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US20170292129A1 - Treatment of c9ftd/als by targeting rna expanded repeat sequences - Google Patents

Treatment of c9ftd/als by targeting rna expanded repeat sequences Download PDF

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US20170292129A1
US20170292129A1 US15/503,524 US201515503524A US2017292129A1 US 20170292129 A1 US20170292129 A1 US 20170292129A1 US 201515503524 A US201515503524 A US 201515503524A US 2017292129 A1 US2017292129 A1 US 2017292129A1
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ggggcc
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Matthew D Disney
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Definitions

  • Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are overlapping neurodegenerative diseases with no effective treatment. Success in developing a treatment will require a well-orchestrated effort that addresses multiple aspects of the drug discovery process, including target identification and validation, as well as the identification of biomarkers to assess efficacy of potential therapies in clinical trials. These endeavors have been hampered by an incomplete understanding of FTD and ALS pathogenesis. However, with the discovery that a GGGGCC repeat expansion in C9ORF72 is the most common genetic cause of FTD and ALS (DeJesus-Hernandez et al., 2011; Renton et al., 2011), a new therapeutic target has come to light.
  • RNA transcribed from the expansion Two putative pathomechanisms of “c9FTD/ALS” involve RNA transcribed from the expansion.
  • these transcripts (termed r(GGGGCC) exp ) may cause toxicity through the formation of nuclear RNA foci that sequester various RNA-binding proteins [for review, see (Gendron et al., 2014)].
  • r(GGGGCC) exp undergoes repeal associated non-ATG (RAN) translation producing “c9RAN proteins” that form neuronal inclusions throughout the central nervous system (Ash et al., 2013; Mori et al., 2013b). Consequently, neutralizing or degrading r(GGGGCC) exp holds promise as a therapeutic approach for c9FTD/ALS.
  • RAN non-ATG
  • antisense oligonucleotides to C9ORF72 transcripts suppress features associated with the repeat expansion in human induced pluripotent stem cell-derived neurons (Donnelly et al., 2013; Sareen et al., 2013).
  • small molecules may offer an attractive option for targeting r(GGGGCC) exp .
  • poly(GP) c9RAN proteins are detected in c9ALS cerebrospinal fluid (CSF)
  • poly(GP) proteins may serve as a pharmacodynamic biomarker to assess efficacy of potential therapies that target r(GGGGCC) exp .
  • the invention is directed, in various embodiments, to small molecules targeting the RNA expanded repeat sequence r(GGGGCC) exp and to the use of the compounds to significantly decrease RAN translation and foci formation in cultured cells expressing r(GGGGCC) 66 and in induced neurons (iNeurons) directly converted from fibroblasts of C9ORF72 repeat expansion carriers.
  • the invention provides a compound of formula
  • the invention provides a method of inhibiting repeat-associated non-ATG (RAN) translation and foci formation in cultured cells expressing r(GGGGCC) 66 and neurons trans-differentiated from fibroblasts of repeat expansion carriers, comprising contacting the cells with an effective amount of a compound of formula 1a, 2, or 3, or a pharmaceutically acceptable salt thereof.
  • RAN repeat-associated non-ATG
  • the invention provides a method of treating a patient afflicted with ALS, comprising administering to the patient an effective dose of a compound of formula 1a, 2, or 3, or a pharmaceutically acceptable salt thereof.
  • FIG. 2 A-B) Synthetic route (A) and analytic HPLC chromatogram (B) for 1a-CA-Biotin.
  • C) (GGGGCC) 66 -expressing cells were treated with DMSO or compounds 1a (25, 50 and 100 ⁇ M) for 24 h.
  • FIG. 3 Human fibroblasts are converted to iNearons following PTB1 knockdown.
  • iNeurons express cytoskeletal neuronal markers MAP2, TUJ1 and neurofilament Smi32, as well as synaptic markers synapsin 1 (SYN1) and post-synaptic density protein 95 (PSD95).
  • C9ORF72+iNeurons Nuclear foci detected in C9ORF72+iNeurons are primarily composed of RNA. C9ORF72+iNeurons were treated with DNase I or RNase A prior to RNA FISH using a 5′TYE563-(CCCCGG) 2.5 -′3 LNA probe. Treatment with RNAse A degraded all foci, but DNAse I only degraded nuclear DNA (observed by loss of Hoechst staining) leaving foci in iNeurons intact. Scale bars, 5 ⁇ m.
