Therapeutic Treatment for Fragile X-Associated Disorders

Abstract

Provided herein, in various embodiments, is a method 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 reduces expression of an abnormal fragile X messenger ribonucleoprotein 1 (FMR1) gene product (e.g., FMR1-2VT). Also provided herein, in various embodiments, is a composition (e.g., a polynucleotide such as an antisense oligonucleotide or a pharmaceutical composition) for reducing expression of the abnormal FMR1 gene product.

Description

[Technical field]

[0001] Related Application(s) This application claims the benefit of U.S. Provisional Application No. 63 / 265,989, filed December 23, 2021. The entire teachings of the above application are incorporated herein by reference.

[0002] Including material by reference in XML This application incorporates by reference a sequence listing contained in the following extensible markup language (XML) file submitted concurrently herewith. a) File name: 54391028001.xml, created on December 22, 2022, size 88,063 bytes.

[0003] Government support This invention was made with Government support under grants GM135087, GM046779, and NS111990 from the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0004] Fragile X syndrome (FXS) is an autism spectrum disorder that is the most frequent inherited form of intellectual disability. FXS affects 1 in 4,000 boys and 1 in 7,000 girls. In addition to intellectual disability, children with FXS exhibit a variety of symptoms including language and developmental delays, perseveration, hyperactivity, aggression, and epilepsy, among other disorders. FXS is caused by a CGG triplet repeat expansion in a single gene, fragile X messenger ribonucleoprotein 1 (FMR1), located on the X chromosome. When the CGG triplet expands to 200 or more, the FMR1 gene becomes methylated and thereby transcriptionally inactivated. Loss of the FMR1 gene product, the protein fragile X messenger ribonucleoprotein (FMRP), is responsible for the disorder.

[0005] Treatments for Fragile X Syndrome (and other autism spectrum disorders) are based primarily on animal models and have had 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)). In fact, there are no broadly applicable therapies that show even a modicum of efficacy for FXS. Summary of the Invention

[0006] It is crucial to develop methods and therapeutic agents for treating Fragile X-associated disorders, such as Fragile X Syndrome (FXS). The present 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 expression of CGG expansion-dependent aberrantly spliced ​​FMR1-217 RNA and restores fragile X messenger ribonucleoprotein (FMRP) to levels observed in cells from typically developing individuals. Thus, the present disclosure generally relates to compositions (e.g., polynucleotides, pharmaceutical compositions) and methods that are useful for treating fragile X-associated disorders.

[0008] In one aspect, the disclosure provides a method of treating fragile X-associated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an agent that reduces expression of an abnormal fragile X messenger ribonucleoprotein 1 (FMR1) gene product, thereby treating the fragile X-associated disorder in the subject.

[0009] In another aspect, the disclosure provides a method of treating fragile X-associated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an agent that modulates splicing of the FMR1 gene (e.g., reduces splicing between exon 1 and exon 2 of FMR1-217), thereby treating fragile X-associated disorder in the subject.

[0010] In another aspect, the disclosure provides a method for reducing expression of an aberrant FMR1 gene product in a cell, comprising contacting the cell with an agent under conditions such that the agent is introduced into the cell, thereby reducing expression of the aberrant FMR1 gene product in the cell.

[0011] In another aspect, the disclosure provides a method for regulating splicing and / or expression of FMR1 in a cell, the method comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions whereby the agent is introduced into the cell, thereby regulating splicing and / or expression of FMR1 in the cell.

[0012] In another aspect, the disclosure provides a method of increasing the level of FMRP in a cell, the method comprising contacting a cell with an agent (e.g., a polynucleotide) under conditions such that the agent is introduced into the cell such that the level of FMRP in the cell is enhanced.

[0013] In another aspect, the disclosure provides a method of enhancing levels of FMRP in a cell, the method comprising contacting the cell with an oligonucleotide that is complementary to at least 8 consecutive 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 disclosure provides a method of reducing a CGG triplet repeat expansion in the FMR1 5'UTR in a cell, comprising contacting the cell with an agent that reduces expression of an aberrant FMR1 gene product under conditions in which the agent is introduced into the cell, thereby reducing the CGG triplet repeat expansion in the cell.

[0015] In some embodiments, the fragile X-associated disorder is FXS.

[0016] In some embodiments, the aberrant FMR1 gene product comprises FMR1-217.

[0017] In some embodiments, the agent is a polynucleotide (eg, any one of the modified polynucleotides disclosed herein).

[0018] In some embodiments, the methods increase expression of fragile X messenger ribonucleoprotein (FMRP) in the subject.

[0019] In another aspect, the disclosure provides an agent that reduces expression of an abnormal FMR1 gene product.

[0020] In another aspect, the disclosure provides agents that modulate splicing and / or expression of the FMR1 gene (e.g., reducing splicing between exon 1 and exon 2 of FMR1-217, or reducing the protein encoded by FMR1-217).

[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 pharma- ceutically acceptable excipients, diluents, or carriers.

[0022] In some embodiments, the agent is a polynucleotide (eg, any one of the modified polynucleotides disclosed herein). [Brief description of the drawings]

[0023] [Figure 1] A genome browser view of FMR1 RNA in seven typically developing ("TD" or "control") and ten fragile X syndrome (FXS) patients sequenced from white blood cells (WBCs) is shown. [Diagram 2] Genome browser maps of exon 1 and intron 1 of FMR1 RNA in seven typically developing individuals and ten fragile X syndrome patients sequenced from white blood cells are shown. [Diagram 3] 1 shows a non-limiting approach using antisense oligonucleotides (ASOs) to block production of isoform 12, increase production of isoform 1, and increase FMRP levels. [Figure 4] Schematic diagrams of FMR1 iso1 and iso12 pre-mRNAs are shown. Numbered boxes (704-714) refer to antisense oligonucleotides spanning the intron1 and iso12 junction and complementary to a region of intron1 within iso12. Iso1_1F, Iso1_1R, Exon1F, Exon1R, and Iso12_1R refer to primers (F, forward; R, reverse) used to detect RNA levels by RT-qPCR. [Diagram 5] RT-qPCR data showing a reduction in iso12 and an increase in iso1 are shown. Asterisks indicate p<0.05. [Figure 6A] RT-qPCR data from a fully methylated FXS cell line (FXS1, GM07365) showing increased FMR1 iso12 upon 5-AzaC treatment and partial rescue of FMR1 iso12 when combined with ASO treatment. Asterisks indicate p<0.05. [Figure 6B] RT-qPCR data from a fully methylated FXS cell line (FXS1, GM07365) is shown, showing an increase in FMR1 iso1 upon 5-AzaC treatment and a further increase when combined with ASO treatment. Asterisks indicate p<0.05. [Figure 7A] FMRP levels are shown. Western blot data of dual FXS1 LCL cell lines showing an increase in FMRP after treatment with 1 μM 5-AzaC and ASO treatment (both antisense oligonucleotides 713 and 714 at 80 nM) when compared to samples treated with DMSO or 5-AzaC only (top panel). Mouse brain (hippocampal tissue) from wild-type and Fmr1 knockout mice was loaded as a control. The bottom panel represents GAPDH protein levels used to normalize the amount of protein loaded in each sample. [Figure 7B] FMRP levels are shown. Quantification of FMRP protein levels relative to GAPDH protein levels as seen in the Western blot in FIG. 7A is shown. [Figure 8A] Figure 1 shows FMR1 iso1 and iso12 levels in fibroblasts from six individuals.Figure 2 shows the number of CGG repeats in the FMR1 RNA 5'UTR from three healthy males and three premutation carrier males with FXS. [Figure 8B] FMR1 iso1 and iso12 levels in fibroblasts from six individuals are shown. RT-qPCR data of FMR1 iso1 levels in fibroblasts from six individuals normalized to GAPDH RNA levels are shown. [Figure 8C] Figure 1 shows FMR1 iso1 and iso12 levels in fibroblasts from six individuals. FMR1 iso12 levels in individual P1 are compared with those in other premutation carriers and healthy control samples. [Figure 9A] Truncated isoforms of FMR1 mRNA identified in a subset of FXS individuals. Integrated Genomics Viewer (IGV) tracks of RNA-seq data for the FMR1 gene in FXS and TD individuals. FMR1 RNA was detected in all TD individuals and in FXS individuals 1-21. The bold box marked on the FMR1 gene shown at the bottom indicates the region in intron 1 with differential reads between TD (1-13) and FXS (1-21) individuals. [Figure 9B] Truncated isoforms of FMR1 mRNA identified in a subset of FXS individuals. Zoomed-in view maps to exons containing the annotated FMR1-217 isoforms. All annotated FMR1 isoforms and sequence data for the FMR1-217 PCR fragment from FXS RNA samples are shown in Table 3 and Figures 9E-9H. H indicates high FMR1 and L indicates low FMR1. [Figure 9C]Truncated isoforms of FMR1 mRNA identified in a subset of FXS individuals. Full-length FMR1 RNA (exon-grey box) and FMR1-217 isoform (exon-grey box) are illustrated with a CGG repeat in the 5'UTR (UTR-black box). The ratio of full-length FMR1 to FMR1-217 was quantified by RT-qPCR in TD, H FMR1 (N=7), and L FMR1 (N=5) individuals. Forward (F) and reverse (R) primers used for q-PCR are shown. Total FMR1 RNA relative to GAPDH RNA levels were significantly reduced in H FMR1 and L FMR1 versus TD (*P<0.05, t-test). Bar graphs show the mean and error bars show + / - SEM. [Figure 9D] Summary table of altered alternative splicing events from L FMR1 versus H FMR1 samples detected by rMATS ( 14 ) at FDR < 5%, and differences in exon inclusion levels (PSI, percent spliced ​​in) between genotypes of ≥ 5% (delta PSI). A schematic of the splicing event categories is shown on the left side of the table. [Figure 9E] The FMR1-217 isoform was identified in an RNA sample generated from leukocytes (individual FXS-05). DNase-treated RNA samples were reverse transcribed using oligo(dT)(20), and PCR products generated using primers Ex1F and 217R were sequenced. [Figure 9F] Predicted protein product of the FMR1-217 isoform. The predicted protein length is 31 amino acids and mass is 3,524 Da. [Figure 9G] Alignment of sequencing data of PCR products using primers Ex1F and 217R to the FMR1 gene is shown. Poly(A) sites were identified by sequencing PCR products with primer 217F and oligo(dT) ( 20 ). [Figure 9H]FMR1 isoforms annotated in the GRCh38.p13 genome assembly. The FMR1-217 isoform (ENST00000621447.1) is marked with a bold box. [Figure 10] Correlation of FXS molecular parameters with IQ. 3D comparison of the indicated parameters. The inset shows a sample with 100% methylation. Increasing size of the dots represents increasing FMRP levels, and darkness from low to high represents increasing IQ levels (see Table 4). [Figure 11A] FMR1-217 is derived from FMR1, requires CGG expansion, and is expressed in human postmortem brain tissue (FXS and premutation carriers) and skin-derived fibroblasts (premutation carriers). Integrated Genomics Viewer (IGV) track of RNA-seq data for the FMR1 gene in FXS and TD individuals (Tran et al., Widespread RNA editing dysregulation in brains from autistic individuals, Nat. Neurosci. (2019)). [Figure 11B] FMR1-217 is derived from FMR1, requires CGG expansion, and is expressed in human postmortem brain tissue (FXS and premutation carriers) and skin-derived fibroblasts (premutation carriers). IGV tracks of selected regions of FMR1 reanalyzed from RNA-seq data from Vershkov et al., FMR1 Reactivating Treatments in Fragile X iPSC-Derived Neural Progenitors In Vitro and In Vivo, Cell Rep. 26:2531-39 (2019), in which Vershkov et al. deleted the FMR1 CGG expansion by CRISPR / Cas9 gene editing. Biological replicates of iPSC-derived neural stem cells (NSCs) from FXS individuals (FXS-NSCs) treated with vehicle or 5-aza-2-deoxycytidine (5-azadC) and isogenic CGG edited samples are shown. FMR1-217 reads are only detected in the 5-azadC treated sample. [Figure 11C] FMR1-217 is derived from FMR1, requires CGG expansion, and is expressed in human postmortem brain tissue (FXS and premutation carriers) and skin-derived fibroblasts (premutation carriers). IGV tracks of selected regions of FMR1 reanalyzed from RNA-seq data in Liu et al. Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene, Cell 172:979-91 (2018), Liu et al. performed targeted FMR1 gene demethylation in FXS iPSCs and iPSC-derived neurons. iPSCs from FXS individuals were incubated with a mock guide RNA (i_mock) or a virus expressing a catalytically inactive Cas9 fused to FMR1 guide RNA and Tet1 demethylase (i_Tet1). iPSC-derived neurons from FXS individuals were treated with mock guide RNA (N1_mock, N2_mock) or catalytically inactive Cas9 fused to FMR1 guide RNA and Tet1 demethylase (N1_Tet1, N2_Tet1, N3_Tet1). All cells were incubated with catalytically inactive Cas9 fused to FMR1 guide RNA and Tet1 demethylase and express FMR1-217. [Figure 11D] FMR1-217 is derived from FMR1, requires CGG expansion, and is expressed in human postmortem brain tissue (FXS and premutation carriers), and skin-derived fibroblasts (premutation carriers). Experimental design for RNA extraction from postmortem cortical tissue obtained from six FXS males (F1-F6) and five typically developing (T1-T5) age-matched males. RT-qPCR data of RNA samples from cortical tissue representing the abundance of FMR1 and FMR1-217 isoforms relative to GAPDH RNA. Each sample was analyzed in duplicate. Primers used for amplification are represented in Figure 9C (**P<0.01, t-test). [Figure 11E]FMR1-217 is derived from FMR1, requires CGG expansion, and is expressed in human postmortem brain tissue (FXS and premutation carriers), and skin-derived fibroblasts (premutation carriers). Schematic diagram of fibroblasts generated from skin biopsies obtained from three male premutation carriers (P1-P3) and three male TD individuals (T1-T3). The table shows the de-identified name of the patient, the genotype, and the number of CGG repeats in the 5'UTR in the FMR1 gene. ND: not determined. qPCR data of fibroblast-derived RNA samples representing the abundance of FMR1 and FMR1-217 isoforms relative to GAPDH RNA. Each sample was analyzed in duplicate. Primers used for amplification are represented in Figure 9C. [Figure 12A] FMR1-217 is expressed in lymphoblastoid cell cultures from FXS individuals. Sample information for lymphoblastoid cell lines (LCLs) (Coriell Institute, NJ) from two FXS and two TD members of the family. FMRP and GAPDH (loading control) levels were determined by Western blot. The ratio of FMRP / GAPDH normalized to FXS1 is shown below the blot. FMRP quantification by Luminex Microplex immunochemistry assay is shown in ng FMRP / μg total protein). [Figure 12B] FMR1-217 is expressed in lymphoblastoid cell cultures from FXS individuals. The ratio of full-length FMR1 to FMR1-217 was quantified using RT-qPCR in TD and FXS2 LCLs relative to GAPDH RNA levels. Primers used for q-PCR are shown in the gene diagram. Total FMR1 RNA was unchanged, but the ratio of FMR1-217 was significantly higher in FXS2 LCLs compared to TD LCLs. [Figure 12C] FMR1-217 is expressed in lymphoblastoid cell cultures from FXS individuals. Schematic of 5-AzadC treatment (1 μM for 7 days) of FXS1 and FXS2 LCLs to determine FMR1 isoform and FMRP levels after demethylation. DMSO-treated cells were used as vehicle control. [Figure 12D]FMR1-217 is expressed in lymphoblastoid cell cultures from FXS individuals. The ratio of full-length FMR1 to FMR1-217 was quantified using RT-qPCR in FXS1 and FXS2 LCLs treated with 5-AzadC versus vehicle, normalized to GAPDH RNA levels (**P<0.001, t-test). [Figure 12E] FMR1-217 is expressed in lymphoblastoid cell cultures from FXS individuals. The ratio of full-length FMR1 to FMR1-217 was quantified using RT-qPCR in FXS1 and FXS2 LCLs treated with 5-AzadC versus vehicle, normalized to GAPDH RNA levels (**P<0.001, t-test). [Figure 12F] FMR1-217 is expressed in lymphoblastoid cell cultures from FXS individuals. FMRP levels were determined using Western blots against GAPDH in FXS1 and FXS2 LCLs treated with DMSO or 5-AzadC (see FIG. 13A). The ratios of FMRP / GAPDH are shown for FXS1 and FXS2 cells, respectively. Histograms show mean values ​​(N=2) and error bars show + / - SEM. [Figure 12G] FMR1-217 is expressed in lymphoblastoid cell cultures from FXS individuals. FMRP levels were determined using Western blots against GAPDH in FXS1 and FXS2 LCLs treated with DMSO or 5-AzadC (see FIG. 13A). The ratios of FMRP / GAPDH are shown for FXS1 and FXS2 cells, respectively. Histograms show mean values ​​(N=2) and error bars show + / - SEM. [Figure 13A] Western blots showing FXS1 and FXS2 cells treated with DMSO or 5-AzadC, respectively (FIG. 12C: treated, FIGS. 12F-12G: quantified). [Figure 13B]The reduction in MALAT1 RNA levels relative to GAPDH RNA was quantified by RT-qPCR in TD1 LCLs treated with MALAT1 ASO (80 nM and 100 nM) for 48 hours. Untreated cells were used as a negative control (* represents P<0.05, t-test). The right panel shows the reduction in MALAT1 RNA levels relative to GAPDH RNA levels quantified using RT-qPCR in TD1 LCLs treated with 80 nM MALAT1 gapmer ASO for 48 or 72 hours. Untreated cells were used as a negative control (* represents P<0.05 using t-test). [Figure 13C] FXS2 LCLs were treated with 80 nM of either ASO704 and 705, 709 and 710, or 713 and 714 for 72 h. RNA levels of FMR1-217 and FMR1 full-length RNA were quantified by RT-qPCR using primers as in Figure 9C. ASO713 and 714 reduced FMR1-217 levels, whereas FMR1 full-length RNA levels increased (* denotes P<0.05, t-test). [Figure 13D] FXS2 LCLs were treated with either ASO713 and 714 at 80 nM or 160 nM, or Malat1 gapmer ASO at 80 nM for 72 h. RNA levels of FMR1-217 and FMR1 full-length RNA were quantified by RT-qPCR using primers as in Figure 9C. ASO713 and 714 reduced FMR1-217 levels at both 80 nM and 160 nM concentrations, whereas FMR1 full-length RNA levels increased. No changes in FMR1 isoform levels were observed upon MALAT1 ASO treatment (* denotes P<0.05, t-test). [Figure 14A] ASOs targeting FMR1-217 restore FMRP levels in FXS LCLs with partial or complete FMR1 gene methylation. Examples of ASOs designed against FMR1-217 RNA are shown (intron-specific: 704-706, intron-exon junction-specific: 707-710, and exon-specific: 711-714). [Figure 14B]ASO targeting FMR1-217 restores FMRP levels in FXS LCLs with partial or complete FMR1 gene methylation. Schematic of ASO treatment (72 h, 80 nM) of FXS2 LCLs to determine FMR1 isoform and FMRP levels after demethylation. DMSO-treated cells were used as vehicle control (****P<0.0001, **P<0.01, t-test). [Figure 14C] ASO targeting FMR1-217 restores FMRP levels in FXS LCLs with partial or complete FMR1 gene methylation. FMRP levels were determined in DMSO (vehicle) and ASO-treated FXS2 LCLs as described in Figure 12A. TD LCLs were also probed for FMRP in the same Western blot. The ratio of FMRP / GAPDH normalized to FXS1 is shown below the blot. [Figure 14D] ASOs targeting FMR1-217 restore FMRP levels in FXS LCLs with partial or complete FMR1 gene methylation. Fully methylated FXS1 LCLs were treated with ASO713 and 714 (80 nM each) followed by the addition of 5-AzadC (1 uM) for 2-9 consecutive days, after which RNA and protein were extracted. FMR1-217 and FMR1 isoforms were assessed using qPCR primers shown in Figure 9C and determined using one-way ANOVA with multiple comparison tests (****P< 0.0001, ***P < 0.001, **P<0.01, *P<0.05). Data information: Bar graphs show the mean and error bars show + / - SEM. [Figure 14E] ASO targeting FMR1-217 restores FMRP levels in FXS LCLs with partial or complete FMR1 gene methylation Western blots of FMRP and GAPDH from FXS1 LCLs treated with DMSO, 5-AzadC, or 5-AzadC and ASO as in Figure 13A. [Figure 14F]ASO targeting FMR1-217 restores FMRP levels in FXS LCLs with partial or complete FMR1 gene methylation. Histograms showing quantification of Western blots of 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 (****P<0.0001, Data information: Bars show mean, error bars show + / - SEM). [Figure 15A] ASOs targeting FMR1-217 restore FMRP levels in FXS fibroblasts with an inactive FMR1 gene treated with 5-AzadC. Skin fibroblasts from an FXS individual (GM05131B, Coriell Institute) were cultured with 5-AzadC for 8 days and then treated with ASO713 / 714 (100 nM each) for 72 hours before RNA and protein extraction. [Figure 15B] ASO targeting FMR1-217 restores FMRP levels in FXS fibroblasts with an inactive FMR1 gene treated with 5-AzadC. RT-qPCR analysis of FMR1-217, FMR1, and GAPDH RNA in skin fibroblasts treated with DMSO, ASO713 / 714, 5-AzadC, or ASO713 / 714 + 5-AzadC. Quantities of FMR1-217 and FMR1 were plotted against GAPDH. (*P<0.05, **P<0.01, one-way ANOVA with multiple comparison test). [Figure 15C] ASO targeting FMR1-217 restores FMRP levels in FXS fibroblasts with an inactive FMR1 gene treated with 5-AzadC. Western blots of FMRP and GAPDH from treated skin fibroblasts as in FIG. 15B. Quantification of FMRP relative to GAPDH is indicated on the right. *p<0.05. Histograms showing quantification of Western blots (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: Bars show mean, error bars show + / - SEM. [Figure 15D] ASOs targeting FMR1-217 restore FMRP levels in FXS fibroblasts with an inactive FMR1 gene treated with 5-AzadC. Lung fibroblasts from an FXS individual (GM07072, Coriell Institute) were cultured with 5-AzadC for 8 days and then treated with ASO713 / 714 (100 nM each) for 72 hours before RNA and protein extraction. RT-qPCR analysis of FMR1-217, FMR1, and GAPDH RNA in lung fibroblasts treated with DMSO, ASO713 / 714, 5-AzadC, or ASO713 / 714 + 5-AzadC. Quantities of FMR1-217 and FMR1 were plotted against GAPDH. (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA with multiple comparison test). [Figure 15E] ASO targeting FMR1-217 restores FMRP levels in FXS fibroblasts with an inactive FMR1 gene treated with 5-AzadC. Western blots of FMRP and GAPDH from lung fibroblasts treated as in FIG. 15B. Quantification of FMRP relative to GAPDH is shown below (N=2). Significance was determined using one-way ANOVA with multiple comparison test (P<0.0001****, P<0.001, ***P<0.01**, one-way ANOVA with multiple comparison test). Data information: Bars indicate mean, error bars indicate + / - SEM. [Figure 15F] ASOs targeting FMR1-217 restore FMRP levels in FXS fibroblasts with an inactive FMR1 gene treated with 5-AzadC. Models showing active FMR1 transcription in FXS cells (or following treatment with a demethylating agent to activate FMR1 transcription) result in the production of misspliced ​​FMR1-217. Downregulation of FMR1-217 with ASOs results in rescue of correctly spliced ​​FMR1 transcripts and restoration of FMRP protein. [Figure 16A] Additional ASO sequences. [Figure 16B]Treatment with 160 nM of each ASO for 72 hours in lymphoblastoid cell line FXS2. Total RNA was extracted using TRIzol™ Reagent (ThermoFisher Scientific No. 15596026). 1 μg of total RNA was primed with oligo(dT)20 to generate cDNA using QuantiTect cDNA synthesis kit with random hexamers (Figure 9E). qPCR was performed in triplicate on a QuantStudio 3 qPCR instrument using iTaq™ Universal SYBR® Green Supermix. Fold changes of full-length FMR1 and FMR1-217 in ASO-treated cells relative to vehicle (control) were measured using qPCR. RNA levels were normalized to GAPDH RNA (*P values ​​were determined using t-test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The foregoing will be apparent from the following more particular description of exemplary 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 the embodiments.

