PATENT ABSTRACT
Disclosed herein are compositions and methods for reducing expression of C90RF72 mRNA and protein in an animal with C90RF72 specific inhibitors. Such methods are useful to treat, prevent, or ameliorate neurodegenerative diseases in an individual in need thereof. Such C90RF72 specific inhibitors include antisense compounds. Examples of neurodegenerative diseases that can be treated, prevented, and ameliorated with the administration C90RF72 specific inhibitors include amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), corticalbasal degeneration syndrome (CBD), atypical Parkinsonian syndrome, and olivopontocerellar degeneration (OPCD).

PATENT DESCRIPTION
SEQUENCE LISTING 
       [0001]    The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0211WOSEQ.txt created Oct. 14, 2013, which is 184 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. 
       FIELD 
       [0002]    Provided are compositions and methods for reducing expression of C9ORF72 mRNA and protein in an animal. Such methods are useful to treat, prevent, or ameliorate neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), corticalbasal degeneration syndrome (CBD), atypical Parkinsonian syndrome, and olivopontocerellar degeneration (OPCD). 
       BACKGROUND 
       [0003]    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized clinically by progressive paralysis leading to death from respiratory failure, typically within two to three years of symptom onset (Rowland and Shneider, N. Engl. J. Med., 2001, 344, 1688-1700). ALS is the third most common neurodegenerative disease in the Western world (Hintz et al., Neurology, 2007, 68, 326-337), and there are currently no effective therapies. Approximately 10% of cases are familial in nature, whereas the bulk of patients diagnosed with the disease are classified as sporadic as they appear to occur randomly throughout the population (Chio et al., Neurology, 2008, 70, 533-537). There is growing recognition, based on clinical, genetic, and epidemiological data, that ALS and frontotemporal dementia (FTD) represent an overlapping continuum of disease, characterized pathologically by the presence of TDP-43 positive inclusions throughout the central nervous system (Lillo and Hodges, J. Clin. Neurosci., 2009, 16, 1131-1135; Neumann et al., Science, 2006, 314, 130-133). 
         [0004]    To date, a number of genes have been discovered as causative for classical familial ALS, for example, SOD1, TARDBP, FUS, OPTN, and VCP (Johnson et al., Neuron, 2010, 68, 857-864; Kwiatkowski et al., Science, 2009, 323, 1205-1208; Maruyama et al., Nature, 2010, 465, 223-226; Rosen et al., Nature, 1993, 362, 59-62; Sreedharan et al., Science, 2008, 319, 1668-1672; Vance et al., Brain, 2009, 129, 868-876). Recently, linkage analysis of kindreds involving multiple cases of ALS, FTD, and ALS-FTD had suggested that there was an important locus for the disease on the short arm of chromosome 9 (Boxer et al., J. Neurol. Neurosurg. Psychiatry, 2011, 82, 196-203; Morita et al., Neurology, 2006, 66, 839-844; Pearson et al. J. Nerol., 2011, 258, 647-655; Vance et al., Brain, 2006, 129, 868-876). The chromosome 9p21ALS-FTD locus in the last major autosomal-dominant gene whose mutation is causative of ALS. The ALS-FTD causing mutation is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of the C9ORF72 gene (Renton et al., Neuron, 2011, 72, 257-268; DeJesus-Hernandez et al., Neuron, 2011, 72, 245-256). A founder haplotype, covering the C9ORF72 gene, is present in the majority of cases linked to this region (Renton et al., Neuron, 2011, 72, 257-268). This locus on chromosome 9p21 accounts for nearly half of familial ALS and nearly one-quarter of all ALS cases in a cohort of 405 Finnish patients (Laaksovirta et al, Lancet Neurol., 2010, 9, 978-985). 
         [0005]    A founder haplotype, covering the C9ORF72 gene, is present in the majority of cases linked to this region. 
         [0006]    There are currently no effective therapies to treat such neurodegenerative diseases. Therefore, it is an object to provide compositions and methods for the treatment of such neurodegenerative diseases. 
       SUMMARY 
       [0007]    Provided herein are compositions and methods for modulating levels of C9ORF72 mRNA and protein in cells, tissues, and animals. In certain embodiments, C9ORF72 specific inhibitors modulate expression of C9ORF72 mRNA and protein. In certain embodiments, C9ORF72 specific inhibitors are nucleic acids, proteins, or small molecules. 
         [0008]    In certain embodiments, modulation can occur in a cell or tissue. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human. In certain embodiments, C9ORF72 mRNA levels are reduced. In certain embodiments, C9ORF72 protein levels are reduced. In certain embodiments, certain C9ORF72 mRNA variants are preferentially reduced. In certain embodiments, the C9ORF72 mRNA variants preferentially reduced are variants containing intron 1. In certain embodiments, intron 1 contains a hexanucleotide repeat expansion. In certain embodiments, the hexanucleotide repeat expansion is associated with a C9ORF72 associated disease. In certain embodiments, the hexanucleotide repeat expansion is associated with a C9ORF72 hexanucleotide repeat expansion associated disease. In certain embodiments, the hexanucleotide repeat expansion comprises at least 30 GGGGCC repeats. In certain embodiments, the hexanucleotide repeat expansion is associated with nuclear foci. In certain embodiments, the compositions and methods described herein are useful for reducing C9ORF72 mRNA levels, C9ORF72 protein levels, and nuclear foci. Such reduction can occur in a time-dependent manner or in a dose-dependent manner. 
         [0009]    Also provided are methods useful for preventing, treating, and ameliorating diseases, disorders, and conditions associated with C9ORF72. In certain embodiments, such diseases, disorders, and conditions associated with C9ORF72 are neurodegenerative diseases. In certain embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), corticalbasal degeneration syndrome (CBD), atypical Parkinsonian syndrome, and olivopontocerellar degeneration (OPCD). 
         [0010]    Such diseases, disorders, and conditions can have one or more risk factors, causes, or outcomes in common. Certain risk factors and causes for development of a neurodegenerative disease, and, in particular, ALS and FTD, include genetic predisposition and older age. 
         [0011]    In certain embodiments, methods of treatment include administering a C9ORF72 specific inhibitor to an individual in need thereof. In certain embodiments, the C9ORF72 specific inhibitor is a nucleic acid. In certain embodiments, the nucleic acid is an antisense compound. In certain embodiments, the antisense compound is a single-stranded antisense oligonucleotide. In certain embodiments, the single-stranded antisense oligonucleotide is complementary to a C9ORF72 nucleic acid. 
