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(12) Patent Application Publication (10) Pub. No.: US 2016/0024496 A1 Bennett Et Al US 2016.0024496A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0024496 A1 Bennett et al. (43) Pub. Date: Jan. 28, 2016 (54) METHODS FORMONITORING C90RF72 Related U.S. Application Data EXPRESSION (60) Provisional application No. 61/714,142, filed on Oct. (71) Applicants: ISIS PHARMACEUTICALS, INC., 15, 2012. Carlsbad, CA (US): THE JOHNS HOPKINS UNIVERSITY, Baltimore, MD (US) Publication Classification (72) Inventors: C. Frank Bennett, Carlsbad, CA (US); (51) Int. Cl. Susan M. Freier, San Diego, CA (US); CI2N IS/II3 (2006.01) Jeffrey D. Rothstein, Catonsville, MD A613 L/7088 (2006.01) (US); Christopher Donnelly, Newtown (52) U.S. Cl. Square, PA (US); Rita G. Sattler, CPC .......... CI2N 15/I 13 (2013.01); A61K3I/7088 Baltimore, MD (US) (2013.01); C12N 23 10/11 (2013.01); C12N 23 10/11 1 (2013.01); C12N 23 10/341 (2013.01); (73) Assignees: The Johns Hopkins University, CI2N 23.10/315 (2013.01); C12N 23.10/3341 Baltimore, MD (US); Isis (2013.01); C12N 23.10/3231 (2013.01); C12N Pharmaceuticals, Inc., Carlsbad, CA 23.10/321 (2013.01) (US) (21) Appl. No.: 14/436,039 (57) ABSTRACT (22) PCT Fled: Oct. 15, 2013 Disclosed herein are methods for monitoring expression of (86) PCT NO.: PCT/US13A65131 C9ORF72 mRNA and protein in an animal with C90RF72 S371 (c)(1), specific inhibitors. Such C9CRF72 specific inhibitors include (2) Date: Apr. 15, 2015 antisense compounds. Patent Application Publication Jan. 28, 2016 Sheet 1 of 9 US 2016/0024496 A1 'GGGGCc &epseat NM_0183252:-- g NP 060795.1 NM 145.0054 - HINP 6591122 Legend: ! s UTR - intron i Y CDs as ASO a 19-20 nt GAPners : - 5'8, 3' 5 nt 2'O-methyl RNA bases 1 10 nt phosphothioate backbones &uSRimasamanmaauwman Control 1 2 3 4 5 -RT C90RF72 mRN GAPDH mRNA ::::::::::::::::: Control Fibroblasts Figure 1 a-b Patent Application Publication Jan. 28, 2016 Sheet 2 of 9 US 2016/0024496 A1 Human Cerebellum coorf72 as / control / ENPP2 mRNAs Frio fixtixias NP2 sewgis RSPO3 in RNA GAPDH rRNA s k peos vs. Gortraitroalasts c s f, p<0.05 vs. CSORF2 fibroblasts Figure 2 a-b Patent Application Publication Jan. 28, 2016 Sheet 3 of 9 US 2016/0024496 A1 A si. * 3. COR2A Ciria is tria in their tria Air Figure 3 a-b Patent Application Publication Jan. 28, 2016 Sheet 4 of 9 US 2016/0024496 A1 control Fibroblasts 9oRF72 Fibroblast 6 O O 2 O. O Figure 4a Patent Application Publication Jan. 28, 2016 Sheet 5 of 9 US 2016/0024496 A1 1 2 O control Aso M o C9 OR72. As 4.26 OoOO O Figure 4b Patent Application Publication Jan. 28, 2016 Sheet 6 of 9 US 2016/0024496 A1 3 O 2 O 10 Figure 5 Patent Application Publication Jan. 28, 2016 Sheet 7 of 9 US 2016/0024496 A1 -2 5 SABIC DE Figure 6 Patent Application Publication Jan. 28, 2016 Sheet 8 of 9 US 2016/0024496 A1 x 8: W s: xxxx N & xxxxxxxx N 2 36% x: s O (O. O. O. O. O. 3 st xsi -Wese o co st (JOS 60 Ol e/\eel) SeNe WN Patent Application Publication Jan. 28, 2016 Sheet 9 of 9 US 2016/0024496 A1 f E. 2 S N:5&O 8x: 2. XxxxxXXXXXXXX s i E.Fix a SeAe WN US 2016/0024496 A1 Jan. 28, 2016 METHODS FORMONITORING C90F72 nearly half of familial ALS and nearly one-quarter of all ALS EXPRESSION cases in a cohort of 405 Finnish patients (Laaksovirta et al. Lancet Neurol., 2010, 9,978-985). SEQUENCE LISTING 0005. A founder haplotype, covering the C9CRF72 gene, 0001. The present application is being filed along with a is present in the majority of cases linked to this region. Sequence Listing in electronic format. The Sequence Listing 0006. There are currently no effective therapies to treat is provided as a file entitled BIOLO215 WOSEQ.txt created Such neurodegenerative diseases. Therefore, it is an object to Oct. 15, 2013, which is 120Kb in size. The information in the provide compositions and methods for the treatment of Such electronic format of the sequence listing is incorporated neurodegenerative diseases. herein by reference in its entirety. SUMMARY FIELD 0007 Disclosed herein are biomarkers and methods for 0002 Provided are methods for reducing expression of moritoring expression of C9CRF72 mRNA and protein in an C9ORF72 mRNA and protein in an animal. Such methods are animal with C90RF72 specific inhibitors. Such C90RF72 useful to treat, prevent, or ameliorate neurodegenerative dis specific inhibitors include antisense compounds. eases, including amyotrophic lateral Sclerosis (ALS), fronto 0008 Provided herein are methods for modulating levels temporal dementia (FTD), corticalbasal degeneration syn of C9CRF72 mRNA and protein in cells, tissues, and ani drome (CBD), atypical Parkinsonian syndrome, and mals. In certain embodiments, C9CRF72 specific inhibitors olivopontocerellar degeneration (OPCD). modulate expression of C9CRF72 mRNA and protein. In certain embodiments, C9CRF72 specific inhibitors are BACKGROUND nucleic acids, proteins, or Small molecules. 