 18-100, 30-80, or 50-85 years of age. In some embodiments, a subject is about 24-68 months of age. In some embodiments, a subject is about 42 months to 16 years of age. In some embodiments, a subject is 12 years or older, and less than 18 years of age. Samples [00123] In some embodiments, a sample is a sample from or derived from a human subject. In some embodiments, a sample comprises a single cell or cell lysate, a population of cells, a cell culture, a tissue, or a bodily fluid. [00124] In some embodiments, a sample comprises a bodily fluid sample, a hair sample (e.g., from hair follicles), a nasal sample (e.g., nasal swab), a buccal sample (e.g., buccal swab), or a skin sample. [00125] In some embodiments, a sample comprises a bodily fluid sample. Non-limiting examples of bodily fluids include cerebrospinal fluid (CSF), blood (e.g., whole blood and derivatives and fractions of blood, such as plasma or serum), bone marrow aspirates, extracted galls, gingival crevicular fluid (GCF), milk, prostate fluid, pus, saliva (including whole saliva, individual gland secretions, oral rinse), skin scrapes, sputum, surface washings, tears (liquid secreted by lacrimal glands), and urine. In some embodiments, a bodily fluid comprises CSF, blood, saliva, sputum, tears, urine or semen, or a combination thereof. In some embodiments, a bodily fluid comprises CSF, blood, serum, plasma, or urine, or a combination thereof. In some embodiments, a bodily fluid comprises CSF. [00126] In some embodiments, a sample comprises a bodily fluid selected from cerebrospinal fluid (CSF), blood, serum, plasma, or urine. In some embodiments, a sample comprises peripheral blood mononuclear cells (PBMCs). [00127] In some embodiments, a sample comprises a tissue sample. In some embodiments, a sample comprises a brain sample. In some embodiments, a sample comprises a non-brain sample. In some embodiments, a sample comprises a non-neural sample. [00128] In some embodiments, a sample comprises a cell sample. In some embodiments, a - 29 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 sample comprises a blood cell (e.g., peripheral blood mononuclear cell (PBMC) or white blood cell), a fetal cell (e.g., circulating fetal cell), a blastomere, a trophectoderm cell, a stem cell (e.g., induced pluripotent stem cell (iPSC) or derived stem cell), a fibroblast (e.g., a dermal derived fibroblast cell or lung-derived fibroblast cell), a modified fibroblast, a leukocyte, a pluripotent cell, or a cultured cell, or a combination thereof. In some embodiments, a sample comprises a PBMC. In some embodiments, a sample comprises a white blood cell. In some embodiments, a sample comprises transformed lymphoblastoid B-cells (e.g., human lymphoblastoid cell line). [00129] In some embodiments, a sample comprises a brain sample (e.g., a human brain sample), a CSF sample (e.g., a human CSF sample), a PBMC sample (e.g., a human PBMC sample), or transformed lymphoblastoid B-cells (e.g., transformed human lymphoblastoid B- cells), or a combination thereof. In some embodiments, a sample comprises a brain sample, a PBMC sample, or a peripheral blood lymphocyte sample, or a combination thereof. [00130] In some embodiments, a method further comprises enriching (e.g., isolating) a cellular compartment or organelle from a sample. In some embodiments, a method further comprises enriching a cellular compartment (e.g., cytoplasm) from a sample. In some embodiments, a method further comprises enriching an organelle from a sample. Non-limiting examples of organelles include mitochondria, nuclei, Golgi apparatus, endoplasmic reticulum (ER), and ribosomes. In some embodiments, a method further comprises enriching mitochondria from a sample. [00131] In some embodiments, a sample is substantially free of macromolecules, e.g., molecules having molecular weights of greater than about 1,500 Daltons, for example, greater than about: 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Daltons. FXS-Associated Metabolites [00132] In some embodiments, a method comprises determining, in a sample from a subject, a presence, an absence, or a level of an FXS-associated metabolite. [00133] In some embodiments, a method comprises determining, in a sample from a subject, an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite. - 30 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00134] In some embodiments, a method comprises determining, in a sample from a subject, a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites. [00135] In some embodiments, a method comprises determining a presence, absence, amount, and/or alteration of at least one FXS-associated metabolite (e.g., an FXS-associated metabolite). [00136] In some embodiments, an FXS-associated metabolite is present in aberrant amount in a FXS subject, is potentially involved in FXS initiation, progression, and/or prediction, or both. In some embodiments, an FXS-associated metabolite is present in aberrant amount in a FXS subject. In some embodiments, an FXS-associated metabolite is potentially involved in FXS initiation, progression, and/or prediction. [00137] In some embodiments, a method comprises determining a presence of at least one FXS-associated metabolite. In some embodiments, a method comprises determining an absence of at least one FXS-associated metabolite. In some embodiments, a method comprises determining an amount of at least one FXS-associated metabolite. In some embodiments, a method comprises determining an alteration of at least one FXS-associated metabolite. [00138] In other embodiments, an FXS-associated metabolite is absent in a sample (e.g., a CSF sample) from a subject (e.g., a human), but present in a control sample and/or a reference. [00139] In other embodiments, an FXS-associated metabolite is present in a sample (e.g., a CSF sample) from a subject (e.g., a human), but absent in a control sample and/or a reference. [00140] In some embodiments, at least one metabolite has an altered level in a sample (e.g., a CSF sample) from a subject (e.g., a human), relative to a control sample and/or a reference (e.g., reference level). In some embodiments, at least two (e.g., at least three, four, five, six, seven, eight, nine, or ten) metabolites have altered levels in a sample (e.g., a CSF sample) from a subject (e.g., a human), relative to a control sample and/or a reference. [00141] In some embodiments, a control sample comprises a sample from one or more age- matched typically-developing individuals (e.g., two or more age-matched typically-developing individuals), a baseline sample from a subject (e.g., prior to the onset of the disease state), or a sample derived from healthy cells (e.g., not affected by the disease state and from a similar origin), or any combination thereof. In some embodiments, a control sample is a sample from one or more age-matched typically-developing individuals. In some embodiments, a control - 31 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 sample is a baseline sample from a subject. In some embodiments, a control sample is a sample derived from healthy cells. [00142] In some embodiments, the reference level is based on: a sample from an age-matched typically-developing individual, or a baseline sample from the subject. [00143] In some embodiments, a level of at least one metabolite is compared to a level of a reference (e.g., standard). In some embodiments, a reference (e.g., reference level) is a theoretical value obtained from a population, e.g., a general population (e.g., from the same tissue, organ, bodily fluid, cell, organelle, and/or cellular compartment as a FXS sample). In some embodiments, a reference is a theoretical value obtained from one or more FXS subjects who were treated successfully. [00144] In some embodiments, the term “metabolite” (e.g., an FXS-associated metabolite) excludes molecules having molecular weights over 2,000 Daltons. In some embodiments, the term “metabolite” excludes molecules having molecular weights over 1,500 Daltons. In some embodiments, the term “metabolite” excludes molecules having molecular weights over 1,000 Daltons. [00145] In some embodiments, an FXS-associated metabolite comprises a signaling molecule. In some embodiments, an FXS-associated metabolite comprises an amino acid, fatty acid, nucleotide, sugar, or an intermediate of a cellular process, or a combination thereof. [00146] In some embodiments, at least one metabolite (e.g., an FXS-associated metabolite) having an altered level in a sample from a subject, relative to a control sample and/or a reference, comprises n-acetylneuraminate, D-alanine, L-alanine arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, L-carnitine, carnosine, citrulline, creatine, cystine, cytosine, fumarate, alpha-D-glucose, glucuronate, oxidized glutathione (GSSG), reduced glutathione (GSH), glycerol, glycine, guanine, 3-methyl-L-histidine, homoserine, hypoxanthine, indoleacetaldehyde, isoleucine, kynurenate, leucine, lysine, malate, methionine, methyl acetoacetate, methylmalonate, mevalonate, n-formyl-L-methionine, nicotinamide, phenylalanine, proline, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, D-sedoheptulose, serine, succinate, threonine, allothreonine, tryptophan, tyramine, tyrosine, uracil, urate, valine, norvaline, xanthine, phosphoenolpyruvate, orotate, guanosine, trehalose, or a combination thereof. - 32 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00147] In some embodiments, at least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference, comprises n-acetylneuraminate, D-alanine, L-alanine arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, L-carnitine, carnosine, citrulline, creatine, cystine, cytosine, fumarate, alpha-D-glucose, glucuronate, oxidized glutathione (GSSG), reduced glutathione (GSH), glycerol, glycine, guanine, 3-methyl-L-histidine, homoserine, hypoxanthine, indoleacetaldehyde, isoleucine, kynurenate, leucine, lysine, malate, methionine, methyl acetoacetate, methylmalonate, mevalonate, n-formyl-L-methionine, nicotinamide, phenylalanine, proline, cis-4-hydroxy-D- proline, trans-4-hydroxy-L-proline, D-sedoheptulose, serine, succinate, threonine, allothreonine, tryptophan, tyramine, tyrosine, uracil, urate, valine, norvaline, or xanthine, or a combination thereof. [00148] In some embodiments, at least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference, comprises: a) lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L- carnitine, arginine, cystine, urate, GSH, phosphoenolpyruvate, GSSG, orotate, or guanosine, or a combination thereof, b) argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-formyl-L-methionine, arginine, azelate, trehalose, or D- sedoheptulose, or a combination thereof, or c) valine, norvaline, nicotinamide, D-alanine, creatine, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha- D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n-acetylneuraminate, asparagine, or L-alanine, or a combination thereof, or a combination thereof. [00149] In some embodiments, at least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference, comprises lysine, proline, glycine, - 33 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L-carnitine, arginine, cystine, urate, GSH, phosphoenolpyruvate, GSSG, orotate, or guanosine, or a combination thereof. [00150] In some embodiments, at least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference, comprises argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-formyl-L-methionine, arginine, azelate, trehalose, or D-sedoheptulose, or a combination thereof. [00151] In some embodiments, at least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference, comprises valine, norvaline, nicotinamide, D-alanine, creatine, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha-D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n-acetylneuraminate, asparagine, or L-alanine, or a combination thereof. [00152] In some embodiments, at least one metabolite having an altered level in a sample from a subject, relative to a control sample and/or a reference, comprises arginine, leucine, lysine, or D-sedoheptulose, or a combination thereof. [00153] In some embodiments, at least one metabolite has a reduced level in a sample from a subject, relative to a control sample and/or a reference. In some embodiments, the FXS- associated metabolite has a reduced level in the sample from the subject, relative to a reference level of the same metabolite. [00154] In some embodiments, at least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference, comprises: a) lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L- carnitine, arginine, cystine, urate, or GSH, or a combination thereof, b) argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, - 34 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 glucuronate, n-formyl-L-methionine, arginine, or azelate, or a combination thereof, or c) valine, norvaline, nicotinamide, D-alanine, creatine, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha- D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n-acetylneuraminate, asparagine, or L-alanine, or a combination thereof, or a combination thereof. [00155] In some embodiments, a FXS-associated metabolite comprises n-acetylneuraminate, D-alanine, L-alanine arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, L-carnitine, carnosine, citrulline, creatine, cystine, cytosine, fumarate, alpha-D-glucose, glucuronate, oxidized glutathione (GSSG), reduced glutathione (GSH), glycerol, glycine, guanine, 3-methyl-L-histidine, homoserine, hypoxanthine, indoleacetaldehyde, isoleucine, kynurenate, leucine, lysine, malate, methionine, methyl acetoacetate, methylmalonate, mevalonate, n-formyl-L-methionine, nicotinamide, phenylalanine, proline, cis-4-hydroxy-D- proline, trans-4-hydroxy-L-proline, D-sedoheptulose, serine, succinate, threonine, allothreonine, tryptophan, tyramine, tyrosine, uracil, urate, valine, norvaline, xanthine, phosphoenolpyruvate, orotate, guanosine, trehalose, or a combination thereof. [00156] In some embodiments, a FXS-associated metabolite comprises: a) lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L- carnitine, arginine, cystine, urate, or reduced glutathione (GSH), or a combination thereof, b) argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-formyl-L-methionine, arginine, or azelate, or a combination thereof, or c) valine, norvaline, nicotinamide, D-alanine, creatine, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha- - 35 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n-acetylneuraminate, asparagine, or L-alanine, or a combination thereof, or a combination thereof. [00157] In some embodiments, a FXS-associated metabolite comprises arginine, leucine, or lysine, or a combination thereof. [00158] In some embodiments, a FXS-associated metabolite comprises: a) phosphoenolpyruvate, oxidized glutathione disulfide (GSSG), orotate, or guanosine, or a combination thereof, or b) trehalose, or D-sedoheptulose, or both, or a combination thereof. [00159] In some embodiments, at least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference, comprises lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L-carnitine, arginine, cystine, urate, or GSH, or a combination thereof. [00160] In some embodiments, at least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference, comprises argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-formyl-L-methionine, arginine, or azelate, or a combination thereof. [00161] In some embodiments, at least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference, comprises valine, norvaline, nicotinamide, D-alanine, creatine, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha-D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n-acetylneuraminate, asparagine, or L-alanine, or a combination thereof. [00162] In some embodiments, at least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference, comprises arginine, leucine, - 36 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 lysine, or D-sedoheptulose, or a combination thereof. In some embodiments, at least one metabolite having a reduced level in a sample from a subject, relative to a control sample and/or a reference, comprises arginine, leucine, or lysine, or a combination thereof. [00163] In some embodiments, the alteration of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, is a log2 fold reduction of from about -30 to about 0 (e.g., from about -20 to about 0, from about -20 to about -0.90, from about -10 to about 0, from about -10 to about -0.90, from about -9 to about 0, from about -8 to about 0, from about - 7 to about 0, from about -6 to about 0, from about -5 to about 0, from about -4 to about 0, from about -3 to about 0, from about -2 to about 0, from about -1 to about 0, from about -0.5 to about 0, etc.) or a log2 fold increase of from about 0 to about 30 (e.g., from about 0 to about 20, from about 0.90 to about 20, from about 0 to about 10, from about 0.90 to about 10, from about 0 to about 9, from about 0 to about 8, from about 0 to about 7, from about 0 to about 6, from about 0 to about 5, from about 0 to about 4, from about 0 to about 3, from about 0 to about 2, from about 0 to about 1, from about 0 to about 0.5, etc.). In some embodiments, the alteration of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, is a log2 fold reduction of from about -10 to about 0 or a log2 fold increase of from about 0 to about 10. [00164] In some embodiments, the alteration of a ratio between levels of two FXS-associated metabolites relative to a reference ratio of the two FXS-associated metabolites is a decrease of at least about 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500% etc.). For example, the ratio between levels of two FXS-associated metabolites is smaller than a reference ratio of the same two metabolites by at least about 10%. In some embodiments, the