     
    
     DETAILED DESCRIPTION 
       [0012]    It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Additionally, as used herein, the use of “and” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise. 
         [0013]    The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this disclosure, including, but not limited to, patents, patent applications, published patent applications, articles, books, treatises, and GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety. 
       DEFINITIONS 
       [0014]    Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. 
         [0015]    Unless otherwise indicated, the following terms have the following meanings: 
         [0016]    “2′-O-methoxyethyl group” (also 2′-MOE and 2′-OCH 2 CH 2 —OCH 3  and MOE) refers to an O-methoxy-ethyl modification of the 2′ position of a furanosyl ring. A 2′-O-methoxyethyl modified sugar is a modified sugar. 
         [0017]    “2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-O-methoxyethyl group. 
         [0018]    “5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. A 5-methylcytosine is a modified nucleobase. 
         [0019]    “About” means within ±7% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of C9ORF72”, it is implied that the C9ORF72 levels are inhibited within a range of 63% and 77%. 
         [0020]    “Administered concomitantly” refers to the co-administration of two pharmaceutical agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both pharmaceutical agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both pharmaceutical agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive. 
         [0021]    “Administering” means providing a pharmaceutical agent to an animal, and includes, but is not limited to administering by a medical professional and self-administering. 
         [0022]    “Amelioration” or “ameliorate” or “ameliorating” refers to a lessening of at least one indicator, sign, or symptom of a disease, disorder, or condition. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art. 
         [0023]    “Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees. 
         [0024]    “Antibody” refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, Fab region, and Fc region. 
         [0025]    “Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid. 
         [0026]    “Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds. Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms, including, without limitiation uniform modified oligonucleotides. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms. 
         [0027]    “Antisense inhibition” means reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound Inhibition may be any means including RNase H degradation, such as with a gapmer, and steric blockage, such as with a uniformly modified oligonucleotide. 
         [0028]    “Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding segment of a target nucleic acid. 
         [0029]    “Bicyclic sugar” means a furanosyl ring modified by the bridging of two atoms. A bicyclic sugar is a modified sugar. 
         [0030]    “Bicyclic nucleoside” (also BNA) means a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. 
         [0031]    “C9ORF72 associated disease” means any disease associated with any C9ORF72 nucleic acid or expression product thereof. Such diseases may include a neurodegenerative disease. Such neurodegenerative diseases may include ALS and FTD. 
         [0032]    “C9ORF72 hexanucleotide repeat expansion associated disease” means any disease associated with a C9ORF72 nucleic acid containing a hexanucleotide repeat expansion. In certain embodiments, the hexanucleotide repeat expansion may comprise GGGGCC, GGGGGG, GGGGGC, or GGGGCG repeated at least 30 times. Such diseases may include a neurodegenerative disease. Such neurodegenerative diseases may include ALS and FTD. 
         [0033]    “C9ORF72 nucleic acid” means any nucleic acid encoding C9ORF72. For example, in certain embodiments, a C9ORF72 nucleic acid includes a DNA sequence encoding C9ORF72, an RNA sequence transcribed from DNA encoding C9ORF72 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding C9ORF72. “C9ORF72 mRNA” means an mRNA encoding a C9ORF72 protein. 
         [0034]    “C9ORF72 specific inhibitor” refers to any agent capable of specifically inhibiting the expression of C9ORF72 mRNA and/or C9ORF72 protein at the molecular level. For example, C9ORF72 specific inhibitors include nucleic acids (including antisense compounds), siRNAs, aptamers, antibodies, peptides, small molecules, and other agents capable of inhibiting the expression of C9ORF72 mRNA and/or C9ORF72 protein. Similarly, in certain embodiments, C9ORF72 specific inhibitors may affect other molecular processes in an animal. 
         [0035]    “Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound. 
         [0036]    “cEt” or “constrained ethyl” means a bicyclic nucleoside having a sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH 3 )—O-2′. 
         [0037]    “Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH 3 )—O-2′ bridge. 
         [0038]    “Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleosides is chemically distinct from a region having nucleosides without 2′-O-methoxyethyl modifications. 
         [0039]    “Chimeric antisense compound” means an antisense compound that has at least two chemically distinct regions. 
         [0040]    “Co-administration” means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration. 
         [0041]    “Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid. 
         [0042]    “Contiguous nucleobases” means nucleobases immediately adjacent to each other. 
         [0043]    “Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition may be a liquid, e.g. saline solution. 
         [0044]    “Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month. 
         [0045]    “Effective amount” means the amount of pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the pharmaceutical agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual&#39;s medical condition, and other relevant factors. 
         [0046]    “Expression” means conversion of the information from a C9ORF72 gene into mRNA via transcription and then to protein via translation. Expression may result in a phenotypic manifestation of the C9ORF72 gene. 
         [0047]    “Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid. 
         [0048]    “Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as a “gap” and the external regions may be referred to as the “wings.” 
         [0049]    “Gap-narrowed” means a chimeric antisense compound having a gap segment of 9 or fewer contiguous 2′-deoxyribonucleosides positioned between and immediately adjacent to 5′ and 3′ wing segments having from 1 to 6 nucleosides. 
         [0050]    “Gap-widened” means a chimeric antisense compound having a gap segment of 12 or more contiguous 2′-deoxyribonucleosides positioned between and immediately adjacent to 5′ and 3′ wing segments having from 1 to 6 nucleosides. 
         [0051]    “Hexanucleotide repeat expansion” means a series of six bases (for example, GGGGCC, GGGGGG, GGGGCG, or GGGGGC) repeated at least twice. In certain embodiments, the hexanucleotide repeat expansion may be located in intron 1 of a C9ORF72 nucleic acid. In certain embodiments, a pathogenic hexanucleotide repeat expansion includes at least 30 repeats of GGGGCC, GGGGGG, GGGGCG, or GGGGGC in a C9ORF72 nucleic acid and is associated with disease. In certain embodiments, the repeats are consecutive. In certain embodiments, the repeats are interrupted by 1 or more nucleobases. In certain embodiments, a wild-type hexanucleotide repeat expansion includes 23 or fewer repeats of GGGGCC, GGGGGG, GGGGCG, or GGGGGC in a C9ORF72 nucleic acid. In certain embodiments, the repeats are consecutive. In certain embodiments, the repeats are interrupted by 1 or more nucleobases. 
         [0052]    “Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid. 