0003 Amyotrophic lateral sclerosis (ALS) is a fatal neu 0009. In certain embodiments, modulation can occur in a rodegenerative disease characterized clinically by progres cellor tissue. In certain embodiments, the cellor tissue is in an sive paralysis leading to death from respiratory failure, typi animal. In certain embodiments, the animal is a human. In cally within two to three years of symptom onset (Rowland certain embodiments, C90RF72 mRNA levels are reduced. and Shneider, N. Engl. J. Med., 2001,344, 1688-1700). ALS In certain embodiments, C9CRF72 protein levels are is the third most common neurodegenerative disease in the reduced. In certain embodiments, certain C9CRF72 mRNA Western world (Hintz et al., Neurology, 2007, 68, 326-337), variants are preferentially reduced. In certain embodiments, and there are currently no effective therapies. Approximately the C9CRF72 mRNA variants preferentially reduced are 10% of cases are familial in nature, whereas the bulk of variants containing intron 1. In certain embodiments, intron 1 patients diagnosed with the disease are classified as sporadic contains a hexanucleotide repeat expansion. In certain as they appear to occur randomly throughout the population embodiments, the hexanucleotide repeat expansion is associ (Chio et al., Neurology, 2008, 70,533-537). There is growing ated with a C90RF72 associated disease. In certain embodi recognition, based on clinical, genetic, and epidemiological ments, the hexanucleotide repeat expansion is associated with data, that ALS and frontotemporal dementia (FTD) represent a C9ORF72 hexanucleotide repeat expansion associated dis an overlapping continuum of disease, characterized patho ease. In certain embodiments, the hexanucleotide repeat logically by the presence of TDP-43 positive inclusions expansion comprises at least 30 GGGGCC repeats. In certain throughout the central nervous system (Lillo and Hodges, J. embodiments, the hexanucleotide repeat expansion is associ Clin. Neurosci., 2009, 16, 1131-1135; Neumann et al., Sci ated with nuclear foci. In certain embodiments, the hexa ence, 2006, 314, 130-133). nucleotide repeat expansion is associated with misregulated 0004 To date, a number of genes have been discovered as expression of various genes. In certain embodiments, the causative for classical familial ALS, for example, SOD1, hexanucleotide repeat expansion is associated with nuclear TARDBP, FUS, OPTN, and VCP (Johnson et al., Neuron, retention of various proteins. In certain embodiments, the 2010, 68,857-864; Kwiatkowski et al., Science, 2009, 323, methods described herein are useful for reducing C9CRF72 1205-1208; Maruyama et al., Nature, 2010, 465, 223-226; mRNA, C90RF72 protein levels, and nuclear foci. Such Rosen et al., Nature, 1993, 362, 59-62: Sreedharan et al., reduction can occur in a time-dependent manner or in a dose Science, 2008, 319, 1668-1672; Vance et al., Brain, 2009, dependent manner. In certain embodiments, the methods 129,868-876). Recently, linkage analysis of kindreds involv described herein are useful for normalizing expression of ing multiple cases of ALS, FTD, and ALS-FTD had sug various genes and reducing nuclear retention of various pro gested that there was an important locus for the disease on the teins. short arm of chromosome 9 (Boxer et al., J. Neurol. Neuro 0010 Also provided are methods useful for preventing, Surg. Psychiatry, 2011, 82, 196-203: Morita et al., Neurology, treating, and ameliorating diseases, disorders, and conditions 2006, 66, 839-844; Pearson et al. J. Nerol., 2011, 258, 647 associated with C90RF72. In certain embodiments, such dis 655: Vance et al., Brain, 2006, 129,868-876). The chromo eases, disorders, and conditions associated with C90RF72 some 9p21 ALS-FTD locus in the last major autosomal-domi are neurodegenerative diseases. In certain embodiments, the nant gene whose mutation is causative of ALS. The ALS-FTD neurodegenerative disease is ALS, FTD, corticalbasal degen causing mutation is a large hexanucleotide (GGGGCC) eration syndrome (CBD), atypical Parkinsonian syndrome, repeat expansion in the first intron of the C9CRF72 gene and olivopontocerellar degeneration (OPCD). (Renton et al., Neuron, 2011, 72,257-268; DeJesus-Hernan 0011 Such diseases, disorders, and conditions can have dez et al., Neuron, 2011, 72,245-256). A founder haplotype, one or more risk factors, causes, or outcomes in common. covering the C9CRF72 gene, is present in the majority of Certain risk factors and causes for development of a neuro cases linked to this region (Renton et al., Neuron, 2011, 72, degenerative disease, and, in particular, ALS and FTD, 257-268). This locus on chromosome 9p21 accounts for include genetic predisposition and older age. US 2016/0024496 A1 Jan. 28, 2016 0012. In certain embodiments, methods of treatment comprising one unit and elements and components that com include administering a C9CRF72 specific inhibitor to an prise more than one subunit, unless specifically stated other individual in need thereof. In certain embodiments, the wise.
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