  • FIG. 4 Validation of Poly(GP) MSD sandwich immunoassay.
  • a sandwich MSD immunoassay using rabbit polyclonal anti-GP was developed.
  • synthetic peptides representing each possible c9RAN protein translated from sense or antisense transcripts of the expanded C9ORF72 repeat were diluted in Tris-buffered saline (TBS) and assayed (200 ng/ml, 50 ⁇ l per well in duplicate wells).
  • Response values correspond to intensity of emitted light upon electrochemical stimulation of the assay plate using the MSD Sector Imager 2400, from which the background response in wells containing only TBS was subtracted.
  • B) Poly(OP) protein expression in frontal cortical homogenates from 6 c9FTD/ALS patients and 4 patients without the C9ORF72 repeat expansion were analyzed by poly(GP) MSD immunoassay. Response values correspond to the intensity of emitted light upon electrochemical stimulation of the assay plate, from which the average background response measured in brain lysates lacking the C9ORF72 mutation was subtracted. ### P 0.0002 (non-paired, two-tailed t test).
  • c9RAN proteins are detected in c9ALS patient cerebrospinal fluid.
  • r(GGGGCC) 8 Preserves a Hairpin Structure with Periodically Repeating 1 ⁇ 1 Nucleotide GG Internal Loops in Equilibrium with a G-Quadruplex
  • r(GGGGCC) forms intra- and intermolecular G-quadruplex structures (Fratta et al., 2012; Reddy et al., 2013), with another suggesting r(GGGGCC) repeats adopt both G-quadruplex and hairpin structures (Haeusler et al., 2014).
  • spectroscopic circular dichroism (CD) and optical melting
  • chemical modification with dimethyl sulfate (DMS)
  • RNA samples were heated at 95° C. in 10 mM Tris HCl buffer, pH 7.4 and 100 mM KCl prior to completing optical melting experiments, the RNA was too stable to observe melting at highest temperature tested (95° C.) and thermodynamic parameters were not calculated. All data was recorded in duplicate and presented as mean ⁇ SD.
  • r(GGGGCC) 8 was next examined using enzymatic and chemical mapping in the presence of Li + or K + , the latter known to stabilize G-quadruplex formation (Hardin et al., 1992). Enzymatic mapping revealed an alternating pattern of cleavage by enzymes that specifically cleave paired or non-canonically paired nucleotides, suggesting that some populations form a hairpin structure. These findings were confirmed using the chemical modification reagent DMS.
  • RNAs that form quadruplexes can form alternative structures, including hairpins (Bugaut et al., 2012).
  • Small molecule leads can be further optimized by chemical similarity searching, which identifies compounds that are chemically similar to the leads.
  • small molecule 1a binds 1 ⁇ 1 GG internal loops present in r(CGG) exp and improves fragile X-associated tremor/ataxia syndrome (FXTAS)-associated defects (Disney et al., 2012).
  • FXTAS fragile X-associated tremor/ataxia syndrome
  • 1a and compounds chemically similar to it might bind r(GGGGCC) exp .
  • Three lead compounds (1a, 2 and 3) were identified ( FIG. 1A ; Table 2) and further characterized.
  • fibroblasts with or without the C9ORF72 repeat expansion were directly converted to a neuronal lineage by repressing polypyrimidine-tract-binding protein (PTB1), as recently described (Xue et al., 2013).
  • PTB1 depletion caused fibroblasts to adopt a neuronal morphology with reduced soma size and neurite formation ( FIG. 3A ).
  • These iNeurons expressed neuronal and synaptic markers, including MAP2, TUJ1, PSD95, Synapsin I and Drebrin FIG. 3B ).
  • Nuclear foci, degraded by RNase A but resistant to DNase 1 FIG. 3C ), were present in both C9ORF72+fibroblasts ( FIG.
  • c9RAN proteins As a pathological hallmark of c9FTD/ALS, and one that is influenced by r(GGGGCC) exp -targeting small molecules, c9RAN proteins have potential to serve as clinically relevant biomarkers.
  • poly(GP) is detectable specifically in c9ALS CSF could facilitate identification of C9ORF72 repeat expansion carriers in the course of standard diagnostic work-ups, and also pave the way in determining whether changes in c9RAN protein levels in CSF correlate with disease severity or progression.