         [0053]    “Identifying an animal having a C9ORF72 associated disease” means identifying an animal having been diagnosed with a C9ORF72 associated disease or predisposed to develop a C9ORF72 associated disease. Individuals predisposed to develop a C9ORF72 associated disease include those having one or more risk factors for developing a C9ORF72 associated disease, including, having a personal or family history or genetic predisposition of one or more C9ORF72 associated diseases. Such identification may be accomplished by any method including evaluating an individual&#39;s medical history and standard clinical tests or assessments, such as genetic testing. 
         [0054]    “Immediately adjacent” means there are no intervening elements between the immediately adjacent elements. 
         [0055]    “Individual” means a human or non-human animal selected for treatment or therapy. 
         [0056]    “Inhibiting C9ORF72” means reducing expression of C9ORF72 mRNA and/or protein levels in the presence of a C9ORF72 specific inhibitor, including a C9ORF72 antisense oligonucleotide, as compared to expression of C9ORF72 mRNA and/or protein levels in the absence of a C9ORF72 specific inhibitor, such as a C9ORF72 antisense oligonucleotide. 
         [0057]    “Internucleoside linkage” refers to the chemical bond between nucleosides. 
         [0058]    “Linked nucleosides” means adjacent nucleosides which are bonded together. 
         [0059]    “Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid. 
         [0060]    “Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e., a phosphodiester internucleoside bond). 
         [0061]    “Modified nucleobase” refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). 
         [0062]    “Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase. A “modified nucleoside” means a nucleoside having, independently, a modified sugar moiety or modified nucleobase. 
         [0063]    “Modified oligonucleotide” means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, or a modified nucleobase. 
         [0064]    “Modified sugar” refers to a substitution or change from a natural sugar. 
         [0065]    “Motif” means the pattern of chemically distinct regions in an antisense compound. 
         [0066]    “Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage. 
         [0067]    “Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH). 
         [0068]    “Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). 
         [0069]    “Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid. 
         [0070]    “Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification. 
         [0071]    “Nucleoside” means a nucleobase linked to a sugar. 
         [0072]    “Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo, or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. 
         [0073]    “Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside. 
         [0074]    “Oligomeric compound” or “oligomer” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule. 
         [0075]    “Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another. 
         [0076]    “Parenteral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebroventricular administration. 
         [0077]    “Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins. 
         [0078]    “Pharmaceutical agent” means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, in certain embodiments an antisense oligonucleotide targeted to C9ORF72 is a pharmaceutical agent. 
         [0079]    “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more pharmaceutical agents and a sterile aqueous solution. 
         [0080]    “Pharmaceutically acceptable derivative” encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein. 
         [0081]    “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto. 
         [0082]    “Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage (P═S) is a modified internucleoside linkage. 
         [0083]    “Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. 
         [0084]    In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound. 
         [0085]    “Prevent” or “preventing” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or condition. 
         [0086]    “Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form within the body or cells thereof by the action of endogenous enzymes or other chemicals or conditions. 
         [0087]    “Side effects” means physiological responses attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. 
         [0088]    “Single-stranded oligonucleotide” means an oligonucleotide which is not hybridized to a complementary strand. 
         [0089]    “Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments. 
         [0090]    “Targeting” or “targeted” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect. 
         [0091]    “Target nucleic acid,” “target RNA,” and “target RNA transcript” all refer to a nucleic acid capable of being targeted by antisense compounds. 
         [0092]    “Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment. 
         [0093]    “Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual. 
         [0094]    “Treat” or “treating” refers to administering a pharmaceutical composition to effect an alteration or improvement of a disease, disorder, or condition. 
         [0095]    “Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside). 
       CERTAIN EMBODIMENTS 
       [0096]    Certain embodiments provide methods for decreasing C9ORF72 mRNA and protein expression. 
         [0097]    Certain embodiments provide methods for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with C9ORF72 in an individual in need thereof. Also contemplated are methods for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with C9ORF72. C9ORF72 associated diseases, disorders, and conditions include neurodegenerative diseases. In certain embodiments, the neurodegenerative disease may be ALS or FTD. In certain embodiments, the neurodegenerative disease may be familial or sporadic. 
         [0098]    Certain embodiments provide for the use of a C9ORF72 specific inhibitor for treating, preventing, or ameliorating a C9ORF72 associated disease. Certain embodiments provide for the use of a C9ORF72 specific inhibitor for treating, preventing, or ameliorating a C9ORF72 hexanucleotide repeat expansion associated disease. In certain embodiments, the hexanucleotide repeat expansion may comprise GGGGCC, GGGGGG, GGGGGC, or GGGGCG. In certain embodiments, C9ORF72 specific inhibitors are nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of C9ORF72 mRNA and/or C9ORF72 protein. 
         [0099]    Described herein are compounds comprising a single-stranded antisense oligonucleotide complementary to a C9ORF72 nucleic acid or a C9ORF72 homolog nucleic acid. 
         [0100]    In certain embodiments, the C9ORF72 nucleic acid is a human C9ORF72 nucleic acid. 
         [0101]    In certain embodiments, the C9ORF72 nucleic acid contains a hexanucleotide repeat expansion. 
         [0102]    In certain embodiments, the C9ORF72 nucleic acid does not contain a hexanucleotide repeat expansion. 
         [0103]    In certain embodiments, the single-stranded antisense oligonucleotide is specifically hybridizable to a human C9ORF72 nucleic acid. 
         [0104]    In certain embodiments, the single-stranded antisense oligonucleotide is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to an equal length portion of a human C9ORF72 nucleic acid. 
         [0105]    In certain embodiments, the single-stranded antisense oligonucleotide is complementary to any of exon, an intron, the 5′ UTR, the 3′ UTR, a repeat region, a splice junction, an exon:exon splice junction, an exonic splicing silencer (ESS), an exonic splicing enhancer (ESE), exon 1a, exon 1b, exon 1c, exon 1d, exon 1e, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, or intron 10 of a human C9ORF72 nucleic acid. 
         [0106]    Described herein are compounds comprising a single-stranded antisense oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases of SEQ ID NO: 30-369. 
         [0107]    In certain embodiments, the single-stranded antisense oligonucleotide comprises at least one modification. 
         [0108]    In certain embodiments, the single-stranded antisense oligonucleotide comprises at least one modified internucleoside linkage. 
         [0109]    In certain embodiments, each internucleoside linkage of the single-stranded antisense oligonucleotide is a modified internucleoside linkage. 
         [0110]    In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. 
         [0111]    In certain embodiments, the single-stranded antisense oligonucleotide comprises at least one modified nucleoside. 
         [0112]    In certain embodiments, the single-stranded antisense oligonucleotide comprises at least one modified nucleoside having a modified sugar. 
