WO2023122800A1 - Therapeutic treatment for fragile x-associated disorder - Google Patents

Therapeutic treatment for fragile x-associated disorder Download PDF

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Publication number
WO2023122800A1
WO2023122800A1 PCT/US2022/082380 US2022082380W WO2023122800A1 WO 2023122800 A1 WO2023122800 A1 WO 2023122800A1 US 2022082380 W US2022082380 W US 2022082380W WO 2023122800 A1 WO2023122800 A1 WO 2023122800A1
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fmri
seq
aso
polynucleotide
rna
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PCT/US2022/082380
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French (fr)
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Joel D. Richter
Sneha Shah
Jonathan Watts
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University Of Massachusetts
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • Fragile X syndrome is an autism spectrum disorder that is the most frequent inherited form of intellectual impairment. FXS afflicts 1 in 4,000 boys and 1 in 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 (FMRI), which resides on the X chromosome. When the CGG triplet expands to 200 or more, the FMRI gene is methylated and thereby transcriptionally inactivated. The loss of the FMRI gene product, the protein fragile X messenger ribonucleoprotein (FMRP), is the cause of the disorder.
  • FMRI fragile X messenger ribonucleoprotein 1
  • the disclosure provided herein is based, in part, on the discovery that, in FXS cells, ASO treatment reduces the expression of the CGG expansion-dependent aberrantly spliced FMRI -217 RNA and restores fragile X messenger ribonucleoprotein (FMRP) to levels observed in cells from typically developing individuals. Accordingly, the disclosure generally relates to compositions (e.g., polynucleotides, pharmaceutical compositions) and methods that are useful for treating a fragile X-associated disorder.
  • compositions e.g., polynucleotides, pharmaceutical compositions
  • the present disclosure provides a method of treating a fragile X- associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that decreases expression of an aberrant fragile X messenger ribonucleoprotein 1 (FMRI') gene product, thereby treating the fragile X-associated disorder in the subject.
  • FMRI' fragile X messenger ribonucleoprotein 1
  • the present disclosure provides a method of treating a fragile X- associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that modulates splicing of an FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMRI -217), thereby treating the fragile X-associated disorder in the subject.
  • a therapeutically effective amount of an agent that modulates splicing of an FMRI gene e.g., decreasing splicing between Exons 1 and 2 of FMRI -217
  • the present disclosure provides a method of decreasing expression of an aberrant FMRI gene product in a cell, comprising contacting the cell with an agent under conditions whereby the agent is introduced into the cell, thereby decreasing expression of the aberrant FMRI gene product in the cell.
  • the present disclosure provides a method of modulating FMRI splicing and/or expression in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions whereby the agent is introduced into the cell, thereby modulating FMRI splicing and/or expression in the cell.
  • an agent e.g., a polynucleotide
  • the present disclosure provides a method of increasing the level of FMRP in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions whereby the agent is introduced into the cell, such that the level of FMRP in the cell is enhanced.
  • an agent e.g., a polynucleotide
  • the present disclosure provides a method of enhancing the level of FMRP in a cell, comprising contacting the cell with an oligonucleotide which is complementary to at least 8 contiguous nucleotides of a sequence set forth in SEQ ID NOs:24-42, such that the level of FMRP in the cell is enhanced.
  • the present disclosure provides a method of reducing CGG triplet repeat expansion in FMRI 5’ UTR in a cell, comprising contacting the cell with an agent that reduces expression of an aberrant FMRI gene product under conditions whereby the agent is introduced into the cell, thereby reducing CGG triplet repeat expansion in the cell.
  • the fragile X-associated disorder is FXS.
  • the aberrant FMRI gene product comprises FMRI -217.
  • the agent is a polynucleotide (e.g., any one of the modified polynucleotides disclosed herein).
  • the method increases expression of fragile X messenger ribonucleoprotein (FMRP) in the subject.
  • FMRP fragile X messenger ribonucleoprotein
  • the present disclosure provides an agent that decreases expression of an aberrant FMRI gene product.
  • the present disclosure provides an agent that modulates splicing and/or expression of an FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMR1- 217 or decreasing a protein encoded by FMRI -217).
  • an FMRI gene e.g., decreasing splicing between Exons 1 and 2 of FMR1- 217 or decreasing a protein encoded by FMRI -217.
  • the present disclosure provides a pharmaceutical composition, comprising any one or more of the agents disclosed herein, and one or more pharmaceutically acceptable excipients, diluents, or carriers.
  • the agent is a polynucleotide (e.g., any one of the modified polynucleotides disclosed herein).
  • FIG. 1 shows a genome browser view of FMRI RNA in 7 typically developing (“TD” or “control”) and 10 fragile X syndrome (FXS) patients sequenced from white blood cells (WBCs).
  • FIG. 2 shows a genome browser view of exon 1 and intron 1 of FMRI RNA in 7 typically developing individuals and 10 fragile X syndrome patients sequenced from white blood cells.
  • FIG. 3 illustrates a non-limiting approach, using antisense oligonucleotides (ASOs), for blocking isoform 12 production, increasing isoform 1 production, and increasing FMRP levels.
  • ASOs antisense oligonucleotides
  • FIG. 4 shows a schematic of FMRI isol and isol2 pre-mRNAs.
  • the numbered boxes (704-714) refer to antisense oligonucleotides complementary to regions in intron 1, that span intron 1 and iso 12 junction, and within isol2.
  • Isol l F, Isol l R, Exonl F, Exonl R, and Isol2_l R refer to primers (F, forward; R, reverse) that were used to detect RNA levels by RT- qPCR.
  • FIG. 5 shows RT-qPCR data demonstrating a reduction in isol2 and increase in isol.
  • the asterisk refers to p ⁇ 0.05.
  • FIGs. 6A-6B show RT-qPCR data from a fully methylated FXS cell line (FXS1, GM07365).
  • FIG. 6A shows an increase in FMRI isol2 upon 5-AzaC treatment and a partial rescue of the FMRI isol2 increase when combined with the ASO treatment.
  • FIG. 6B shows an increase in FMRI isol upon 5-AzaC treatment and a further increase when combined with the ASO treatment.
  • the asterisks refer to p ⁇ 0.05.
  • FIGs. 7A-7B show FMRP levels.
  • FIG. 7A shows western blot data for an FXS1 LCL cell line in duplicates (the upper panel), demonstrating an increase in FMRP after treatment with IpM 5-AzaC and ASO treatment (80nM of both antisense oligonucleotides 713 and 714) when compared to DMSO or 5-AzaC only treated samples.
  • the mouse brains hippocampus tissue
  • the bottom panel represents GAPDH protein levels used to normalize the protein amounts loaded in each sample.
  • FIG. 7B shows quantification of the FMRP protein levels relative to GAPDH protein levels as seen on the western blot in FIG. 7A.
  • FIGs. 8A-8C show FMRJ isol and isol2 levels in fibroblast cells from six individuals.
  • FIG. 8A is a table showing the number of CGG repeats in the FMRI RNA 5’ UTR from three healthy males and three premutation carrier males for FXS.
  • FIG. 8B shows RT-qPCR data of FMRI isol levels in fibroblast cells from the six individuals, normalized to GAPDH RNA levels.
  • FIG. 8C compares the FMRI isol2 level in individual Pl to those in the other premutation carriers and healthy control samples.
  • FIGs. 9A-9C A truncated isoform of FMRI mRNA identified in a subset of FXS individuals.
  • FIG. 9A-9C A truncated isoform of FMRI mRNA identified in a subset of FXS individuals.
  • FIG. 9A Integrative Genomics Viewer (IGV) tracks of RNA-seq data for FXS and TD individuals for the FMRI gene.
  • FMRI RNA was detected in all TD individuals, and FXS individuals 1-21.
  • the thick-lined box marked on the FMRI gene illustrated at the bottom shows the region of intron 1 with differential reads between TD (1-13) and FXS (1-21) individuals.
  • FIG. 9B Expanded view map to an exon that comprises the annotated FMRI -217 isoform. All annotated FMRI isoforms and sequence data for FMRI -2 1 PCR fragments from FXS RNA sample are shown in Table 3 and FIGs. 9E-9H. H refers to high and L refers to low FMRI.
  • FIG. 9C is
  • the full length FMRI RNA (exons-grey boxes) and the FMR1-2Y1 isoform (exons-grey boxes) are illustrated with the CGG repeats in the 5’UTR (UTRs-black boxes).
  • the forward (F) and reverse (R) primers used for q-PCR are shown.
  • the total FMRI RNA relative to GAPDH RNA levels was significantly reduced in H FMRI and L FMRI vs TD (*P ⁇ 0.05, t test). Bar graphs indicate mean, and error bars indicate +/- SEM.
  • FIG. 9D Summary table of changes in alternative splicing events from L FMRI vs H FMRI samples detected by rMATS (14) at an FDR ⁇ 5% and a difference in the exon inclusion levels (PSI, Percent spliced-in) between the genotypes (deltaPSI) of > 5%.
  • PSI Exon inclusion levels
  • deltaPSI Percent spliced-in
  • Schematic for the splicing event categories is shown at the left of the table.
  • FIG. 9E FMRI -217 isoform was identified in RNA samples generated from leukocytes (individual FXS-05). DNase treated RNA samples were reverse transcribed using an oligo(dT)(20), and the PCR product, generated using primers ExlF and 217R, was sequenced.
  • FIG. 9E FMRI -217 isoform was identified in RNA samples generated from leukocytes (individual FXS-05). DNase treated RNA samples were reverse transcribed using an
  • FIG. 9F The predicted protein product of the FMR1-2V1 isoform.
  • the predicted protein length is 31 amino acids, with a mass of 3,524 Da.
  • FIG. 9G Alignment of the sequencing data of the PCR product using primers ExlF and 217R to FMRI gene is displayed. The poly(A) site was identified by sequencing the PCR product of primer 217F and oligo(dT)(20).
  • the FMRI-217 isoform (ENST00000621447.1) is marked with a thick-lined box.
  • FIG. 10 Correlation of FXS molecular parameters with IQ. Three-dimensional comparison of indicated parameters. The inset shows samples with 100% methylation. The increasing size of the dots represent increase in FMRP levels, and the darkness from low to high represent increase in IQ levels (see Table 4).
  • FIGs. 11A-11E FMRI -217 is derived from FMRI, requires the CGG expansion, and is expressed in human postmortem brain tissues (FXS and premutation carriers), and in skin- derived fibroblasts (premutation carrier).
  • FIG. 11A Integrative Genomics Viewer (IGV) tracks of RNA-seq data (Tran et al., Widespread RNA editing dysregulation in brains from autistic individuals, Nat. Neurosci. (2019)) for FXS and TD individuals for the FMRI gene.
  • FIG. 11A Integrative Genomics Viewer (IGV) tracks of RNA-seq data (Tran et al., Widespread RNA editing dysregulation in brains from autistic individuals, Nat. Neurosci. (2019)) for FXS and TD individuals for the FMRI gene.
  • iPSCs derived from FXS individuals were incubated with viruses expressing a mock guide RNA (i_mock), or an FMRI guide RNA and catalytically inactive Cas9 fused to the Tetl demethylase (i Tetl).
  • iPSC-derived neurons from to FXS individuals were treated with a mock guide RNA (Nl_mock, N2_mock), or an FMRI guide RNA and catalytically inactive Cas9 fused to the Tetl demethylase (Nl_Tetl, N2_Tetl, N3_Tetl). All cells were incubated with an FMRI guide RNA and catalytically inactive Cas9 fused to the Tetl demethylase express FMRI -217.
  • FIG. 11D Experimental design for RNA extraction from postmortem cortical tissue obtained from 6 FXS males (F1-F6) and 5 typically developing (T1-T5) age-matched males.
  • FIG. 9C RT-qPCR data for cortical tissue-derived RNA samples representing abundance for FMRI and FMR1-2V1 isoforms relative to GAPDH RNA. Each sample was analyzed in duplicate. Primers used for amplification are represented in FIG. 9C (**P ⁇ 0.01, t test).
  • FIG. HE Schematic diagram of fibroblast generated from skin biopsies obtained from three male premutation carriers (P1-P3) and three male TD individuals (T1-T3). The table shows patient de-identified designation, genotypes, and CGG repeat numbers in the 5’UTR in the FMRI gene. ND: not determined.
  • FIGs. 12A-12G FMRI -217 is expressed in lymphoblast cell cultures from FXS individuals.
  • FIG. 12A Sample information for lymphoblast cell lines (LCLs) (Coriell Institute, NJ) from two FXS and two TD members of a family. FMRP and GAPDH (loading control) levels were determined by western blots. Ratios of FMRP/GAPDH normalized to FXS1 are shown below the blot. FMRP quantification by Luminex Microplex immunochemistry assay are shown in ng FMRP/pg total protein).
  • FIG. 12A Sample information for lymphoblast cell lines (LCLs) (Coriell Institute, NJ) from two FXS and two TD members of a family. FMRP and GAPDH (loading control) levels were determined by western blots. Ratios of FMRP/GAPDH normalized to FXS1 are shown below the blot. FMRP quantification by Luminex Microplex immunochemistry assay are shown in ng FMRP/p
  • FIG. 12B Schematic diagram of the 5-AzadC treatment (1
  • FIG. 13A Western blots showing FXS1 and FXS2 cells respectively treated with DMSO or 5-AzadC (as treated in FIG. 12C and quantified in FIGs. 12F-12G).
  • FIG. 13B The decrease in MALAT1 RNA levels relative to GAPDH RNA was quantified by RT-qPCR in the TD1 LCL treated with MALAT1 ASO (80nM and lOOnM) for 48hrs. Untreated cells were used as negative controls (* represents P ⁇ 0.05, t test).
  • the right panel shows a decrease in MALAT1 RNA levels compared to GAPDH KN A levels, quantified using RT-qPCR in the TD1 LCL treated with 80nM MALAT1 gapmer ASO for 48hrs or 72 hrs. Untreated cells were used as negative controls (* represents P ⁇ 0.05 using t test).
  • FIG. 13C FXS2 LCLs were treated with either 80nM of ASOs 704 and 705, 709 and 710 or 713 and 714 for 72 hrs. RNA levels for FMRI -217 and FMRI full length RNA were quantified by RT-qPCR using primers as in FIG. 9C.
  • FIG. 13D FXS2 LCLs were treated with either 80nM or 160nM of ASOs 713 and 714 or 80nM of Malatl gapmer ASO for 72 hrs. RNA levels for FMRI -217 and FMRI full length RNA were quantified by RT-qPCR using primers as in FIG. 9C. ASOs 713 and 714 reduced FMRI-217 levels at both 80nM and 160nM concentrations whereas FMRI full length RNA levels were increased. No change in the FMRI isoform levels was observed upon MALAT1 ASO treatment (* represents P ⁇ 0.05, t test).
  • FIGs. 14A-14F ASOs targeting FMR1-2Y1 restore FMRP levels in FXS LCLs with partial or complete FMRI gene methylation.
  • FIG. 14A ASOs designed against the FMRI -217 RNA are illustrated. (Intron specific: 704-706, intron-exon junction specific: 707-710 and exon specific: 711-714).
  • FIG. 14B Schematic diagram of the ASO treatment (80nM for 72 hours) of the FXS2 LCLs to determine FMRI isoform and FMRP levels after demethylation. DMSO treated cells were used as a vehicle control (****p ⁇ 0.0001, **P ⁇ 0.01, / test).
  • FIG. 14A ASOs targeting FMR1-2Y1 restore FMRP levels in FXS LCLs with partial or complete FMRI gene methylation.
  • FIG. 14A ASOs designed against the FMRI -217 RNA are illustrated. (Intron specific: 704-706, intron-exon junction specific: 707-710 and exon
  • FIG. 14C FMRP levels were determined for FXS2 LCLs treated with DMSO (vehicle) and ASOs as described in FIG. 12A. TD LCLs were also probed for FMRP on the same western blots. Ratios of FMRP/GAPDH normalized to FXS1 are shown below the blot.
  • FIG. 14D Fully methylated FXS1 LCLs were treated with ASOs 713 and 714 (80nM each) followed by 5-AzadC (luM) added on consecutive days 2-9 after which RNA and protein were extracted. FMRI -217 and FMRI isoforms were assessed using qPCR primers as shown in FIG.
  • FIG. 14E Western blot of FMRP and GAPDH from FXS1 LCLs treated with DMSO, 5-AzadC, or 5- AzadC plus ASOs as in FIG. 13 A.
  • FIGs. 15A-15F ASOs targeting FMR1-2V1 restore FMRP levels in FXS fibroblasts with an inactive FMRI gene treated with 5-AzadC.
  • FIG. 15A Dermal fibroblasts derived from a FXS individual (GM05131B, Cori ell Institute) were cultured with 5-AzadC for 8 days and then treated with ASOs 713/714 (100 nM each) for 72 hours prior to RNA and protein extraction.
  • FIG. 15B RT-qPCR analysis of FMRI-217, FMRI, and GAPDH RNAs in dermal fibroblasts treated with DMSO, ASOs 713/714, 5-AzadC, or the ASOs 713/714 plus 5-AzadC.
  • 15D Lung fibroblasts derived from a FXS individual were cultured with 5-AzadC for 8 days and then treated with ASOs 713/714 (100 nM each) for 72 hours prior to RNA and protein extraction.
  • the amounts of FMRI-217 and FMRI were made relative to GAPDH. (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, one way ANOVA with multiple comparisons test).
  • FIG. 15F Model depicting active FMRI transcription in FXS cells for after treatment with demethylating agents to activate FMRI transcription) result in production of mis-spliced FMRI-217. Down-regulation of FMRI-217 with an ASO results in rescue of correctly spliced FMRI transcripts and restoration of FMRP protein.
  • FIG. 16A Additional ASO sequences.
  • FIG. 16B 72-hour treatment with 160 nM of each ASO in lymphoblastoid cell line FXS2.
  • Total RNA was extracted using TRIzolTM Reagent (ThermoFisher Scientific # 15596026).
  • One pg of total RNA was primed with oligo(dT)20 to generate cDNA with a QuantiTect cDNA synthesis kit using random hexamers (FIG. 9E).
  • qPCR was performed using the iTaqTM Universal SYBR® Green Supermix on a QuantStudio 3 qPCR machine in triplicate.
  • the fold change of full length FMRI and FMR1-2Y1 in ASO treated cells relative to vehicle (control) was measured using qPCR.
  • the RNA levels were normalized to GAPDH RNA (*P values measured using t test).
  • “about” can mean within an acceptable standard deviation, per the practice in the art.
  • “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 24-96 hours,” the term “about” should be read as applying to both of the given values of the range, such that “about 24-96 hours” means about 24 hours to about 96 hours.
  • 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.”
  • an agent includes a single agent, and two or more agents. Further the two or more agents can be the same or different as, for example, in embodiments wherein a first agent comprises a polynucleotide (e.g., ASO) of a first sequence and a second agent comprises a polynucleotide (e.g., ASO) of a second sequence.
  • ASO polynucleotide
  • 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.
  • 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 compounds described herein include salts derived from suitable inorganic and organic acids, and suitable inorganic and organic bases.
  • 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.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • 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, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxy-ethanesulfonate, hydroxymaleate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate
  • 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.
  • 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 + ((Ci-C4)alkyl)4 salts.
  • inorganic bases such as alkali metal, alkaline earth metal, and ammonium bases
  • salts derived from aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline, or N + ((Ci-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.
  • the present disclosure provides a method of treating a fragile X- associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that decreases expression of an aberrant fragile X messenger ribonucleoprotein 1 (FMRI') gene product, thereby treating the fragile X-associated disorder in the subject.
  • An agent that decreases expression of an aberrant FMRI gene product in a method disclosed herein can be any one or more of the agents disclosed herein.
  • the present disclosure provides a method of treating a fragile X- associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that modulates splicing of an FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMRI -217), thereby treating the fragile X-associated disorder in the subject.
  • an agent that modulates splicing of an FMRI gene in a method disclosed herein can be any one or more of the agents disclosed herein.
  • the present disclosure provides a method of decreasing expression of an aberrant FMRI gene product in a cell, comprising contacting the cell with an agent under conditions whereby the agent is introduced into the cell, thereby decreasing expression of the aberrant FMRI gene product in the cell.
  • the present disclosure provides a method of modulating FMRI splicing and/or expression in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions whereby the agent is introduced into the cell, thereby modulating FMRI splicing and/or expression in the cell.
  • an agent e.g., a polynucleotide
  • An agent that modulates FMRI splicing and/or expression in a method disclosed herein can be any one or more of the agents disclosed herein.
  • the present disclosure provides a method of increasing the level of fragile X messenger ribonucleoprotein (FMRP) in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions whereby the agent is introduced into the cell, such that the level of FMRP in the cell is enhanced.
  • an agent e.g., a polynucleotide
  • the present disclosure provides a method of reducing CGG triplet repeat expansion in FMRI 5’ UTR in a cell, comprising contacting the cell with an agent that reduces expression of an aberrant FMRI gene product under conditions whereby the agent is introduced into the cell, thereby reducing CGG triplet repeat expansion in the cell.
  • Fragile X-associated disorders are caused by mutation of the fragile X messenger ribonucleoprotein 1 (FMRI, previously known as fragile X mental retardation 7) gene, located in the q27.3 locus of the X chromosome.
  • FMRI fragile X messenger ribonucleoprotein 1
  • the expansion of the trinucleotide CGG above the normal range (greater than 54 repeats) in the non-coding region of the FMRI gene has been associated with the development of fragile X-associated disorders.
  • the trinucleotide CGG can range from 55-200 CGG repeats.
  • a fragile X-associated disorder described herein is linked to greater than 77 CGG repeats in FMRI, e.g., greater than 98 CGG repeats in FMRI. In some embodiments, the fragile X-associated disorder is linked to at least 140 CGG repeats in FMRI. In some embodiments, the fragile X-associated disorder is linked to at least 201 CGG repeats in FMRI.
  • 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 described herein is fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI), or fragile X-associated tremor/ataxia syndrome (FXTAS), or a combination thereof.
  • the fragile X-associated disorder is FXS.
  • a FMRI gene encodes a fragile X messenger ribonucleoprotein (FMRP, previously known as fragile X mental retardation protein).
  • FMRP fragile X messenger ribonucleoprotein
  • an FMRI gene described herein is a human FMRI gene (e.g., corresponding to GenBank reference number NC 000023.11), a mouse FMRI gene (e.g., NC_000086.8), a ra FMRl gene (e.g, NC_051356.1), a golden hamster FMRI gene (e.g, NW_024429188.1), a Chinese hamster FMRI gene (e.g., NW_003614110.1), a Ao FMRl gene (e.g., NC_051843.1), a FMRl gene (e.g., NC_046383.1), or a monkey FMRI gene (e.g., NC 041774.1).
  • a human FMRI gene e.g., corresponding to GenBank reference number NC 000023.11
  • a mouse FMRI gene e.g., NC_000086.8
  • a ra FMRl gene e.g, NC_051356.1
  • the FMRI gene is a human FMRI gene.
  • the human FMRI 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 FMRI gene product refers to an FMRI gene product elevated in a subject who has, or is predisposed to have a fragile X-associated disorder.
  • an aberrant FMRI gene product described herein is elevated in a subject who is being treated, or has been treated, for a fragile X-associated disorder.
  • the aberrant FMRI gene product is elevated in a subject having at least 55 CGG repeats in the 5’ untranslated region of an FMRI 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 FMRI gene.
  • the aberrant FMRI gene product is elevated in a subject having at least 201 CGG repeats in the 5’ untranslated region of an FMRI gene.
  • an aberrant FMRI gene product described herein is not expressed in typically developing subjects (e.g., typically developing humans).
  • the aberrant FMRI gene product is elevated in a subject who is a premutation carrier for FXS. In some embodiments, the aberrant FMRI gene product is elevated in a subject who has FXS.
  • an aberrant FMRI gene product described herein is produced from a CGG expansion-dependent mis-splicing of a. FMRI gene.
  • an aberrant FMRI gene product described herein contributes to pathology of a fragile X-associated disorder described herein.
  • an aberrant FMRI transcript, its protein product, or both contribute to pathology of the fragile X- associated disorder.
  • an aberrant FMRI transcript described herein contributes to pathology of the fragile X-associated disorder.
  • a protein encoded by an aberrant FMRI transcript described herein contributes to pathology of the fragile X-associated disorder.
  • an aberrant FMRI transcript and its protein product contribute to pathology of the fragile X-associated disorder.
  • an aberrant FMRI gene product described herein comprises FMRI -217, its protein product, or both.
  • the aberrant FMRI gene product comprises FMRI -217.
  • the aberrant FMRI gene product comprises the protein product of FMRI -217.
  • the aberrant FMRI gene product comprises FMRI -217 and its protein product.
  • FMR1-2Y1 also referred to as “isoform 12” or “isol2,” is a transcript corresponding to A0A087X1M7 (ENST00000621447.1, 1,832 nucleotides).
  • FMR1-2Y1 has 2 exons, and the splicing between Exon 1 of FMR1-2Y1 (between base pairs 147,912,123 and 147,912,230, SEQ ID NO:23) and Exon 2 of FMR1-2Y1 (between base pairs 147,912,728 and 147,914,451, SEQ ID NO:21) is considered aberrant FMRI RNA splicing.
  • FMR1-2V1 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.
  • FMR1-2V1 encodes a 31-amino acid protein (SEQ ID NO:22)).
  • a method disclosed herein increases the level of expression of FMRP in a subject described herein. In some embodiments, a method disclosed herein increases the level of expression of FMRP in a cell described herein.
  • a method disclosed herein increases a normal FMRI gene product (e.g., a normal FMRI transcript, its protein product, or both) in a subject and/or cell described herein.
  • a normal FMRI gene product e.g., a normal FMRI transcript, its protein product, or both
  • Several normal FMRI gene products are expressed in typically developing subjects (e.g., humans who do not have FXS).
  • Non-limiting examples of “normal” human FMRI gene products include: a transcript corresponding to Q06787 (FMR1-2QF, ENST00000370475.9, 4,441 nucleotides), and its protein product (a 632-amino acid protein (NP 002015.1)), 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 normal FMRI gene product described herein comprises a transcript corresponding to Q06787 (FMR1-2Q5, ENST00000370475.9, 4,441 nucleotides), and its protein product (a 632-amino acid protein (NP_002015.1)).
  • FMR1-2Q5 also referred to as “isoform 1” or “isol”, is produced in typical developing individuals and a subpopulation of FXS subjects.
  • FMRI -205 has 17 exons, and the splicing between Exon 1 of FMRI -205 (between base pairs 147,911,919 and 147,912,230, SEQ ID NO: 19) and Exon 2 ofFMRJ-205 (between base pairs 147,921,933 and 147,921,985, SEQ ID NO:20) is considered normal FMRI RNA splicing.
  • CTCAGTCAGGCGCTCAGCTCCGTTTCGGTTTCACTTCCGGTGGAGGGCCGC CTCTGAGCGGCGGGCCGACGGCGAGCGCGGGCGGCGGCGGTGACGGAGGCG CCGCTGCCAGGGGGCGTGCGGCAGCGCGGCGGCGGCGGCGGCGGCGGCGGCGGCG GAGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCG GAGGCGGCGGCGGCGGCGGCGGCGGCTGGGCCTCGAGCGCCCGCAGCCC ACCTCTCGGGGGCGGGCTCCCGGCGCTAGCAGGGCTGAAGAAGATGGAGGAGCT GGTGGTGGAAGTGCGGGGCTCCAATGGCGCTTTCTACAAG (SEQ ID NO: 19).
  • GCATTTGTAAAGGATGTTCATGAAGATTCAATAACAGTTGCATTTGAAAA CAA (SEQ ID NO:20).
  • the present disclosure provides an agent that modulates splicing and/or expression of FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMRI -217 or decreasing a protein encoded by FMRI -217).
  • FMRI gene e.g., decreasing splicing between Exons 1 and 2 of FMRI -217 or decreasing a protein encoded by FMRI -217.
  • the present disclosure provides an agent that modulates splicing and/or expression of FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMRI -217 or decreasing a protein encoded by FMRI -217).
  • the present disclosure provides an agent that decreases expression of an aberrant FMRI gene product.
  • the term “decreasing,” “decrease,” “reducing” or “reduce” refers to modulation that results in a lower level of the aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product), relative to a reference (e.g., the level prior to or in an absence of modulation by an agent disclosed herein).
  • an agent disclosed herein decreases expression of an aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product), relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
  • an aberrant FMRI gene product e.g., FMRI -217 and/or its protein product
  • an agent disclosed herein decreases expression of an aberrant FMRI transcript, decreases expression of an aberrant /’A7 7 -encoded protein, or both.
  • an agent disclosed herein decreases expression of an aberrant FMRI transcript (e.g., FMRI -217).
  • the agent decreases expression of the aberrant FMRI transcript, relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
  • an agent disclosed herein decreases expression of an aberrant /’A7/? /-encoded protein (e.g., the protein product of FMRI -217).
  • the agent decreases expression of the aberrant /’A7/? /-encoded protein, relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
  • an agent disclosed herein decreases expression of an aberrant FMRI transcript and an aberrant FMR1- encoded protein (e.g., FMRI -217 and its protein product).
  • the agent decreases expression of the aberrant FMRI transcript and the aberrant /’A7/? /-encoded protein, relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
  • An agent disclosed herein may decrease expression of an aberrant FMRI gene product directly or indirectly, for example, by altering transcription initiation, transcription elongation, transcription termination, RNA splicing, RNA processing, RNA stability, translation initiation, post-translational modification, protein stability, protein degradation, or a combination of the foregoing.
  • an agent disclosed herein decreases splicing of an aberrant FMRI transcript (e.g., between Exons 1 and 2 of 7-217).
  • the agent decreases splicing of the aberrant FMRI transcript, relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
  • an agent disclosed herein increases the level of expression of FMRP.
  • increasing or “increase” refers to modulation that results in a higher level of FMRP, relative to a reference (e.g., the level prior to or in an absence of modulation by an agent disclosed herein).
  • an agent disclosed herein increases FMRP expression, relative to a reference, by at least 5%, e.g., by at least: 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%.
  • an agent disclosed herein increases expression of a normal FMRI gene product, relative to a reference, by at least 5%, e.g., by at least: 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%.
  • the agent increases expression of a normal FMRI gene product to at least 5% of the level observed in in typically developing subjects (e.g., human), e.g., at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, of the level observed in the typically developing subject.
  • the agent increases expression of a normal FMRI gene product to at least 30% of the level observed in in typically developing subjects (e.g., human).
  • an agent disclosed herein e.g., an anti-sense RNA polynucleotide increases expression of a normal FMRI transcript, a normal FA 7?7-encoded protein, or both.
  • an agent disclosed herein increases expression of a normal FMRI transcript (e.g., FMRI -IQ ').
  • the agent increases expression of the normal FMRI transcript, relative to a reference, by at least 5%, e.g., by at least: 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%.
  • an agent disclosed herein increases expression of a normal FMRI -encoded protein (e.g., a protein encoded by FMR1-2QS).
  • the agent increases expression of the normal FMR1- encoded protein, relative to a reference, by at least 5%, e.g., by at least: 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%.
  • an agent disclosed herein increases expression of a normal FMRI transcript and a normal FMRI -encoded protein (e.g., FMRI -205 and its protein product).
  • the agent increases expression of the normal FMRI transcript and the normal FW7?7-encoded protein, relative to a reference, by at least 5%, e.g., by at least: 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%.
  • an agent disclosed herein increases splicing of a normal FMRI transcript (e.g., between Exons 1 and 2 of FMRl- QF).
  • the agent increases splicing of the normal FMRI transcript, relative to a reference, by at least 5%, e.g., by at least: 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%.
  • an agent disclosed herein decreases expression of an aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product) and increases expression of FMRP.
  • an aberrant FMRI gene product e.g., FMRI -217 and/or its protein product
  • an agent disclosed herein decreases expression of an aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product) and increases expression of a normal FMRI gene product (e.g., FMRI -205 and/or its protein product).
  • an agent disclosed herein decreases expression of an aberrant FMRI transcript, decreases expression of an aberrant FMRI -encoded protein, increases expression of a normal FMRI transcript, increases expression of a normal FW7?7-encoded protein, or a combination thereof.
  • an agent disclosed herein decreases expression of an aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product), relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%; and increases expression of a normal FMRI gene product (e.g., FMRI -205 and/or its protein product), relative to a reference, by at least 5%, e.g., by at least: 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%.
  • an aberrant FMRI gene product e.g., FMRI -217 and/or its protein product
  • a normal FMRI gene product
  • an agent disclosed herein decreases splicing of an aberrant FMRI transcript (e.g., between Exons 1 and 2 of FMR1- 217), relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%; and increases splicing of a normal FMRI transcript (e.g., between Exons 1 and 2 of FMR1- 205), relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, 105%, 110%, 120%
  • a level of an FMRI gene product e.g., an aberrant FMRI transcript, an aberrant FA 7?7-encoded protein, a normal FMRI transcript, a normal FMR1- encoded protein, or a combination thereof
  • an agent disclosed herein is administered to a subject, e.g., for at least: 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 months or 6 months, after a treatment with an agent disclosed herein has begun.
  • a level an FMRI gene product is measured in a tissue or a cell. In some embodiments, a level an FMRI gene product is measured in a white blood cell. In some embodiments, a level an FMRI gene product is measured in a leukocyte. In some embodiments, a level an FMRI gene product is measured in a fibroblast cell e.g., a dermal derived fibroblast cell or a lung-derived fibroblast cell). In some embodiments, a level an FMRI gene product is measured in a cortex tissue e.g., a brain biopsy of superficial cortex).
  • an agent disclosed herein e.g., an antisense oligonucleotide (ASO) promotes exclusion of an aberrant FMRI exon.
  • the agent promotes exclusion of Exon 2 of FMRI -217.
  • an agent disclosed herein e.g., an ASO targets (indirectly, or directly, e.g., binds) a primary aberrant transcript (pre-mRNA) of an FMRI gene.
  • pre-mRNA a primary aberrant transcript of an FMRI gene.
  • target refers to a preliminary mRNA region, and specifically, to a region identified by Exon 2, and the adjacent intron 1-2 regions of FMRI -217 , which is responsible for the splicing associated with FMRI -217.
  • a target sequence refers to a portion of the target RNA against which a polynucleotide e.g., an ASO) is directed, that is, the sequence to which the polynucleotide will hybridize by Watson-Crick base pairing of a complementary sequence.
  • a polynucleotide e.g., an ASO
  • the agent targets a contiguous nucleotide sequence within pre- mRNA of FMRI -217, wherein the contiguous nucleotide sequence is at least 8 nucleotides in length. In some embodiments, the contiguous nucleotide sequence is at least 9 nucleotides in length, for example, at least: 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 in length. In some embodiments, the contiguous nucleotide sequence is at least 12 nucleotides in length.
  • the contiguous nucleotide sequence is about 8-80 nucleotides in length, for example, about: 10- 60, 10-40, 10-30, 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 length. In some embodiments, the contiguous nucleotide sequence is about 10-30 nucleotides in length.
  • the agent e.g., an ASO
  • targets a contiguous nucleotide sequence within SEQ ID NO:24 e.g., within any one or more of SEQ ID NOs:25-42
  • the contiguous nucleotide sequence is at least 8 nucleotides in length
  • the agent e.g., an ASO
  • the contiguous nucleotide sequence is at least 9 nucleotides in length, for example, at least: 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 in length.
  • the agent e.g., an ASO
  • the agent e.g., an ASO
  • the agent targets a contiguous nucleotide sequence within any one or more of SEQ ID NOs:37-42, wherein the contiguous nucleotide sequence is at least 8 nucleotides in length.
  • the contiguous nucleotide sequence is selected from a polynucleotide sequence set forth in any one of SEQ ID NOs:28-42.
  • the contiguous nucleotide sequence is selected from a polynucleotide sequence set forth in any one of SEQ ID NOs:37-42.
  • an agent disclosed herein comprises at least one polynucleotide disclosed herein. In some embodiments, the agent comprises at least two polynucleotides disclosed herein.
  • the present disclosure provides a polynucleotide capable of decreasing expression of an aberrant FMRI gene product.
  • the present disclosure provides a polynucleotide capable of decreasing splicing of FMR1-2Y1.
  • the present disclosure provides a method of enhancing the level of FMRP in a cell, comprising contacting the cell with an oligonucleotide which is complementary to at least 8 contiguous nucleotides of a sequence set forth in SEQ ID NOs:24-42, such that the level of FMRP in the cell is enhanced.
  • 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 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.
  • a nucleotide analog comprises a modification to the sugar portion of the nucleotide.