  • CSF c9RAN proteins could serve as an enrollment stratification tool in clinical trials, and a pharmacodynamic biomarker to assess efficacy of therapies that target r(GGGGCC) exp ( FIG. 4B ).
  • r(GGGGCC) 8 Preserves a Hairpin Structure with Periodically Repeating 1 ⁇ 1 Nucleotide GG Internal Loops in Equilibrium with a G-Quadruplex
  • r(GGGGCC) 8 The folding of r(GGGGCC) 8 was next examined using enzymatic and chemical mapping in the presence of Li + or K + , the latter known to stabilize G-quadruplex formation (Ehresmann et al., 1987; Hardin et al., 1992). Enzymatic mapping was performed using S1 (cleaves single stranded and non-canonically paired nucleotides), T1 (cleaves single stranded and non-canonically paired G's), and V1 (cleaves base pairs). If r(GGGGCC) 8 forms a quadruplex, G residues should be protected from cleavage by T1 and S1 (Todd and Neidle. 2011).
  • r(GGGGCC) 8 forms a hairpin structure with internal loops in the stem, an alternating pattern of T1/S1 cleavage and V1 cleavage should be observed. Indeed, our mapping studies revealed such a pattern, suggesting that some population forms a hairpin structure. Enzymatic mapping data were used to construct a model of r(GGGGCC) 8 's structure using the program RNAstructure (Mathews et al., 2004), affording a hairpin with GG internal loops. Our hypothesis that r(GGGGCC) 8 forms a hairpin was further investigated by chemically probing the RNA's structure by reaction with dimethyl sulfate (DMS).
  • DMS dimethyl sulfate
  • DMS methylates the N7 position of G's (Ehresmann et al., 1987).
  • the N7 position of G's in quadruplexes are hydrogen bonded and thus protected from methylation (Todd and Neidle, 2011).
  • the majority of guanine N7 positions were susceptible to DMS modification and the pattern did not change in the presence of Li + or K + .
  • r(GGGGCC) 8 was explored by analyzing its 1D 1 H NMR spectra. Guanines in non-canonically paired conformations (internal loops, quadruplexes) typically give rise to imino proton signals from 10 to 12 ppm whereas the resonances from G's in base pairs appear from 12 to 14 ppm (Bugaut et al., 2012). r(GGGGCC) 8 prepared in 10 mM Tris HCl and 100 mM KCl was heated at 37° C., 60° C., or 95° C., followed by slow cooling and equilibration at room temperature for 2 h.
  • ATP adenosine triphosphate
  • BLI biolayer interferometry
  • bp base pair
  • BSA bovine serum albumin
  • CA chlorambucil
  • CD circular dichroism
  • DCM dichloromethane
  • DIPEA N,N-diisopropylethylamine
  • DMF N,N-dimethylformamide
  • DMS dimethyl sulfate
  • DMSO dimethyl sulfoxide
  • DNA deoxyribonucleic acid
  • DEPC-PBS DEPC-treated PBS
  • DPBS Dulbecco's phosphate buffered saline
  • EDTA ethylenediaminetetraacetic acid
  • EtOAC ethyl acetate
  • FBS fetal bovine serum
  • HBTU O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
  • HEPES 4-(
  • RNA oligonucleotides were deprotected per the manufacturer's recommended protocol and desalted using a PD-10 gel filtration column (GE Healthcare). Concentrations were determined by measuring absorbance at 260 nm using a Beckman Coulter DU800 UV-Vis spectrophotometer equipped with a Peltier temperature controller unit.
  • RNA oligonucleotides were radioactively labelled at the 5′ end using T4 polynucleotide kinase (New England Biolabs) and [ ⁇ - 32 P] ATP (PerkinElmer) and purified by either passing through a Sephadex G-25 column (Promega) or by PAGE as previously described (Disney et al., 2000). DNA oligonucleotides (Integrated DNA Technologies, Inc. (IDT)) were used without further purification.