alteration of a ratio between levels of two FXS-associated metabolites relative to a reference ratio of the two FXS-associated metabolites is a decrease of at least about 20%. [00165] In some embodiments, the alteration of a ratio between levels of two FXS-associated metabolites relative to a reference ratio of the two FXS-associated metabolites is a decrease of from about 10% to about 500% (e.g., about 10% to about 300%, about 10% to about 200%, about 10% about 100%, about 10% to about 80%, about 20% to about 80%, about 30% to about 70%, about 40% to about 70%, etc.). In some embodiments, the alteration of a ratio between levels of two FXS-associated metabolites relative to a reference ratio of the two FXS-associated metabolites is a decrease of from about 20% to about 80%. - 37 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00166] In some embodiments, the alteration of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, is a log2 fold reduction of equal to or less than about -2.0 (e.g., -1.9, -1.8, -1.7, -1.6, -1.5, -1.4, -1.3, -1.2, -1.1, -1.0, -0.95, -0.90, -0.85, - 0.80. -0.75, -0.70, -0.65, -0.60, -0.55, -0.50, -0.45, -0.40, -0.35, -0.30, -0.25, -0.20, -0.15, -0.10, etc.) or a log2 fold increase of equal to or greater than about 0.10 (e.g., 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, etc). In some embodiments, the alteration of a level of an FXS- associated metabolite, relative to a reference level of the same metabolite, is a log2 fold reduction of equal to or less than about -0.50 or a log2 fold increase of equal to or greater than about 0.50. [00167] In some embodiments, at least one FXS-associated metabolite has a log2 fold reduction of ≤(-0.10) in a sample from a subject, relative to a control sample and/or a reference, for example, ≤(-0.15), ≤(-0.20), ≤(-0.25), ≤(-0.30), ≤(-0.35), ≤(-0.40), ≤(-0.45), ≤(-0.50), ≤(- 0.55), ≤(-0.60), ≤(-0.65), ≤(-0.70), ≤(-0.75), ≤(-0.80), ≤(-0.85), ≤(-0.90), ≤(-0.95), ≤(-1.0), ≤(- 1.1), ≤(-1.2), ≤(-1.3), ≤(-1.4), ≤(-1.5), ≤(-1.6), ≤(-1.7), ≤(-1.8), ≤(-1.9), or ≤(-2.0), relative to a control sample and/or a reference. [00168] In some embodiments, at least one FXS-associated metabolite has a log2 fold reduction of ≤(-0.20) in a sample from a subject, relative to a control sample and/or a reference. In some embodiments, at least one FXS-associated metabolite has a log2 fold reduction of ≤(-0.50) in a sample from a subject, relative to a control sample and/or a reference. In some embodiments, at least one FXS-associated metabolite has a log2 fold reduction of ≤(-0.90) in a sample from a subject, relative to a control sample and/or a reference. [00169] In some embodiments, level of at least one FXS-associated metabolite is at least about 5% lower in a sample from a subject, relative to a control sample and/or a reference. For example, level of at least one FXS-associated metabolite is at least about: 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower in a sample from a subject, relative to a control sample and/or a reference. [00170] In some embodiments, reduction of a level of at least one FXS-associated metabolite in a sample from a subject, relative to a control sample and/or a reference, is statistically significant. In some embodiments, p-value is less than 0.05. In some embodiments, p-value is less than 0.01. In some embodiments, p-value is less than 0.001. - 38 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00171] In some embodiments, determining: an alteration or a ratio of a level of an FXS- associated metabolite, relative to a reference level of the same metabolite; or a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, comprises determining the statistical significance of the alteration based on a significance threshold. [00172] In some embodiments, the significance threshold is a p-value equal to or less than about 0.05. In some embodiments, p-value is less than 0.01. In some embodiments, p-value is less than 0.001. [00173] In some embodiments, at least one metabolite has an increased level in a sample from a subject, relative to a control sample and/or a reference. [00174] In some embodiments, at least one metabolite having an increased level in a sample from a subject, relative to a control sample and/or a reference, comprises: a) phosphoenolpyruvate, GSSG, orotate, or guanosine, or a combination thereof, or b) trehalose, or D-sedoheptulose, or both, or a combination thereof. [00175] In some embodiments, at least one metabolite having an increased level in a sample from a subject, relative to a control sample and/or a reference, comprises phosphoenolpyruvate, GSSG, orotate, or guanosine, or a combination thereof. [00176] In some embodiments, at least one metabolite having an increased level in a sample from a subject, relative to a control sample and/or a reference, comprises trehalose, or D- sedoheptulose, or both. [00177] In some embodiments, at least one FXS-associated metabolite has a log2 fold increase of ≥0.10 in a sample from a subject, relative to a control sample and/or a reference, for example, ≥0.15, ≥0.20, ≥0.25, ≥0.30, ≥0.35, ≥0.40, ≥0.45, ≥0.50, ≥0.55, ≥0.60, ≥0.65, ≥0.70, ≥0.75, ≥0.80, ≥0.85, ≥0.90, ≥0.95, ≥1.0, ≥1.1, ≥1.2, ≥1.3, ≥1.4, ≥1.5, ≥1.6, ≥1.7, ≥1.8, ≥1.9, or ≥2.0, relative to a control sample and/or a reference. [00178] In some embodiments, at least one FXS-associated metabolite has a log2 fold increase of ≥0.20 in a sample from a subject, relative to a control sample and/or a reference. In some embodiments, at least one FXS-associated metabolite has a log2 fold increase of ≥0.50 in a sample from a subject, relative to a control sample and/or a reference. In some embodiments, at - 39 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 least one FXS-associated metabolite has a log2 fold increase of ≥0.90 in a sample from a subject, relative to a control sample and/or a reference. [00179] In some embodiments, level of at least one FXS-associated metabolite is at least about 5% higher in a sample from a subject, relative to a control sample and/or a reference. For example, level of at least one FXS-associated metabolite is at least about: 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, or 200%, higher in a sample from a subject, relative to a control sample and/or a reference. [00180] In some embodiments, increase of a level of at least one FXS-associated metabolite in a sample from a subject, relative to a control sample and/or a reference, is statistically significant. In some embodiments, p-value is less than 0.05. In some embodiments, p-value is less than 0.01. In some embodiments, p-value is less than 0.001. [00181] In some embodiments, a first metabolite and a second metabolite have an altered ratio in a sample from a subject (e.g., a human), relative to a control sample and/or a reference. [00182] In some embodiments, an altered ratio is indicative of increased oxidative stress in a sample from a subject. In some embodiments, a first metabolite is reduced glutathione (GSH), and a second metabolite is oxidized glutathione disulfide (GSSG). [00183] In some embodiments, an altered ratio is indicative of suppressed mitochondrial metabolism in a sample from a subject. In some embodiments, a first metabolite is adenosine triphosphate (ATP), and a second metabolite is adenosine diphosphate (ADP). [00184] In some embodiments, the ratio between the levels of a pair of FXS-associated metabolites is the ratio of reduced glutathione (GSH) to glutathione disulfide (GSSG) or the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). In some embodiments, the ratio between the levels of a pair of FXS-associated metabolites is the ratio of reduced glutathione (GSH) to glutathione disulfide (GSSG). In some embodiments, the ratio between the levels of a pair of FXS-associated metabolites is the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). [00185] In some embodiments, a first metabolite and a second metabolite have a reduced ratio in a sample from a subject (e.g., a human), relative to a control sample and/or a reference. - 40 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00186] In some embodiments, a reduced ratio is indicative of increased oxidative stress in a sample from a subject. In some embodiments, a first metabolite is reduced glutathione (GSH), and a second metabolite is oxidized glutathione disulfide (GSSG). [00187] In some embodiments, a reduced ratio is indicative of suppressed mitochondrial metabolism in a sample from a subject. In some embodiments, a first metabolite is adenosine triphosphate (ATP), and a second metabolite is adenosine diphosphate (ADP). [00188] In some embodiments, the methods and teachings disclosed herein can be used to assess efficacy of treatment (e.g., therapeutics such as ASO therapeutics), e.g., in the CSF. In some embodiments, the method comprises assessing the metabolome of a subject, e.g., for restoration of biology in the brain, following ASO treatment, e.g., ASO treatment comprising intra-thecal injection. In some embodiments, the method comprises assessing the metabolome in a sample (e.g., CSF) of a subject with FSX, before and after treatment, e.g., ASO treatment. FXS-Associated Metabolites in the Brain [00189] In some embodiments, at least one metabolite has an altered level in a brain sample from a subject (e.g., a human), relative to a control brain sample. In some embodiments, at least one metabolite having an altered level in a brain sample from a subject, relative to a control brain sample, comprises arginine, L-carnitine, cystine, GSSG, GSH, glycine, guanine, guanosine, homoserine, isoleucine, leucine, lysine, methionine, orotate, phenylalanine, phosphoenolpyruvate, proline, D-sedoheptulose, serine, threonine, tryptophan, tyrosine, or urate, or a combination thereof. [00190] In some embodiments, at least one metabolite has a reduced level in a brain sample from a subject (e.g., a human), relative to a control brain sample. In some embodiments, at least one metabolite having a reduced level in a brain sample from a subject, relative to a control brain sample, comprises lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L-carnitine, arginine, cystine, urate, or GSH, or a combination thereof. [00191] In some embodiments, at least one metabolite has an increased level in a brain sample from a subject, relative to a control brain sample. In some embodiments, at least one metabolite having an increased level in a brain sample from a subject, relative to a control brain sample comprises phosphoenolpyruvate, GSSG, orotate, or guanosine, or a combination thereof. - 41 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00192] In some embodiments, a first metabolite and a second metabolite have an altered ratio in a brain sample from a subject (e.g., a human), relative to a control brain sample. In some embodiments, an altered ratio is indicative of increased oxidative stress in a brain sample from a subject. In some embodiments, a first metabolite is reduced glutathione (GSH), and a second metabolite is oxidized glutathione disulfide (GSSG). [00193] In some embodiments, a first metabolite and a second metabolite have a reduced ratio in a brain sample from a subject (e.g., a human), relative to a control brain sample. In some embodiments, a reduced ratio is indicative of increased oxidative stress in a brain sample from a subject. In some embodiments, a first metabolite is reduced glutathione (GSH), and a second metabolite is oxidized glutathione disulfide (GSSG). FXS-Associated Metabolites in Peripheral Blood Mononuclear Cell (PBMC) Samples [00194] In some embodiments, at least one metabolite has an altered level in a PBMC sample from a subject (e.g., a human), relative to a control PBMC sample. In some embodiments, at least one metabolite having an altered level in a PBMC sample from a subject, relative to a control PBMC sample, comprises methyl acetoacetate, arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, carnosine, cytosine, fumarate, glucuronate, glycerol, indoleacetaldehyde, kynurenate, leucine, lysine, 3-methyl-L-histidine, malate, n-formyl-L- methionine, mevalonate, D-sedoheptulose, trehalose, tyramine, uracil, or xanthine, or a combination thereof. [00195] In some embodiments, at least one metabolite has a reduced level in a PBMC sample from a subject (e.g., a human), relative to a control PBMC sample. In some embodiments, at least one metabolite having a reduced level in a PBMC sample from a subject, relative to a control PBMC sample, comprises argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-formyl-L-methionine, arginine, or azelate, or a combination thereof. [00196] In some embodiments, at least one metabolite has an increased level in a PBMC sample from a subject (e.g., a human), relative to a control PBMC sample. In some embodiments, at least one metabolite having an increased level in a PBMC sample from a subject, relative to a control PBMC sample, comprises trehalose, or D-sedoheptulose, or both. - 42 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00197] In some embodiments, a first metabolite and a second metabolite have an altered ratio in a PBMC sample from a subject (e.g., a human), relative to a control PBMC sample. [00198] In some embodiments, an altered ratio is indicative of suppressed mitochondrial metabolism in a PBMC sample from a subject. In some embodiments, a first metabolite is adenosine triphosphate (ATP), and a second metabolite is adenosine diphosphate (ADP). [00199] In some embodiments, a first metabolite and a second metabolite have a reduced ratio in a PBMC sample from a subject (e.g., a human), relative to a control sample and/or a reference. In some embodiments, a reduced ratio is indicative of suppressed mitochondrial metabolism in a PBMC sample from a subject. In some embodiments, a first metabolite is adenosine triphosphate (ATP), and a second metabolite is adenosine diphosphate (ADP). FXS-Associated Metabolites in Peripheral Blood Lymphocyte Samples [00200] In some embodiments, at least one metabolite has an altered level in a peripheral blood lymphocyte sample from a subject (e.g., a human), relative to a control peripheral blood lymphocyte sample. In some embodiments, at least one metabolite having an altered level in a peripheral blood lymphocyte sample from a subject, relative to a control peripheral blood lymphocyte sample, comprises valine, norvaline, nicotinamide, D-alanine, creatine, cis-4- hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha-D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n- acetylneuraminate, asparagine, or L-alanine, or a combination thereof. [00201] In some embodiments, at least one metabolite has a reduced level in a peripheral blood lymphocyte sample from a subject (e.g., a human), relative to a control peripheral blood lymphocyte sample. In some embodiments, at least one metabolite having a reduced level in a peripheral blood lymphocyte sample from a subject, relative to a control peripheral blood lymphocyte sample, comprises valine, norvaline, nicotinamide, D-alanine, creatine, cis-4- hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, - 43 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 alpha-D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n- acetylneuraminate, asparagine, or L-alanine, or a combination thereof. Metabolites Detection & Quantification [00202] A single technique or a combination of techniques known in the art may be used for separating, identifying, and/or quantifying metabolites. Non-limiting examples of techniques include mass spectroscopy, nuclear magnetic resonance spectroscopy (NMR), refractive index spectroscopy (RI), ultra-violet spectroscopy (UV), near-infrared spectroscopy (Near-IR), electrochemical analysis, fluorescent analysis, radiochemical analysis, and light scattering analysis (LS). [00203] In some embodiments, determining a presence, absence, amount, or alteration of a FXS-associated metabolite, or an alteration of a FXS-associated ratio comprises performing a spectrophotometric detection of one or more metabolites. [00204] In some embodiments, methods of the present disclosure employ a chromatography technique. [00205] In some embodiments, a chromatography technique is employed to determine, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing. [00206] In some embodiments, the chromatography technique is liquid chromatography-mass spectrometry (LC-MS). In other embodiments, the chromatography technique is gas chromatography-mass spectrometry (GC-MS). In other embodiments, the chromatography technique is high-performance liquid chromatography (HPLC). [00207] In some embodiments, determining a presence, absence, amount, or alteration of a FXS-associated metabolite, or an alteration of a FXS-associated ratio comprises performing liquid chromatography-mass spectrometry (LC-MS). - 44 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00208] In some embodiments, determining a presence, absence, amount, or alteration of a FXS-associated metabolite, or an alteration of a FXS-associated ratio comprises performing gas chromatography-mass spectrometry (GC-MS). [00209] In some embodiments, a method comprises determining a level, amount, and/or concentration of a metabolite in a sample (e.g., tissue, bodily fluid, cell, cellular compartment, or organelle). Treatments [00210] In some embodiments, a method of treating fragile X syndrome (FXS) in a subject is disclosed herein, the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and administering to the subject a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product. [00211] In some embodiments, a method disclosed herein further comprises treating FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, or the alteration of the FXS-associated ratio, in a sample. [00212] In some embodiments, treating FXS in a subject in need thereof comprises administering