  • 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 Ci-Ce alkyl, alkenyl, alkynyl or aryl.
  • a phosphate group of the nucleotide is modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates).
  • the 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.
  • a polynucleotide disclosed herein binds a target sequence described herein.
  • a targeting polynucleotide disclosed herein has near or substantial complementarity to a target sequence described herein.
  • the polynucleotide is formed of contiguous complementary sequences (to the target sequence).
  • the polynucleotide sequence is formed of non-contiguous complementary sequences (to the target sequence), for example, when placed together, constitute sequence that spans the target sequence.
  • a polynucleotide disclosed herein comprises a nucleotide sequence that is complementary (e.g., fully complementary or partially complementary) to a target sequence described herein (such that the polynucleotide is capable of hybridizing or annealing to target sequence, e.g., under physiological conditions).
  • 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 (z.e., hydrogen bonding) with a residue of the second region, thus forming a hydrogen-bonded duplex.
  • a polynucleotide disclosed herein specifically hybridizes to a target polynucleotide described herein (e.g., contiguous nucleotides of a sequence set forth in SEQ ID NOs:24-42), for example, under physiological conditions, with a 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.
  • the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide at a given ionic strength and pH.
  • specific hybridization corresponds to stringent hybridization conditions.
  • specific hybridization occurs with near complementary of the antisense oligomer to the target sequence. In some embodiments, specific hybridization occurs with substantial complementary of the antisense oligomer to the target sequence. In some embodiments, specific hybridization occurs with exact complementary of the antisense oligomer to the target sequence.
  • a polynucleotide disclosed herein comprises a nucleotide sequence that is complementary to a contiguous nucleotide sequence (e.g, 10 to 30 nucleotides) of pre-mRNA of an aberrant FMRI transcript.
  • a polynucleotide disclosed herein comprises a nucleotide sequence that is complementary to a contiguous nucleotide sequence (e.g., 10 to 30 nucleotides) of pre-mRNA of FMR1-2Y1.
  • the polynucleotide comprises a nucleotide sequence that is complementary to a target sequence within any one of SEQ ID NOs:24-42 (e.g., any one of SEQ ID NOs:24-27, any one of SEQ ID NOs:28-42, or a combination thereof).
  • a polynucleotide disclosed herein is an antisense oligonucleotide (ASO).
  • the polynucleotide is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense DNA, an antisense RNA, a microRNA (miRNA), an antagomir, a guide RNA (gRNA).
  • the polynucleotide 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.
  • LNA locked nucleic acid
  • a polynucleotide disclosed herein comprises a nucleotide sequence specifically hybridizes to (e.g., having near, substantial, or exact complementarity to) at least a portion of X chromosome between base pairs 147,911,919 and 147,921,985 (e.g., a target sequence within X chromosome between base pairs 147,911,919 and 147,921,985), for example, 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 147,914,451, between 147,912,231 and 147,91
  • a polynucleotide disclosed herein has exact complementarity to at least a portion of X chromosome between base pairs 147,911,919 and 147,921,985, for example, 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,
  • the polynucleotide comprises a nucleotide sequence specifically hybridizes to (e.g., having near, substantial, or exact complementarity to) at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766, for example, having at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reverse and complementary sequence of the at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766.
  • 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.
  • sequence alignment and comparison typically one sequence is designated as a reference sequence, to which a 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 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.
  • the polynucleotide comprises a nucleotide sequence having at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766.
  • the polynucleotide comprises a nucleotide sequence having about 70-100% sequence identity to at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%.
  • the polynucleotide comprises a nucleotide sequence that is identical to at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766.
  • a polynucleotide disclosed herein comprises a nucleotide sequence specifically hybridizes to (e.g., having near, substantial, or exact complementarity to) at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766, for example, having at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reverse and complementary sequence of the at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766.
  • the polynucleotide comprises a nucleotide sequence having at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766.
  • the polynucleotide comprises a nucleotide sequence having about 70-100% sequence identity to at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%.
  • the polynucleotide comprises a nucleotide sequence that is identical to at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766.
  • a polynucleotide disclosed herein comprises a nucleotide sequence having at least 70% sequence identity to, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: l-l l and SEQ ID NOs:43-50.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50.
  • the polynucleotide (e.g., ASO) has about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96- 100%, 97-100%, 98-100% or 99-100%.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50.
  • each nucleobase shown as T may independently be T or U.
  • 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.
  • AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705).
  • CTAGACCGGAAAAGAGAAGCCA SEQ ID NO:3 (W-706).
  • ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707).
  • CAATGCTAGACCGGAAAAGA SEQ ID NO:5
  • AAGTCCCAATGCTAGACCGGA (SEQ ID NO:6) (W-709).
  • AGAACAGTGGAGCTCTCCGA (SEQ ID NO:9) (W-712).
  • CAGTGGAGCTCTCCGAAGTCC (SEQ ID NO:43) (2831).
  • CCCAGAACAGTGGAGCTCTCC (SEQ ID NO:44) (2832).
  • TTCTTCACAGCCCTCGCCCA (SEQ ID NO:46) (2834).
  • a polynucleotide disclosed herein comprises a nucleotide sequence having at least 70% sequence identity to, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 10-11 and SEQ ID NOs:43-46.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50.
  • the polynucleotide (e.g., ASO) has about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 10-11 and SEQ ID NOs:43-46.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 10-11 and SEQ ID NOs:43-46, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to a sequence set forth in any one of SEQ ID NOs: 10-11 and SEQ ID NOs:43-46.
  • an agent disclosed herein comprises a first polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to SEQ ID NO: 10, and a second polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to SEQ ID NO: 11.
  • ASO polynucleotide
  • the first polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10
  • the second polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11.
  • the first polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to SEQ ID NO: 10, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99- 100%, sequence identity to SEQ ID NO: 10; and the second polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to SEQ ID NO: 11, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100% sequence identity to SEQ ID NO: 11.
  • the first polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to SEQ ID NO: 10
  • the second polynucleotide comprises a nucleotide sequence that is identical to SEQ ID NO: 11.
  • a polynucleotide disclosed herein comprises a nucleotide sequence having at least 70% sequence identity to, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 51-69.
  • the polynucleotide comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:51-69.
  • the polynucleotide (e.g., ASO) has about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:51-69.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to a sequence set forth in any one of SEQ ID NOs:51-69, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to a sequence set forth in any one of SEQ ID NOs: 51-69.
  • AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:52) (W-705).
  • AUGCUAGACCGGAAAAGAGAA (SEQ ID NO:54) (W-707).
  • AGAACAGUGGAGCUCUCCGA (SEQ ID NO:59) (W-712).
  • CGCCCAGAACAGUGGAGCUC (SEQ ID NO:60) (W-713).
  • CCUCGCCCAGAACAGUGGAG (SEQ ID NO:61) (W-714).
  • CAGUGGAGCUCUCCGAAGUCC (SEQ ID NO:62) (2831).
  • CCCAGAACAGUGGAGCUCUCC (SEQ ID NO:63) (2832).
  • CACAGCCCUCGCCCAGAACA (SEQ ID NO:64) (2833).
  • UUCUUCACAGCCCUCGCCCA (SEQ ID NO:65) (2834).
  • a polynucleotide disclosed herein comprises a nucleotide sequence having at least 70% sequence identity to, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:60-65.
  • the polynucleotide e.g., ASO
  • the polynucleotide (e.g., ASO) has about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:60-65.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to a sequence set forth in any one of SEQ ID NOs:60-65, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to a sequence set forth in any one of SEQ ID NOs:60-65.
  • an agent disclosed herein comprises a first polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to SEQ ID NO:60, and a second polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to SEQ ID NO:61.
  • ASO polynucleotide
  • the first polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:60
  • the second polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:61.
  • the first polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to SEQ ID NO:60
  • the second polynucleotide comprises a nucleotide sequence that is identical to SEQ ID NO:61.
  • the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is at least about 70% identical to a sequence within X chromosome region between
  • 147,912,230 and 147,914,451 e.g., between 147,912,230 and 147,912,728 or between 147,912,728 and 147,914,451
  • the polynucleotide comprises a nucleotide sequence that is about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence within X chromosome region between 147,912,230 and 147,914,451 (e.g., between 147,912,230 and 147,912,728 or between 147,912,728 and 147,914,451).
  • the polynucleotide comprises a nucleotide sequence having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence within X chromosome region between 147,912,230 and 147,914,451 (e.g., between 147,912,230 and 147,912,728 or between 147,912,728 and 147,914,451).
  • the polynucleotide comprises a nucleotide sequence having about 70-100% sequence identity to a sequence within X chromosome region between 147,912,230 and 147,914,451 (e.g., between
  • 147,912,230 and 147,912,728 or between 147,912,728 and 147,914,451) for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96- 100%, 97-100%, 98-100% or 99-100%.
  • the polynucleotide (e.g., ASO) is at least about 70% complimentary to at least a portion of an FMRI gene transcript, for example, at least about: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to at least a portion of an FMRI gene transcript. In some embodiments, the polynucleotide is about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to at least a portion of an FMRI gene transcript.
  • the polynucleotide is about 70-100% complimentary to at least a portion of an FMRI gene transcript, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100% complimentary to at least a portion of an FMRI gene transcript.
  • a polynucleotide disclosed herein has a length of at least about 8 nucleotides, for example, 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.
  • the polynucleotide has a length of about 8-80 nucleotides, for example, 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, the polynucleotide has a length of about 10-30 or 12-30 nucleotides.
  • the polynucleotide has a length of about: 8, 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.
  • a polynucleotide disclosed herein has a length of at least about 12 nucleotides, for example, at least about: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
  • the polynucleotide has a length of about 12- 40 nucleotides, for example, about: 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, the polynucleotide has a length of about 15-25 nucleotides. In some embodiments, the polynucleotide has a length of about: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
  • a polynucleotide is an oligonucleotide. In some embodiments, the length of the polynucleotide is about 18-22 nucleotides.
  • a polynucleotide disclosed herein 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 nucleotide sequences.
  • the polynucleotide is an isolated DNA polynucleotide.
  • the polynucleotide is an isolated RNA polynucleotide.
  • Polynucleotides of the disclosure 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.
  • a polynucleotide of the disclosure is a recombinant polynucleotide.
  • the present disclosure provides a polynucleotide capable of increasing the expression of a functional FMRI gene product.
  • the polynucleotide is any one of the polynucleotides, modified or unmodified, disclosed herein. In some embodiments, the polynucleotide is any one of the modified polynucleotides disclosed herein.
  • a polynucleotide of the disclosure comprises one or more modified nucleotides.
  • one or more modified nucleotides each independently comprises a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.
  • Chemical modifications can be chosen to, e.g., increase nuclease resistance of a polynucleotide (e.g., oligonucleotide), to prevent RNase H cleavage of a polynucleotide (e.g., a complementary RNA strand), or to increase cellular uptake of a polynucleotide.
  • a variety of compatible chemical modifications are available and will be familiar to those skilled in the art.
  • each modification of a ribose group comprises 2’-(9-methyl, 2’-fluoro, 2’-deoxy, 2’- ⁇ -(2-methoxyethyl) (MOE), 2’-O-alkyl, 2’-O-alkoxy, 2’-O-alkylamino, 2’-NH2, or a constrained nucleotide, or a combination thereof.
  • a substituted RNA analogue disclosed herein comprises a methoxy ethyl group on the 2’ OH.
  • a constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2’-(5)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2’-(9,4’-C-aminomethylene bridged nucleic acid (2’,4’-BNANC), an alpha- L-locked nucleic acid, and a tricyclo-DNA, or a combination thereof.
  • LNA locked nucleic acid
  • S-cEt 2’-(5)-constrained ethyl nucleotide
  • constrained MOE a 2’-(9,4’-C-aminomethylene bridged nucleic acid (2’,4’-BNANC
  • 2’,4’-BNANC 2’-(9,4’-C-aminomethylene bridged nucleic acid
  • modification of a ribose group comprises a 2’ -O-(2- methoxyethyl) (MOE) modification.
  • every nucleotide of a polynucleotide e.g., oligonucleotide
  • MOE 2-methoxyethyl
  • modification of a ribose group comprises a tricyclo-DNA modification.
  • every nucleotide of a polynucleotide e.g., antisense oligonucleotide
  • comprises a tricyclo-DNA modification e.g., antisense oligonucleotide
  • modification of a ribose group comprises a 2’-deoxy modification.
  • 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.
  • each modification of a phosphate group comprises a phosphoramidate.
  • modification of a phosphate group comprises a phosphorothioate modification.
  • every nucleotide of a polynucleotide e.g., oligonucleotide
  • a polynucleotide is a phosphorothioate-modified polynucleotide.
  • a sugar-phosphate backbone is replaced with a phosphorodiamidate morpholino (PMO) backbone.
  • PMO phosphorodiamidate morpholino
  • a sugar-phosphate backbone is replaced with a peptide nucleic acid or other pseudopeptide backbone.
  • each modification of a nucleobase comprises 2-thiouridine, 4- thiouridine, N 6 -methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5- methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or a combination thereof.
  • modification of a nucleobase group comprises a 5- methylcytosine modification.
  • a polynucleotide comprises a mixture of modified nucleotides.
  • a mixture of modified nucleotides comprise two or more modifications selected from the group consisting of 2’-(9-methyl, 2’-deoxy, 2’ -O-(2- methoxyethyl) (MOE), LNA, and tricyclo-DNA.
  • a polynucleotide comprises 4 or fewer consecutive 2’ -deoxy modified nucleotides.
  • a mixture of modified nucleotides comprise one or more -O- methyl modified nucleotides and one or more LNA modified nucleotides.
  • a mixture of modified nucleotides comprise one or more -O- (2-methoxy ethyl) (MOE) modified nucleotides and one or more LNA modified nucleotides.
  • MOE (2-methoxy ethyl)
  • each ribose group of a polynucleotide disclosed herein comprises 2’ -O-(2 -methoxy ethyl) (MOE) and/or each phosphate group of the polynucleotide comprises a phosphorothioate.
  • each ribose group of the polynucleotide e.g., ASO
  • each phosphate group of the polynucleotide comprises a phosphorothioate.
  • each ribose group of a polynucleotide disclosed herein comprises 2’- O-(2-methoxyethyl) (MOE), and each phosphate group of the polynucleotide comprises a phosphorothi oate .
  • an agent disclosed herein comprises a polypeptide.
  • 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 oc-amino acids (e.g., alanine, glycine, valine), non-oc-amino acids (e.g., [3-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 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), Mm ethyl - oc-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).
  • a polypeptide disclosed herein is an isolated polypeptide. In some embodiments, a polypeptide disclosed herein is a recombinant polypeptide.
  • the polypeptide is an inhibitor e.g., a direct inhibitor or an indirect inhibitor) of expression of an aberrant FMRI gene product e.g., FMRI -217, and/or its protein product).
  • the polypeptide is an activator e.g., a direct activator or an indirect activator) of expression of a normal FMRI gene product e.g., FMRI -2Q5, and/or its protein product).
  • the polypeptide reduces expression of an aberrant FMRI gene product e.g., FMRI -217, and/or its protein product) and increases expression of a normal FMRI gene product e.g., FMRI -2Q5, and/or its protein product).
  • a polypeptide disclosed herein is an immunoglobulin molecule.
  • the immunoglobulin molecule an antibody.
  • the antibody is an antagonist antibody that binds an FMRI transcript, or isoform, associated with a fragile X-associated disorder e.g., FXS).
  • the 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.
  • the antibody is primatized e.g., humanized).
  • the antibody is a polyclonal antibody.
  • the antibody is a monoclonal antibody.
  • the antibody e.g., monoclonal antibody is multispecific, e.g., bi-, tri-, or quad-specific.
  • a polypeptide disclosed herein 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.
  • the 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.
  • a polypeptide disclosed herein is an antibody mimetic.
  • antibody mimetic refers to polypeptides capable of mimicking an antibody’s ability to bind an antigen, but structurally differ from native antibody structures.
  • 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.
  • a target antigen e.g., an FMRI transcript, or isoform, associated with a fragile X-associated disorder, such as FXS
  • a target antigen e.g., an FMRI transcript, or isoform, 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. Greenfield eds., 2d ed.
  • an agent disclosed herein comprises a gene editing system.
  • the gene editing system produces a deletion of nucleotides, a substitution of nucleotides, an addition of nucleotides or a combination of the foregoing, in the FMRI gene.
  • the gene editing system produces a partial or complete deletion in Exon 2 of FMR1-2V1 (e.g., pseudo exon between base pairs 147,911,919 and 147,914,451 in the human FMRI gene).
  • the gene editing system is a CRISPR/Cas system, a transposon-based gene editing system, or a transcription activator-like effector nuclease (TALEN) system.
  • the gene editing system is a CRISPR/Cas system.
  • the gene editing system is a class II CRISPR/Cas system.
  • the gene editing system comprises a single Cas endonuclease or a polynucleotide encoding the single Cas endonuclease.
  • the single Cas endonuclease is Cas9, Cpfl, C2C1 or C2C3.
  • the single Cas endonuclease is Cas9 (e.g., of Streptococcus Pyogenes).
  • the single Cas endonuclease is Cpfl.
  • the Cpfl is AsCpfl (Com Acidaminococcus sp.) or LbCpfl (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.
  • the type II Cas endonuclease is Cas 9 (e.g., of Streptococcus pyogenes).
  • the modified Cas 9 is nickase Cas9, dead Cas9 (dCas9) or eSpCas9.
  • the nickase Cas9 is Cas9 D10A.
  • the dCas9 is D10A or H840A.
  • the 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).
  • Various CRISPR/Cas9 plasmids are publicly available from the Addgene repository (Addgene, Cambridge, MA: addgene . org/ cri spr/) .
  • 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, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616.
  • Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 Al.
  • 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).
  • the agent comprises a small molecule.
  • the small molecule binds to a protein capable of modulating the splicing and/or expression of FMRI or a fragment thereof.
  • the small molecule is an inhibitor of the target protein (e.g., a direct inhibitor, an indirect inhibitor).
  • the small molecule is an activator of the target protein (e.g., a direct activator, and indirect activator).
  • Nonlimiting examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic, organometallic or inorganic compounds.
  • 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 subject comprises a CGG repeat expansion between about 55 and about 200 repeats in the 5’ untranslated region of an FMRI gene.
  • the subject comprises a CGG repeat expansion exceeding 200 repeats in the 5’ untranslated region of an FMRI gene.
  • the subject comprises a CGG repeat expansion that is partially methylated.
  • the subject comprises a CGG repeat expansion that is fully methylated.
  • the subject has an increased level of isoform 12 of FMRI, a decreased level of isoform 1 of FMRI, or a combination thereof.
  • the subject has one X chromosome and one Y chromosome. In some embodiments, the subject has two X chromosomes. In some embodiments, the subject has two X chromosomes and one Y chromosome. In some embodiments, the subject has one X chromosome and two Y chromosomes.
  • the subject is a human male. In some embodiments the subject is human female.
  • the subject is at least about 1 month of age, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18 or 21 months of age, or 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.
  • the subject is about: 1-100, 1-80, 1-60, 1-30, 1-24, 1-20, 1-18, 1-12, 1-10, 1-8, 1- 6, 2-100, 2-80, 2-60, 2-30, 2-24, 2-20, 2-18, 2-12, 2-10, 2-8, 2-6, 3-100, 3-80, 3-60, 3-30, 3-24, 3-20, 3-18, 3-12, 3-10, 3-8, 3-6, 4-100, 4-80, 4-60, 4-30, 4-24, 4-20, 4-18, 4-12, 4-10, 4-8, 4-6, 5-100, 5-80, 5-60, 5-30, 5-24, 5-20, 5-18, 5-12, 5-10, 5-8, 6-100, 6-80, 6-60, 6-30, 6-24, 6-20, 6- 18, 6-12, 6-10, 8-100, 8-80, 8-60, 8-30, 8-24, 8-20, 8-18, 8-12, 10-100, 10-80, 10-60, 10-30, 10-
  • the subject is 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, 50, 55, 60, 65, 70, 75, 80 or 100 years of age.
  • the subject is about 12-38 years of age.
  • the subject is a fetus.
  • the subject is a neonatal subject.
  • the subject is 18 years of age or older, e.g., 18 to less than 40 years of age, 18 to less than 45 years of age, 18 to less than 50 years of age, 18 to less than 55 years of age, 18 to less than 60 years of age, 18 to less than 65 years of age, 18 to less than 70 years of age, 18 to less than 75 years of age, 40 to less than 75 years of age, 45 to less than 75 years of age, 50 to less than 75 years of age, 55 to less than 75 years of age, 60 to less than 75 years of age, 65 to less than 75 years of age, 60 to less than 75 years of age, 40 years of age or older, 45 years of age or older, 50 years of age or older, 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older or 90 years of age or older.
  • the subject is 50 years of age or older. In some embodiments, the subject is a child. In some embodiments, the subject is 18 years of age or younger, e.g., 0-18 years of age, 0-12 years of age, 0-16 years of age, 0-17 years of age, 2-12 years of age, 2-16 years of age, 2-17 years of age, 2-18 years of age, 3-12 years of age, 3-16 years of age, 3-17 years of age, 3-18 years of age, 4-12 years of age, 4-16 years of age, 4-17 years of age, 4-18 years of age, 6-12 years of age, 6-16 years of age, 6-17 years of age, 6-18 years of age, 9-12 years of age, 9-16 years of age, 9-17 years of age, 9-18 years of age, 12-16 years of age, 12-17 years of age or 12-18 years of age.
  • 0-18 years of age 0-12 years of age, 0-16 years of age, 0-17 years of age, 2-12 years of age, 2-16 years of age, 2-17 years of
  • the subject is about 2-11, 4-17, 12-18, 18-50, 18-90 or 50-90 years of age.
  • a subject is a human.
  • the human subject has, or is predisposed to have a fragile X-associated disorder.
  • the human subject has, or is predisposed to have, FXS, FXPOI, FXTAS, or a combination thereof.
  • the human subject has, or is predisposed to have FXS.
  • the subject is a human (e.g., about 50 years of age or older) who has, or is predisposed to have, FXTAS.
  • the 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.
  • Nonlimiting 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.
  • the subject is a female with an IQ score of less than 115, 110, 105, 100, 95 or 90.
  • the subject is a male with an IQ score of less than 60, 55, 50 or 45.
  • the 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”).
  • the subject has one or more of the following: intention tremor, parkinsonism, ataxia, memory loss, white matter lesion involving middle cerebellar peduncles, and cognitive decline.
  • 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.
  • the method further comprises assessing the efficacy of the agent (e.g., polynucleotide such as ASO) (outcome measure) for treatment of the fragile X- associated disorder (e.g., FXS) in the subject, comprising assaying a biological sample from the subject for the presence and/or level of FMRI RNA isoform 1, FMRI RNA isoform 12, or a combination thereof.
  • the agent e.g., polynucleotide such as ASO
  • FXS fragile X- associated disorder
  • treating a fragile X-associated disorder includes slowing progression of the fragile X-associated disorder (e.g., FXS), alleviating one or more signs or symptoms of the fragile X-associated disorder (e.g., FXS), preventing one or more signs or symptoms of the fragile X-associated disorder (e.g., FXS), or a combination thereof.
  • Non-limiting examples of treatment benefits include improvements in speech and motor development; a reduction in or prevention of cognitive disabilities, ranging from learning disabilities to intellectual disability; alleviating or preventing physical and medical features such as 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 mitral valve prolapse; reducing or preventing behaviors of hyperactivity, short attention span, hand biting or hand flapping, poor eye contact and social skills, shyness, anxiety, delayed speech and/or motor development, repetitive speech, and/or sensitivity to sensory stimulation (including a hypersensitivity to being touched).
  • treatment may include modulation of or improvement in language, fragile X behaviors, brain activity, clinical impression, inattention, safety, social avoidance, cognition, hyperactivity, executive function, irritability, eye contact, or memory.
  • treatment results in an intelligence quotient (IQ) score of at least about 40, for example, at least about: 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130.
  • treatment results in an IQ score between about: 40-110, 40-100, 50-105, 60-80, 65-90, 70-80, 75-95, or 70-100.
  • treatment results in an IQ score of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130. In some embodiments, treatment results in an increase in IQ score of at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 points. In some embodiments, treatment results in an increase in IQ score of between about: 1-10, 1-15, 2-20, 2- 15, 2-10, 5-15, 5-10, 10-20, or 15-20 points. In some embodiments, treatment results in an increase in IQ score of about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 points.
  • treatment can include reducing or preventing absent or irregular menses, fertility problems, elevated FSH (follicle-stimulating hormone) levels, premature ovarian failure, primary ovarian insufficiency, and/or hot flashes.
  • treating may include reducing or preventing intention tremors, parkinsonism, ataxia, memory loss, white matter lesions involving middle cerebellar peduncles, and/or cognitive decline.
  • treatment may reduce or prevent neuropathy of extremities, mood instability, irritability, explosive outbursts, personality changes, autonomic function problems such as impotence, loss of bladder or bowel functions. Treatment may also include reducing or preventing high blood pressure, thyroid disorders, or fibromyalgia.
  • “Therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • 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.
  • an agent disclosed herein is in a form of a pharmaceutical composition, or a pharmaceutically acceptable salt thereof.
  • 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.
  • “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.
  • a pharmaceutical composition disclosed herein is formulated as a solution.
  • “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.
  • 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.
  • compositions may 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. The concentration will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the mode of administration.
  • a pharmaceutical composition suitable for use in methods disclosed herein further comprises one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable carrier includes, but is not limited to, such as those widely employed in the art of drug manufacturing.
  • the carrier may be a diluent, adjuvant, excipient, or vehicle with which the agent is administered.
  • 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.
  • 0.4% saline and 0.3% glycine may 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 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, e.g., from less than about 0.5%, usually to at least about 1% to as much as 15%, 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 particular mode of administration selected.
  • Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin are described, for example, in e.g., 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 pp 691- 1092, see especially pp. 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 that may be used 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 antioxidants that may be used are ascorbic acid, methionine, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, lecithin, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol and tartaric acid.
  • Non-limiting examples of amino acids that may be used are histidine, isoleucine, methionine, glycine, arginine, lysine, L-leucine, tri-leucine, alanine, glutamic acid, L-threonine, and 2-phenylamine.
  • Non-limiting examples of surfactants that may be used are polysorbates (e.g., polysorbate-20 or polysorbate-80); poly oxamers (e.g., pol oxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; 1 auroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-,
  • Non-limiting examples of preservatives that may be used 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.
  • Non-limiting examples of saccharides that may be used 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.
  • Non-limiting examples of salts that may be used 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, phenylsubstituted 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.
  • the salt is sodium chloride (NaCl).
  • Agents e.g., polynucleotides
  • disclosed 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).
  • an agent disclosed herein e.g., ASO
  • controlled or sustained-release delivery systems e.g., capsules, biodegradable matrices.
  • sustained-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.
  • polynucleotides 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.
  • 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.
  • 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
  • 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.
  • 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 parahydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.
  • Administration of the agent to the subject can be by parenteral or non-parenteral means.
  • an agent disclosed herein e.g., ASO
  • administration refers to taking steps to deliver an agent to a subject, such as a mammal, in need thereof. 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 selfadministration, and indirect administration, including an act of prescribing a drug or directing a subject to consume an agent.
  • one (e.g., a physician) who instructs a subject (e.g., a 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.
  • Administration of an agent can be once in a day or more than once in a day (e.g., twice a day or more).
  • Administration of the agent 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.
  • an agent disclosed herein e.g., polynucleotide such as ASO
  • ASO a polynucleotide
  • This can include intrathecal or intraventricular injections, including the use of a catheter or Ommaya reservoir.
  • agents e.g., drugs
  • an agent disclosed herein e.g., polynucleotide such as ASO
  • the agent e.g., polynucleotide such as ASO
  • the agent 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.
  • the agent e.g., polynucleotide such as ASO
  • the agent e.g., polynucleotide such as ASO
  • the agent e.g., polynucleotide such as ASO
  • an agent disclosed herein e.g, polynucleotide such as ASO
  • the agent e.g., polynucleotide such as ASO
  • 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 e.g., polynucleotide such as ASO
  • Other methods of enhancing bioavailability in the central nervous system after systemic administration will be known to one skilled in the art.
  • a method disclosed herein comprises administering to the subject two or more polynucleotides, for example, 2, 3, 4, or 5 polynucleotides.
  • the two or more polynucleotides are administered together. In other embodiments, the two or more polynucleotides are administered separately.
  • a first polynucleotide disclosed herein comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65.
  • the first polynucleotide comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. In some embodiments, the first polynucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs: 51-65.
  • a second polynucleotide disclosed herein comprises a nucleotide sequence having at least: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65.
  • the second polynucleotide comprises a nucleotide sequence having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. In some embodiments, the second polynucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65.
  • a method disclosed herein comprises administering to a subject a third, fourth, or fifth polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65.
  • ASO polynucleotide
  • the third, fourth, or fifth polynucleotide comprises a nucleotide sequence having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65.
  • the third, fourth, or fifth polynucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65.
  • the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 1, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:2, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:6, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:7, or both.
  • the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 10, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 11, or both.
  • the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:51, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:52, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:56, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:57, or both.
  • the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:60, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:61, or both.
  • an agent e.g., a polynucleotide such as an antisense oligonucleotide, a pharmaceutical composition thereof, or a pharmaceutically acceptable salt of the foregoing
  • an agent e.g., a polynucleotide such as an antisense oligonucleotide, a pharmaceutical composition thereof, or a pharmaceutically acceptable salt of the foregoing
  • a modulator of DNA methylation e.g., an agent that inhibits DNA methylation or promotes DNA demethylation, see for example, the section of “DNA demethylation”
  • a modulator of DNA methylation e.g., an agent that inhibits DNA methylation or promotes DNA demethylation, see for example, the section of “DNA demethylation”
  • mGluR5 modulators e.g., Basimglurant or Mavoglurant
  • GAB AB receptor activator e.g., arbaclofen
  • GABAA or GAB AB receptor activator e.g., acamprosate
  • AMPAkine e.g., AX516)
  • CB1 inhibitor e.g., rimonabant
  • RAS signaling inhibitor e.g., lovastatin
  • STEP inhibitor S6K inhibitor
  • PAK inhibitor e.g., lovastatin
  • the term “combination therapy” refers to the administration of two or more therapeutic agents to treat a disease, disorder or condition described herein. Such administration encompasses co-administration of the 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 reconstituted or diluted to a desired dose prior to administration. Typically, the treatment regimen will provide beneficial effects of a drug combination in treating diseases, conditions or disorders described herein.
  • a method of treatment disclosed herein further comprises administering to the subject a therapeutically effective amount of a DNA-dem ethylating compound or DNA demethylase, prior to, during, or after, administering an agent disclosed herein (e.g., polynucleotide such as an ASO).
  • the method of treatment further comprises administering to the subject a therapeutically effective amount of a DNA- demethylating compound or DNA demethylase after administering an agent disclosed herein (e.g., polynucleotide such as an ASO).
  • 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'-deoxy cytidine, Hydralazine, RG108, procainamide, and SGI- 1027.
  • the DNA-demethylating compound is a nucleoside analogue.
  • the DNA-demethylating compound is a non-nucleoside analogue.
  • the DNA demethylase e.g., DNA methylation modification enzymes Dnmt or Tet (dCas9-Dnmt/Tet) is fused to a catalytically inactivate Cas9.
  • a single guide RNA sgRNA
  • the dCas9-Tetl demethylates the FMRI locus and promoter region when FMRI has an expanded CGG repeat of 200 or more.
  • the DNA-demethylating compound or DNA demethylase is in an amount sufficient to demethylate at least about 5% of an FMRI 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% of the FMRI gene.
  • the DNA-demethylating compound or DNA demethylase is in an amount sufficient to demethylate 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% of an FMRI gene.
  • a DNA demethylase is in an amount sufficient to demethylate 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 m FMRI gene.
  • a DNA-demethylating compound or DNA demethylase is in an amount sufficient to demethylate about 25-50% of an FMRI gene.
  • a method of modulating FMRI splicing and/or expression further comprising contacting the cell with a DNA-demethylating compound or DNA demethylase, prior to, during, or after, contacting the cell with the agent (e.g., polynucleotide).
  • a method of treatment disclosed herein further comprises decreasing (e.g., shortening or deleting) FMRI CGG expansion (e.g., by CRISPR/Cas9 gene editing) in the subject, prior to, during, or after, administering an agent disclosed herein (e.g., polynucleotide such as an ASO).
  • the method of treatment further comprises decreasing (e.g., shortening or deleting) FMRI CGG expansion prior to administering an agent disclosed herein (e.g., polynucleotide such as an ASO).
  • the present disclosure provides a method of modulating FMRI splicing and/or expression in a cell, comprising contacting the cell with an agent (e.g., polynucleotide) under conditions whereby the agent is introduced into the cell, thereby modulates FMRI splicing and/or expression in the cell.
  • an agent e.g., polynucleotide
  • the agent can be any one of the agents disclosed herein.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • FMRI gene increases splicing of isoform 1 (between X chromosome base pairs 147,912,230 and 147,921,933)
  • decreases expression of isoform 12 of the FMRI gene decreases splicing of isoform 12 (between X chromosome between base pairs 147,912,230 and 147,912,728), or a combination thereof.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the agent decreases the splicing and/or expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
  • the agent decreases the splicing of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
  • the agent decreases the expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • isoform 1 or “isol” refers to normal FMRI RNA with exon 1 spliced to exon 2.
  • Isoform 12 or “isol2” refers to missplicing of FMRI RNA, where exon 1 is spliced to a pseudo exon located within intron 1. Isoform 12 would generate a 31-amino acid protein, which probably would have no biological function.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • increases isoform 1 of FMRI by at least about 5% relative to a reference, 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% relative to the reference.
  • the agent e.g., polynucleotide
  • increases isoform 1 of the FMRI gene by about 75%.
  • the agent decreases isoform 12 of FMRI by at least about 5% relative to a reference, 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% relative to the reference.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the level of splicing and/or expression of FMRI or a fragment thereof is measured after the agent is contacted with the cell for at least about 1 day, e.g., 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 months or 6 months.
  • the agent comprises, consists essentially of or consists of any one of the polypeptides, polynucleotides, gene editing systems or small molecules disclosed herein.
  • the agent comprises at least one of the polynucleotides of the disclosure. In some embodiments, the agent comprises two or more of the polynucleotides of the disclosure.
  • the cell is 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, a modified fibroblast, a pluripotent cell, or a cultured cell.
  • a fetal cell e.g., circulating fetal cell
  • a blastomere e.g., circulating fetal cell
  • a trophectoderm cell e.g., a trophectoderm cell
  • a stem cell e.g., induced pluripotent stem cell (iPSC) or derived stem cell
  • a fibroblast e.g., induced pluripotent stem cell (iPSC) or derived stem cell
  • a fibroblast e.g., induced pluripotent stem cell (iPSC) or derived stem cell
  • the cell is an in vitro cell or an ex vivo cell.
  • the cell is an iPSC-derived neuron from a human who has or is predisposed to have FXS, a primary human cell, or a cell line.
  • the cell is a cell of any one of the subjects disclosed herein.
  • the cell of the subject is allogeneic.
  • the cell of the subject is autologous or syngeneic.
  • the present disclosure provides a method of reducing CGG triplet repeat expansion in FMRI 5’ UTR in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide disclosed herein, an agent that modulates DNA methylation, or a combination thereof) under conditions whereby the agent is introduced into the cell, thereby reducing CGG triplet repeat expansion in the cell.
  • an agent e.g., a polynucleotide disclosed herein, an agent that modulates DNA methylation, or a combination thereof
  • the agent can be any one of the agents disclosed herein.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • increases expression of isoform 1 of the FMRI gene increases splicing of isoform 1 (between X chromosome between base pairs 147,912,230 and 147,921,933), decreases expression of isoform 12 of WIQ FMRI gene, decreases splicing of isoform 12 (between X chromosome between base pairs 147,912,230 and 147,912,728), or a combination thereof.
  • the agent e.g., a polynucleotide disclosed herein, an agent that modulates DNA methylation, or a combination thereof
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the agent decreases the splicing and/or expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
  • the agent decreases the splicing of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
  • the agent decreases the expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • isoform 1 or “isol” refers to normal FMRI RNA with exon 1 spliced to exon 2.
  • Isoform 12 or “iso!2” refers to missplicing of FMRI RNA, where exon 1 is spliced to a pseudo exon located within intron 1. Isoform 12 would generate a 31-amino acid protein, which probably would have no biological function.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the agent decreases isoform 12 of FMRI by at least about 5% relative to a reference, 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% relative to the reference.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the agent decreases CGG triplet repeat expansion in FMRI 5’ UTR in the cell by at least about 5% relative to a reference, 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% relative to the reference.