  • T4 polynucleotide kinase New England Biolabs
  • [ ⁇ - 32 P] ATP PerkinElmer
  • Mass spectra were collected using an ABI 4800 MALDI-TOF or Varian 500-MS IT mass spectrometer. Reverse-phase HPLC was completed using a Waters 1525 binary HPLC pump equipped with a Waters 2487 dual absorbance detector system. Optical melting spectra were acquired using a Beckman Coulter DU800 UV-Vis spectrometer connected to a Peltier heater. Circular dichroism experiments were performed on a Jasco J-815 spectrometer equipped with a Jasco Peltier temperature controller. TO-PRO-1 displacement assays were performed on a PerkinElmer Envision® multilabel reader. Gel images were acquired using a Molecular Dynamics Typhoon 9410 variable mode imager. BLI experiments were performed on ForteBio Octet RED.
  • RNA concentration was determined using a Thermo Scientific Nanodrop 2000C spectrophotometer. qRT-PCR analyses were performed on an ABI 7900 HT Real-Time PCR System. 1 H NMR spectra of RNA were recorded at 10° C. using a 700 MHz Bruker Avance TCI spectrometer equipped with a cryogenic TCI ATM probe, water suppression was achieved using excitation sculpting. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra for compound characterization were recorded at 25° C. on a 400 MHz Bruker Avance spectrometer.
  • RNA samples (4 ⁇ M) were folded in 1 ⁇ CD Buffer (10 mM Tris HCl, pH 7.4 containing no monovalent cation or 100 mM LiCl, NaCl or KCl) by heating at 95° C. for 5 min and then slowly cooling to room temperature. Samples were then transferred into a 1 mL quartz cell with a pathlength of 1 mm.
  • CD spectra were recorded at 20° C. by measuring ellipticity from 220 to 320 nm at a rate of 50 nm/min, a 2 second digital integration time (D.I.T.), 1 nm data pitch, and 1 nm band width. The background was subtracted from each spectrum, which were smoothed and normalized to zero at the starting point (320 nm).
  • Enzymatic digestions using T1 (0.01 U/ ⁇ L) under denaturing conditions (1 ⁇ RNA Sequencing Buffer, Life Technologies), T1 (1 U/ ⁇ L), V1 (0.001 U/ ⁇ L) and S1 (0.1 U/ ⁇ L) under non-denaturing condition were carried out at room temperature for 15 min and quenched by the addition of 1 ⁇ Loading Buffer (1 mM Tris HCl, pH 7.5, 10 mM EDTA, and 4 M urea) and incubation at 95° C. for 2 min. Cleavage products were separated on a denaturing 20% polyacrylamide gel and visualized by autoradiography. Sites of cleavage were used as restraints in secondary structure prediction by free energy minimization (RNAstructure, version 5.4) (Bellaousov et al., 2013).
  • DMS footprinting experiments were completed as previously described (Ziehler and Engelke, 2001). Briefly, 5′ end- 32 P-labeled r(GGGGCC) 8 was folded in 10 mM Tris HCl, pH 7.4, containing 185 mM KC, NaCl, or LiCl by heating at 95° C. and slowly cooling to room temperature. To the samples were added DMS (dissolved in 1:1 EtOH:H 2 O) to a final concentration of 3% (v/v), and the samples were incubated for 2 min. Reactions were quenched by ethanol precipitation; the resulting pellets were washed once with 70% ethanol and briefly dried in a vacuum concentrator.
  • RNA samples were dissolved in 1 M Tris HCl, pH 8 followed by addition of 0.1 M NaBH 4 and incubation on ice for 30 min in the dark. The reactions were quenched by ethanol precipitation as described above. Aniline cleavage of the modified RNA was completed by dissolving the RNA in freshly prepared 1 M aniline in 0.3 M NaOAc, pH 4.5 followed by incubation at 60° C. for 20 min. The samples were ethanol precipitated and dissolved in 1 ⁇ Loading Buffer. Fragments were separated on a denaturing 20% polyacrylamide gel and visualized by autoradiography.
  • RNA of interest (1-35 ⁇ M) was folded in 10 mM Tris HCl, pH 7.4 and 100 mM NaCl or 100 mM KCl by heating at 95° C. for 5 min and slowly cooled to room temperature.
  • 1 ⁇ M RNA was folded as described above followed by addition of 3 ⁇ M compound and incubation at room temperature for 15 min.
  • Absorbance versus temperature spectra were then acquired at 260 nm and 295 nm at a rate of 1° C./min. Melting curves were fit to a self-complementary model using MeltWin (http://www.meltwin.com). The program fits each curve and calculates thermodynamic parameters and melting temperature (T m ) (see Table 1, Table 3, FIG. 1C ).