to the subject a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product, restoring metabolite homeostasis, or both. Agents [00213] In some embodiments, administering an agent to a subject: a) decreases splicing of an aberrant FMR1 transcript (e.g., between Exons 1 and 2 of FMR1-217), - 45 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 b) decreases level of an aberrant FMR1 gene product (e.g., an aberrant FMR1 transcript such as FMR1-217, and/or an aberrant FMR1-encoded protein), c) increases splicing of a normal FMR1 transcript (e.g., between Exons 1 and 2 of FMR1-205), d) increases level of a normal FMR1 gene product (e.g., a normal FMR1 transcript such as FMR1-205 and/or a normal FMR1-encoded protein), or e) increases level of FMRP, or any combination thereof, in the subject. [00214] In some embodiments, contacting an agent to a cell: a) decreases splicing of an aberrant FMR1 transcript (e.g., between Exons 1 and 2 of FMR1-217), b) decreases level of an aberrant FMR1 gene product (e.g., an aberrant FMR1 transcript such as FMR1-217, and/or an aberrant FMR1-encoded protein), c) increases splicing of a normal FMR1 transcript (e.g., between Exons 1 and 2 of FMR1-205), d) increases level of a normal FMR1 gene product (e.g., a normal FMR1 transcript such as FMR1-205 and/or a normal FMR1-encoded protein), or e) increases level of FMRP, or any combination thereof, in the cell. Polynucleotide Agents [00215] In some embodiments, an agent comprises a polynucleotide or an analog thereof. In some embodiments, an agent comprises at least two (e.g., three, four, or more) polynucleotides. [00216] In some embodiments, a polynucleotide is an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense DNA, an antisense RNA, a microRNA (miRNA), an antagomir, or a guide RNA (gRNA). In some embodiments, a polynucleotide is an ASO. [00217] In some embodiments, a polynucleotide comprises a sequence that is (fully or partially) complementary to a target sequence (such that a polynucleotide is capable of hybridizing or annealing to target sequence). In some embodiments, a polynucleotide comprises a sequence that is (fully or partially) complementary to a target sequence under physiological - 46 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 conditions, for example, with a melting temperature (Tm) of at least 45°C, e.g., at least: 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. Tm is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide at a given ionic strength and pH. [00218] In some embodiments, specific hybridization corresponds to stringent hybridization conditions. In some embodiments, specific hybridization occurs with near complementary of a polynucleotide (e.g., ASO) to a target sequence. In some embodiments, specific hybridization occurs with near or substantial complementary of a polynucleotide to a target sequence. In some embodiments, specific hybridization occurs with exact complementary of a polynucleotide to a target sequence. [00219] In some embodiments, a polynucleotide is formed of contiguous complementary sequences (to a target sequence). In some embodiments, the polynucleotide sequence is formed of non-contiguous complementary sequences (to a target sequence), for example, when placed together, constitute sequence that spans the target sequence. [00220] In some embodiments, a polynucleotide has a length of at least about 8 nucleotides, e.g., at least about: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 nucleotides, or about: 10-60, 10-40, 12-80, 12-60, 12- 40, 12-38, 12-30, 13-38, 13-36, 14-36, 14-34, 15-80, 15-60, 15-40, 15-34, 15-32, 16-32, 16-30, 17-30, 17-28, 18-28, 18-26, 19-26, 19-24, 20-80, 20-60, 20-40, 20-30, 20-24, or 20-22 nucleotides. In some embodiments, a polynucleotide has a length of about 10-30 or 12-30 nucleotides. [00221] In some embodiments, a polynucleotide has a length of at least about 12 nucleotides, e.g., at least about: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or about: 12-40, 12-35, 12-30, 12-25, 13-40, 13-35, 13-30, 13-25, 14-40, 14-35, 14- 30, 14-25, 15-40, 15-35, 15-30, or 15-25 nucleotides. In some embodiments, a polynucleotide has a length of about 15-25 nucleotides. In some embodiments, a polynucleotide has a length of about 18-22 nucleotides. [00222] In some embodiments, a polynucleotide is an isolated polynucleotide. An “isolated polynucleotide” refers to a polynucleotide that has been separated from other cellular components normally associated with native nucleotide polymers, including proteins and other - 47 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 nucleotide sequences. In some embodiments, a polynucleotide is an isolated DNA polynucleotide. In some embodiments, a polynucleotide is an isolated RNA polynucleotide. [00223] Polynucleotides described herein can be produced recombinantly or synthetically, using methods, techniques, and reagents that are well known in the art, such as routine and well- known molecular cloning techniques and solid-phase synthesis techniques. In some embodiments, a polynucleotide is a recombinant polynucleotide. [00224] In some embodiments, a polynucleotide comprises a sequence that is complementary to at least 8 contiguous nucleotides (e.g., about 10-30 nucleotides) of a sequence set forth in any one of SEQ ID NOs:6-9, SEQ ID NOs:10-24, or a combination thereof. [00225] In some embodiments, a polynucleotide comprises a sequence that specifically hybridizes to (e.g., having near, substantial, or exact reverse complementarity to) a portion of a sense strand on X chromosome between base pairs 147,911,919 and 147,921,985, e.g., between 147,911,919 and 147,921,933, between 147,911,919 and 147,912,230, between 147,911,919 and 147,912,123, between 147,911,919 and 147,914,451, between 147,911,919 and 147,912,728, between 147,912,231 and 147,921,932, between 147,912,231 and 147,914,451, between 147,912,231 and 147,912,727, between 147,912,728 and 147,914,451, between 147,912,694 and 147,912,727, between 147,912,710 and 147,912,745, between 147,912,731 and 147,912,766, or between 147,912,694 and 147,912,766. In some embodiments, a polynucleotide has exact reverse complementarity to a portion of a sense strand on X chromosome between base pairs 147,911,919 and 147,921,985. [00226] In some embodiments, a polynucleotide has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reverse and complementary sequence of a portion of a sense strand on X chromosome between base pairs 147,912,694 and 147,912,766 (e.g., between base pairs 147,912,731 and 147,912,766). In some embodiments, a polynucleotide has 100% sequence identity to the reverse and complementary sequence of a portion of a sense strand on X chromosome between base pairs 147,912,694 and 147,912,766 (e.g., between base pairs 147,912,731 and 147,912,766). [00227] In some embodiments, a polynucleotide (e.g., ASO) has at least about 70% sequence identity to the reverse and complementary sequence of a portion of X chromosome between base pairs 147,912,230 and 147,914,451 (e.g., between 147,912,230 and 147,912,728 or between - 48 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 147,912,728 and 147,914,451), for example, having at least about: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, or having about: 75-99%, 75-98%, 80-98%, 80-97%, 85-97%, 85-96%, 90-96%, or 90-95% sequence identity to the reverse and complementary sequence of a portion of X chromosome between base pairs 147,912,230 and 147,912,728. In some embodiments, a polynucleotide comprises a sequence having 100% sequence identity to the reverse and complementary sequence of a portion of X chromosome between base pairs 147,912,230 and 147,914,451. [00228] In some embodiments, a polynucleotide comprises a sequence that is reverse complementary to a contiguous nucleotide sequence (e.g., at least 8 nucleotides, for example, about 10-30 nucleotides) of pre-mRNA of an aberrant FMR1 transcript (e.g., pre-mRNA of FMR1-217). [00229] In some embodiments, a polynucleotide comprises a sequence that is reverse complementary to at least 8 contiguous nucleotides of a sequence set forth in SEQ ID NOs:6-24. [00230] UCAGGUCUCCUUUGGCUUCUCUUUUCCGGUCUAGCAUUGGGACUUCGG AGAGCUCCACUGUUCUGGGCGAGGGCUGUGAAGAAAGA (SEQ ID NO:6). [00231] UCAGGUCUCCUUUGGCUUCUCUUUUCCGGUCUAGCAUUGGGACUUCGG AGA (SEQ ID NO:7) [00232] CAUUGGGACUUCGGAGAGCUCCACUGUUCUGGGCGAGGGCUGUGAAGA AAGA (SEQ ID NO:8) [00233] UGGGACUUCGGAGAGCUCCACUGUUCUGGGCGAGGGCUGUGAAGAA (SEQ ID NO:9) [00234] UCAGGUCUCCUUUGGCUUCU (SEQ ID NO:10) [00235] GUCUCCUUUGGCUUCUCUUU (SEQ ID NO:11) [00236] UGGCUUCUCUUUUCCGGUCUAG (SEQ ID NO:12) [00237] UUCUCUUUUCCGGUCUAGCAU (SEQ ID NO:13) [00238] UCUUUUCCGGUCUAGCAUUG (SEQ ID NO:14) [00239] UCCGGUCUAGCAUUGGGACUU (SEQ ID NO:15) [00240] UAGCAUUGGGACUUCGGAGA (SEQ ID NO:16) [00241] UGGGACUUCGGAGAGCUC (SEQ ID NO:17) - 49 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00242] UCGGAGAGCUCCACUGUUCU (SEQ ID NO:18) [00243] GAGCUCCACUGUUCUGGGCG (SEQ ID NO:19) [00244] CUCCACUGUUCUGGGCGAGG (SEQ ID NO:20) [00245] GGACUUCGGAGAGCUCCACUG (SEQ ID NO:21) [00246] GGAGAGCUCCACUGUUCUGGG (SEQ ID NO:22) [00247] UGUUCUGGGCGAGGGCUGUG (SEQ ID NO:23) [00248] UGGGCGAGGGCUGUGAAGAA (SEQ ID NO:24) [00249] In some embodiments, a polynucleotide (e.g., ASO) comprises a sequence having at least about 70% sequence identity to a sequence set forth in any one of SEQ ID NOs:25-43, for example, having at least about: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, or having about: 75-99%, 75-98%, 80-98%, 80-97%, 85-97%, 85-96%, 90-96%, or 90- 95% sequence identity to a sequence set forth in any one of SEQ ID NOs:25-43. In some embodiments, a polynucleotide comprises a sequence having at least about 85% sequence identity to a sequence set forth in any one of SEQ ID NOs:25-43. In some embodiments, a polynucleotide comprises a sequence having 100% sequence identity to a sequence set forth in any one of SEQ ID NOs:25-43. [00250] AGAAGCCAAAGGAGACCTGA (SEQ ID NO:25) (W-704). [00251] AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:26) (W-705). [00252] CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:27) (W-706). [00253] ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:28) (W-707). [00254] CAATGCTAGACCGGAAAAGA (SEQ ID NO:29) (W-708). [00255] AAGTCCCAATGCTAGACCGGA (SEQ ID NO:30) (W-709). [00256] TCTCCGAAGTCCCAATGCTA (SEQ ID NO:31) (W-710). [00257] GAGCTCTCCGAAGTCCCA (SEQ ID NO:32) (W-711). [00258] AGAACAGTGGAGCTCTCCGA (SEQ ID NO:33) (W-712). [00259] CGCCCAGAACAGTGGAGCTC (SEQ ID NO:34) (W-713). [00260] CCTCGCCCAGAACAGTGGAG (SEQ ID NO:35) (W-714). [00261] CAGTGGAGCTCTCCGAAGTCC (SEQ ID NO:36) (2831). [00262] CCCAGAACAGTGGAGCTCTCC (SEQ ID NO:37) (2832). - 50 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00263] CACAGCCCTCGCCCAGAACA (SEQ ID NO:38) (2833). [00264] TTCTTCACAGCCCTCGCCCA (SEQ ID NO:39) (2834). [00265] TCTTTCTTCACAGCCCTCGCCCAGAACAGTGGAGCTCTCCGAAGTCCCAAT GCTAGACCGGAAAAGAGAAGCCAAAGGAGACCTGA (SEQ ID NO:40). [00266] TCTCCGAAGTCCCAATGCTAGACCGGAAAAGAGAAGCCAAAGGAGACCT GA (SEQ ID NO:41). [00267] TCTTTCTTCACAGCCCTCGCCCAGAACAGTGGAGCTCTCCGAAGTCCCAAT G (SEQ ID NO:42). [00268] TTCTTCACAGCCCTCGCCCAGAACAGTGGAGCTCTCCGAAGTCCCA (SEQ ID NO:43). [00269] In some embodiments, a polynucleotide (e.g., ASO) comprises a sequence having at least about 70% sequence identity to a sequence set forth in any one of SEQ ID NOs:34-39, for example, having at least about: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, or having about: 75-99%, 75-98%, 80-98%, 80-97%, 85-97%, 85-96%, 90-96%, or 90- 95% sequence identity to a sequence set forth in any one of SEQ ID NOs:34-39. In some embodiments, a polynucleotide comprises a sequence having at least about 85% sequence identity to a sequence set forth in any one of SEQ ID NOs:34-39. In some embodiments, a polynucleotide comprises a sequence having 100% sequence identity to a sequence set forth in any one of SEQ ID NOs:34-39. [00270] In the sequences (e.g., any one of SEQ ID NOs:25-43), each nucleobase shown as T may independently be T or U. Similarly, each C nucleotide may independently be C or a C analogue such as 5-methyl C, or other substituted C analogue. Other modified nucleobases with equivalent Watson-Crick base pairing properties will be known to one of skill in the art and would also be appropriate for use in the polynucleotides of the instant invention. [00271] In some embodiments, an agent comprises a first polynucleotide (e.g., ASO) comprising a sequence having at least 70% sequence identity to SEQ ID NO:34, and a second polynucleotide (e.g., ASO) comprising a sequence having at least 70% sequence identity to SEQ ID NO:35. - 51 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Modification of Polynucleotides [00272] Polynucleotides described herein may be modified, including with one or more locked nucleic acid (LNA) nucleotides, one or more 2’-modified ribonucleotides, one or more morpholino nucleotides, or a combination thereof. [00273] Chemical modifications can be chosen to, e.g., increase nuclease resistance of a polynucleotide (e.g., ASO), to prevent RNase H cleavage of a polynucleotide (e.g., a complementary RNA strand), or to increase cellular uptake of a polynucleotide. For each of these goals, a variety of compatible chemical modifications are available and will be familiar to those skilled in the art. [00274] In some embodiments, a polynucleotide comprises one or more modified nucleotides. In some embodiments, one or more modified nucleotides each independently comprises a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof. In some embodiments, a polynucleotide (e.g., ASO) is chemically modified such that a sugar- phosphate backbone is replaced with a phosphorodiamidate mopholino (PMO), a peptide nucleic acid or another pseudopeptide backbone, or a combination thereof. [00275] In some embodiments, a polynucleotide (e.g., ASO) is chemically modified to comprise: a) a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2’-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2’-O,4’-C-aminomethylene bridged nucleic acid (2’,4’-BNA(NC)), an alpha-L-locked nucleic acid, a tricyclo- DNA, or a combination thereof, b) a ribose group comprising 2’-O-methyl, 2’-fluoro, 2’-deoxy, 2’-O-(2- methoxyethyl) (MOE), 2’-O-alkyl, 2’-O-alkoxy, 2’-O-alkylamino, 2’-NH2, a constrained nucleotide, a tricyclo-DNA modification, or a combination thereof, c) a phosphate group comprising a phosphorothioate, a phosphoramidate, a phosphorodiamidate, a phosphorodithioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a phosphotriester, or a combination thereof, d) a nucleobase comprising 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5- - 52 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or a combination thereof, e) a sugar-phosphate backbone is replaced with a phosphorodiamidate mopholino (PMO), a peptide nucleic acid or another pseudopeptide backbone, or a combination thereof. [00276] In some embodiments, modification (e.g., each modification) of a ribose group comprises 2’-O-methyl, 2’-fluoro, 2’-deoxy, 2’-O-(2-methoxyethyl) (MOE), 2’-O-alkyl, 2’-O- alkoxy, 2’-O-alkylamino, 2’-NH2, or a constrained nucleotide, or a combination thereof. [00277] In some embodiments, a constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2’-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2’-O,4’-C-aminomethylene bridged nucleic acid (2’,4’-BNANC), an alpha- L-locked nucleic acid, and a tricyclo-DNA, or a combination thereof. [00278] In some embodiments, modification of a ribose group comprises a 2’-O-(2- methoxyethyl) (MOE) modification. In some embodiments, every nucleotide of a polynucleotide (e.g., ASO) comprises a 2’-O-(2-methoxyethyl) (MOE) modification. [00279] In some embodiments, modification of a ribose group comprises a tricyclo-DNA modification. In some embodiments, every nucleotide of a polynucleotide (e.g., ASO) comprises a tricyclo-DNA modification. [00280] In some embodiments, modification of a ribose group comprises a 2’-deoxy modification. In some embodiments, a polynucleotide comprises four or fewer consecutive 2’- deoxy modified nucleotides. [00281] In some embodiments, each modification of a phosphate group comprises a phosphorothioate, a phosphoramidate, a phosphorodiamidate, a phosphorodithioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a phosphotriester, or a combination thereof. In some embodiments, each modification of a phosphate group comprises a phosphoramidate. [00282] In some embodiments, modification of a phosphate group comprises a phosphorothioate modification. In some embodiments, every nucleotide of a polynucleotide (e.g., oligonucleotide) comprises a phosphorothioate modification. In some embodiments, a polynucleotide (e.g., ASO) is a phosphorothioate-modified polynucleotide. - 53 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00283] In some embodiments, a phosphate group of a nucleotide is modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates). In some embodiments, a polynucleotide (e.g., ASO) is a phosphorothioate- modified polynucleotide, such as a polynucleotide where each internucleotide linkage is a phosphorothioate, or where at least half of the internucleotide linkages are phosphorothioate. In some embodiments, at least half of internucleotide linkages of a polynucleotide (e.g., ASO) are phosphorothioate. In some embodiments, each internucleotide linkage of a polynucleotide (e.g., ASO) is a phosphorothioate. [00284] In some embodiments, each modification of a nucleobase comprises 2-thiouridine, 4- thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5- methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or a combination thereof. In some embodiments, modification of a nucleobase group comprises a 5-methylcytosine modification. [00285] In some embodiments, a sugar-phosphate backbone is replaced with a phosphorodiamidate morpholino (PMO) backbone. In other