  • the agent e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof
  • the level CGG triplet repeat in FMRI 5’ UTR in the cell is measured after the agent is contacted with the cell for at least about 1 day, e.g., 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 months or 6 months.
  • the agent comprises, consists essentially of or consists of any one of the polypeptides, polynucleotides, gene editing systems or small molecules disclosed herein. [00310] In some embodiments, the agent comprises at least one of the polynucleotides disclosed herein. In some embodiments, the agent comprises two or more of the polynucleotides disclosed herein.
  • the cell is 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, a modified fibroblast, a pluripotent cell, or a cultured cell.
  • a fetal cell e.g., circulating fetal cell
  • a blastomere e.g., circulating fetal cell
  • a trophectoderm cell e.g., a trophectoderm cell
  • a stem cell e.g., induced pluripotent stem cell (iPSC) or derived stem cell
  • a fibroblast e.g., induced pluripotent stem cell (iPSC) or derived stem cell
  • a fibroblast e.g., induced pluripotent stem cell (iPSC) or derived stem cell
  • the cell is an in vitro cell or an ex vivo cell.
  • the cell is an iPSC-derived neuron from a human who has or is predisposed to have FXS, a primary human cell, or a cell line.
  • the cell is a cell of any one of the subjects disclosed herein.
  • the cell of the subject is allogeneic.
  • the cell of the subject is autologous or syngeneic.
  • the present disclosure provides a polynucleotide capable of reducing expression of an aberrant FMRI gene product.
  • the polynucleotide is any one of the polynucleotides, modified or unmodified, disclosed herein. In some embodiments, the polynucleotide is any one of the modified polynucleotides disclosed herein.
  • the present disclosure provides an agent that modulates splicing and/or expression of FMRI gene.
  • the agent is a polynucleotide.
  • the agent is any one of the modified polynucleotides disclosed herein.
  • the present disclosure provides a pharmaceutical composition, comprising any one of the agents described herein, and one or more pharmaceutically acceptable excipients, diluents, or carriers.
  • RNAlater® RNA Stabilization Solution (Thermo Fisher Scientific, Waltham, MA). The residual RNAlater® was expelled from the LeukoLOCKTM filter and the filters were capped and stored at -80°C.
  • RNA sample was sent to Novogene (Beijing, China) for a directional mRNA library preparation using polyA enrichment.
  • the libraries were sequenced on the NovaSeq platform to generate paired end, 150bp reads.
  • the ratio between reads including or excluding exons also known as “Percent Spliced In” (PSI) indicates how efficiently sequences of interest are spliced into transcripts.
  • PSI Percent Spliced In
  • FDR False Discovery Rate
  • the Percent Spliced In (PSI) levels or the exon inclusion levels were calculated by rMATS using a hierarchical framework. To calculate the difference in PSI between genotypes, a likelihood-ratio test was used. AS events with an FDR ⁇ 5% and
  • Isol2_lReverse (Isol2_l R): 5’ CAGTGGAGCTCTCCGAAGTC 3’ (SEQ ID NO: 1
  • Isol l Forward (Isol l F): 5’ AGAAGATGGAGGAGCTGGTG 3’ (SEQ ID NO: 1
  • a white blood cell line derived from an FXS patient who expressed isol2 was transfected with antisense oligonucleotides (ASOs) pairs 705/705, 709/710, and 713/714.
  • RNA was extracted 48 hours later and subjected to RT-qPCR to detect isol (primers Isol l Forward/ Isol l Reverse) or total FMRI isoforms (isol + iso 12) (primers Exonl Forward and Exonl Reverse) and isol2 (primers Isol l Forward/Isol2_l Reverse). Each assay was performed in triplicate and normalized against non-transfected cells.
  • Lymphoblastoid cell lines were obtained from Coriell Institute from two FXS individuals (GM07365 (FXS1), GM06897(FXS2)) and two typically developing control males (GM07174 (WT3), GM06890 (WT4)).
  • Cells were cultured in RPMI 1640 medium (Sigma- Aldrich, St. Louis, MO), supplemented with 15% fetal bovine serum (FBS) and 2.5% L- glutamine at 37°C with 5% CO2 in T25 flasks.
  • FBS fetal bovine serum
  • Fibroblast cells derived from patient skin samples were cultured in DMEM (15-017- CV) media supplemented with 10% FBS and lx antibiotic-antimitotic, lx L-glutamine in T25 culture flasks at 37°C with 5% CO2.
  • ASOs Antisense oligonucleotides
  • Membranes were washed three times for 10 minutes with 1XTBST and incubated with antirabbit or anti-mouse secondary antibodies (Jackson, 1 : 10000) at room temperature for Ihour. Membranes were washed three times for 10 minutes with 1XTBST, developed with ECL-Plus (Piece), and scanned with GE Amersham Imager.
  • FXS is caused by a CGG triplet repeat expansion in a single gene, FMRI, which resides on the X chromosome.
  • FMRI CGG triplet repeat expansion in a single gene
  • the FMRI gene is methylated and thereby transcriptionally inactivated.
  • the loss of the FMRI gene product, the protein FMRP, is the cause of the disorder.
  • FIG. 1 shows that there was robust expression of FMRI in all 7 typically developing (TD) individuals. There was also FMRI expression in FXS patients 1-5 (+FMRR), but no FMRI expression was detected in FXS patients 6-10 (-FMRF). Therefore, 50% of FXS individuals express FMRI RNA, likely due to incomplete methylation.
  • FMRI exon 1 (chrX: 147,911,919 - 147,912,230) is spliced to FMRI exon 2 (chrX: 147,921,933 - 147,921,985), which produces “isoform 1” or “Isol.”
  • FMRI exon 2 (chrX: 147,921,933 - 147,921,985)
  • FIG. 2 shows an expanded view of FMRI exon 1 and intron 1. Note that although none of the typically developing individuals expresses isoform 12, the five FXS patients who expressed FMRI RNA (+FMR1) all express FMRI isoform 12.
  • Isoform 12 is derived from missplicing, detected only when there was a CGG repeat expansion and when there was incomplete methylation. Isoform 12 does not produce full-length or functional FMRP. Instead, isoform 12 generates a 30-amino acid protein, which probably has no biological function.
  • FMRI RNA not only can be used for diagnosing an individual as having FXS, or having a propensity to develop FXS, but also can be used for stratifying FXS individuals.
  • the identification FMRI RNA isoform 12 enables stratification of FXS individuals into two subpopulations, those who express isoform 12 and those who do not.
  • FMRI RNA, such as isoform 12 may provide novel therapeutic targets for FXS.
  • a reduction of aberrant splicing to isoform 12, alone or commensurate with an increase of proper splicing to isoform 1 may increase FMRP levels and thereby mitigate FXS in patients who express FMRI RNA.
  • FIG. 3 shows a non-limiting example approach for blocking isoform 12 production, increasing isoform 1 production, and increasing FMRP levels using antisense oligonucleotides (ASOs).
  • ASOs were designed to be complementary to regions within intron 1 and upstream of isoform 12, the junction spanning intron 1 and isoform 12, or within isoform 12 (Table 1).
  • FIG. 4 shows a schematic illustration of FMRI isol, iso!2, and relative positions of ASOs complementary to intron 1 (704, 705, and 706), the junction of intron 1 and isol2 (707, 708, 709, and 710), and within isol2 (711, 712, 713, and 714).
  • ASOs 704-714 were chemically modified to increase the nuclease resistance of the ASOs (e.g., reduce RNase H cleavage), increase cellular uptake, and enhance base-pairing capabilities (reduce off-target effects).
  • the ribose groups comprised 2’ -O-(2 -methoxy ethyl) (MOE), and the phosphate groups comprised a phosphorothioate.
  • ASOs of the disclosure may be used singly or in combination.
  • a WBC line derived from a FXS patient who expressed isol2 was transfected with ASOs 704/705, 709/710 or 713/714.
  • RNA was extracted 48 hours later and subjected to RT-qPCR to detect isol (primers Isol l Forward and Isol l Reverse) and isol2 (primers Isol l Forward and Isol2_l Reverse).
  • Each assay was performed in triplicate.
  • FIG. 5 illustrates that ASOs 713 and 714, both of which are complementary to internal regions of isol2, reduced the isol2 level by -30% and increased the isol level by -75%.
  • ASOs may be a potent and specific therapeutic to treat a subpopulation of FXS individuals that express isoform 12.
  • agents such as ASOs, directed against FMRI isoform 12 may provide novel therapeutic treatment to FXS by reducing improper splicing to isoform 12, increasing proper splicing of isoform 1 and increasing FMRP levels.
  • This approach is entirely novel in the fragile X field. It is predicted to be a significant improvement over the prior art because all other treatments for FXS elicit only modest improvements at best. Additionally, all other therapies treat FXS patients as one large cohort, whereas these studies have identified a particular subpopulation - those who express isol2 - and may be particularly amenable to therapeutics, such as ASOs that target isol2.
  • Example 3 Experiments illustrated in Example 3 have been and will be performed in cells with different methylation status.
  • FIG. 6A shows RT-qPCR data from a fully methylated FXS cell line (FXS1, GM07365).
  • FXS1, GM07365 The FMRI locus in this cell line is silenced and thus the FMRI RNA (isol and iso 12) and FMRP protein levels are very low compared to the FXS2 cell line with an unmethylated FMRI gene.
  • Treatment with the demethylating agent 5-AzaC resulted in demethylation of the FMRI gene to allow expression of the FMRI RNA isoforms.
  • FIG. 6B demonstrates an increase in FMRI isol upon 5-AzaC treatment (p ⁇ 0.05) and a further increase when the ASO treatment (80nM of both antisense oligonucleotides 713 and 714) was combined with 5-AzaC treatment (p ⁇ 0.05).
  • FIG. 7 A shows western blot data for FXS1 LCL cell line in duplicates, demonstrating an increase in FMRP after treatment with IpM 5-AzaC and ASO treatment (80nM of both antisense oligonucleotides 713 and 714) when compared to DMSO or 5-AzaC only treated samples.
  • the mouse brains hippocampus tissue
  • the FMRP protein from mouse tissues ran higher on the gel compared to the human FMRP.
  • the bottom panel represents GADPH protein levels used to normalize the protein amounts loaded in each sample.
  • FIG. 7B shows quantification of the FMRP protein levels relative to GAPDH protein levels as seen on the western blot in FIG. 7A.
  • FIG. 8 A is a table demonstrating the CGG repeats in the FMRI RNA 5’ UTR from three healthy males and three premutation carrier males for FXS.
  • the premutation carriers had 55-200 CGG repeats in the 5’UTR of FMRI gene, whereas greater than 200 CGG repeats would lead to FXS, and less than 55 CGG repeats are usually present in healthy individuals.
  • Premutation carriers have a propensity to develop FXTAS (Fragile X-associated tremor/ataxia syndrome) after the age of 50yrs.
  • FIG. 8B shows RT-qPCR data demonstrating the presence of similar FMRI isol levels in fibroblast cells from all six individuals normalized to GN 7797/ RNA levels.
  • FIG. 8C shows the presence of increased FMRI isol2 levels in individual Pl compared to the other premutation carriers and healthy control samples. All premutation carriers expressed similar FMRI isol levels as compared to the healthy controls. However, only individual Pl with higher CGG repeats (140, see FIG. 8 A) expressed FMRI iso 12.
  • ASO treatment will be determined in an animal model. Neural progenitor cells, derived from human FXS patients with partially methylated FMRI and isol2 expression, will be injected into NOD-.sc/t/ IL2Ry tlu11 mouse pups as described by Windrem etal., J Neurosci 34: 16153-16161 (2014) and Liu etal., Cell 172:979-92 (2016). Modified ASOs, such as those described above will be injected into the brain or via intraperitoneal injection (IP). The RNA will be extracted from the brains, and human FMRI isol and isol2 will be quantified by RT-qPCR. This experiment will determine the safety and efficacy of ASO treatment in inhibiting FMRI isol2 production and promoting isol formation in an animal model. FMRP in human neurons will be assessed by immunocytochemistry.
  • Fragile X Syndrome is a neuro-developmental disorder causing a range of maladies including intellectual disability, speech and developmental delays, social deficits, repetitive behavior, attention deficits, and anxiety.
  • FMRI' Fragile X Messenger Ribonucleoprotein 1
  • FMRP Fragile X Messenger Ribonucleoprotein
  • FMRP is an RNA-binding protein that interacts with >1000 mRNAs in the mouse brain and human neurons, predominantly through coding region associations (1-3).
  • FMRP inhibits protein synthesis (4), subsequent high-resolution methods showed that FMRP promotes as well as inhibits translation (5-8).
  • One mechanism by which FMRP inhibits translation is stalling ribosome translocation on mRNAs (9, 10).
  • mRNAs associated with FMRP-stalled ribosomes were identified, one of which encodes SETD2, an epigenetic enzyme that trimethylates histone H3 lysine 36 (H3K36me3) (11).
  • SETD2 was elevated in Fmrl -deficient hippocampus, which resulted in an altered H3K36me3 chromatin landscape.
  • H3K36me3 resides in gene bodies and influences alternative pre-mRNA splicing (12), and indeed multiple mRNAs were mis-spliced in Fmrl -deficient mouse hippocampus. Many of these mis-splicing events were also detected in the human postmortem autism spectrum disorder (ASD) brain and blood tissues 7"/-7S), indicating a convergence of FXS and ASD (11, 13).
  • ASD human postmortem autism spectrum disorder
  • RNA mis-splicing might also be prevalent in human FXS patient tissues (blood and brain). Accordingly, leukocytes were isolated from freshly obtained blood from 29 FXS males and 13 typically developing (TD) age-matched males, and RNA sequencing was performed. The analysis revealed widespread and statistically robust mis-regulation of alternative splicing and RNA abundance of greater than 1,000 mRNAs. Mis-regulated RNA expression and processing in FXS postmortem brain were also found.
  • RNA-seq data unexpectedly revealed that FMRI RNA was expressed in 21 of 29 FXS leukocyte samples, some nearly as high as FMRI transcript levels from TD individuals. Because all FXS samples were from individuals with >200 CGG repeats, this was a surprising result because the FMRI locus, which was purported to be silent under these conditions, was transcriptionally active in patients even when the gene appeared to be fully methylated in standard assays. However, the highest FMRI RNA expressing FXS individuals were mosaic (CGG repeat number mosaicism or partial methylation of a full expansion).
  • Fibroblasts from some FXS premutation i.e., -55-200 CGG repeats
  • FXS premutation i.e., -55-200 CGG repeats
  • FMRI -217 as well as full-length FMRI RNA, indicating that mis-splicing may be widespread in other disorders linked to CGG expansions in FMRI.
  • ASO treatment may offer a novel therapeutic approach to mitigate FXS.
  • Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders.
  • the Fragile X Messenger Ribonucleoprotein 1 FMRI gene was transcribed in >70% of the FXS tissues, in many instances even when the gene was fully methylated.
  • FMRI RNA itself was mis-spliced in a CGG expansion-dependent manner to generate the little-known FMR1-2V1 RNA isoform, which is comprised of FMRI exon 1 and a pseudo-exon in intron 1.
  • FMRI -217 was also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissue. It was shown that in cells aberrantly expressing mis-spliced FMRI, antisense oligonucleotide (ASO) treatment reduced FMRI -217, rescued full-length FMRI RNA, and restored Fragile X Messenger Ribonucleoprotein (FMRP) to normal levels. Notably, FMRI gene reactivation in transcriptionally silent FXS cells using 5-aza-2'-deoxycytidine (5-AzadC), which prevented DNA methylation, increased FMRI -217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescued full-length FMRI expression and restored FMRP. These findings indicate that in FXS individuals (e.g., those expressing FMRI -217), ASO treatment may offer a new therapeutic approach to mitigate the disorder.
  • ASO antisense oligonucleotide
  • the Adaptive Behavior Composite (ABC) standard score was the measure of overall adaptive functioning based on scores assessing the following domains: communication, daily living skills, and socialization.
  • FXS patients were aged 16-38 years with FXS phenotypes, a z-deviation IQ range of 20-52 and ABC standard score range of 20-41.
  • Age matched TD individuals for the study were aged 22-29 with a normal IQ and no known neuropsychiatric conditions.
  • CGG repeat size determination in the 5’ UTR of the FMRI gene DNA isolated from whole blood was analyzed using the Asuragen FMRI AmplideX PCR Kit. Methylation status was determined using the Asuragen FMRI methylation PCR Kit and/or Southern blot analysis.
  • FMRP levels were quantified by generating dried blood spots (DBS) from the samples. To generate DBS, 12- 50 pl spots were put on each blood card and allowed to dry. The blood cards were then stored at -80°C. Discs were punched using a 6-mm punch and incubated in lysis buffer. Extracted sample was centrifuged, and FMRP was quantified using the Luminex Microplex immunochemistry assay. FMRP levels were normalized to 1,000 WBCs per sample. Additionally, FMRP levels were also quantified by using peripheral blood mononuclear cell (PBMC) samples. PBMCs were isolated from whole blood using Cell Preparation (CPT) blood tubes.
  • PBMC peripheral blood mononuclear cell
  • Isolated PBMC were lysed and quantified for total protein concentration using a spectrophotometer, and FMRP was quantified using a Luminex Microplex immunochemistry assay. FMRP levels were normalized to total protein. Both methods produced comparable levels of FMRP in the samples assessed.
  • RNA Extraction and Sequencing of Tissue Samples from FXS and TD Individuals [00373] Leukocytes
  • RNAlater® phosphate buffered saline
  • PBS phosphate buffered saline
  • RNAlater® 3 ml of phosphate buffered saline
  • the residual RNAlater® was expelled from the LeukoLOCKTM filter, and the filters were capped and stored in -80°C.
  • the filters were thawed at room temperature for 5 minutes, and then the remaining few drops of RNAlater were removed.
  • the filter was flushed with 4 ml of TRIzolTM LS Reagent (ThermoFisher Scientific #10296028), and the lysate was collected in a 15-ml tube.
  • RNA integrity number (RIN) >7.3 was assessed using a 5300 Fragment Analyzer instrument. Three milligrams (mgs) of RNA sample were used for directional mRNA library preparation using polyA enrichment (Novogene Co), and the libraries were sequenced on the NovaSeq platform to generate paired end, 150-bp reads at a sequencing depth of 60-90 million reads per sample.
  • RNA was primed with oligo(dT)2o to generate cDNA with a QuantiTect cDNA synthesis kit (Qiagen, #205311) using random hexamers (Table 3).
  • qPCR was performed using the iTaqTM Universal SYBR® Green Supermix (BIO-RAD #1725122) on a QuantStudio 3 qPCR machine in duplicate.
  • FASTQ files were uploaded to the DolphinNext platform (55) at the UMass Chan Medical School Bioinformatics Core for mapping and quantification. The reads were subjected to FastQC (vO.11.8) analysis, and the quality of reads was assessed. Reads were mapped to the genome assembly GRCh38 (hg38) version 34 using the STAR (v2.5.3a) aligner. Gene and isoform expression levels were quantified by salmon vl.5.2.
  • Lymphoblast cell lines were obtained from Coriell Institute from two FXS individuals (GM07365 (FXS1), GM06897(FXS2)) and two typically developing control males (GM07174 (WT3), GM06890 (WT4)).
  • 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.
  • FBS fetal bovine serum
  • the skin explants were removed from the culture flask, and fibroblasts were trypsinized and spread evenly in the flask. The media were changed after overnight incubation with trypsin. Fibroblast culture medium was added (complete media (500 ml DMEM (15-017- CV) with 10% FBS and IX antibiotic-antimitotic, 5 ml lx L-glutamine)) twice a week to cells in a T25 culture flasks at 37° C with 5% CO2.
  • Fibroblast cell lines were obtained from Coriell Institute from two FXS individuals (GM05131, and GM07072). A control fibroblast line derived from a skin sample of a typically developing male was used. Cells were cultured in DMEM medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS) and 2.5% L-glutamine, at 37°C with 5% CO2.
  • FBS fetal bovine serum
  • ASOs were synthesized on a Dr. Oligo 48 synthesizer. 2’-O-methoxyethyl (MOE)- modified phosphoramidites were coupled for 8 minutes. Oligonucleotides were deprotected in concentrated aqueous ammonia (30% in water) at 55°C for 16 hours and characterized by liquid chromatography-mass spectrometry. Final desalting was effected by diafiltration (3x water wash) in a 3-kDa cutoff Amicon centrifugal filter.
  • MOE methoxyethyl
  • ASOs Antisense oligonucleotides
  • LCL cell lines were dissolved in ultrapure distilled water to a final concentration of 10 pM. Before use, the ASOs were heated to 55°C for 15 minutes and cooled at room temperature. ASOs were added, individually or in combinations, to LCL cell lines at a final concentration of 80 nM or 160 nM using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778030) and incubated at 37°C with 5% CO2 for 16 hours in reduced serum medium. RPMI 1640 medium (Sigma- Aldrich), supplemented with 15% fetal bovine serum (FBS) was added for a total of 72 hours. The cells were collected after 72 hours of ASO treatment for RNA and protein extraction.
  • FBS fetal bovine serum
  • Proteins (10 pg) were diluted in SDS-bromophenol blue reducing buffer with 40 mM DTT and analyzed using western blotting with the following antibodies: FMRP (Millipore, mAb2160, 1 : 1,000), FMRP (Abeam, ab 17722, 1 : 1,000) and GAPDH (14C10, Cell Signaling Technology, mAb 2118, 1 :2,000), diluted in IX TBST with 5% non-fat milk.
  • Membranes were washed three times for 10 minutes with 1XTBST and incubated with anti-rabbit or anti-mouse secondary antibodies (Jackson, 1 : 10,000) at room temperature for 1 hour. Membranes were washed three times for 10 minutes with 1XTBST, developed with ECL-Plus (Piece), and scanned with GE Amersham Imager.
  • the PBMCs were rinsed with IX Dulbecco’s phosphate buffered saline without calcium or magnesium (D-PBS) (Invitrogen #14190-094).
  • the PBMC pellet was resuspended in 250 pL ice-cold D-PBS with protease inhibitors.
  • FMRP levels in PBMCs were quantified using a Luminex Microplex immunochemistry assay. Chromatin isolation and sequencing were performed as previously described (11). Briefly, the cells were cross-linked with 1% formaldehyde and quenched with 150 mM glycine. After centrifugation at 2,000 g for 10 minutes at 4°C, the cells were lysed.
  • the nuclei were harvested by centrifugation at 2,000 g for 5 minutes at 4°C.
  • the nuclei were lysed by incubating for 20 minutes on ice in nuclear lysis buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 mM EGTA). 0.5% SDS was added, and the samples were sonicated on a Bioruptor® sonicator at high power settings (sonication: 30 seconds on, 90 seconds off) for 9 cycles of 15 minutes each at 4°C.
  • the samples were centrifuged and diluted to adjust the SDS concentration to ⁇ 0.1%. 10% of each sample was used as input.
  • the library was PCR amplified using multiplexing barcoded primers.
  • the libraries were pooled with equal molar ratios, denatured, diluted, and sequenced with NextSeq 500/550 High Output Kit v2.5 (Illumina, 75-bp paired-end runs) on a Nextseq500 sequencer (Illumina). [00404] ChlP-Seq analysis
  • FXS individuals 1-21 expressed relatively high FMRI levels (with a cutoff of 0.6 transcript per million (TPM)) (H FMRI), compared to FXS individuals 22-29 who expressed low or undetectable FMRI levels (L FMRI (Table 2 and FIGs. 9A-9B).
  • RNA reads in intron 1 of FMRI displayed strong RNA reads in intron 1 of FMRI (thick-lined black box in FIG. 9A, enlarged in FIG. 9B).
  • RNA reads in this intronic region were not detected in any TD individuals even though FMRI RNA was strongly expressed (FIGs. 9A-9B).
  • the FA7 7 locus expresses multiple alternatively spliced RNA isoforms (Table 3).
  • the RNA reads detected in FMRI intron 1 correspond to the second exon of the FMRI -217 RNA isoform.
  • FMR1-2V1 (ENST00000621447.1) is a 1.8-kb transcript comprised of two exons, and is predicted to encode a 31-amino acid polypeptide (Table 3). Notably, most of the total FMRI RNA in the FXS samples was comprised of the aberrantly spliced FMRI-211 transcript, which was absent in samples from TD individuals (Table 2). TPMs of all 14 FMRI isoforms detected in the TD and FXS patient samples were obtained (data not shown).
  • RT-PCR was used to detect the FMR1-2Y1 isoform in the FXS leukocyte samples (reverse transcription primed with oligodT(20)), and the amplified product was sequenced using primers specific to the FMR1-2Y1 exon-exon junction. Aligning this sequence to FMRI confirmed that this transcript is polyadenylated and is a spliced product of FMRI exon 1 and FMR1-2Y1 exon 2 (FIG. 9C).
  • Table 2 shows normalized gene counts (transcripts per million, TPM) obtained from RNA-seq data analysis for total FMRI (all isoforms), FMR1-2Q5 (encoding the full-length, 632 amino acid FMRP), FMR1-2Y1 (a mis-spliced RNA), and FXR2, a paralogue of FMRI.
  • Table 4 presents determinations of methylation status of the FMRI gene (by PCR), FMRP levels (ng/pg protein), CGG repeat number, FMRI-217, full-length FMRI -205, all detected FMRI isoforms, and IQ (Stanford-Binet test).
  • FMRI gene methylation in percent as determined by PCR analysis; FMRP levels: ng/ug total protein; FMRI', all isoforms; IQ: Stanford-Binet; N/A: not available.
  • Table 5 presents correlation coefficients for pairwise comparisons of the measurements noted above. Methylation of the FMRI gene is negatively correlated with FMR1- 217 and FMRI -2Q5 expression. More interesting is the moderately positive correlation of IQ with FMRP protein levels. Somewhat surprisingly, FMR1-2Q5, which encodes full-length FMRP, has no correlation with IQ. However, it is noted that while FMR1-2Q5 encodes the complete 632- amino acid FMRP, other FMRI isoforms, which vary in abundance, encode truncated FMRP proteins (Table 3). Without presupposing functionality of truncated FMRP proteins, the canonical FMRI isoform, FMR1-2Q5, was used for further comparisons.
  • FIG. 10 displays a 3- dimensional comparison of all the parameters noted above.
  • the inset shows that some FXS patients with a fully methylated FMRI gene expressed FMRI RNA and FMRP. Taken together, these results show several important findings. First, the FMRI locus is frequently transcribed even when the FMRI gene with a full CGG expansion is fully methylated. Second, FMRP levels in WBCs are positively correlated with IQ.
  • the negative correlation of FMRI -217 with IQ suggests that the process of mis-splicing, the 31 -amino acid polypeptide derived from FMR1- 217, the FMR1-2Y1 RNA itself, or a combination thereof (e.g., all three), impart some toxic effect manifest in the brain (e.g., IQ).
  • FMR1-2V1 is Expressed in Human FXS and Pre-Mutation Carrier Postmortem Brain.
  • FMRI -217 is expressed in FXS brain
  • publicly available RNA-seq data of post-mortem frontal cortex tissues from FXS individuals (CGG repeats >200), FXS carriers (CGG repeats 55-200), and TD individuals (CGG repeats ⁇ 55) (16) were analyzed.
  • FMRI RNA (TPM) levels were highest in pre-mutation carriers (Table 6).
  • the FXS sample UMB5746 which displayed CGG repeat number mosaicism, displayed high levels of FMRI RNA (Table 6 and FIG. 11A) and to a lesser extent, FMRP (16).
  • FMR1-2Y1 RNA may only be expressed in the brains of a subset of FXS individuals and premutation carriers.
  • Table 6 shows sample information for postmortem FXS frontal cortex, premutation FXS carriers and TD individuals (derived from (16)).
  • RNA-seq datasets GSE107867 (NIH samples) and GSE117776 were reanalyzed for DGE and DAS.
  • the TPM for FMRI RNA in the samples is shown.
  • a BLAST analysis showed that /’A7 7-217 aligned only with intron 1 of FMRI and with no other region of the genome. Additional data showed unequivocally that /’A7 7-217 is derived from FMRI, and that its synthesis is dependent the CGG expansion in this gene.
  • Vershkov et al. (17) used CRISPR/Cas9 to delete the CGG expansion from FMRI in FXS iPSC- derived neural stem cells (NSCs). Additional FXS NSCs were incubated with 5-AzadC, a nucleoside analogue that prevents DNA methylation. RNA sequencing from these samples, as well as from FXS NSCs incubated with vehicle, was then performed. The RNA-seq data from Vershkov et al. (17) was reanalyzed, some of which is presented in FIG. 1 IB, and FMRI transcript quantification (TPM) in Table 7. RNA-seq reads corresponding to FA7 7-2 I 7 were clearly evident in the FXS-NSCs incubated with 5-AzadC, but not in the other samples.
  • TPM FMRI transcript quantification
  • the CGG edited cells which were isogenic to the unedited FXS NSCs, had no FMR1- 217 reads, but instead robust expression of full-length FMRI.
  • Quantification of the RNA-seq reads showed strong total FMRI and FMR1-2Q5 expression in the CGG-edited and 5- AzadC-treated cells but not in vehicle-treated cells. More importantly, s ⁇ xon FMRl-217 expression was observed only in the 5-AzadC-treated cells. Therefore, FMRI-217 is derived from the FMRI locus and requires a CGG expansion.
  • FMRI-211 RNA was significantly reduced in the FXS individuals compared to that in the TD individuals.
  • TD typically developing
  • 3 or 4 of the 6 FXS individuals expressed varying levels of the FMRI full-length RNA as well as /’ 7-2 I 7 RNA (1031-09LZ, 1001-18DL and 1033-08WS) (FIG. 11D).
  • FMRI -217 RNA was detected in only one of the two premutation carrier samples.
  • FXS carrier tissue CGG repeats between 55-200
  • skin biopsies were obtained from 3 additional premutation carriers and 3 TD individuals (FIG. 1 IE).
  • the skin samples were cultured in vitro to generate fibroblast cell lines for RNA analysis.
  • FMRI -217 was detected in one premutation carrier (C172) with 140 CGG repeats but not in samples with 77 or 98 CGG repeats (FIG. 1 IE).
  • FMRI -217 was detected in one premutation carrier (C172) with 140 CGG repeats but not in samples with 77 or 98 CGG repeats (FIG. 1 IE).
  • FMR1-2V1 may be linked to the number of CGG repeats in the FMRI gene.
  • FMRI -217 RNA is expressed in lymphoblast cell cultures from FXS individuals [00419] DNA methylation of the CpG island upstream of the FMRI gene promoter in FXS individuals (MFM, methylated full mutation) contributes to transcriptional silencing of the locus and loss of FMRP. FMRI transcription can be reactivated by treatment with the nucleoside analogue 5-AzadC (5-aza-2 '-deoxy cytidine), which inhibits DNA methylation (21, 22). Consequently, whether re-activating FMRI transcription in cells from FXS individuals with a completely silenced and presumably fully methylated FMRI locus results in FMRI -217 expression was investigated.
  • 5-AzadC 5-aza-2 '-deoxy cytidine
  • lymphoblast cell lines derived from a FXS individual with a fully methylated locus (MFM) that was transcriptionally inactive (FXS1, GM07365), a FXS individual with a presumably partially methylated locus (UFM) that expressed some FMRI RNA (FXS2, GM06897), and two typically developing individuals (TD1, GM07174, and TD2, GM06890), were used (all samples from Coriell Institute, NJ, USA) (FIG. 12A). Western blot analysis showed that modest levels of FMRP were detected in FXS2, but not FXS1 cell lines.
  • FMRP was strongly expressed in TD1 and TD2 cells (ratios of FMRP/GAPDH relative to TD2 were shown below the blot) (FIG. 12A). Similar ratios of FMRP protein expression in these cell lines were obtained by the Luminex Microplex immunochemistry assay (FMRP levels in ng FMRP/pg total protein) (FIG. 12A). Using RT-qPCR, it was found that FMRI -217 RNA is expressed in FXS2 LCLs and comprises 56% of the total FMRI RNA compared to only 9% in TD cells (FIG. 12B). It is noteworthy that although total FMRI RNA levels in FXS2 cells were similar to those in TD cells, FMRP levels were much lower (FIGs. 12A-12B).
  • FXS1 and FXS2 cell lines were treated with 5-AzadC, and then FMRI RNA and FMRP levels were measured (FIG. 12C).
  • FXS1 cell line treatment with 5-AzadC for seven days resulted in significant increases of both full-length FMRI and FMRI -217 RNAs relative to DMSO-treated cells (FIG. 12D).
  • FXS2 cells 5-AzadC treatment resulted in an increase of only full-length FMRI RNA (FIG. 12E).
  • 5-AzadC treatment induce a significant increase in FMRP, suggesting either a longer treatment time or a higher concentration of 5-AzadC may be needed to induce FMRP expression (FIGs. 12F-12G and FIG.
  • FMR1-2Y1 was expressed in the UFM FXS2 cells and after demethylation of MFM FXS1 cells. At the time points tested, although full-length FMRI increased in both FXS LCLs after 5-AzadC treatment, FMRP was unchanged. To test whether blocking the formation of FMR1-2V1 could lead to an increase in full-length FMRI and concomitantly an increase in FMRP, 11 2 '-O-m ethoxy ethyl (MOE)-modified antisense oligonucleotides (ASOs) tiling across intron 1, the intron 1-exon 1 junction, or within exon 2 of FMR1-2V1 RNA were generated (FIG. 14A).
  • MOE ethoxy ethyl
  • an ASO targeting MALAT1 RNA (23) was used in LCL cultures to optimize treatment conditions and serves as a marker of transfection efficiency.
  • LCLs cultured with 80nM MALAT1 ASO for 72 hrs led to -60% decrease in MALAT1 RNA levels (FIG. 13B), confirming that the transfection conditions were appropriate.
  • FXS2 FXS2
  • FIG. 13C the combination of ASO 713 and 714 (80nM each) led to a significant decrease in FMR1-2V1 and an increase in full-length FMRI (FIG. 14B, FIGs. 13C-13D).
  • FIG. 14C shows that 80nM or 160nM of ASOs 713 and 714 completely restored FMRP when compared to TD levels. Therefore, ASO treatment of cells from at least certain FXS individuals, which suggests a possible therapeutic path forward through FMRP restoration.
  • FXS1 LCL In the fully methylated FXS1 LCL, a 7-day treatment with 5-AzadC resulted in the expression of FMR and full-length FMRI but did not affect FMRP levels. Thus, whether treatment of FXS1 LCLs with a combination of 5-AzadC and ASOs (713 and 714) could restore FMRP was addressed.
  • FXS1 LCLs were incubated with 80nM each of ASO 713 and 714, 24 hrs preceding the addition of IpM of 5-AzadC every day for seven days prior to sample collection (FIG. 14D). FMRI RNA isoform expression and FMRP levels were tested in these samples.
  • FXS patient-derived fibroblast cell lines were incubated with 5-AzadC and the ASOs to determine FMRI splicing rescue as well as restoration of FMRP.
  • a dermal cell line from a FXS individual (5131b) with CGG repeat numbers of 800, 166 (24), and previously shown to harbor a transcriptionally active FMRI locus, was treated with 5-AzadC and then ASOs 713/714 for 72 hours before RNA and protein extraction (FIG. 15A).
  • RT-qPCR of FMRI and FMRI -217 showed an ASO-dependent decrease in FMR1-2V1 and a subsequent increase in FMRI levels (FIG. 15B).
  • RNA changes may be useful as biomarkers to assess therapeutic outcomes, disease prognosis, and cognitive abilities (28-30).
  • biomarkers Unlike protein-based biomarkers for FXS (31-33), blood derived RNA biomarkers are more sensitive and specific and can easily be translated into the clinic.
  • FMRI gene promoter When it contains an expansion of 200 or more CGG repeats, the FMRI gene promoter is methylated and transcriptionally silenced. It was therefore surprising that FMRI RNA was detected in 19 of 29 FXS blood samples and in 5 of 10 FXS post-mortem brain samples. Most of these FXS individuals appeared fully mutated with >200 CGG repeats and methylated in standard assays. Remarkably, in >70% of these FXS cells and tissues, the FMRI RNA was also mis-spliced to generate the FMRI -2X1 isoform, a highly truncated RNA that could encode a 31 amino acid peptide. FMRI-217 RNA was not detected in any TD sample.