  • a 600 ⁇ M sample of r(GGGGCC) 8 was prepared in 10 mM Tris HCl, pH 7.4 and 100 mM KCl and annealed at the appropriate temperature for 5 min. The sample was then slowly cooled to room temperature. After equilibration at room temperature for 2 h, the sample was transferred to a 3 mm Shigemi D 2 O NMR tube, and NMR spectra were recorded at 10° C.
  • r(GGGGCC) 8 (36 nM) was folded in 8 mM Na 2 HPO 4 , pH 7.0, 185 mM NaCl, and 1 mM EDTA by heating at 95° C. for 5 min and slowly cooling to room temperature.
  • TO-PRO-1 and BSA were then added to final concentrations of 10 nM and 40 ⁇ g/mL, respectively, and the samples were incubated at room temperature for 15 min.
  • the compound of interest 100 ⁇ M was added, and the samples were incubated for an additional 15 min at room temperature. After incubation, fluorescence intensity was recorded and converted to the percentage of dye-RNA complex using equation 1:
  • I is the observed fluorescence intensity
  • I 0 is the fluorescence intensity in the absence of RNA
  • I max is the fluorescence intensity in the absence of compound.
  • BLI was used to measure the binding affinities of 1a, 2, and 3 for three different RNAs, including 5′-Biotin-r(GGGGCC) 8 , 5′-Biotin-r(CGG) 12 , and a hairpin containing all GC pairs in the stem and a GAAA tetraloop (see FIG. 1B ).
  • RNA 100 nM was folded in 1 ⁇ Kinetics Buffer (ForteBio; 1 ⁇ PBS, 0.1% (w/v) BSA, 0.02% (v/v) Tween20, and 0.05% (w/v) sodium azide) supplemented with no cation, 100 mM NaCl, or 100 mM KCl by heating at 95° C. for 5 min and slowly cooling to room temperature.
  • the RNA 200 ⁇ L aliquots was then added to a black 96-well plate (Greiner Bio-One).
  • the compound of interest (16-100 ⁇ M; 200 ⁇ L aliquots; 2-fold serial dilutions; 7 samples total) was dissolved in 1 ⁇ Kinetics Buffer. A sample with no compound was used as background. All experiments were performed at 30° C. with agitation set to 1000 rpm.
  • RNA was loaded onto the surface of streptavidin biosensors (SA) for 660 s. Optimal response levels were between 0.5 and 2 nm, and variability within a row of eight tips did not exceed 0.2 nm. Biosensors were then washed in 1 ⁇ Kinetics Buffer for 300 s followed by association of the compound (analyte) for 5000 s. Finally, dissociation of the ligand-analyte interaction was analyzed for 5000 s. The resulting curves were corrected by subtracting the response recorded on a sensor loaded with ligand (RNA) but incubated with no analyte (compound). Data analyses and curve fitting were completed using Octet Data Analysis, version 7.0.
  • Chlorambucil (26 mg. 0.085 mmoles), HBTU (43 mg, 0.11 mmoles), HOBT (17 mg 0.11 mmoles) and DIPEA (58 mg, 0.44 mmoles) in 1 mL DMF were stirred at room temperature for 40 min.
  • r(GGGGCC) 2 , r(GGGGCC) 20 and r(GGGGCC) 66 expression vectors was previously reported (Gendron et al., 2013).
  • genomic DNA from muscle or spleen from a C9ORF72 expanded repeat carrier was used as a template in a nested PCR strategy using ThermalAce DNA Polymerase (Invitrogen) to amplify, the (GGGGCC) repeat region, including 113 bp of 5′ and 99 bp of 3′ flanking sequence.
  • the PCR products were cloned into the pAG3 expression vector. These constructs contain 3 upstream stop codons in each reading frame.
  • Clones containing r(GGGGCC) 2 , r(GGGGCC) 20 and r(GGGGCC) 66 were verified by hairpin sequence analysis.
  • COS7 cells were grown as monolayers in a 75 cm 2 flask to ⁇ 95% confluency and then transfected with r(GGGGCC) 66 using Lipofectamine 2000 (Invitrogen) per the manufacturer's recommended protocol. Approximately 16 h post-transfection, 1a-CA-Biotin and the compound of interest, or vehicle, were added to the cells, and the samples were incubated at 37° C. for 20-24 h. Total RNA was extracted using Trizol reagent (Ambion) according to the manufacturer's protocol.