         [0113]    In certain embodiments, the single-stranded antisense oligonucleotide comprises at least one modified nucleoside comprising a bicyclic sugar. 
         [0114]    In certain embodiments, the bicyclic sugar comprises a 4′ to 2′ bridge selected from among: 4′-(CH 2 ) n —O-2′ bridge, wherein n is 1 or 2; and 4′-CH 2 —O—CH 2 -2′. 
         [0115]    In certain embodiments, the bicyclic sugar comprises a 4′-CH(CH 3 )—O-2′ bridge. 
         [0116]    In certain embodiments, the at least one modified nucleoside having a modified sugar comprises a non-bicyclic 2′-modified modified sugar moiety. 
         [0117]    In certain embodiments, the 2′-modified sugar moiety comprises a 2′-O-methoxyethyl group. 
         [0118]    In certain embodiments, the 2′-modified sugar moiety comprises a 2′-O-methyl group. 
         [0119]    In certain embodiments, the at least one modified nucleoside having a modified sugar comprises a sugar surrogate. 
         [0120]    In certain embodiments, the sugar surrogate is a morpholino. 
         [0121]    In certain embodiments, the sugar surrogate is a peptide nucleic acid. 
         [0122]    In certain embodiments, each nucleoside is modified. 
         [0123]    In certain embodiments, the single-stranded antisense oligonucleotide comprises at least one modified nucleobase. 
         [0124]    In certain embodiments, the modified nucleobase is a 5′-methylcytosine. 
         [0125]    In certain embodiments, the single-stranded antisense oligonucleotide comprises: 
         [0000]    a gap segment consisting of linked deoxynucleosides;
 
a 5′ wing segment consisting of linked nucleosides;
 
a 3′ wing segment consisting of linked nucleosides;
 
wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
 
         [0126]    In certain embodiments, the single-stranded antisense oligonucleotide comprises: 
         [0000]    a gap segment consisting of ten linked deoxynucleosides;
 
a 5′ wing segment consisting of five linked nucleosides;
 
a 3′ wing segment consisting of five linked nucleosides;
 
wherein the gap segment is positioned immediately adjacent and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.
 
         [0127]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 15 linked nucleosides. 
         [0128]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 16 linked nucleosides. 
         [0129]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 17 linked nucleosides. 
         [0130]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 18 linked nucleosides. 
         [0131]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 19 linked nucleosides. 
         [0132]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 20 linked nucleosides. 
         [0133]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 21 linked nucleosides. 
         [0134]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 22 linked nucleosides. 
         [0135]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 23 linked nucleosides. 
         [0136]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 24 linked nucleosides. 
         [0137]    In certain embodiments, the single-stranded antisense oligonucleotide consists of 25 linked nucleosides. 
         [0138]    Described herein are uses of the compound for the manufacture of a medicament for treating a neurodegenerative disease. 
         [0139]    Provided herein are methods of preferentially inhibiting expression of mRNA transcripts containing a hexanucleotide repeat expansion by contacting a cell with an antisense oligonucleotide targeting upstream of exon 1B. 
       Antisense Compounds 
       [0140]    Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. 
         [0141]    In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. 
         [0142]    In certain embodiments, an antisense compound targeted to a C9ORF72 nucleic acid is 12 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits. In certain embodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 linked subunits. In certain embodiments, the antisense compounds are 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleosides. 
         [0143]    In certain embodiments antisense oligonucleotides targeted to a C9ORF72 nucleic acid may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a C9ORF72 nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end. 
         [0144]    When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end. 
         [0145]    It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches. 
         [0146]    Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo. 
         [0147]    Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides. 
       Antisense Compound Motifs 
       [0148]    In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases. 
         [0149]    Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex. 
         [0150]    Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE, and 2′-O—CH 3 , among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH 2 )n-O-2′ bridge, where n=1 or n=2 and 4′-CH 2 —O—CH 2 -2′). Preferably, each distinct region comprises uniform sugar moieties. The wing-gap-wing motif is frequently described as “X—Y—Z”, where “X” represents the length of the 5′ wing region, “Y” represents the length of the gap region, and “Z” represents the length of the 3′ wing region. As used herein, a gapmer described as “X—Y—Z” has a configuration such that the gap segment is positioned immediately adjacent to each of the 5′ wing segment and the 3′ wing segment. Thus, no intervening nucleotides exist between the 5′ wing segment and gap segment, or the gap segment and the 3′ wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments, X and Z are the same, in other embodiments they are different. In a preferred embodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, gapmers described herein include, but are not limited to, for example 5-10-5, 5-10-4, 4-10-4, 4-10-3, 3-10-3, 2-10-2, 5-9-5, 5-9-4, 4-9-5, 5-8-5, 5-8-4, 4-8-5, 5-7-5, 4-7-5, 5-7-4, or 4-7-4. 
         [0151]    In certain embodiments, the antisense compound has a “wingmer” motif, having a wing-gap or gap-wing configuration, i.e. an X—Y or Y—Z configuration as described above for the gapmer configuration. Thus, wingmer configurations described herein include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, 5-13, 5-8, or 6-8. 
         [0152]    In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid possess a 5-10-5 gapmer motif. 
         [0153]    In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid possess a 5-10-4 gapmer motif. 
         [0154]    In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid possess a 4-10-4 gapmer motif. 
         [0155]    In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid possess a 4-10-3 gapmer motif. 
         [0156]    In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid possess a 5-9-5 gapmer motif. 
         [0157]    In certain embodiments, an antisense compound targeted to a C9ORF72 nucleic acid has a gap-narrowed motif. In certain embodiments, a gap-narrowed antisense oligonucleotide targeted to a C9ORF72 nucleic acid has a gap segment of 9, 8, 7, or 6 2′-deoxynucleotides positioned immediately adjacent to and between wing segments of 5, 4, 3, 2, or 1 chemically modified nucleosides. In certain embodiments, the chemical modification comprises a bicyclic sugar. In certain embodiments, the bicyclic sugar comprises a 4′ to 2′ bridge selected from among: 4′-(CH 2 ) n —O-2′ bridge, wherein n is 1 or 2; and 4′-CH 2 —O—CH 2 -2′. In certain embodiments, the bicyclic sugar is comprises a 4′-CH(CH 3 )—O-2′ bridge. In certain embodiments, the chemical modification comprises a non-bicyclic 2′-modified sugar moiety. In certain embodiments, the non-bicyclic 2′-modified sugar moiety comprises a 2′-O-methylethyl group or a 2′-O-methyl group. 