embodiments, a sugar-phosphate backbone is replaced with a peptide nucleic acid or other pseudopeptide backbone. [00286] In some embodiments, a polynucleotide comprises a mixture of modified nucleotides. [00287] In some embodiments, a mixture of modified nucleotides comprise two or more modifications selected from the group consisting of: 2’-O-methyl, 2’-deoxy, 2’-O-(2- methoxyethyl) (MOE), LNA, and tricyclo-DNA. [00288] In some embodiments, a mixture of modified nucleotides comprise one or more 2’-O- methyl modified nucleotides and one or more LNA modified nucleotides. [00289] In some embodiments, a mixture of modified nucleotides comprises one or more 2’- O-(2-methoxyethyl) (MOE) modified nucleotides and one or more LNA modified nucleotides. [00290] In some embodiments, each ribose group of a polynucleotide (e.g., ASO) comprises 2’-O-(2-methoxyethyl) (MOE) and/or each phosphate group of the polynucleotide comprises a phosphorothioate. In some embodiments, each ribose group of a polynucleotide comprises 2’-O- (2-methoxyethyl) (MOE). In some embodiments, each phosphate group of a polynucleotide comprises a phosphorothioate. In some embodiments, each ribose group of a polynucleotide comprises 2’-O-(2-methoxyethyl) (MOE), and each phosphate group of the polynucleotide - 54 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 comprises a phosphorothioate. [00291] In some embodiments, a nucleotide analog comprises a modification to the sugar portion of the nucleotide. For example, the 2’ OH— group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, wherein R is substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl or aryl. [00292] In some embodiments, a substituted RNA analogue disclosed herein comprises a methoxyethyl group on the 2’OH. Polypeptide Agents [00293] In some embodiments, an agent comprises a polypeptide. In some embodiments, a polypeptide is an isolated polypeptide. In some embodiments, a polypeptide is a recombinant polypeptide. [00294] In some embodiments, a polypeptide inhibits (directly or indirectly) expression of an aberrant FMR1 gene product (e.g., FMR1-217, and/or its protein product). In some embodiments, a polypeptide activates (directly or indirectly) expression of a normal FMR1 gene product (e.g., FMR1-205, and/or its protein product). In some embodiments, a polypeptide reduces level of an aberrant FMR1 gene product and increases level of a normal FMR1 gene product. [00295] In some embodiments, a polypeptide is an immunoglobulin molecule. In some embodiments, an immunoglobulin molecule an antibody. In some embodiments, an antibody is an antagonist antibody that binds an FMR1 transcript or protein product associated with FXS. An antibody can be of any species, such as a rodent (e.g., murine, rat, guinea pig) antibody, a primate (e.g., human) antibody, or a chimeric antibody. In some embodiments, an antibody is primatized (e.g., humanized). In some embodiments, an antibody is a polyclonal antibody. In some embodiments, an antibody is a monoclonal antibody. In some embodiments, an antibody (e.g., monoclonal antibody) is multispecific, e.g., bi-, tri-, or quad-specific. [00296] In some embodiments, a polypeptide is an antigen-binding fragment of an immunoglobulin molecule (e.g., an antibody), that retains the antigen binding properties of the parental full-length immunoglobulin molecule. In some embodiments, an antigen-binding fragment is a Fab, Fab’, F(ab’)2, Fd, Fv, disulfide-linked Fvs (sdFv, e.g., diabody, triabody or tetrabody), scFv, SMIP or rlgG. [00297] In some embodiments, a polypeptide is an antibody mimetic. - 55 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00298] Techniques, assays, and reagents for making and using therapeutic antibodies, or antigen-binding fragments thereof, against a target antigen (e.g., an FMR1 transcript or protein product associated with a fragile X-associated disorder, such as FXS) are known in the art. See, e.g., Therapeutic Monoclonal Antibodies: From Bench to Clinic (Zhiqiang An eds., 1st ed. 2009); Antibodies: A Laboratory Manual (Edward A. Greenfield eds., 2d ed.2013); Ferrara et al., Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker, PLoS ONE 7(11): e49535 (2012), for techniques and methods of screening, making, purifying, storing, labeling, and characterizing antibodies. Small Molecule Agents [00299] In some embodiments, an agent comprises a small molecule. In some embodiments, a small molecule binds to a protein capable of modulating the splicing and/or expression of FMR1 or a fragment thereof. In some embodiments, a small molecule is an inhibitor of the target protein (e.g., a direct inhibitor, an indirect inhibitor). In some embodiments, a small molecule is an activator of the target protein (e.g., a direct activator, and indirect activator). Non-limiting examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic, organometallic or inorganic compounds. [00300] In some embodiments, an agent comprises a phosphodiesterase-4D (PDE4D) allosteric inhibitor. In some embodiments, a PDE4D allosteric inhibitor is BPN14770. For additional information on BPN14770, see, e.g., Berry-Kravis et al., Inhibition of phosphodiesterase-4D in adults with fragile X syndrome: a randomized, placebo-controlled, phase 2 clinical trial, Nat Med.27(5):862-70 (2021), the entire teachings of which are incorporated herein by reference. Gene Editing Systems [00301] In some embodiments, an agent comprises a gene editing system. In some embodiments, a gene editing system produces deletion, substitution, and/or addition of one or more nucleotides in the FMR1 gene. In some embodiments, a gene editing system produces a partial or complete deletion in Exon 2 of FMR1-217 (e.g., pseudo-exon between base pairs 147,911,919 and 147,914,451 in the human FMR1 gene). - 56 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00302] In some embodiments, a gene editing system (e.g., a CRISPR/Cas9 gene editing system) is used to decrease FMR1 CGG expansion (e.g., shortening CGG repeats) in a subject, prior to, during, or after, administering a polynucleotide (e.g., ASO) described herein. In some embodiments, a gene editing system is used to decrease FMR1 CGG expansion in a subject prior to administering a polynucleotide. [00303] In some embodiments, a gene editing system is a CRISPR/Cas system, a transposon- based gene editing system, or a transcription activator-like effector nuclease (TALEN) system. In some embodiments, a gene editing system is a CRISPR/Cas system. In some embodiments, the gene editing system is a class II CRISPR/Cas system. [00304] In some embodiments, a gene editing system comprises a Cas endonuclease or a polynucleotide encoding a Cas endonuclease. In some embodiments, a Cas endonuclease is Cas9, Cpf1, C2C1, or C2C3. In some embodiments, a Cas endonuclease is Cas9 (e.g., of Streptococcus Pyogenes). In some embodiments, a Cas endonuclease is Cpf1. In some embodiments, Cpf1 is AsCpf1 (from Acidaminococcus sp.) or LbCpf1 (from Lachnospiraceae sp.). The choice of nuclease and gRNA(s) will typically be determined according to whether a deletion, a substitution, or an addition of nucleotide(s) to a targeted sequence is desired. [00305] In some embodiments, a type II Cas endonuclease is Cas9 (e.g., of Streptococcus pyogenes). In some embodiments, a modified Cas 9 is nickase Cas9, dead Cas9 (dCas9) or eSpCas9. In some embodiments, a nickase Cas9 is Cas9 D10A. In some embodiments, a dCas9 is D10A or H840A. [00306] In some embodiments, a gene editing system comprises a double nickase Cas9 (e.g., to achieve more accurate genome editing; see, e.g., Ran et al.., Cell 154: 1380-89 (2013). Wild- type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA. Nickase Cas9 generates only a single-strand break. dCas9 is catalytically inactive. In some embodiments, dCas9 is fused to a nuclease (e.g., a FokI to generate DSBs at target sequences homologous to two gRNAs). Various CRISPR/Cas9 plasmids are publicly available from the Addgene repository (Addgene, Cambridge, MA: addgene.org/crispr/). [00307] CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016/0138008A1 and US2015/0344912A1, and in US Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, - 57 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpf1 endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 A1. CRISPR technology for generating mtDNA dysfunction in the mitochondrial genome is disclosed in Jo et al.., BioMed Res. Int.2015: 305716 (2015). Co-delivery of Cas9 and sgRNA with nanoparticles is disclosed in Mout et al.., ACS Nano 11(3): 2452-58 (2017). Formulation and Administration [00308] In some embodiments, an agent (e.g., ASO) is in a form of a pharmaceutical composition, or a pharmaceutically acceptable salt thereof. [00309] In some embodiments disclosed herein are pharmaceutical compositions comprising ASOs of the present disclosure, and a pharmaceutically acceptable carrier. In some embodiments, pharmaceutical compositions of the present disclosure further comprise one or more additional therapeutic agents. Non-limiting examples of additional therapeutic agents include a modulator of DNA methylation (e.g., by inhibiting DNA methylation and/or promoting DNA demethylation), a metabotropic glutamate receptor 5 (mGluR5) modulator (e.g., Basimglurant or Mavoglurant), a GABAB receptor activator (e.g., arbaclofen), a GABAA or GABAB receptor activator (e.g., acamprosate), an AMPAkine (e.g.,AX516), a CB1 inhibitor (e.g., rimonabant), a RAS signaling inhibitor (e.g., lovastatin), a STEP inhibitor, an S6K inhibitor, a PAK inhibitor (e.g., FRAX486), an MMP9 inhibitor (e.g., minocycline), a GSK3β inhibitor (e.g., lithium), and combinations thereof. [00310] Pharmaceutical compositions of the present disclosure may be administered (e.g., to a subject in need thereof) in a number of ways (e.g., depending upon whether local or systemic treatment is desired and upon the area to be treated). Administration may be topical (including but not limited to ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes but are no limited to intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, - 58 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be desirable. [00311] For oral administration, an agent (e.g., a polynucleotide such as ASO) or a composition may be in the form of, for example, a tablet, capsule, suspension, or liquid. A polynucleotide is preferably made in the form of a dosage unit containing a therapeutically effective amount of an active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, tablets and capsules can contain, in addition to an active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica, or talc; disintegrants, for example potato starch; flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally in the form of aqueous or oily solutions, suspensions, emulsions, syrups, or elixirs may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents, and flavoring agents. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid. [00312] Pharmaceutical compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. Compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for non- limiting example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. In some embodiments, a pharmaceutical composition described herein is formulated as a solution. [00313] Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. Pharmaceutical compositions and formulations of the present disclosure may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients. - 59 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00314] Emulsions include but are not limited to heterogenous systems of one liquid dispersed in another in the form of droplets (e.g., droplets exceeding 0.1 μm in diameter). Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as embodiments of the present disclosure. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. [00315] Formulations of the present disclosure include but are not limited to liposomal formulations. As used in the present disclosure, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells. [00316] Pharmaceutical formulations and compositions of the present disclosure may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. [00317] In some embodiments, various penetration enhancers are employed to effect the efficient delivery of an agent (e.g., ASO) or a composition of the present disclosure. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. [00318] In some embodiments, an agent (e.g., ASO) is administered at a dosage of about 4-20 mg per administration, for example, about: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg per administration. In some embodiments, an agent is administered at a dosage of about - 60 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 12 mg per administration. In some embodiments, an agent is administered at a dosage of about, e.g., up to 50 or 100 mg per injection. [00319] Administration of an agent or a composition of the present disclosure can be once in a day or more than once in a day (e.g., twice a day or more). Administration of the agent or a composition can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose. [00320] In some embodiments, a method disclosed herein comprises administering to a subject two or more polynucleotides (e.g., ASOs), for example, 2, 3, 4, or 5 polynucleotides. In some embodiments, two or more polynucleotides are administered together. In other embodiments, two or more polynucleotides are administered separately. [00321] Administration of an agent or a composition of the present disclosure to a subject can be by parenteral or non-parenteral means. In some embodiments, an agent (e.g., ASO) is administered intravenously, intra-arterially, intrathecally, intraventricularly, intramuscularly, intradermally, subcutaneously, intracranially, or spinally. [00322] In some embodiments, an agent (e.g., ASO) or a composition of the present disclosure is delivered locally to the central nervous system. This can include intrathecal or intraventricular injections, including the use of a catheter or Ommaya reservoir. Other methods of delivering agents (e.g., drugs) directly to the cerebrospinal fluid or central nervous system will be known to one skilled in the art. [00323] In some embodiments, an agent or a composition of the present disclosure is delivered using an approach that enhances bioavailability in the central nervous system after systemic administration. These approaches can include modification of the sugars or phosphate linkages, delivering as a duplex with a ligand-conjugated RNA molecule, formulation into an artificial exosome, liposome, polymer nanoparticle or lipid nanoparticle, or conjugation to lipids, antibodies, peptides, sugars, neuroactive molecules, or other moieties that enhance delivery to the central nervous system. In some embodiments, the agent is delivered after transiently - 61 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 disrupting the blood-brain barrier. Other methods of enhancing bioavailability in the central nervous system after systemic administration will be known to one skilled in the art. [00324] In some embodiments, an agent (e.g., ASO) or a composition of the present disclosure is administered as intrathecal bolus injection. In some embodiments, an agent or a composition is delivered systemically, such as via intravenous or subcutaneous injection. [00325] In some embodiments, an agent (e.g., ASO) or a composition of the present disclosure is delivered using one or more controlled or sustained-release delivery systems (e.g., capsules, biodegradable matrices). Non-limiting example delayed-release delivery systems for drug delivery that would be suitable for administration of a composition described herein are described in U.S. Patent Nos. US 5,990,092 (issued to Walsh); 5,039,660 (issued to Leonard); 4,452,775 (issued to Kent); and 3,854,480 (issued to Zaffaroni), the entire teachings of which are incorporated herein by reference. Therapeutically Effective Amounts [00326] In some embodiments, a therapeutically effective amount of an agent is sufficient to: a) decrease splicing of an aberrant FMR1 transcript (e.g., between Exons 1 and 2 of FMR1-217), b) decrease level of an aberrant FMR1 gene product (e.g., an aberrant FMR1 transcript such as FMR1-217, and/or an aberrant FMR1-encoded protein), c) increase splicing of a normal FMR1 transcript (e.g., between Exons 1 and 2 of FMR1-205), d) increase level of a normal FMR1 gene product (e.g., a normal FMR1 transcript such as FMR1-205 and/or a normal FMR1-encoded protein), or e) increase level of FMRP, or any combination thereof, relative to a reference. [00327] In some embodiments, a therapeutically effective amount of an agent is sufficient to decrease splicing of an aberrant FMR1 transcript, relative to a reference, for example, by at least about 5% (e.g., by at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%). [00328] In some embodiments, a therapeutically effective amount of an agent is sufficient to decrease level of an aberrant FMR1 gene product, relative to a reference, for example, by at least - 62 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 about 5% (e.g., by at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%). In some embodiments, a therapeutically effective amount of an agent is sufficient to decrease level of an aberrant FMR1 gene product, relative to a reference, by at least about 25%. [00329] In some embodiments, a therapeutically effective amount of an agent is sufficient to increase splicing of a normal FMR1 transcript, relative to a reference, for example, by at least about 5% (e.g., by at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, 105%, 110%, 120%, or 125%). [00330] In some embodiments, a therapeutically effective amount of an agent is sufficient to increase level of a normal FMR1 gene product, relative to a