  • RNA-1a-CA-Biotin adducts were released from beads by heating in 50 ⁇ l 1 ⁇ Elution Buffer (95% formamide, 10 mM EDTA, pH 8.2) at 65° C. for 5 min. The concentration of the bound RNA was quantified by UV absorbance.
  • cDNA was generated from 50 ng of RNA using a qScript cDNA Synthesis Kit (Quanta Biosciences) per the manufacturer's protocol. Power SYBR® Green PCR Master Mix (Applied Biosystems) was used to quantify the amount of r(GGGGCC) 66 according to the manufacturer's protocol. The amount of the r(GGGGCC) 66 was normalized relative to 18S rRNA. Primer sequences for r(GGGGCC) 66 (C9down-F and C9down-R), 18S rRNA (18S-F and 18S-R), and ⁇ -actin
  • Membranes were washed three times for 10 min in TBST and incubated with donkey anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (1:5000; Jackson ImmunoResearch) for 1 h. Protein expression was visualized by enhanced chemiluminescence treatment and exposure to film. Immunoassay Analysis of c9RAN Proteins.
  • Poly(GP) proteins were also measured in lysates from cultured cells (10-35 ⁇ g of protein per well) prepare as described above, or from RIPA-soluble homogenates from frozen frontal cortical tissues (35 ⁇ g of protein per well). Brain homogenates were prepared as previously described (Almeida et al., 2013). In brief, tissue was lysed in cold RIPA buffer and sonicated on ice. Lysates were cleared by centrifugation at 100,000 g for 30 min at 4° C. The supernatant was collected and protein concentration was determined by BCA assay.
  • Poly(OP) protein expression was similarly evaluated in CSF (90 ⁇ l per well, in duplicate or triplicate wells) from 5 healthy controls, 25 ALS patients without the C9ORF72 repeat expansion, and 14 ALS patients with the expansion (see Table 4 for patient information and the section on Human Samples below for additional information on CSF collection).
  • a second MSD sandwich immunoassay was developed for the detection of poly(PR) proteins using polyclonal anti-PR as capture and detection antibodies.
  • lysates from cells transfected to express each possible protein RAN translated from the sense or antisense transcripts of the expanded C9ORF72 repeat [GFP-(GA) 5 , GFP-(GR) 5 , GFP-(GP) 5 , GFP-(PA) 5 , GFP-(PR) 5 ] were assayed, as were lysates from cells expressing (CCCCGG) 66 ( FIG. 2F ).
  • RNA Fluorescence In situ Hybridization FISH of (GGGGCC) n -Expressing Cells.
  • HEK293T cells grown on glass coverslips in 24-well plates were transfected with 0.5 ⁇ g r(GGGGCC) 2 , r(GGGGCC) 20 or r(GGGGCC) 66 vectors. After 24 h, cells were fixed in 4% paraformaldehyde for 20 min, permeabilized in ice-cold methanol for 10 min, and washed 3 times with DEPC-treated PBS (DEPC-PBS).
  • DEPC-PBS DEPC-treated PBS
  • GGCCCC denatured Cy3-conjugated 4 probe (2 ng/ ⁇ l) in hybridization buffer (50% formamide, 10% dextran sulfate, 0.1 mg/mL yeast tRNA, 2 ⁇ SSC, 50 mM sodium phosphate) overnight at 37° C. Cells were then washed once with 40% formamide/1 ⁇ SSC for 30 min at 37° C. and twice with DEPC-PBS at room temperature for 5 min, followed by counterstaining with Hoechst 33258 (1 ⁇ g/ml, Invitrogen). Immunostained cells were visualized using a Zeiss Axiovert Fluorescence Microscope with apotome module.
  • HEK293 cells grown on glass coverslips in 24-well plates were transfected with 0.6 ⁇ g of r(GGGGCC) 66 vector.
  • cells were treated with DMSO or compound (1a, 2 or 3) for 24 h, and then subjected to FISH as described above.
  • coverslips mounted on slides were scanned by Aperio ScanScope. Ten fields were randomly selected under 20 ⁇ magnification. For each field, the number of foci-positive nuclei and the total number of nuclei were counted using MetaMorph software. These counts were used to determine the average percentage of foci-positive cells for each condition.