         [0158]    In certain embodiments, an antisense compound targeted to a C9ORF72 nucleic acid is uniformly modified. In certain embodiments, the antisense compound comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides. In certain embodiments, each nucleosides is chemically modified. In certain embodiments, the chemical modification comprises a non-bicyclic 2′-modified sugar moiety. In certain embodiments, the 2′-modified sugar moiety comprises a 2′-O-methoxyethyl group. In certain embodiments, the 2′-modified sugar moiety comprises a 2′-O-methyl group. In certain embodiments, uniformly modified antisense compounds may target C9ORF72, or any portion thereof, such as a hexanucleotide repeat expansion. In certain embodiments, targeting the hexanucleotide repeat expansion with a unformily modified antisense compound reduces the repeat RNA by blocking the interaction with RNA binding proteins. In certain embodiments, this results in the toxic RNA being absent from foci and benig degraded instead. 
       Target Nucleic Acids, Target Regions and Nucleotide Sequences 
       [0159]    Nucleotide sequences that encode C9ORF72 include, without limitation, the following: the complement of GENBANK Accession No. NM — 001256054.1 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT — 008413.18 truncated from nucleobase 27535000 to 27565000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No. BQ068108.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. NM — 018325.3 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No. DN993522.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No. NM — 145005.5 (incorporated herein as SEQ ID NO: 6), GENBANK Accession No. DB079375.1 (incorporated herein as SEQ ID NO: 7), GENBANK Accession No. BU194591.1 (incorporated herein as SEQ ID NO: 8), Sequence Identifier 4141 — 014_A (incorporated herein as SEQ ID NO: 9), and Sequence Identifier 4008 — 73_A (incorporated herein as SEQ ID NO: 10). 
         [0160]    It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif. 
         [0161]    In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for C9ORF72 can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the same target region. 
         [0162]    Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid. 
         [0163]    A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain emodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5′ target sites or 3′ target sites listed herein. 
         [0164]    Suitable target segments may be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifcally exclude a certain structurally defined region such as the start codon or stop codon. 
         [0165]    The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences). 
         [0166]    There may be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within a target region. In certain embodiments, reductions in C9ORF72 mRNA levels are indicative of inhibition of C9ORF72 expression. Reductions in levels of a C9ORF72 protein are also indicative of inhibition of target mRNA expression. Reduction in the presence of expanded C9ORF72 RNA foci are indicative of inhibition of C9ORF72 epxression. Further, phenotypic changes are indicative of inhibition of C9ORF72 expression. For example, improved motor function and respiration may be indicative of inhibition of C9ORF72 expression. 
       Hybridization 
       [0167]    In some embodiments, hybridization occurs between an antisense compound disclosed herein and a C9ORF72 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules. 
         [0168]    Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized. 
         [0169]    Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a C9ORF72 nucleic acid. 
       Complementarity 
       [0170]    An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a C9ORF72 nucleic acid). 
         [0171]    Non-complementary nucleobases between an antisense compound and a C9ORF72 nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a C9ORF72 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). 
         [0172]    In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a C9ORF72 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods. 
         [0173]    For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489). 
         [0174]    In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e., 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a C9ORF72 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence. 
         [0175]    The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e., linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide. 
         [0176]    In certain embodiments, antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a C9ORF72 nucleic acid, or specified portion thereof. 
         [0177]    In certain embodiments, antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a C9ORF72 nucleic acid, or specified portion thereof. 
         [0178]    The antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values. 
       Identity 
       [0179]    The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared. 
         [0180]    In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein. 
         [0181]    In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid. 
         [0182]    In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid. 
       Modifications 
       [0183]    A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide. 
         [0184]    Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity. 
         [0185]    Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides. 
       Modified Internucleoside Linkages 
       [0186]    The naturally occuring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases. 
         [0187]    Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known. 
         [0188]    In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are interspersed throughout the antisense compound. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage. 
       Modified Sugar Moieties 
       [0189]    Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R 1 )(R 2 ) (R, R 1  and R 2  are each independently H, C 1 -C 12  alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group). 
         [0190]    Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH 3 , 2′-OCH 2 CH 3 , 2′-OCH 2 CH 2 F and 2′-O(CH 2 ) 2 OCH 3  substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C 1 -C 10  alkyl, OCF 3 , OCH 2 F, O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 —O—N(R m )(R n ), O—CH 2 —C(═O)—N(R m )(R n ), and O—CH 2 —C(═O)—N(R 1 )—(CH 2 ) 2 —N(R m )(R n ), where each R l , R m  and R n  is, independently, H or substituted or unsubstituted C 1 -C 10  alkyl. 
         [0191]    As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4′-(CH 2 )—O-2′ (LNA); 4′-(CH 2 )—S-2; 4′-(CH 2 ) 2 —O-2′ (ENA); 4′-CH(CH 3 )—O-2′ and 4′-CH(CH 2 OCH 3 )—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH 3 )(CH 3 )—O-2′ (and analogs thereof see published International Application WO/2009/006478, published Jan. 8, 2009); 4′-CH 2 —N(OCH 3 )-2′ (and analogs thereof see published International Application WO/2008/150729, published Dec. 11, 2008); 4′-CH 2 —O—N(CH 3 )-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH 2 —N(R)—O-2′, wherein R is H, C 1 -C 12  alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH 2 —C(H)(CH 3 )-2′ (see Chattopadhyaya et al.,  J. Org. Chem.,  2009, 74, 118-134); and 4′-CH 2 —C—(═CH 2 )-2′ (and analogs thereof see published International Application WO 2008/154401, published on Dec. 8, 2008). 
         [0192]    Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al.,  Chem. Commun.,  1998, 4, 455-456; Koshkin et al.,  Tetrahedron,  1998, 54, 3607-3630; Wahlestedt et al.,  Proc. Natl. Acad. Sci. U.S.A.,  2000, 97, 5633-5638; Kumar et al.,  Bioorg. Med. Chem. Lett.,  1998, 8, 2219-2222; Singh et al.,  J. Org. Chem.,  1998, 63, 10035-10039; Srivastava et al.,  J. Am. Chem. Soc.,  2007, 129(26) 8362-8379; Elayadi et al.,  Curr. Opinion Invest. Drugs,  2001, 2, 558-561; Braasch et al.,  Chem. Biol.,  2001, 8, 1-7; and Orum et al.,  Curr. Opinion Mol. Ther.,  2001, 3, 239-243; U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos. 60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; and 61/099,844; Published PCT International applications WO 1994/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; and WO 2009/006478. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226). 