[0025] A description of an exemplary embodiment follows.

[0026] Some aspects of the present disclosure are described below with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are described in order to provide a thorough understanding of the present disclosure. However, one of ordinary skill in the art will readily recognize that the present disclosure can be practiced without one or more of the specific details, or can be practiced with other methods, protocols, reagents, cell lines, and animals. The present disclosure is not limited by the order of acts or events described, as some acts may occur in different orders and / or simultaneously 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 by those of ordinary skill in the art and are commonly employed using conventional methodology.

[0027] Unless otherwise defined, all terms, notations, and other scientific or technical terms of the art used herein are intended to have the meaning commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is commonly 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 consistent with the meaning in the context of the relevant art and / or the meaning otherwise defined herein.

[0028] The terminology used herein is for the purpose of describing some embodiments only and is not intended to be limiting.

[0029] As used herein, the indefinite articles "a," "an," and "the" should be understood to include plural references unless the context clearly indicates otherwise.

[0030] Unless the context otherwise requires, throughout this specification and the claims which follow, the word "comprise", as well as variations such as "comprises" and "comprising", will be understood to imply the inclusion of the specified elements or steps, or group of elements or steps, but not the exclusion of any other elements or steps, or group of elements or steps. As used herein, the term "comprising" can be substituted with the terms "containing" or "including".

[0031] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art. Typically, the acceptable error range for a particular value depends, at least in part, on how the value is measured or determined, e.g., on the limitations of the measurement system. For example, "about" may mean within an acceptable standard deviation, as practiced in the art. Alternatively, "about" may mean within a range of ±20%, e.g., ±10%, ±5%, or ±1% of the given value. It is to be understood that the term "about" may precede any particular value specified herein, except for the particular values ​​used in the examples. When "about" precedes a range, such as "about 24-96 hours," the term "about" should be read as applying to both of the given values ​​in the range, such that "about 24-96 hours" means about 24 hours to about 96 hours.

[0032] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of an aspect or embodiment of the invention, any of the terms "comprising," "containing," "including," and "having" can, in some embodiments, be replaced with the terms "consisting of" or "consisting essentially of" to vary the scope of the present disclosure.

[0033] As used herein, the connector term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to fall within the meaning and thus meets the requirements of the term "and / or" as used herein. Two or more of the options being applicable simultaneously is also understood to fall within the meaning and thus meets the requirements of the term "and / or."

[0034] When lists are presented, unless otherwise stated, 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" should be interpreted to include the embodiments, "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0035] 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. Furthermore, 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, as well as two or more agents. Furthermore, the two or more agents may be the same or different, for example, in embodiments where a first agent comprises a polynucleotide of a first sequence (e.g., an ASO) and a second agent comprises a polynucleotide of a second sequence (e.g., an ASO).

[0036] The phrase "pharmacologically acceptable" means that the substance or composition it 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 is commensurate with a reasonable benefit / risk ratio.

[0037] As used herein, the term "pharmaceutically acceptable salts" refers to those salts that are within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, etc., and 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.

[0038] Examples of salts derived from suitable acids include salts of amino groups formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acids, or using other methods used in the art, such as ion exchange. Other pharma- ceutically acceptable salts derived from suitable acids include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogensulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrogen iodide, hydroxybenzoate, 2-hydroxy-ethanesulfonate, hydrazine ... Examples of the salts include roximaleate, 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, and valerate.

[0039] Either the mono-, di-, or triacid salts can be formed, and such salts can exist in either hydrated, solvated or substantially anhydrous form.

[0040] Salts derived from appropriate bases include salts derived from inorganic bases such as alkali metal, alkaline earth metal, and ammonium bases, as well as salts derived from aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and picoline, or salts derived from N +Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium, and the like. Additionally, pharma- ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxyls, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0041] In one aspect, the 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 reduces expression of an abnormal fragile X messenger ribonucleoprotein 1 (FMR1) gene product, thereby treating the fragile X-associated disorder in the subject. The agent that reduces expression of an abnormal FMR1 gene product in the methods disclosed herein can be any one or more of the agents disclosed herein.

[0042] In another aspect, the 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 the FMR1 gene (e.g., reduces splicing between exon 1 and exon 2 of FMR1-217), thereby treating the fragile X-associated disorder in the subject. The agent that modulates splicing of the FMR1 gene in the methods disclosed herein can be any one or more of the agents disclosed herein.

[0043] In another aspect, the disclosure provides a method for reducing expression of an aberrant FMR1 gene product in a cell, comprising contacting the cell with an agent under conditions such that the agent is introduced into the cell, thereby reducing expression of the aberrant FMR1 gene product in the cell.

[0044] In another aspect, the disclosure provides a method of modulating splicing and / or expression of FMR1 in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions where the agent is introduced into the cell, thereby modulating splicing and / or expression of FMR1 in the cell. The agent that modulates splicing and / or expression of FMR1 in the methods disclosed herein can be any one or more of the agents disclosed herein.

[0045] In another aspect, the disclosure provides a method of increasing the level of Fragile X messenger ribonucleoprotein (FMRP) in a cell, the method comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions such that the agent is introduced into the cell such that the level of FMRP in the cell is enhanced.

[0046] In another aspect, the disclosure provides a method of reducing a CGG triplet repeat expansion in the FMR1 5'UTR in a cell, comprising contacting the cell with an agent that reduces expression of an aberrant FMR1 gene product under conditions in which the agent is introduced into the cell, thereby reducing the CGG triplet repeat expansion in the cell.

[0047] Fragile X-associated disorder Fragile X-associated disorders are caused by mutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1, formerly known as Fragile X Mental Retardation 1) gene located at the q27.3 locus of the X chromosome. Expansion of the trinucleotide CGG (more than 54 repeats) above the normal range in the non-coding region of the FMR1 gene is associated with the development of Fragile X-associated disorders. For example, in those with premutations, the trinucleotide CGG can range from 55 to 200 CGG repeats. In some embodiments, the Fragile X-associated disorders described herein are associated with more than 77 CGG repeats in FMR1, such as more than 98 CGG repeats in FMR1. In some embodiments, the Fragile X-associated disorders are associated with at least 140 CGG repeats in FMR1. In some embodiments, the Fragile X-associated disorders are associated with at least 201 CGG repeats in FMR1.

[0048] 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 disorder (FXAND), and fragile X syndrome (FXS). In some embodiments, the 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.

[0049] FMR1 gene product The FMR1 gene encodes the fragile X messenger ribonucleoprotein (FMRP, formerly known as fragile X mental retardation protein).

[0050] In some embodiments, the FMR1 gene described herein is a human FMR1 gene (e.g., corresponding to GenBank reference number NC_000023.11), a mouse FMR1 gene (e.g., NC_000086.8), a rat FMR1 gene (e.g., NC_051356.1), a golden hamster FMR1 gene (e.g., NW_024429188.1), a Chinese hamster FMR1 gene (e.g., NW_003614110.1), a dog FMR1 gene (e.g., NC_051843.1), a pig FMR1 gene (e.g., NC_046383.1), or a monkey FMR1 gene (e.g., NC_041774.1). In some embodiments, the FMR1 gene is a human FMR1 gene. The human FMR1 gene (ensemble: ENSG00000102081.16) is located within chromosome band Xq27.3 at base pairs 147,911,919 to 147,951,125 (numbering refers to Genome Reference Consortium Human Build 38 (GRCh38)).

[0051] As used herein, "abnormal FMR1 gene product" refers to an FMR1 gene product that is elevated in a subject who has or is prone to have a fragile X-associated disorder. In some embodiments, the aberrant FMR1 gene product described herein is elevated in a subject who is being or has been treated for a fragile X-associated disorder. In some embodiments, the aberrant FMR1 gene product is elevated in a subject who has at least 55 CGG repeats in the 5' untranslated region of the FMR1 gene, e.g., at least 77, at least 78, at least 98, at least 99, at least 140, or at least 201 CGG repeats in the 5' untranslated region of the FMR1 gene. In some embodiments, the aberrant FMR1 gene product is elevated in a subject who has at least 201 CGG repeats in the 5' untranslated region of the FMR1 gene. In some embodiments, the aberrant FMR1 gene product described herein is not expressed in a typically developing subject (e.g., a typically developing human). In some embodiments, the aberrant FMR1 gene product is elevated in a subject who is a premutation carrier of FXS. In some embodiments, the abnormal FMR1 gene product is elevated in subjects with FXS.

[0052] In some embodiments, the aberrant FMR1 gene products described herein are produced from CGG expansion-dependent mis-splicing of the FMR1 gene.

[0053] In some embodiments, the aberrant FMR1 gene products described herein contribute to the pathology of the fragile X-associated disorders described herein. In some embodiments, the aberrant FMR1 transcript, its protein product, or both, contribute to the pathology of the fragile X-associated disorders. In some embodiments, the aberrant FMR1 transcripts described herein contribute to the pathology of the fragile X-associated disorders. In some embodiments, the proteins encoded by the aberrant FMR1 transcripts described herein contribute to the pathology of the fragile X-associated disorders. In some embodiments, the aberrant FMR1 transcripts and their protein products contribute to the pathology of the fragile X-associated disorders.

[0054] In some embodiments, the aberrant FMR1 gene product described herein comprises FMR1-217, its protein product, or both. In some embodiments, the aberrant FMR1 gene product comprises FMR1-217. In some embodiments, the aberrant FMR1 gene product comprises the protein product of FMR1-217. In some embodiments, the aberrant FMR1 gene product comprises FMR1-217 and its protein product.

[0055] In humans, FMR1-217, also referred to as "isoform 12" or "iso12", is a transcript corresponding to A0A087X1M7 (ENST00000621447.1, 1,832 nucleotides). FMR1-217 has two exons, and splicing between exon 1 of FMR1-217 (base pairs 147,912,123 to 147,912,230, SEQ ID NO:23) and exon 2 of FMR1-217 (base pairs 147,912,728 to 147,914,451, SEQ ID NO:21) is considered aberrant FMR1 RNA splicing. FMR1-217 is detected in a subpopulation of subjects with fragile X-associated disorder, including a subpopulation of FXS patients and a subpopulation of FXS premutation carriers.

[0056] CGCCCGCAGCCCACCTCTCGGGGGCGGGCTCCCGGCGCTAGCAGGGCTGAAGAGAAGATGGAGGAGCTGGTGGTGGAAGTGCGGGGCTCCAATGGCGCTTTCTACAAG (SEQ ID NO: 23).

[0057]

[0058] FMR1-217 encodes a 31 amino acid protein (SEQ ID NO:22).

[0059] MEELVVEVRGSNGAFYKHWDFGELHCSGRGL (sequence number 22).

[0060] Additional information regarding FMR1-217 and its protein products can be found at the following web addresses, the contents of which are incorporated herein by reference in their entireties:

[0061] useast.ensembl.org / Homo_sapiens / Transcript / Summary?db=core;g=ENSG00000102081;r=X:147911951-147951125;t=ENST00000621447.

[0062] In some embodiments, the methods disclosed herein increase the expression level of FMRP in a subject as described herein.In some embodiments, the methods disclosed herein increase the expression level of FMRP in a cell as described herein.

[0063] In some embodiments, the methods disclosed herein increase a normal FMR1 gene product (e.g., a normal FMR1 transcript, its protein product, or both) in a subject and / or cell described herein.

[0064] Several normal FMR1 gene products are expressed in typically developing subjects (e.g., humans without FXS). Non-limiting examples of "normal" human FMR1 gene products include: The transcript corresponding to Q06787 (FMR1-205, ENST00000370475.9, 4,441 nucleotides) and its protein product (632 amino acid protein (NP_002015.1)), The transcript corresponding to NM_001185075.2 (4,170 nucleotides) and its protein product (537 amino acid protein (NP_001172004.1)), The transcript corresponding to NM_001185076.2 (4,378 nucleotides) and its protein product (611 amino acid protein (NP_001172005.1)), The transcript corresponding to NM_001185082.2 (4,303 nucleotides) and its protein product (586 amino acid protein (NP_001172011.1)), The transcript corresponding to NM_001185081.2 (4,107 nucleotides) and its protein product (516 amino acid protein (NP_001172010.1)), The transcript corresponding to Q06787-9 (FMR1-201, ENST00000218200.12, 4,333 nucleotides) and its protein product (611 amino acid protein), The transcript corresponding to Q06787-8 (FMR1-208, ENST00000440235.6, 4,271 nucleotides) and its protein product (586 amino acid protein), A transcript corresponding to X5D907 (FMR1-223, ENST00000687593.1, 4,159 nucleotides) and its protein product (594 amino acid protein); The transcript corresponding to Q06787-10 (FMR1-204, ENST00000370471.7, 4,125 nucleotides) and its protein product (537 amino acid protein), The transcript corresponding to G3V0J0 (FMR1-207, ENST00000439526.6, 3,699 nucleotides) and its protein product (592 amino acid protein), A transcript corresponding to A8MQB8 (FMR1-206, ENST00000370477.5, 3,437 nucleotides) and its protein product (582 amino acid protein); A transcript corresponding to A0A087WY29 (FMR1-212, ENST00000495717.6, 2,874 nucleotides) and its protein product (561 amino acid protein); A transcript corresponding to A0A087WXI3 (FMR1-214, ENST00000616382.5, 2,799 nucleotides) and its protein product (536 amino acid protein), and The transcript corresponding to R9WNI0 ("FMR1-218", ENST00000621453.5, 1,827 nucleotides) and its protein product (548 amino acid protein) are included.

[0065] In some embodiments, normal FMR1 gene products as described herein include the transcript corresponding to Q06787 (FMR1-205, ENST00000370475.9, 4,441 nucleotides) and its protein product (632 amino acid protein (NP_002015.1)). FMR1-205, also referred to as "isoform 1" or "iso1", is produced in a subpopulation of typically developing individuals and FXS subjects. FMR1-205 has 17 exons, and splicing between exon 1 of FMR1-205 (base pairs 147,911,919-147,912,230, SEQ ID NO: 19) and exon 2 of FMR1-205 (base pairs 147,921,933-147,921,985, SEQ ID NO: 20) is considered normal FMR1 RNA splicing. Additional information regarding FMR1-205 and its protein product can be found at the following web address, 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:147911951-147951125;t=ENST00000370475.

[0066] CTCAGTCAGGCGCTCAGCTCCGTTTCGGTTTCACTTCCGGTGGAGGGCCGCCTCTGAGCGGGCGGCGGGCCGACGGCGAGCGCGGGCGGCGGCGGTGACGGAGGCGCCGCTGCCAGGGGGCGTGCGGCAGCGCGGCGGCGGCGGCGGCGGCGGCGGCGGC GGAGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCTGGGCCTCGAGCGCCGCAGCCCACCTCTCGGGGGCGGGCTCCCGGCGCTAGCAGGGCTGAAGAGAAGATGGAGGAGCTGGTGGTGGAAGTGCGGGGCTCCAATGGCGCTTTCTACAAG (SEQ ID NO: 19).

[0067] GCATTTGTAAAGGATGTTCATGAAGATTCAATAACAGTTGCATTTGAAAACAA (sequence number 20).

[0068] Drugs In another aspect, the disclosure provides agents that modulate splicing and / or expression of the FMR1 gene (e.g., reducing splicing between exon 1 and exon 2 of FMR1-217, or reducing the protein encoded by FMR1-217).

[0069] In another aspect, the disclosure provides agents that modulate splicing and / or expression of the FMR1 gene (e.g., reducing splicing between exon 1 and exon 2 of FMR1-217, or reducing the protein encoded by FMR1-217).

[0070] In another aspect, the disclosure provides an agent that reduces expression of an abnormal FMR1 gene product.

[0071] As used herein, the terms "reducing," "reducing," "reducing," or "reducing" refer to modulation that results in lower levels of an abnormal FMR1 gene product (e.g., FMR1-217 and / or its protein product) relative to a reference (e.g., levels before or in the absence of modulation by an agent disclosed herein).

[0072] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein reduces expression of an aberrant FMR1 gene product (e.g., FMR1-217 and / or its protein product) by at least 5%, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to a reference.

[0073] In some embodiments, an agent disclosed herein (e.g., an antisense RNA polynucleotide) reduces expression of an aberrant FMR1 transcript, reduces expression of an aberrant FMR1-encoded protein, or both.

[0074] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein reduces expression of an aberrant FMR1 transcript (e.g., FMR1-217). In some embodiments, the agent reduces expression of the aberrant FMR1 transcript by at least 5%, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, relative to a reference.

[0075] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein reduces expression of an aberrant FMR1-encoded protein (e.g., the protein product of FMR1-217). In some embodiments, the agent reduces expression of the aberrant FMR1-encoded protein by at least 5%, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to a reference.

[0076] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein reduces expression of aberrant FMR1 transcripts and aberrant FMR1 encoded proteins (e.g., FMR1-217 and its protein products). In some embodiments, the agent reduces expression of aberrant FMR1 transcripts and aberrant FMR1 encoded proteins by at least 5%, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to a reference.

[0077] The agents disclosed herein may directly or indirectly reduce expression of an aberrant FMR1 gene product, 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.

[0078] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein reduces splicing of the aberrant FMR1 transcript (e.g., between exon 1 and exon 2 of FMR1-217). In some embodiments, the agent reduces splicing of the aberrant FMR1 transcript by at least 5%, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, relative to a reference.

[0079] In some embodiments, an agent disclosed herein (e.g., an antisense RNA polynucleotide) increases the expression level of FMRP. As used herein, the term "increasing" or "increasing" refers to modulation that results in a higher level of FMRP relative to a reference (e.g., the level before or in the absence of modulation by an agent disclosed herein).

[0080] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein increases FMRP expression by at least 5%, e.g., 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%, relative to a reference.

[0081] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein increases expression of a normal FMR1 gene product by at least 5%, e.g., 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%, relative to a reference. In some embodiments, the agent increases expression of a normal FMR1 gene product to at least 5% of the level observed in a typically developing subject (e.g., a 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 a typically developing subject. In some embodiments, the agent increases expression of a normal FMR1 gene product to at least 30% of the level observed in a typically developing subject (e.g., a human).

[0082] In some embodiments, an agent disclosed herein (eg, an antisense RNA polynucleotide) increases expression of a normal FMR1 transcript, a normal FMR1-encoded protein, or both.

[0083] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein increases expression of a normal FMR1 transcript (e.g., FMR1-205). In some embodiments, the agent increases expression of a normal FMR1 transcript by at least 5%, e.g., 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%, relative to a reference.

[0084] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein increases expression of a normal FMR1 encoded protein (e.g., a protein encoded by FMR1-205). In some embodiments, the agent increases expression of a normal FMR1 encoded protein by at least 5%, e.g., 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%, relative to a reference.

[0085] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein increases expression of a normal FMR1 transcript and a normal FMR1 encoded protein (e.g., FMR1-205 and its protein product). In some embodiments, the agent increases expression of a normal FMR1 transcript and a normal FMR1 encoded protein by at least 5%, e.g., 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%, relative to a reference.

[0086] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein increases splicing of the normal FMR1 transcript (e.g., between exon 1 and exon 2 of FMR1-205). In some embodiments, the agent increases splicing of the normal FMR1 transcript by at least 5%, e.g., 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%, relative to a reference.

[0087] In some embodiments, an agent disclosed herein (e.g., an antisense RNA polynucleotide) reduces expression of an abnormal FMR1 gene product (e.g., FMR1-217 and / or its protein product) and increases expression of FMRP.

[0088] In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein reduces expression of an aberrant FMR1 gene product (e.g., FMR1-217 and / or its protein product) and increases expression of a normal FMR1 gene product (e.g., FMR1-205 and / or its protein product). In some embodiments, an agent (e.g., an antisense RNA polynucleotide) disclosed herein reduces expression of an aberrant FMR1 transcript, reduces expression of an aberrant FMR1 encoded protein, increases expression of a normal FMR1 transcript, increases expression of a normal FMR1 encoded protein, or a combination thereof.

[0089] In some embodiments, the agents disclosed herein (e.g., antisense RNA polynucleotides) reduces expression of an aberrant FMR1 gene product (e.g., FMR1-217 and / or its protein product) by at least 5%, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to a reference, and Relative to a reference, expression of a normal FMR1 gene product (e.g., FMR1-205 and / or its protein product) is increased by at least 5%, e.g., 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%.