reference, for example, by at least about 5% (e.g., by at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, 105%, 110%, 120%, or 125%). [00331] In some embodiments, a therapeutically effective amount of an agent is sufficient to increase level of FMRP, relative to a reference, for example, by at least about 5% (e.g., by at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, 105%, 110%, 120%, or 125%). In some embodiments, a therapeutically effective amount of an agent is sufficient to increase level of FMRP, relative to a reference, by at least about 25%. [00332] In some embodiments, a therapeutically effective amount of an agent is sufficient to increase a level of FMRP in a FXS subject to at least about 5% of a level observed in typically developing individuals, e.g., to at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, of a level observed in typically developing individuals. In some embodiments, a therapeutically effective amount of an agent is sufficient to increase level of FMRP in a FXS subject to at least about 30% of a level observed in typically developing individuals. [00333] In some embodiments, splicing of an aberrant FMR1 transcript, level of an aberrant FMR1 gene product, splicing of a normal FMR1 transcript, level of a normal FMR1 gene product, level of FMRP, or any combination thereof, is measured at least about 1 day after an agent is administered to a subject, e.g., for at least: about: 2 days, 3 days, 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 - 63 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 months, or 6 months, after administration. [00334] In some embodiments, splicing of an aberrant FMR1 transcript, level of an aberrant FMR1 gene product, splicing of a normal FMR1 transcript, level of a normal FMR1 gene product, level of FMRP, or any combination thereof, is measured a tissue or a cell, for example, a white blood cell, a leukocyte, a fibroblast cell (e.g., a dermal derived fibroblast cell or a lung- derived fibroblast cell), a cortex tissue (e.g., a brain biopsy of superficial cortex), or a combination thereof. [00335] In some embodiments, a therapeutically effective amount of an agent is sufficient to slow progression of FXS, and/or alleviate (e.g., prevent) one or more signs or symptoms of FXS. [00336] In some embodiments, a therapeutically effective amount of an agent is sufficient to: a) improve speech and motor development, b) reduce (e.g., prevent) one or more cognitive disabilities (e.g., learning and/or intellectual disability), c) alleviate (e.g., prevent) one or more physical and medical features (e.g., a long face, prominent ears and chin, arched palate, large testicles at puberty, low muscle tone, flat feet, hyperextensible joints, sleep problems, seizures, recurrent ear infections, and/or mitral valve prolapse), or d) reduce (e.g., prevent) one or more FXS-associated behaviors (e.g., hyperactivity, short attention span, hand biting, hand flapping, poor eye contact, poor social skills, shyness, anxiety, delayed speech, delayed motor development, repetitive speech, sensitivity to sensory stimulation (e.g., touch)), or a combination thereof. [00337] In some embodiments, a therapeutically effective amount of an agent is sufficient to raise an intelligence quotient (IQ) score, for example, to at least about 40, e.g., to at least about: 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130, to between about: 40-110, 40-100, 50-105, 60-80, 65-90, 70-80, 75-95, or 70-100, or to about: 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130. [00338] In some embodiments, a therapeutically effective amount of an agent is sufficient to raise an IQ score by at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or - 64 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 20 points, by 1-10, 1-15, 2-20, 2-15, 2-10, 5-15, 5-10, 10-20, or 15-20 points, or by about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 points. [00339] In some embodiments, a therapeutically effective amount of an agent is sufficient to reduce (e.g., prevent) absent or irregular menses, fertility problems, elevated FSH (follicle- stimulating hormone) levels, premature ovarian failure, primary ovarian insufficiency, and/or hot flashes. In some embodiments, a therapeutically effective amount of an agent is sufficient to reduce (e.g., prevent) intention tremors, parkinsonism, ataxia, memory loss, white matter lesions involving middle cerebellar peduncles, and/or cognitive decline. In some embodiments, a therapeutically effective amount of an agent is sufficient to reduce (e.g., prevent) neuropathy of extremities, mood instability, irritability, explosive outbursts, personality changes, and/or autonomic function problems such as impotence or loss of bladder or bowel functions. In some embodiments, a therapeutically effective amount of an agent is sufficient to reduce (e.g., prevent) high blood pressure, thyroid disorders, and/or fibromyalgia. Additional Therapeutic Agents [00340] In some embodiments, one or more additional therapeutic agents are administered to a subject. Non-limiting examples of additional therapeutic agents include a modulator of DNA methylation (e.g., by inhibiting DNA methylation and/or promoting DNA demethylation), a metabotropic glutamate receptor 5 (mGluR5) modulator (e.g., Basimglurant or Mavoglurant), a GABAB receptor activator (e.g., arbaclofen), a GABAA or GABAB receptor activator (e.g., acamprosate), an AMPAkine (e.g.,AX516), a CB1 inhibitor (e.g., rimonabant), a RAS signaling inhibitor (e.g., lovastatin), a STEP inhibitor, an S6K inhibitor, a PAK inhibitor (e.g., FRAX486), an MMP9 inhibitor (e.g., minocycline), a GSK3β inhibitor (e.g., lithium), and combinations thereof. [00341] In some embodiments, treating a subject comprises providing the subject with a ketogenic (“keto”) diet. [00342] In some embodiments, a therapeutically effective amount of a DNA-demethylating compound or DNA demethylase is administered to a subject, prior to, during, or after, administering an agent (e.g., ASO) or a composition described herein to the subject. In some embodiments, a therapeutically effective amount of a DNA-demethylating compound or DNA - 65 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 demethylase is administered to a subject after administering an agent or a composition disclosed herein to the subject. [00343] Non-limiting examples of DNA-demethylating compounds include 5-Azacytidine (5- Aza-CR) and 5-aza-2′-deoxycytidine (5-Aza-CdR), dihydro-5-azacytidine (DHAC), zebularine, 5-fluoro-2'-deoxycytidine, Hydralazine, RG108, procainamide, and SGI-1027. In some embodiments, a DNA-demethylating compound is a nucleoside analogue. In some embodiments, a DNA-demethylating compound is a non-nucleoside analogue. [00344] In some embodiments, a DNA demethylase (e.g., DNA methylation modification enzymes Dnmt or Tet) is fused to a catalytically inactivate Cas9 (dCas9-Dnmt/Tet). Under the guidance of a single guide RNA (sgRNA), a dCas9-Tet1 demethylates the FMR1 locus and promoter region when FMR1 has an expanded CGG repeat of 200 or more. [00345] In some embodiments, a therapeutically effective amount of a DNA-demethylating compound or DNA demethylase is sufficient to demethylate at least about 5% of an FMR1 gene, for example, at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, about: 10-100%, 10-90%, 15-90%, 15-80%, 15-75%, 20-75%, 20-70%, 25-60%, 25-55%, 25-50%, 30-40%, or 30-35%, or about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an FMR1 gene. In some embodiments, a therapeutically effective amount of a DNA-demethylating compound or DNA demethylase is sufficient to demethylate about 25-50% of an FMR1 gene. [00346] The term “combination therapy” refers to the administration of two or more therapeutic agents (e.g., one or more agents and additional therapeutic agents described herein) to treat a disease, disorder, or condition described herein. Such administration encompasses co- administration of two or more therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses co-administration in multiple, or in separate containers (e.g., capsules, powders, and liquids) for each active ingredient. Such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. Therapeutic agents in a combination therapy can be administered via the same administration route or via different administration routes. Powders and/or liquids may be - 66 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 reconstituted or diluted to a desired concentration (e.g., appropriate for dosage) prior to administration. Typically, a treatment regimen will provide beneficial effects of a drug combination in treating diseases, conditions, or disorders described herein. [00347] In some embodiments, a method is used for monitoring (e.g., in a clinical trial and/or in a drug screen) effectiveness of an agent. In some embodiments, a method comprises obtaining level of a metabolite from a subject before administering an agent, and monitoring changes in the level as an indication of the effectiveness of the agent in the subject. In some embodiments, a level of a metabolite of a subject is compared to a predetermined reference (e.g., standard). [00348] In some embodiments, a method comprises altering (e.g., increasing or decreasing) the administration of an agent to a subject. In some embodiments, increased administration of an agent is desirable to increase effectiveness of an agent. In some embodiments, decreased administration of an agent is desirable to decrease effect(s) of an agent. Methods of Diagnosing, Subclassifying or Predicting a Treatment Outcome of Fragile X- Associated Disorders [00349] The disclosure provides, among other things, methods of predicting a treatment outcome of, diagnosing, prognosing, or subclassifying fragile X syndrome (FXS) in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing. [00350] The disclosure also provides, among other things, methods of diagnosing FXS in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, - 67 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing. [00351] The disclosure also provides, among other things, methods of prognosing FXS in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing. [00352] The disclosure also provides, among other things, methods of subclassifying FXS in a subject, the methods comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing. [00353] The disclosure also provides, among other things, methods of diagnosing, subclassifying or predicting a treatment outcome of FXS in a subject, the methods comprising: a) determining a presence, absence, amount, or alteration of at least one FXS- associated metabolite, or b) determining an alteration of an FXS-associated ratio of two metabolites, or both a) and b), in a sample from a subject, wherein the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS- associated ratio, in the sample is indicative of the presence, risk, progression, severity, or treatment outcome of FXS. [00354] The disclosure also provides, among other things, methods of predicting a treatment outcome of FXS in a subject, the methods comprising: - 68 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 a) determining: i. a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii. an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject, and b) predicting the treatment outcome of FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the sample. [00355] The disclosure also provides, among other things, methods of diagnosing FXS in a subject, the method comprising: a) determining: i. a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii. an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject, and b) diagnosing the subject as having or having a propensity to develop FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the sample. [00356] The disclosure also provides, among other things, methods of prognosing FXS in a subject, the methods comprising: a) determining: i. a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii. an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject, and b) prognosing the subject as having a propensity to have a poorer prognosis of FXS based on the presence, absence, amount, or alteration of the at least one FXS- associated metabolite, or the alteration of the FXS-associated ratio, in the sample. [00357] For example, if the ratio between levels of two FXS-associated metabolites in a sample from the subject is determined to be 1:1 and the reference ratio of the two FXS- - 69 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 associated metabolites is 1:5, the subject may be prognosed as having a propensity to have a poorer prognosis of FXS, compared to another subject with a ratio of 1:4. [00358] A subject is any one or more subjects described herein. [00359] At least one FXS-associated metabolite is any one or more FXS-associated metabolites disclosed herein. [00360] In some embodiments, a sample is any one or more samples described herein. [00361] In some embodiments, an FXS-associated ratio is any one or more FXS-associated ratios disclosed herein. [00362] In some embodiments, a treatment outcome is any one or more treatment outcomes described herein. [00363] In some embodiments, an aberrant metabolite level (e.g., an increased level or a reduced level of a metabolite, a presence of a metabolite not usually present, or an absence of a metabolite usually present) indicates a presence of a disease state. [00364] In some embodiments, a method disclosed herein is used to identify a subject having or at risk for developing FXS. [00365] In some embodiments, a method is used to determine whether to administer an agent to a subject. [00366] In some embodiments, the subject is treated when the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS- associated ratio, in the subject’s sample, is determined or detected. In some embodiments, the subject’s treatment is altered when the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the subject’s sample, is determined or detected. [00367] In some embodiments, a method is used to determine whether a subject is effectively treated with an agent. In some embodiments, a method provides information useful in prognostication, staging and/or management of FXS. In some embodiments, a method provides information useful for designing a treatment regimen. [00368] In some embodiments, a method is used for monitoring (e.g., in a clinical trial and/or in a drug screen) effectiveness of an agent. In some embodiments, a method comprises obtaining level of a metabolite from a subject before administering an agent, and monitoring changes in the - 70 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 level as an indication of the effectiveness of the agent in the subject. In some embodiments, a level of a metabolite of a subject is compared to a predetermined reference (e.g., standard). [00369] In some embodiments, the subject is treated when the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS- associated ratio, in the subject’s sample, is determined or detected, for example, in a method disclosed herein. In some embodiments, the subject’s treatment is altered when the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the subject’s sample, is determined or detected for example, in a method disclosed herein. [00370] While having described the invention in general terms, the embodiments of the invention will be further disclosed in the following example that should not be construed as limiting the scope of the claims. Exemplification Example 1. Metabolite Levels Associated with Fragile X Syndrome (FXS) [00371] FXS is the most frequently inherited form of intellectual impairment and most common single-gene cause of autism. There are about 83,000 FXS cases in the United States; 1 in approximately 4,000 boys and 1 in approximately 7,000 girls are afflicted. FXS children present a range of symptoms beyond intellectual impairment, including speech and developmental delays, perseveration, hyperactivity, aggression, and epilepsy, among other maladies. FXS is caused by a CGG triplet repeat expansion in a single gene, fragile X messenger ribonucleoprotein 1 (FMR1), which resides on the X chromosome. When the CGG triplet repeat expands to 200 or more, the FMR1 gene is methylated and thereby transcriptionally inactivated. The loss of the FMR1 gene product, the protein fragile X messenger ribonucleoprotein protein (FMRP), is the cause of the disorder. [00372] Treatments for FXS or indeed most other autism spectrum disorders, which are primarily based on animal models, have met with very limited success in human clinical trials (Hagerman et al., Nature Rev Disease Primers 3:17065 (2017); Berry-Kravis et al., Nature Rev Drug Disc.17:280-299 (2018)). [00373] There is no widely available therapy that shows even modest efficacy for FXS. Moreover, there are no established molecular readouts with statistical rigor of any FXS - 71 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 treatment. Because any FXS treatment would be targeted, directly or indirectly, to the brain, a therapeutic biomarker may entail measurement of brain tissue, or a brain-derived fluid – particularly cerebral spinal fluid (CSF), which is derived from the brain ventricles. [00374] In all FXS model systems so far examined in the field (Drosophila, mouse, iPSC- derived neurons), a common theme is dysregulated mitochondrial function. These observations suggest that mitochondrial or indeed other metabolites, molecules weighing <1000 Daltons, might have altered levels in FXS brain/tissue/CSF/cells compared to control (i.e., non-FXS). Metabolites may be determined with extreme accuracy and with strong statistical rigor using liquid chromatography-mass spectrometry (LC-MS). [00375] Human post-mortem brain cortex samples (University of California at Davis Brain Repository), six FXS and six