         [0193]    In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R a )(R b )] n —, —C(R a )═C(R b )—, —C(R a )═N—, —C(═O)—, —C(═NR a )—, —C(═S)—, —O—, —Si(R a ) 2 —, —S(═O) x —, and —N(R a )—; 
         [0194]    wherein: 
         [0195]    x is 0, 1, or 2; 
         [0196]    n is 1, 2, 3, or 4; 
         [0197]    each R a  and R b  is, independently, H, a protecting group, hydroxyl, C 1 -C 12  alkyl, substituted C 1 -C 12  alkyl, C 2 -C 12  alkenyl, substituted C 2 -C 12  alkenyl, C 2 -C 12  alkynyl, substituted C 2 -C 12  alkynyl, C 5 -C 20  aryl, substituted C 5 -C 20  aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C 5 -C 7  alicyclic radical, substituted C 5 -C 7  alicyclic radical, halogen, OJ 1 , NJ 1 J 2 , SJ 1 , N 3 , COOJ 1 , acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O) 2 -J 1 ), or sulfoxyl (S(═O)-J 1 ); and 
         [0198]    each J 1  and J 2  is, independently, H, C 1 -C 12  alkyl, substituted C 1 -C 12  alkyl, C 2 -C 12  alkenyl, substituted C 2 -C 12  alkenyl, C 2 -C 12  alkynyl, substituted C 2 -C 12  alkynyl, C 5 -C 20  aryl, substituted C 5 -C 20  aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C 1 -C 12  aminoalkyl, substituted C 1 -C 12  aminoalkyl or a protecting group. 
         [0199]    In certain embodiments, the bridge of a bicyclic sugar moiety is —[C(R a )(R b )] n —, —[C(R a )(R b )] n —O—, —C(R a R b )—N(R)—O— or —C(R a R b )—O—N(R)—. In certain embodiments, the bridge is 4′-CH 2 -2′, 4′-(CH 2 ) 2 -2′, 4′-(CH 2 ) 3 -2′, 4′-CH 2 —O-2′, 4′-(CH 2 ) 2 —O-2′, 4′-CH 2 —O—N(R)-2′ and 4′-CH 2 —N(R)—O-2′- wherein each R is, independently, H, a protecting group or C 1 -C 12  alkyl. 
         [0200]    In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH 2 —O-2′) BNA&#39;s have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al.,  Nucleic Acids Research,  2003, 21, 6365-6372). 
         [0201]    In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4′-CH 2 —O-2′) BNA, (B) β-D-methyleneoxy (4′-CH 2 —O-2′) BNA, (C) ethyleneoxy (4′-(CH 2 ) 2 —O-2′) BNA, (D) aminooxy (4‘-CH 2 —O—N(R)-2’) BNA, (E) oxyamino (4′-CH 2 —N(R)—O-2′) BNA, and (F) methyl(methyleneoxy) (4′-CH(CH 3 )—O-2′) BNA, (G) methylene-thio (4′-CH 2 —S-2′) BNA, (H) methylene-amino (4′-CH 2 —N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH 2 —CH(CH 3 )-2′) BNA, and (J) propylene carbocyclic (4′-(CH 2 ) 3 -2′) BNA as depicted below. 
         [0000]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
         [0000]    wherein Bx is the base moiety and R is independently H, a protecting group or C 1 -C 12  alkyl. 
         [0202]    In certain embodiments, bicyclic nucleosides are provided having Formula I: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein: 
         [0203]    Bx is a heterocyclic base moiety; 
         [0204]    -Q a -Q b -Q c - is —CH 2 —N(R c )—CH 2 —, —C(═O)—N(R c )—CH 2 —, —CH 2 —O—N(R c )—, —CH 2 —N(R c )—O— or —N(R c )—O—CH 2 ; 
         [0205]    R c  is C 1 -C 12  alkyl or an amino protecting group; and 
         [0206]    T a  and T b  are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium. 
         [0207]    In certain embodiments, bicyclic nucleosides are provided having Formula II: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein: 
         [0208]    Bx is a heterocyclic base moiety; 
         [0209]    T a  and T b  are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; 
         [0210]    Z a  is C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, substituted C 1 -C 6  alkyl, substituted C 2 -C 6  alkenyl, substituted C 2 -C 6  alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio. 
         [0211]    In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ c , NJ c J d , SJ c , N 3 , OC(═X)J c , and NJ e C(═X)NJ c J d , wherein each J c , J d  and J e  is, independently, H, C 1 -C 6  alkyl, or substituted C 1 -C 6  alkyl and X is O or NJ c . 
         [0212]    In certain embodiments, bicyclic nucleosides are provided having Formula III: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein: 
         [0213]    Bx is a heterocyclic base moiety; 
         [0214]    T a  and T b  are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; 
         [0215]    Z b  is C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, substituted C 1 -C 6  alkyl, substituted C 2 -C 6  alkenyl, substituted C 2 -C 6  alkynyl or substituted acyl (C(═O)—). 
         [0216]    In certain embodiments, bicyclic nucleosides are provided having Formula IV: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein: 
         [0217]    Bx is a heterocyclic base moiety; 
         [0218]    T a  and T b  are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; 
         [0219]    R d  is C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, C 2 -C 6  alkenyl, substituted C 2 -C 6  alkenyl, C 2 -C 6  alkynyl or substituted C 2 -C 6  alkynyl; 
         [0220]    each q a , q b , q c  and q d  is, independently, H, halogen, C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, C 2 -C 6  alkenyl, substituted C 2 -C 6  alkenyl, C 2 -C 6  alkynyl or substituted C 2 -C 6  alkynyl, C 1 -C 6  alkoxyl, substituted C 1 -C 6  alkoxyl, acyl, substituted acyl, C 1 -C 6  aminoalkyl or substituted C 1 -C 6  aminoalkyl; 
         [0221]    In certain embodiments, bicyclic nucleosides are provided having Formula V: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein: 
         [0222]    Bx is a heterocyclic base moiety; 
         [0223]    T a  and T b  are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; 
         [0224]    q a , q b , q e  and q f  are each, independently, hydrogen, halogen, C 1 -C 12  alkyl, substituted C 1 -C 12  alkyl, C 2 -C 12  alkenyl, substituted C 2 -C 12  alkenyl, C 2 -C 12  alkynyl, substituted C 2 -C 12  alkynyl, C 1 -C 12  alkoxy, substituted C 1 -C 12  alkoxy, OJ j , SJ j , SOJ j , SO 2 J j , NJ j J k , N 3 , CN, C(═O)OJ j , C(═O)NJ j J k , C(═O)J j , O—C(═O)NJ j J k , N(H)C(═NH)NJ j J k , N(H)C(═O)NJ j J k  or N(H)C(═S)NJ j J k ; 
         [0225]    or q e  and q f  together are ═C(q g )(q h ); 
         [0226]    q g  and q h  are each, independently, H, halogen, C 1 -C 12  alkyl or substituted C 1 -C 12  alkyl. 