[0090] In some embodiments, the agents disclosed herein (e.g., antisense RNA polynucleotides) reduces splicing of the aberrant FMR1 transcript (e.g., between exon 1 and exon 2 of FMR1-217) by at least 5%, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to a reference, and Increases splicing of the normal FMR1 transcript (e.g., between exon 1 and exon 2 of FMR1-205) by at least 5%, e.g., 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% relative to a reference.

[0091] In some embodiments, the level of an FMR1 gene product (e.g., an aberrant FMR1 transcript, an aberrant FMR1-encoded protein, a normal FMR1 transcript, a normal FMR1-encoded protein, or a combination thereof) is measured at least one day after an agent disclosed herein is administered to the subject, e.g., 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 treatment with an agent disclosed herein is initiated.

[0092] In some embodiments, the level of the FMR1 gene product is measured in a tissue or cell. In some embodiments, the level of the FMR1 gene product is measured in a white blood cell. In some embodiments, the level of the FMR1 gene product is measured in a leukocyte. In some embodiments, the level of the FMR1 gene product is measured in a fibroblast (e.g., a skin-derived fibroblast or a lung-derived fibroblast). In some embodiments, the level of the FMR1 gene product is measured in a cortical tissue (e.g., a superficial cortical brain biopsy).

[0093] Target sequence In some embodiments, the agents disclosed herein (e.g., antisense oligonucleotides (ASOs)) promote the exclusion of aberrant FMR1 exons. In some embodiments, the agents promote the exclusion of exon 2 of FMR1-217.

[0094] In some embodiments, the agents (e.g., ASOs) disclosed herein target (e.g., bind, indirectly or directly) the primary aberrant transcript (pre-mRNA) of the FMR1 gene. As used herein, the term "target" refers to a pre-mRNA region, specifically the region specified by exon 2 and the adjacent intron 1-2 region of FMR1-217 that are responsible for splicing associated with FMR1-217. In some embodiments, a target sequence refers to a portion of a target RNA to which a polynucleotide (e.g., ASO) is directed, i.e., the sequence to which the polynucleotide hybridizes by Watson-Crick base pairing of complementary sequences.

[0095] In some embodiments, the agent targets a contiguous nucleotide sequence in the pre-mRNA of FMR1-217, and 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, e.g., 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.

[0096] In some embodiments, an 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), and the contiguous nucleotide sequence is at least 8 nucleotides in length. In some embodiments, an agent (e.g., an ASO) targets a contiguous nucleotide sequence within SEQ ID NO:27, and 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, e.g., 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.

[0097] UCAGGUCUCCUUUGGCUUCUCUUUUCCGGUCUAGCAUUGGGACUUCGGAGAGCUCCACUGUUCUGGGCGAGGGCUGUGAAGAAAGA (sequence number 24).

[0098] UCAGGUCUCCUUUGGCUUCUCUUUUCCGGUCUAGCAUUGGGACUUCGGAGA (SEQ ID NO: 25)

[0099] CAUUGGGACUUCGGAGAGCUCCACUGUUCUGGGCGAGGGCUGUGAAGAAAGA (SEQ ID NO: 26)

[0100] UGGGACUUCGGAGAGCUCCACUGUUCUGGGCGAGGGCUGUGAAGAA (SEQ ID NO: 27)

[0101] In some embodiments, the agent (e.g., ASO) targets a contiguous nucleotide sequence within exon 2 of FMR1-217, introns 1-2 of FMR1-217, the junction between exon 2 and introns 1-2 of FMR1-217, or a combination thereof. In some embodiments, the agent (e.g., ASO) targets a contiguous nucleotide sequence of any one or more of SEQ ID NOs: 28-42, the contiguous nucleotide sequence being at least 8 nucleotides in length. In some embodiments, the agent (e.g., ASO) targets a contiguous nucleotide sequence of any one or more of SEQ ID NOs: 37-42, the contiguous nucleotide sequence being 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.

[0102] UCAGGUCUCCUUUGGCUUCU (SEQ ID NO: 28)

[0103] GUCUCCUUUGGCUUCUCUUU (SEQ ID NO: 29)

[0104] UGGCUUCUCUUUCCGGUCUAG (SEQ ID NO: 30)

[0105] UUCUCUUUUCCGGUCUAGCAU (SEQ ID NO:31)

[0106] UCUUUUCCGGUCUAGCAUUG (SEQ ID NO: 32)

[0107] UCCGGUCUAGCAUUGGGACUU (SEQ ID NO: 33)

[0108] UAGCAUUGGGACUUCGGAGA (SEQ ID NO: 34)

[0109] UGGGACUUCGGAGAGCUC (SEQ ID NO: 35)

[0110] UCGGAGAGCUCCACUGUUCU (SEQ ID NO: 36)

[0111] GAGCUCCACUGUUCUGGGCG (SEQ ID NO: 37)

[0112] CUCCACUGUUCUGGGCGAGG (SEQ ID NO: 38)

[0113] GGACUUCGGAGAGCUCCACUG (SEQ ID NO: 39)

[0114] GGAGAGCUCCACUGUUCUGGG (SEQ ID NO: 40)

[0115] UGUUCUGGGCGAGGGCUGUG (SEQ ID NO: 41)

[0116] UGGGCGAGGGCUGUGAAGAA (SEQ ID NO: 42)

[0117] Polynucleotides (polynucleotide agents) In some embodiments, the agent disclosed herein comprises at least one polynucleotide disclosed herein. In some embodiments, the agent comprises at least two polynucleotides disclosed herein.

[0118] In another aspect, the disclosure provides polynucleotides capable of reducing expression of an aberrant FMR1 gene product.

[0119] In another aspect, the disclosure provides polynucleotides capable of decreasing splicing of FMR1-217.

[0120] In another aspect, the disclosure provides a method of enhancing levels of FMRP in a cell, the method comprising contacting the cell with an oligonucleotide that is complementary to at least 8 consecutive nucleotides of a sequence set forth in SEQ ID NOs: 24-42, such that the level of FMRP in the cell is enhanced.

[0121] 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, the polynucleotides disclosed herein comprise deoxyribonucleotides. In some embodiments, the polynucleotides comprise ribonucleotides. Non-limiting examples of polynucleotides include single-stranded, double-stranded, or multi-stranded DNA or RNA, DNA-RNA hybrids (e.g., each "T" position may be independently replaced by "U" or vice versa), or polymers that contain purine and pyrimidine bases, or other natural, chemically, or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may comprise sugars and phosphate groups, modified or substituted sugars or phosphate groups, polymers of synthetic subunits such as phosphoramidates, or combinations thereof.

[0122] 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. Nucleotide analogs can be modified at any position to change certain chemical properties of the nucleotide but still retain the ability to perform its intended function. Non-limiting examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, and 5-propenyluridine, the 6-position, e.g., 6-(2-amino)propyluridine, the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, and 8-fluoroguanosine. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine, O- and N-modified (e.g., alkylated or N6-methyladenosine) nucleotides.

[0123] In some embodiments, the nucleotide analog comprises a modification to the sugar portion of the nucleotide, for example, the 2'OH-group can be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, or aryl.

[0124] In some embodiments, the phosphate group of the nucleotide is modified, for example, by replacing one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioate). In some embodiments, the ASO is a phosphorothioate-modified polynucleotide, such as a polynucleotide in which each internucleotide linkage is phosphorothioate, or a polynucleotide in which at least half of the internucleotide linkages are phosphorothioate.

[0125] In some embodiments, the polynucleotides (e.g., ASOs) disclosed herein bind to a target sequence described herein.

[0126] In some embodiments, the target polynucleotides (e.g., ASOs) disclosed herein have near or substantial complementarity to the target sequences described herein. In some embodiments, the polynucleotides are formed of contiguous complementary sequences (to the target sequence). In some embodiments, the polynucleotide sequences are formed of non-contiguous complementary sequences (to the target sequence), e.g., when placed together, they constitute a sequence that spans the target sequence.

[0127] In some embodiments, the polynucleotides (e.g., ASOs) disclosed herein comprise a nucleotide sequence that is complementary (e.g., fully complementary or partially complementary) to a target sequence described herein (e.g., such that the polynucleotide can hybridize or anneal to the target sequence 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 nucleic acid if at least one nucleotide residue of the first region can base pair (i.e., hydrogen bond) with a residue of the second region when the two regions are arranged in an antiparallel manner, thereby forming a hydrogen-bonded duplex.

[0128] In some embodiments, a polynucleotide (e.g., ASO) disclosed herein specifically hybridizes to a target polynucleotide (e.g., consecutive nucleotides of a sequence set forth in SEQ ID NOs: 24-42) described herein, e.g., under physiological conditions, at 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 the 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 when the antisense oligomer is nearly complementary to the target sequence. In some embodiments, specific hybridization occurs when the antisense oligomer is substantially complementary to the target sequence. In some embodiments, specific hybridization occurs when the antisense oligomer is exactly complementary to the target sequence.

[0129] In some embodiments, a polynucleotide (e.g., an ASO) disclosed herein comprises a nucleotide sequence that is complementary to a contiguous nucleotide sequence (e.g., 10-30 nucleotides) of the pre-mRNA of an aberrant FMR1 transcript.

[0130] In some embodiments, a polynucleotide (e.g., ASO) disclosed herein comprises a nucleotide sequence that is complementary to a contiguous nucleotide sequence (e.g., 10-30 nucleotides) of the pre-mRNA of FMR1-217. 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).

[0131] In some embodiments, the 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 to include one or more locked nucleic acid (LNA) nucleotides, one or more 2'-modified ribonucleotides, one or more morpholino nucleotides, or a combination thereof.

[0132] In some embodiments, the polynucleotides (e.g., ASOs) disclosed herein target sequences within the X chromosome at base pairs 147,911,919 to 147,921,985 (e.g., target sequences within the X chromosome at base pairs 147,911,919 to 147,921,985), e.g., 147,911,919 to 147,921,933, 147,911,919 to 147,912,230, 147,911,919 to 147,912,123, 147,911,919 to 147,914,451, 147,911,919 to 147,912,728, 147,912,231 to 1 147,912,731 to 147,912,766, or 147,912,694 to 147,912,766. In some embodiments, the polynucleotides (e.g., ASOs) disclosed herein are nucleotides having a length of 147,911,919 to 147,921,985 base pairs, e.g., 147,911,919 to 147,921,933, 147,911,919 to 147,912,230, 147,911,919 to 147,912,123, 147,911,919 to 147,914,451, 147,911,919 to 147,912,728, 147,912,231 to 147, 921,932, 147,912,231 to 147,914,451, 147,912,231 to 147,912,727, 147,912,728 to 147,914,451, 147,912,694 to 147,912,727, 147,912,710 to 147,912,745, 147,912,731 to 147,912,766, or 147,912,694 to 147,912,766.

[0133] In some embodiments, the polynucleotide comprises a nucleotide sequence that specifically hybridizes (e.g., has near, substantial, or exact complementarity) to at least a portion of the X chromosome from base pairs 147,912,694 to 147,912,766, e.g., has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reverse and complementary sequences of at least a portion of the X chromosome from base pairs 147,912,694 to 147,912,766.

[0134] As used herein, the term "sequence identity" refers to the degree to which two nucleotide sequences have the same residues at the same positions when the sequences are aligned to achieve a maximum 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 sequence is compared. The sequence identity between a reference sequence and a test sequence is expressed as a percentage of positions over the entire length of the reference sequence where the reference sequence and the test sequence share the same nucleotide or amino acid when the reference sequence and the test sequence are aligned to achieve a maximum level of identity. As an example, if the test sequence has the same nucleotide residues at 70% of the same positions over the entire length of the reference sequence when aligned to achieve a maximum level of identity, the two sequences are considered to have 70% sequence identity.

[0135] Alignment of sequences for comparison to achieve maximum levels of identity can be readily performed by one of skill in the art using an appropriate alignment method or algorithm. In some cases, the alignment can include gaps introduced to provide maximum levels 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, e.g., generally, Ausubel et al., Current Protocols in Molecular Biology).

[0136] 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 the X chromosome from base pairs 147,912,694 to 147,912,766. 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 the X chromosome from base pairs 147,912,694 to 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence having about 70-100%, e.g., 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 at least a portion of the X chromosome between base pairs 147,912,694 and 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence that is identical to at least a portion of the X chromosome between base pairs 147,912,694 and 147,912,766.

[0137] In some embodiments, the polynucleotides disclosed herein comprise a nucleotide sequence that specifically hybridizes (e.g., has near, substantial, or exact complementarity) to at least a portion of the X chromosome from base pairs 147,912,731 to 147,912,766, e.g., has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reverse and complementary sequences of at least a portion of the X chromosome from base pairs 147,912,731 to 147,912,766.

[0138] 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 the X chromosome from base pairs 147,912,731 to 147,912,766. 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 the X chromosome from base pairs 147,912,731 to 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence having about 70-100%, e.g., 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 at least a portion of the X chromosome from base pairs 147,912,731 to 147,912,766. In some embodiments, the polynucleotide comprises a nucleotide sequence that is identical to at least a portion of the X chromosome from base pairs 147,912,731 to 147,912,766.

[0139] In some embodiments, a polynucleotide (e.g., ASO) disclosed herein comprises a nucleotide sequence having at least 70% sequence identity, e.g., 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: 1-11 and 43-50. In certain embodiments, a 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 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 any one of SEQ ID NOs: 1-11 and 43-50. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100%, e.g., 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 any one of SEQ ID NOs: 1-11 and 43-50. In some embodiments, a polynucleotide (e.g., an ASO) comprises a nucleotide sequence identical to a sequence set forth in any one of SEQ ID NOs: 1-11 and 43-50. In the sequence, each nucleobase designated as T may independently be T or U. Similarly, each C nucleotide may independently be C or a C analog such as 5-methyl C or other substituted C analog. Other modified nucleobases having equivalent Watson-Crick base pairing properties are known to those of skill in the art and are also suitable for use in the polynucleotides of the invention.

[0140] AGAAGCCAAAGGAGACCTGA (sequence number 1) (W-704).

[0141] AAAGAGAAGCCAAAGGAGAC (sequence number 2) (W-705).

[0142] CTAGACCGGAAAAGAGAAGCCA (sequence number 3) (W-706).

[0143] ATGCTAGACCGGAAAAGAGAA (sequence number 4) (W-707).

[0144] CAATGCTAGACCGGAAAAGA (sequence number 5) (W-708).

[0145] AAGTCCCAATGCTAGACCGGA (sequence number 6) (W-709).

[0146] TCTCCGAAGTCCCAATGCTA (sequence number 7) (W-710).

[0147] GAGCTCTCCGAAGTCCCA (sequence number 8) (W-711).

[0148] AGAACAGTGGAGCTCTCCGA (sequence number 9) (W-712).

[0149] CGCCCAGAACAGTGGAGCTC (sequence number 10) (W-713).

[0150] CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714).

[0151] CAGTGGAGCTCTCCGAAGTCC (sequence number 43) (2831).

[0152] CCCAGAACAGTGGAGCTCTCC (sequence number 44) (2832).

[0153] CACAGCCCTCGCCCAGAACA (sequence number 45) (2833).

[0154] TTCTTCACAGCCCTCGCCCA (sequence number 46) (2834).

[0155] TCTTTCTTCACAGCCCTCGCCCAGAACAGTGGAGCTCTCCGAAGTCCCAATGCTAGACCGGAAAAGAGAAGCCAAAGGAGACCTGA (SEQ ID NO: 47).

[0156] TCTCCGAAGTCCCAATGCTAGACCGGAAAAGAGAAGCCAAAGGAGACCTGA (sequence number 48).

[0157] TCTTTCTTCACAGCCCTCGCCCAGAACAGTGGAGCTCTCCGAAGTCCCAATG (sequence number 49).

[0158] TTCTTCACAGCCCTCGCCCAGAACAGTGGAGCTCTCCGAAGTCCCA (sequence number 50).

[0159] In some embodiments, a polynucleotide (e.g., ASO) disclosed herein comprises a nucleotide sequence having at least 70% sequence identity, e.g., 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 43-46. In certain embodiments, a 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 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 any one of SEQ ID NOs: 10-11 and 43-46. In some embodiments, the polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100%, 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 any one of SEQ ID NOs: 10-11 and 43-46. In some embodiments, the polynucleotide (eg, ASO) comprises a nucleotide sequence identical to the sequence set forth in any one of SEQ ID NOs:10-11 and 43-46.

[0160] In some embodiments, the agents disclosed herein include a first polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:10, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, and a second polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:11, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. 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) has about 70-100% sequence identity to SEQ ID NO:10, e.g., 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. In some embodiments, the first polynucleotide (e.g., ASO) comprises a nucleotide sequence having about 70-100% sequence identity to SEQ ID NO: 11, e.g., 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 identical to SEQ ID NO: 10, and the second polynucleotide comprises a nucleotide sequence identical to SEQ ID NO: 11.

[0161] In some embodiments, a polynucleotide (e.g., ASO) disclosed herein comprises a nucleotide sequence having at least 70% sequence identity, e.g., 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, a 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, a 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%, e.g., 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 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 identical to a sequence set forth in any one of SEQ ID NOs: 51-69.

[0162] AGAAGCCAAAGGAGACCUGA (sequence number 51) (W-704).

[0163] AAAGAGAAGCCAAAGGAGAC (sequence number 52) (W-705).

[0164] CUAGACCGGAAAAGAGAAGCCA (sequence number 53) (W-706).

[0165] AUGCUAGACCGGAAAAGAGAA (sequence number 54) (W-707).

[0166] CAAUGCUAGACCGGAAAAGA (sequence number 55) (W-708).

[0167] AAGUCCCAAUGCUAGACCGGA (sequence number 56) (W-709).

[0168] UCUCCGAAGUCCCAAUGCUA (sequence number 57) (W-710).

[0169] GAGCUCUCCGAAGUCCCA (sequence number 58) (W-711).

[0170] AGAACAGUGGAGCUCUCCGA (sequence number 59) (W-712).

[0171] CGCCCAGAACAGUGGAGCUC (sequence number 60) (W-713).

[0172] CCUCGCCCAGAACAGUGGAG (sequence number 61) (W-714).

[0173] CAGUGGAGCUCUCCGAAGUCC (sequence number 62) (2831).

[0174] CCCAGAACAGUGGAGCUCUCC (sequence number 63) (2832).

[0175] CACAGCCCUCGCCCAGAACA (sequence number 64) (2833).

[0176] UUCUUCACAGCCCUCGCCCA (sequence number 65) (2834).

[0177] UCUUUCUUCACAGCCCUCGCCCAGAACAGUGGAGCUCUCCGAAGUCCCAAUGCUAGACCGGAAAAGAGAAGCCAAAGGAGACCUGA (SEQ ID NO: 66).

[0178] UCUCCGAAGUCCCAAUGCUAGACCGGAAAAGAGAAGCCAAAGGAGACCUGA (sequence number 67).

[0179] UCUUUCUUCACAGCCCUCGCCCAGAACAGUGGAGCUCUCCGAAGUCCCAAUG (sequence number 68).

[0180] UUCUUCACAGCCCUCGCCCAGAACAGUGGAGCUCUCCGAAGUCCCA (sequence number 69).

[0181] In some embodiments, the polynucleotides (e.g., ASOs) disclosed herein comprise a nucleotide sequence having at least 70% sequence identity, e.g., 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 polynucleotides (e.g., ASOs) comprise 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 polynucleotides (e.g., ASOs) comprise 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%, e.g., 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 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 identical to a sequence set forth in any one of SEQ ID NOs: 60-65.

[0182] In some embodiments, the agents disclosed herein include a first polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:60, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, and a second polynucleotide (e.g., ASO) comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:61, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. 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) has about 70-100% sequence identity to SEQ ID NO:60, e.g., 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, e.g., 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 identical to SEQ ID NO: 60 and the second polynucleotide comprises a nucleotide sequence identical to SEQ ID NO: 61.

[0183] In some embodiments, the polynucleotide (e.g., the ASO) comprises a nucleotide sequence that is at least about 70% identical to a sequence within the X chromosome region of 147,912,230 to 147,914,451 (e.g., 147,912,230 to 147,912,728 or 147,912,728 to 147,914,451), e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence within the X chromosome region of 147,912,230 to 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 the X chromosome region of 147,912,230 to 147,914,451 (e.g., 147,912,230 to 147,912,728 or 147,912,728 to 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 the X chromosome region of 147,912,230 to 147,914,451 (e.g., 147,912,230 to 147,912,728 or 147,912,728 to 147,914,451). In some embodiments, the polynucleotide comprises a nucleotide sequence having about 70% to 100%, e.g., about 75 to 100%, 75 to 99%, 80 to 100%, 80 to 98%, 85 to 100%, 85 to 97%, 90 to 100%, 90 to 96%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, or 99 to 100% sequence identity to a sequence within the X chromosome region of 147,912,230 to 147,914,451 (e.g., 147,912,230 to 147,912,728 or 147,912,728 to 147,914,451).

[0184] In some embodiments, the polynucleotide (e.g., ASO) is at least about 70% complementary to at least a portion of the FMR1 gene transcript, e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to at least a portion of the FMR1 gene transcript. In some embodiments, the polynucleotide is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to at least a portion of the FMR1 gene transcript. In some embodiments, the polynucleotide is about 70-100% complementary to at least a portion of the FMR1 gene transcript, e.g., 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% complementary to at least a portion of the FMR1 gene transcript.

[0185] In some embodiments, the polynucleotides disclosed herein have a length of at least about 8 nucleotides, e.g., at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 nucleotides. In some embodiments, the polynucleotide has a length of about 8 to 80 nucleotides, e.g., about 10 to 60, 10 to 40, 12 to 80, 12 to 60, 12 to 40, 12 to 38, 12 to 30, 13 to 38, 13 to 36, 14 to 36, 14 to 34, 15 to 80, 15 to 60, 15 to 40, 15 to 34, 15 to 32, 16 to 32, 16 to 30, 17 to 30, 17 to 28, 18 to 28, 18 to 26, 19 to 26, 19 to 24, 20 to 80, 20 to 60, 20 to 40, 20 to 30, 20 to 24, or 20 to 22 nucleotides. In some embodiments, the polynucleotide has a length of about 10 to 30 or 12 to 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.