  • FMR1-2V1 FMRI mis-splicing to generate the FMR1-2Y1 isoform in FXS clearly requires a CGG expansion, although some evidence suggests that CGG repeat number may be a critical determinant for mis-splicing.
  • FMR1-2Y1 RNA expression was detected in FXS premutation carrier-derived fibroblasts with 140 CGG repeats, but not lesser amounts (77 or 98 CGG repeats) or cells from TD individuals ( ⁇ 55 CGG repeats).
  • a second more critical point is that while FMRI -217 is generated in FXS iPSC-derived NPCs incubated with 5- AzadC, it is not produced when the CGG expansion is deleted by CRISPR/Cas9 (17),' FIG. 11C and Table 8). Therefore, the CGG expansion drives mis-spliced 7-277 generation.
  • ASO treatment can be a viable therapeutic option.
  • an ASO-based treatment would be more complex.
  • demethylation of the locus by a chemical compound or a CRISPR/Cas9-anchored demethylating enzyme (17, 22, 34), or ASO-mediated blocking of CGG RNA translation (35, 36) have met with limited success in restoring FMRP.
  • CRISPR/Cas9-mediated gene editing of the CGG repeats (37-40) have resulted in a nearly 70% restoration of FMRP levels.
  • a method of treating a fragile X-associated disorder comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that modulates splicing of Fragile X Mental Retardation 1 (FMRI gene, thereby treating the fragile X- associated disorder in the subject.
  • FMRI Fragile X Mental Retardation 1
  • the fragile X-associated disorder is fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI), or fragile X-associated tremor/ataxia syndrome (FXTAS).
  • FXS fragile X syndrome
  • FXPOI fragile X-associated primary ovarian insufficiency
  • FXTAS fragile X-associated tremor/ataxia syndrome
  • Embodiment 3 The method of Embodiment 1 or 2, wherein the agent increases splicing and/or expression of isoform 1 of the FMRI gene, decreases splicing and/or expression of isoform 12 of the FMRI gene, or a combination thereof.
  • the polynucleotide is 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).
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • gRNA guide RNA
  • the length of the polynucleotide is about 18-22 nucleotides.
  • the polynucleotide comprises a nucleotide sequence that is complementary to a portion of the FMRI gene transcript.
  • the method of Embodiment 10 wherein the polynucleotide comprises a nucleotide sequence that is at least 80% identical to at least a portion of the pseudo exon of the FMRI gene (SEQ ID NO: 19), at least 80% identical to at least a portion of the junction of intron 1 and the pseudo exon, or both.
  • the method of Embodiment 11 wherein the nucleotide sequence is at least 80% identical to:
  • AGAAGCCAAAGGAGACCTGA (SEQ ID NO: 1) (W-704), AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705), CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:3) (W-706), ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707), CAATGCTAGACCGGAAAAGA (SEQ ID NO: 5) (W-708), AAGTCCCAATGCTAGACCGGA(SEQ ID NO: 6) (W-709), TCTCCGAAGTCCCAATGCTA (SEQ ID NO:7) (W-710), GAGCTCTCCGAAGTCCCA (SEQ ID NO: 8) (W-711), AGAACAGTGGAGCTCTCCGA (SEQ ID NO: 9) (W-712), CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713), or CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714).
  • Embodiment 12 wherein the nucleotide sequence is identical to: AGAAGCCAAAGGAGACCTGA (SEQ ID NO: 1) (W-704), AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705), CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:3) (W-706), ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707), CAATGCTAGACCGGAAAAGA (SEQ ID NO: 5) (W-708), AAGTCCCAATGCTAGACCGGA(SEQ ID NO: 6) (W-709), TCTCCGAAGTCCCAATGCTA (SEQ ID NO:7) (W-710), GAGCTCTCCGAAGTCCCA (SEQ ID NO: 8) (W-711), AGAACAGTGGAGCTCTCCGA (SEQ ID NO: 9) (W-712), CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713), or
  • polynucleotide comprising the nucleotide sequence of CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714), or both.
  • LNA locked nucleic acid
  • the method of Embodiment 15, wherein the modification is a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.
  • the method of Embodiment 15, wherein the polynucleotide is chemically modified to increase the nuclease resistance, to prevent RNase H cleavage of the complementary RNA strand, to increase cellular uptake, or a combination thereof.
  • the polynucleotide is chemically modified to comprise 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.
  • LNA locked nucleic acid
  • S-cEt 2’-(S)- constrained ethyl
  • MOE a constrained MOE
  • 2'-O,4'-C-aminomethylene bridged nucleic acid 2',4'-BNANC
  • alpha-L-locked nucleic acid and a tricyclo- DNA, or a combination thereof.
  • the chemical modification is a modification of a phosphate group and wherein the modification of the 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.
  • the modification of the 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.
  • the method of Embodiment 16 wherein the chemical modification is a modification of a nucleobase and wherein the modification of the 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.
  • the method of Embodiment 15, wherein the chemical modification is a modification of the polynucleotide sugar-phosphate backbone.
  • Embodiment 22 wherein the sugar-phosphate backbone is replaced with a phosphorodiamidate mopholino (PMO), a peptide nucleic acid or other pseudopeptide backbone.
  • PMO phosphorodiamidate mopholino
  • Embodiment 25 wherein the subject comprises a CGG repeat expansion exceeding 200 repeats in the 5’ untranslated region of the FMRI gene.
  • the method of any one of Embodiments 1-36, wherein treating FXS includes slowing progression of FXS, alleviating one or more signs or symptoms of FXS, preventing one or more signs or symptoms of FXS, or a combination thereof.
  • a method of modulating Fragile X Mental Retardation 1 (FMRI) splicing and/or expression in a cell comprising contacting the cell with a polynucleotide under conditions whereby the polynucleotide is introduced into the cell, wherein the polynucleotide increases splicing and/or expression of isoform 1 of the /’A7 7 gene, decreases splicing and/or expression of isoform 12 of the FMRI gene, or a combination thereof.
  • the method of Embodiment 38, wherein the cell is an in vitro cell or an ex vivo cell.
  • Embodiment 39 wherein the cell is an induced pluripotent stem cell (iPSC)-derived neuron from a human who has or is predisposed to have FXS, a primary human cell, or a cell line.
  • iPSC induced pluripotent stem cell
  • the method of Embodiment 40 wherein the cell is a cell of a subject.
  • the method of Embodiment 41 wherein the cell is allogeneic.
  • the method of Embodiment 41, wherein the cell is autologous or syngeneic.
  • a polynucleotide comprising a nucleotide sequence that is complementary to a portion of the FMRI gene transcript.
  • the polynucleotide of Embodiment 44 wherein the nucleotide sequence is at least 80% identical to at least a portion of isol2 of the FMRI gene, at least 80% identical to at least a portion of the junction of intron 1 and isol2 of the FMRI gene, or both.
  • the polynucleotide of Embodiment 45 wherein the nucleotide sequence is at least 80% identical to:
  • AGAAGCCAAAGGAGACCTGA (SEQ ID NO:1) (W-704), AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705), CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:3) (W-706), ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707), CAATGCTAGACCGGAAAAGA (SEQ ID NO: 5) (W-708), AAGTCCCAATGCTAGACCGGA(SEQ ID NO: 6) (W-709), TCTCCGAAGTCCCAATGCTA (SEQ ID NO:7) (W-710), GAGCTCTCCGAAGTCCCA (SEQ ID NO: 8) (W-711), AGAACAGTGGAGCTCTCCGA (SEQ ID NO: 9) (W-712), CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713), or CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714).
  • a pharmaceutical composition comprising the polynucleotide of any one of Embodiments 44-46, and one or more pharmaceutically acceptable excipients, diluents, or carriers.
  • a microarray for the detection of a fragile X-associated disorder comprising at least one nucleic acid probe immobilized on a solid substrate, said probe comprising a nucleic acid sequence complementary to a portion of the FMRI gene transcript.
  • microarray of Embodiment 48 wherein the nucleotide sequence has at least 80% sequence identity to at least a portion of Exon 2 of FMRI -217, at least a portion of the junction of intron 1-2 and Exon 2 of FMR1-2Y1 , or both.
  • microarray of Embodiment 49 wherein the nucleotide sequence is at least 80% identical to:
  • AGAAGCCAAAGGAGACCTGA (SEQ ID NO: 1) (W-704), AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705), CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:3) (W-706), ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707), CAATGCTAGACCGGAAAAGA (SEQ ID NO: 5) (W-708), AAGTCCCAATGCTAGACCGGA(SEQ ID NO: 6) (W-709), TCTCCGAAGTCCCAATGCTA (SEQ ID NO:7) (W-710), GAGCTCTCCGAAGTCCCA (SEQ ID NO: 8) (W-711), AGAACAGTGGAGCTCTCCGA (SEQ ID NO: 9) (W-712), CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713), or CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714).

Abstract

Provided herein, in various embodiments, are methods of treating a fragile X-associated disorder (e.g., fragile X syndrome), comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that decreases expression of an aberrant fragile X messenger ribonucleoprotein 1 (FMR1) gene product (e.g., FMR1-2VT). Also provided herein, in various embodiments, are compositions (e.g., polynucleotides such as antisense oligonucleotides or pharmaceutical compositions) for decreasing expression of an aberrant FMR1 gene product.

Description

Therapeutic Treatment for Fragile X-Associated Disorder
RELATED APPLICATION(S)
[0001] This application claims the benefit of U.S. Provisional Application No. 63/265,989, filed on December 23, 2021. The entire teachings of the above application are incorporated herein by reference.
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: a) File name: 54391028001.xml; created December 22, 2022, 88,063 Bytes in size.
GOVERNMENT SUPPORT
[0003] This invention was made with government support under GM135087, GM046779 and NS 111990 from the 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 frequent inherited form of intellectual impairment. FXS afflicts 1 in 4,000 boys and 1 in 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 (FMRI), which resides on the X chromosome. When the CGG triplet expands to 200 or more, the FMRI gene is methylated and thereby transcriptionally inactivated. The loss of the FMRI gene product, the protein fragile X messenger ribonucleoprotein (FMRP), is the cause of the disorder.
[0005] 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)). Indeed, there is no widely applicable therapy that shows even modest efficacy for FXS. SUMMARY
[0006] There is a critical need to develop methods and therapeutic agents for treating fragile X-associated disorders such as fragile X syndrome (FXS). The disclosure provides such methods and therapeutic agents.
[0007] The disclosure provided herein is based, in part, on the discovery that, in FXS cells, ASO treatment reduces the expression of the CGG expansion-dependent aberrantly spliced FMRI -217 RNA and restores fragile X messenger ribonucleoprotein (FMRP) to levels observed in cells from typically developing individuals. Accordingly, the disclosure generally relates to compositions (e.g., polynucleotides, pharmaceutical compositions) and methods that are useful for treating a fragile X-associated disorder.
[0008] In one aspect, the present disclosure provides a method of treating a fragile X- associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that decreases expression of an aberrant fragile X messenger ribonucleoprotein 1 (FMRI') gene product, thereby treating the fragile X-associated disorder in the subject.
[0009] In another aspect, the present disclosure provides a method of treating a fragile X- associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that modulates splicing of an FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMRI -217), thereby treating the fragile X-associated disorder in the subject.
[0010] In another aspect, the present disclosure provides a method of decreasing expression of an aberrant FMRI gene product in a cell, comprising contacting the cell with an agent under conditions whereby the agent is introduced into the cell, thereby decreasing expression of the aberrant FMRI gene product in the cell.
[0011] In another aspect, the present disclosure provides a method of modulating FMRI splicing and/or expression in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions whereby the agent is introduced into the cell, thereby modulating FMRI splicing and/or expression in the cell.
[0012] In another aspect, the present disclosure provides a method of increasing the level of FMRP in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions whereby the agent is introduced into the cell, such that the level of FMRP in the cell is enhanced. [0013] In another aspect, the present disclosure provides a method of enhancing the level of FMRP in a cell, comprising contacting the cell with an oligonucleotide which is complementary to at least 8 contiguous nucleotides of a sequence set forth in SEQ ID NOs:24-42, such that the level of FMRP in the cell is enhanced.
[0014] In another aspect, the present disclosure provides a method of reducing CGG triplet repeat expansion in FMRI 5’ UTR in a cell, comprising contacting the cell with an agent that reduces expression of an aberrant FMRI gene product under conditions whereby the agent is introduced into the cell, thereby reducing CGG triplet repeat expansion in the cell.
[0015] In some embodiments, the fragile X-associated disorder is FXS.
[0016] In some embodiments, the aberrant FMRI gene product comprises FMRI -217.
[0017] In some embodiments, the agent is a polynucleotide (e.g., any one of the modified polynucleotides disclosed herein).
[0018] In some embodiments, the method increases expression of fragile X messenger ribonucleoprotein (FMRP) in the subject.
[0019] In another aspect, the present disclosure provides an agent that decreases expression of an aberrant FMRI gene product.
[0020] In another aspect, the present disclosure provides an agent that modulates splicing and/or expression of an FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMR1- 217 or decreasing a protein encoded by FMRI -217).
[0021] In yet another aspect, the present disclosure provides a pharmaceutical composition, comprising any one or more of the agents disclosed herein, and one or more pharmaceutically acceptable excipients, diluents, or carriers.
[0022] In some embodiments, the agent is a polynucleotide (e.g., any one of the modified polynucleotides disclosed herein).
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] 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.
[0024] FIG. 1 shows a genome browser view of FMRI RNA in 7 typically developing (“TD” or “control”) and 10 fragile X syndrome (FXS) patients sequenced from white blood cells (WBCs). [0025] FIG. 2 shows a genome browser view of exon 1 and intron 1 of FMRI RNA in 7 typically developing individuals and 10 fragile X syndrome patients sequenced from white blood cells.
[0026] FIG. 3 illustrates a non-limiting approach, using antisense oligonucleotides (ASOs), for blocking isoform 12 production, increasing isoform 1 production, and increasing FMRP levels.
[0027] FIG. 4 shows a schematic of FMRI isol and isol2 pre-mRNAs. The numbered boxes (704-714) refer to antisense oligonucleotides complementary to regions in intron 1, that span intron 1 and iso 12 junction, and within isol2. Isol l F, Isol l R, Exonl F, Exonl R, and Isol2_l R refer to primers (F, forward; R, reverse) that were used to detect RNA levels by RT- qPCR.
[0028] FIG. 5 shows RT-qPCR data demonstrating a reduction in isol2 and increase in isol. The asterisk refers to p<0.05.
[0029] FIGs. 6A-6B show RT-qPCR data from a fully methylated FXS cell line (FXS1, GM07365). FIG. 6A shows an increase in FMRI isol2 upon 5-AzaC treatment and a partial rescue of the FMRI isol2 increase when combined with the ASO treatment. FIG. 6B shows an increase in FMRI isol upon 5-AzaC treatment and a further increase when combined with the ASO treatment. The asterisks refer to p<0.05.
[0030] FIGs. 7A-7B show FMRP levels. FIG. 7A shows western blot data for an FXS1 LCL cell line in duplicates (the upper panel), demonstrating an increase in FMRP after treatment with IpM 5-AzaC and ASO treatment (80nM of both antisense oligonucleotides 713 and 714) when compared to DMSO or 5-AzaC only treated samples. The mouse brains (hippocampus tissue) from a wild-type mouse and an Fmrl knock-out mouse were loaded as controls. The bottom panel represents GAPDH protein levels used to normalize the protein amounts loaded in each sample. FIG. 7B shows quantification of the FMRP protein levels relative to GAPDH protein levels as seen on the western blot in FIG. 7A.
[0031] FIGs. 8A-8C show FMRJ isol and isol2 levels in fibroblast cells from six individuals. FIG. 8A is a table showing the number of CGG repeats in the FMRI RNA 5’ UTR from three healthy males and three premutation carrier males for FXS. FIG. 8B shows RT-qPCR data of FMRI isol levels in fibroblast cells from the six individuals, normalized to GAPDH RNA levels. FIG. 8C compares the FMRI isol2 level in individual Pl to those in the other premutation carriers and healthy control samples. [0032] FIGs. 9A-9C A truncated isoform of FMRI mRNA identified in a subset of FXS individuals. FIG. 9A Integrative Genomics Viewer (IGV) tracks of RNA-seq data for FXS and TD individuals for the FMRI gene. FMRI RNA was detected in all TD individuals, and FXS individuals 1-21. The thick-lined box marked on the FMRI gene illustrated at the bottom shows the region of intron 1 with differential reads between TD (1-13) and FXS (1-21) individuals. FIG. 9B Expanded view map to an exon that comprises the annotated FMRI -217 isoform. All annotated FMRI isoforms and sequence data for FMRI -2 1 PCR fragments from FXS RNA sample are shown in Table 3 and FIGs. 9E-9H. H refers to high and L refers to low FMRI. FIG. 9C. The full length FMRI RNA (exons-grey boxes) and the FMR1-2Y1 isoform (exons-grey boxes) are illustrated with the CGG repeats in the 5’UTR (UTRs-black boxes). The proportion of full length FMRI to FMRI -217 was quantified by RT-qPCR in TD, H FMRI (N=7), and L FMRI (N=5) individuals. The forward (F) and reverse (R) primers used for q-PCR are shown. The total FMRI RNA relative to GAPDH RNA levels was significantly reduced in H FMRI and L FMRI vs TD (*P <0.05, t test). Bar graphs indicate mean, and error bars indicate +/- SEM. FIG. 9D Summary table of changes in alternative splicing events from L FMRI vs H FMRI samples detected by rMATS (14) at an FDR < 5% and a difference in the exon inclusion levels (PSI, Percent spliced-in) between the genotypes (deltaPSI) of > 5%. Schematic for the splicing event categories is shown at the left of the table. FIG. 9E FMRI -217 isoform was identified in RNA samples generated from leukocytes (individual FXS-05). DNase treated RNA samples were reverse transcribed using an oligo(dT)(20), and the PCR product, generated using primers ExlF and 217R, was sequenced. FIG. 9F The predicted protein product of the FMR1-2V1 isoform. The predicted protein length is 31 amino acids, with a mass of 3,524 Da. FIG. 9G Alignment of the sequencing data of the PCR product using primers ExlF and 217R to FMRI gene is displayed. The poly(A) site was identified by sequencing the PCR product of primer 217F and oligo(dT)(20). FIG. 9H FMRI isoforms annotated in the GRCh38.pl3 genome assembly. The FMRI-217 isoform (ENST00000621447.1) is marked with a thick-lined box.
[0033] FIG. 10 Correlation of FXS molecular parameters with IQ. Three-dimensional comparison of indicated parameters. The inset shows samples with 100% methylation. The increasing size of the dots represent increase in FMRP levels, and the darkness from low to high represent increase in IQ levels (see Table 4).
[0034] FIGs. 11A-11E FMRI -217 is derived from FMRI, requires the CGG expansion, and is expressed in human postmortem brain tissues (FXS and premutation carriers), and in skin- derived fibroblasts (premutation carrier). FIG. 11A Integrative Genomics Viewer (IGV) tracks of RNA-seq data (Tran et al., Widespread RNA editing dysregulation in brains from autistic individuals, Nat. Neurosci. (2019)) for FXS and TD individuals for the FMRI gene. FIG. 11B IGV tracks of selected regions of FMRI re-analyzed from the RNA-seq data of Vershkov et al., FMRI Reactivating Treatments in Fragile X iPSC-Derived Neural Progenitors In Vitro and In Vivo, Cell Rep. 26: 2531-39 (2019), who deleted the FMRI CGG expansion by CRISPR/Cas9 gene editing. Biologic duplicate of iPSC-derived neural stem cells (NSCs) from FXS individuals (FXS-NSC) treated with vehicle or 5-aza-2-deoxy cytidine (5-azadC) as well as isogenic CGG- edited samples are shown. FMRI-217 reads are detected only in the 5-azadC-treated samples. FIG. 11C IGV tracks of selected regions of FMRI re-analyzed from the RNA-seq data of Liu et al. Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMRI Gene, Cell 172: 979-91 (2018), who performed targeted FMRI gene demethylation in FXS iPSCs and iPSC-derived neurons. iPSCs derived from FXS individuals were incubated with viruses expressing a mock guide RNA (i_mock), or an FMRI guide RNA and catalytically inactive Cas9 fused to the Tetl demethylase (i Tetl). iPSC-derived neurons from to FXS individuals were treated with a mock guide RNA (Nl_mock, N2_mock), or an FMRI guide RNA and catalytically inactive Cas9 fused to the Tetl demethylase (Nl_Tetl, N2_Tetl, N3_Tetl). All cells were incubated with an FMRI guide RNA and catalytically inactive Cas9 fused to the Tetl demethylase express FMRI -217. FIG. 11D Experimental design for RNA extraction from postmortem cortical tissue obtained from 6 FXS males (F1-F6) and 5 typically developing (T1-T5) age-matched males. RT-qPCR data for cortical tissue-derived RNA samples representing abundance for FMRI and FMR1-2V1 isoforms relative to GAPDH RNA. Each sample was analyzed in duplicate. Primers used for amplification are represented in FIG. 9C (**P <0.01, t test). FIG. HE Schematic diagram of fibroblast generated from skin biopsies obtained from three male premutation carriers (P1-P3) and three male TD individuals (T1-T3). The table shows patient de-identified designation, genotypes, and CGG repeat numbers in the 5’UTR in the FMRI gene. ND: not determined. qPCR data for fibroblast-derived RNA samples representing abundance for FMRI and FMR1-2V1 isoforms relative to GAPDH RNA. Each sample was analyzed in duplicate. Primers used for amplification are represented in FIG. 9C.
[0035] FIGs. 12A-12G FMRI -217 is expressed in lymphoblast cell cultures from FXS individuals. FIG. 12A Sample information for lymphoblast cell lines (LCLs) (Coriell Institute, NJ) from two FXS and two TD members of a family. FMRP and GAPDH (loading control) levels were determined by western blots. Ratios of FMRP/GAPDH normalized to FXS1 are shown below the blot. FMRP quantification by Luminex Microplex immunochemistry assay are shown in ng FMRP/pg total protein). FIG. 12B The proportion of full length FMRI to FMR1- 217 was quantified using RT-qPCR in the TD and FXS2 LCLs relative to GAPDH KN A levels. Primers used for q-PCR are shown in the gene illustration. The total FMRI RNA was unchanged but the proportion of FMRI -217 was significantly higher in FXS2 LCL compared to TD LCL. FIG. 12C Schematic diagram of the 5-AzadC treatment (1|1M for 7 days) of the FXS1 and FXS2 LCLs to determine FMRI isoforms and FMRP levels after demethylation. DMSO treated cells were used as a vehicle control. FIGs. 12D-12E The proportion of full length FMRI to FMR1- 217 was quantified using RT-qPCR in the FXS1 and FXS2 LCLs treated with 5-AzadC relative to vehicle, normalized to GAPDH RNA levels (**P <0.001, / test). FIGs. 12F-12G FMRP levels were determined using western blots relative to GAPDH in FXS1 and FXS2 LCLs treated with DMSO or 5-AzadC (See FIG. 13 A) Ratios of FMRP/GAPDH are shown for FXS1 and FXS2 cells, respectively. Histograms indicate mean values (N=2); error bars indicate +/- SEM.
[0036] FIG. 13A Western blots showing FXS1 and FXS2 cells respectively treated with DMSO or 5-AzadC (as treated in FIG. 12C and quantified in FIGs. 12F-12G). FIG. 13B The decrease in MALAT1 RNA levels relative to GAPDH RNA was quantified by RT-qPCR in the TD1 LCL treated with MALAT1 ASO (80nM and lOOnM) for 48hrs. Untreated cells were used as negative controls (* represents P <0.05, t test). The right panel shows a decrease in MALAT1 RNA levels compared to GAPDH KN A levels, quantified using RT-qPCR in the TD1 LCL treated with 80nM MALAT1 gapmer ASO for 48hrs or 72 hrs. Untreated cells were used as negative controls (* represents P <0.05 using t test). FIG. 13C FXS2 LCLs were treated with either 80nM of ASOs 704 and 705, 709 and 710 or 713 and 714 for 72 hrs. RNA levels for FMRI -217 and FMRI full length RNA were quantified by RT-qPCR using primers as in FIG. 9C. ASOs 713 and 714 reduced FMRl-2\2 levels whereas FMRI full length RNA levels were increased (* represents P <0.05, t test). FIG. 13D FXS2 LCLs were treated with either 80nM or 160nM of ASOs 713 and 714 or 80nM of Malatl gapmer ASO for 72 hrs. RNA levels for FMRI -217 and FMRI full length RNA were quantified by RT-qPCR using primers as in FIG. 9C. ASOs 713 and 714 reduced FMRI-217 levels at both 80nM and 160nM concentrations whereas FMRI full length RNA levels were increased. No change in the FMRI isoform levels was observed upon MALAT1 ASO treatment (* represents P <0.05, t test).
[0037] FIGs. 14A-14F ASOs targeting FMR1-2Y1 restore FMRP levels in FXS LCLs with partial or complete FMRI gene methylation. FIG. 14A ASOs designed against the FMRI -217 RNA are illustrated. (Intron specific: 704-706, intron-exon junction specific: 707-710 and exon specific: 711-714). FIG. 14B Schematic diagram of the ASO treatment (80nM for 72 hours) of the FXS2 LCLs to determine FMRI isoform and FMRP levels after demethylation. DMSO treated cells were used as a vehicle control (****p < 0.0001, **P <0.01, / test). FIG. 14C FMRP levels were determined for FXS2 LCLs treated with DMSO (vehicle) and ASOs as described in FIG. 12A. TD LCLs were also probed for FMRP on the same western blots. Ratios of FMRP/GAPDH normalized to FXS1 are shown below the blot. FIG. 14D Fully methylated FXS1 LCLs were treated with ASOs 713 and 714 (80nM each) followed by 5-AzadC (luM) added on consecutive days 2-9 after which RNA and protein were extracted. FMRI -217 and FMRI isoforms were assessed using qPCR primers as shown in FIG. 9C was determined using one way ANOVA with multiple comparisons test (****p < 0.0001, ***P <0.001, **P <0.01, *P <0.05). Data information: bar graphs indicate mean, error bars indicate +/- SEM. FIG. 14E Western blot of FMRP and GAPDH from FXS1 LCLs treated with DMSO, 5-AzadC, or 5- AzadC plus ASOs as in FIG. 13 A. FIG. 14F Histogram depicting quantification of western blot for FXS1 cells treated with DMSO, 5-AzadC and ASO or 5-AzadC alone (N=3). Significance was determined using one way ANOVA with multiple comparisons test ((**** < 0.0001, Data information: bar graphs indicate mean, error bars indicate +/- SEM.
[0038] FIGs. 15A-15F ASOs targeting FMR1-2V1 restore FMRP levels in FXS fibroblasts with an inactive FMRI gene treated with 5-AzadC. FIG. 15A Dermal fibroblasts derived from a FXS individual (GM05131B, Cori ell Institute) were cultured with 5-AzadC for 8 days and then treated with ASOs 713/714 (100 nM each) for 72 hours prior to RNA and protein extraction. FIG. 15B RT-qPCR analysis of FMRI-217, FMRI, and GAPDH RNAs in dermal fibroblasts treated with DMSO, ASOs 713/714, 5-AzadC, or the ASOs 713/714 plus 5-AzadC. The amounts of FMRI -217 and FMRI were made relative to GAPDH. (* P <0.05, ** P <0.01, one way ANOVA with multiple comparisons test). FIG. 15C Western blots of FMRP and GAPDH from the dermal fibroblasts treated as in FIG. 15B. Quantification of FMRP relative to GAPDH is noted at right. *p<0.05. Histogram depicting quantification of western blot (N=3). Significance was determined using one way ANOVA with multiple comparisons test (*p<0.05, one way ANOVA with multiple comparisons test). Data information: bar graphs indicate mean, error bars indicate +/- SEM. FIG. 15D Lung fibroblasts derived from a FXS individual (GM07072, Coriell Institute) were cultured with 5-AzadC for 8 days and then treated with ASOs 713/714 (100 nM each) for 72 hours prior to RNA and protein extraction. RT-qPCR analysis of FMRI-217, FMRI, and GAPDH RNAs in lung fibroblasts treated with DMSO, ASOs 713/714, 5-AzadC, or the ASOs 713/714 plus 5-AzadC. The amounts of FMRI-217 and FMRI were made relative to GAPDH. (*p<0.05, **p<0.01, ***p<0.001, one way ANOVA with multiple comparisons test). FIG. 15E Western blots of FMRP and GAPDH from the lung fibroblasts treated as in FIG. 15B. Quantification of FMRP relative to GAPDH is noted below (N=2). Significance was determined using one way ANOVA with multiple comparisons test (P < 0.0001****, P <0.001, *** P <0.01**, one way ANOVA with multiple comparisons test). Data information: bar graphs indicate mean, error bars indicate +/- SEM. FIG. 15F Model depicting active FMRI transcription in FXS cells for after treatment with demethylating agents to activate FMRI transcription) result in production of mis-spliced FMRI-217. Down-regulation of FMRI-217 with an ASO results in rescue of correctly spliced FMRI transcripts and restoration of FMRP protein.
[0039] FIG. 16A Additional ASO sequences. FIG. 16B 72-hour treatment with 160 nM of each ASO in lymphoblastoid cell line FXS2. Total RNA was extracted using TRIzol™ Reagent (ThermoFisher Scientific # 15596026). One pg of total RNA was primed with oligo(dT)20 to generate cDNA with a QuantiTect cDNA synthesis kit using random hexamers (FIG. 9E). qPCR was performed using the iTaq™ Universal SYBR® Green Supermix on a QuantStudio 3 qPCR machine in triplicate. The fold change of full length FMRI and FMR1-2Y1 in ASO treated cells relative to vehicle (control) was measured using qPCR. The RNA levels were normalized to GAPDH RNA (*P values measured using t test).
DETAILED DESCRIPTION
[0040] A description of example embodiments follows.
[0041] Several aspects of the disclosure 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 disclosure. One having ordinary skill in the relevant art, however, will readily recognize that the disclosure can be practiced without one or more of the specific details or practiced with other methods, protocols, reagents, cell lines and animals. The present disclosure 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 disclosure. 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.
[0042] 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 invention 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.
[0043] The terminology used herein is for the purpose of describing some embodiments only and is not intended to be limiting.
[0044] As used herein, the indefinite articles “a,” “an” and “the” should be understood to include plural reference unless the context clearly indicates otherwise.
[0045] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “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.” [0046] “About” means within an acceptable error range for the 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 24-96 hours,” the term “about” should be read as applying to both of the given values of the range, such that “about 24-96 hours” means about 24 hours to about 96 hours.
[0047] As used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “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 of the invention, can in some embodiments, be replaced with the term “consisting of,” or “consisting essentially of’ to vary scopes of the disclosure.
[0048] 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.”
[0049] 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 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.”
[0050] 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. Thus, for example, unless the context clearly indicates otherwise, “an agent” includes a single agent, and two or more agents. Further the two or more agents can be the same or different as, for example, in embodiments wherein a first agent comprises a polynucleotide (e.g., ASO) of a first sequence and a second agent comprises a polynucleotide (e.g., ASO) of a second sequence.
[0051] 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.
[0052] 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 compounds described herein include salts derived from suitable inorganic and organic acids, and suitable inorganic and organic bases.
[0053] 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, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxy-ethanesulfonate, hydroxymaleate, lactobionate, lactate, laurate, 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.
[0054] 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.
[0055] 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+((Ci-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.
[0056] In one aspect, the present disclosure provides a method of treating a fragile X- associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that decreases expression of an aberrant fragile X messenger ribonucleoprotein 1 (FMRI') gene product, thereby treating the fragile X-associated disorder in the subject. An agent that decreases expression of an aberrant FMRI gene product in a method disclosed herein can be any one or more of the agents disclosed herein.
[0057] In another aspect, the present disclosure provides a method of treating a fragile X- associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that modulates splicing of an FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMRI -217), thereby treating the fragile X-associated disorder in the subject. An agent that modulates splicing of an FMRI gene in a method disclosed herein can be any one or more of the agents disclosed herein.
[0058] In another aspect, the present disclosure provides a method of decreasing expression of an aberrant FMRI gene product in a cell, comprising contacting the cell with an agent under conditions whereby the agent is introduced into the cell, thereby decreasing expression of the aberrant FMRI gene product in the cell.
[0059] In another aspect, the present disclosure provides a method of modulating FMRI splicing and/or expression in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions whereby the agent is introduced into the cell, thereby modulating FMRI splicing and/or expression in the cell. An agent that modulates FMRI splicing and/or expression in a method disclosed herein can be any one or more of the agents disclosed herein.
[0060] In another aspect, the present disclosure provides a method of increasing the level of fragile X messenger ribonucleoprotein (FMRP) in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions whereby the agent is introduced into the cell, such that the level of FMRP in the cell is enhanced.
[0061] In another aspect, the present disclosure provides a method of reducing CGG triplet repeat expansion in FMRI 5’ UTR in a cell, comprising contacting the cell with an agent that reduces expression of an aberrant FMRI gene product under conditions whereby the agent is introduced into the cell, thereby reducing CGG triplet repeat expansion in the cell.
Fragile X-Associated Disorders
[0062] Fragile X-associated disorders are caused by mutation of the fragile X messenger ribonucleoprotein 1 (FMRI, previously known as fragile X mental retardation 7) gene, located in the q27.3 locus of the X chromosome. The expansion of the trinucleotide CGG above the normal range (greater than 54 repeats) in the non-coding region of the FMRI gene has been associated with the development of fragile X-associated disorders. For example, in those carrying the premutation, the trinucleotide CGG can range from 55-200 CGG repeats. In some embodiments, a fragile X-associated disorder described herein is linked to greater than 77 CGG repeats in FMRI, e.g., greater than 98 CGG repeats in FMRI. In some embodiments, the fragile X-associated disorder is linked to at least 140 CGG repeats in FMRI. In some embodiments, the fragile X-associated disorder is linked to at least 201 CGG repeats in FMRI.
[0063] 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 described herein 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, the fragile X-associated disorder is FXS.
FMRI Gene Products
[0064] A FMRI gene encodes a fragile X messenger ribonucleoprotein (FMRP, previously known as fragile X mental retardation protein).
[0065] In some embodiments, an FMRI gene described herein is a human FMRI gene (e.g., corresponding to GenBank reference number NC 000023.11), a mouse FMRI gene (e.g., NC_000086.8), a ra FMRl gene (e.g, NC_051356.1), a golden hamster FMRI gene (e.g, NW_024429188.1), a Chinese hamster FMRI gene (e.g., NW_003614110.1), a Ao FMRl gene (e.g., NC_051843.1), a FMRl gene (e.g., NC_046383.1), or a monkey FMRI gene (e.g., NC 041774.1). In some embodiments, the FMRI gene is a human FMRI gene. The human FMRI 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)).
[0066] As used herein, “an aberrant FMRI gene product” refers to an FMRI gene product elevated in a subject who has, or is predisposed to have a fragile X-associated disorder. In some embodiments, an aberrant FMRI gene product described herein is elevated in a subject who is being treated, or has been treated, for a fragile X-associated disorder. In some embodiments, the aberrant FMRI gene product is elevated in a subject having at least 55 CGG repeats in the 5’ untranslated region of an FMRI 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 FMRI gene. In some embodiments, the aberrant FMRI gene product is elevated in a subject having at least 201 CGG repeats in the 5’ untranslated region of an FMRI gene. In some embodiments, an aberrant FMRI gene product described herein is not expressed in typically developing subjects (e.g., typically developing humans). In some embodiments, the aberrant FMRI gene product is elevated in a subject who is a premutation carrier for FXS. In some embodiments, the aberrant FMRI gene product is elevated in a subject who has FXS.
[0067] In some embodiments, an aberrant FMRI gene product described herein is produced from a CGG expansion-dependent mis-splicing of a. FMRI gene. [0068] In some embodiments, an aberrant FMRI gene product described herein contributes to pathology of a fragile X-associated disorder described herein. In some embodiments, an aberrant FMRI transcript, its protein product, or both contribute to pathology of the fragile X- associated disorder. In some embodiments, an aberrant FMRI transcript described herein contributes to pathology of the fragile X-associated disorder. In some embodiments, a protein encoded by an aberrant FMRI transcript described herein contributes to pathology of the fragile X-associated disorder. In some embodiments, an aberrant FMRI transcript and its protein product contribute to pathology of the fragile X-associated disorder.
[0069] In some embodiments, an aberrant FMRI gene product described herein comprises FMRI -217, its protein product, or both. In some embodiments, the aberrant FMRI gene product comprises FMRI -217. In some embodiments, the aberrant FMRI gene product comprises the protein product of FMRI -217. In some embodiments, the aberrant FMRI gene product comprises FMRI -217 and its protein product.