  • RNA FISH was then performed using hybridization buffer containing the Cy3-(GGGGCC) 4 RNA probe and either DMSO or 1a in excess of 20 times the molar concentration of the probe.
  • COS7 cells were grown as monolayers in 96-well plates in growth medium (1 ⁇ DMEM, 10% FBS, and 1 ⁇ GlutaMax (Invitrogen)). After the cells reached 90-95% confluency, they were transfected with 200 ng of plasmid using Lipofectamine 2000 (Invitrogen) per the manufacturer's standard protocol. Compound 1a was added to the transfection cocktail, which was then applied to the cells.
  • the transfection cocktail was replaced with growth medium containing 1a approximately 5 h post transfection, and the cells were incubated at 37° C. for 18 h.
  • Cells were lysed in the plate using 100 ⁇ l/well of MPER Mammalian Protein Extraction Reagent (Pierce Biotechnology) containing 1 ⁇ l of Halt Protease Inhibitor cocktail (Thermo Scientific).
  • Cellular proteins were separated by SDS-PAGE (10% polyacrylamide) and then transferred to a PVDF membrane by wet transfer method.
  • Protein content was analyzed by Western blotting by using anti-GFP (Santa Cruz) or anti- ⁇ -actin (Sigma Aldrich) as primary antibodies and anti-IgG-horseradish peroxidase conjugate as the secondary antibody.
  • Chemiluminescent signal was generated by SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific), and the blot was imaged with X-ray film (Phenix Research).
  • Frozen frontal cortex tissue used for biochemical analysis included samples from 6 FTD/ALS cases with the C9ORF72 expansion, and 4 FTD/ALS cases without the expansion.
  • Fibroblasts were derived from skin sampled by punch biopsy on the anterior aspect of the forearm. Skin biopsies were obtained from six individuals, which included three control participants (control 1: female diagnosed with sixth nerve palsy, 61 years of age at the time of biopsy; control 2: healthy female, 64 years of age at the time of biopsy; control 3: healthy female, 38 years of age at the time of biopsy) and three repeat expansion carriers (carrier 1: 28 year old female at the time of biopsy; carrier 2: female diagnosed with ALS at 49 years of age, 50 years of age at the time of biopsy; carrier 3: male diagnosed with ALS/FTD at 41 years of age, 43 years of age at the time of biopsy). Fibroblasts were generated by ReGen Theranostics Inc (Rochester, Minn.).
  • CSF was obtained from healthy controls or ALS patients seen at the ALS Center at Mayo Clinic Florida, the National Institutes of Health (NIH), the IRCCS Istituto Auxologico Italiano (Milan. Italy), the University of Massachusetts Medical School, and Massachusetts General Hospital (Table 4).
  • CSF was collected via standard lumbar puncture, aliquoted and stored at ⁇ 80° C.
  • ALS patients had El Escorial clinically definite, probable, laboratory supported probable or possible ALS of ⁇ 5 years' duration.
  • Patients received lumbar puncture generally in the diagnostic early phase of the disease. Patients receiving tracheostomy ventilation or non-invasive mechanical ventilation for >23 h/day were excluded.
  • PCR polymerase chain reaction
  • Fibroblasts were maintained in Dulbecco's modified Eagle's medium (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin (Gibco) at 37° C., in an atmosphere containing 5% CO 2 and 95% air.
  • Lentiviral shRNA against human PTBP1 (shPTB) cloned into pLKO.1 was a kind gift from Dr. Fu (University of California, San Diego).
  • shPTB and non-silencing shRNA in the pLKO.1 vector were packaged in HEK293FT cells using Virapower (Invitrogen) packaging mix. Viral particles were collected 48 and 72 h after transfection.
  • fibroblasts were seeded on a poly-D-lysine-coated surface and were transduced with pLKO.1 coding for shPTB1 or non-silencing control shRNA for 12-18 h in the presence of 5 ⁇ g/ml polybrene.
  • pLKO.1 coding for shPTB1 or non-silencing control shRNA for 12-18 h in the presence of 5 ⁇ g/ml polybrene.
  • Two days post-infection cells were selected with 1.5 ⁇ g/ml puromycin for 48 h.
  • 10 ng/ml basic fibroblast growth factor (bFGF, GenScript) was added to the medium for two days.