[0186] In some embodiments, the polynucleotides disclosed herein have a length of at least about 12 nucleotides, e.g., at least about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the polynucleotides have a length of about 12-40 nucleotides, e.g., 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 polynucleotides have 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, the polynucleotide is an oligonucleotide. In some embodiments, the polynucleotide is about 18-22 nucleotides in length.

[0187] In some embodiments, the polynucleotides (e.g., oligonucleotides) disclosed herein are isolated polynucleotides. An "isolated polynucleotide" refers to a polynucleotide that is separated from other cellular components that are normally associated with a naturally occurring nucleotide polymer, 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.

[0188] Polynucleotides of the disclosure may be produced recombinantly or synthetically using methods, techniques, and reagents well known in the art, such as routine and well known molecular cloning and solid phase synthesis techniques, hi some embodiments, a polynucleotide of the disclosure is a recombinant polynucleotide.

[0189] In another aspect, the present disclosure provides a polynucleotide that can increase the expression of a functional FMR1 gene product. The polynucleotide is any one of the modified or unmodified polynucleotides disclosed herein. In some embodiments, the polynucleotide is any one of the modified polynucleotides disclosed herein.

[0190] Polynucleotide Modifications In some embodiments, a polynucleotide of the disclosure comprises one or more modified nucleotides, in some embodiments, the one or more modified nucleotides each independently comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.

[0191] Chemical modifications may be selected, for example, to increase the nuclease resistance of a polynucleotide (e.g., an oligonucleotide), to prevent RNase H cleavage of a polynucleotide (e.g., a complementary RNA strand), or to increase cellular uptake of the polynucleotide. For each of these purposes, a variety of compatible chemical modifications are available and will be familiar to those of skill in the art.

[0192] In some embodiments, each modification of the ribose group comprises 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'-O-(2-methoxyethyl) (MOE), 2'-O-alkyl, 2'-O-alkoxy, 2'-O-alkylamino, 2'-NH2, or constrained nucleotides, or combinations thereof.

[0193] In some embodiments, the substituted RNA analogs disclosed herein comprise a methoxyethyl group on the 2'OH.

[0194] In some embodiments, the constrained nucleotides include locked nucleic acids (LNA), ethyl constrained nucleotides, 2'-(S) constrained ethyl (S-cEt) nucleotides, constrained MOE, 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC), alpha-L-locked nucleic acid, and tricycloDNA, or combinations thereof.

[0195] In some embodiments, the modification of the ribose group comprises a 2'-O-(2-methoxyethyl) (MOE) modification. In some embodiments, all nucleotides of a polynucleotide (e.g., an oligonucleotide) comprise a 2'-O-(2-methoxyethyl) (MOE) modification.

[0196] In some embodiments, the modification of the ribose group comprises a tricycloDNA modification. In some embodiments, all nucleotides of the polynucleotide (e.g., the antisense oligonucleotide) comprise a tricycloDNA modification.

[0197] In some embodiments, the modification of the ribose group comprises a 2'-deoxy modification.

[0198] In some embodiments, each 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. In some embodiments, each modification of the phosphate group comprises a phosphoramidate.

[0199] In some embodiments, the modification of the phosphate group comprises a phosphorothioate modification. In some embodiments, all nucleotides of a polynucleotide (e.g., an oligonucleotide) comprise a phosphorothioate modification. In some embodiments, the polynucleotide is a phosphorothioate modified polynucleotide.

[0200] In some embodiments, the sugar phosphate backbone is replaced with a phosphorodiamidate morpholino (PMO) backbone, hi other embodiments, the sugar phosphate backbone is replaced with a peptide nucleic acid or other pseudopeptide backbone.

[0201] In some embodiments, each modification of the nucleobase is 2-thiouridine, 4-thiouridine, N 6 -methyl adenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidines, isoguanine, isocytosine, a halogenated aromatic group, or a combination thereof.

[0202] In some embodiments, the nucleobase group modification comprises a 5-methylcytosine modification.

[0203] In some embodiments, a polynucleotide comprises a mixture of modified nucleotides.

[0204] In some embodiments, the mixture of modified nucleotides comprises two or more modifications selected from the group consisting of 2'-O-methyl, 2'-deoxy, 2'-O-(2-methoxyethyl) (MOE), LNA, and tricyclo-DNA.

[0205] In some embodiments, the polynucleotide comprises no more than four contiguous 2'-deoxy modified nucleotides.

[0206] In some embodiments, the mixture of modified nucleotides comprises one or more 2'-O-methyl modified nucleotides and one or more LNA modified nucleotides.

[0207] In some embodiments, the mixture of modified nucleotides comprises one or more 2'-O-(2-methoxyethyl) (MOE) modified nucleotides and one or more LNA modified nucleotides.

[0208] In some embodiments, each ribose group of a polynucleotide (e.g., ASO) disclosed herein comprises 2'-O-(2-methoxyethyl) (MOE) and / or each phosphate group of the polynucleotide comprises phosphorothioate. In some embodiments, each ribose group of a polynucleotide (e.g., ASO) comprises 2'-O-(2-methoxyethyl) (MOE). In some embodiments, each phosphate group of a polynucleotide comprises phosphorothioate. In some embodiments, each ribose group of a polynucleotide (e.g., ASO) disclosed herein comprises 2'-O-(2-methoxyethyl) (MOE) and each phosphate group of the polynucleotide comprises phosphorothioate.

[0209] Polypeptides In some embodiments, the agents disclosed herein include polypeptides. The term "polypeptide" as used herein refers to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). A polypeptide may include any suitable L-amino acid and / or D-amino acid, such as common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statins), and unconventional amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). Amino, carboxyl, and / or other functional groups on a polypeptide may 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, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis," John Wiley and Sons, 1991. 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 hapten) using methods known in the art. A polypeptide can optionally contain one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclization modifications), N-methyl-α-amino group substitutions). In addition, a polypeptide can be an analog of a known and / or naturally occurring peptide, e.g., a peptide analog having conservative amino acid residue substitution(s).

[0210] In some embodiments, the polypeptides disclosed herein are isolated polypeptides. In some embodiments, the polypeptides disclosed herein are recombinant polypeptides.

[0211] In some embodiments, the polypeptide is an inhibitor (e.g., a direct or indirect inhibitor) of expression of an abnormal FMR1 gene product (e.g., FMR1-217 and / or its protein product). In some embodiments, the polypeptide is an activator (e.g., a direct or indirect activator) of expression of a normal FMR1 gene product (e.g., FMR1-205 and / or its protein product). In some embodiments, the polypeptide reduces expression of an abnormal FMR1 gene product (e.g., FMR1-217 and / or its protein product) and increases expression of a normal FMR1 gene product (e.g., FMR1-205 and / or its protein product).

[0212] In some embodiments, the polypeptides disclosed herein are immunoglobulin molecules. In some embodiments, the immunoglobulin molecules are antibodies. In some embodiments, the antibodies are antagonistic antibodies that bind to FMR1 transcripts or isoforms associated with fragile X-associated disorders (e.g., FXS). The antibodies can be of any species, such as rodent (e.g., mouse, rat, guinea pig) antibodies, primate (e.g., human) antibodies, or chimeric antibodies. In some embodiments, the antibodies are primatized (e.g., humanized). In some embodiments, the antibodies are polyclonal antibodies. In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibodies (e.g., monoclonal antibodies) are multispecific, e.g., bispecific, trispecific, or tetraspecific.

[0213] In some embodiments, the polypeptides disclosed herein are antigen-binding fragments of immunoglobulin molecules (e.g., antibodies) that retain antigen-binding properties of the parent 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., diabodies, triabodies, or tetrabodies), scFv, SMIP, or rlgG.

[0214] In some embodiments, the polypeptides disclosed herein are antibody mimetics. The term "antibody mimic" refers to a polypeptide that can mimic the ability of an antibody to bind to an antigen, but is structurally distinct from the natural antibody structure. Examples of antibody mimetics include, but are not limited to, adnectins, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, nanobodies, nanoCLAMPs, and versabodies.

[0215] Techniques, assays, and reagents for making and using therapeutic antibodies or antigen-binding fragments thereof against target antigens (e.g., FMR1 transcripts or isoforms associated with fragile X-associated disorders such as FXS) are known in the art. For example, see Therapeutic Monoclonal Antibodies: From Bench to Clinic (Zhiqiang An eds., 1st ed. 2009), Antibodies: A Laboratory Manual (Edward A. Greenfield eds., 2d ed. 2013), Ferrara et al., Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker, PLoS ONE 7(11):e49535 (2012) for techniques and methods for screening, making, purifying, storing, labeling, and characterizing antibodies.

[0216] Gene editing In some embodiments, the agents disclosed herein include a gene editing system. In some embodiments, the gene editing system produces a nucleotide deletion, a nucleotide substitution, a nucleotide addition, or a combination of the above in the FMR1 gene. In some embodiments, the gene editing system produces a partial or complete deletion in exon 2 of FMR1-217 (e.g., a pseudoexon at base pairs 147,911,919 to 147,914,451 of the human FMR1 gene).

[0217] 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.

[0218] In some embodiments, the gene editing system comprises a single Cas endonuclease or a polynucleotide encoding a single Cas endonuclease. In some embodiments, the single Cas endonuclease is Cas9, Cpf1, C2C1, or C2C3. In some embodiments, the single Cas endonuclease is Cas9 (e.g., from Streptococcus Pyogenes). In some embodiments, the single Cas endonuclease is Cpf1. In some embodiments, Cpf1 is AsCpf1 (from Acidaminococcus sp.) or LbCpf1 (from Lachnospiraceae sp.). The selection of nuclease and gRNA(s) is typically determined according to whether deletion, substitution, or addition of nucleotide(s) to the target sequence is desired.

[0219] In some embodiments, the type II Cas endonuclease is Cas9 (e.g., from Streptococcus pyogenes). In some embodiments, the modified Cas9 is a nickase Cas9, a dead Cas9 (dCas9), or an 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 (see, e.g., Ran et al., Cell 154:1380-89 (2013) to achieve more precise genome editing. Wild-type Cas9 generates a double-stranded break (DSB) at the specific DNA sequence targeted by the gRNA. Nickase Cas9 generates only a single-stranded break. dCas9 is catalytically inactive. In some embodiments, dCas9 is fused to a nuclease (e.g., FokI, which generates a DSB at the target sequence homologous to the two gRNAs). Various CRISPR / Cas9 plasmids are publicly available from the Addgene repository (Addgene, Cambridge, MA: addgene.org / crispr / ).

[0220] CRISPR technology for editing eukaryotic genes has been disclosed in U.S. Patent Application Publication Nos. 2016 / 0138008(A1) and 2015 / 0344912(A1), as well as U.S. Patent Nos. 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. Cpf1 endonuclease and corresponding guide RNA and PAM sites are disclosed in US Patent Application Publication No. 2016 / 0208243(A1). CRISPR technology for generating mtDNA dysfunction in the mitochondrial genome is disclosed in Jo et al., BioMed Res. Int. 2015:305716 (2015). Co-delivery of Cas9 and sgRNA with nanoparticles is disclosed in Mout et al., ACS Nano 11(3):2452-58 (2017).

[0221] In some embodiments, the agent comprises a small molecule. In some embodiments, the small molecule binds to a protein that can regulate splicing and / or expression of FMR1 or a fragment thereof. In some embodiments, the small molecule is an inhibitor (e.g., direct inhibitor, indirect inhibitor) of the target protein. In some embodiments, the small molecule is an activator (e.g., direct activator, and indirect activator) of the target protein. Non-limiting examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic, organometallic, or inorganic compounds.

[0222] subject The term "subject" refers to a mammalian subject, preferably a human, diagnosed with or suspected of having a fragile X-associated disorder (eg, FXS).

[0223] In some embodiments, the subject comprises a CGG repeat expansion of about 55 to about 200 repeats in the 5' untranslated region of the FMR1 gene. In some embodiments, the subject comprises a CGG repeat expansion of more than 200 repeats in the 5' untranslated region of the FMR1 gene. In some embodiments, the subject comprises a partially methylated CGG repeat expansion. In some embodiments, the subject comprises a fully methylated CGG repeat expansion. In some embodiments, the subject has increased levels of isoform 12 of FMR1, decreased levels of isoform 1 of FMR1, or a combination thereof.

[0224] In some embodiments, the subject has one X 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 and two Y chromosomes.

[0225] In some embodiments, the subject is a male human. In some embodiments, the subject is a female human.

[0226] In some embodiments, the subject is at least about 1 month old, e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 21 months old, 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 old. In some embodiments, the subject is at 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 old.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 old. In some embodiments, the subject is about 12-38 years old. In other embodiments, the subject is a fetus. In some embodiments, the subject is a neonatal subject.

[0227] In some embodiments, the subject is 18 years of age or older, e.g., 18 to under 40 years of age, 18 to under 45 years of age, 18 to under 50 years of age, 18 to under 55 years of age, 18 to under 60 years of age, 18 to under 65 years of age, 18 to under 70 years of age, 18 to under 75 years of age, 40 to under 75 years of age, 45 to under 75 years of age, 50 to under 75 years of age, 55 to under 75 years of age, 60 to under 75 years of age, 65 to under 75 years of age, 60 to under 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.

[0228] In some embodiments, the subject is about 2-11, 4-17, 12-18, 18-50, 18-90, or 50-90 years old.

[0229] In some embodiments, the subject is a human. In some embodiments, the human subject has or is prone to have fragile X-associated disorder. In some embodiments, the human subject has or is prone to have FXS, FXPOI, FXTAS, or a combination thereof. In some embodiments, the human subject has or is prone to have FXS. In some embodiments, the subject is a human (e.g., about 50 years of age or older) who has or is prone to have FXTAS.

[0230] In some embodiments, the subject has one or more of the physical and / or medical characteristics associated with fragile X-associated disorders (e.g., FXS). Non-limiting examples of physical characteristics associated with FXS include long face, prominent ears and chin, arched palate, large testes at puberty, low muscle tone, flat feet, and hyperextensible joints. Non-limiting examples of medical or behavioral characteristics associated with FXS include sleep disorders, seizures, recurrent ear infections, mitral valve drop, hyperactive behavior, short attention span, hand biting or hand flapping, poor eye contact and social skills, shyness, anxiety, autism, epilepsy, aggression, delayed language and / or motor development, repetitive speech, sensitivity to sensory stimuli (including hypersensitivity to touch, light, or 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.

[0231] In some embodiments, the subject has one or more of the following: irregular menstruation, infertility problems, elevated FSH (follicle stimulating hormone) levels, premature ovarian failure, primary ovarian insufficiency, and vasomotor symptoms (e.g., "hot flashes"). In some embodiments, the subject has one or more of the following: intention tremor, parkinsonism, ataxia, memory loss, white matter lesions involving the middle cerebellar peduncle, and cognitive decline.

[0232] treatment "Treate," "treating," or "treatment" refers to therapeutic procedures in which the purpose is to slow (alleviate) undesirable physiological changes or diseases, such as the onset or progression of 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, whether detectable or undetectable, reduction in the extent of disease, a stable (i.e., non-worsening) state of disease, a delay or slowing of disease progression, or an improvement or remission of the condition.

[0233] In some embodiments, the method further comprises evaluating the efficacy (an outcome measure) of an agent (e.g., a polynucleotide such as an ASO) for the treatment of 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 FMR1 RNA isoform 1, FMR1 RNA isoform 12, or a combination thereof.

[0234] In some embodiments, treating a fragile X-associated disorder (e.g., FXS) includes slowing the progression of a fragile X-associated disorder (e.g., FXS), alleviating one or more signs or symptoms of a fragile X-associated disorder (e.g., FXS), preventing one or more signs or symptoms of a fragile X-associated disorder (e.g., FXS), or a combination thereof.

[0235] Non-limiting examples of treatment benefits include improved language and motor development; reduction or prevention of cognitive impairments ranging from learning disabilities to intellectual disability; reduction or prevention of physical and medical characteristics such as long face, prominent ears and chin, arched palate, large testes during puberty, low muscle tone, flat feet, hyperextensible joints, sleep disorders, seizures, recurrent ear infections, and mitral valve drop; reduction or prevention of behaviors such as hyperactive behavior, short attention span, hand biting or hand flapping, poor eye contact and social skills, shyness, anxiety, delayed language and / or motor development, repetitive speech, and sensitivity to sensory stimuli (including hypersensitivity to touch).

[0236] In some embodiments, treatment may include modulation or improvement of language, fragile X behavior, brain activity, clinical impression, inattention, safety, social avoidance, cognition, hyperactive behavior, executive function, irritability, eye contact, or memory.

[0237] In some embodiments, the treatment results in an intelligence quotient (IQ) score of at least about 40, e.g., 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, the treatment results in an IQ score of about 40-110, 40-100, 50-105, 60-80, 65-90, 70-80, 75-95, or 70-100. In some embodiments, the 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, the 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, the treatment results in an increase in IQ score of about 1-10, 1-15, 2-20, 2-15, 2-10, 5-15, 5-10, 10-20, or 15-20 points. In some embodiments, the 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.

[0238] In yet other embodiments, treatment may include reducing or preventing absent or irregular menstruation, infertility problems, elevated FSH (follicle stimulating hormone) levels, premature ovarian failure, primary ovarian insufficiency, and / or hot flashes. In yet another embodiment, treatment may include reducing or preventing intention tremor, parkinsonism, ataxia, memory loss, white matter lesions involving the middle cerebellar peduncle, and / or cognitive decline. In some embodiments, treatment may reduce or prevent problems with autonomic function, such as limb neuropathy, mood instability, irritability, explosive emotional outbursts, personality changes, impotence, loss of bladder or bowel function. Treatment may also include reducing or preventing high blood pressure, thyroid disorders, or fibromyalgia.

[0239] Formulation and Administration A "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" can vary depending on factors such as the medical condition, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual.

[0240] In some embodiments, the agent (e.g., ASO) disclosed herein is in the form of a pharmaceutical composition, or a pharma- ceutical 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 a subject, e.g., a human. In some embodiments, a pharmaceutical composition may include one or more pharma- ceutical acceptable excipients, diluents, or carriers. A "pharma- ceutical acceptable carrier, diluent, or excipient" includes any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the United States Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0241] In some embodiments, the pharmaceutical compositions disclosed herein are formulated as a solution.

[0242] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some embodiments, a carrier can be a diluent, adjuvant, excipient, or vehicle administered with a drug (e.g., a polynucleotide). Such vehicles can 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 can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions can contain pharma-ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and coloring agents. The concentration of the drug in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5% by weight to at least about 1% by weight, or even up to 15% or 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight. The concentration is selected primarily based on the required dose, fluid volume, viscosity, etc., according to the mode of administration. Suitable vehicles and formulations, including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing: 691-1092 (e.g., pages 958-89).

[0243] In some embodiments, pharmaceutical compositions suitable for use in the methods disclosed herein further comprise one or more pharma- ceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is non-toxic to a subject and should not interfere with the effectiveness of the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, those commonly used in the field of drug manufacturing. A carrier can be a diluent, adjuvant, excipient, or vehicle with which a drug is administered. Such vehicles can 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 can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The composition may contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickening agents, lubricants, and coloring agents. The concentration of the drug in such pharmaceutical formulations may vary widely, for example, from less than about 0.5% by weight, usually at least about 1% by weight, up to 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight. The concentration is selected primarily based on the required dose, fluid volume, viscosity, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, Pa 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see in particular pp. 958-89.

[0244] Non-limiting examples of pharma- ceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., such as salts, buffers, antioxidants, sugars, aqueous or nonaqueous carriers, preservatives, wetting agents, surfactants, or emulsifying agents, or combinations thereof.

[0245] Non-limiting examples of buffers that can be used are acetate, citrate, formate, succinate, phosphate, carbonate, malate, aspartate, histidine, borate, Tris buffer, HEPPSO, and HEPES.

[0246] Non-limiting examples of antioxidants that can be used are ascorbic acid, methionine, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, lecithin, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, and tartaric acid.

[0247] Non-limiting examples of amino acids that can be used are histidine, isoleucine, methionine, glycine, arginine, lysine, L-leucine, tri-leucine, alanine, glutamic acid, L-threonine, and 2-phenylamine.

[0248] Non-limiting examples of surfactants that may be used include polysorbates (e.g., polysorbate-20 or polysorbate-80); poloxamers (e.g., poloxamer 188); triton; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine. lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauramidopropyl); myristamidopropyl dimethylamine; palmidopropyl dimethylamine; or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and the MONAQUA™ series (Mona Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl col, and copolymers of ethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.).

[0249] Non-limiting examples of preservatives that may be used are phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof.

[0250] Non-limiting examples of sugars that may be used are monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars such as glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, syritol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactose, maltulose, glucitol, maltitol, lactitol, or isomaltulose.

[0251] Non-limiting examples of salts that can be used are acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorus acid, and those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and those derived from non-toxic 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).

[0252] The agents (e.g., polynucleotides) disclosed herein can be prepared in accordance with standard procedures and administered in dosages selected to reduce, prevent, or eliminate the condition being treated, or to slow or halt its progression (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, NY, the contents of which are incorporated herein by reference for a general description of methods for administering various agents for human therapy).

[0253] In some embodiments, the agents disclosed herein (e.g., ASOs) are delivered using controlled release or sustained release delivery systems (e.g., capsules, biodegradable matrices). Exemplary delayed release delivery systems for drug delivery that may be suitable for administration of the compositions described herein are described in U.S. Patent Nos. 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.

[0254] For oral administration, the polynucleotide may be in the form of, for example, a tablet, capsule, suspension, or liquid. The polynucleotide is preferably made in the form of a dosage unit containing a therapeutically effective amount of active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, tablets and capsules may contain, in addition to the active ingredient, conventional carriers such as binders, for example, gum acacia, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, corn starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica, or talc; disintegrants, for example, potato starch, flavorings or colorings, or acceptable wetting agents. In general, oral liquid preparations 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, colorings, and flavorings. Examples of excipients for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methylcellulose, methyl or propyl parahydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.