[0070] In humans, FMR1-2Y1, also referred to as “isoform 12” or “isol2,” is a transcript corresponding to A0A087X1M7 (ENST00000621447.1, 1,832 nucleotides). FMR1-2Y1 has 2 exons, and the splicing between Exon 1 of FMR1-2Y1 (between base pairs 147,912,123 and 147,912,230, SEQ ID NO:23) and Exon 2 of FMR1-2Y1 (between base pairs 147,912,728 and 147,914,451, SEQ ID NO:21) is considered aberrant FMRI RNA splicing. FMR1-2V1 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.
[0071] CGCCCGCAGCCCACCTCTCGGGGGCGGGCTCCCGGCGCTAGCAGGGCTGA AGAGAAGATGGAGGAGCTGGTGGTGGAAGTGCGGGGCTCCAATGGCGCTTTCTACA AG (SEQ ID NO:23).
[0072] CATTGGGACTTCGGAGAGCTCCACTGTTCTGGGCGAGGGCTGTGAAGAAA GAGTAGTAAGAAGCGGTAGTCGGCACCAAATCACAATGGCAACTGATTTTTAGTGG CTTCTCTTTGTGGATTTCGGAGGAGATTTTAGATCCAAAAGTTTCAGGAAGACCCTA ACATGGCCCAGCAGTGCATTGAAGAAGTTGATCATCGTGAATATTCGCGTCCCCCTT TTTGTTAAACGGGGTAAATTCAGGAATGCACATGCTTCAGCGTCTAAAACCATTAGC AGCGCTGCTACTTAAAAATTGTGTGTGTGTGTTTAAGTTTCCAAAGACCTAAATATA TGCCATGAAACTTCAGGTAATTAACTGAGAGTATATTATTACTAGGGCATTTTTTTTT TAACTGAGCGAAAATATTTTTGTGCCCCTAAGAACTTGACCACATTTCCTTTGAATTT GTGGTGTTGCAGTGGACTGAATTGTTGAGGCTTTATATAGGCATTCATGGGTTTACT GTGCTTTTTAAAGTTACACCATTGCAGATCAACTAACACCTTTCAGTTTTAAAAGGA AGATTTACAAATTTGATGTAGCAGTAGTGCGTTTGTTGGTATGTAGGTGCTGTATAA ATTCATCTATAAATTCTCATTTCCTTTTGAATGTCTATAACCTCTTTCAATAATATCCC ACCTTACTACAGTATTTTGGCAATAGAAGGTGCGTGTGGAAGGAAGGCTGGAAAAT AGCTATTAGCAGTGTCCAACACAATTCTTAAATGTATTGTAGAATGGCTTGAATGTT TCAGACAGGACACGTTTGGCTATAGGAAAATAAACAATTGACTTTATTCTGTGTTTA CCAATTTTATGAAGACATTTGGAGATCAGTATATTTCATAAATGAGTAAAGTATGTA AACTGTTCCATACTTTGAGCACAAAGATAAAGCCTTTTGCTGTAAAAGGAGGCAAA AGGTAACCCCGCGTTTATGTTCTTAACAGTCTCATGAATATGAAATTGTTTCAGTTG ACTCTGCAGTCAAAATTTTAATTTCATTGATTTTATTGATCCATAATTTCTTCTGGTG AGTTTGCGTAGAATCGTTCACGGTCCTAGATTAGTGGTTTTGGTCACTAGATTTCTGG CACTAATAACTATAATACATATACATATATATGTGTGAGTAACGGCTAATGGTTAGG CAAGATTTTGATTGACCTGTGATATAAACTTAGATTGGATGCCACTAAAGTTTGCTT ATCACAGAGGGCAAGTAGCACATTATGGCCTTGAAGTACTTATTGTTCTCTTCCAGC AACTTATGATTTGCTCCAGTGATTTTGCTTGCACACTGACTGGAATATAAGAAATGC CTTCTATTTTTGCTATTAATTCCCTCCTTTTTTGTTTTGTTTTGTAACGAAGTTGTTTA ACTTGAAGGTGAATGAAGAATAGGTTGGTTGCCCCTTAGTTCCCTGAGGAGAAATGT TAATACTTGAACAAGTGTGTGTCAGACAAATTGCTGTTATGTTTATTTAATTAAGTTT GATTTCTAAGAAAATCTCAAATGGTCTGCACTGATGGAAGAACAGTTTCTGTAACAA AAAAGCTTGAAATTTTTATATGACTTATAATACTGCTGTGAGTTTTAAAAGTAAAGC AAAAGTAAACTGAGTTGCTTGTCCAGTGGGATGGACAGGAAAGATGTGAAATAAAA ACCAATGAAAAATGAA (SEQ ID NO:21).
[0073] FMR1-2V1 encodes a 31-amino acid protein (SEQ ID NO:22)).
[0074] MEELVVEVRGSNGAFYKHWDFGELHCSGRGL (SEQ ID NO: 22).
[0075] Additional information on FMRI -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:
[0076] useast.ensembl.org/Homo_sapiens/Transcript/Summary?db=core;g=ENSG00000102 081;r=X:147911951-147951125;t=ENST00000621447.
[0077] In some embodiments, a method disclosed herein increases the level of expression of FMRP in a subject described herein. In some embodiments, a method disclosed herein increases the level of expression of FMRP in a cell described herein.
[0078] In some embodiments, a method disclosed herein increases a normal FMRI gene product (e.g., a normal FMRI transcript, its protein product, or both) in a subject and/or cell described herein. [0079] Several normal FMRI gene products are expressed in typically developing subjects (e.g., humans who do not have FXS). Non-limiting examples of “normal” human FMRI gene products include: a transcript corresponding to Q06787 (FMR1-2QF, ENST00000370475.9, 4,441 nucleotides), and its protein product (a 632-amino acid protein (NP 002015.1)), 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 (FMRI-201, ENST00000218200.12, 4,333 nucleotides), and its protein product (a 611-amino acid protein), a transcript corresponding to Q06787-8 (FMRI-208, ENST00000440235.6, 4,271 nucleotides), and its protein product (a 586-amino acid protein), a transcript corresponding to X5D907 (FMRI-223, ENST00000687593.1, 4,159 nucleotides), and its protein product (a 594-amino acid protein), a transcript corresponding to Q06787-10 (FMRI-204, ENST00000370471.7, 4,125 nucleotides), and its protein product (a 537-amino acid protein), a transcript corresponding to G3V0J0 (FMRI-207. ENST00000439526.6, 3,699 nucleotides), and its protein product (a 592-amino acid protein), a transcript corresponding to A8MQB8 (FMRI-206, ENST00000370477.5, 3,437 nucleotides), and its protein product (a 582-amino acid protein), a transcript corresponding to A0A087WY29 (FMRI-212, ENST00000495717.6, 2,874 nucleotides), and its protein product (a 561-amino acid protein), a transcript corresponding to A0A087WXI3 (FMRI-214, ENST00000616382.5, 2,799 nucleotides), and its protein product (a 536-amino acid protein), and a transcript corresponding to R9WNI0 ‘FMRI-218”, ENST00000621453.5, 1,827 nucleotides), and its protein product (a 548-amino acid protein).
[0080] In some embodiments, a normal FMRI gene product described herein comprises a transcript corresponding to Q06787 (FMR1-2Q5, ENST00000370475.9, 4,441 nucleotides), and its protein product (a 632-amino acid protein (NP_002015.1)). FMR1-2Q5, also referred to as “isoform 1” or “isol”, is produced in typical developing individuals and a subpopulation of FXS subjects. FMRI -205 has 17 exons, and the splicing between Exon 1 of FMRI -205 (between base pairs 147,911,919 and 147,912,230, SEQ ID NO: 19) and Exon 2 ofFMRJ-205 (between base pairs 147,921,933 and 147,921,985, SEQ ID NO:20) is considered normal FMRI RNA splicing. Additional information on FMRI -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.
[0081] CTCAGTCAGGCGCTCAGCTCCGTTTCGGTTTCACTTCCGGTGGAGGGCCGC CTCTGAGCGGGCGGCGGGCCGACGGCGAGCGCGGGCGGCGGCGGTGACGGAGGCG CCGCTGCCAGGGGGCGTGCGGCAGCGCGGCGGCGGCGGCGGCGGCGGCGGCGGCG GAGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCTGGGCCTCGAGCGCCCGCAGCCC ACCTCTCGGGGGCGGGCTCCCGGCGCTAGCAGGGCTGAAGAGAAGATGGAGGAGCT GGTGGTGGAAGTGCGGGGCTCCAATGGCGCTTTCTACAAG (SEQ ID NO: 19).
[0082] GCATTTGTAAAGGATGTTCATGAAGATTCAATAACAGTTGCATTTGAAAA CAA (SEQ ID NO:20).
Agents
[0083] In another aspect, the present disclosure provides an agent that modulates splicing and/or expression of FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMRI -217 or decreasing a protein encoded by FMRI -217).
[0084] In another aspect, the present disclosure provides an agent that modulates splicing and/or expression of FMRI gene (e.g., decreasing splicing between Exons 1 and 2 of FMRI -217 or decreasing a protein encoded by FMRI -217).
[0085] In another aspect, the present disclosure provides an agent that decreases expression of an aberrant FMRI gene product.
[0086] As used herein, the term “decreasing,” “decrease,” “reducing” or “reduce” refers to modulation that results in a lower level of the aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product), relative to a reference (e.g., the level prior to or in an absence of modulation by an agent disclosed herein).
[0087] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) decreases expression of an aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product), relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
[0088] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) decreases expression of an aberrant FMRI transcript, decreases expression of an aberrant /’A7 7 -encoded protein, or both.
[0089] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) decreases expression of an aberrant FMRI transcript (e.g., FMRI -217). In some embodiments, the agent decreases expression of the aberrant FMRI transcript, relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
[0090] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) decreases expression of an aberrant /’A7/? /-encoded protein (e.g., the protein product of FMRI -217). In some embodiments, the agent decreases expression of the aberrant /’A7/? /-encoded protein, relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
[0091] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) decreases expression of an aberrant FMRI transcript and an aberrant FMR1- encoded protein (e.g., FMRI -217 and its protein product). In some embodiments, the agent decreases expression of the aberrant FMRI transcript and the aberrant /’A7/? /-encoded protein, relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
[0092] An agent disclosed herein may decrease expression of an aberrant FMRI gene product directly or indirectly, for example, by altering transcription initiation, transcription elongation, transcription termination, RNA splicing, RNA processing, RNA stability, translation initiation, post-translational modification, protein stability, protein degradation, or a combination of the foregoing.
[0093] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) decreases splicing of an aberrant FMRI transcript (e.g., between Exons 1 and 2 of 7-217). In some embodiments, the agent decreases splicing of the aberrant FMRI transcript, relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%.
[0094] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) increases the level of expression of FMRP. As used herein, the term “increasing” or “increase” refers to modulation that results in a higher level of FMRP, relative to a reference (e.g., the level prior to or in an absence of modulation by an agent disclosed herein).
[0095] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) increases FMRP expression, relative to a reference, by at least 5%, e.g., by at least: 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%.
[0096] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) increases expression of a normal FMRI gene product, relative to a reference, by at least 5%, e.g., by at least: 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, the agent increases expression of a normal FMRI gene product to at least 5% of the level observed in in typically developing subjects (e.g., human), e.g., at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, of the level observed in the typically developing subject. In some embodiments, the agent increases expression of a normal FMRI gene product to at least 30% of the level observed in in typically developing subjects (e.g., human).
[0097] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) increases expression of a normal FMRI transcript, a normal FA 7?7-encoded protein, or both.
[0098] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) increases expression of a normal FMRI transcript (e.g., FMRI -IQ '). In some embodiments, the agent increases expression of the normal FMRI transcript, relative to a reference, by at least 5%, e.g., by at least: 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%.
[0099] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) increases expression of a normal FMRI -encoded protein (e.g., a protein encoded by FMR1-2QS). In some embodiments, the agent increases expression of the normal FMR1- encoded protein, relative to a reference, by at least 5%, e.g., by at least: 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%.
[00100] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) increases expression of a normal FMRI transcript and a normal FMRI -encoded protein (e.g., FMRI -205 and its protein product). In some embodiments, the agent increases expression of the normal FMRI transcript and the normal FW7?7-encoded protein, relative to a reference, by at least 5%, e.g., by at least: 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%.
[00101] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) increases splicing of a normal FMRI transcript (e.g., between Exons 1 and 2 of FMRl- QF). In some embodiments, the agent increases splicing of the normal FMRI transcript, relative to a reference, by at least 5%, e.g., by at least: 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%.
[00102] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) decreases expression of an aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product) and increases expression of FMRP.
[00103] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) decreases expression of an aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product) and increases expression of a normal FMRI gene product (e.g., FMRI -205 and/or its protein product). In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide) decreases expression of an aberrant FMRI transcript, decreases expression of an aberrant FMRI -encoded protein, increases expression of a normal FMRI transcript, increases expression of a normal FW7?7-encoded protein, or a combination thereof.
[00104] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide): decreases expression of an aberrant FMRI gene product (e.g., FMRI -217 and/or its protein product), relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%; and increases expression of a normal FMRI gene product (e.g., FMRI -205 and/or its protein product), relative to a reference, by at least 5%, e.g., by at least: 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%.
[00105] In some embodiments, an agent disclosed herein (e.g., an anti-sense RNA polynucleotide): decreases splicing of an aberrant FMRI transcript (e.g., between Exons 1 and 2 of FMR1- 217), relative to a reference, by at least 5%, e.g., by at least: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%; and increases splicing of a normal FMRI transcript (e.g., between Exons 1 and 2 of FMR1- 205), relative to a reference, by at least 5%, e.g., by at least: 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%.
[00106] In some embodiments, a level of an FMRI gene product e.g., an aberrant FMRI transcript, an aberrant FA 7?7-encoded protein, a normal FMRI transcript, a normal FMR1- encoded protein, or a combination thereof), is measured at least 1 day after an agent disclosed herein is administered to a subject, e.g., for at least: 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 months or 6 months, after a treatment with an agent disclosed herein has begun.
[00107] In some embodiments, a level an FMRI gene product is measured in a tissue or a cell. In some embodiments, a level an FMRI gene product is measured in a white blood cell. In some embodiments, a level an FMRI gene product is measured in a leukocyte. In some embodiments, a level an FMRI gene product is measured in a fibroblast cell e.g., a dermal derived fibroblast cell or a lung-derived fibroblast cell). In some embodiments, a level an FMRI gene product is measured in a cortex tissue e.g., a brain biopsy of superficial cortex).
Target Sequences
[00108] In some embodiments, an agent disclosed herein e.g., an antisense oligonucleotide (ASO)) promotes exclusion of an aberrant FMRI exon. In some embodiments, the agent promotes exclusion of Exon 2 of FMRI -217.
[00109] In some embodiments, an agent disclosed herein e.g., an ASO) targets (indirectly, or directly, e.g., binds) a primary aberrant transcript (pre-mRNA) of an FMRI gene. As used herein, the term “target” refers to a preliminary mRNA region, and specifically, to a region identified by Exon 2, and the adjacent intron 1-2 regions of FMRI -217 , which is responsible for the splicing associated with FMRI -217. In some embodiments, a target sequence refers to a portion of the target RNA against which a polynucleotide e.g., an ASO) is directed, that is, the sequence to which the polynucleotide will hybridize by Watson-Crick base pairing of a complementary sequence.
[00110] In some embodiments, the agent targets a contiguous nucleotide sequence within pre- mRNA of FMRI -217, wherein the contiguous nucleotide sequence is at least 8 nucleotides in length. In some embodiments, the contiguous nucleotide sequence is at least 9 nucleotides in length, for example, at least: 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 in length. In some embodiments, the contiguous nucleotide sequence is at least 12 nucleotides in length. In some embodiments, the contiguous nucleotide sequence is about 8-80 nucleotides in length, for example, about: 10- 60, 10-40, 10-30, 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 length. In some embodiments, the contiguous nucleotide sequence is about 10-30 nucleotides in length.
[00111] In some embodiments, the agent (e.g., an ASO) targets a contiguous nucleotide sequence within SEQ ID NO:24 (e.g., within any one or more of SEQ ID NOs:25-42), wherein the contiguous nucleotide sequence is at least 8 nucleotides in length. In some embodiments, the agent (e.g., an ASO) targets a contiguous nucleotide sequence within SEQ ID NO:27, wherein the contiguous nucleotide sequence is at least 8 nucleotides in length. In some embodiments, the contiguous nucleotide sequence is at least 9 nucleotides in length, for example, at least: 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 in length.
[00112] UCAGGUCUCCUUUGGCUUCUCUUUUCCGGUCUAGCAUUGGGACUUCGG AGAGCUCCACUGUUCUGGGCGAGGGCUGUGAAGAAAGA (SEQ ID NO:24).
[00113] UCAGGUCUCCUUUGGCUUCUCUUUUCCGGUCUAGCAUUGGGACUUCGG AGA (SEQ ID NO :25)
[00114] CAUUGGGACUUCGGAGAGCUCCACUGUUCUGGGCGAGGGCUGUGAAGA AAGA (SEQ ID NO:26)
[00115] UGGGACUUCGGAGAGCUCCACUGUUCUGGGCGAGGGCUGUGAAGAA (SEQ ID NO:27)
[00116] In some embodiments, the agent (e.g., an ASO) targets a contiguous nucleotide sequence within FMR1-2V1 Exon 2, FMR1-2V1 Intron 1-2, the junction between Exon 2 and Intron 1-2 of FMR1-2Y1 , or a combination thereof. In some embodiments, the agent (e.g., an ASO) targets a contiguous nucleotide sequence within any one or more of SEQ ID NOs:28-42, wherein the contiguous nucleotide sequence is at least 8 nucleotides in length. In some embodiments, the agent (e.g., an ASO) targets a contiguous nucleotide sequence within any one or more of SEQ ID NOs:37-42, wherein the contiguous nucleotide sequence is at least 8 nucleotides in length. In some embodiments, the contiguous nucleotide sequence is selected from a polynucleotide sequence set forth in any one of SEQ ID NOs:28-42. In some embodiments, the contiguous nucleotide sequence is selected from a polynucleotide sequence set forth in any one of SEQ ID NOs:37-42.
[00117] UCAGGUCUCCUUUGGCUUCU (SEQ ID NO:28)
[00118] GUCUCCUUUGGCUUCUCUUU (SEQ ID NO:29)
[00119] UGGCUUCUCUUUUCCGGUCUAG (SEQ ID NO:30)
[00120] UUCUCUUUUCCGGUCUAGCAU (SEQ ID NO:31)
[00121] UCUUUUCCGGUCUAGCAUUG (SEQ ID NO:32)
[00122] UCCGGUCUAGCAUUGGGACUU (SEQ ID NO:33)
[00123] UAGCAUUGGGACUUCGGAGA (SEQ ID NO:34)
[00124] UGGGACUUCGGAGAGCUC (SEQ ID NO:35)
[00125] UCGGAGAGCUCCACUGUUCU (SEQ ID NO:36)
[00126] GAGCUCCACUGUUCUGGGCG (SEQ ID NO:37)
[00127] CUCCACUGUUCUGGGCGAGG (SEQ ID NO:38)
[00128] GGACUUCGGAGAGCUCCACUG (SEQ ID NO:39)
[00129] GGAGAGCUCCACUGUUCUGGG (SEQ ID NO :40)
[00130] UGUUCUGGGCGAGGGCUGUG (SEQ ID NO:41)
[00131] UGGGCGAGGGCUGUGAAGAA (SEQ ID NO:42)
Polynucleotides (Polynucleotide Agents)
[00132] In some embodiments, an agent disclosed herein comprises at least one polynucleotide disclosed herein. In some embodiments, the agent comprises at least two polynucleotides disclosed herein.
[00133] In another aspect, the present disclosure provides a polynucleotide capable of decreasing expression of an aberrant FMRI gene product.
[00134] In another aspect, the present disclosure provides a polynucleotide capable of decreasing splicing of FMR1-2Y1.
[00135] In another aspect, the present disclosure provides a method of enhancing the level of FMRP in a cell, comprising contacting the cell with an oligonucleotide which is complementary to at least 8 contiguous nucleotides of a sequence set forth in SEQ ID NOs:24-42, such that the level of FMRP in the cell is enhanced.
[00136] 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 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.
[00137] 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.
[00138] 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 Ci-Ce alkyl, alkenyl, alkynyl or aryl.
[00139] In some embodiments, a phosphate group of the 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, the 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. [00140] In some embodiments, a polynucleotide disclosed herein (e.g., ASO) binds a target sequence described herein.
[00141] In some embodiments, a targeting polynucleotide disclosed herein (e.g., ASO) has near or substantial complementarity to a target sequence described herein. In some embodiments, the polynucleotide is formed of contiguous complementary sequences (to the target sequence). In some embodiments, the polynucleotide sequence is formed of non-contiguous complementary sequences (to the target sequence), for example, when placed together, constitute sequence that spans the target sequence.
[00142] In some embodiments, a polynucleotide disclosed herein (e.g., ASO) comprises a nucleotide sequence that is complementary (e.g., fully complementary or partially complementary) to a target sequence described herein (such that the polynucleotide is capable of hybridizing or annealing to target sequence, e.g., under physiological conditions). As used herein, “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 (z.e., hydrogen bonding) with a residue of the second region, thus forming a hydrogen-bonded duplex.
[00143] In some embodiments, a polynucleotide disclosed herein (e.g., ASO) specifically hybridizes to a target polynucleotide described herein (e.g., contiguous nucleotides of a sequence set forth in SEQ ID NOs:24-42), for example, under physiological conditions, with a 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. The Tm is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide at a given ionic strength and pH. In some embodiments, specific hybridization corresponds to stringent hybridization conditions. In some embodiments, specific hybridization occurs with near complementary of the antisense oligomer to the target sequence. In some embodiments, specific hybridization occurs with substantial complementary of the antisense oligomer to the target sequence. In some embodiments, specific hybridization occurs with exact complementary of the antisense oligomer to the target sequence.
[00144] In some embodiments, a polynucleotide disclosed herein (e.g, ASO) comprises a nucleotide sequence that is complementary to a contiguous nucleotide sequence (e.g, 10 to 30 nucleotides) of pre-mRNA of an aberrant FMRI transcript.
[00145] In some embodiments, a polynucleotide disclosed herein (e.g., ASO) comprises a nucleotide sequence that is complementary to a contiguous nucleotide sequence (e.g., 10 to 30 nucleotides) of pre-mRNA of FMR1-2Y1. In some embodiments, the polynucleotide comprises a nucleotide sequence that is complementary to a target sequence within any one of SEQ ID NOs:24-42 (e.g., any one of SEQ ID NOs:24-27, any one of SEQ ID NOs:28-42, or a combination thereof). [00146] In some embodiments, a polynucleotide disclosed herein is an antisense oligonucleotide (ASO). In some embodiments, the polynucleotide is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense DNA, an antisense RNA, a microRNA (miRNA), an antagomir, a guide RNA (gRNA). The polynucleotide 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.
[00147] In some embodiments, a polynucleotide disclosed herein (e.g., ASO) comprises a nucleotide sequence specifically hybridizes to (e.g., having near, substantial, or exact complementarity to) at least a portion of X chromosome between base pairs 147,911,919 and 147,921,985 (e.g., a target sequence within X chromosome between base pairs 147,911,919 and 147,921,985), for example, 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 disclosed herein (e.g., ASO) has exact complementarity to at least a portion of X chromosome between base pairs 147,911,919 and 147,921,985, for example, 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.
[00148] In some embodiments, the polynucleotide comprises a nucleotide sequence specifically hybridizes to (e.g., having near, substantial, or exact complementarity to) at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766, for example, having at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reverse and complementary sequence of the at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766.
[00149] 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 a 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.
[00150] 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 el al., Current Protocols in Molecular Biology).
[00151] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence having about 70-100% sequence identity to at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%. In some embodiments, the polynucleotide comprises a nucleotide sequence that is identical to at least a portion of X chromosome between base pairs 147,912,694 and 147,912,766.
[00152] In some embodiments, a polynucleotide disclosed herein comprises a nucleotide sequence specifically hybridizes to (e.g., having near, substantial, or exact complementarity to) at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766, for example, having at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reverse and complementary sequence of the at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766.
[00153] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence having about 70-100% sequence identity to at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%. In some embodiments, the polynucleotide comprises a nucleotide sequence that is identical to at least a portion of X chromosome between base pairs 147,912,731 and 147,912,766.
[00154] In some embodiments, a polynucleotide disclosed herein (e.g., ASO) comprises a nucleotide sequence having at least 70% sequence identity to, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: l-l l and SEQ ID NOs:43-50. In certain embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50. In some embodiments, the polynucleotide (e.g., ASO) has about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96- 100%, 97-100%, 98-100% or 99-100%. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50. In the sequences, 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.
[00155] AGAAGCCAAAGGAGACCTGA (SEQ ID NO: 1) (W-704).
[00156] AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705).
[00157] CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:3) (W-706).
[00158] ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707).
[00159] CAATGCTAGACCGGAAAAGA (SEQ ID NO:5) (W-708).
[00160] AAGTCCCAATGCTAGACCGGA (SEQ ID NO:6) (W-709).
[00161] TCTCCGAAGTCCCAATGCTA (SEQ ID NO:7) (W-710).
[00162] GAGCTCTCCGAAGTCCCA (SEQ ID NO:8) (W-711).
[00163] AGAACAGTGGAGCTCTCCGA (SEQ ID NO:9) (W-712).
[00164] CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713).
[00165] CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714).
[00166] CAGTGGAGCTCTCCGAAGTCC (SEQ ID NO:43) (2831).
[00167] CCCAGAACAGTGGAGCTCTCC (SEQ ID NO:44) (2832).
[00168] CACAGCCCTCGCCCAGAACA (SEQ ID NO:45) (2833).
[00169] TTCTTCACAGCCCTCGCCCA (SEQ ID NO:46) (2834).
[00170] TCTTTCTTCACAGCCCTCGCCCAGAACAGTGGAGCTCTCCGAAGTCCCAAT GCTAGACCGGAAAAGAGAAGCCAAAGGAGACCTGA (SEQ ID NO:47).
[00171] TCTCCGAAGTCCCAATGCTAGACCGGAAAAGAGAAGCCAAAGGAGACCT GA (SEQ ID NO:48).
[00172] TCTTTCTTCACAGCCCTCGCCCAGAACAGTGGAGCTCTCCGAAGTCCCAAT G (SEQ ID NO:49).
[00173] TTCTTCACAGCCCTCGCCCAGAACAGTGGAGCTCTCCGAAGTCCCA (SEQ ID NO:50).
[00174] In some embodiments, a polynucleotide disclosed herein (e.g., ASO) comprises a nucleotide sequence having at least 70% sequence identity to, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 10-11 and SEQ ID NOs:43-46. In certain embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11 and SEQ ID NOs:43-50. In some embodiments, the polynucleotide (e.g., ASO) has about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 10-11 and SEQ ID NOs:43-46. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 10-11 and SEQ ID NOs:43-46, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to a sequence set forth in any one of SEQ ID NOs: 10-11 and SEQ ID NOs:43-46.
[00175] In some embodiments, an agent disclosed herein comprises a first polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to SEQ ID NO: 10, and a second polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to SEQ ID NO: 11. In some embodiments, the first polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10, and the second polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the first polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to SEQ ID NO: 10, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99- 100%, sequence identity to SEQ ID NO: 10; and the second polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to SEQ ID NO: 11, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100% sequence identity to SEQ ID NO: 11. In some embodiments, the first polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to SEQ ID NO: 10, and the second polynucleotide comprises a nucleotide sequence that is identical to SEQ ID NO: 11.
[00176] In some embodiments, a polynucleotide disclosed herein (e.g., ASO) comprises a nucleotide sequence having at least 70% sequence identity to, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 51-69. In certain embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:51-69. In some embodiments, the polynucleotide (e.g., ASO) has about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:51-69. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to a sequence set forth in any one of SEQ ID NOs:51-69, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to a sequence set forth in any one of SEQ ID NOs: 51-69.
[00177] AGAAGCCAAAGGAGACCUGA (SEQ ID NO:51) (W-704).
[00178] AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:52) (W-705).
[00179] CUAGACCGGAAAAGAGAAGCCA (SEQ ID NO: 53) (W-706).
[00180] AUGCUAGACCGGAAAAGAGAA (SEQ ID NO:54) (W-707).
[00181] CAAUGCUAGACCGGAAAAGA (SEQ ID NO:55) (W-708). [00182] AAGUCCCAAUGCUAGACCGGA (SEQ ID NO:56) (W-709). [00183] UCUCCGAAGUCCCAAUGCUA (SEQ ID NO:57) (W-710). [00184] GAGCUCUCCGAAGUCCCA (SEQ ID NO:58) (W-711).
[00185] AGAACAGUGGAGCUCUCCGA (SEQ ID NO:59) (W-712). [00186] CGCCCAGAACAGUGGAGCUC (SEQ ID NO:60) (W-713). [00187] CCUCGCCCAGAACAGUGGAG (SEQ ID NO:61) (W-714). [00188] CAGUGGAGCUCUCCGAAGUCC (SEQ ID NO:62) (2831). [00189] CCCAGAACAGUGGAGCUCUCC (SEQ ID NO:63) (2832). [00190] CACAGCCCUCGCCCAGAACA (SEQ ID NO:64) (2833). [00191] UUCUUCACAGCCCUCGCCCA (SEQ ID NO:65) (2834).
[00192] UCUUUCUUCACAGCCCUCGCCCAGAACAGUGGAGCUCUCCGAAGUCCCA AUGCUAGACCGGAAAAGAGAAGCCAAAGGAGACCUGA (SEQ ID NO: 66).
[00193] UCUCCGAAGUCCCAAUGCUAGACCGGAAAAGAGAAGCCAAAGGAGACC UGA (SEQ ID NO:67).
[00194] UCUUUCUUCACAGCCCUCGCCCAGAACAGUGGAGCUCUCCGAAGUCCCA AUG (SEQ ID NO :68).
[00195] UUCUUCACAGCCCUCGCCCAGAACAGUGGAGCUCUCCGAAGUCCCA (SEQ ID NO: 69).
[00196] In some embodiments, a polynucleotide disclosed herein (e.g., ASO) comprises a nucleotide sequence having at least 70% sequence identity to, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:60-65. In certain embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:60-65. In some embodiments, the polynucleotide (e.g., ASO) has about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:60-65. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to a sequence set forth in any one of SEQ ID NOs:60-65, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100%. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to a sequence set forth in any one of SEQ ID NOs:60-65.
[00197] In some embodiments, an agent disclosed herein comprises a first polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to SEQ ID NO:60, and a second polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity, for example, at least: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, to SEQ ID NO:61. In some embodiments, the first polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:60, and the second polynucleotide (e.g., ASO) comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:61. In some embodiments, the first polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to SEQ ID NO:60, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99- 100%, sequence identity to SEQ ID NO: 60; and the second polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to SEQ ID NO:61, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100% sequence identity to SEQ ID NO:61. In some embodiments, the first polynucleotide (e.g., ASO) comprises a nucleotide sequence that is identical to SEQ ID NO:60, and the second polynucleotide comprises a nucleotide sequence that is identical to SEQ ID NO:61. [00198] In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence that is at least about 70% identical to a sequence within X chromosome region between
147,912,230 and 147,914,451 (e.g., between 147,912,230 and 147,912,728 or between 147,912,728 and 147,914,451), for example, at least about: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence within X chromosome region between
147,912,230 and 147,912,728. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence within X chromosome region between 147,912,230 and 147,914,451 (e.g., between 147,912,230 and 147,912,728 or between 147,912,728 and 147,914,451). In some embodiments, the polynucleotide comprises a nucleotide sequence having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence within X chromosome region between 147,912,230 and 147,914,451 (e.g., between 147,912,230 and 147,912,728 or between 147,912,728 and 147,914,451). In some embodiments, the polynucleotide comprises a nucleotide sequence having about 70-100% sequence identity to a sequence within X chromosome region between 147,912,230 and 147,914,451 (e.g., between
147,912,230 and 147,912,728 or between 147,912,728 and 147,914,451), for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96- 100%, 97-100%, 98-100% or 99-100%.
[00199] In some embodiments, the polynucleotide (e.g., ASO) is at least about 70% complimentary to at least a portion of an FMRI gene transcript, for example, at least about: 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to at least a portion of an FMRI gene transcript. In some embodiments, the polynucleotide is about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to at least a portion of an FMRI gene transcript. In some embodiments, the polynucleotide is about 70-100% complimentary to at least a portion of an FMRI gene transcript, for example, about: 75-100%, 75-99%, 80-100%, 80-98%, 85-100%, 85-97%, 90-100%, 90-96%, 95-100%, 96-100%, 97-100%, 98-100% or 99-100% complimentary to at least a portion of an FMRI gene transcript.
[00200] In some embodiments, a polynucleotide disclosed herein has a length of at least about 8 nucleotides, for example, 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. In some embodiments, the polynucleotide has a length of about 8-80 nucleotides, for example, 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, the polynucleotide has a length of about 10-30 or 12-30 nucleotides. In some embodiments, the polynucleotide has a length of about: 8, 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.
[00201] In some embodiments, a polynucleotide disclosed herein has a length of at least about 12 nucleotides, for example, at least about: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29 or 30 nucleotides. In some embodiments, the polynucleotide has a length of about 12- 40 nucleotides, for example, about: 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, the polynucleotide has a length of about 15-25 nucleotides. In some embodiments, the polynucleotide has a length of about: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 35 or 40 nucleotides. In some embodiments, a polynucleotide is an oligonucleotide. In some embodiments, the length of the polynucleotide is about 18-22 nucleotides.
[00202] In some embodiments, a polynucleotide disclosed herein (e.g., oligonucleotide) 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 nucleotide sequences. In some embodiments, the polynucleotide is an isolated DNA polynucleotide. In some embodiments, the polynucleotide is an isolated RNA polynucleotide.
[00203] Polynucleotides of the disclosure 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 of the disclosure is a recombinant polynucleotide.
[00204] In another aspect, the present disclosure provides a polynucleotide capable of increasing the expression of a functional FMRI gene product. The polynucleotide is any one of the polynucleotides, modified or unmodified, disclosed herein. In some embodiments, the polynucleotide is any one of the modified polynucleotides disclosed herein.
Modification of Polynucleotides
[00205] In some embodiments, a polynucleotide of the disclosure 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. [00206] Chemical modifications can be chosen to, e.g., increase nuclease resistance of a polynucleotide (e.g., oligonucleotide), 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.
[00207] In some embodiments, each modification of a ribose group comprises 2’-(9-methyl, 2’-fluoro, 2’-deoxy, 2’-< -(2-methoxyethyl) (MOE), 2’-O-alkyl, 2’-O-alkoxy, 2’-O-alkylamino, 2’-NH2, or a constrained nucleotide, or a combination thereof.
[00208] In some embodiments, a substituted RNA analogue disclosed herein comprises a methoxy ethyl group on the 2’ OH.
[00209] In some embodiments, a constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2’-(5)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2’-(9,4’-C-aminomethylene bridged nucleic acid (2’,4’-BNANC), an alpha- L-locked nucleic acid, and a tricyclo-DNA, or a combination thereof.
[00210] 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., oligonucleotide) comprises a 2’-< -(2-methoxyethyl) (MOE) modification.
[00211] In some embodiments, modification of a ribose group comprises a tricyclo-DNA modification. In some embodiments, every nucleotide of a polynucleotide (e.g., antisense oligonucleotide) comprises a tricyclo-DNA modification.
[00212] In some embodiments, modification of a ribose group comprises a 2’-deoxy modification.
[00213] 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.
[00214] 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 is a phosphorothioate-modified polynucleotide. [00215] 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.
[00216] 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.
[00217] In some embodiments, modification of a nucleobase group comprises a 5- methylcytosine modification.
[00218] In some embodiments, a polynucleotide comprises a mixture of modified nucleotides. [00219] In some embodiments, a mixture of modified nucleotides comprise two or more modifications selected from the group consisting of 2’-(9-methyl, 2’-deoxy, 2’ -O-(2- methoxyethyl) (MOE), LNA, and tricyclo-DNA.
[00220] In some embodiments, a polynucleotide comprises 4 or fewer consecutive 2’ -deoxy modified nucleotides.
[00221] In some embodiments, a mixture of modified nucleotides comprise one or more -O- methyl modified nucleotides and one or more LNA modified nucleotides.