         [0227]    The synthesis and preparation of the methyleneoxy (4′-CH 2 —O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al.,  Tetrahedron,  1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226. 
         [0228]    Analogs of methyleneoxy (4′-CH 2 —O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar et al.,  Bioorg. Med. Chem. Lett.,  1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al.,  J. Org. Chem.,  1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA&#39;s have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported. 
         [0229]    In certain embodiments, bicyclic nucleosides are provided having Formula VI: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein: 
         [0230]    Bx is a heterocyclic base moiety; 
         [0231]    T a  and T b  are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; 
         [0232]    each q i , q j , q k  and q l  is, independently, H, halogen, C 1 -C 12  alkyl, substituted C 1 -C 12  alkyl, C 2 -C 12  alkenyl, substituted C 2 -C 12  alkenyl, C 2 -C 12  alkynyl, substituted C 2 -C 12  alkynyl, C 1 -C 12  alkoxyl, substituted C 1 -C 12  alkoxyl, OJ j , SJ j , SOJ j , SO 2 J j , NJ j J k , N 3 , CN, C(═O)OJ j , C(═O)NJ j J k , C(═O)J j , O—C(═O)NJ j J k , N(H)C(═NH)NJ j J k , N(H)C(═O)NJ j J k  or N(H)C(═S)NJ j J k ; and 
         [0233]    q i  and q j  or q l  and q k  together are ═C(q g )(q h ), wherein q g  and q h  are each, independently, H, halogen, C 1 -C 12  alkyl or substituted C 1 -C 12  alkyl. 
         [0234]    One carbocyclic bicyclic nucleoside having a 4′-(CH 2 ) 3 -2′ bridge and the alkenyl analog bridge 4′-CH═CH—CH 2 -2′ have been described (Freier et al.,  Nucleic Acids Research,  1997, 25(22), 4429-4443 and Albaek et al.,  J. Org. Chem.,  2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al.,  J. Am. Chem. Soc.,  2007, 129(26), 8362-8379). 
         [0235]    As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring. 
         [0236]    As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position. 
         [0237]    As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n F, O(CH 2 ) n ONH 2 , OCH 2 C(═O)N(H)CH 3 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C 1 -C 12  alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, F, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modifed nucleosides comprise a 2′-MOE side chain (Baker et al.,  J. Biol. Chem.,  1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin,  Helv. Chim. Acta,  1995, 78, 486-504; Altmann et al.,  Chimia,  1996, 50, 168-176; Altmann et al.,  Biochem. Soc. Trans.,  1996, 24, 630-637; and Altmann et al.,  Nucleosides Nucleotides,  1997, 16, 917-926). 
         [0238]    As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann,  Bioorg. Med. Chem.,  2002, 10, 841-854), fluoro HNA (F-HNA) or those compounds having Formula VII: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII: 
         [0239]    Bx is a heterocyclic base moiety; 
         [0240]    T a  and T b  are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T a  and T b  is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T a  and T b  is H, a hydroxyl protecting group, a linked conjugate group or a 5′ or 3′-terminal group; 
         [0241]    q 1 , q 2 , q 3 , q 4 , q 5 , q 6  and q 7  are each independently, H, C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, C 2 -C 6  alkenyl, substituted C 2 -C 6  alkenyl, C 2 -C 6  alkynyl or substituted C 2 -C 6  alkynyl; and each of R 1  and R 2  is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJ 1 J 2 , SJ 1 , N 3 , OC(═X)J 1 , OC(═X)NJ 1 J 2 , NJ 3 C(═X)NJ 1 J 2  and CN, wherein X is O, S or NJ 1  and each J 1 , J 2  and J 3  is, independently, H or C 1 -C 6  alkyl. 
         [0242]    In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q 1 , q 2 , q 3 , q 4 , q 5 , q 6  and q 7  are each H. In certain embodiments, at least one of q 1 , q 2 , q 3 , q 4 , q 5 , q 6  and q 7  is other than H. In certain embodiments, at least one of q 1 , q 2 , q 3 , q 4 , q 5 , q 6  and q 7  is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R 1  and R 2  is fluoro. In certain embodiments, R 1  is fluoro and R 2  is H; R 1  is methoxy and R 2  is H, and R 1  is H and R 2  is methoxyethoxy. 
         [0243]    As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′ substituents, such as allyl, amino, azido, thio, O-allyl, O—C 1 -C 10  alkyl, —OCF 3 , O—(CH 2 ) 2 —O—CH 3 , 2′-O(CH 2 ) 2 SCH 3 , O—(CH 2 ) 2 —O—N(R m )(R n ), or O—CH 2 —C(═O)—N(R m )(R n ), where each R m  and R n  is, independently, H or substituted or unsubstituted C 1 -C 10  alkyl. 2′-modifed nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase. 
         [0244]    As used herein, “2′-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2′ position. 
         [0245]    As used herein, “2′-OMe” or “2′-OCH 3 ” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH 3  group at the 2′ position of the sugar ring. 
         [0246]    As used herein, “MOE” or “2′-MOE” or “2′-OCH 2 CH 2 OCH 3 ” or “2′-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a —OCH 2 CH 2 OCH 3  group at the 2′ position of the sugar ring. 
         [0247]    As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA). 
         [0248]    Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann,  Bioorg. Med. Chem.,  2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity. 
         [0249]    Methods for the preparations of modified sugars are well known to those skilled in the art. 
         [0250]    In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target. 
         [0251]    In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4′-CH(CH 3 )—O-2′) bridging group. In certain embodiments, the (4′-CH(CH 3 )—O-2′) modified nucleosides are arranged throughout the wings of a gapmer motif. 
       Compositions and Methods for Formulating Pharmaceutical Compositions 
       [0252]    Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered. 
         [0253]    An antisense compound targeted to a C9ORF72 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a C9ORF72 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide. 
         [0254]    Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. 
         [0255]    A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound. 
       Conjugated Antisense Compounds 
       [0256]    Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. 
         [0257]    Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003. 