[0255] Administration of an agent to a subject may be by parenteral or non-parenteral means. In some embodiments, an agent (e.g., ASO) disclosed herein is administered intravenously, intraarterially, intrathecally, intracerebroventricularly, intramuscularly, intradermally, subcutaneously, intracranially, or spinally. As used herein, "administering" or "administration" refers to taking steps to deliver an agent to a subject, such as a mammal in need thereof. Administration may be performed, for example, once, multiple times, and / or over one or more extended periods of time. Administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug or instructing a subject to take a drug. For example, as used herein, a person (e.g., a physician) who instructs a subject (e.g., a patient) to self-administer an agent (e.g., a drug) or to be administered by another person and / or who provides a prescription for a drug to a patient administers the agent to the subject. Administration of an agent may be once per day or more than once per day (e.g., more than once per day). Administration of the agent may be repeated after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, or more. As with chronic administration, repeated courses of treatment are also possible. Repeated administration may be at the same dose or at different doses.

[0256] In some embodiments, the agents disclosed herein (e.g., polynucleotides such as ASOs) are delivered locally to the central nervous system. This may include intrathecal or intraventricular injection, including using a catheter or Ommaya reservoir. Other methods of delivering agents (e.g., drugs) directly to the cerebrospinal fluid or central nervous system are known to those skilled in the art.

[0257] In some embodiments, the agent disclosed herein (e.g., a polynucleotide such as an ASO) is administered as an intrathecal bolus injection. In some embodiments, the agent (e.g., a polynucleotide such as an ASO) is administered at a dosage of about 4-20 mg per administration, e.g., 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., a polynucleotide such as an ASO) is administered at a dosage of about 12 mg per administration. In some embodiments, the agent (e.g., a polynucleotide such as an ASO) is administered at a dosage of about, e.g., up to 50 or 100 mg per injection.

[0258] In some embodiments, the agents disclosed herein (e.g., polynucleotides such as ASOs) are delivered systemically, such as via intravenous or subcutaneous injection. In some embodiments, the agents (e.g., polynucleotides such as ASOs) are delivered using approaches that enhance bioavailability in the central nervous system after systemic administration. These approaches may include modification of sugar or phosphate linkages, delivery as duplexes with ligand-conjugated RNA molecules, formulation into artificial exosomes, liposomes, polymeric or lipid nanoparticles, or conjugation to lipids, antibodies, peptides, sugars, neuroactive molecules, or other moieties that enhance delivery to the central nervous system. In some embodiments, the agents (e.g., polynucleotides such as ASOs) are 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 those skilled in the art.

[0259] In some embodiments, the methods disclosed herein include administering to a subject two or more polynucleotides, e.g., 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.

[0260] In some embodiments, the first polynucleotide (e.g., ASO) disclosed herein comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-11, 43-46, and 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 any one of SEQ ID NOs: 1-11, 43-46, and 51-65. In some embodiments, the first polynucleotide comprises a nucleotide sequence having any one of SEQ ID NOs: 1-11, 43-46, and 51-65.

[0261] In some embodiments, the second polynucleotide (e.g., ASO) disclosed herein comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-11, 43-46, and 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 any one of SEQ ID NOs: 1-11, 43-46, and 51-65. In some embodiments, the second polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-11, 43-46, and 51-65.

[0262] In some embodiments, the methods disclosed herein include administering to a subject a third, fourth, or fifth polynucleotide (e.g., an ASO) comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-11, 43-46, and 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 any one of SEQ ID NOs: 1-11, 43-46, and 51-65. In yet other embodiments, the third, fourth, or fifth polynucleotide comprises a nucleotide sequence having any one of SEQ ID NOs: 1-11, 43-46, and 51-65.

[0263] In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:1, an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:2, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:6, an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:7, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:10, an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:11, or both.

[0264] In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:51, an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:52, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:56, an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:57, or both. In some embodiments, the method comprises administering to the subject an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:60, an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO:61, or both.

[0265] In some embodiments, it may be advantageous to administer an agent of the present disclosure (e.g., a polynucleotide such as an antisense oligonucleotide, a pharmaceutical composition thereof, or a pharma- ceutical acceptable salt of the foregoing) in combination with one or more additional therapeutic agents. For example, it may be advantageous to administer a compound of the present disclosure (e.g., an antisense oligonucleotide, a pharmaceutical composition thereof, or a pharma- ceutically acceptable salt thereof) in combination with one or more additional therapeutic agents, such as a modulator of DNA methylation (e.g., an agent that inhibits DNA methylation or promotes DNA demethylation, see, e.g., the "DNA demethylation" section), a metabotropic glutamate receptor 5 (mGluR5) modulator (e.g., basignant or mavoglurant), a GABAB receptor activator (e.g., arbaclofen), a GABAA or GABAB receptor activator (e.g., acamprosate), an AMPAkine (e.g., AX516), a CB1 inhibitor (e.g., rimonabant), a RAS signaling inhibitor (e.g., lovastatin), a STEP inhibitor, an S6K inhibitor, a PAK inhibitor (e.g., FRAX486), an MMP9 inhibitor (e.g., minocycline), and a GSK3β inhibitor (e.g., lithium). In some embodiments, treating the subject comprises providing the subject with a ketogenic ("keto") diet.

[0266] 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 includes co-administration of the therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration in multiple or separate containers (e.g., capsules, powders, and liquids) for each active ingredient. Such administration also includes the use of each type of therapeutic agent sequentially, either at about the same time or at different times. The therapeutic agents in the combination therapy may be administered via the same route of administration or via different routes of administration. The powders and / or liquids may be reconstituted or diluted to the desired dose before administration. Typically, the treatment regimen provides a beneficial effect of the drug combination in the treatment of a disease, condition, or disorder described herein.

[0267] In some embodiments, the methods of treatment disclosed herein further comprise administering to the subject a therapeutically effective amount of a DNA demethylating compound or a DNA demethylase enzyme before, during, or after administering an agent disclosed herein (e.g., a polynucleotide such as an ASO). In some embodiments, the methods of treatment further comprise administering to the subject a therapeutically effective amount of a DNA demethylating compound or a DNA demethylase enzyme after administering an agent disclosed herein (e.g., a polynucleotide such as an ASO).

[0268] Non-limiting examples of DNA demethylating compounds include 5-azacytidine (5-aza-CR) and 5-aza-2'-deoxycytidine (5-aza-CdR), dihydro-5-azacytidine (DHAC), zebularine, 5-fluoro-2'-deoxycytidine, hydralazine, RG108, procainamide, and SGI-1027. In some embodiments, the DNA demethylating compound is a nucleoside analog. In some embodiments, the DNA demethylating compound is a non-nucleoside analog.

[0269] In some embodiments, a DNA demethylase (e.g., the DNA methylation modification enzyme Dnmt or Tet (dCas9-Dnmt / Tet)) is fused to a catalytically inactivated Cas9. Under the guidance of a single guide RNA (sgRNA), dCas9-Tet1 demethylates the FMR1 locus and promoter region when FMR1 has an expansion of 200 or more CGG repeats.

[0270] In some embodiments, the DNA demethylating compound or DNA demethylase is in an amount sufficient to demethylate at least about 5% of the FMR1 gene, e.g., 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 FMR1 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 the FMR1 gene. In some embodiments, the DNA demethylase is in an amount sufficient to demethylate 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 FMR1 gene. In some embodiments, the DNA demethylating compound or DNA demethylase is in an amount sufficient to demethylate about 25-50% of the FMR1 gene.

[0271] In some embodiments, the method of modulating splicing and / or expression of FMR1 further comprises contacting the cell with a DNA demethylating compound or a DNA demethylating enzyme before, during, or after contacting the cell with the agent (e.g., a polynucleotide).

[0272] In some embodiments, the therapeutic methods disclosed herein further comprise reducing (e.g., truncating or deleting) the FMR1 CGG expansion in the subject (e.g., by CRISPR / Cas9 gene editing) before, during, or after administering an agent (e.g., a polynucleotide such as an ASO) disclosed herein. In some embodiments, the therapeutic methods further comprise reducing (e.g., truncating or deleting) the FMR1 CGG expansion before administering an agent (e.g., a polynucleotide such as an ASO) disclosed herein.

[0273] Methods for modulating splicing and / or expression of FMR1 In another aspect, the disclosure provides a method of modulating splicing and / or expression of FMR1 in a cell, comprising contacting the cell with an agent (e.g., a polynucleotide) under conditions where the agent is introduced into the cell, thereby modulating splicing and / or expression of FMR1 in the cell. The agent can be any one of the agents disclosed herein.

[0274] 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 FMR1 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 FMR1 gene, decreases splicing of isoform 12 (between X chromosome base pairs 147,912,230 and 147,912,728), or a combination thereof.

[0275] 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 FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases splicing of FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference. 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 FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference.

[0276] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces splicing and / or expression of FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide) reduces splicing of FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces expression of FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference.

[0277] In some embodiments, an 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 FMR1, decreases splicing and / or expression of isoform 12 of FMR1, or a combination thereof. "Isoform 1" or "iso1" refers to normal FMR1 RNA with exon 1 spliced ​​to exon 2. "Isoform 12" or "iso12" refers to a missplicing of FMR1 RNA, where exon 1 is spliced ​​to a pseudoexon located within intron 1. Isoform 12 would generate a 31 amino acid protein that likely has no biological function.

[0278] In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) increases isoform 1 of FMR1 by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide) increases isoform 1 of the FMR1 gene by about 75%.

[0279] In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces isoform 12 of FMR1 by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces isoform 12 of the FMR1 gene by about 30%.

[0280] In some embodiments, the level of splicing and / or expression of FMR1 or a fragment thereof is measured after the agent has contacted the cells 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.

[0281] 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.

[0282] In some embodiments, the agent comprises at least one of the polynucleotides of the present disclosure. In some embodiments, the agent comprises two or more of the polynucleotides of the present disclosure.

[0283] In some embodiments, the cell is a fetal cell (e.g., a circulating fetal cell), a blastomere, a trophectoderm cell, a stem cell (e.g., an induced pluripotent stem cell (iPSC) or an induced stem cell), a fibroblast, a modified fibroblast, a pluripotent cell, or a cultured cell.

[0284] 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, a primary human cell, or a cell line from a human having or prone to having FXS. In some embodiments, the cell is a cell of any one of the subjects disclosed herein. In some embodiments, the subject's cells are allogeneic. In some embodiments, the subject's cells are autologous or syngeneic.

[0285] Methods for reducing CGG triplet repeat expansion in the FMR1 5'UTR In another aspect, the disclosure provides a method of reducing a CGG triplet repeat expansion in the FMR1 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 in which the agent is introduced into the cell, thereby reducing a CGG triplet repeat expansion in the cell. The agent can be any one of the agents disclosed herein.

[0286] 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 FMR1 gene, increases splicing of isoform 1 (between base pairs 147,912,230 and 147,921,933 on the X chromosome), decreases expression of isoform 12 of the FMR1 gene, decreases splicing of isoform 12 (between base pairs 147,912,230 and 147,912,728 on the X chromosome), or a combination thereof.

[0287] In some embodiments, the agent (e.g., a polynucleotide disclosed herein, an agent that modulates DNA methylation, or a combination thereof) increases splicing and / or expression of FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) increases splicing of FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference. 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 FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference.

[0288] In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces splicing and / or expression of FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces splicing of FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide of the disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces expression of FMR1 or a fragment thereof by at least about 5% relative to a reference, e.g., 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 a reference.

[0289] In some embodiments, an 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 FMR1, decreases splicing and / or expression of isoform 12 of FMR1, or a combination thereof. "Isoform 1" or "iso1" refers to normal FMR1 RNA with exon 1 spliced ​​to exon 2. "Isoform 12" or "iso12" refers to a missplicing of FMR1 RNA, where exon 1 is spliced ​​to a pseudoexon located within intron 1. Isoform 12 would generate a 31 amino acid protein that likely has no biological function.

[0290] In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) increases isoform 1 of FMR1 by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) increases isoform 1 of the FMR1 gene by about 75%.

[0291] In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces isoform 12 of FMR1 by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces isoform 12 of the FMR1 gene by about 30%.

[0292] In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces the CGG triplet repeat expansion in the FMR1 5'UTR in a cell by at least about 5% relative to a reference, e.g., 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 a reference. In some embodiments, the agent (e.g., a polynucleotide of the present disclosure, an agent that modulates DNA methylation, or a combination thereof) reduces the CGG triplet repeat expansion in the FMR1 5'UTR in a cell by at least about 10% relative to a reference.

[0293] In some embodiments, the level of CGG triplet repeats in the FMR1 5'UTR in the cells is measured after the agent has contacted the cells 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.

[0294] 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.

[0295] 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.

[0296] In some embodiments, the cell is a fetal cell (e.g., a circulating fetal cell), a blastomere, a trophectoderm cell, a stem cell (e.g., an induced pluripotent stem cell (iPSC) or an induced stem cell), a fibroblast, a modified fibroblast, a pluripotent cell, or a cultured cell.

[0297] 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, a primary human cell, or a cell line from a human having or prone to having FXS. In some embodiments, the cell is a cell of any one of the subjects disclosed herein. In some embodiments, the subject's cells are allogeneic. In some embodiments, the subject's cells are autologous or syngeneic.

[0298] In another aspect, the present disclosure provides a polynucleotide that can reduce the expression of an abnormal FMR1 gene product. The polynucleotide is any one of the modified or unmodified polynucleotides disclosed herein. In some embodiments, the polynucleotide is any one of the modified polynucleotides disclosed herein.

[0299] In another aspect, the present disclosure provides an agent that modulates splicing and / or expression of the FMR1 gene. In some embodiments, the agent is a polynucleotide. In some embodiments, the agent is any one of the modified polynucleotides disclosed herein.

[0300] In yet another aspect, the disclosure provides a pharmaceutical composition comprising any one of the agents described herein and one or more pharma- ceutically acceptable excipients, diluents, or carriers.

[0301] Example Most FXS studies have focused on Fmr1 knockout (KO) mouse models. Shah et al. have shown that Fmr1 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)).

[0302] The new data show that missplicing in FMRP KO mice occurs in all brain regions and peripheral tissues examined, and therefore, because FMRP is likely present in all cells, missplicing probably also occurs in all cells.

[0303] Example 1. Method RNA extraction and sequencing RNA was extracted from patient leukocytes using the LeukoLOCK™ Total RNA Isolation System (AM1923, Thermo Fisher Scientific, Waltham, MA). 10 mL of fresh blood was collected from male FXS patients (N=10) and age-matched typically developing males (N=7) (controls) into anticoagulant-containing tubes, and RNA was extracted using the LeukoLOCK™ Fractionation and Stabilization Kit (AM1933, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Briefly, blood samples were passed through a LeukoLOCK™ filter, and the filter was washed using 3 mL of phosphate-buffered saline (PBS), followed by 3 mL of RNAlater® RNA stabilization solution (Thermo Fisher Scientific, Waltham, MA). Residual RNAlater® was drained from the LeukoLOCK™ filter, and the filter was capped and stored at -80°C.

[0304] To extract RNA, the filters were thawed at room temperature for 5 min, and then the remaining RNAlater® was removed. The filters were flushed with 4 ml of TRI Reagent, and the lysates were collected in 15 ml tubes. 800 μl of 1-bromo-3-chloropropane (BCP) was added to each tube and vortexed vigorously for 30 seconds. The tubes were then incubated at room temperature for 5 min. The aqueous phase was collected after centrifugation at approximately 2,000×g for 10 min at 4° C. To collect the long RNA fraction, 0.5 volumes of 100% ethanol were added and mixed well. The RNA was then collected using an RNA Cleaning and Concentrator Kit. DNase treatment was performed using Turbo™ DNase (Thermo Fisher Scientific, Waltham, MA), and the resulting RNA was resuspended in RNAse-free water and stored at −80° C. 1 μg of RNA was used for cDNA synthesis using the QuantiTect® Reverse Transcription Kit (Qiagen, Hilden, Germany) and qPCR was used to assess depletion of globin mRNA and confirm exclusion of red blood cells from the preparation. 3 μg of RNA samples were sent to Novogene (Beijing, China) for directional mRNA library preparation using polyA enrichment. Libraries were sequenced on the NovaSeq platform to generate paired-end, 150 bp reads.

[0305] RNA-Seq data analysis Fastq files were uploaded to the DolphinNext platform at the UMMS Bioinformatics Core for mapping and quantification (Yukselen et al., BMC Genomics 21(1):310(2020)). Reads were subjected to the fastqc pipeline to assess read quality. 9 nt molecular labels were trimmed from both 5' ends of paired-end reads and quality filtered with Trimmomatic (0.32). Reads that mapped to human rRNA were filtered out by Bowtie2 (2.1.0). Cleaned reads were then mapped to the Refseq (V38) human transcriptome and quantified by RSEM (1.2.11). Estimated counts for each gene were used for differential gene expression analysis by DESeq2 (1.16.1). After normalization by the median ratio method, only genes with a minimum count average of 5 across all samples were retained for differential gene expression analysis. An FDR (padj) cutoff of <5% was used. The generated TDF files were uploaded into the Integrative Genomics Viewer for visualization.

[0306] The ratio between reads that include or exclude an exon, also known as "percent spliced ​​in" (PSI), indicates how efficiently a sequence of interest is spliced ​​into a transcript. False discovery rate (FDR) is a way to conceptualize the rate of Type I error in null hypothesis testing when performing multiple comparisons.

[0307] Alternative splicing analysis RNA-seq data generated from leukocytes from male FXS patients (N=10) and age-matched typically developing males (N=7) were used to analyze alternative splicing (AS) using the rMATS package v3.2.5 (Shen et al., Proc Natl Acad Sci USA. 111(51):E5593-601(2014)) with default parameters. Percent splicing in (PSI) levels or exon inclusion levels were calculated by rMATS using a hierarchical framework. A likelihood ratio test was used to calculate differences in PSI between genotypes. AS events with FDR<5% and |delta PSI| ≥5% identified using rMATS were used for further analysis.

[0308] Primer sets for detecting FMR1 isoforms Iso1_1 forward (Iso1_1F): 5' AGAAGATGGAGGAGCTGGTG 3' (SEQ ID NO: 12)

[0309] Iso12_1 reverse (Iso12_1R): 5' CAGTGGAGCTCTCCGAAGTC 3' (SEQ ID NO: 13)

[0310] Iso12_2 forward 5' CCAGCAGTGCATTGAAGAAG 3' (SEQ ID NO: 14)

[0311] Iso12_2 reverse 5' CTGAAGCATGTGCATTCCTG 3' (SEQ ID NO: 15)

[0312] Iso1_1 forward (Iso1_1F): 5' AGAAGATGGAGGAGCTGGTG 3' (SEQ ID NO: 12)

[0313] Iso1_1 reverse (Iso1_1R): 5' TTCATGAACATCCTTTACAAATGC 3' (SEQ ID NO: 16)

[0314] Exon 1 forward (Exon 1F): 5' TAGCAGGGCTGAAGAGAA 3' (SEQ ID NO: 17)

[0315] Exon 1 reverse (Exon 1R): 5' CTTGTAGAAAGCGCCATTG 3' (SEQ ID NO: 18)

[0316] Detection of FMR1 isoforms Leukocyte lines derived from FXS patients that expressed iso12 were transfected with antisense oligonucleotide (ASO) pairs 705 / 705, 709 / 710, and 713 / 714. RNA was extracted 48 hours later and subjected to RT-qPCR to detect iso1 (primers Iso1_1 forward / Iso1_1 reverse) or total FMR1 isoforms (iso1+iso12) (primers exon 1 forward and exon 1 reverse) and iso12 (primers Iso1_1 forward / Iso12_1 reverse). Each assay was performed in triplicate and normalized to non-transfected cells.

[0317] cell culture Cell lines and treatments Lymphoblastoid cell lines (LCLs) were obtained from the Coriell Institute from two FXS individuals (GM07365 (FXS1), GM06897 (FXS2)) and two typically developing control males (GM07174 (WT3), GM06890 (WT4)). Cells were cultured in T25 flasks at 37°C with 5% CO2 in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 15% fetal bovine serum (FBS) and 2.5% L-glutamine.

[0318] Fibroblasts derived from patient skin samples were cultured in T25 culture flasks at 37°C with 5% CO2 in DMEM (15-017-CV) medium supplemented with 10% FBS and 1x antibiotic-antimitotic, 1x L-glutamine.

[0319] ASO Processing Antisense oligonucleotides (ASOs) were dissolved in ultrapure distilled water to a final concentration of 10 μM. Before use, ASOs were heated to 55° C. for 15 min and cooled at room temperature. ASOs were added individually or in combination to LCL cell lines at a final concentration of 80 nM using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, 13778030) and incubated in reduced serum medium at 5% CO2 and 37° C. for 16 h. RPMI1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 15% FBS was added for a total of 48 h. Cells were harvested 48 h after ASO treatment for RNA and protein extraction.

[0320] 5-AzaC treatment For each cell culture, 30 x 10 5 Cells / ml were added in a final volume of 20 ml medium (RPMI1640 medium (Sigma-Aldrich) supplemented with 15% FBS and 2.5% L-glutamine, at 37°C and 5% CO2) per T25 flask. 5-Aza-2'-deoxycytidine (5-AzaC) (Sigma-Aldrich, A3656) was added to the cell cultures (final concentration 1 μM) for 7 consecutive days. A 2 mM stock of 5-AzaC was made in DMSO. For each cell line, two independent treatments were performed (n=2). For untreated controls of each cell line, DMSO was added to the flask. For samples with both 5-AzaC and ASO treatment, 80 nM ASO or vehicle was added on day 1, and either 5-AzaC or DMSO was added daily from day 2 up to day 9 at a final concentration of 1 μM. On day 9, cells were harvested in 1× phosphate buffered saline and proceeded with RNA extraction or Western blot.

[0321] Western blot Cells were homogenized in RIPA buffer at 4°C, incubated on ice for 10 min and dissociated by pipetting. Extracts were centrifuged at 13,200 rpm at 4°C for 10 min and the supernatant was collected. Protein concentration was determined by BCA reagent. Proteins (10 μg) were diluted in SDS-bromophenol blue reducing buffer with 40 mM DTT and analyzed using Western blot on 10% SDS-PAGE gels with the following antibodies: FMRP (Abcam, 1:2000) and GAPDH (Cell signaling, 1:2000) diluted in 1X TBST with 5% non-fat milk. Membranes were washed 3 times for 10 min with 1X TBST and incubated with anti-rabbit or anti-mouse secondary antibodies (Jackson, 1:10000) for 1 h at room temperature. The membrane was washed 3 times for 10 min with 1×TBST, developed with ECL-Plus (Piece) and scanned on a GE Amersham Imager.