[00222] In some embodiments, a mixture of modified nucleotides comprise one or more -O- (2-methoxy ethyl) (MOE) modified nucleotides and one or more LNA modified nucleotides.
[00223] In some embodiments, each ribose group of a polynucleotide disclosed herein (e.g., ASO) comprises 2’ -O-(2 -methoxy ethyl) (MOE) and/or each phosphate group of the polynucleotide comprises a phosphorothioate. In some embodiments, each ribose group of the polynucleotide (e.g., ASO) comprises 2’-O-(2-methoxyethyl) (MOE). In some embodiments, each phosphate group of the polynucleotide comprises a phosphorothioate. In some embodiments, each ribose group of a polynucleotide disclosed herein (e.g., ASO) comprises 2’- O-(2-methoxyethyl) (MOE), and each phosphate group of the polynucleotide comprises a phosphorothi oate .
Polypeptides
[00224] In some embodiments, an agent disclosed herein comprises a polypeptide. 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 oc-amino acids (e.g., alanine, glycine, valine), non-oc-amino acids (e.g., [3-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 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), Mm ethyl - oc-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).
[00225] In some embodiments, a polypeptide disclosed herein is an isolated polypeptide. In some embodiments, a polypeptide disclosed herein is a recombinant polypeptide.
[00226] In some embodiments, the polypeptide is an inhibitor e.g., a direct inhibitor or an indirect inhibitor) of expression of an aberrant FMRI gene product e.g., FMRI -217, and/or its protein product). In some embodiments, the polypeptide is an activator e.g., a direct activator or an indirect activator) of expression of a normal FMRI gene product e.g., FMRI -2Q5, and/or its protein product). In some embodiments, the polypeptide reduces expression of an aberrant FMRI gene product e.g., FMRI -217, and/or its protein product) and increases expression of a normal FMRI gene product e.g., FMRI -2Q5, and/or its protein product).
[00227] In some embodiments, a polypeptide disclosed herein is an immunoglobulin molecule. In some embodiments, the immunoglobulin molecule an antibody. In some embodiments, the antibody is an antagonist antibody that binds an FMRI transcript, or isoform, associated with a fragile X-associated disorder e.g., FXS). The 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, the antibody is primatized e.g., humanized). In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody e.g., monoclonal antibody) is multispecific, e.g., bi-, tri-, or quad-specific.
[00228] In some embodiments, a polypeptide disclosed herein 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, the 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.
[00229] In some embodiments, a polypeptide disclosed herein is an antibody mimetic. 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.
[00230] Techniques, assays and reagents for making and using therapeutic antibodies, or antigen-binding fragments thereof, against a target antigen (e.g., an FMRI transcript, or isoform, 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 el 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.
Gene Editing Systems
[00231] In some embodiments, an agent disclosed herein comprises a gene editing system. In some embodiments, the gene editing system produces a deletion of nucleotides, a substitution of nucleotides, an addition of nucleotides or a combination of the foregoing, in the FMRI gene. In some embodiments, the gene editing system produces a partial or complete deletion in Exon 2 of FMR1-2V1 (e.g., pseudo exon between base pairs 147,911,919 and 147,914,451 in the human FMRI gene).
[00232] In some embodiments, the 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, the gene editing system is a CRISPR/Cas system. In some embodiments, the gene editing system is a class II CRISPR/Cas system.
[00233] In some embodiments, the gene editing system comprises a single Cas endonuclease or a polynucleotide encoding the single Cas endonuclease. In some embodiments, the single Cas endonuclease is Cas9, Cpfl, C2C1 or C2C3. In some embodiments, the single Cas endonuclease is Cas9 (e.g., of Streptococcus Pyogenes). In some embodiments, the single Cas endonuclease is Cpfl. In some embodiments, the Cpfl is AsCpfl (Com Acidaminococcus sp.) or LbCpfl (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.
[00234] In some embodiments, the type II Cas endonuclease is Cas 9 (e.g., of Streptococcus pyogenes). In some embodiments, the modified Cas 9 is nickase Cas9, dead Cas9 (dCas9) or eSpCas9. In some embodiments, the nickase Cas9 is Cas9 D10A. In some embodiments, the dCas9 is D10A or H840A. In some embodiments, the 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/ cri spr/) .
[00235] 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, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 Al. 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).
[00236] In some embodiments, the agent comprises a small molecule. In some embodiments, the small molecule binds to a protein capable of modulating the splicing and/or expression of FMRI or a fragment thereof. In some embodiments, the small molecule is an inhibitor of the target protein (e.g., a direct inhibitor, an indirect inhibitor). In some embodiments, the small molecule is an activator of the target protein (e.g., a direct activator, and indirect activator). Nonlimiting examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic, organometallic or inorganic compounds.
Subjects
[00237] The term “subject” refers to a mammalian subject, preferably human, diagnosed with or suspected of having a fragile X-associated disorder (e.g., FXS). [00238] In some embodiments, the subject comprises a CGG repeat expansion between about 55 and about 200 repeats in the 5’ untranslated region of an FMRI gene. In some embodiments, the subject comprises a CGG repeat expansion exceeding 200 repeats in the 5’ untranslated region of an FMRI gene. In some embodiments, the subject comprises a CGG repeat expansion that is partially methylated. In some embodiments, the subject comprises a CGG repeat expansion that is fully methylated. In some embodiments, the subject has an increased level of isoform 12 of FMRI, a decreased level of isoform 1 of FMRI, or a combination thereof.
[00239] In some embodiments, the subject has one X chromosome and one Y chromosome. In some embodiments, the subject has two X chromosomes. In some embodiments, the subject has two X chromosomes and one Y chromosome. In some embodiments, the subject has one X chromosome and two Y chromosomes.
[00240] In some embodiments, the subject is a human male. In some embodiments the subject is human female.
[00241] In some embodiments, the subject is at least about 1 month of age, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18 or 21 months of age, or 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, the subject is about: 1-100, 1-80, 1-60, 1-30, 1-24, 1-20, 1-18, 1-12, 1-10, 1-8, 1- 6, 2-100, 2-80, 2-60, 2-30, 2-24, 2-20, 2-18, 2-12, 2-10, 2-8, 2-6, 3-100, 3-80, 3-60, 3-30, 3-24, 3-20, 3-18, 3-12, 3-10, 3-8, 3-6, 4-100, 4-80, 4-60, 4-30, 4-24, 4-20, 4-18, 4-12, 4-10, 4-8, 4-6, 5-100, 5-80, 5-60, 5-30, 5-24, 5-20, 5-18, 5-12, 5-10, 5-8, 6-100, 6-80, 6-60, 6-30, 6-24, 6-20, 6- 18, 6-12, 6-10, 8-100, 8-80, 8-60, 8-30, 8-24, 8-20, 8-18, 8-12, 10-100, 10-80, 10-60, 10-30, 10-
24, 10-20, 10-18, 12-100, 12-80, 12-38, 12-60, 12-50, 12-40, 12-30, 12-24, 12-20, 12-18, 18- 100, 18-80, 18-60, 18-50, 18-40, 18-30, 18-24, 20-100, 20-80, 20-60, 20-50, 20-40, 20-30, 20-
25, 30-100, 30-80, 30-60, 30-55, 30-50, 30-45, 30-40, 40-100, 40-80, 40-60, 40-55 or 40-50 years of age. In some embodiments, the subject is 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, 50, 55, 60, 65, 70, 75, 80 or 100 years of age. In some embodiments, the subject is about 12-38 years of age. In other embodiments, the subject is a fetus. In some embodiments, the subject is a neonatal subject.
[00242] In some embodiments, the subject is 18 years of age or older, e.g., 18 to less than 40 years of age, 18 to less than 45 years of age, 18 to less than 50 years of age, 18 to less than 55 years of age, 18 to less than 60 years of age, 18 to less than 65 years of age, 18 to less than 70 years of age, 18 to less than 75 years of age, 40 to less than 75 years of age, 45 to less than 75 years of age, 50 to less than 75 years of age, 55 to less than 75 years of age, 60 to less than 75 years of age, 65 to less than 75 years of age, 60 to less than 75 years of age, 40 years of age or older, 45 years of age or older, 50 years of age or older, 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older or 90 years of age or older. In some embodiments, the subject is 50 years of age or older. In some embodiments, the subject is a child. In some embodiments, the subject is 18 years of age or younger, e.g., 0-18 years of age, 0-12 years of age, 0-16 years of age, 0-17 years of age, 2-12 years of age, 2-16 years of age, 2-17 years of age, 2-18 years of age, 3-12 years of age, 3-16 years of age, 3-17 years of age, 3-18 years of age, 4-12 years of age, 4-16 years of age, 4-17 years of age, 4-18 years of age, 6-12 years of age, 6-16 years of age, 6-17 years of age, 6-18 years of age, 9-12 years of age, 9-16 years of age, 9-17 years of age, 9-18 years of age, 12-16 years of age, 12-17 years of age or 12-18 years of age.
[00243] In some embodiments, the subject is about 2-11, 4-17, 12-18, 18-50, 18-90 or 50-90 years of age.
[00244] In some embodiments, a subject is a human. In some embodiments, the human subject has, or is predisposed to have a fragile X-associated disorder. In some embodiments the human subject has, or is predisposed to have, FXS, FXPOI, FXTAS, or a combination thereof. In some embodiments, the human subject has, or is predisposed to have FXS. In some embodiments, the subject is a human (e.g., about 50 years of age or older) who has, or is predisposed to have, FXTAS.
[00245] In some embodiments, the 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. Nonlimiting 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, the subject is a female with an IQ score of less than 115, 110, 105, 100, 95 or 90. In some embodiments, the subject is a male with an IQ score of less than 60, 55, 50 or 45. [00246] In some embodiments, the 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, the subject has one or more of the following: intention tremor, parkinsonism, ataxia, memory loss, white matter lesion involving middle cerebellar peduncles, and cognitive decline.
Treatments
[00247] “ 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.
[00248] In some embodiments, the method further comprises assessing the efficacy of the agent (e.g., polynucleotide such as ASO) (outcome measure) for treatment of the fragile X- associated disorder (e.g., FXS) in the subject, comprising assaying a biological sample from the subject for the presence and/or level of FMRI RNA isoform 1, FMRI RNA isoform 12, or a combination thereof.
[00249] In some embodiments, treating a fragile X-associated disorder (e.g., FXS) includes slowing progression of the fragile X-associated disorder (e.g., FXS), alleviating one or more signs or symptoms of the fragile X-associated disorder (e.g., FXS), preventing one or more signs or symptoms of the fragile X-associated disorder (e.g., FXS), or a combination thereof.
[00250] Non-limiting examples of treatment benefits include improvements in speech and motor development; a reduction in or prevention of cognitive disabilities, ranging from learning disabilities to intellectual disability; alleviating or preventing physical and medical features such as 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 mitral valve prolapse; reducing or preventing behaviors of hyperactivity, short attention span, hand biting or hand flapping, poor eye contact and social skills, shyness, anxiety, delayed speech and/or motor development, repetitive speech, and/or sensitivity to sensory stimulation (including a hypersensitivity to being touched). [00251] In some embodiments, treatment may include modulation of or improvement in language, fragile X behaviors, brain activity, clinical impression, inattention, safety, social avoidance, cognition, hyperactivity, executive function, irritability, eye contact, or memory. [00252] In some embodiments, treatment results in an intelligence quotient (IQ) score of at least about 40, for example, at least about: 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130. In some embodiments, treatment results in an IQ score between about: 40-110, 40-100, 50-105, 60-80, 65-90, 70-80, 75-95, or 70-100. In some embodiments, treatment results in an IQ score of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130. In some embodiments, treatment results in an increase in IQ score of at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 points. In some embodiments, treatment results in an increase in IQ score of between about: 1-10, 1-15, 2-20, 2- 15, 2-10, 5-15, 5-10, 10-20, or 15-20 points. In some embodiments, treatment results in an increase in IQ score of about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 points.
[00253] In still other embodiments, treatment can include reducing or preventing absent or irregular menses, fertility problems, elevated FSH (follicle-stimulating hormone) levels, premature ovarian failure, primary ovarian insufficiency, and/or hot flashes. In still further embodiments, treating may include reducing or preventing intention tremors, parkinsonism, ataxia, memory loss, white matter lesions involving middle cerebellar peduncles, and/or cognitive decline. In some embodiments, treatment may reduce or prevent neuropathy of extremities, mood instability, irritability, explosive outbursts, personality changes, autonomic function problems such as impotence, loss of bladder or bowel functions. Treatment may also include reducing or preventing high blood pressure, thyroid disorders, or fibromyalgia.
Formulation and Administration
[00254] “Therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. 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.
[00255] In some embodiments, an agent disclosed herein (e.g., ASO) is in a form of a pharmaceutical composition, or a pharmaceutically acceptable salt thereof. 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. “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.
[00256] In some embodiments, a pharmaceutical composition disclosed herein is formulated as a solution.
[00257] “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 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). [00258] In some embodiments, a pharmaceutical composition suitable for use in methods disclosed herein further comprises one or more pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject and should not interfere with the efficacy of the active ingredient. A pharmaceutically acceptable carrier includes, but is not limited to, such as those widely employed in the art of drug manufacturing. The carrier may be a diluent, adjuvant, excipient, or vehicle with which the agent 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 may 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 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, e.g., from less than about 0.5%, usually to at least about 1% to as much as 15%, 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 particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691- 1092, see especially pp. 958-89.
[00259] 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.
[00260] Non-limiting examples of buffers that may be used 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.
[00261] Non-limiting examples of antioxidants that may be used are ascorbic acid, methionine, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, lecithin, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol and tartaric acid.
[00262] Non-limiting examples of amino acids that may be used are histidine, isoleucine, methionine, glycine, arginine, lysine, L-leucine, tri-leucine, alanine, glutamic acid, L-threonine, and 2-phenylamine.
[00263] Non-limiting examples of surfactants that may be used are polysorbates (e.g., polysorbate-20 or polysorbate-80); poly oxamers (e.g., pol oxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; 1 auroamidopropyl-, 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., Paterson, N. J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.). [00264] Non-limiting examples of preservatives that may be used 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.
[00265] Non-limiting examples of saccharides that may be used 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. [00266] Non-limiting examples of salts that may be used 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, phenylsubstituted 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).
[00267] Agents (e.g., polynucleotides) disclosed 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).
[00268] In some embodiments, an agent disclosed herein (e.g., ASO) is delivered using controlled or sustained-release delivery systems (e.g., capsules, biodegradable matrices). 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.
[00269] For oral administration, polynucleotides 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 parahydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.
[00270] Administration of the agent to the subject can be by parenteral or non-parenteral means. In some embodiments, an agent disclosed herein (e.g., ASO) is administered intravenously, intra-arterially, intrathecally, intraventricularly, intramuscularly, intradermally, subcutaneously, intracranially, or spinally. “Administering” or “administration” as used herein, refers to taking steps to deliver an agent to a subject, such as a mammal, in need thereof. 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 selfadministration, 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 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. Administration of an agent can be once in a day or more than once in a day (e.g., twice a day or more). Administration of the agent 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.
[00271] In some embodiments, an agent disclosed herein (e.g., polynucleotide such as ASO) 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.
[00272] In some embodiments, an agent disclosed herein (e.g., polynucleotide such as ASO) is administered as intrathecal bolus injection. In some embodiments, the agent (e.g., polynucleotide such as 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, the agent (e.g., polynucleotide such as ASO) is administered at a dosage of about 12 mg per administration. In some embodiments, the agent (e.g., polynucleotide such as ASO) is administered at a dosage of about, e.g., up to 50 or 100 mg per injection.
[00273] In some embodiments, an agent disclosed herein (e.g, polynucleotide such as ASO) is delivered systemically, such as via intravenous or subcutaneous injection. In some embodiments, the agent (e.g., polynucleotide such as ASO) 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 (e.g., polynucleotide such as ASO) is delivered after transiently 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.
[00274] In some embodiments, a method disclosed herein comprises administering to the subject two or more polynucleotides, for example, 2, 3, 4, or 5 polynucleotides. In some embodiments, the two or more polynucleotides are administered together. In other embodiments, the two or more polynucleotides are administered separately.
[00275] In some embodiments, a first polynucleotide disclosed herein (e.g., ASO) comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. In some embodiments, the first polynucleotide comprises a nucleotide sequence having about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. In some embodiments, the first polynucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs: 51-65.
[00276] In some embodiments, a second polynucleotide disclosed herein (e.g., ASO) comprises a nucleotide sequence having at least: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. In some embodiments, the second polynucleotide comprises a nucleotide sequence having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. In some embodiments, the second polynucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. [00277] In some embodiments, a method disclosed herein comprises administering to a subject a third, fourth, or fifth polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. In some embodiments, the third, fourth, or fifth polynucleotide comprises a nucleotide sequence having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. In still other embodiments, the third, fourth, or fifth polynucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-11, SEQ ID NOs:43-46, SEQ ID NOs:51-65. [00278] In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 1, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:2, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:6, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:7, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 10, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 11, or both.
[00279] In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:51, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:52, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:56, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:57, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:60, an antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO:61, or both.
[00280] In some embodiments, it may be advantageous to administer an agent (e.g., a polynucleotide such as an antisense oligonucleotide, a pharmaceutical composition thereof, or a pharmaceutically acceptable salt of the foregoing) of the present disclosure in combination with one or more additional therapeutic agent(s). For example, it may be advantageous to administer a compound of the present disclosure (e.g., an antisense oligonucleotide, or a pharmaceutical composition thereof, or a pharmaceutically acceptable salt of the foregoing) in combination with one or more additional therapeutic agents, e.g., a modulator of DNA methylation (e.g., an agent that inhibits DNA methylation or promotes DNA demethylation, see for example, the section of “DNA demethylation”) a metabotropic glutamate receptor 5 (mGluR5) modulators (e.g., Basimglurant or Mavoglurant), GAB AB receptor activator (e.g., arbaclofen), GABAA or GAB AB receptor activator (e.g., acamprosate), AMPAkine (e.g., AX516), CB1 inhibitor (e.g., rimonabant), RAS signaling inhibitor (e.g., lovastatin), STEP inhibitor, S6K inhibitor, PAK inhibitor (e.g., FRAX486), MMP9 inhibitor (e.g., minocycline), and GSK3P inhibitor (e.g., lithium). In some embodiments, treating the subject comprises providing the subject with a ketogenic (“keto”) diet.
[00281] The term “combination therapy” refers to the administration of two or more therapeutic agents to treat a disease, disorder or condition described herein. Such administration encompasses co-administration of the 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 reconstituted or diluted to a desired dose prior to administration. Typically, the treatment regimen will provide beneficial effects of a drug combination in treating diseases, conditions or disorders described herein.
[00282] In some embodiments, a method of treatment disclosed herein further comprises administering to the subject a therapeutically effective amount of a DNA-dem ethylating compound or DNA demethylase, prior to, during, or after, administering an agent disclosed herein (e.g., polynucleotide such as an ASO). In some embodiments, the method of treatment further comprises administering to the subject a therapeutically effective amount of a DNA- demethylating compound or DNA demethylase after administering an agent disclosed herein (e.g., polynucleotide such as an ASO).
[00283] 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'-deoxy cytidine, Hydralazine, RG108, procainamide, and SGI- 1027. In some embodiments, the DNA-demethylating compound is a nucleoside analogue. In some embodiments, the DNA-demethylating compound is a non-nucleoside analogue.
[00284] In some embodiments, the DNA demethylase (e.g., DNA methylation modification enzymes Dnmt or Tet (dCas9-Dnmt/Tet) is fused to a catalytically inactivate Cas9. Under the guidance of a single guide RNA (sgRNA), the dCas9-Tetl demethylates the FMRI locus and promoter region when FMRI has an expanded CGG repeat of 200 or more.
[00285] In some embodiments, the DNA-demethylating compound or DNA demethylase is in an amount sufficient to demethylate at least about 5% of an FMRI 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% of the FMRI gene. In some embodiments, the DNA-demethylating compound or DNA demethylase is in an amount sufficient to demethylate 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% of an FMRI gene. In some embodiments, a DNA demethylase is in an amount sufficient to demethylate 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 m FMRI gene. In some embodiments, a DNA-demethylating compound or DNA demethylase is in an amount sufficient to demethylate about 25-50% of an FMRI gene. [00286] In some embodiments, a method of modulating FMRI splicing and/or expression further comprising contacting the cell with a DNA-demethylating compound or DNA demethylase, prior to, during, or after, contacting the cell with the agent (e.g., polynucleotide). [00287] In some embodiments, a method of treatment disclosed herein further comprises decreasing (e.g., shortening or deleting) FMRI CGG expansion (e.g., by CRISPR/Cas9 gene editing) in the subject, prior to, during, or after, administering an agent disclosed herein (e.g., polynucleotide such as an ASO). In some embodiments, the method of treatment further comprises decreasing (e.g., shortening or deleting) FMRI CGG expansion prior to administering an agent disclosed herein (e.g., polynucleotide such as an ASO).
Methods of Modulating FMRI Splicing and/or Expression
[00288] In another aspect, the present disclosure provides a method of modulating FMRI splicing and/or expression in a cell, comprising contacting the cell with an agent (e.g., polynucleotide) under conditions whereby the agent is introduced into the cell, thereby modulates FMRI splicing and/or expression in the cell. The agent can be any one of the agents disclosed herein.
[00289] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases expression of isoform 1 of xe. FMRI gene, increases splicing of isoform 1 (between X chromosome base pairs 147,912,230 and 147,921,933), decreases expression of isoform 12 of the FMRI gene, decreases splicing of isoform 12 (between X chromosome between base pairs 147,912,230 and 147,912,728), or a combination thereof.
[00290] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases the splicing and/or expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases the splicing of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases the expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
[00291] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases the splicing and/or expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference. In some embodiments, the agent (e.g., polynucleotide) decreases the splicing of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference. In some embodiments, the agent (e.g, a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases the expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
[00292] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases splicing and/or expression of isoform 1 of FMRI, decrease splicing and/or expression of isoform 12 of FMRI, or a combination thereof. “Isoform 1” or “isol” refers to normal FMRI RNA with exon 1 spliced to exon 2. “Isoform 12” or “isol2” refers to missplicing of FMRI RNA, where exon 1 is spliced to a pseudo exon located within intron 1. Isoform 12 would generate a 31-amino acid protein, which probably would have no biological function.
[00293] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases isoform 1 of FMRI by at least about 5% relative to a reference, 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% relative to the reference. In some embodiments, the agent (e.g., polynucleotide) increases isoform 1 of the FMRI gene by about 75%.
[00294] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases isoform 12 of FMRI by at least about 5% relative to a reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases isoform 12 of the FMRI gene by about 30%.
[00295] In some embodiments, the level of splicing and/or expression of FMRI or a fragment thereof, is measured after the agent is contacted with the cell for at least about 1 day, e.g., 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 months or 6 months.
[00296] In some embodiments, the agent comprises, consists essentially of or consists of any one of the polypeptides, polynucleotides, gene editing systems or small molecules disclosed herein.
[00297] In some embodiments, the agent comprises at least one of the polynucleotides of the disclosure. In some embodiments, the agent comprises two or more of the polynucleotides of the disclosure.
[00298] In some embodiments, the cell is 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, a modified fibroblast, a pluripotent cell, or a cultured cell.
[00299] In some embodiments, the cell is an in vitro cell or an ex vivo cell. In some embodiments, the cell is an iPSC-derived neuron from a human who has or is predisposed to have FXS, a primary human cell, or a cell line. In some embodiments, the cell is a cell of any one of the subjects disclosed herein. In some embodiments, the cell of the subject is allogeneic. In some embodiments, the cell of the subject is autologous or syngeneic.
Methods of Reducing CGG triplet repeat expansion in FMRI 5’ UTR
[00300] In another aspect, the present disclosure provides a method of reducing CGG triplet repeat expansion in FMRI 5’ UTR in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide disclosed herein, an agent that modulates DNA methylation, or a combination thereof) under conditions whereby the agent is introduced into the cell, thereby reducing CGG triplet repeat expansion in the cell. The agent can be any one of the agents disclosed herein.
[00301] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases expression of isoform 1 of the FMRI gene, increases splicing of isoform 1 (between X chromosome between base pairs 147,912,230 and 147,921,933), decreases expression of isoform 12 of WIQ FMRI gene, decreases splicing of isoform 12 (between X chromosome between base pairs 147,912,230 and 147,912,728), or a combination thereof. [00302] In some embodiments, the agent (e.g., a polynucleotide disclosed herein, an agent that modulates DNA methylation, or a combination thereof) increases the splicing and/or expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases the splicing of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases the expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
[00303] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases the splicing and/or expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases the splicing of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases the expression of FMRI or a fragment thereof, by at least about 5% relative to the reference, 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% relative to the reference.
[00304] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases splicing and/or expression of isoform 1 of FMRI, decrease splicing and/or expression of isoform 12 of FMRI, or a combination thereof. “Isoform 1” or “isol” refers to normal FMRI RNA with exon 1 spliced to exon 2. “Isoform 12” or “iso!2” refers to missplicing of FMRI RNA, where exon 1 is spliced to a pseudo exon located within intron 1. Isoform 12 would generate a 31-amino acid protein, which probably would have no biological function.
[00305] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases isoform 1 of FMRI by at least about 5% relative to a reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases isoform 1 of the FMRI gene by about 75%.
[00306] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases isoform 12 of FMRI by at least about 5% relative to a reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases isoform 12 of the FMRI gene by about 30%.
[00307] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases CGG triplet repeat expansion in FMRI 5’ UTR in the cell by at least about 5% relative to a reference, 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% relative to the reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) decreases CGG triplet repeat expansion in FMRI 5’ UTR in the cell by at least about 10%, relative to a reference.
[00308] In some embodiments, the level CGG triplet repeat in FMRI 5’ UTR in the cell, is measured after the agent is contacted with the cell for at least about 1 day, e.g., 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 months or 6 months.
[00309] In some embodiments, the agent comprises, consists essentially of or consists of any one of the polypeptides, polynucleotides, gene editing systems or small molecules disclosed herein. [00310] In some embodiments, the agent comprises at least one of the polynucleotides disclosed herein. In some embodiments, the agent comprises two or more of the polynucleotides disclosed herein.
[00311] In some embodiments, the cell is 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, a modified fibroblast, a pluripotent cell, or a cultured cell.
[00312] In some embodiments, the cell is an in vitro cell or an ex vivo cell. In some embodiments, the cell is an iPSC-derived neuron from a human who has or is predisposed to have FXS, a primary human cell, or a cell line. In some embodiments, the cell is a cell of any one of the subjects disclosed herein. In some embodiments, the cell of the subject is allogeneic. In some embodiments, the cell of the subject is autologous or syngeneic.
[00313] In another aspect, the present disclosure provides a polynucleotide capable of reducing expression of an aberrant FMRI gene product. The polynucleotide is any one of the polynucleotides, modified or unmodified, disclosed herein. In some embodiments, the polynucleotide is any one of the modified polynucleotides disclosed herein.
[00314] In another aspect, the present disclosure provides an agent that modulates splicing and/or expression of FMRI gene. In some embodiments, the agent is a polynucleotide. In some embodiments, the agent is any one of the modified polynucleotides disclosed herein.
[00315] In yet another aspect, the present disclosure provides a pharmaceutical composition, comprising any one of the agents described herein, and one or more pharmaceutically acceptable excipients, diluents, or carriers.
Exemplification
[00316] Most FXS studies are focused on Fmrl knockout (KO) mouse models. Shah et al. shows that Fmrl KO mice have dysregulated pre-mRNA splicing in the brain (Shah et al., FMRP Control of Ribosome Translocation Promotes Chromatin Modifications and Alternative Splicing of Neuronal Genes Linked to Autism, Cell Rep. 30(13):4459-72 (2020)).
[00317] New data show that missplicing in the FMRP KO mouse occurs in all brain regions and peripheral tissues tested. Therefore, because FMRP is likely present in all cells, missplicing probably also occurs in all cells.
Example 1. Methods
[00318] RNA Extraction and Sequencing [00319] RNA was extracted from patient leukocytes using the LeukoLOCK™ total RNA isolation system (AM1923, Thermo Fisher Scientific, Waltham, MA). Ten mL fresh blood was collected from FXS male patients (N=10) and age-matched typically developing males (N=7) (controls) in an anti-coagulant containing tube, and RNA was extracted using a LeukoLOCK™ fractionation & stabilization kit (AM1933, Thermo Fisher Scientific, Waltham, MA), per the manufacturer’s instructions. Briefly, the blood sample was passed through a LeukoLOCK™ filter and 3 mL phosphate buffered saline (PBS) was used to rinse the filter followed by 3 mL of RNAlater® RNA Stabilization Solution (Thermo Fisher Scientific, Waltham, MA). The residual RNAlater® was expelled from the LeukoLOCK™ filter and the filters were capped and stored at -80°C.
[00320] To extract RNA, the filters were thawed at room temperature for 5 minutes and then the remaining RNAlater® was removed. The filter was flushed with 4 ml of TRI Reagent, and the lysate was collected in a 15-ml tube. 800pl l-Bromo-3 -chloropropane (BCP) was added to each tube and vortexed vigorously for 30 seconds. The tube was then incubated at room temperature for 5 minutes. After centrifugation for 10 minutes at 4°C at -2,000 x g, the aqueous phase was recovered. To recover long RNA fractions, 0.5 volumes of 100% ethanol were added and mixed well. The RNA was then recovered using the RNA clean and concentrator kit. DNase treatment was performed using Turbo™ DNase (Thermo Fisher Scientific, Waltham, MA), and the RNA obtained was resuspended in RNAse free water and stored at -80°C. Ipg of the RNA was used for cDNA synthesis using the QuantiTect® reverse transcription kit (Qiagen, Hilden, Germany) to assess for depletion of the Globin mRNA using qPCR, to confirm exclusion of red blood cells from the prep. 3pg of RNA sample was sent to Novogene (Beijing, China) for a directional mRNA library preparation using polyA enrichment. The libraries were sequenced on the NovaSeq platform to generate paired end, 150bp reads.
[00321] RNA-Seq Data Analysis
[00322] Fastq files were uploaded to the DolphinNext platform (Yukselen et al., BMC Genomics 21(l):310 (2020)) at the UMMS Bioinformatics Core for mapping and quantification. The reads were subjected to fastqc pipeline, and the quality of reads was assessed. 9-nt molecular labels were trimmed from both 5’ends of the pair-end reads and quality-filtered with Trimmomatic (0.32). Reads mapped to human rRNA by Bowtie2 (2.1.0) were filtered out. Cleaned reads were next mapped to the Refseq (V38) human transcriptome and quantified by RSEM (1.2.11). Estimated counts on each gene were used for the differential gene expression analysis by DESeq2 (1.16.1). After the normalization by median of ratios method, only the genes with minimal 5 counts average across all samples were kept for the Differential Gene expression analysis. The FDR (padj) cut-off < 5% was used. The TDF files generated were uploaded on the Integrative Genomics Viewer for visualization.
[00323] The ratio between reads including or excluding exons, also known as “Percent Spliced In” (PSI), indicates how efficiently sequences of interest are spliced into transcripts. The False Discovery Rate (FDR) is a method of conceptualizing the rate of type I errors in null hypothesis testing when conducting multiple comparisons.
[00324] Alternative Splicing Analysis
[00325] RNA-seq data generated from leukocytes from FXS male patients (N=10) and age- matched typically developing males (N=7) was used to analyze alternative splicing (AS) using the rMATS package v3.2.5 (Shen et al., Proc Natl Acad Sci U S A. 11 l(51):E5593-601 (2014)) with default parameters. The Percent Spliced In (PSI) levels or the exon inclusion levels were calculated by rMATS using a hierarchical framework. To calculate the difference in PSI between genotypes, a likelihood-ratio test was used. AS events with an FDR < 5% and |deltaPSI| > 5% as identified using rMATS were used for further analysis.
[00326] Primer Sets for Detecting FMRI Isoforms
[00327] Isol lForward (Isol l F): 5’ AGAAGATGGAGGAGCTGGTG 3’ (SEQ ID
NO: 12)
[00328] Isol2_lReverse (Isol2_l R): 5’ CAGTGGAGCTCTCCGAAGTC 3’ (SEQ ID
NO:13)
[00329] Isol2_2Forward: 5’ CCAGCAGTGCATTGAAGAAG 3’ (SEQ ID NO:14)
[00330] Isol2_2Reverse: 5’ CTGAAGCATGTGCATTCCTG 3’ (SEQ ID NO: 15)
[00331] Isol l Forward (Isol l F): 5’ AGAAGATGGAGGAGCTGGTG 3’ (SEQ ID
NO: 12)
[00332] Isol l Reverse (Isol l R): 5’ TTCATGAACATCCTTTACAAATGC 3’ (SEQ
ID NO: 16)
[00333] Exonl Forward (Exonl F): 5’ TAGCAGGGCTGAAGAGAA 3’ (SEQ ID
NO: 17)
[00334] Exonl Reverse (Exonl R): 5’ CTTGTAGAAAGCGCCATTG 3’ (SEQ ID
NO: 18) [00335] Detection of FMRI Isoforms
[00336] A white blood cell line derived from an FXS patient who expressed isol2 was transfected with antisense oligonucleotides (ASOs) pairs 705/705, 709/710, and 713/714. RNA was extracted 48 hours later and subjected to RT-qPCR to detect isol (primers Isol l Forward/ Isol l Reverse) or total FMRI isoforms (isol + iso 12) (primers Exonl Forward and Exonl Reverse) and isol2 (primers Isol l Forward/Isol2_l Reverse). Each assay was performed in triplicate and normalized against non-transfected cells.
Cell culture
Cell lines and treatments
[00337] Lymphoblastoid cell lines (LCL) were obtained from Coriell Institute from two FXS individuals (GM07365 (FXS1), GM06897(FXS2)) and two typically developing control males (GM07174 (WT3), GM06890 (WT4)). Cells were cultured in RPMI 1640 medium (Sigma- Aldrich, St. Louis, MO), supplemented with 15% fetal bovine serum (FBS) and 2.5% L- glutamine at 37°C with 5% CO2 in T25 flasks.
[00338] Fibroblast cells derived from patient skin samples were cultured in DMEM (15-017- CV) media supplemented with 10% FBS and lx antibiotic-antimitotic, lx L-glutamine in T25 culture flasks at 37°C with 5% CO2.
ASO treatment
[00339] Antisense oligonucleotides (ASOs) were dissolved in ultrapure distilled water to a final concentration of lOpM. Before use, the ASOs were heated to 55°C for 15 minutes and cooled at room temperature. ASOs were added individually or in combinations to LCL cell lines at a final concentration of 80nM using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, #13778030) and incubated at 37°C with 5% CO2 for 16hrs in reduced serum medium. RPMI 1640 medium (Sigma- Aldrich, St. Louis, MO), supplemented with 15% FBS was added for a total of 48hrs. The cells were collected after 48hrs of ASO treatment for RNA and protein extraction.
5-AzaC treatment
[00340] For each cell culture, 30* 105 cells/ml were added in a final volume of 20 ml media (RPMI 1640 medium (Sigma-Aldrich), supplemented with 15% FBS and 2.5% L-glutamine at 37°C with 5% CO2) per T25 flask. 5-Aza-2'-deoxycytidine (5-AzaC) (Sigma-Aldrich, A3656) was added to the cell cultures (final concentration 1 pM) for 7 consecutive days. A 2mM stock of 5-AzaC was made in DMSO. For each cell line, two independent treatments were performed (n = 2). For the no treatment controls for each cell line, DMSO was added to the flasks. For samples with both 5-AzaC and ASO treatment, 80nM ASOs or vehicle were added on Day 1 and either 5-AzaC or DMSO was added each day from Day 2 up to Day 9 at a final concentration of IpM. On Day 9 the cells were collected in lx Phosphate buffered saline to proceed with RNA extraction or Western blotting.
Western Blotting
[00341] Cells were homogenized at 4°C in RIPA buffer with incubation on ice for 10 minutes and dissociation by pipetting. The extract was centrifuged at 13,200 rpm for 10 minutes at 4°C and the supernatant collected. Protein concentration was determined by BCA reagent. Proteins (10 pg) were diluted in SDS-bromophenol blue reducing buffer with 40 mM DTT and analyzed using western blotting on a 10% SDS-PAGE gel with the following antibodies: FMRP (Abeam, 1 : 2000) and GAPDH (Cell signaling, 1 :2000) diluted in IX TBST with 5% non-fat milk. Membranes were washed three times for 10 minutes with 1XTBST and incubated with antirabbit or anti-mouse secondary antibodies (Jackson, 1 : 10000) at room temperature for Ihour. Membranes were washed three times for 10 minutes with 1XTBST, developed with ECL-Plus (Piece), and scanned with GE Amersham Imager.