  • DMEM/F12 medium containing 2% FBS, 25 mg/ml insulin (Sigma-Aldrich), 100 nM putrescine (Sigma-Aldrich), 50 mg/ml transferrin (Sigma-Aldrich), 30 nM sodium selenite (Sigma-Aldrich) and 15 ng/ml bFGF.
  • the medium was enriched with B27 supplement (Gibco) and a cocktail of neurotrophic factors, including 10 ng/ml each of BDNF, GDNF (R&D Systems), NT3 (Peprotech), and CNTF (Sigma). Immunocytochemical analysis was performed 2-6 days later.
  • Secondary fluorescent antibodies (Invitrogen) were used at 1:1000 in 5% skim milk/TBS-T. Confocal microscopy was performed using Zeiss LSM 510 microscope.
  • fibroblasts were transduced with shPTB1 or non-silencing control shRNA. Five days later, cell lysates were prepared and analysed by Western blot using an antibody to PTB1.
  • fibroblasts were converted to iNeurons in 96-well plates and treated with compound 1a (2 or 4 ⁇ M) or DMSO for four days to analyze their effect on the accumulation of poly(GP) or poly(PR) protein inclusions.
  • Serial pictures were generated using the BD Pathway Bioimager. For each condition, the percentage of cells containing poly(GP) or poly(PR) inclusions was calculated from 3-6 wells for each of 3 independent experiments.
  • RNA Fluorescent In Situ Hybridization in Fibroblasts and iNearons.
  • RNA FISH of fibroblasts and iNeurons treated with DMSO or compound 1a (2 ⁇ M) for four days was performed as previously described (Lagier-Tourenne et al., 2013) with some modifications. Briefly, plated cells were fixed in 4% PFA/DEPC-PBS, permeabilized with 0.2% Triton X-100/DEPC-PBS, washed twice with DEPC-PBS, dehydrated through 70% and 100% ethanol, and air dried.
  • iNeurons were treated with RNase A (2.5 ⁇ M; Qiagen) for 15 min at 37° C., or with DNase I (3 U/ml; Invitrogen) for 30 minutes at room temperature, prior to dehydration.
  • Cells were incubated in hybridization buffer (10% dextran sulfate, 50% formamide, 50 mM sodium phosphate buffer (pH 7), 2 ⁇ SSC) at 66° C. for 20-60 min.
  • hybridization buffer 10% dextran sulfate, 50% formamide, 50 mM sodium phosphate buffer (pH 7), 2 ⁇ SSC) at 66° C. for 20-60 min.
  • the locked nucleic acid probe (5TYE563-CCCCGCCCCGGCCCC-3′, Batch #612968, Exiqon) was denatured at 80° C. for 75 s and diluted to 40 nM with hybridization buffer.
  • RNA foci in iNeurons were visualized and quantified using a Zeiss Axiovert Fluorescence Microscope with apotome module. For each of 3 cell lines, 3 fields were randomly selected per condition. For each field, the number of foci-positive nuclei and the total number of nuclei were counted to determine the average percentage of foci-positive cells.
  • RNA Extraction and Quantitative PCR (qRT-PCR) of C9ORF72.
  • iNeurons three different cell lines, in triplicate were harvested in 1 ml of Trizol after treatment with DMSO or 1a (4 ⁇ M, 4 d).
  • RNA was extracted using the Direct-Zol RNA kit combined with in-column DNase I digestion, as per the manufacturer's instructions (Zymo Research, Irvine, Calif., USA).
  • RNA integrity was obtained using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA).
  • cDNA was obtained after reverse transcription polymerase chain reactions (RT-PCR) using approximately 500 ng of RNA with random primers and the High Capacity cDNA Transcription Kit (Applied Biosystems, Foster City, Calif., USA) as per the manufacturer's instructions.
  • qRT-PCR was conducted in triplicates for all samples using inventoried TaqMan gene expression assays for total C9ORF72 [transcript variants 1 (NM_145005.5), 2 (NM_018325.3), 3 (NM_001256054.1) (Hs00376619)], the long form of C9ORF72 [variants 2, 3 (Hs00945132)], and GAPDH (Hs00266705) (Applied Biosystems) on an ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems). Relative quantification of C9ORF72 variants was determined using the ⁇ Ct method and normalized to GAPDH.

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