[0322] Example 2. FMR1 isoform 12 detected in a subpopulation of FXS patients FXS is caused by a CGG triplet repeat expansion in a single gene, FMR1, located on the X chromosome. When the CGG triplet expands to more than 200, the FMR1 gene becomes methylated and thereby transcriptionally inactivated. Loss of the FMR1 gene product, the protein FMRP, is responsible for the disorder.

[0323] Bioinformatic analysis showed that one-half of FXS patients expressed detectable levels of FMR1 RNA. This was unexpected given that all patients had >200 CGG repeats and were clinically diagnosed with fragile X syndrome. This detection of FMR1 RNA in one-half of FXS patients indicated that these individuals had incomplete DNA methylation of FMR1, as it is DNA methylation that silences genes. Figure 1 shows that there was robust expression of FMR1 in all seven typically developing (TD) individuals. There was also expression of FMR1 in FXS patients 1–5 (+FMR1), but no expression of FMR1 was detected in FXS patients 6–10 (-FMR1). Thus, 50% of FXS individuals express FMR1 RNA, likely due to incomplete methylation.

[0324] In fragile X syndrome patients who expressed FMR1 RNA, further bioinformatic analysis showed that the FMR1 RNA was mis-spliced; that is, instead of or in addition to proper FMR1 splicing, there was a little-known isoform resulting from mis-splicing. Normally, FMR1 exon 1 (chrX:147,911,919-147,912,230) is spliced ​​to FMR1 exon 2 (chrX:147,921,933-147,921,985), which produces "isoform 1" or "Iso1". However, within intron 1, there is a pseudoexon (chrX:147,912,728-147,914,451), and splicing between FMR1 exon 1 and this pseudoexon produces "isoform 12" or "Iso12". Figure 2 shows a close-up of FMR1 exon 1 and intron 1. Note that none of the typically developing individuals express isoform 12, whereas all five FXS patients who expressed FMR1 RNA (+FMR1) express FMR1 isoform 12.

[0325] Isoform 12 results from mis-splicing and is only detected when there is a CGG repeat expansion and when there is incomplete methylation. Isoform 12 does not produce full-length or functional FMRP. Instead, it produces a 30 amino acid protein that likely has no biological function.

[0326] These findings suggest that FMR1 RNA may be used not only to diagnose individuals with or prone to developing FXS, but also to stratify FXS individuals. The specific FMR1 RNA isoform 12 allows for the stratification of FXS individuals into two subpopulations: those that express isoform 12 and those that do not.

[0327] These findings further suggest that FMR1 RNA, such as isoform 12, may provide a novel therapeutic target for FXS. For example, reducing aberrant splicing to isoform 12 (i.e., normal FMR1 RNA with exon 1 spliced ​​to exon 2), alone or in combination with increasing proper splicing to isoform 1, may increase FMRP levels, thereby alleviating FXS in patients who express FMR1 RNA. In patients who do not express FMR1 RNA, it may be feasible to generate isoform 12 using a therapeutically effective amount of a DNA demethylating compound or DNA demethylating enzyme, which would ideally involve a targeted approach to partially demethylate the FMR1 gene without inducing generalized, widespread DNA demethylation.

[0328] Example 3. Reducing the production of isoform 12 and increasing the production of isoform 1 Figure 3 shows a non-limiting exemplary approach to block production of isoform 12, increase production of isoform 1, and increase FMRP levels using antisense oligonucleotides (ASOs). ASOs were designed to be complementary to regions within intron 1 and upstream of isoform 12, at the junction spanning intron 1 and isoform 12, or within isoform 12 (Table 1). Figure 4 shows a schematic of the relative locations of ASOs complementary within FMR1 iso1, iso12, and intron 1 (704, 705, and 706), the junction of intron 1 and iso12 (707, 708, 709, and 710), and iso12 (711, 712, 713, and 714).

[0329] ASO704-714 were chemically modified to increase the nuclease resistance of the ASO (e.g., reduce RNase H cleavage), increase cellular uptake, and enhance base-pairing ability (reduce off-target effects). The ribose group contained 2'-O-(2-methoxyethyl) (MOE) and the phosphate group contained phosphorothioate.

[0330] The ASOs of the present disclosure may be used alone or in combination. WBC lines derived from FXS patients that expressed iso12 were transfected with ASO704 / 705, 709 / 710, or 713 / 714. RNA was extracted 48 hours later and subjected to RT-qPCR to detect iso1 (primers Iso1_1 forward and Iso1_1 reverse) and iso12 (primers Iso1_1 forward and Iso12_1 reverse). Each assay was performed in triplicate. Figure 5 shows that ASO713 and 714, both of which are complementary to the internal region of iso12, reduced iso12 levels by about 30% and increased iso1 levels by about 75%. These data indicate that ASOs can be used to reduce the expression of isoform 12. More importantly, these data indicate that ASOs can be used to increase the expression of FMR1 isoform 1, which can then increase FMRP levels and alleviate FXS.

[0331] These data suggest that ASOs may be a potent and specific therapeutic agent for treating the subpopulation of the FXS population that expresses isoform 12. This finding further supports that agents such as ASOs against FMR1 isoform 12 may provide a novel therapeutic treatment for FXS by reducing inappropriate 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, as all other treatments for FXS induce only modest improvements at best. Furthermore, while all other therapies treat FXS patients as one large cohort, these studies have identified a specific subpopulation (the subpopulation that expresses isoform 12) that may be particularly amenable to therapeutic agents such as ASOs targeting iso12.

[0332] Example 4. Partial demethylation of FMR1 DNA The experiments exemplified in Example 3 have been and will be performed in cells with different methylation states.

[0333] Figure 6A shows RT-qPCR data from a fully methylated FXS cell line (FXS1, GM07365). The FMR1 locus in this cell line is silenced, and therefore FMR1 RNA (iso1 and iso12) and FMRP protein levels are very low compared to the FXS2 cell line, which has an unmethylated FMR1 gene. Treatment with the demethylating agent 5-AzaC resulted in demethylation of the FMR1 gene to allow expression of the FMR1 RNA isoform. The data show an increase in FMR1 iso12 upon 5-AzaC treatment (p<0.05) and partial rescue of FMR1 iso12 when 5-AzaC treatment is combined with ASO treatment (both antisense oligonucleotides 713 and 714 at 80 nM) (p<0.05). FIG. 6B shows an increase in FMR1 iso1 upon 5-AzaC treatment (p<0.05), and a further increase (p<0.05) when ASO treatment (80 nM of both antisense oligonucleotides 713 and 714) was combined with 5-AzaC treatment.

[0334] These data demonstrate that in fully methylated FXS cell lines, demethylation of the locus resulted in expression of both FMR1 RNA isoforms. However, when demethylation was combined with ASO against FMR1 isoform 12, an increase in FMR1 isoform 1 mRNA was found. Thus, the combination of demethylation and ASO treatment may be useful for FXS patients with a fully methylated FMR1 locus.

[0335] The top panel of Figure 7A shows western blot data of the dual FXS1 LCL cell line, showing an increase in FMRP after treatment with 1 μM 5-AzaC and ASO treatment (both antisense oligonucleotides 713 and 714 at 80 nM) when compared to samples treated with DMSO or 5-AzaC only. Mouse brain (hippocampal tissue) from wild-type and Fmr1 knockout mice was loaded as a control. FMRP protein from mouse tissue ran higher on the gel compared to human FMRP. The bottom panel represents GADPH protein levels used to normalize the amount of protein loaded in each sample. Figure 7B shows quantification of FMRP protein levels relative to GAPDH protein levels as seen in the western blot of Figure 7A.

[0336] These data show FMRP protein levels from samples analyzed for FMR1 RNA levels in Figures 6A-6B. In parallel with ASO treatment against FMR1 iso12, treatment of the FXS1 cell line (fully methylated FMR1 locus) with a demethylating agent (5-AzaC) resulted in a significant increase in FMRP protein levels compared to untreated FXS1 cells or cells treated with 5-AzaC alone. In comparison, the levels of FMRP protein expressed with this treatment combination were similar to those found in wild-type mouse brain tissue (see Figures 7A-7B).

[0337] FIG. 8A is a table showing CGG repeats in the FMR1 RNA 5'UTR from three healthy males and three premutation carrier males with FXS. Premutation carriers had between 55 and 200 CGG repeats in the 5'UTR of the FMR1 gene, with more than 200 CGG repeats leading to FXS and less than 55 CGG repeats usually present in healthy individuals. Premutation carriers have a propensity to develop FXTAS (Fragile X-associated Tremor / Ataxia Syndrome) after the age of 50. FIG. 8B shows RT-qPCR data showing the presence of similar FMR1 iso1 levels in fibroblasts from all six individuals, normalized to GAPDH RNA levels. FIG. 8C shows the presence of increased FMR1 iso12 levels in individual P1 compared to other premutation carriers and healthy control samples. All premutation carriers expressed similar FMR1 iso1 levels compared to healthy controls. However, only individual P1, which has a higher CGG repeat (140, see FIG. 8A), expressed FMR1 iso12.

[0338] These data indicate that FMR1 iso12 may be expressed in premutation carriers with a higher number of CGG repeats, and in some embodiments, ASO treatment in these individuals may be therapeutically beneficial by increasing FMRP protein levels.

[0339] Prophetic Examples In the first set of experiments, various ASOs, alone or in combination, are introduced into a human FXS WBC line that is partially methylated and therefore expresses some FMR1 RNA. Levels of FMR1 iso1, FMR1 iso12, and FMRP are assessed at various time points, e.g., about 24, 48, 72, 96, 120, 144, and 168 hours after transfection.

[0340] In a second set of experiments, human FXS WBC lines with complete methylation of FMR1 DNA and no expression of FMR1 RNA are incubated with various amounts of DNA demethylating agents, such as 5-aza-2-deoxycytidine (5-azadC) (Sigma A3656), to partially demethylate FMR1 DNA. Various ASOs are then introduced, alone or in combination, into DNA demethylase-treated cells. At various time points, such as about 24, 48, 72, 96, 120, 144, and 168 hours after transfection, the levels of FMR1 iso1, FMR1 iso12, and FMRP are evaluated.

[0341] In the third set of experiments, various ASOs are introduced into primary fibroblasts derived from partially methylated FXS patients, either alone or in combination. At various time points, for example, about 24, 48, 72, 96, 120, 144, and 168 hours after transfection, the levels of FMR1 iso1, FMR1 iso12, and FMRP are evaluated. In primary fibroblasts derived from patients with fully methylated FMR1 locus, cells are incubated with various amounts of DNA demethylating agent, for example, 5-aza-2-deoxycytidine (5-azadC) (Sigma A3656), to partially demethylate FMR1 DNA. Then, various ASOs are introduced into DNA demethylase-treated cells, either alone or in combination.

[0342] Example 5. Safety and efficacy in animal models. The safety and efficacy of ASO treatment will be determined in animal models. Neural progenitor cells derived from human FXS patients with partially methylated FMR1 and iso12 expression were transfected with NOD-scid IL2Rγ as described by Windrem et al., J Neurosci 34:16153-16161 (2014) and Liu et al., Cell 172:979-92 (2018). nullMouse pups were injected with modified ASOs such as those described above into the brain or via intraperitoneal injection (IP). RNA was extracted from the brain and human FMR1 iso1 and iso12 were quantified by RT-qPCR. This experiment determines the safety and efficacy of ASO therapy in inhibiting FMR1 iso12 production and promoting iso1 formation in animal models. FMRP in human neurons was assessed by immunocytochemistry. [Table 1]

[0343] Examples 6 to 10 Fragile X syndrome (FXS) is a neurodevelopmental disorder that causes a variety of disorders, including intellectual disability, language and developmental delay, social deficits, repetitive behaviors, attention deficits, and anxiety. Previous studies have shown that an expansion of more than 200 CGG triplets in the 5'UTR of fragile X messenger ribonucleoprotein 1 (FMR1) 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 more than 1000 mRNAs in mouse brain and human neurons, primarily through coding region association (1-3). Previous studies suggested that FMRP inhibits protein synthesis (4), but subsequent high-resolution methods have shown that FMRP not only promotes but also inhibits translation (5-8). One mechanism by which FMRP inhibits translation is by 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 Fmr1-deficient hippocampus, resulting in altered H3K36me3 chromatin landscape. H3K36me3 is present in gene bodies and affects alternative pre-mRNA splicing (12), and indeed, multiple mRNAs were misspliced ​​in Fmr1-deficient mouse hippocampus. Many of these missplicing events were also detected in human postmortem autism spectrum disorder (ASD) brain and blood tissue (14-18), indicating the convergence of FXS and ASD (11, 13).

[0344] Because mRNA missplicing was widespread in Fmr1-deficient mouse brains and individuals with FXS are often on the autism spectrum, we speculated that RNA missplicing might also be widespread in human FXS patient tissues (blood and brain). Therefore, we isolated leukocytes from freshly obtained blood from 29 FXS males and 13 typically developing (TD) age-matched males and performed RNA sequencing. This analysis revealed widespread and statistically robust misregulation of alternative splicing and RNA presence of over 1,000 mRNAs. We also found misregulation of RNA expression and processing in FXS postmortem brains.

[0345] Further analysis of the RNA-seq data unexpectedly revealed that FMR1 RNA was expressed in 21 of 29 FXS leukocyte samples, some nearly as high as FMR1 transcript levels from TD individuals. This was a surprising result, since all FXS samples were from individuals with more than 200 CGG repeats, and the FMR1 locus, which was assumed to be silent under these conditions, was transcriptionally active in patients even when the gene appeared fully methylated by standard assays. However, the FXS individuals expressing the highest FMR1 RNA were mosaic (either CGG repeat number mosaic or partial methylation of the full expansion). Furthermore, much of the FMR1 mRNA in FXS individuals was found to be itself misspliced ​​to generate a little-known 1.8 kb isoform, FMR1-217, composed of FMR1 exon 1 and a pseudoexon within FMR1 intron 1. This isoform is predicted to encode a truncated 31 amino acid polypeptide with unknown function, if any. Further analysis revealed that FMR1-217 was detected in FXS skin- and lung-derived fibroblasts and in five of seven FXS postmortem cortical samples, further demonstrating the predominance of FMR1 missplicing in the FXS population and, most importantly, that this altered processing event occurs in the brain and leukocytes. Fibroblasts from male carriers of several FXS premutations (i.e., approximately 55-200 CGG repeats) also expressed FMR1-217 and full-length FMR1 RNA, indicating that missplicing may be widespread in other disorders associated with CGG expansions in FMR1.

[0346] These findings suggest that modulation of FMR1 missplicing is a suitable approach to increase FMRP levels in individuals expressing FMR1-217. To investigate further, eleven 2'-O-methoxyethyl (MOE) / phosphorothioate-containing antisense oligonucleotides (ASOs) against several regions of FMR1-217 were generated and transfected into an established FXS lymphoblast cell line expressing this transcript. Single ASOs or a combination of two ASOs blocked inappropriate FMR1 splicing, rescued proper FMR1 splicing, and restored FMRP to TD levels. Furthermore, application of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC) to a second FXS lymphoblast cell line, as well as to an FXS fibroblast cell line that does not normally express any FMR1, resulted in the synthesis of both FMR1 and FMR1-217 RNA, but little or no FMRP. However, treatment of these cells with both 5-AzadC and ASO resulted in strong FMRP upregulation. These studies showed, first, that in cells from FXS but not TD individuals, a significant proportion of FMR1 RNA was misspliced ​​to produce the FMR1-217 isoform, and, second, that ASO treatment to reduce FMR1-217 levels restored FMRP to TD levels. Thus, ASO treatment may provide a novel therapeutic approach to alleviate FXS.

[0347] Aberrant alternative splicing of mRNA leads to dysregulation of gene expression in multiple neurological disorders. Strikingly, the fragile X messenger ribonucleoprotein 1 (FMR1) gene was transcribed in over 70% of FXS tissues, often even when the gene was fully methylated. In all FMR1-expressing FXS tissues, FMR1 RNA itself was misspliced ​​in a CGG expansion-dependent manner to generate the little-known FMR1-217 RNA isoform, which is composed of a pseudoexon within FMR1 exon 1 and intron 1. FMR1-217 was also expressed in skin fibroblasts and brain tissue from FXS premutation carriers. In cells abnormally expressing misspliced ​​FMR1, antisense oligonucleotide (ASO) treatment was shown to reduce FMR1-217, rescue full-length FMR1 RNA, and restore fragile X messenger ribonucleoprotein (FMRP) to normal levels. Notably, FMR1 gene reactivation in transcriptionally silent FXS cells using 5-aza-2'-deoxycytidine (5-AzadC), which prevented DNA methylation, increased FMR1-217 but not FMRP RNA levels. ASO treatment of cells prior to 5-AzadC application rescued full-length FMR1 expression and restored FMRP. These findings indicate that in FXS individuals (e.g., individuals expressing FMR1-217), ASO treatment may provide a new therapeutic approach to alleviate the disorder.

[0348] Example 6. Materials and Methods Human FXS participant study All participants were Caucasian males with FMR1 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 for the study. This 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), applying the z-deviation method to avoid floor effects in people with intellectual disabilities (53). Participants' adaptive skills were determined using semi-structured interviews and measured using the Vineland Adaptive Behavior Skills Inventory (Vineland-3, (54)). The Adaptive Behavior Composite (ABC) standard score (SS) was a measure of global adaptive functioning, based on scores assessing the following domains: communication, daily living skills, and socialization. FXS patients were aged 16-38 years with FXS phenotype, z-deviation IQ range of 20-52, and ABC standard score range of 20-41. Age-matched TD individuals in this study were aged 22-29 years with normal IQ and no known neuropsychiatric conditions. For determination of CGG repeat size in the 5'UTR of the FMR1 gene, DNA isolated from whole blood was analyzed using the Asuragen FMR1 AmplideX PCR kit. Methylation status was determined using the Asuragen FMR1 Methylation PCR kit and / or Southern blot analysis. FMRP levels were quantified by generating dried blood spots (DBS) from the samples. To generate DBS, a 12-50 μl spot was placed 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 samples were centrifuged and FMRP was quantified using a Luminex Microplex immunochemistry assay. FMRP levels were normalized to 1,000 WBC per sample. In addition, 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 PBMCs were lysed, total protein concentration was quantified 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 evaluated.

[0349] Frozen postmortem brain tissue was obtained from the Brain Repository at the University of California at Davis from FXS male individuals (N=6) and age-matched typically developing (TD) males (N=5).

[0350] RNA extraction and sequencing of tissue samples from FXS and TD individuals white blood cells Eight milliliters (ml) of fresh blood was collected from FXS male individuals (N=29) and age-matched typically developing (TD) males (N=13) in BD Vacutainer cell preparation tubes (CPT, sodium citrate-blue top tubes, Becton Dickinson #REF362761) and RNA was extracted using the LeukoLOCK™ Fractionation and Stabilization Kit (Ambion #1933) after white blood cell collection with a LeukoLOCK™ filter according to the manufacturer's instructions. Briefly, blood samples were passed through a LeukoLOCK™ filter and then rinsed with 3 ml of phosphate-buffered saline (PBS), followed by 3 ml of RNAlater™. Residual RNAlater™ was drained from the LeukoLOCK™ filter, and the filter was capped and stored at -80°C. To extract RNA, the filter was thawed at room temperature for 5 minutes and then the remaining few drops of RNAlater were removed. The filters were flushed with 4 ml of TRIzol™ LS Reagent (ThermoFisher Scientific #10296028) and the lysates were collected in 15 ml tubes. 800 μl of Bromo-3-chloropropane (BCP) (Sigma #B9673) was added to each tube and vortexed vigorously for 30 seconds. The tubes were then incubated at room temperature for 5 minutes and centrifuged at approximately 2,000×g, 4° C. for 10 minutes. The aqueous phase containing the RNA was collected. To collect the long RNA fraction, 0.5 volumes of 100% ethanol was added and mixed well. The RNA was then collected 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. RNA quality (RNA integrity number (RIN)>7.3) was assessed using a 5300 Fragment Analyzer instrument.Three milligrams (mg) of RNA samples were used for directional mRNA library preparation using polyA enrichment (Novogene Co), and 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.

[0351] Brain tissue Postmortem frozen cortical tissue 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 No. 15596026) and lysates were collected. Total RNA was extracted using BCP, collected as above, and stored at -80°C.

[0352] cDNA synthesis and qPCR One microgram (μg) of total RNA was diluted with oligo(dT) 20 cDNA was generated using the QuantiTect cDNA Synthesis Kit (Qiagen, no. 205311) primed with 1000 ng / ml of ...

[0353] RNA-Seq data analysis FASTQ files were uploaded to the DolphinNext platform (55) at the UMass Chan Medical School Bioinformatics Core for mapping and quantification. Reads were subjected to FastQC (v0.11.8) analysis to assess read quality. Reads were mapped to genome assembly GRCh38 (hg38) version 34 using the STAR (v2.5.3a) aligner. Gene and isoform expression levels were quantified by salmon v1.5.2.

[0354] Differential gene expression analysis: DESeq2 (v3.9) was used to obtain differentially expressed genes from the estimated count table. After normalization by the median ratio method, genes with a minimum of 5 count average across all samples were retained for differential gene expression analysis. P<0.0002 was used as the cutoff. The generated TDF files were uploaded and autoscaled into Integrated Genomics Viewer (2.6.2) for visualization.

[0355] Alternative splicing analysis: To analyze differential alternative splicing (AS), the rMATS package v3.2.5 (14) was used with default parameters. Percent splicing in (PSI) levels or exon inclusion levels were calculated by rMATS using a hierarchical framework. A likelihood ratio test was used to calculate the difference in PSI between genotypes. AS events with FDR < 5% and |delta PSI| ≥ 5% identified using rMATS were used for further analysis. Genes with significantly skipped exons were used for validation using RT-qPCR analysis. 1 μg of RNA was used to generate cDNA using the QuantiTect cDNA synthesis kit. Primers were designed to overlap the skipped / inclusive exon junctions and qPCR was performed using Bio-Rad SYBR reagents on a Quantstudio3 instrument.