age-matched typically-developing (TD) controls, were frozen at -80°C and powdered with a mortar and pestle. The metabolites were extracted from ~10 mg of powder material. The samples were vortexed in 800 µL 60% LC-MS-grade methanol in LC-MS- grade water for 10 minutes at 4°C. Five hundred µL of LC-MS-grade chloroform was added to each sample tube and vortexed for 10 minutes at 4°C. The samples were centrifuged at 22,000 x g for 10 minutes at 4°C, creating three layers. The top layer contained polar metabolites, the middle layer was the protein interface, and the bottom layer contained nonpolar metabolites. The polar metabolites were transferred into a new tube and the fluid evaporated in a refrigerated CENTRIVAP® attached to a -105°C cold trap (Labconco Corporation, Kansas City, MO). The bottom layer was discarded, and the protein interface was used to quantify protein. Briefly, 1 mL of RIPA buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 150mM NaCl) was added to the tube containing the protein interface and samples were vortexed 10 minutes at 4°C. The samples were centrifuged at 22,000 x g for 10 minutes at 4°C and protein was quantified with a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA). Dried down polar metabolite pellets were resuspended in LC-MS-grade water to a concentration of 0.5 µg/µL and the material was vortexed for 10 minutes at 4°C. The samples were centrifuged at 22,000 x g for 10 minutes at 4°C and 20 µL was moved into LC-MS vials. - 72 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00376] Ten mL fresh blood was collected from participants to isolate peripheral blood mononuclear cells (PBMCs). All participants were Caucasian males with a FMR1 full mutation (CGG repeats >200) or typically-developing (TD) individuals (CGG repeats < 55) as confirmed by DNA analysis. All participants or their legal guardians, as appropriate, signed informed consent to the study. The project was approved by the Rush University Medical Center Institutional Review Board. FXS patients were aged 16–38 years. Age-matched TD individuals for the study were aged 22-29 years. PBMCs were isolated from whole blood using a Cell Preparation (BD VACUTAINER® CPT™) blood tube (BD Biosciences, Franklin Lakes, NJ). PBMCs were pelleted and washed twice with 1X PBS. The PBMCs were cultured in RPMI-1640 medium (Sigma-Aldrich, Burlington, MA), supplemented with 15% fetal bovine serum (FBS) and 2.5% L-glutamine, at 37℃ with 5% CO2 in T25 flasks for 4 days. PBMCs were collected by centrifugation and pellets were frozen at -80℃. For LC-MS, the cells were pelleted and washed twice with 1X PBS. Samples were resuspended in 800 µL 80% LC-MS-grade methanol in LC- MS-grade water and vortexed for 10 minutes at 4°C. Samples were centrifuged at 22,000 x g for 10 minutes at 4°C and supernatants were dried down in a refrigerated CENTRIVAP® attached to a -105°C cold trap (Labconco Corporation). Dried polar metabolite pellets were resuspended in 100 µL of LC-MS-grade water and vortexed for 10 minutes at 4°C. Samples were centrifuged at 22,000 x g for 10 minutes at 4°C and 20 µL was moved into LC-MS vials. [00377] Lymphoblast cell lines (LCLs) were obtained from Coriell Institute (Camden, NJ) from two FXS individuals (GM07365 and GM06897) and two typically-developing control males (GM07174 and GM06890). Cells were cultured in RPMI-1640 medium (Sigma-Aldrich), supplemented with 15% fetal bovine serum (FBS) and 2.5% L-glutamine, at 37℃ with 5% CO2 in T25 flasks. LCLs were collected by centrifugation and pellets were frozen at -80℃. Samples were resuspended in 800 µL 80% LC-MS-grade methanol in LC-MS-grade water and vortexed for 10 minutes at 4°C. Samples were centrifuged at 22,000 x g for 10 minutes at 4°C and supernatants were dried down in a refrigerated CENTRIVAP® attached to a -105°C cold trap (Labconco Corporation). Dried polar metabolite pellets were resuspended in 100 µL of LC-MS- grade water and vortexed for 10 minutes at 4°C. Samples were centrifuged at 22,000 x g for 10 minutes at 4°C and 20 µL was moved into LC-MS vials. - 73 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00378] Cerebrospinal fluid was collected from non-Fragile X individuals at Rush University Medical Center (Chicago, IL). It was shipped to UMass Chan Medical School and used for LC- MS analysis. [00379] A Q EXACTIVE® Plus Hybrid Quadrupole-ORBITRAP® mass spectrometer (Thermo Fisher Scientific) equipped with an Ion Max source and a HESI II probe coupled to a VANQUISH® Horizon UHPLC System (Thermo Fisher Scientific) was used to measure polar metabolites on post-mortem brain samples. Prior to operation, the instrument underwent mass calibration for positive and negative ion mode using Calmix (Thermo Fisher Scientific). Two µL of resuspended polar metabolite samples were injected onto a SeQuant ZIC®-pHILIC 5μm 150 x 2.1 mm analytical column equipped with a 2.1 x 20 mm guard column (MilliporeSigma, Burlington, MA). The column oven was held at 25°C and the autosampler tray was held at 4°C. Buffer A was comprised of 20 mM ammonium carbonate, 0.1% ammonium hydroxide. Buffer B was comprised of 100% acetonitrile. The chromatographic gradient was run at a flow rate of 0.150 mL/min as follows: 0-20 min: linear gradient from 80-20% buffer B; 20-20.5 min: linear gradient from 20-80% buffer B; 20.5-28 min: hold at 80% buffer B. The mass spectrometer was operated in full-scan, polarity switching mode, with the spray voltage set to 4.0 kV and the heated capillary at 350°C. The sheath gas flow was 10 units, the auxiliary gas flow was 1 unit, and the sweep gas flow was 1 unit. Mass spectrometry data was collected in a range of m/z = 70- 1000. The resolution was set at 70,000, the automatic gain control (AGC) target at 1x106, and the maximum injection time at 20 msec. [00380] Changes in polar metabolite levels between post-mortem cortex tissue samples from six FXS males and six age-matched typically-developing (TD) males were detected by liquid chromatography-mass spectrometry (LC-MS) (see FIGs.1A-1B and Table 1). A significant increase in levels of 4 metabolites in the FXS samples compared to TD samples was detected. The ratio of glutathione (GSH) to glutathione disulfide (GSSG) levels compared between the FXS and TD samples used in FIG.1A reflected increased oxidative stress in the FXS samples. Table 1. Changes in Polar Metabolite Levels in Post-Mortem FXS Cortex Tissue Polar metabolite log2(fold change) -log10(p-value)
Figure imgf000075_0001
- 74 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) D-ALANINE 0.003405714 0.024242254
Figure imgf000076_0001
- 75 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) S-ADENOSYLMETHIONINE 0.600873559 0.574377604
Figure imgf000077_0001
- 76 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) PHOSPHOENOLPYRUVATE 0.769126219 1.99061885
Figure imgf000078_0001
g p - p p mononuclear cell (PBMC) samples. Changes in polar metabolite levels between PBMC samples from six FXS males and five age-matched TD males were captured using LC-MS. A significant decrease in levels of 24 metabolites in the FXS samples compared to TD samples was detected (see FIGs.2A-2B and Table 2). The ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) levels compared between the FXS and TD samples reflected altered mitochondrial metabolism in the FXS samples. Table 2. Metabolic Changes in FXS Patient-Derived PBMC Samples Polar metabolite log2(fold change) -log10(p-value)
Figure imgf000078_0002
- 77 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) 3-METHYLGLUTACONATE -0.2408179 0.12009894
Figure imgf000079_0001
- 78 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) TAURINE -0.7380303 0.45335216
Figure imgf000080_0001
- 79 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) URIDINE MONOPHOSPHATE -0.5254017 0.67543034
Figure imgf000081_0001
- 80 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) ITACONATE -0.7745421 0.92155176
Figure imgf000082_0001
- 81 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) GLUCURONATE -1.9551136 1.31391138 [0
Figure imgf000083_0001
g g p p ortex (brain) samples affected some of the same metabolites. Four polar metabolites exhibited significantly changed levels in both PBMC and cortex samples from FXS males compared to TD males (see FIGs.3A-3C). [00383] Metabolic changes were observed in FXS patient-derived lymphoblastoid cell lines (LCLs). Changes in polar metabolite levels in two human male FXS LCLs (GM05131 and GM07072) compared to two TD LCLs (GM07174 and GM06890) were captured using LC-MS. A significant decrease in levels of 21 metabolites in the FXS samples compared to TD samples was detected (see FIG.4 and Table 3). [00384] Finally, metabolites in cerebrospinal fluid (CSF) detected by LC/MS from individuals who have no known brain or CSF disorder are listed in Table 4. - 82 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Table 3. Metabolic Changes in FXS Patient-Derived Lymphoblastoid Cell Lines (LCLs) Polar metabolite log2(fold change) -log10(p-value) 1-AMINOCYCLOPROPANECARBOXYLATE -0.5605937 0.32782845
Figure imgf000084_0001
- 83 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) CIS-4-HYDROXY-D-PROLINE -0.9104972 1.68225823
Figure imgf000085_0001
- 84 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) GLUTATHIONE 7.84116294 0.37547061
Figure imgf000086_0001
- 85 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) METHYLMALONATE -1.0445177 1.54990777
Figure imgf000087_0001
- 86 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Polar metabolite log2(fold change) -log10(p-value) SUCCINATE -1.0445177 1.54990777
Figure imgf000088_0001
Table 4. CSF Metabolites CSF (Relative concentration)
Figure imgf000088_0002
- 87 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 CSF (Relative concentration) 3-METHYL-L-HISTIDINE 4625664.062
Figure imgf000089_0001
- 88 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 CSF (Relative concentration) L-ALANINE 3557331.371
Figure imgf000090_0001
- 89 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 CSF (Relative concentration) SORBITOL 1566487.649
Figure imgf000091_0001
[00385] Example 2 [00386] Fragile X Syndrome (FXS) is a neurodevelopmental disorder characterized by a triplet repeat expansion mutation (>200 CGG repeats) in the FMR1 gene, leading to the loss of FMRP protein expression. This absence results in dysregulated protein synthesis, mitochondrial deficits, gene expression abnormalities, and altered neuronal function, culminating in symptoms like anxiety, seizures, and intellectual disability. (J. D. Richter, X. Zhao, The molecular biology of FMRP: new insights into fragile X syndrome Nat. Rev. Neurosci. (2021), doi:10.1038/s41583-021-00432-0). Most individuals with FXS also exhibit features consistent with autism spectrum disorder (ASD), highlighting shared molecular deficits between the two conditions. (S. Shah, J. D. Richter, Do Fragile X Syndrome and Other Intellectual Disorders Converge at Aberrant Pre-mRNA Splicing? Front. Psychiatry 12, 715346 (2021)). - 90 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00387] In FXS patients, repeat expansion in the FMR1 locus triggers DNA methylation, leading to gene silencing and a subsequent deficiency in FMR1 RNA production. However, recent research challenges this conventional view by revealing that approximately 70% of FXS individuals express FMR1 RNA, albeit in a mis-spliced form (FMR1-217), rendering it non- functional (S. Shah et al.., Antisense oligonucleotide rescue of CGG expansion–dependent FMR1 mis-splicing in fragile X syndrome restores FMRP. Proc. Natl. Acad. Sci.120, e2302534120 (2023)). (FIG.5A). To address FMR1 splicing mis-regulation, splice-switching antisense oligonucleotides (ASOs) were selected over RNAseH recruiting ASOs, which degrade transcripts. Treatment with ASOs in FXS cells led to decreased FMR1-217 levels (FIG.5B), increased full-length FMR1 RNA (FIG.5C), and most importantly restored FMRP protein (FIG. 5D). Thus, ASO treatments in FXS cells can be used to restore endogenous FMRP expression. [00388] Previous studies have investigated mitochondrial dysfunction and energy deficits in FXS (B. Liu et al., Regulatory discrimination of mRNAs by FMRP controls mouse adult neural stem cell differentiation. Proc. Natl. Acad. Sci.115, 201809588 (2018); P. Licznerski et al.., ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome. Cell 182, 1170-1185.e9 (2020); J. Geng et al., Deregulation of ER-mitochondria contact formation and mitochondrial calcium homeostasis mediated by VDAC in fragile X syndrome. Dev. Cell 58, 597-615.e10 (2023); M. Shen et al.., Species-specific FMRP regulation of RACK1 is critical for prenatal cortical development. Neuron 111, 3988-4005.e11 (2023); M. Shen et al.., Reduced mitochondrial fusion and Huntingtin levels contribute to impaired dendritic maturation and behavioral deficits in Fmr1-mutant mice. Nat. Neurosci.22, 386–400 (2019)), suggesting their potential impact on brain metabolomics). [00389] Liquid chromatography/mass spectrometry (LC/MS) for polar metabolite analysis was used in post-mortem brain tissues (cortex and cerebellum) from FXS and TD individuals (6 male samples per genotype, age-matched). Reductions in various metabolites were observed, particularly amino acids and their derivatives as highlighted in FIGs.6A-D. Specifically, 14 of the 20 free amino acids were reduced in the FXS cerebellum tissues, 13 of 20 in the human cortex samples. These reductions encompassed both essential and non-essential amino acids, with notable examples such as decreases in the branched chain amino acids (BCAA) leucine and lysine. Moreover, by utilizing white blood cell (WBC)-derived cell lines from FXS patients - 91 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 exhibiting different FMRP expression levels, partial FMRP expression might dampen metabolomic disruptions in FXS cells compared to complete loss of FMRP (FIG.7). [00390] This initial evidence implies that restoring FMRP in FXS cells may alleviate metabolomic deficits in FXS. Amino acid homeostasis is tightly regulated and reduction in amino acids levels can have multiple consequences such as compromised protein synthesis, disrupted neurotransmitter balance and alterations of numerous signaling pathways and energy production. Neurological symptoms manifest in numerous hereditary disorders associated with amino acid metabolism, emphasizing the need for maintaining amino acid homeostasis in the brain (P. Licznerski, et al.., ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome. Cell 182, 1170-1185.e9 (2020)). Recent studies revealed that mutations of amino acid transporters result in neuronal death and microencephaly in patients with autism. (D. C. Tărlungeanu et al.., Impaired Amino Acid Transport at the Blood Brain Barrier Is a Cause of Autism Spectrum Disorder. Cell 167, 1481-1494.e18 (2016); L. S. Knaus et al.., Large neutral amino acid levels tune perinatal neuronal excitability and survival. Cell 186, 1950-1967.e25 (2023)). In a developing brain, amino acids are the main source of energy for neurons, prior to the switch to glucose in a mature brain and, thus, can underlie neurodevelopmental intellectual disability. Thus, alterations in metabolic profiles, specifically amino acid levels, could be an underlying feature of intellectual disability in several neurodevelopmental, neurodegenerative, and metabolic disorders. Thus, the amino acid imbalance observed in the FXS brain may indeed play a significant role in its pathophysiology. The study of how this imbalance occurs and whether it can be reversed by altering FMRP levels is described herein. [00391] Reduced levels of free amino acids may result from the loss of FMRP expression in FXS. Factors contributing to amino acid imbalances in FXS cells will be explored. Subsequently, whether the restoration of FMRP expression can alleviate amino acid dysregulation in FXS cells will be assessed. To achieve these objectives, established FXS cell lines and ASO-mediated rescue strategies will be utilized, as well as LC-MS. [00392] One aspect of this study investigates Fragile X syndrome as a metabolic disorder using an ASO-based FMRP rescue paradigm (S. Shah et al.., Antisense oligonucleotide rescue of CGG expansion–dependent FMR1 mis-splicing in fragile X syndrome restores FMRP. Proc. Natl. Acad. Sci.120, e2302534120 (2023)) to restore metabolic imbalances in FXS. - 92 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Furthermore, analysis of metabolites from bodily fluids can provide an objective measure of therapeutic efficacy in FXS patients. [00393] Approach [00394] Preliminary studies: [00395] LC-MS (polar analysis, <1000kDa) was conducted on post-mortem human brain tissue (cerebellum and cortex) (N=6 males), comparing FXS to age-matched typically developing (TD) individuals. In FIGs.6A-B, a volcano plots display metabolites that exhibit statistical significance (-log(p>1.3)) in A. cerebellum tissue and B. cortex. FIG.6C. depicts a bar graph displaying changes in leucine and lysine. Free amino acids are denoted in green, while their derivatives are highlighted in red, constituting most observed changes. The most significant reductions in brain tissues were observed in free amino acids, with 14 out of 20 reduced in the cerebellum and 13 out of 20 in the cortex. Essential neuroactive amino acids crucial for neurotransmission and neuronal function, including glycine and serine, were reduced in both tissues, while aspartate, taurine, and Gamma-aminobutyric acid (GABA) were depleted specifically in the FXS cerebellum. [00396] Whether FMRP levels might influence the metabolomic disruptions in FXS was studied. The metabolome in white blood cells (WBC) from FXS patients was analyzed, specifically Lymphoblastoid cell lines (LCLs), and compared to a WBC line from a typically developing (TD) individual. These LCLs were cultured in a complete, nutrient-rich