Example 2. FMRI Isoform 12 Detected in a Subpopulation of FXS Patients
[00342] FXS is caused by a CGG triplet repeat expansion in a single gene, FMRI, which resides on the X chromosome. When the CGG triplet expands to 200 or more, the FMRI gene is methylated and thereby transcriptionally inactivated. The loss of the FMRI gene product, the protein FMRP, is the cause of the disorder.
[00343] Bioinformatic analysis showed that one-half of the FXS patients expressed detectable levels of FMRI RNA, which was unexpected given that all patients had greater than 200 CGG repeats and had been clinically diagnosed with fragile X syndrome. This detection of FMRI RNA in one-half of the FXS patients indicated that these individuals had incomplete DNA methylation of FMRI, because it is DNA methylation that silences the gene. FIG. 1 shows that there was robust expression of FMRI in all 7 typically developing (TD) individuals. There was also FMRI expression in FXS patients 1-5 (+FMRR), but no FMRI expression was detected in FXS patients 6-10 (-FMRF). Therefore, 50% of FXS individuals express FMRI RNA, likely due to incomplete methylation. [00344] In the fragile X syndrome patients who did express FMRI RNA, further bioinformatic analysis showed that the FMRI RNA was misspliced. That is, instead of, or in addition to proper FMRI splicing, there was a little-known isoform derived from missplicing. Normally, FMRI exon 1 (chrX: 147,911,919 - 147,912,230) is spliced to FMRI exon 2 (chrX: 147,921,933 - 147,921,985), which produces “isoform 1” or “Isol.” However, within intron 1, there is a pseudo exon (chrX: 147,912,728 - 147,914,451), and splicing between FMRI exon 1 and this pseudo exon produces “isoform 12” or “Isol2.” FIG. 2 shows an expanded view of FMRI exon 1 and intron 1. Note that although none of the typically developing individuals expresses isoform 12, the five FXS patients who expressed FMRI RNA (+FMR1) all express FMRI isoform 12.
[00345] Isoform 12 is derived from missplicing, detected only when there was a CGG repeat expansion and when there was incomplete methylation. Isoform 12 does not produce full-length or functional FMRP. Instead, isoform 12 generates a 30-amino acid protein, which probably has no biological function.
[00346] These findings suggest that FMRI RNA not only can be used for diagnosing an individual as having FXS, or having a propensity to develop FXS, but also can be used for stratifying FXS individuals. The identification FMRI RNA isoform 12 enables stratification of FXS individuals into two subpopulations, those who express isoform 12 and those who do not. [00347] These findings further suggest that FMRI RNA, such as isoform 12, may provide novel therapeutic targets for FXS. For example, a reduction of aberrant splicing to isoform 12, alone or commensurate with an increase of proper splicing to isoform 1 (/.< ., normal FMRI RNA with exon 1 spliced to exon 2), may increase FMRP levels and thereby mitigate FXS in patients who express FMRI RNA. In patients who does not express FMRI RNA, it may be feasible to generate isoform 12 with a therapeutically effective amount of a DNA-demethylating compound or DNA demethylase, which could ideally include a targeted approach to partially demethylate the FMRI gene without inducing general, widespread DNA demethylation.
Example 3. Reducing Isoform 12 Production and Increasing Isoform 1 Production
[00348] FIG. 3 shows a non-limiting example approach for blocking isoform 12 production, increasing isoform 1 production, and increasing FMRP levels using antisense oligonucleotides (ASOs). ASOs were designed to be complementary to regions within intron 1 and upstream of isoform 12, the junction spanning intron 1 and isoform 12, or within isoform 12 (Table 1). FIG. 4 shows a schematic illustration of FMRI isol, iso!2, and relative positions of ASOs complementary to intron 1 (704, 705, and 706), the junction of intron 1 and isol2 (707, 708, 709, and 710), and within isol2 (711, 712, 713, and 714).
[00349] ASOs 704-714 were chemically modified to increase the nuclease resistance of the ASOs (e.g., reduce RNase H cleavage), increase cellular uptake, and enhance base-pairing capabilities (reduce off-target effects). The ribose groups comprised 2’ -O-(2 -methoxy ethyl) (MOE), and the phosphate groups comprised a phosphorothioate.
[00350] ASOs of the disclosure may be used singly or in combination. A WBC line derived from a FXS patient who expressed isol2 was transfected with ASOs 704/705, 709/710 or 713/714. RNA was extracted 48 hours later and subjected to RT-qPCR to detect isol (primers Isol l Forward and Isol l Reverse) and isol2 (primers Isol l Forward and Isol2_l Reverse). Each assay was performed in triplicate. FIG. 5 illustrates that ASOs 713 and 714, both of which are complementary to internal regions of isol2, reduced the isol2 level by -30% and increased the isol level by -75%. These data indicate that ASOs can be used to reduce isoform 12 expression. More importantly, these data indicate that ASOs can be used to elevate FMRI isoform 1 expression, which may in turn increase FMRP levels and mitigate FXS.
[00351] These data suggest that ASOs may be a potent and specific therapeutic to treat a subpopulation of FXS individuals that express isoform 12. The findings provide further support that agents, such as ASOs, directed against FMRI isoform 12 may provide novel therapeutic treatment to FXS by reducing improper splicing to isoform 12, increasing proper splicing of isoform 1 and increasing FMRP levels. This approach is entirely novel in the fragile X field. It is predicted to be a significant improvement over the prior art because all other treatments for FXS elicit only modest improvements at best. Additionally, all other therapies treat FXS patients as one large cohort, whereas these studies have identified a particular subpopulation - those who express isol2 - and may be particularly amenable to therapeutics, such as ASOs that target isol2.
Example 4. Partial Demethylation of FMRI DNA
[00352] Experiments illustrated in Example 3 have been and will be performed in cells with different methylation status.
[00353] FIG. 6A shows RT-qPCR data from a fully methylated FXS cell line (FXS1, GM07365). The FMRI locus in this cell line is silenced and thus the FMRI RNA (isol and iso 12) and FMRP protein levels are very low compared to the FXS2 cell line with an unmethylated FMRI gene. Treatment with the demethylating agent 5-AzaC resulted in demethylation of the FMRI gene to allow expression of the FMRI RNA isoforms. The data demonstrate an increase in FMRI isol2 upon 5-AzaC treatment (p<0.05) and a partial rescue of the FMRI isol2 increase when the 5-AzaC treatment was combined with the ASO treatment (80nM of both antisense oligonucleotides 713 and 714) (p<0.05). FIG. 6B demonstrates an increase in FMRI isol upon 5-AzaC treatment (p<0.05) and a further increase when the ASO treatment (80nM of both antisense oligonucleotides 713 and 714) was combined with 5-AzaC treatment (p<0.05).
[00354] These data demonstrate that in a fully methylated FXS cell line, demethylation of the locus resulted in expression of both FMRI RNA isoforms. However, when demethylation was combined with an ASO against FMRI isoform 12, an increase in the FMRI isoform 1 mRNA was found. Thus, a combination of demethylation and ASO treatment may be useful for FXS patients with a fully methylated FMRI locus.
[00355] The upper panel of FIG. 7 A shows western blot data for FXS1 LCL cell line in duplicates, demonstrating an increase in FMRP after treatment with IpM 5-AzaC and ASO treatment (80nM of both antisense oligonucleotides 713 and 714) when compared to DMSO or 5-AzaC only treated samples. The mouse brains (hippocampus tissue) from a wild-type mouse and an Fmrl knock-out mouse were loaded as controls. The FMRP protein from mouse tissues ran higher on the gel compared to the human FMRP. The bottom panel represents GADPH protein levels used to normalize the protein amounts loaded in each sample. FIG. 7B shows quantification of the FMRP protein levels relative to GAPDH protein levels as seen on the western blot in FIG. 7A.
[00356] These data demonstrate the FMRP protein levels from the samples analyzed for FMRI RNA levels in FIGs. 6A-6B. Treatment of the FXS1 cell line (fully methylated FMRI locus) with a demethylating agent (5-AzaC) alongside the ASO treatment against FMRI isol2, resulted in a significant increase in FMRP protein levels as against the untreated FXS1 cells or the 5-AzaC treatment cells alone. As a comparison, the levels of FMRP protein expressed with this combination of treatment was similar to that seen in wild-type mouse brain tissues (see FIGs. 7A-7B).
[00357] FIG. 8 A is a table demonstrating the CGG repeats in the FMRI RNA 5’ UTR from three healthy males and three premutation carrier males for FXS. The premutation carriers had 55-200 CGG repeats in the 5’UTR of FMRI gene, whereas greater than 200 CGG repeats would lead to FXS, and less than 55 CGG repeats are usually present in healthy individuals. Premutation carriers have a propensity to develop FXTAS (Fragile X-associated tremor/ataxia syndrome) after the age of 50yrs. FIG. 8B shows RT-qPCR data demonstrating the presence of similar FMRI isol levels in fibroblast cells from all six individuals normalized to GN 7797/ RNA levels. FIG. 8C shows the presence of increased FMRI isol2 levels in individual Pl compared to the other premutation carriers and healthy control samples. All premutation carriers expressed similar FMRI isol levels as compared to the healthy controls. However, only individual Pl with higher CGG repeats (140, see FIG. 8 A) expressed FMRI iso 12.
[00358] These data demonstrate that the FMRI iso 12 might be expressed in premutation carriers with a higher CGG repeat number, and, in some embodiments, ASO treatment in these individuals can be therapeutically beneficial by increasing FMRP protein levels.
[00359] Prophetic Examples
[00360] In a first set of experiments, various ASOs will be introduced, singly or in combination, into human FXS WBC lines that are partially methylated and hence express some FMRI RNA. At various time points, for example, about 24, 48, 72, 96, 120, 144 and 168 hours after transfection, levels of FMRI isol, FMRI isol2, and FMRP will be assessed.
[00361] In a second set of experiments, human FXS WBC lines that have full methylation of FMRI DNA and express no FMRI RNA will be incubated with varying amounts of DNA demethylation agent, for example, 5-aza-2-deoxycytidine (5-azadC) (Sigma A3656), to partially demethylate the FMRI DNA. Then, various ASOs will be introduced, singly or in combination, into the DNA demethylase-treated cells. At various time points, for example, about 24, 48, 72, 96, 120, 144 and 168 hours after transfection, levels of FMRI isol, FMRI isol2, and FMRP will be assessed.
[00362] In a third set of experiments, various ASOs will be introduced, singly or in combination, into primary fibroblasts from FXS patients that are partially methylated. At various time points, for example, about 24, 48, 72, 96, 120, 144 and 168 hours after transfection, levels of FMRI isol, FMRI isol2, and FMRP will be assessed. In the primary fibroblasts from patients with a completely methylated FMRI locus, the cells will be incubated with varying amounts of DNA demethylation agent, for example, 5-aza-2-deoxycytidine (5-azadC) (Sigma A3656), to partially demethylate the FMRI DNA. Then, various ASOs will be introduced, singly or in combination, into the DNA demethylase-treated cells.
Example 5. Safety and Efficacy in an Animal Model.
[00363] The safety and efficacy of ASO treatment will be determined in an animal model. Neural progenitor cells, derived from human FXS patients with partially methylated FMRI and isol2 expression, will be injected into NOD-.sc/t/ IL2Rytlu11 mouse pups as described by Windrem etal., J Neurosci 34: 16153-16161 (2014) and Liu etal., Cell 172:979-92 (2018). Modified ASOs, such as those described above will be injected into the brain or via intraperitoneal injection (IP). The RNA will be extracted from the brains, and human FMRI isol and isol2 will be quantified by RT-qPCR. This experiment will determine the safety and efficacy of ASO treatment in inhibiting FMRI isol2 production and promoting isol formation in an animal model. FMRP in human neurons will be assessed by immunocytochemistry.
Table 1. Non-limiting Examples of ASOs and Other Pertinent Information.
Oligo # SEQ Sequence Scale nt s MW Vol Cone. pMol nmol/pL ID NO (pMol) Count (L/mol*cm) (g/mol) (gL) (pM)
W-704 1 AGAAGCCAAAG 1 20 216990 8035.67 500 614.68 0.31 0.61
GAGACCTGA
W-705 2 AAAGAGAAGCC 1 20 231300 8054.99 500 598.53 0.30 0.60
AAAGGAGAC
W-706 3 CTAGACCGGAAA 1 22 236430 8832.38 500 663.28 0.33 0.66
AGAGAAGCCA
W-707 4 ATGCTAGACCGG 1 21 233100 8439.7 500 582.75 0.29 0.58
AAAAGAGAA
W-708 5 CAATGCTAGACC 1 20 214470 8010.4 500 610.06 0.31 0.61
GGAAAAGA
W-709 6 AAGTCCCAATGC 1 21 205740 8384.38 500 561.92 0.28 0.56
TAGACCGGA
W-710 7 TCTCCGAAGTCC 1 20 178560 7920.39 500 603.77 0.30 0.60
CAATGCTA
W-711 8 GAGCTCTCCGAA 1 18 159390 7148.33 500 605.31 0.30 0.61
GTCCCA
W-712 9 AGAACAGTGGA 1 20 196650 8007.03 500 617.65 0.31 0.62
GCTCTCCGA
W-713 10 CGCCCAGAACAG 1 20 186120 7996.35 500 669.57 0.33 0.67
TGGAGCTC
W-714 11 CCTCGCCCAGAA 1 20 186120 7996.35 500 576.78 0.29 0.58
CAGTGGAG
Examples 6-10
[00364] Fragile X Syndrome (FXS) is a neuro-developmental disorder causing a range of maladies including intellectual disability, speech and developmental delays, social deficits, repetitive behavior, attention deficits, and anxiety. Previous studies have shown an expansion of >200 CGG triplets in the 5’UTR of Fragile X Messenger Ribonucleoprotein 1 (FMRI') induces gene methylation and transcriptional silencing, loss of the encoded FMRP, and FXS. Fragile X Messenger Ribonucleoprotein (FMRP) is an RNA-binding protein that interacts with >1000 mRNAs in the mouse brain and human neurons, predominantly through coding region associations (1-3). Although earlier studies suggested that FMRP inhibits protein synthesis (4), subsequent high-resolution methods showed that FMRP promotes as well as inhibits translation (5-8). One mechanism by which FMRP inhibits translation is stalling ribosome translocation on mRNAs (9, 10). Previously, several mRNAs associated with FMRP-stalled ribosomes were identified, one of which encodes SETD2, an epigenetic enzyme that trimethylates histone H3 lysine 36 (H3K36me3) (11). SETD2 was elevated in Fmrl -deficient hippocampus, which resulted in an altered H3K36me3 chromatin landscape. H3K36me3 resides in gene bodies and influences alternative pre-mRNA splicing (12), and indeed multiple mRNAs were mis-spliced in Fmrl -deficient mouse hippocampus. Many of these mis-splicing events were also detected in the human postmortem autism spectrum disorder (ASD) brain and blood tissues 7"/-7S), indicating a convergence of FXS and ASD (11, 13).
[00365] Because mis-splicing of mRNAs is widespread in Fmrl -deficient mouse brain, and because individuals with FXS are often on the autism spectrum, it was surmised that RNA mis- splicing might also be prevalent in human FXS patient tissues (blood and brain). Accordingly, leukocytes were isolated from freshly obtained blood from 29 FXS males and 13 typically developing (TD) age-matched males, and RNA sequencing was performed. The analysis revealed widespread and statistically robust mis-regulation of alternative splicing and RNA abundance of greater than 1,000 mRNAs. Mis-regulated RNA expression and processing in FXS postmortem brain were also found.
[00366] Further analysis of the RNA-seq data unexpectedly revealed that FMRI RNA was expressed in 21 of 29 FXS leukocyte samples, some nearly as high as FMRI transcript levels from TD individuals. Because all FXS samples were from individuals with >200 CGG repeats, this was a surprising result because the FMRI locus, which was purported to be silent under these conditions, was transcriptionally active in patients even when the gene appeared to be fully methylated in standard assays. However, the highest FMRI RNA expressing FXS individuals were mosaic (CGG repeat number mosaicism or partial methylation of a full expansion). Furthermore, it was found that much of the FMRI mRNA in the FXS individuals was itself misspliced to generate FMR1-2Y1 , a little-known 1.8 kb isoform comprised of FMRI exon 1 and a pseudo-exon within FMRI intron 1. This isoform is predicted to encode a truncated, 31 amino acid polypeptide whose function, if any, is unknown. Additional analysis revealed that f- 217 was detected in FXS dermal and lung-derived fibroblasts as well as in five of seven FXS postmortem cortex samples, further indicating the preponderance of FMRI mis-splicing in FXS populations and, most importantly, that this altered processing event occurs in the brain as well as leukocytes. Fibroblasts from some FXS premutation (i.e., -55-200 CGG repeats) male carriers also expressed FMRI -217 as well as full-length FMRI RNA, indicating that mis-splicing may be widespread in other disorders linked to CGG expansions in FMRI.
[00367] These findings suggest that modulation of FMRI mis-splicing is a suitable approach to increase FMRP levels in individuals expressing FMRI-217. To investigate further, eleven 2'- (9-methoxyethyl (MOE)/phosphorothioate-containing antisense oligonucleotides (ASOs) against several regions of FMRI -217 were generated and transfected into an established FXS lymphoblast cell line that expresses this transcript. Single ASOs or a combination of two ASOs blocked improper FMRI splicing, rescued proper FMRI splicing, and restored FMRP to TD levels. Moreover, application of the DNA methylation inhibitor 5-aza-2'-deoxy cytidine (5- AzadC) to a second FXS lymphoblast line as well as FXS fibroblast lines that normally do not express any FMRI resulted in synthesis of both FMRI and FMR1-2Y1 RNAs but little or no FMRP. However, treatment of these cells with both 5-AzadC and the ASOs produced strong FMRP up-regulation. These studies demonstrated that first, in cells from FXS but not TD individuals, a significant proportion of the FMRI RNA was mis-spliced to produce the FMR1- 217 isoform; and second, ASO treatment to reduce FMRI -217 levels resulted in FMRP restoration to TD levels. Therefore, ASO treatment may offer a novel therapeutic approach to mitigate FXS. [00368] Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Surprisingly, the Fragile X Messenger Ribonucleoprotein 1 (FMRI gene was transcribed in >70% of the FXS tissues, in many instances even when the gene was fully methylated. In all FMRI expressing FXS tissues, FMRI RNA itself was mis-spliced in a CGG expansion-dependent manner to generate the little-known FMR1-2V1 RNA isoform, which is comprised of FMRI exon 1 and a pseudo-exon in intron 1. FMRI -217 was also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissue. It was shown that in cells aberrantly expressing mis-spliced FMRI, antisense oligonucleotide (ASO) treatment reduced FMRI -217, rescued full-length FMRI RNA, and restored Fragile X Messenger Ribonucleoprotein (FMRP) to normal levels. Notably, FMRI gene reactivation in transcriptionally silent FXS cells using 5-aza-2'-deoxycytidine (5-AzadC), which prevented DNA methylation, increased FMRI -217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescued full-length FMRI expression and restored FMRP. These findings indicate that in FXS individuals (e.g., those expressing FMRI -217), ASO treatment may offer a new therapeutic approach to mitigate the disorder.
Example 6. Materials and Methods
[00369] Human FXS Participant Studies
[00370] All participants were Caucasian males with a FMRI full mutation (CGG repeats >200) or typically developing 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. Intelligence quotient (IQ) scores were obtained using the Stanford-Binet Scale — Fifth Edition (SB5) (52) and applying the z-deviation method to avoid floor effects in persons with intellectual disability (53). The adaptive skills of participants were determined using an semi-structured interview and measured using the Vineland Adaptive Behavior skills (Vineland-3, (54) . The Adaptive Behavior Composite (ABC) standard score (SS) was the measure of overall adaptive functioning based on scores assessing the following domains: communication, daily living skills, and socialization. FXS patients were aged 16-38 years with FXS phenotypes, a z-deviation IQ range of 20-52 and ABC standard score range of 20-41. Age matched TD individuals for the study were aged 22-29 with a normal IQ and no known neuropsychiatric conditions. For CGG repeat size determination in the 5’ UTR of the FMRI gene, DNA isolated from whole blood was analyzed using the Asuragen FMRI AmplideX PCR Kit. Methylation status was determined using the Asuragen FMRI methylation PCR Kit and/or Southern blot analysis. FMRP levels were quantified by generating dried blood spots (DBS) from the samples. To generate DBS, 12- 50 pl spots were put on each blood card and allowed to dry. The blood cards were then stored at -80°C. Discs were punched using a 6-mm punch and incubated in lysis buffer. Extracted sample was centrifuged, and FMRP was quantified using the Luminex Microplex immunochemistry assay. FMRP levels were normalized to 1,000 WBCs per sample. Additionally, FMRP levels were also quantified by using peripheral blood mononuclear cell (PBMC) samples. PBMCs were isolated from whole blood using Cell Preparation (CPT) blood tubes. Isolated PBMC were lysed and quantified for total protein concentration using a spectrophotometer, and FMRP was quantified using a Luminex Microplex immunochemistry assay. FMRP levels were normalized to total protein. Both methods produced comparable levels of FMRP in the samples assessed. [00371] Frozen post-mortem brain tissues were obtained from University of California at Davis Brain Repository from FXS male individuals (N=6) and age-matched typically developing (TD) males (N=5).
[00372] RNA Extraction and Sequencing of Tissue Samples from FXS and TD Individuals [00373] Leukocytes
[00374] Eight milliliters (ml) of fresh blood were collected from FXS male individuals (N=29) and age-matched typically developing (TD) males (N=13) in a BD vacutainer Cell Preparation Tube (CPT, with sodium citrate- blue top tube, Becton Dickinson #REF362761), and the leukocytes were collected on a LeukoLOCK™ filter, prior to RNA extraction using a LeukoLOCK™ Fractionation & Stabilization Kit (Ambion #1933) as per the manufacturer’s instructions. Briefly, the blood samples were passed through LeukoLOCK™ filters that were then rinsed with 3 ml of phosphate buffered saline (PBS), followed by 3 ml of RNAlater®. The residual RNAlater® was expelled from the LeukoLOCK™ filter, and the filters were capped and stored in -80°C. To extract RNA, the filters were thawed at room temperature for 5 minutes, and then the remaining few drops of RNAlater were removed. The filter was flushed with 4 ml of TRIzol™ LS Reagent (ThermoFisher Scientific #10296028), and the lysate was collected in a 15-ml tube. 800 pl bromo-3 -chloro-propane (BCP) (Sigma #B9673) was added to each tube and vortexed vigorously for 30 seconds. The tube was then incubated at room temperature for 5 minutes and centrifuged for 10 minutes at 4°C at -2,000 x g; the aqueous phase containing the RNA was recovered. To recover the long RNA fraction, 0.5 volume of 100% ethanol was added and mixed well. The RNA was then recovered using an RNA clean and concentrator kit (Zymo Research, #11-325 / R1015), DNase-treated with TURBO™ DNase (Invitrogen # AM2238), resuspended in RNase-free water, and stored at -80°C. The quality of RNA (RNA integrity number (RIN) >7.3) was assessed using a 5300 Fragment Analyzer instrument. Three milligrams (mgs) of RNA sample were used for directional mRNA library preparation using polyA enrichment (Novogene Co), and the libraries were sequenced on the NovaSeq platform to generate paired end, 150-bp reads at a sequencing depth of 60-90 million reads per sample.
[00375] Brain Tissue
[00376] The post-mortem frozen cortical tissues from FXS male individuals (N=6) and age- matched typically developing (TD) males (N=5) were powdered in liquid nitrogen using a mortar and pestle. The fine powder was then homogenized on ice in a Dounce homogenizer using TRIzol™ Reagent (ThermoFisher Scientific # 15596026), and the lysates were collected. Total RNA was extracted using BCP, recovered as described above, and stored at -80°C.
[00377] cDNA Synthesis and qPCR
[00378] One microgram (pg) of total RNA was primed with oligo(dT)2o to generate cDNA with a QuantiTect cDNA synthesis kit (Qiagen, #205311) using random hexamers (Table 3). qPCR was performed using the iTaq™ Universal SYBR® Green Supermix (BIO-RAD #1725122) on a QuantStudio 3 qPCR machine in duplicate.
[00379] RNA-Seq Data Analysis
[00380] FASTQ files were uploaded to the DolphinNext platform (55) at the UMass Chan Medical School Bioinformatics Core for mapping and quantification. The reads were subjected to FastQC (vO.11.8) analysis, and the quality of reads was assessed. Reads were mapped to the genome assembly GRCh38 (hg38) version 34 using the STAR (v2.5.3a) aligner. Gene and isoform expression levels were quantified by salmon vl.5.2.
[00381] Differential gene expression analysis: DESeq2 (v3.9) was used to obtain differentially expressed genes from the estimated counts table. After normalization by the median of ratios method, genes with minimal 5 counts average across all samples were kept for the Differential Gene expression analysis. P <0.0002 was used as a cutoff. The TDF files generated were uploaded on the Integrative Genomics Viewer (2.6.2) and autoscaled for visualization.
[00382] Alternative splicing analysis: To analyze differential alternative splicing (AS), the rMATS package v3.2.5 (14) was used with default parameters. The Percent Spliced In (PSI) levels or the exon inclusion levels were calculated by rMATS using a hierarchical framework. To calculate the difference in PSI between genotypes, a likelihood-ratio test was used. AS events with an FDR < 5% and |deltaPSI| > 5% as identified using rMATS were used for further analysis. The genes with significant skipped exons were used for validation using RT-qPCR analysis. One pg of RNA was used to generate cDNA using the QuantiTect cDNA synthesis kit. Primers were designed to overlap skipped/inclusion exon junctions, and qPCR was performed using the Bio-Rad SYBR reagent on a Quantstudio3 instrument.
[00383] Cell Culture
[00384] Lymphoblast Cell Lines
[00385] Lymphoblast cell lines (LCL) were obtained from Coriell Institute from two FXS individuals (GM07365 (FXS1), GM06897(FXS2)) and two typically developing control males (GM07174 (WT3), GM06890 (WT4)). 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% CO2 in T25 flasks.
[00386] Fibroblast Cells
[00387] Skin biopsies from participants were collected in a 15-cc tube with transfer culture media (DMEM with 5% Gentamicin). The biopsy was then removed from the transfer media with tweezers onto a sterile tissue culture dish and dissected into approximately 6-7 pieces using sterile tweezers and scissors in the culture hood. Three to four pieces of skin explants were kept on the bottom of a T25 flask, and 3 ml CHANG AMNIO culture media was added. The flask was then incubated at 37°C with 5% CO2 for 10 days. The culture media was changed after cells started growing out from the skin explants. After the cells had grown to 5-6 layers around the skin explants, the skin explants were removed from the culture flask, and fibroblasts were trypsinized and spread evenly in the flask. The media were changed after overnight incubation with trypsin. Fibroblast culture medium was added (complete media (500 ml DMEM (15-017- CV) with 10% FBS and IX antibiotic-antimitotic, 5 ml lx L-glutamine)) twice a week to cells in a T25 culture flasks at 37° C with 5% CO2.
[00388] Fibroblast cell lines were obtained from Coriell Institute from two FXS individuals (GM05131, and GM07072). A control fibroblast line derived from a skin sample of a typically developing male was used. Cells were cultured in DMEM medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS) and 2.5% L-glutamine, at 37°C with 5% CO2.
[00389] ASO Synthesis and Treatment
[00390] ASO Synthesis
[00391] ASOs were synthesized on a Dr. Oligo 48 synthesizer. 2’-O-methoxyethyl (MOE)- modified phosphoramidites were coupled for 8 minutes. Oligonucleotides were deprotected in concentrated aqueous ammonia (30% in water) at 55°C for 16 hours and characterized by liquid chromatography-mass spectrometry. Final desalting was effected by diafiltration (3x water wash) in a 3-kDa cutoff Amicon centrifugal filter.
[00392] ASO Treatment
[00393] Antisense oligonucleotides (ASOs) were dissolved in ultrapure distilled water to a final concentration of 10 pM. Before use, the ASOs were heated to 55°C for 15 minutes and cooled at room temperature. ASOs were added, individually or in combinations, to LCL cell lines at a final concentration of 80 nM or 160 nM using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778030) and incubated at 37°C with 5% CO2 for 16 hours in reduced serum medium. RPMI 1640 medium (Sigma- Aldrich), supplemented with 15% fetal bovine serum (FBS) was added for a total of 72 hours. The cells were collected after 72 hours of ASO treatment for RNA and protein extraction.
[00394] 5-AzadC Treatment
[00395] For each cell culture, 30* 105 cells/ml were added to a final volume of 20 ml media (RPMI 1640 medium (Sigma- Aldrich) supplemented with 15% fetal bovine serum (FBS) and 2.5% L-glutamine at 37°C with 5% CO2) per T25 flask. 5-Aza-2’ -deoxy cytidine (5-AzadC) (Sigma-Aldrich, A3656) was added to the cell cultures (final concentration 1 pM) for 7 consecutive days. A 2mM stock of 5-AzadC was made in DMSO. For each cell line, two independent treatments were performed (n = 2). For the no treatment controls for each cell line, DMSO was added to the flasks. For samples with both 5-AzadC and ASO treatment, 80nM or 160 nM ASOs or vehicle were added on Day 1 and either 5-AzadC or DMSO was added each day from Day 2 up to Day 9 at a final concentration of IpM. On Day 9 the cells were collected in IX phosphate buffered saline to proceed with RNA extraction or Western blotting.
[00396] Western Blotting
[00397] Cells were homogenized at 4°C in RIPA buffer, with incubation on ice for 10 minutes and dissociation by pipetting. The extract was centrifuged at 13,200 rpm for 10 minutes at 4°C, and the supernatant collected. Protein concentration was determined using BCA reagent. Proteins (10 pg) were diluted in SDS-bromophenol blue reducing buffer with 40 mM DTT and analyzed using western blotting with the following antibodies: FMRP (Millipore, mAb2160, 1 : 1,000), FMRP (Abeam, ab 17722, 1 : 1,000) and GAPDH (14C10, Cell Signaling Technology, mAb 2118, 1 :2,000), diluted in IX TBST with 5% non-fat milk. Membranes were washed three times for 10 minutes with 1XTBST and incubated with anti-rabbit or anti-mouse secondary antibodies (Jackson, 1 : 10,000) at room temperature for 1 hour. Membranes were washed three times for 10 minutes with 1XTBST, developed with ECL-Plus (Piece), and scanned with GE Amersham Imager.
[00398] Quantification and Statistical Analysis
[00399] All grouped data were presented as mean ± s.e.m. All tests used to compare the samples were mentioned in the respective figure legends and corresponding text. When exact P values were not indicated, they were represented as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, P value <0.0001; n.s., p > 0.05.
[00400] Data and Code Availability
[00401] Codes and scripts used for quantification analysis were written in Python or R and will be provided upon request. Data Resources Sequencing datasets generated in this study have been deposited into the Gene Expression Omnibus (GEO) database under the accession number: Super series GSE202179. The sub series GSE202177 comprise the raw data for the RNA-seq and GSE202178 for the ChlP-Seq experiments. [00402] Chromatin immunoprecipitation Sequencing (ChlP-Seq)
[00403] Eight ml of fresh blood was collected from FXS male (N=10) and age-matched typically developing males (N=7) individuals in a BD vacutainer CPT (Cell Preparation Tube with sodium citrate- blue top tube, Becton Dickinson #REF362761). The tube was gently inverted 5 times, and the sample was centrifuged for 25 minutes at 1,500-1,800 RCF at room temperature. The tubes were then inverted to collect the lymphocytes and other mononuclear cells resuspended in the upper liquid phase in a new 15-ml tube. The samples were centrifuged again for 10 minutes at 300 RCF to obtain the PBMC pellet. The PBMCs were rinsed with IX Dulbecco’s phosphate buffered saline without calcium or magnesium (D-PBS) (Invitrogen #14190-094). The PBMC pellet was resuspended in 250 pL ice-cold D-PBS with protease inhibitors. FMRP levels in PBMCs were quantified using a Luminex Microplex immunochemistry assay. Chromatin isolation and sequencing were performed as previously described (11). Briefly, the cells were cross-linked with 1% formaldehyde and quenched with 150 mM glycine. After centrifugation at 2,000 g for 10 minutes at 4°C, the cells were lysed. After homogenization, the nuclei were harvested by centrifugation at 2,000 g for 5 minutes at 4°C. The nuclei were lysed by incubating for 20 minutes on ice in nuclear lysis buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 mM EGTA). 0.5% SDS was added, and the samples were sonicated on a Bioruptor® sonicator at high power settings (sonication: 30 seconds on, 90 seconds off) for 9 cycles of 15 minutes each at 4°C. The samples were centrifuged and diluted to adjust the SDS concentration to <0.1%. 10% of each sample was used as input. The remainder of the samples were divided into two and incubated with protein G dynabeads coupled overnight at 4°C with antibodies against H3K36me3 (Abeam ab9050, 5pg per ChIP) or H3K4me3 (Active Motif- 39159, 5 pg per ChIP). After IP, the beads were washed, and chromatin de-crosslinked overnight at 65°C. After RNase and proteinase K treatment, the DNA was purified. ChlP-Seq libraries were prepared by performing the following steps: ends repair using T4 DNA polymerase, A’ base addition by Klenow polymerase, and Illumina adapter ligation using T4 Polynucleotide kinase from New England Biolabs (NEB). The library was PCR amplified using multiplexing barcoded primers. The libraries were pooled with equal molar ratios, denatured, diluted, and sequenced with NextSeq 500/550 High Output Kit v2.5 (Illumina, 75-bp paired-end runs) on a Nextseq500 sequencer (Illumina). [00404] ChlP-Seq analysis
[00405] For ChlP-seq data analysis, alignments were performed with Bowtie2 (2.1.0) using the GRCh38 (hg38) version 34 genome, duplicates were removed with Picard and TDF files for Genomics Viewer (IGV), viewing were generated using a ChlP-seq pipeline from DolphinNext (55). The broad peaks for H3K36me3 ChlP-Seq were called using the broad peak parameter MACS2. Narrow peaks for H3K4me3 ChIP were called using the narrow parameter in MACS2. deepTools2 (57) was used to plot heatmaps and profiles for genic distribution of H3K36me3 and H3K4me3 ChIP signals over input. IGV tools (2.6.2) were used for visualizing TDF files, and all tracks shown were normalized for total read coverage.
Example 7. FMRI RNA is Expressed and Mis-Spliced in a Subset of FXS Individuals.
[00406] Expansion of >200 CGG repeats in FMRI induces gene methylation, transcriptional silencing, loss of FMRP, and FXS. It was therefore surprising that in leukocytes of 21 of 29 FXS individuals, FMRI RNA was detected, and in four individuals, the level of all isoforms of this RNA were similar to, or even higher than, those in the TD individuals (Table 2, FMRI RNA TPM levels). When only full-length FMRI encoding 632 amino acid FMRP (FMRI -IQ ') was examined (FIG. 9H, Table 3), WBCs from 6 individuals had levels of this transcript that were similar to those of TD (Table 2). For comparison, the levels of the FMRI paralog FXR2 were similar in all individuals (Table 2). Visualizing the RNA reads at the FMRI locus with the Integrated Genome Viewer (IGV) made it evident that exonic reads were detected at robust levels in TD individuals, and that the exonic reads were also detected in FXS individuals (FIGs. 9A-9B). FXS individuals 1-21 expressed relatively high FMRI levels (with a cutoff of 0.6 transcript per million (TPM)) (H FMRI), compared to FXS individuals 22-29 who expressed low or undetectable FMRI levels (L FMRI (Table 2 and FIGs. 9A-9B). Remarkably, the \ -FMR1 FXS individuals displayed strong RNA reads in intron 1 of FMRI (thick-lined black box in FIG. 9A, enlarged in FIG. 9B). Notably, RNA reads in this intronic region were not detected in any TD individuals even though FMRI RNA was strongly expressed (FIGs. 9A-9B). The FA7 7 locus expresses multiple alternatively spliced RNA isoforms (Table 3). The RNA reads detected in FMRI intron 1 correspond to the second exon of the FMRI -217 RNA isoform. FMR1-2V1 (ENST00000621447.1) is a 1.8-kb transcript comprised of two exons, and is predicted to encode a 31-amino acid polypeptide (Table 3). Notably, most of the total FMRI RNA in the FXS samples was comprised of the aberrantly spliced FMRI-211 transcript, which was absent in samples from TD individuals (Table 2). TPMs of all 14 FMRI isoforms detected in the TD and FXS patient samples were obtained (data not shown). RT-PCR was used to detect the FMR1-2Y1 isoform in the FXS leukocyte samples (reverse transcription primed with oligodT(20)), and the amplified product was sequenced using primers specific to the FMR1-2Y1 exon-exon junction. Aligning this sequence to FMRI confirmed that this transcript is polyadenylated and is a spliced product of FMRI exon 1 and FMR1-2Y1 exon 2 (FIG. 9C).