[0356] cell culture Lymphoblastoid cell lines Lymphoblastoid cell lines (LCLs) were obtained from the Coriell Institute from two FXS individuals (GM07365 (FXS1), GM06897 (FXS2)) and two typically developing control males (GM07174 (WT3), GM06890 (WT4)). Cells were cultured in T25 flasks at 37°C with 5% CO2 in RPMI1640 medium (Sigma-Aldrich) supplemented with 15% fetal bovine serum (FBS) and 2.5% L-glutamine.

[0357] Fibroblasts Skin biopsies from participants were collected in 15cc tubes containing explant culture medium (DMEM with 5% gentamicin). The biopsies were then transferred from the explant medium using tweezers into a sterile tissue culture dish and dissected into approximately 6-7 pieces using sterile tweezers and scissors in a culture hood. 3-4 skin explants were held at the bottom of a T25 flask and 3ml of CHANG AMNIO culture medium was added. The flask was then incubated at 37°C with 5% CO2 for 10 days. After cells began to grow out of the skin explants, the culture medium was changed. After the cells had grown in 5-6 layers around the skin explant, the skin explant was removed from the culture flask and the fibroblasts were trypsinized and spread evenly in the flask. After overnight incubation with trypsin, the medium was changed. Fibroblast culture medium (complete medium (500 ml of DMEM (15-017-CV) containing 10% FBS and 1× antibiotic-antimitotic agent, 5 ml of 1× L-glutamine)) was added twice a week to cells in T25 culture flasks at 37° C. with 5% CO2.

[0358] Fibroblast cell lines were obtained from the Coriell Institute from two FXS individuals (GM05131, and GM07072). A control fibroblast cell line derived from a typically developing male skin sample 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.

[0359] ASO synthesis and processing ASO synthesis ASOs were synthesized on a Dr. Oligo48 synthesizer. 2'-O-Methoxyethyl (MOE)-modified phosphoramidites were coupled for 8 min. Oligonucleotides were deprotected in concentrated aqueous ammonia (30% in water) at 55°C for 16 h and characterized by liquid chromatography-mass spectrometry. Final desalting was performed by diafiltration (three water washes) in 3 kDa cut-off Amicon centrifugal filters.

[0360] ASO Processing Antisense oligonucleotides (ASOs) were dissolved in ultrapure distilled water to a final concentration of 10 μM. Before use, ASOs were heated to 55°C for 15 min and cooled at room temperature. ASOs were added individually or in combination 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 in reduced serum medium at 5% CO2 and 37°C for 16 h. RPMI1640 medium (Sigma-Aldrich) supplemented with 15% fetal bovine serum (FBS) was added for a total of 72 h. Cells were harvested 72 h after ASO treatment for RNA and protein extraction.

[0361] 5-AzadC treatment For each cell culture, 30 x 10 5 Cells / ml were added in a final volume of 20 ml medium (RPMI1640 medium (Sigma-Aldrich) supplemented with 15% fetal bovine serum (FBS) and 2.5% L-glutamine, at 37°C and 5% CO2) per T25 flask. 5-Aza-2'-deoxycytidine (5-AzadC) (Sigma-Aldrich, A3656) was added to the cell cultures (final concentration 1 μM) for 7 consecutive days. A 2 mM stock of 5-AzadC was made in DMSO. For each cell line, two independent treatments were performed (n=2). For untreated controls of each cell line, DMSO was added to the flasks. For samples with both 5-AzadC and ASO treatment, 80 nM or 160 nM ASO or vehicle was added on day 1, and either 5-AzadC or DMSO was added at a final concentration of 1 μM daily from day 2 up to day 9. On day 9, cells were harvested in 1× phosphate buffered saline and proceeded with RNA extraction or Western blot.

[0362] Western blot Cells were incubated on ice for 10 min and homogenized in RIPA buffer at 4°C with dissociation by pipetting. Extracts were centrifuged at 13,200 rpm for 10 min at 4°C and the supernatant was collected. Protein concentration was determined using BCA reagent. Protein (10 μg) was diluted in SDS-bromophenol blue reducing buffer with 40 mM DTT and analyzed using Western blot with the following antibodies: FMRP (Millipore, mAb2160, 1:1,000), FMRP (Abcam, ab17722, 1:1,000), and GAPDH (14C10, Cell Signaling Technology, mAb2118, 1:2,000) diluted in 1X TBST with 5% nonfat milk. The membrane was washed 3 times for 10 min with 1XTBST and incubated with anti-rabbit or anti-mouse secondary antibody (Jackson, 1:10,000) for 1 hour at room temperature. The membrane was washed 3 times for 10 min with 1XTBST, developed with ECL-Plus (Piece) and scanned with a GE Amersham Imager.

[0363] Quantification and statistical analysis All grouped data were presented as mean ± sem. All tests used to compare samples were described in the respective figure legends and corresponding text. When exact P values ​​were not given, they were represented as follows: *, p<0.05; **, p<0.01; ***, p<0.001; ****, P value<0.0001; ns, p>0.05.

[0364] Availability of data and code Codes and scripts used for quantitative analysis are written in Python or R and are available upon request. The datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) database under the following accession numbers: superseries GSE202179; subseries GSE202177 includes the raw data of RNA-seq and GSE202178 of the ChIP-Seq experiment.

[0365] Chromatin immunoprecipitation sequencing (ChIP-Seq) Eight milliliters of fresh blood was collected from FXS males (N=10) and age-matched typically developing males (N=7) individuals in BD Vacutainer CPT (cell preparation tubes with sodium citrate-blue top tubes, Becton Dickinson #REF362761). The tubes were gently inverted five times and the samples were centrifuged at 1,500-1,800 RCF for 25 minutes at room temperature. The tubes were then inverted to collect lymphocytes and other mononuclear cells that were resuspended in the upper liquid phase in a new 15 ml tube. The samples were centrifuged again at 300 RCF for 10 minutes to obtain a PBMC pellet. The PBMCs were washed with 1X Dulbecco's phosphate buffered saline (D-PBS) without calcium or magnesium (Invitrogen #14190-094). The PBMC pellet was resuspended in 250 μL of ice-cold D-PBS with protease inhibitors. FMRP levels in PBMCs were quantified using a Luminex Microplex immunochemical assay. Chromatin isolation and sequencing were performed as previously described (11). Briefly, cells were cross-linked with 1% formaldehyde and quenched with 150 mM glycine. Cells were lysed after centrifugation at 2,000 g for 10 min at 4°C. After homogenization, nuclei were harvested by centrifugation at 2,000 g for 5 min at 4°C. Nuclei were lysed by incubation on ice for 20 min in nuclear lysis buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 mM EGTA). 0.5% SDS was added and samples were sonicated in a Bioruptor® sonicator at high power setting (sonication: 30 s on, 90 s off) for 9 cycles of 15 min each at 4°C. Samples were centrifuged and diluted to adjust the SDS concentration to less than 0.1%. 10% of each sample was used as input. The remaining samples were split into two and incubated with coupled protein G Dynabeads overnight at 4°C with antibodies against H3K36me3 (Abcam ab9050, 5 μg per ChIP) or H3K4me3 (Active Motif-39159, 5 μg per ChIP). After IP, beads were washed and chromatin was reverse cross-linked overnight at 65°C. After RNase and proteinase K treatment, DNA was purified.ChIP-Seq libraries were prepared by performing the following steps: end repair using T4 DNA polymerase, A' base addition with Klenow polymerase, and Illumina adaptor ligation using T4 polynucleotide kinase (New England Biolabs (NEB)). Libraries were PCR amplified using multiplexed barcoded primers. Libraries were pooled in equimolar ratios, denatured, diluted, and sequenced on a Nextseq500 sequencer (Illumina) with the NextSeq 500 / 550 High Output Kit v2.5 (Illumina, 75 bp paired end run).

[0366] ChIP-Seq analysis For ChIP-seq data analysis, alignments were performed with Bowtie2 (2.1.0) using GRCh38 (hg38) version 34 genomes, duplicates were removed with Picard and TDF files in Genomics Viewer (IGV), and figures were generated using the ChIP-seq pipeline from DolphinNext (55). Broad peaks for H3K36me3 ChIP-Seq were called using MACS2 with broad peak parameters. Narrow peaks for H3K4me3 ChIP were called using MACS2 with narrow parameters. Heatmaps and profiles of gene distribution of H3K36me3 and H3K4me3 ChIP signals relative to input were plotted using deepTools2 (57). IGV tools (2.6.2) was used to visualize TDF files, and all tracks shown were normalized to total read coverage.

[0367] Example 7. FMR1 RNA is expressed and misspliced ​​in a subset of FXS individuals. The 200+ CGG repeat expansion in FMR1 induces gene methylation, transcriptional silencing, loss of FMRP, and FXS. It was therefore surprising that FMR1 RNA was detected in leukocytes from 21 of 29 FXS individuals, and in 4 individuals, the levels of all isoforms of this RNA were similar or higher than those in TD individuals (Table 2, FMR1 RNA TPM levels). When only full-length FMR1, encoding the 632 amino acid FMRP (FMR1-205), was examined (Figure 9H, Table 3), WBCs from 6 individuals had levels of this transcript similar to those in TD (Table 2). For comparison, levels of the FMR1 paralog FXR2 were similar in all individuals (Table 2). Visualization of RNA reads at the FMR1 locus using the Integrated Genome Viewer (IGV) revealed that exonic reads were detected at robust levels in TD individuals, and exonic reads were also detected in FXS individuals (Figures 9A-9B). FXS individuals 1-21 expressed relatively high FMR1 levels (0.6 transcripts per million (TPM)) compared to FXS individuals 22-29, who expressed low or undetectable FMR1 levels (L FMR1) (Table 2 and Figures 9A-9B). Notably, H-FMR1 FXS individuals showed strong RNA reads in intron 1 of FMR1 (bold black box in Figure 9A, expanded in Figure 9B). Notably, RNA reads in this intronic region were not detected in any of the TD individuals, even though FMR1 RNA was strongly expressed (Figures 9A-9B). The FMR1 locus expresses multiple alternatively spliced ​​RNA isoforms (Table 3). The RNA reads detected in FMR1 intron 1 correspond to the second exon of the FMR1-217 RNA isoform. FMR1-217 (ENST00000621447.1) is a 1.8 kb transcript composed of two exons and predicted to encode a 31 amino acid polypeptide (Table 3). Notably, the majority of total FMR1 RNA in FXS samples consisted of the aberrantly spliced ​​FMR1-217 transcript, which was absent in samples from TD individuals (Table 2).TPMs were obtained for all 14 FMR1 isoforms detected in TD and FXS patient samples (data not shown). RT-PCR was used to detect the FMR1-217 isoform in FXS leukocyte samples (reverse transcription primed with oligo T(20)) and the amplified product was sequenced using primers specific for the FMR1-217 exon-exon junction. Alignment of this sequence to FMR1 confirmed that the transcript was polyadenylated and a spliced ​​product of FMR1 exon 1 and FMR1-217 exon 2 (Figure 9C). [Table 2]

[0368] Table 2 shows normalized gene counts (transcripts per million, TPM) obtained from RNA-seq data analysis for total FMR1 (all isoforms), FMR1-205 (full-length, encoding the 632 amino acid FMRP), FMR1-217 (misspliced ​​RNA), and FXR2, a paralog of FMR1. [Table 3]

[0369] The proportion of full-length FMR1 RNA relative to FMR1-217 RNA in TD or FXS leukocytes was then assessed. In TD samples, 95% of the total FMR1 RNA (primers Ex1F and Ex1R) represented full-length molecules (primers Ex1F and Ex2R), whereas in H FMR1 samples, 75% of the total FMR1 RNA was full-length and 25% was FMR1-217 (primers Ex1F and 217R) (Figure 9C). In L FMR1 samples, both isoforms were barely detectable. Total FMR1 RNA levels in all samples were normalized to GAPDH RNA expression (* indicates P value < 0.05). Importantly, all FXS individuals in this study showed typical FXS symptoms, regardless of FMR1 expression, suggesting that functional FMRP may be absent or present in very low amounts even in patients with high FMR1 expression (FMRP protein levels were quantified for available samples (data not shown)).

[0370] We investigated whether the stratification of FXS individuals based on relatively high (H) or low (L) amounts of FMR1 (using a cutoff of 0.6 TPM, Table 2) was reflected in RNA alterations across the transcriptome. By reanalyzing the RNA-seq data of FXS leukocytes and comparing significant RNA alterations between these two groups, we found hundreds of aberrant splicing events tracked by the amount of this misspliced ​​transcript (Figure 9D and data not shown). We investigated whether parameters measured in WBCs correlated with IQ. Table 4 presents the determination of the methylation status of the FMR1 gene (by PCR), FMRP levels (ng / μg protein), number of CGG repeats, FMR1-217, full-length FMR1-205, all detected FMR1 isoforms, and IQ (Stanford-Binet test). [Table 4]

[0371] In Table 4, it is as follows: FMR1 gene methylation (MPCR): in percentage determined by PCR analysis; FMRP levels: ng / μg total protein; FMR1: all isoforms; IQ: Stanford-Binet; N / A: not available.

[0372] Table 5 shows the correlation coefficients for pairwise comparison of the above measurements. Methylation of the FMR1 gene is negatively correlated with expression of FMR1-217 and FMR1-205. More interesting is the moderate positive correlation between IQ and FMRP protein levels. Somewhat surprisingly, FMR1-205, which encodes the full-length FMRP, does not correlate with IQ. However, it is noted that while FMR1-205 encodes the complete 632 amino acid FMRP, other FMR1 isoforms with different abundances encode truncated FMRP proteins (Table 3). Without assuming functionality of the truncated FMRP protein, the canonical FMR1 isoform, FMR1-205, was used for further comparison. FMR1-217 correlates negatively with IQ, indicating the deleterious effect of this isoform. Figure 10 shows a three-dimensional comparison of all the above parameters. The inset shows that some FXS patients with a fully methylated FMR1 gene expressed FMR1 RNA and FMRP. Taken together, these results indicate several important findings. First, the FMR1 locus is frequently transcribed even when the FMR1 gene with a complete CGG expansion is fully methylated. Second, FMRP levels in WBCs are positively correlated with IQ. Third, the negative correlation of FMR1-217 with IQ suggests that the process of mis-splicing, the 31 amino acid polypeptide derived from FMR1-217, the FMR1-217 RNA itself, or a combination of them (e.g., all three) confers some toxic effects manifested in the brain (e.g., IQ). In any case, additional transcriptome-wide changes in FMR1-217 expression levels, as well as RNA processing events, will likely form the basis of molecular stratification of FXS individuals. [Table 5]

[0373] In Table 5, it is as follows: + / -0~0.1: no correlation, + / -0.1~0.29: weak correlation, + / -0.3~0.49: moderate correlation, + / -0.5~1: strong correlation.

[0374] Example 8. FMR1-217 is expressed in postmortem brains of human FXS and premutation carriers. To investigate whether FMR1-217 is expressed in FXS brains, we analyzed publicly available RNA-seq data (16) of postmortem frontal cortex tissue from FXS individuals (CGG repeats >200), FXS carriers (CGG repeats 55-200), and TD individuals (CGG repeats <55). FMR1 RNA (TPM) levels were highest in premutation carriers (Table 6). Interestingly, FXS sample UMB5746, which showed CGG repeat number mosaicism, showed high levels of FMR1 RNA (Table 6 and Figure 11A) and, to a lesser extent, FMRP (16). Analysis showed that this individual expressed FMR1-217, similar to FXS carrier UMB5212, who had fragile X-associated tremor / ataxia syndrome (FXTAS) (Table 6 and Figure 11A). None of the TD individuals had RNA reads corresponding to FMR1-217 (Table 6 and Figure 11A). Thus, FMR1-217 RNA may only be expressed in the brains of a subset of FXS individuals and premutation carriers. [Table 6]

[0375] Table 6 shows sample information for postmortem FXS frontal cortex, premutation FXS carriers and TD individuals (from (16)). RNA-seq datasets GSE107867 (NIH sample) and GSE117776 were reanalyzed for DGE and DAS. The TPM of FMR1 RNA in the samples is shown.

[0376] BLAST analysis showed that FMR1-217 aligned only with intron 1 of FMR1 and not with other regions of the genome. Additional data clearly demonstrated that FMR1-217 is derived from FMR1 and its synthesis depends on a CGG expansion in this gene. Vershkov et al. (17) used CRISPR / Cas9 to delete the CGG expansion from FMR1 in FXS iPSC-derived neural stem cells (NSCs). Additional FXS NSCs were incubated with 5-AzadC, a nucleoside analog that prevents DNA methylation. RNA sequencing was then performed from these samples and from FXS NSCs incubated with vehicle. The RNA-seq data from Vershkov et al. (17) was reanalyzed, some of which are presented in Figure 11B, and FMR1 transcript quantification (TPM) is presented in Table 7. RNA-seq reads corresponding to FMR1-217 were clearly evident in FXS-NSCs incubated with 5-AzadC, but not in other samples. Furthermore, CGG-edited cells were isogenic to unedited FXS NSCs, with no FMR1-217 reads and instead robust expression of full-length FMR1. Quantification of RNA-seq reads (TPM) showed strong total FMR1 and FMR1-205 expression in CGG-edited and 5-AzadC-treated cells, but not in vehicle-treated cells. More importantly, strong FMR1-217 expression was only observed in 5-AzadC-treated cells. Thus, FMR1-217 originates from the FMR1 locus and requires CGG expansion. [Table 7]

[0377] In a complementary study, Liu et al. (18) performed targeted FMR1 gene demethylation experiments by incubating FXS iPSCs and neurons derived from FXS iPSCs with FMR1 small guide RNA and catalytically inactive Cas9 fused to Tet1 demethylase sequences. Subsequent reanalysis of RNA-seq data is shown in Figure 11C, and FMR1 transcript quantification (TPM) is shown in Table 8. Their experimental paradigm showed that the FMR1-217 sequence was only evident when the gene was demethylated in FXS cells. Quantification of the relevant transcripts in Table 8 showed that strong FMR1 and FMR1-205 expression was detected in Tet1-treated samples (but, puzzlingly, FMR1-205 was absent in sample N1_Tet1), as well as FMR1-217 expression was detected in all Tet1-treated samples. Thus, these data again indicate that FMR1-217 originates from the FMR1 locus and requires CGG expansion. [Table 8]

[0378] To confirm the expression of FMR1-217 RNA in FXS brain tissue, frozen postmortem cortical samples were obtained from six FXS males and five age-matched typically developing (TD) males (UC Davis Health). Using RT-qPCR, FMR1 full-length RNA was found to be significantly reduced in FXS individuals compared to TD individuals. However, three or four of the six FXS individuals expressed various levels of FMR1 full-length RNA and FMR1-217 RNA (1031-09LZ, 1001-18DL, and 1033-08WS) (Figure 11D). A previous study of FXS sample 1031-09LZ noted similar expression of FMR1 RNA to that in TD individuals, despite the presence of a methylated, fully mutated FMR1 locus (19). However, no detectable FMRP was found in FXS brain sample 1031-09LZ (20). Also consistent with these studies, RNA-seq data from Tran et al. showed no FMR1 RNA in FXS tissue samples (1031-08GP and JS03) (Table 6 and Figure 11A), indicating the absence of FMRP (16).

[0379] FMR1-217 RNA was detected in only one of the two premutation carrier samples. To better understand the relationship of FMR1-217 FXS carrier tissues (55-200 CGG repeats), skin biopsies were obtained from three additional premutation carriers and three TD individuals (Figure 11E). Skin samples were cultured in vitro to generate fibroblast cell lines for RNA analysis. Interestingly, using RT-qPCR, FMR1-217 was detected in one premutation carrier (C172) with 140 CGG repeats, but not in samples with 77 or 98 CGG repeats (Figure 11E). There was no change in total FMR1 RNA levels between samples (Figure 11E). Thus, the generation of FMR1-217 may be associated with the number of CGG repeats in the FMR1 gene.

[0380] Example 9. FMR1-217 RNA is expressed in lymphoblast cell cultures derived from FXS individuals. DNA methylation of the CpG island upstream of the FMR1 gene promoter in FXS individuals (MFM, fully methylated) contributes to transcriptional silencing of the locus and loss of FMRP. FMR1 transcription can be reactivated by treatment with the nucleoside analog 5-AzadC (5-aza-2'-deoxycytidine), which inhibits DNA methylation (21,22). Therefore, we investigated whether reactivation of FMR1 transcription in cells derived from FXS individuals with a fully silenced and presumably fully methylated FMR1 locus would result in FMR1-217 expression. In these experiments, lymphoblastoid cell lines (LCLs) derived from an FXS individual with a fully methylated locus (MFM) that was transcriptionally inactive (FXS1, GM07365), an FXS individual with a possibly partially methylated locus (UFM) that expressed some FMR1 RNA (FXS2, GM06897), and two typically developing individuals (TD1, GM07174, and TD2, GM06890) were used (all samples were from the Coriell Institute, NJ, USA) (Figure 12A). Western blot analysis showed that trace levels of FMRP were detected in FXS2 cell lines, but not in FXS1 cell lines. FMRP was strongly expressed in TD1 and TD2 cells (the ratio of FMRP / GAPDH for TD2 is shown below the blot) (Figure 12A). Similar ratios of FMRP protein expression in these cell lines were obtained by Luminex Microplex immunochemical assay (FMRP levels in ng FMRP / μg total protein) (Figure 12A). Using RT-qPCR, FMR1-217 RNA was found to be expressed in FXS2 LCLs and comprised 56% of the total FMR1 RNA, compared to only 9% in TD cells (Figure 12B). It is noteworthy that total FMR1 RNA levels in FXS2 cells were similar to those in TD cells, whereas FMRP levels were much lower (Figures 12A-12B). FXS1 and FXS2 cell lines were then treated with 5-AzadC, and then FMR1 RNA and FMRP levels were measured (Figure 12C).In the FXS1 cell line, treatment with 5-AzadC for 7 days resulted in a significant increase in both full-length FMR1 and FMR1-217 RNA relative to DMSO-treated cells (Figure 12D). However, in FXS2 cells, 5-AzadC treatment resulted in an increase in full-length FMR1 RNA only (Figure 12E). In both cell lines, treatment with 5-AzadC did not induce a significant increase in FMRP, suggesting that longer treatment times or higher concentrations of 5-AzadC may be required to induce expression of FMRP (Figures 12F-12G and 13A). However, a previous study showed that longer treatment of FXS LCLs with 5-AzadC (36 days) only restored FMR1 RNA up to 40% compared to TD cells, which still produced lower levels of FMRP (22). Thus, transcriptional activation of the normally silenced FMR1 by demethylation induces expression of full-length FMR1 and FMR1-217 RNA, but does not correspondingly induce FMRP expression.