medium and then subjected to LC-MS analysis. Two FXS patient-derived cell lines with varying FMRP expression levels were utilized: the FXS2 line displayed 50% FMRP compared to TD cells, whereas the FXS1 line exhibited undetectable FMRP levels via western blot analysis (FIG.7A). The PCA plot showed distinct clustering based on genotype, with FXS1 further apart from TD compared to FXS2, suggesting that partial FMRP expression in FXS2 cells influences the metabolite profile towards TD (FIG.7B). The findings revealed a depletion of the free amino acid pool in both FXS LCL cultures (FIG.7C, D), mirroring the observations in FXS brain tissues (FIG.6). Indeed, upon comparing the FXS2 vs. FXS1 metabolite profiles, a few amino acids exhibit higher expression levels, which may correlate with the increased FMRP levels in FXS2 line (FIG.7E). Examples of amino acids changes are depicted in FIG.7F. These findings suggest that restoring FMRP levels could potentially ameliorate metabolic deficits in FXS. - 93 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00397] Research Strategy: [00398] Methods: [00399] a) Cell culture- LCL lines will be grown in RPMI media with 15% FBS as per instructions from Coriell Institute, NJ for all experiments pertaining to 2.1 and 2.2. To assess the metabolome in FXS neurons and perform the ASO rescue paradigm, iPSC derived forebrain neurons via reprogramming of the LCL lines (FIG.8) have been generated. The forebrain neurons will be cultured in maturation media (STEMCELL Technologies) for a period of 9 days. ASOs resuspended in water will be applied to cultures at 5uM concentration for a minimum of 7 days before testing. [00400] b) Drug treatments- FXS and TD LCLs will be treated with inhibitors such as Rapamycin, MG-132 or Bortezomib, 3-methyladenine (3-MA) and dexpramipexole (DEX) or vehicle control. LC-MS will be performed on the samples. [00401] c) Protein assays- Western blots will be performed using antibodies against FMRP, GAPDH, phospho-mTOR, mTOR, anti–LC3-I/II, ubiquitin, Hexokinase I. [00402] d) qPCR- To measure levels of charged vs uncharged tRNAs, the itrap method will be used. This technique involves demethylases to remove hindering base methylations and a β- elimination reaction targeting the 3' end of adenine residues in uncharged tRNA, followed by TaqMan MGB qRT-PCR to distinguish between charged and uncharged tRNA cDNA. [00403] e) LC-MS- Metabolites will be extracted using an acetonitrile:methanol:water based solution and subjected to a quadrupole orbitrap mass spectrometer coupled to a Vanquish UHPLC system with electrospray ionization. [00404] f) Data analysis- The LC-MS data will be analyzed using the MAVEN software and Compound Discoverer software. [00405] g) Power and statistical analysis- All experiments will be performed using at least three biological replicates. Controls will include cells from typically developing individuals cultured parallel to FXS cell lines and treated with the inhibitors or ASOs as well as their respective vehicle treatments. All samples within an experiment will be run simultaneously for LC-MS. All qPCR and western blot data will be analyzed using one-way ANOVA with Tukey’s multiple comparisons test. [00406] 2.1. Investigation of the pathways contributing to metabolome deficits in FXS - 94 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00407] The amino acid pool is influenced by protein synthesis, degradation, autophagy, mitochondrial function, amino acid uptake, and catabolism. Drug treatments will be used to perturb the proteostasis and mitochondrial function in FXS cells and test their effects on the amino acid pool (FIG.9). [00408] 2.1A: Effect of increased protein synthesis on amino acid levels in FXS [00409] Activation of the mTOR (mammalian target of rapamycin) pathway in FXS enhances translation initiation, increases ribosomal biogenesis and alters expression of synaptic proteins (A. Sharma et al.., Dysregulation of mTOR signaling in fragile X syndrome. J. Neurosci.30, 694–702 (2010)). Thus, this study will block mTOR activation using Rapamycin in FXS cells and studying metabolome changes using LC-MS. TD cells will be treated as controls and western blots for phospho-mTOR and mTOR will be used to test the effect of Rapamycin. Reduced amino acids could also alter charged tRNA: uncharged tRNA ratios and impact protein synthesis. A qPCR-based method, i-trap (individual tRNA acetylation PCR), will be used to measure tRNA charging (Y. Tsukamoto et al.., i-tRAP (individual tRNA acylation PCR): a convenient method for selective quantification of tRNA charging. Rna 29, 111–112 (2023)). This method includes a demethylation step which makes uncharged tRNAs’ 3′-end CC while charged tRNAs’ 3′-end CCA, and using TaqMan-MGB probes designed to recognize tRNAs of interest as well as the CC and CCA polymorphisms the charged tRNA fraction will be quantified. [00410] 2.1B: Effect of protein degradation deficits on amino acid levels in FXS [00411] Increased activity of the ubiquitin proteasome system (UPS) in FXS model mice, elevates protein degradation. Normalizing proteasome activity with bortezomib or MG-132 inhibitors has been shown to correct FXS phenotypes such as excessive hippocampal neuronal firing (S. R. Louros et al.., Excessive proteostasis contributes to pathology in fragile X syndrome. Neuron 111, 508-525.e7 (2023)). Altered protein degradation regulates recycling of free amino acids and hence, whether inhibitors such as MG-132 or bortezomib can restore changes in free amino acids will be tested by LC-MS. Decreased autophagosis is another feature of FXS which can result in reduced breakdown of proteins (J. Yan et al.., Activation of autophagy rescues synaptic and cognitive deficits in fragile X mice. Proc. Natl. Acad. Sci. U. S. A.115, E9707–E9716 (2018)). Inhibitors such as 3-methyladenine (3-MA) may be used to alter autophagy and its effect tested using LC-MS. - 95 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 [00412] 2.1C: Study of whether restoring mitochondrial deficits can influence amino acid levels in FXS cells [00413] Mitochondrial deficits, including inner membrane proton leaks, may exacerbate amino acid imbalances by altering their synthesis via the increased flux in the TCA cycle that can be ameliorated by inhibitors such as dexpramipexole (DEX) (P. Licznerski et al.., ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome. Cell 182, 1170-1185.e9 (2020)) To assess the activity of DEX, western blot will be used against proteins such as HEXOKINASE I and II to measure TCA activity. FXS and TD cells may be treated with dexpramipexole (DEX) and testing the amino acid levels using LC-MS. [00414] 2.2: Study of whether restoring FMRP can rescue metabolome deficits in FXS cells Preliminary data using FXS LCL lines indicates an influence of differential FMRP expression on the free amino acid levels (FIGs.7A-F). In some embodiments, FMRP may be restored in FXS cells and metabolome changes measured using LC-MS (FIG.10). [00415] 2.2A: Study of whether FMRP rescue can restore amino acid levels in FXS LCLs [00416] In FXS2 cells that express the FMR1-217 mis-spliced RNA, restoration of FMRP levels using antisense oligonucleotides (ASOs) have been shown ( S. Shah, et al.., Antisense oligonucleotide rescue of CGG expansion–dependent FMR1 mis-splicing in fragile X syndrome restores FMRP. Proc. Natl. Acad. Sci.120, e2302534120 (2023)). This paradigm in FXS cells may be used to restore FMRP and in TD cells (as controls) and the samples subjected to LC-MS. FMRP rescue upon ASO treatment will be examined using western blots. [00417] 2.2B: Study of whether FMRP rescue can restore amino acid levels in FXS patient derived forebrain neurons [00418] FXS is a neurodevelopmental disorder. Thus, FXS LCLs have been re-programmed (FIGs.7 and 8) to derive forebrain excitatory mature neurons. LC-MS will be used to determine whether the metabolome changes observed in FXS patient derived brain tissues and LCLs (FIGs. 6 and 7) are mirrored in the iPSC derived neurons in culture. Next, neurons will be treated with ASOs (as in 2.2A) and whether the metabolome deficits are rescued by LC-MS will be determined. [00419] The compounds and strategies proposed in 2.1 and 2.2 will be optimized in FXS LCL lines. Studies of the FXS human tissue samples have provided a list of metabolites other than the - 96 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 amino acids which can be studied further. Other than increase in proteostasis and mitochondrial dysfunction, there are additional factors, such as cellular uptake of amino acids, catabolism and synthesis of downstream nitrogen compounds that could influence the free amino acid pool. [00420] Tables 5-10 show the raw data of all the metabolites in the LCL cell lines (TD, FXS1, FXS2). Table 5. Metabolic changes comparing FXS1 to TD LCLs (cell pellet) Metabolite log2(fold change) -log10(p-value) HISTIDINOL -44532700 35617310
Figure imgf000098_0001
- 97 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Metabolite log2(fold change) -log10(p-value) TYROSINE -0.9744300 2.3462150
Figure imgf000099_0001
Table 6. Metabolic changes comparing FXS1 to TD LCLs (culture media) Metabolite log2(fold change) -log10(p-value)
Figure imgf000099_0002
- 98 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Metabolite log2(fold change) -log10(p-value) CARNOSINE -0.1463210 1.5141962
Figure imgf000100_0001
- 99 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Metabolite log2(fold change) -log10(p-value) METHIONINE -0.9095272 3.4960272
Figure imgf000101_0001
- 100 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Table 7. Metabolic changes comparing FXS2 to TD LCLs (cell pellet) Metabolite log2(fold change) -log10(p-value) O-PHOSPHOETHANOLAMINE -2.6872600 3.3875600
Figure imgf000102_0001
- 101 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Table 8. Metabolic changes comparing FXS2 to TD LCLs (culture media) Metabolite log2(fold change) -log10(p-value) 3-METHYLGLUTACONATE -1.3559872 3.764998
Figure imgf000103_0001
Table 9. Metabolic changes comparing FXS2 to FXS1 (cell pellet) Metabolite log2(fold change) -log10(p-value)
Figure imgf000103_0002
Table 10. Metabolic changes comparing FXS2 to FXS1 (culture media) - 102 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Metabolite log2(fold change) -log10(p-value) AICAR 0.0704180 1.3042006 [00421] E
Figure imgf000104_0001
xamp e [00422] Wild-type (WT) and FXS2 cells were incubated with RPMI media (without glutamine) + FBS + 2mM of 13C5,15N2 glutamine isotope for 8hrs. The cells and media were collected post incubation, and LC-MS was performed. Increased glutamine incorporation into citrate in FXS (FIG.16A) is indicative of increased reverse carboxylation of glutamine in the citric acid cycle. In FXS cells, increased percent of glutamine incorporation into glutamate was observed which accumulated more in the extracellular media than intracellularly (FIGs.16B-E). Increased ion count of glutamine-derived glutamate was observed in media in FXS cultures (FIGs.16B and 16D) but a smaller increase was observed intracellularly (FIGs.16C and 16E). EMBODIMENTS 1. A method of diagnosing, subclassifying, or predicting a treatment outcome of fragile X syndrome (FXS) in a subject, the method comprising: a) determining a presence, absence, amount, or alteration of at least one FXS- associated metabolite, or b) determining an alteration of an FXS-associated ratio of two metabolites, - 103 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 or both a) and b), in a sample from the subject, wherein the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS- associated ratio, in the sample is indicative of the presence, risk, progression, severity, or treatment outcome of FXS. 2. A method of predicting a treatment outcome of fragile X syndrome (FXS) in a subject, comprising: a) determining: i) a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii) an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject, and b) predicting the treatment outcome of FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the sample. 3. A method of diagnosing fragile X syndrome (FXS) in a subject, comprising: a) determining: i) a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii) an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject, and b) diagnosing the subject as having or having a propensity to develop FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the sample. 4. A method of prognosing fragile X syndrome (FXS) in a subject, the method comprising: a) determining: i) a presence, absence, amount, or alteration of at least one FXS-associated metabolite, or ii) an alteration of an FXS-associated ratio of two metabolites, or both i) and ii), in a sample from the subject, and - 104 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 b) prognosing the subject as having a propensity to have a poorer prognosis of FXS based on the presence, absence, amount, or alteration of the at least one FXS- associated metabolite, and/or the alteration of the FXS-associated ratio, in the sample. 5. A method of treating fragile X syndrome (FXS) in a subject, the method comprising: a) determining a presence, absence, amount, or alteration of at least one FXS- associated metabolite, or an alteration of an FXS-associated ratio of two metabolites, in a sample from the subject, and b) treating FXS based on the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, and/or the alteration of the FXS-associated ratio, in the sample. 6. The method of Embodiment 1, 2, or 5, wherein treating FXS comprises administering to the subject a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product. 7. The method of Embodiment 6, wherein the therapeutically effective amount of the agent decreases an aberrant FMR1 transcript, a protein encoded by the aberrant FMR1 transcript, or both. 8. The method of Embodiment 6 or 7, wherein the aberrant FMR1 gene product comprises FMR1-217. 9. The method of any one of Embodiments 6-8, wherein the therapeutically effective amount of the agent decreases FMR1-217 by at least 25%. 10. The method of any one of Embodiments 6-9, wherein the therapeutically effective amount of the agent increases the expression of fragile X messenger ribonucleoprotein (FMRP) by at least 25%. - 105 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 11. The method of any one of Embodiments 6-10, wherein the agent targets a contiguous nucleotide sequence in a polynucleotide sequence set forth in any one of SEQ ID NOs:6- 24. 12. The method of Embodiment 11, wherein the contiguous nucleotide sequence is at least 12 nucleotides in length. 13. The method of any one of Embodiments 6-12, wherein the agent is an antisense oligonucleotide (ASO) comprising a nucleotide sequence having at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOs:25-43. 14. The method of Embodiment 13, wherein the ASO comprises a nucleotide sequence set forth in any one of SEQ ID NOs:25-43. 15. The method of Embodiment 13, wherein the ASO comprises a nucleotide sequence having at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOs:34-39. 16. The method of Embodiment 15, wherein the ASO comprises a nucleotide sequence set forth in any one of SEQ ID NOs:34-39. 17. The method of any one of Embodiments 13-16, wherein the ASO is about 18-22 nucleotides in length. 18. The method of any one of Embodiments 13-17, wherein the ASO comprises a modification of a ribose group, a modification of a phosphate group, a modification of a nucleobase, or a combination thereof. 19. The method of any one of Embodiments 13-17, wherein the ASO is chemically modified to comprise: a) a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2’-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2’-O,4’-C-aminomethylene bridged nucleic acid (2’,4’-BNA(NC)), an alpha-L-locked nucleic acid, or a tricyclo-DNA, or a combination thereof, - 106 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 b) a ribose group comprising 2’-O-methyl, 2’-fluoro, 2’-deoxy, 2’-O-(2- methoxyethyl) (MOE), 2’-O-alkyl, 2’-O-alkoxy, 2’-O-alkylamino, 2’-NH2, a constrained nucleotide, or a tricyclo-DNA modification, or a combination thereof, c) a phosphate group comprising a phosphorothioate, a phosphoramidate, a phosphorodiamidate, a phosphorodithioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, or a phosphotriester, or a combination thereof, d) a nucleobase comprising 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5- substituted pyrimidine, isoguanine, isocytosine, or halogenated aromatic groups, or a combination thereof, e) a phosphorodiamidate mopholino (PMO), a peptide nucleic acid, or another pseudopeptide backbone, or a combination of the foregoing. 20. The method of any one of Embodiments 13-19, wherein the ASO is a phosphorothioate- modified polynucleotide. 21. The method of any one of Embodiments 13-20, wherein at least half of the internucleotide linkages of the ASO are phosphorothioate. 22. The method of any one of Embodiments 13-21, wherein each internucleotide linkage of the ASO is a phosphorothioate. 23. The method of any one of Embodiments 1-22, wherein the sample comprises a bodily fluid selected from cerebrospinal fluid, blood, serum, plasma, or urine. 24. The method of any one of Embodiments 1-23, wherein the sample comprises cerebrospinal fluid (CSF). 25. The method of any one of Embodiments 1-24, wherein the sample comprises peripheral blood mononuclear cells (PBMCs). - 107 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 26. The method of any one of Embodiments 1-25, comprising determining an alteration of at least one FXS-associated metabolite in the sample. 27. The method of any one of Embodiments 1-26, wherein the at least one metabolite has an altered level in a sample from a subject, relative to a control sample and/or reference level. 