Table 2. FMRI RNA TPM levels
Figure imgf000080_0001
Figure imgf000081_0001
[00407] Table 2 shows normalized gene counts (transcripts per million, TPM) obtained from RNA-seq data analysis for total FMRI (all isoforms), FMR1-2Q5 (encoding the full-length, 632 amino acid FMRP), FMR1-2Y1 (a mis-spliced RNA), and FXR2, a paralogue of FMRI.
Table 3. FMRI Transcript Identification & Corresponding Predicted Amino Acid Numbers of Encoded Proteins from ENSEMBL (56)
Figure imgf000081_0002
Figure imgf000082_0001
[00408] Next, the proportion of full-length FMRI RNA to FMRI -211 RNA in TD or FXS leukocytes was assessed. In the TD samples, 95% of the total FMRI RNA (primers ExlF and ExlR) represented full-length molecules (primers ExlF and Ex2R), whereas in the H FMRI samples, 75% of the total FMRI RNA was full-length and 25% was FMR1-2Y1 (primers ExlF and 217R) (FIG. 9C). In the L FMRI samples, both isoforms were just barely detected. The total FMRI RNA levels in all the samples were normalized to GAPDHTFNA expression (* denotes P values <0.05). Importantly, all FXS individuals in this study, irrespective of FMRI expression, displayed typical FXS symptoms, suggesting that even in patients with high FA7 7 expression, functional FMRP may not be present or is present at very low amounts (FMRP protein levels were quantified for available samples (data not shown)).
[00409] Whether stratification of FXS individuals, based on relatively high (H) or low (L) amounts of FMRI (using a cutoff of 0.6 TPM, Table 2), was reflected in transcriptome-wide RNA changes was examined. By reanalyzing FXS leukocyte RNA-seq data to compare significant RNA alterations between these two groups, hundreds of aberrant splicing events that tracked with the amount of this mis-spliced transcript were found (FIG. 9D and data not shown). Whether the parameters measured in WBCs correlated with IQ was investigated. Table 4 presents determinations of methylation status of the FMRI gene (by PCR), FMRP levels (ng/pg protein), CGG repeat number, FMRI-217, full-length FMRI -205, all detected FMRI isoforms, and IQ (Stanford-Binet test).
Table 4. Characterizing Leukocytes of Each FXS Individual
Figure imgf000083_0001
[00410] In Table 4, FMRI gene methylation (MPCR): in percent as determined by PCR analysis; FMRP levels: ng/ug total protein; FMRI', all isoforms; IQ: Stanford-Binet; N/A: not available.
[00411] Table 5 presents correlation coefficients for pairwise comparisons of the measurements noted above. Methylation of the FMRI gene is negatively correlated with FMR1- 217 and FMRI -2Q5 expression. More intriguing is the moderately positive correlation of IQ with FMRP protein levels. Somewhat surprisingly, FMR1-2Q5, which encodes full-length FMRP, has no correlation with IQ. However, it is noted that while FMR1-2Q5 encodes the complete 632- amino acid FMRP, other FMRI isoforms, which vary in abundance, encode truncated FMRP proteins (Table 3). Without presupposing functionality of truncated FMRP proteins, the canonical FMRI isoform, FMR1-2Q5, was used for further comparisons. FMR1-2Y1 has a negative correlation with IQ, indicating a deleterious effect of this isoform. FIG. 10 displays a 3- dimensional comparison of all the parameters noted above. The inset shows that some FXS patients with a fully methylated FMRI gene expressed FMRI RNA and FMRP. Taken together, these results show several important findings. First, the FMRI locus is frequently transcribed even when the FMRI gene with a full CGG expansion is fully methylated. Second, FMRP levels in WBCs are positively correlated with IQ. Third, the negative correlation of FMRI -217 with IQ suggests that the process of mis-splicing, the 31 -amino acid polypeptide derived from FMR1- 217, the FMR1-2Y1 RNA itself, or a combination thereof (e.g., all three), impart some toxic effect manifest in the brain (e.g., IQ). In any event, the levels of FMRI -217 expression, as well as additional transcriptome-wide changes in RNA processing events, likely form the basis for molecular stratification of FXS individuals.
Table 5. Correlation coefficients for pairwise comparisons for indicated parameters
Figure imgf000084_0001
[00412] In Table 5, +/- 0-0.1 : no correlation; +/- 0.1-0.29: weak correlation; +/- 0.3-0.49: moderate correlation; +/- 0.5-1 : strong correlation.
Example 8. FMR1-2V1 is Expressed in Human FXS and Pre-Mutation Carrier Postmortem Brain. [00413] To investigate whether FMRI -217 is expressed in FXS brain, publicly available RNA-seq data of post-mortem frontal cortex tissues from FXS individuals (CGG repeats >200), FXS carriers (CGG repeats 55-200), and TD individuals (CGG repeats <55) (16) were analyzed. FMRI RNA (TPM) levels were highest in pre-mutation carriers (Table 6). Interestingly, the FXS sample UMB5746, which displayed CGG repeat number mosaicism, displayed high levels of FMRI RNA (Table 6 and FIG. 11A) and to a lesser extent, FMRP (16). The analysis showed that this individual expressed FMR1-2Y1, as did FXS carrier UMB5212, who had Fragile X- associated tremor/ataxia syndrome (FXTAS) (Table 6 and FIG. 11 A). Neither TD individual had any RNA reads corresponding to FMR1-2Y1 (Table 6 and FIG. 11 A). Thus, FMR1-2Y1 RNA may only be expressed in the brains of a subset of FXS individuals and premutation carriers.
Table 6
Figure imgf000085_0001
[00414] Table 6 shows sample information for postmortem FXS frontal cortex, premutation FXS carriers and TD individuals (derived from (16)). RNA-seq datasets GSE107867 (NIH samples) and GSE117776 were reanalyzed for DGE and DAS. The TPM for FMRI RNA in the samples is shown.
[00415] A BLAST analysis showed that /’A7 7-217 aligned only with intron 1 of FMRI and with no other region of the genome. Additional data showed unequivocally that /’A7 7-217 is derived from FMRI, and that its synthesis is dependent the CGG expansion in this gene.
Vershkov et al. (17) used CRISPR/Cas9 to delete the CGG expansion from FMRI in FXS iPSC- derived neural stem cells (NSCs). Additional FXS NSCs were incubated with 5-AzadC, a nucleoside analogue that prevents DNA methylation. RNA sequencing from these samples, as well as from FXS NSCs incubated with vehicle, was then performed. The RNA-seq data from Vershkov et al. (17) was reanalyzed, some of which is presented in FIG. 1 IB, and FMRI transcript quantification (TPM) in Table 7. RNA-seq reads corresponding to FA7 7-2 I 7 were clearly evident in the FXS-NSCs incubated with 5-AzadC, but not in the other samples.
Moreover, the CGG edited cells, which were isogenic to the unedited FXS NSCs, had no FMR1- 217 reads, but instead robust expression of full-length FMRI. Quantification of the RNA-seq reads (TPM) showed strong total FMRI and FMR1-2Q5 expression in the CGG-edited and 5- AzadC-treated cells but not in vehicle-treated cells. More importantly, s\xon FMRl-217 expression was observed only in the 5-AzadC-treated cells. Therefore, FMRI-217 is derived from the FMRI locus and requires a CGG expansion.
Table 7. FMRI (Total, -205 or -217) reads (TPM) of the samples in FIG. 11B
Figure imgf000086_0001
[00416] In a complementary study, Liu et al. (18) performed a targeted FMRI gene demethylation experiment by incubating FXS iPSC and FXS iPSC-derived neurons with a FMRI small guide RNA and a catalytically inactive Cas9 fused to Tetl demethylase sequences. Reanalysis of the subsequent RNA-seq data is shown in FIG. 11C, and FMRI transcript quantification (TPM) in Table 8. Their experimental paradigm showed that /’A7 7-217 sequences were evident only when the gene was demethylated in the FXS cells. Quantification of the relevant transcripts in Table 8 showed that strong FMRI and 7-205 expression was detected in the Tetl-treated samples (but inexplicably, no F 7-205 in sample Nl_Tetl), and FMR1-2V1 expression in all Tetl-treated samples. These data therefore show once again that 7-217 is derived from the FMRI locus and requires a CGG expansion. Table 8. FMRI (Total, 205 or 217) reads (TPM) of the samples in FIG. 11C.
Figure imgf000087_0001
[00417] To confirm expression of FMRI-211 RNA in FXS brain tissue, frozen post-mortem cortex samples were obtained from six FXS males and five age-matched typically developing (TD) males (UC Davis Health). Using RT-qPCR, it was found that the FMRI full-length RNA was significantly reduced in the FXS individuals compared to that in the TD individuals. However, 3 or 4 of the 6 FXS individuals expressed varying levels of the FMRI full-length RNA as well as /’ 7-2 I 7 RNA (1031-09LZ, 1001-18DL and 1033-08WS) (FIG. 11D). Previous studies on the FXS sample 1031-09LZ had noted expression of FMRI RNA similar to that in TD individuals, despite the presence of a methylated fully mutated FMRI locus (19). However, no detectable FMRP was found in the FXS brain sample 1031-09LZ (20). Also, in agreement with these studies, RNA-seq data from Tran et al. showed no FMRI RNA in the FXS tissue samples (1031-08GP and JS03) (Table 6 and FIG. 11 A) as well as an absence of FMRP (16).
[00418] FMRI -217 RNA was detected in only one of the two premutation carrier samples. To gain greater insight into the relationship of FMRI -217 FXS carrier tissue (CGG repeats between 55-200), skin biopsies were obtained from 3 additional premutation carriers and 3 TD individuals (FIG. 1 IE). The skin samples were cultured in vitro to generate fibroblast cell lines for RNA analysis. Interestingly, using RT-qPCR, FMRI -217 was detected in one premutation carrier (C172) with 140 CGG repeats but not in samples with 77 or 98 CGG repeats (FIG. 1 IE). There was no change in total FMRI RNA levels among the samples (FIG. 1 IE). Thus, generation of FMR1-2V1 may be linked to the number of CGG repeats in the FMRI gene.
Example 9. FMRI -217 RNA is expressed in lymphoblast cell cultures from FXS individuals [00419] DNA methylation of the CpG island upstream of the FMRI gene promoter in FXS individuals (MFM, methylated full mutation) contributes to transcriptional silencing of the locus and loss of FMRP. FMRI transcription can be reactivated by treatment with the nucleoside analogue 5-AzadC (5-aza-2 '-deoxy cytidine), which inhibits DNA methylation (21, 22). Consequently, whether re-activating FMRI transcription in cells from FXS individuals with a completely silenced and presumably fully methylated FMRI locus results in FMRI -217 expression was investigated. For these experiments, lymphoblast cell lines (LCLs) derived from a FXS individual with a fully methylated locus (MFM) that was transcriptionally inactive (FXS1, GM07365), a FXS individual with a presumably partially methylated locus (UFM) that expressed some FMRI RNA (FXS2, GM06897), and two typically developing individuals (TD1, GM07174, and TD2, GM06890), were used (all samples from Coriell Institute, NJ, USA) (FIG. 12A). Western blot analysis showed that modest levels of FMRP were detected in FXS2, but not FXS1 cell lines. FMRP was strongly expressed in TD1 and TD2 cells (ratios of FMRP/GAPDH relative to TD2 were shown below the blot) (FIG. 12A). Similar ratios of FMRP protein expression in these cell lines were obtained by the Luminex Microplex immunochemistry assay (FMRP levels in ng FMRP/pg total protein) (FIG. 12A). Using RT-qPCR, it was found that FMRI -217 RNA is expressed in FXS2 LCLs and comprises 56% of the total FMRI RNA compared to only 9% in TD cells (FIG. 12B). It is noteworthy that although total FMRI RNA levels in FXS2 cells were similar to those in TD cells, FMRP levels were much lower (FIGs. 12A-12B). Next, FXS1 and FXS2 cell lines were treated with 5-AzadC, and then FMRI RNA and FMRP levels were measured (FIG. 12C). In the FXS1 cell line, treatment with 5-AzadC for seven days resulted in significant increases of both full-length FMRI and FMRI -217 RNAs relative to DMSO-treated cells (FIG. 12D). However, in FXS2 cells, 5-AzadC treatment resulted in an increase of only full-length FMRI RNA (FIG. 12E). In neither cell line did 5-AzadC treatment induce a significant increase in FMRP, suggesting either a longer treatment time or a higher concentration of 5-AzadC may be needed to induce FMRP expression (FIGs. 12F-12G and FIG. 13A). However, previous studies showed that longer treatment (36 days) of FXS LCLs with 5-AzadC restored FMRI RNA only up to 40% and produced an even lower level FMRP compared to that in TD cells (22). Thus, transcriptional activation of normally silenced FMRI by demethylation induces expression of full-length FMRI and FMRI-217 RNAs but does not commensurately induce FMRP expression. Example 10. ASOs targeting FMR1-2V1 restored FMRP levels in FXS LCLs with partial or complete FMRI gene methylation.
[00420] FMR1-2Y1 was expressed in the UFM FXS2 cells and after demethylation of MFM FXS1 cells. At the time points tested, although full-length FMRI increased in both FXS LCLs after 5-AzadC treatment, FMRP was unchanged. To test whether blocking the formation of FMR1-2V1 could lead to an increase in full-length FMRI and concomitantly an increase in FMRP, 11 2 '-O-m ethoxy ethyl (MOE)-modified antisense oligonucleotides (ASOs) tiling across intron 1, the intron 1-exon 1 junction, or within exon 2 of FMR1-2V1 RNA were generated (FIG. 14A). First, an ASO targeting MALAT1 RNA (23) was used in LCL cultures to optimize treatment conditions and serves as a marker of transfection efficiency. LCLs cultured with 80nM MALAT1 ASO for 72 hrs led to -60% decrease in MALAT1 RNA levels (FIG. 13B), confirming that the transfection conditions were appropriate. Among the ASOs tested in FXS2 (FIG. 13C), the combination of ASO 713 and 714 (80nM each) led to a significant decrease in FMR1-2V1 and an increase in full-length FMRI (FIG. 14B, FIGs. 13C-13D). ASOs 713 and 714, at 80 nM or 160nM each, for 72 hours elicited similar decreases in FMR1-2V1 and increases in full-length FMRI RNA (FIG. 13D). The MALAT1 ASO had no effect on FMRI isoform levels (FIG. 13D). Next, whether FMRP was restored in FXS2 cells following ASO treatment was assessed. FIG. 14C shows that 80nM or 160nM of ASOs 713 and 714 completely restored FMRP when compared to TD levels. Therefore, ASO treatment of cells from at least certain FXS individuals, which suggests a possible therapeutic path forward through FMRP restoration.
[00421] In the fully methylated FXS1 LCL, a 7-day treatment with 5-AzadC resulted in the expression of FMR and full-length FMRI but did not affect FMRP levels. Thus, whether treatment of FXS1 LCLs with a combination of 5-AzadC and ASOs (713 and 714) could restore FMRP was addressed. FXS1 LCLs were incubated with 80nM each of ASO 713 and 714, 24 hrs preceding the addition of IpM of 5-AzadC every day for seven days prior to sample collection (FIG. 14D). FMRI RNA isoform expression and FMRP levels were tested in these samples. Treatment with 5-AzadC alone led to the expected increase in FMRI full length and FMRI -217 RNA compared to the DMSO control (FIG. 14D). Also, treatment with the ASOs alone did not affect FMRI isoform levels, because the locus was completely methylated. However, treatment of cells with a combination of 5-AzadC and the ASOs rescued FMRI -217 RNA levels and further increased the full-length FMRI compared to 5-AzadC treatment alone (FIG. 14D). Although FMRP levels were unaffected by 5-AzadC alone, FMRP was restored after treatment with a combination of 5-AzadC and the ASOs (FIGs. 14E-14F). These data showed that in FXS patient-derived cells with a UFM, treatment with FMRI -217 targeting ASOs restored FMRP levels while in MFM cells, a combinatorial treatment of demethylation (5-AzadC treatment) and ASOs restored FMRP.
[00422] Finally, two FXS patient-derived fibroblast cell lines were incubated with 5-AzadC and the ASOs to determine FMRI splicing rescue as well as restoration of FMRP. A dermal cell line from a FXS individual (5131b) with CGG repeat numbers of 800, 166 (24), and previously shown to harbor a transcriptionally active FMRI locus, was treated with 5-AzadC and then ASOs 713/714 for 72 hours before RNA and protein extraction (FIG. 15A). RT-qPCR of FMRI and FMRI -217 showed an ASO-dependent decrease in FMR1-2V1 and a subsequent increase in FMRI levels (FIG. 15B). The western blot in FIG. 15C showed while 5-AzadC treatment had no effect on FMRP levels, the ASOs alone or in combination with 5-AzadC significantly increased FMRP levels. In a similar experiment with lung fibroblasts from another FXS individual with a fully methylated FMRI locus, incubation with 5-AzadC in the absence or presence of ASOs 713/714 resulted in increased FMRI and FMRI-217 (FIG. 15D). The western blot in FIG. 15E showed, as with the dermal fibroblasts, ASO treatment resulted in a significant increase of FMRP, albeit lesser than that in the TD fibroblast line.
[00423] To summarize, it was found that in most FXS patient samples tested, the FMRI locus was active but predominantly expressed a mis-spliced FA/7?7-277 isoform as well as very modest levels of FMRP. In the FXS cells that are transcriptionally silent, application of demethylating agents induced FMRI transcription, which resulted in FMRI-217 expression. In both cases, treatment of cells with ASOs to block FMR1-2V1 production resulted in partial to complete restoration of FMRP (FIG. 15F).
[00424] Defects in alternative splicing of mRNAs alter the transcript and protein repertoire of cells and occur in many neurological disorders such as autism, schizophrenia, and bipolar disorder (25-27). In fragile X syndrome model (e.g., Fmrl knockout) mice, hundreds of dysregulated alternative splicing events were detected, a number of which appeared to be linked to an altered epigenetic histone H3 lysine 36 trimethylation (H3K36me3) landscape (11). In this study, >1000 RNA mis-splicing events were detected in human FXS white blood cells, but interestingly, they do not correlate with H3K36me3, which is unaffected in FXS blood. The large number of white blood cell RNA changes, if correlated with certain pathologies of FXS, may be useful as biomarkers to assess therapeutic outcomes, disease prognosis, and cognitive abilities (28-30). Unlike protein-based biomarkers for FXS (31-33), blood derived RNA biomarkers are more sensitive and specific and can easily be translated into the clinic.
[00425] When it contains an expansion of 200 or more CGG repeats, the FMRI gene promoter is methylated and transcriptionally silenced. It was therefore surprising that FMRI RNA was detected in 19 of 29 FXS blood samples and in 5 of 10 FXS post-mortem brain samples. Most of these FXS individuals appeared fully mutated with >200 CGG repeats and methylated in standard assays. Remarkably, in >70% of these FXS cells and tissues, the FMRI RNA was also mis-spliced to generate the FMRI -2X1 isoform, a highly truncated RNA that could encode a 31 amino acid peptide. FMRI-217 RNA was not detected in any TD sample. Moreover, in FXS individuals with a fully methylated and silenced FMRI locus, abrogation of DNA methylation by 5-AzadC treatment results in FMR1-2V1 expression. FMRI mis-splicing to generate the FMR1-2Y1 isoform in FXS clearly requires a CGG expansion, although some evidence suggests that CGG repeat number may be a critical determinant for mis-splicing. For example, FMR1-2Y1 RNA expression was detected in FXS premutation carrier-derived fibroblasts with 140 CGG repeats, but not lesser amounts (77 or 98 CGG repeats) or cells from TD individuals (< 55 CGG repeats).
[00426] An important point is the non-linear relationship between FMRI levels and FMRP expression in FXS tissue samples. The data show that although total FMRI levels are similar in UFM FXS2 LCLs to that of the TD LCLs, FMRP expression is much lower. Likewise, high FMRI expression does not ensure proper FMRP levels in FXS brain tissue samples 1031-09LZ and UMB5746 (16, 20). Similarly, in FXS LCLs and fibroblasts treated with 5-AzadC, a robust increase in FMRI RNA, but not FMRP, ensues. Interestingly, all FXS samples that express FMRI full-length RNA, or after 5-AzadC-mediated transcriptional activation, the FMR1-2V1 mis-spliced RNA was expressed. This relationship between aberrant FMRI expression in FXS cells an<3 FMRl-217 was also evident in FXS iPSC-derived cells. Although the reanalysis of an RNA-seq dataset from FXS neurons with a full CGG expansion show that FMRI-217 was not produced, they did so when the FMRI gene is specifically targeted for demethylation by CRISPR/inactive Cas9 fused to Tetl demethylase (18),' FIG. 11C and Table 8). A second more critical point is that while FMRI -217 is generated in FXS iPSC-derived NPCs incubated with 5- AzadC, it is not produced when the CGG expansion is deleted by CRISPR/Cas9 (17),' FIG. 11C and Table 8). Therefore, the CGG expansion drives mis-spliced 7-277 generation.
[00427] Intellectual impairment is a major characteristic of FXS. The measurements of leukocyte full-length FMRI -205, FMRI-217, FMRP, and FMRI gene methylation allowed correlating these molecular parameters with IQ. FMRP was moderately correlated with a higher IQ, whereas FMRI-217 was weakly correlated with a lower IQ. Based on these correlations, whether abrogating FMR1-2V1 RNA could elevate FMRI and restore FMRP levels were considered. Accordingly, it was found that ASOs targeting the second exon of the FMR1-2Y1 RNA reduced its levels in UFM FXS cells, rescued full-length FMRI and importantly restored FMRP levels similar to TD cells. Therefore, in FXS individuals that express FMRI-217, ASO treatment can be a viable therapeutic option. In individuals with a fully methylated FMRI locus, an ASO-based treatment would be more complex. Consider that in FXS cells with a silenced FMRI, demethylation of the locus by a chemical compound or a CRISPR/Cas9-anchored demethylating enzyme (17, 22, 34), or ASO-mediated blocking of CGG RNA translation (35, 36) have met with limited success in restoring FMRP. CRISPR/Cas9-mediated gene editing of the CGG repeats (37-40) have resulted in a nearly 70% restoration of FMRP levels. However, we show that in FXS cells with silenced FMRI, DNA demethylation combined with ASO treatment restores FMRP. Therefore, treatments that combine DNA demethylation with an ASO approach can be a useful therapeutic strategy for individuals with a fully silenced FMRI gene. [00428] These data demonstrate that 7-217 RNA is an underlying factor inhibiting FMRP expression in FMRI RNA permissive FXS cells.
[00429] The findings suggest that ASOs can be used to correct dysregulated alternative splicing of FMRI and restore FMRP in individuals with FXS, thereby offering a novel therapeutic strategy to treat the disorder. EMBODIMENTS
1. A method of treating a fragile X-associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that modulates splicing of Fragile X Mental Retardation 1 (FMRI gene, thereby treating the fragile X- associated disorder in the subject.
2. The method of Embodiment 1, wherein the fragile X-associated disorder is fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI), or fragile X-associated tremor/ataxia syndrome (FXTAS).
3. The method of Embodiment 1 or 2, wherein the agent increases splicing and/or expression of isoform 1 of the FMRI gene, decreases splicing and/or expression of isoform 12 of the FMRI gene, or a combination thereof.
4. The method of Embodiment 3, wherein the agent increases isoform 1 of the FMRI gene by about 75%.
5. The method of Embodiment 3 or 4, wherein the agent decreases isoform 12 of the FMRI gene by about 30%.
6. The method of any one of Embodiments 1-5, wherein the agent is a polynucleotide, optionally, wherein the polynucleotide is an antisense oligonucleotide (ASO).
7. The method of Embodiment 6, wherein the polynucleotide is a DNA polynucleotide or an RNA polynucleotide.
8. The method of Embodiment 6, wherein the polynucleotide is 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).
9. The method of any one of Embodiments 6-8, wherein the length of the polynucleotide is about 18-22 nucleotides. The method of any one of Embodiments 6-9, wherein the polynucleotide comprises a nucleotide sequence that is complementary to a portion of the FMRI gene transcript. The method of Embodiment 10, wherein the polynucleotide comprises a nucleotide sequence that is at least 80% identical to at least a portion of the pseudo exon of the FMRI gene (SEQ ID NO: 19), at least 80% identical to at least a portion of the junction of intron 1 and the pseudo exon, or both. The method of Embodiment 11, wherein the nucleotide sequence is at least 80% identical to:
AGAAGCCAAAGGAGACCTGA (SEQ ID NO: 1) (W-704), AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705), CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:3) (W-706), ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707), CAATGCTAGACCGGAAAAGA (SEQ ID NO: 5) (W-708), AAGTCCCAATGCTAGACCGGA(SEQ ID NO: 6) (W-709), TCTCCGAAGTCCCAATGCTA (SEQ ID NO:7) (W-710), GAGCTCTCCGAAGTCCCA (SEQ ID NO: 8) (W-711), AGAACAGTGGAGCTCTCCGA (SEQ ID NO: 9) (W-712), CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713), or CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714). The method of Embodiment 12, wherein the nucleotide sequence is identical to: AGAAGCCAAAGGAGACCTGA (SEQ ID NO: 1) (W-704), AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705), CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:3) (W-706), ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707), CAATGCTAGACCGGAAAAGA (SEQ ID NO: 5) (W-708), AAGTCCCAATGCTAGACCGGA(SEQ ID NO: 6) (W-709), TCTCCGAAGTCCCAATGCTA (SEQ ID NO:7) (W-710), GAGCTCTCCGAAGTCCCA (SEQ ID NO: 8) (W-711), AGAACAGTGGAGCTCTCCGA (SEQ ID NO: 9) (W-712), CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713), or
CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714). The method of Embodiment 13, comprising administering to the subject a polynucleotide comprising the nucleotide sequence of CGCCCAGAACAGTGGAGCTC (SEQ ID
NO: 10) (W-713), a polynucleotide comprising the nucleotide sequence of CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714), or both. The method of any one of Embodiments 6-14, wherein the polynucleotide is modified, optionally, wherein the polynucleotide is modified with one or more locked nucleic acid (LNA) nucleotides, one or more 2’-modified ribonucleotides, one or more morpholino nucleotides, or a combination thereof. The method of Embodiment 15, wherein the modification is a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof. The method of Embodiment 15, wherein the polynucleotide is chemically modified to increase the nuclease resistance, to prevent RNase H cleavage of the complementary RNA strand, to increase cellular uptake, or a combination thereof. The method of Embodiment 15, wherein the polynucleotide is chemically modified to comprise 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. The method of Embodiment 16, wherein the chemical modification is a modification of a ribose group and wherein the modification of the ribose group comprises 2'-O-methyl, 2’- fluoro, 2’-deoxy, 2 ’-O-(2 -methoxy ethyl) (MOE), 2’-O-alkyl, 2’-O-alkoxy, 2’-O- alkylamino, 2’-NH2, a constrained nucleotide, a tricyclo-DNA modification, or a combination thereof. The method of Embodiment 16, wherein the chemical modification is a modification of a phosphate group and wherein the modification of the 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. The method of Embodiment 16, wherein the chemical modification is a modification of a nucleobase and wherein the modification of the 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. The method of Embodiment 15, wherein the chemical modification is a modification of the polynucleotide sugar-phosphate backbone. The method of Embodiment 22, wherein the sugar-phosphate backbone is replaced with a phosphorodiamidate mopholino (PMO), a peptide nucleic acid or other pseudopeptide backbone. The method of Embodiment 15, wherein the polynucleotide is a phosphorothioate- modified polynucleotide, such as a polynucleotide where each intemucleotide linkage is a phosphorothioate, or wherein at least half of the intemucleotide linkages are phosphorothi oate . The method of any one of Embodiments 1-24, wherein the subject is a human who has, or is predisposed to have, FXS. The method of Embodiment 25, wherein the subject comprises a CGG repeat expansion exceeding 200 repeats in the 5’ untranslated region of the FMRI gene. The method of any one of Embodiments 1-24, wherein the subject is a human who has, or is predisposed to have, FXTAS. The method of Embodiment 27, wherein the subject comprises a CGG repeat expansion of about 50 to about 200 repeats in the 5’ untranslated region of the FMRI gene. The method of any one of Embodiments 25-28, wherein the CGG repeat expansion is partially methylated. The method of any one of Embodiments 25-28, wherein the CGG repeat expansion is fully methylated. The method of any one of Embodiments 25-30, wherein the subject has an increased level of isoform 12 of the FMRI gene. The method of any one of Embodiments 25-31, wherein the human is a male. The method of any one of Embodiments 25-32, wherein the subject is about 2-11, 4-17, 12-18, or 18-50 years of age. The method of any one of Embodiments 6-33, wherein the polynucleotide is administered intravenously, intra-arterially, intrathecally, intraventricularly, intramuscularly, intradermally, subcutaneously, intracranially, or spinally. The method of any one of Embodiments 1-34, further comprising administering to the subject a therapeutically effective amount of a DNA-dem ethylating compound or DNA demethylase prior to administering the polynucleotide. The method of Embodiment 35, wherein the DNA-demethylating compound or DNA demethylase is administered in an amount sufficient to demethylate about 25-50% of FMRI gene. The method of any one of Embodiments 1-36, wherein treating FXS includes slowing progression of FXS, alleviating one or more signs or symptoms of FXS, preventing one or more signs or symptoms of FXS, or a combination thereof. A method of modulating Fragile X Mental Retardation 1 (FMRI) splicing and/or expression in a cell, comprising contacting the cell with a polynucleotide under conditions whereby the polynucleotide is introduced into the cell, wherein the polynucleotide increases splicing and/or expression of isoform 1 of the /’A7 7 gene, decreases splicing and/or expression of isoform 12 of the FMRI gene, or a combination thereof. The method of Embodiment 38, wherein the cell is an in vitro cell or an ex vivo cell. The method of Embodiment 39, wherein the cell is an induced pluripotent stem cell (iPSC)-derived neuron from a human who has or is predisposed to have FXS, a primary human cell, or a cell line. The method of Embodiment 40, wherein the cell is a cell of a subject. The method of Embodiment 41, wherein the cell is allogeneic. The method of Embodiment 41, wherein the cell is autologous or syngeneic. A polynucleotide, comprising a nucleotide sequence that is complementary to a portion of the FMRI gene transcript. The polynucleotide of Embodiment 44, wherein the nucleotide sequence is at least 80% identical to at least a portion of isol2 of the FMRI gene, at least 80% identical to at least a portion of the junction of intron 1 and isol2 of the FMRI gene, or both. The polynucleotide of Embodiment 45, wherein the nucleotide sequence is at least 80% identical to:
AGAAGCCAAAGGAGACCTGA (SEQ ID NO:1) (W-704), AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705), CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:3) (W-706), ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707), CAATGCTAGACCGGAAAAGA (SEQ ID NO: 5) (W-708), AAGTCCCAATGCTAGACCGGA(SEQ ID NO: 6) (W-709), TCTCCGAAGTCCCAATGCTA (SEQ ID NO:7) (W-710), GAGCTCTCCGAAGTCCCA (SEQ ID NO: 8) (W-711), AGAACAGTGGAGCTCTCCGA (SEQ ID NO: 9) (W-712), CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713), or CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714).
47. A pharmaceutical composition, comprising the polynucleotide of any one of Embodiments 44-46, and one or more pharmaceutically acceptable excipients, diluents, or carriers.
48. A microarray for the detection of a fragile X-associated disorder, comprising at least one nucleic acid probe immobilized on a solid substrate, said probe comprising a nucleic acid sequence complementary to a portion of the FMRI gene transcript.
49. The microarray of Embodiment 48, wherein the nucleotide sequence has at least 80% sequence identity to at least a portion of Exon 2 of FMRI -217, at least a portion of the junction of intron 1-2 and Exon 2 of FMR1-2Y1 , or both.
50. The microarray of Embodiment 49, wherein the nucleotide sequence is at least 80% identical to:
AGAAGCCAAAGGAGACCTGA (SEQ ID NO: 1) (W-704), AAAGAGAAGCCAAAGGAGAC (SEQ ID NO:2) (W-705), CTAGACCGGAAAAGAGAAGCCA (SEQ ID NO:3) (W-706), ATGCTAGACCGGAAAAGAGAA (SEQ ID NO:4) (W-707), CAATGCTAGACCGGAAAAGA (SEQ ID NO: 5) (W-708), AAGTCCCAATGCTAGACCGGA(SEQ ID NO: 6) (W-709), TCTCCGAAGTCCCAATGCTA (SEQ ID NO:7) (W-710), GAGCTCTCCGAAGTCCCA (SEQ ID NO: 8) (W-711), AGAACAGTGGAGCTCTCCGA (SEQ ID NO: 9) (W-712), CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713), or CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714).
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[00430] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
[00431] 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.

Claims

CLAIMS What is claimed is:
1. A method of treating a fragile X-associated disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of an agent that decreases an aberrant fragile X messenger ribonucleoprotein 1 (FMRI') gene product, thereby treating the fragile X-associated disorder in the subject.
2. The method of claim 1, wherein the fragile X-associated disorder is fragile X syndrome (FXS).
3. The method of claim 1 or 2, wherein the therapeutically effective amount of the agent decreases an aberrant FMRI transcript, a protein encoded by the aberrant FMRI transcript, or both.
4. The method of any one of claims 1-3, wherein the aberrant FMRI gene product comprises FMRI -2)1.
5. The method of any one of claims 1-4, wherein the therapeutically effective amount of the agent decreases FMRI -217 by at least 25%.
6. The method of any one of claims 1-5, wherein the therapeutically effective amount of the agent increases the expression of fragile X messenger ribonucleoprotein (FMRP) by at least 25%.
7. The method of any one of claims 1-6, wherein the agent targets a contiguous nucleotide sequence in a polynucleotide sequence set forth in any one of SEQ ID NOs:24-42.
8. The method of any one of claims 1-7, wherein the contiguous nucleotide sequence is at least 12 nucleotides in length. The method of any one of claims 1-8, 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: l-l l, 43-50 and 51-69. 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: 1-11, 43-50 and 51-69. A pharmaceutical composition comprising an antisense oligonucleotide (ASO) and a pharmaceutically acceptable excipient, diluent, or carrier, 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: 1-11, 43-50 and 51-69. The method of Claim 9, the ASO of Claim 10, or the pharmaceutical composition of Claim 11, 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: 10, 11, 43-46 and 60-65. The method of Claim 9, the ASO of Claim 10, or the pharmaceutical composition of Claim 11, wherein the ASO comprises a nucleotide sequence set forth in any one of SEQ ID NOs: l-l l, 43-50 and 51-69. The method of any one of Claims 9, 12 and 13, the ASO of any one of Claims 10, 12 and
13, or the pharmaceutical composition of any one of Claims 11-13, wherein the ASO comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 10, 11, 43-46 and 60-65. The method of any one of Claims 9 and 12-14, the ASO of any one of Claims 10 and 12-
14, or the pharmaceutical composition of any one of Claims 11-14, wherein the ASO is about 18-22 nucleotides in length. The method of any one of Claims 9 and 12-15, the ASO of any one of Claims 10 and 12-
15, or the pharmaceutical composition of any one of Claims 11-15, 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. The method of any one of Claims 9 and 12-16, the ASO of any one of Claims 10 and 12-
16, or the pharmaceutical composition of any one of Claims 11-16, 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’ -0,4 ’-C-aminom ethylene 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- 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 of the foregoing. The method of any one of Claims 9 and 12-17, the ASO of any one of Claims 10 and 12-
17, or the pharmaceutical composition of any one of Claims 11-17, wherein the ASO is a phosphorothioate-modified polynucleotide. The method of any one of Claims 9 and 12-18, the ASO of any one of Claims 10 and 12-
18, or the pharmaceutical composition of any one of Claims 11-18, wherein at least half of the intemucleotide linkages of the polynucleotide are phosphorothioate.
107 The method of any one of Claims 9 and 12-19, the ASO of any one of Claims 10 and 12-
19, or the pharmaceutical composition of any one of Claims 11-19, wherein each internucleotide linkage of the polynucleotide is a phosphorothioate.
108
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