[0381] Example 10. ASO targeting FMR1-217 restored FMRP levels in FXS LCLs with partial or complete FMR1 gene methylation. FMR1-217 was expressed in UFM FXS2 cells and after demethylation in MFM FXS1 cells. Full-length FMR1 was increased in both FXS LCLs after 5-AzadC treatment at the time points examined, whereas FMRP was unchanged. To test whether blocking the formation of FMR1-217 could result in an increase in full-length FMR1 and a concomitant increase in FMRP, eleven 2'-O-methoxyethyl (MOE)-modified antisense oligonucleotides (ASOs) were generated that tile across intron 1, the intron 1-exon 1 junction, or within exon 2 of FMR1-217 RNA (Figure 14A). First, MALAT1 RNA (23)-targeted ASOs were used in LCL cultures to optimize treatment conditions and serve as a marker of transfection efficiency. LCLs cultured with 80 nM MALAT1 ASO for 72 h resulted in a ∼60% reduction in MALAT1 RNA levels (Figure 13B), confirming that the transfection conditions were appropriate. Among the ASOs tested in FXS2 (Figure 13C), the combination of ASO713 and 714 (80 nM each) resulted in a significant reduction in FMR1-217 and an increase in full-length FMR1 (Figure 14B, Figures 13C-13D). ASO713 and 714 at 80 nM or 160 nM each induced a similar reduction in FMR1-217 and an increase in full-length FMR1 RNA over 72 h (Figure 13D). MALAT1 ASO did not affect FMR1 isoform levels (Figure 13D). We then assessed whether FMRP was restored in FXS2 cells after ASO treatment. Figure 14C shows that ASO713 and 714 at 80 nM or 160 nM completely restored FMRP when compared to TD levels, thus suggesting that ASO treatment of cells from at least certain FXS individuals may pave the way for treatment via FMRP restoration.

[0382] In fully methylated FXS1 LCLs, treatment with 5-AzadC for 7 days resulted in expression of FMR1 and full-length FMR1, but did not affect FMRP levels. Therefore, we addressed whether treatment of FXS1 LCLs with a combination of 5-AzadC and ASOs (713 and 714) could restore FMRP. FXS1 LCLs were incubated with 80 nM each of ASOs 713 and 714 24 hours prior to the addition of 1 μM 5-AzadC daily for 7 days prior to sample collection (Figure 14D). FMR1 RNA isoform expression and FMRP levels were examined in these samples. Treatment with 5-AzadC alone resulted in the expected increase in FMR1 full-length and FMR1-217 RNA compared to DMSO controls (Figure 14D). Also, treatment with ASOs alone did not affect FMR1 isoform levels, as the locus was fully methylated. However, treatment of cells with a combination of 5-AzadC and ASO rescued FMR1-217 RNA levels and further increased full-length FMR1 compared to treatment with 5-AzadC alone (Figure 14D). FMRP levels were unaffected by 5-AzadC alone, but FMRP was restored after treatment with a combination of 5-AzadC and ASO (Figures 14E-14F). These data indicated that treatment with FMR1-217 targeting ASO restored FMRP levels in MFM cells, and combined treatment with demethylation (5-AzadC treatment) and ASO restored FMRP in FXS patient-derived cells with UFM.

[0383] Finally, two FXS patient-derived fibroblast cell lines were incubated with 5-AzadC and ASO to determine FMR1 splicing rescue and restoration of FMRP. A dermal cell line from an FXS individual (5131b), which has a CGG repeat number of 800,166 (24) and has previously been shown to harbor a transcriptionally active FMR1 locus, was treated with 5-AzadC and then ASO713 / 714 for 72 h prior to RNA and protein extraction (Figure 15A). RT-qPCR of FMR1 and FMR1-217 showed an ASO-dependent decrease in FMR1-217 and a subsequent increase in FMR1 levels (Figure 15B). Western blots in Figure 15C showed that 5-AzadC treatment did not affect FMRP levels, but ASO alone or in combination with 5-AzadC significantly increased FMRP levels. In a similar experiment using lung fibroblasts from another FXS individual with a fully methylated FMR1 locus, incubation with 5-AzadC in the absence or presence of ASO713 / 714 resulted in an increase in FMR1 and FMR1-217 (Figure 15D). Western blots in Figure 15E show that, similar to dermal fibroblasts, ASO treatment resulted in a significant increase in FMRP, although less than in the TD fibroblast cell line.

[0384] In summary, in most FXS patient samples tested, the FMR1 locus was found to be active but expressed mainly the misspliced ​​FMR1-217 isoform as well as very low levels of FMRP. In transcriptionally silent FXS cells, application of a demethylating agent induced FMR1 transcription, resulting in expression of FMR1-217. In both cases, treatment of cells with ASOs to block FMR1-217 production resulted in partial to full restoration of FMRP (Figure 15F).

[0385] Defects in mRNA alternative splicing alter cellular transcript and protein repertoires and occur in many neurological disorders, such as autism, schizophrenia, and bipolar disorder (25-27). In fragile X syndrome model (e.g., Fmr1 knockout) mice, hundreds of dysregulated alternative splicing events were detected, many of which appeared to be associated with altered epigenetic histone H3 lysine 36 trimethylation (H3K36me3) landscapes (11). In this study, over 1000 RNA missplicing events were detected in human FXS leukocytes, but interestingly, they do not correlate with unaffected H3K36me3 in FXS blood. The alterations in a large number of leukocyte RNAs, if correlated with certain pathologies of FXS, may be useful as biomarkers for assessing treatment outcomes, disease prognosis, and cognitive performance (28-30). Unlike protein-based biomarkers for FXS (31-33), blood-derived RNA biomarkers are more sensitive, specific, and can be easily translated into the clinic.

[0386] The FMR1 gene promoter is methylated and transcriptionally silenced when it contains an expansion of 200 or more CGG repeats. It was therefore surprising that FMR1 RNA was detected in 19 of 29 FXS blood samples and 5 of 10 FXS postmortem brain samples. The majority of these FXS individuals were fully mutated with more than 200 CGG repeats and appeared methylated by standard assays. Notably, in over 70% of these FXS cells and tissues, FMR1 RNA was also mis-spliced ​​to generate the FMR1-217 isoform, a highly truncated RNA that can code for a 31 amino acid peptide. FMR1-217 RNA was not detected in any of the TD samples. Furthermore, in FXS individuals with a fully methylated and silenced FMR1 locus, removal of DNA methylation by 5-AzadC treatment results in expression of FMR1-217. Although FMR1 missplicing to generate the FMR1-217 isoform in FXS apparently requires a CGG expansion, several lines of evidence suggest that the number of CGG repeats may be an important determinant of missplicing. For example, expression of FMR1-217 RNA was detected in fibroblasts from FXS premutation carriers with 140 CGG repeats, but not in cells with fewer CGG repeats (77 or 98 CGG repeats) or from TD individuals (fewer than 55 CGG repeats).

[0387] An important point is the nonlinear relationship between FMR1 levels and FMRP expression in FXS tissue samples. The data show that total FMR1 levels are similar to those in TD LCLs in UFM FXS2 LCLs, but FMRP expression is much lower. Similarly, high FMR1 expression does not guarantee adequate FMRP levels in FXS brain tissue samples 1031-09LZ and UMB5746 (16, 20). Similarly, a robust increase in FMR1 RNA, but not FMRP, occurs in FXS LCLs and fibroblasts treated with 5-AzadC. Interestingly, in all FXS samples expressing FMR1 full-length RNA or after 5-AzadC-mediated transcriptional activation, the misspliced ​​RNA of FMR1-217 was expressed. This relationship between aberrant FMR1 expression and FMR1-217 in FXS cells was also evident in FXS iPSC-derived cells. Reanalysis of RNA-seq datasets from FXS neurons with intact CGG expansions shows that FMR1-217 was not produced when the FMR1 gene was specifically targeted for demethylation by CRISPR / inactive Cas9 fused to Tet1 demethylase ((18), Figure 11C and Table 8). The second and more important point is that FMR1-217 is produced in NPCs derived from FXS iPSCs incubated with 5-AzadC, but not when the CGG expansion is deleted by CRISPR / Cas9 ((17), Figure 11C and Table 8). Thus, the CGG expansion drives the generation of the mis-spliced ​​FMR1-217.

[0388] Intellectual disability is a major feature of FXS. Measurements of leukocyte full-length FMR1-205, FMR1-217, FMRP, and FMR1 gene methylation allowed these molecular parameters to be correlated with IQ. FMRP correlated moderately with higher IQ, whereas FMR1-217 correlated weakly with lower IQ. Based on these correlations, we considered whether ablation of FMR1-217 RNA could elevate FMR1 and restore FMRP levels. Thus, we found that ASOs targeting the second exon of FMR1-217 RNA reduced its levels in UFM FXS cells, rescued full-length FMR1, and importantly restored FMRP levels similar to TD cells. Thus, in FXS individuals expressing FMR1-217, ASO therapy may be a viable treatment option. In individuals with a fully methylated FMR1 locus, ASO-based therapy would be more complicated. Considering that in FXS cells with silenced FMR1, demethylation of the locus with chemical compounds or CRISPR / Cas9-anchored demethylase (17, 22, 34), or ASO-mediated blockade of CGG RNA translation (35, 36) have had limited success in restoring FMRP. CRISPR / Cas9-mediated gene editing of the CGG repeats (37-40) resulted in nearly 70% restoration of FMRP levels. However, we show that DNA demethylation combined with ASO treatment restores FMRP in FXS cells with silenced FMR1. Thus, treatment combining DNA demethylation with an ASO approach may be a useful therapeutic strategy for individuals with a completely silenced FMR1 gene.

[0389] These data indicate that FMR1-217 RNA is the underlying factor inhibiting FMRP expression in FMR1 RNA-permissive FXS cells.

[0390] The present findings suggest that ASOs can be used to correct the dysregulated alternative splicing of FMR1 and restore FMRP in individuals with FXS, thereby providing a novel therapeutic strategy for treating the disorder.

[0391] Embodiment 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 the Fragile X Mental Retardation 1 (FMR1) 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 FMR1 gene, decreases splicing and / or expression of isoform 12 of the FMR1 gene, or a combination thereof. 4. The method of embodiment 3, wherein the agent increases isoform 1 of the FMR1 gene by about 75%. 5. The method of embodiment 3 or 4, wherein the agent reduces isoform 12 of the FMR1 gene by about 30%. 6. The method of any one of embodiments 1-5, wherein the agent is a polynucleotide, and optionally, 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, 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. 10. The method of any one of embodiments 6-9, wherein the polynucleotide comprises a nucleotide sequence that is complementary to a portion of the FMR1 gene transcript. 11. 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 pseudoexon of the FMR1 gene (SEQ ID NO: 19), at least 80% identical to at least a portion of the junction of intron 1 and the pseudoexon, or both. 12. The nucleotide sequence is 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 The method of embodiment 11, wherein the nucleic acid sequence is at least 80% identical to CCTCGCCCAGAACAGTGGAG (SEQ ID NO:11) (W-714). 13. The nucleotide sequence is 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 The method of embodiment 12, wherein the nucleic acid is identical to CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714). 14. The method of embodiment 13, comprising administering to the subject a polynucleotide comprising the nucleotide sequence CGCCCAGAACAGTGGAGCTC (SEQ ID NO: 10) (W-713), a polynucleotide comprising the nucleotide sequence CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714), or both. 15. The method of any one of embodiments 6 to 14, wherein the polynucleotide is modified, optionally with one or more locked nucleic acid (LNA) nucleotides, one or more 2'-modified ribonucleotides, one or more morpholino nucleotides, or a combination thereof. 16. The method of embodiment 15, wherein the modification is a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof. 17. The method of embodiment 15, wherein the polynucleotide is chemically modified to increase nuclease resistance, prevent RNase H cleavage of the complementary RNA strand, increase cellular uptake, or a combination thereof. 18. The method of embodiment 15, wherein the polynucleotide is chemically modified to include locked nucleic acid (LNA), ethyl constrained nucleotides, 2'-(S) constrained ethyl (S-cEt) nucleotides, constrained MOE, 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC), alpha-L-locked nucleic acid, and tricycloDNA, or a combination thereof. 19. The method of embodiment 16, wherein the chemical modification is a modification of the ribose group, and the modification of the ribose group comprises 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'-O-(2-methoxyethyl) (MOE), 2'-O-alkyl, 2'-O-alkoxy, 2'-O-alkylamino, 2'-NH2, constrained nucleotides, tricyclo-DNA modifications, or combinations thereof. 20. The method of embodiment 16, wherein the chemical modification is a modification of a phosphate group, and 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. 21. The method of embodiment 16, wherein the chemical modification is a modification of a nucleobase, and 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, a halogenated aromatic group, or a combination thereof. 22. The method of embodiment 15, wherein the chemical modification is a modification of the polynucleotide sugar-phosphate backbone. 23. The method of embodiment 22, wherein the sugar-phosphate backbone is replaced with a phosphorodiamidato mophorino (PMO), peptide nucleic acid, or other pseudopeptide backbone. 24. The method of embodiment 15, wherein the polynucleotide is a phosphorothioate modified polynucleotide, such as a polynucleotide in which each internucleotide linkage is phosphorothioate, or a polynucleotide in which at least half of the internucleotide linkages are phosphorothioate. 25. The method of any one of embodiments 1-24, wherein the subject is a human having or prone to having FXS. 26. The method of embodiment 25, wherein the subject comprises a CGG repeat expansion of more than 200 repeats in the 5' untranslated region of the FMR1 gene. 27. The method of any one of embodiments 1-24, wherein the subject is a human having or prone to having FXTAS. 28. 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 FMR1 gene. 29. The method of any one of embodiments 25-28, wherein the CGG repeat expansion is partially methylated. 30. The method of any one of embodiments 25-28, wherein the CGG repeat expansion is fully methylated. 31. The method of any one of embodiments 25-30, wherein the subject has an increased level of isoform 12 of the FMR1 gene. 32. The method of any one of embodiments 25-31, wherein the human is a male. 33. The method of any one of embodiments 25-32, wherein the subject is about 2-11, 4-17, 12-18, or 18-50 years old. 34. The method of any one of embodiments 6-33, wherein the polynucleotide is administered intravenously, intraarterially, intrathecally, intracerebroventricularly, intramuscularly, intradermally, subcutaneously, intracranially, or spinally. 35. The method of any one of embodiments 1-34, further comprising administering to the subject a therapeutically effective amount of a DNA demethylating compound or a DNA demethylating enzyme prior to administering the polynucleotide. 36. 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 the FMR1 gene. 37. The method of any one of embodiments 1-36, wherein treating FXS comprises slowing the 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. 38. A method for modulating splicing and / or expression of Fragile X Mental Retardation 1 (FMR1) in a cell, comprising contacting the cell with a polynucleotide under conditions such that the polynucleotide is introduced into the cell, wherein the polynucleotide increases splicing and / or expression of isoform 1 of the FMR1 gene, decreases splicing and / or expression of isoform 12 of the FMR1 gene, or a combination thereof. 39. The method of embodiment 38, wherein the cell is an in vitro cell or an ex vivo cell. 40. The method of embodiment 39, wherein the cell is an artificial pluripotent stem cell (iPSC)-derived neuron, a primary human cell, or a cell line from a human having or prone to having FXS. 41. The method of embodiment 40, wherein the cell is a cell of a subject. 42. The method of embodiment 41, wherein the cells are allogeneic. 43. The method of embodiment 41, wherein the cells are autologous or syngeneic. 44. A polynucleotide comprising a nucleotide sequence that is complementary to a portion of the FMR1 gene transcript. 45. The polynucleotide of embodiment 44, wherein the nucleotide sequence is at least 80% identical to at least a portion of iso12 of the FMR1 gene, at least 80% identical to at least a portion of the junction of intron 1 and iso12 of the FMR1 gene, or both. 46. ​​The nucleotide sequence is 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 46. ​​The polynucleotide of embodiment 45, which is at least 80% identical to CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714). 47. A pharmaceutical composition comprising a polynucleotide according to any one of embodiments 44 to 46 and one or more pharma- ceutically acceptable excipients, diluents or carriers. 48. A microarray for the detection of fragile X-associated disorder, comprising at least one nucleic acid probe immobilized on a solid substrate, the probe comprising a nucleic acid sequence complementary to a portion of an FMR1 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 FMR1-217, at least a portion of the junction of introns 1-2 and exon 2 of FMR1-217, or both. 50. The nucleotide sequence is 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 50. The microarray of embodiment 49, which is at least 80% identical to CCTCGCCCAGAACAGTGGAG (SEQ ID NO: 11) (W-714).

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[0393] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0394] Although 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

1. 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.

2. The ASO of claim 1, 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.

3. 2. The ASO of claim 1, wherein the ASO comprises a nucleotide sequence set forth in any one of i) SEQ ID NOs: 1-11, 43-50, and 51-69, or, optionally, ii) SEQ ID NOs: 10, 11, 43-46, and 60-65.

4. The ASO of claim 1, wherein the ASO is approximately 18 to 22 nucleotides in length.

5. 2. The ASO of claim 1, wherein the ASO comprises a ribose group modification, a phosphate group modification, a nucleobase modification, or a combination thereof.

6. The ASO is a) Locked Nucleic Acids (LNA), Ethyl Constrained Nucleotides, 2'-(S) Constrained Ethyl (S-cEt) Nucleotides, Constrained MOE, 2'-O,4'-C-Aminomethylene Bridged Nucleic Acids (2',4'-BNA(NC)), Alpha-L-Locked Nucleic Acids, TricycloDNA, or combinations thereof; b) a ribose group, including 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'-O-(2-methoxyethyl) (MOE), 2'-O-alkyl, 2'-O-alkoxy, 2'-O-alkylamino, 2'-NH2, constrained nucleotides, tricyclo-DNA modifications, or combinations thereof; c) phosphate groups, including phosphorothioates, phosphoramidates, phosphorodiamidates, phosphorodithioates, phosphonoacetates (PACE), thiophosphonoacetates (thioPACE), amides, triazoles, phosphonates, phosphotriesters, or combinations thereof; d) a nucleobase comprising 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, a halogenated aromatic group, or a combination thereof; e) the sugar-phosphate backbone is replaced with a phosphorodiamidato mophorino (PMO), peptide nucleic acid, or another pseudopeptide backbone; or a combination of the foregoing.

7. The ASO of claim 1, wherein the ASO is a phosphorothioate-modified polynucleotide.

8. The ASO of claim 1, wherein the ASO is a polynucleotide and at least half of the internucleotide linkages of the polynucleotide are phosphorothioate.

9. The ASO of claim 1, wherein the ASO is a polynucleotide and each internucleotide bond of the polynucleotide is phosphorothioate.

10. A pharmaceutical composition comprising the ASO of any one of claims 1 to 9 and a pharmaceutically acceptable excipient, diluent, or carrier.

11. An antisense oligonucleotide (ASO) for use in the treatment of fragile X-associated disorder, comprising administering a therapeutically effective amount of an agent that reduces an abnormal fragile X messenger ribonucleoprotein 1 (FMR1) gene product, thereby treating said fragile X-associated disorder in a subject, wherein said abnormal FMR1 gene product comprises FMR1-217.

12. The ASO for use according to claim 11, wherein the fragile X-associated disorder is fragile X syndrome (FXS).

13. An ASO for use as described in claim 11, wherein the therapeutically effective amount of the agent reduces abnormal FMR1 transcripts, proteins encoded by the abnormal FMR1 transcripts, or both.

14. The ASO for use according to claim 11, wherein the therapeutically effective amount of the agent reduces FMR1-217 by at least 25% or the therapeutically effective amount of the agent increases expression of fragile X messenger ribonucleoprotein (FMRP) by at least 25%.

15. An ASO for use as described in claim 11, wherein the agent targets a consecutive nucleotide sequence in a polynucleotide sequence set forth in any one of SEQ ID NOs: 24 to 42.

16. An antisense oligonucleotide (ASO) for use according to claim 11, wherein the agent is an ASO comprising a nucleotide sequence having at least 85% sequence identity to any one of i) SEQ ID NOs: 1 to 11, 43 to 50, and 51 to 69, or, optionally, ii) SEQ ID NOs: 10, 11, 43 to 46, and 60 to 65.

17. An ASO for use as described in claim 11, wherein the ASO is at least 12 nucleotides in length, or optionally, about 18-22 nucleotides in length.

18. The ASO for use according to claim 11, wherein the ASO comprises a modification of a ribose group, a modification of a phosphate group, a modification of a nucleic acid base, or a combination thereof.

19. The ASO, a) Locked Nucleic Acids (LNA), Ethyl Constrained Nucleotides, 2'-(S) Constrained Ethyl (S-cEt) Nucleotides, Constrained MOE, 2'-O,4'-C-Aminomethylene Bridged Nucleic Acids (2',4'-BNA(NC)), Alpha-L-Locked Nucleic Acids, TricycloDNA, or combinations thereof; b) a ribose group, including 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'-O-(2-methoxyethyl) (MOE), 2'-O-alkyl, 2'-O-alkoxy, 2'-O-alkylamino, 2'-NH2, constrained nucleotides, tricyclo-DNA modifications, or combinations thereof; c) phosphate groups, including phosphorothioates, phosphoramidates, phosphorodiamidates, phosphorodithioates, phosphonoacetates (PACE), thiophosphonoacetates (thioPACE), amides, triazoles, phosphonates, phosphotriesters, or combinations thereof; d) a nucleobase comprising 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, a halogenated aromatic group, or a combination thereof; e) the sugar-phosphate backbone is replaced with a phosphorodiamidato mophorino (PMO), peptide nucleic acid, or another pseudopeptide backbone; or a combination of the foregoing.

Images