28. The method of Embodiment 27, wherein the at least one metabolite comprises n- acetylneuraminate, D-alanine, L-alanine arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, L-carnitine, carnosine, citrulline, creatine, cystine, cytosine, fumarate, alpha-D-glucose, glucuronate, oxidized glutathione (GSSG), reduced glutathione (GSH), glycerol, glycine, guanine, 3-methyl-L-histidine, homoserine, hypoxanthine, indoleacetaldehyde, isoleucine, kynurenate, leucine, lysine, malate, methionine, methyl acetoacetate, methylmalonate, mevalonate, n-formyl-L-methionine, nicotinamide, phenylalanine, proline, cis-4-hydroxy-D-proline, trans-4-hydroxy-L- proline, D-sedoheptulose, serine, succinate, threonine, allothreonine, tryptophan, tyramine, tyrosine, uracil, urate, valine, norvaline, or xanthine, or a combination thereof. 29. The method of any one of Embodiments 1-28, wherein the at least one metabolite comprises: a) lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L- carnitine, arginine, cystine, urate, or reduced glutathione (GSH), or a combination thereof, b) argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-formyl-L-methionine, arginine, or azelate, or a combination thereof, or c) valine, norvaline, nicotinamide, D-alanine, creatine, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha- D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, - 108 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 citrulline, n-acetylneuraminate, asparagine, or L-alanine, or a combination thereof, or a combination thereof. 30. The method of any one of Embodiments 1-29, wherein the at least one metabolite comprises arginine, leucine, or lysine, or a combination thereof. 31. The method of Embodiment 29 or 30, wherein the at least one metabolite has a reduced level in the sample from the subject, relative to a control sample and/or reference level. 32. The method of Embodiment 31, wherein a level of the at least one FXS-associated metabolite has a log2 fold reduction of ≤(-0.50) in the sample, relative to a control sample and/or reference level, optionally, wherein the log2 fold reduction is ≤(-0.90). 33. The method of Embodiment 31 or 32, wherein the control sample: a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject. 34. The method of Embodiment 29 or 30, wherein the at least one metabolite has an increased level in the sample from the subject, relative to a control sample and/or reference level. 35. The method of Embodiment 34, wherein a level of the at least one FXS-associated metabolite has a log2 fold increase of ≥0.50 in the sample, relative to a control sample and/or reference level, optionally, wherein the log2 fold increase is ≥0.90. 36. The method of Embodiment 34 or 35, wherein the control sample: a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject. 37. The method of any one of Embodiments 1-28, wherein the at least one metabolite comprises: a) phosphoenolpyruvate, oxidized glutathione disulfide (GSSG), orotate, or guanosine, or a combination thereof, or - 109 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 b) trehalose, or D-sedoheptulose, or both, or a combination thereof. 38. The method of Embodiment 37, wherein the at least one metabolite has an increased level in the sample from the subject, relative to a control sample and/or reference level. 39. The method of Embodiment 38, wherein a level of at least one FXS-associated metabolite has a log2 fold increase of ≥0.50 in the sample, relative to a control sample and/or reference level, optionally, wherein the log2 fold increase is ≥0.90. 40. The method of Embodiment 38 or 39, wherein the control sample: a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject. 41. The method of Embodiment 37, wherein the at least one metabolite has a reduced level in the sample from the subject, relative to a control sample and/or reference level. 42. The method of Embodiment 41, wherein a level of the at least one FXS-associated metabolite has a log2 fold reduction of ≤(-0.50) in the sample, relative to a control sample and/or reference level, optionally, wherein the log2 fold increase is ≤(-0.90). 43. The method of Embodiment 41 or 42, wherein the control sample: a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject. 44. The method of any one of Embodiments 1-43, wherein the FXS-associated ratio is indicative of increased oxidative stress in the sample from the subject. 45. The method of Embodiment 44, wherein the FXS-associated ratio is a ratio of reduced glutathione (GSH) to oxidized glutathione disulfide (GSSG). 46. The method of any one of Embodiments 1-43, wherein the FXS-associated ratio and/or metabolite level is indicative of suppressed mitochondrial metabolism in the sample from the subject. - 110 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 47. The method of Embodiment 46, wherein the FXS-associated ratio is a ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). 48. The method of any one of Embodiments 44-47, wherein the FXS-associated ratio is reduced in the sample from the subject, relative to a control sample and/or reference level. 49. The method of Embodiment 48, wherein the control sample: a) is a sample from an age-matched typically developing individual, or b) is a baseline sample from the subject. 50. The method of any one of Embodiments 44-47, wherein the FXS-associated ratio is increased in the sample from the subject, relative to a control sample and/or reference level. 51. The method of Embodiment 50, wherein the control sample: a) is a sample from an age-matched typically-developing individual, or b) is a baseline sample from the subject. 52. The method of any one of Embodiments 1-51, wherein the subject is a human male. 53. The method of any one of Embodiments 1-51, wherein the subject is a human female. 54. The method of any one of Embodiments 1-53, wherein determining the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, or the alteration of the FXS-associated ratio, comprises performing liquid chromatography-mass spectrometry (LC-MS). 55. The method of any one of Embodiments 1-53, wherein determining the presence, absence, amount, or alteration of the at least one FXS-associated metabolite, or the alteration of the FXS-associated ratio, comprises performing gas chromatography-mass spectrometry (GC-MS). - 111 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 56. A method of diagnosing, subclassifying, or predicting a treatment outcome of fragile X syndrome (FXS) in a subject, the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing, wherein a), b), or c), or a combination of the foregoing is indicative of the presence, risk, progression, severity, or treatment outcome of FXS. 57. A method of predicting a treatment outcome of fragile X syndrome (FXS) in a subject, the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and predicting the treatment outcome of FXS based on a), b), or c), or a combination of the foregoing. 58. A method of diagnosing fragile X syndrome (FXS) in a subject, the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and - 112 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 diagnosing the subject as having or having a propensity to develop FXS based on a), b), or c), or a combination of the foregoing. 59. A method of prognosing fragile X syndrome (FXS) in a subject, the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and prognosing the subject as having a propensity to have a poorer prognosis of FXS based on a), b), or c), or a combination of the foregoing. 60. A method of treating fragile X syndrome (FXS) in a subject, the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and treating FXS based on a), b), or c), or a combination of the foregoing. REFERENCES 1. J. D. Richter, X. Zhao, The molecular biology of FMRP: new insights into fragile X syndrome, Nat. Rev. Neurosci. (2021), doi:10.1038/s41583-021-00432-0. 2. S. Shah, J. D. Richter, Do Fragile X Syndrome and Other Intellectual Disorders Converge at Aberrant Pre-mRNA Splicing? Front. Psychiatry 12, 715346 (2021). 3. S. Shah, K. J. Sharp, S. Raju Ponny, J. Lee, J. K. Watts, E. Berry-Kravis, J. D. Richter, Antisense oligonucleotide rescue of CGG expansion–dependent FMR1 mis-splicing in fragile X syndrome - 113 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 restores FMRP. Proc. Natl. Acad. Sci.120, e2302534120 (2023). 4. S. Shah, G. Molinaro, B. Liu, R. Wang, K. M. Huber, J. D. Richter, FMRP Control of Ribosome Translocation Promotes Chromatin Modifications and Alternative Splicing of Neuronal Genes Linked to Autism. bioRxiv (2019), doi:10.1101/801076. 5. B. Liu, Y. Li, E. E. Stackpole, A. Novak, Y. Gao, Y. Zhao, X. Zhao, J. D. Richter, Regulatory discrimination of mRNAs by FMRP controls mouse adult neural stem cell differentiation. Proc. Natl. Acad. Sci.115, 201809588 (2018). 6. P. Licznerski, H. A. Park, H. Rolyan, R. Chen, N. Mnatsakanyan, P. Miranda, M. Graham, J. Wu, N. Cruz-Reyes, N. Mehta, S. Sohail, J. Salcedo, E. Song, C. Effman, S. Effman, L. Brandao, G. N. Xu, A. Braker, V. K. Gribkoff, R. J. Levy, E. A. Jonas, ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome. Cell 182, 1170-1185.e9 (2020). 7. J. Geng, T. P. Khaket, J. Pan, W. Li, Y. Zhang, Y. Ping, M. I. Cobos Sillero, B. Lu, Deregulation of ER-mitochondria contact formation and mitochondrial calcium homeostasis mediated by VDAC in fragile X syndrome. Dev. Cell 58, 597-615.e10 (2023). 8. M. Shen, C. L. Sirois, Y. Guo, M. Li, Q. Dong, N. M. Méndez-Albelo, Y. Gao, S. Khullar, L. Kissel, S. O. Sandoval, N. E. Wolkoff, S. X. Huang, Z. Xu, J. E. Bryan, A. M. Contractor, T. Korabelnikov, I. A. Glass, D. Doherty, J. E. Levine, A. M. M. Sousa, Q. Chang, A. Bhattacharyya, D. Wang, D. M. Werling, X. Zhao, Species-specific FMRP regulation of RACK1 is critical for prenatal cortical development. Neuron 111, 3988-4005.e11 (2023). 9. M. Shen, F. Wang, M. Li, N. Sah, M. E. Stockton, J. J. Tidei, Y. Gao, T. Korabelnikov, S. Kannan, J. D. Vevea, E. R. Chapman, A. Bhattacharyya, H. van Praag, X. Zhao, Reduced mitochondrial fusion and Huntingtin levels contribute to impaired dendritic maturation and behavioral deficits in Fmr1-mutant mice. Nat. Neurosci.22, 386–400 (2019). 10. D. C. Tărlungeanu, E. Deliu, C. P. Dotter, M. Kara, P. C. Janiesch, M. Scalise, M. Galluccio, M. Tesulov, E. Morelli, F. M. Sonmez, K. Bilguvar, R. Ohgaki, Y. Kanai, A. Johansen, S. Esharif, T. Ben-Omran, M. Topcu, A. Schlessinger, C. Indiveri, K. E. Duncan, A. O. Caglayan, M. Gunel, J. G. Gleeson, G. Novarino, Impaired Amino Acid Transport at the Blood Brain Barrier Is a Cause of Autism Spectrum Disorder. Cell 167, 1481-1494.e18 (2016). 11. L. S. Knaus, B. Basilico, D. Malzl, M. Gerykova Bujalkova, M. Smogavec, L. A. Schwarz, S. Gorkiewicz, N. Amberg, F. M. Pauler, C. Knittl-Frank, M. Tassinari, N. Maulide, T. Rülicke, J. - 114 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 Menche, S. Hippenmeyer, G. Novarino, Large neutral amino acid levels tune perinatal neuronal excitability and survival. Cell 186, 1950-1967.e25 (2023). 12. A. Sharma, C. A. Hoeffer, Y. Takayasu, T. Miyawaki, S. M. McBride, E. Klann, R. Suzanne Zukin, Dysregulation of mTOR signaling in fragile X syndrome. J. Neurosci.30, 694–702 (2010). 13. Y. Tsukamoto, Y. Nakamura, M. Hirata, R. Sakate, T. Kimura, i-tRAP (individual tRNA acylation PCR): a convenient method for selective quantification of tRNA charging. Rna 29, 111– 112 (2023). 14. S. R. Louros, S. S. Seo, B. Maio, C. Martinez-Gonzalez, M. A. Gonzalez-Lozano, M. Muscas, N. C. Verity, J. C. Wills, K. W. Li, M. F. Nolan, E. K. Osterweil, Excessive proteostasis contributes to pathology in fragile X syndrome. Neuron 111, 508-525.e7 (2023). 15. J. Yan, M. W. Porch, B. Court-Vazquez, M. V. L. Bennett, R. S. Zukin, Activation of autophagy rescues synaptic and cognitive deficits in fragile X mice. Proc. Natl. Acad. Sci. U. S. A. 115, E9707–E9716 (2018). [00423] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. [00424] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims. - 115 - 3935890.v1

Claims

Docket No.5439.1032-002; UMMS 23-42 CLAIMS What is claimed is: 1. A method of predicting a treatment outcome of, diagnosing, prognosing, or subclassifying fragile X syndrome (FXS) in a subject, the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing. 2. A method of treating fragile X syndrome (FXS) in a subject, the method comprising determining, in a sample from the subject: a) a presence, an absence, or a level of an FXS-associated metabolite, b) an alteration or a ratio of a level of an FXS-associated metabolite, relative to a reference level of the same metabolite, c) a ratio between levels of two FXS-associated metabolites, or an alteration thereof relative to a reference ratio of the two FXS-associated metabolites, or a combination of the foregoing; and administering to the subject a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product. 3. The method of claim 1 or 2, wherein the FXS-associated metabolite has a reduced level in the sample from the subject, relative to the reference level of the same metabolite. 4. The method of any one of claims 1-3, wherein the alteration of the level of the FXS- associated metabolite, relative to the reference level of the same metabolite, is a log2 fold reduction of from about -10 to about 0 or a log2 fold increase of from about 0 to about 10. - 116 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 5. The method of any one of claims 1-4, wherein the alteration of the level of the FXS- associated metabolite, relative to the reference level of the same metabolite, is a log2 fold reduction of equal to or less than about -0.50 or a log2 fold increase of equal to or greater than about 0.50. 6. The method of any one of claims 1-5, wherein the alteration of the ratio between levels of two FXS-associated metabolites relative to the reference ratio of the two FXS-associated metabolites is a decrease of at least about 20%. 7. The method of any one of claims 1-6, wherein the ratio between the levels of two FXS- associated metabolites is the ratio of reduced glutathione (GSH) to glutathione disulfide (GSSG) or the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). 8. The method of any one of claims 1-7, wherein the reference level is based on: a sample from an age-matched typically-developing individual, or a baseline sample from the subject. 9. The method of any one of claims 1-8, wherein the sample comprises a bodily fluid selected from cerebrospinal fluid (CSF), blood, serum, plasma, or urine. 10. The method of any one of claims 1-9, wherein the sample comprises peripheral blood mononuclear cells (PBMCs). 11. The method of any one of claims 1-10, wherein the FXS-associated metabolite comprises n-acetylneuraminate, D-alanine, L-alanine arginine, argininosuccinate, asparagine, azelate, 2-hydroxybutyrate, caffeine, L-carnitine, carnosine, citrulline, creatine, cystine, cytosine, fumarate, alpha-D-glucose, glucuronate, oxidized glutathione (GSSG), reduced glutathione (GSH), glycerol, glycine, guanine, 3-methyl-L-histidine, homoserine, hypoxanthine, indoleacetaldehyde, isoleucine, kynurenate, leucine, lysine, malate, methionine, methyl acetoacetate, methylmalonate, mevalonate, n-formyl-L-methionine, nicotinamide, phenylalanine, proline, cis-4-hydroxy-D-proline, trans-4-hydroxy-L- proline, D-sedoheptulose, serine, succinate, threonine, allothreonine, tryptophan, - 117 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 tyramine, tyrosine, uracil, urate, valine, norvaline, xanthine, phosphoenolpyruvate, orotate, guanosine, trehalose, or a combination thereof. 12. The method of any one of claims 1-11, wherein the FXS-associated metabolite comprises: a) lysine, proline, glycine, leucine, serine, phenylalanine, isoleucine, threonine, homoserine, D-sedoheptulose, methionine, guanine, tryptophan, tyrosine, L- carnitine, arginine, cystine, urate, or reduced glutathione (GSH), or a combination thereof, b) argininosuccinate, xanthine, 2-hydroxybutyrate, tyramine, indoleacetaldehyde, glycerol, carnosine, cytosine, fumarate, asparagine, mevalonate, malate, lysine, methyl acetoacetate, uracil, leucine, kynurenate, 3-methyl-L-histidine, caffeine, glucuronate, n-formyl-L-methionine, arginine, or azelate, or a combination thereof, c) valine, norvaline, nicotinamide, D-alanine, creatine, cis-4-hydroxy-D-proline, trans-4-hydroxy-L-proline, azelate, carnosine, methylmalonate, succinate, alpha- D-glucose, allothreonine, threonine, homoserine, hypoxanthine, glycerol, citrulline, n-acetylneuraminate, asparagine, or L-alanine, or a combination thereof, or a combination of a) to c). 13. The method of any one of claims 1-12, wherein the FXS-associated metabolite comprises arginine, leucine, or lysine, or a combination thereof. 14. The method of any one of claims 1-11, wherein the FXS-associated metabolite comprises: a) phosphoenolpyruvate, oxidized glutathione disulfide (GSSG), orotate, or guanosine, or a combination thereof, or a) trehalose, or D-sedoheptulose, or both, or a combination thereof. - 118 - 3935890.v1 Docket No.5439.1032-002; UMMS 23-42 15. The method of any one of claims 2-14, wherein the therapeutically effective amount of the agent decreases an aberrant FMR1 transcript, a protein encoded by the aberrant FMR1 transcript, or both. 16. The method of claim 15, wherein the aberrant FMR1 gene product comprises FMR1-217. 17. The method of any one of claims 2-16, wherein the agent is an antisense oligonucleotide. 18. The method of any one of claims 1-17, wherein determining b) or c) comprises determining the statistical significance of the alteration based on a significance threshold. 19. The method of claim 18, wherein the significance threshold is a p-value equal to or less than about 0.05. 20. The method of any one of claims 1-19, wherein the method employs a chromatography technique. - 119 - 3935890.v1
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