       Cell Culture and Antisense Compounds Treatment 
       [0258]    The effects of antisense compounds on the level, activity or expression of C9ORF72 nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and are cultured according to the vendor&#39;s instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, and primary hepatocytes. 
       In Vitro Testing of Antisense Oligonucleotides 
       [0259]    Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds. 
         [0260]    In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture. 
         [0261]    One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide. 
         [0262]    Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide. 
         [0263]    Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation. 
         [0264]    Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments. 
         [0265]    The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation. 
       RNA Isolation 
       [0266]    RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer&#39;s recommended protocols. 
       Analysis of Inhibition of Target Levels or Expression 
       [0267]    Inhibition of levels or expression of a C9ORF72 nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer&#39;s instructions. 
       Quantitative Real-Time PCR Analysis of Target RNA Levels 
       [0268]    Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer&#39;s instructions. Methods of quantitative real-time PCR are well known in the art. 
         [0269]    Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carried out by methods well known to those skilled in the art. 
         [0270]    Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). Methods of RNA quantification by RIBOGREEN are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence. 
         [0271]    Probes and primers are designed to hybridize to a C9ORF72 nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.). 
       Analysis of Protein Levels 
       [0272]    Antisense inhibition of C9ORF72 nucleic acids can be assessed by measuring C9ORF72 protein levels. Protein levels of C9ORF72 can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Antibodies useful for the detection of mouse, rat, monkey, and human C9ORF72 are commercially available. 
       In Vivo Testing of Antisense Compounds 
       [0273]    Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of C9ORF72 and produce phenotypic changes, such as, improved motor function and respiration. In certain embodiments, motor function is measured by rotarod, grip strength, pole climb, open field performance, balance beam, hindpaw footprint testing in the animal. In certain embodiments, respiration is measured by whole body plethysmograph, invasive resistance, and compliance measurements in the animal. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight. Following a period of treatment with antisense oligonucleotides, RNA is isolated from CNS tissue or CSF and changes in C9ORF72 nucleic acid expression are measured. 
       Targeting C9ORF72 
       [0274]    Antisense oligonucleotides described herein may hybridize to a C9ORF72 nucleic acid in any stage of RNA processing. For example, described herein are antisense oligonucleotides that are complementary to a pre-mRNA or a mature mRNA. Additionally, antisense oligonucleotides described herein may hybridize to any element of a C9ORF72 nucleic acid. For example, described herein are antisense oligonucleotides that are complementary to an exon, an intron, the 5′ UTR, the 3′ UTR, a repeat region, a hexanucleotide repeat expansion, a splice junction, an exon:exon splice junction, an exonic splicing silencer (ESS), an exonic splicing enhancer (ESE), exon 1a, exon 1b, exon 1c, exon 1d, exon 1e, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon11, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, or intron 10 of a C9ORF72 nucleic acid. 
         [0275]    In certain embodiments, antisense oligonucleotides described herein hybridize to all variants of C9ORF72. In certain embodiments, the antisense oligonucleotides described herein selectively hybridize to certain variants of C9ORF72. In certain embodiments, the antisense oligonucleotides described herein selectively hybridize to variants of C9ORF72 containing a hexanucleotide repeat expansion. In certain embodiments, such variants of C9ORF72 containing a hexanucleotide repeat expansion include SEQ ID NO: 1-3 and 6-10. In certain embodiments, such hexanucleotide repeat expansion comprises at least 30 repeats of any of GGGGCC, GGGGGG, GGGGGC, or GGGGCG. 
         [0276]    In certain embodiments, the antisense oligonucleotides described herein inhibit expression of all variants of C9ORF72. In certain embodiments, the antisense oligonucleotides described herein inhibit expression of all variants of C9ORF72 equally. In certain embodiments, the antisense oligonucleotides described herein preferentially inhibit expression of certain variants of C9ORF72. In certain embodiments, the antisense oligonucleotides described herein preferentially inhibit expression of variants of C9ORF72 containing a hexanucleotide repeat expansion. In certain embodiments, such variants of C9ORF72 containing a hexanucleotide repeat expansion include SEQ ID NO: 1-3 and 6-10. In certain embodiments, such hexanucleotide repeat expansion comprises at least 30 repeats of any of GGGGCC, GGGGGG, GGGGGC, or GGGGCG. In certain embodiments, the hexanucleotide repeat expansion forms nuclear foci. In certain embodiments, antisense oligonucleotides described herein are useful for reducing nuclear foci. Nuclear foci may be reduced in terms of percent of cells with foci as well as number of foci per cell. 
         [0277]    Based on earlier studies directed to repeat expansions, it is not possible to predict if antisense oligonucleotides targeting C9ORF72 outside of the hexanucleotide repeat expansion would successfully inhibit expression of C9ORF72 for two reasons. First, the C9ORF72 repeat expansion is located in an intron and it is not known if the RNA in the foci contains only the repeats or also the flanking intronic sequence. For example, an earlier study on myotonic dystrophy type 2 (DM2), which is a disease caused by a CCTG expansion mutation in intron 1 of the ZNF9 gene, determined that large DM2 expansions did not prevent allele-specific pre-mRNA splicing, nuclear export of the transcripts, or steady-state mRNA or protein levels. The study further demonstrated that the ribonuclear inclusions found associated with the disease are enriched for the CCUG expansion, but not the flanking intronic sequences. These data suggest that the downstream molecular effects of the DM2 mutation may be triggered by the accumulation of CCUG repeat tract alone. Therefore, this study implies that targeting the CCUG repeat expansion alone would lead to amelioration of the disease, since targeting the flanking sequences, especially the region downstream of the repeat expansion, would not affect the formation of ribonuclear inclusions (Margolis et al. Hum. Mol. Genet., 2006, 15:1808-1815). Second, it is not known how fast intron 1 of C9ORF72, which contains the repeats, is excised and accumulates in foci. Thus, it is not possible to predict if targeting the pre-mRNA would result in elimination of the repeat RNA and foci. 
       C9OFF72 Features 
       [0278]    Antisense oligonucleotides described herein may hybridize to any C9ORF72 variant at any state of processing within any element of the C9ORF72 gene. For example, antisense oligonucleotides described herein may hybridize to an exon, an intron, the 5′ UTR, the 3′ UTR, a repeat region, a hexanucleotide repeat expansion, a splice junction, an exon:exon splice junction, an exonic splicing silencer (ESS), an exonic splicing enhancer (ESE), exon 1a, exon 1b, exon 1c, exon 1d, exon 1e, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, or intron 10. For example, antisense oligonucleotides may target any of the exons characterized below in Tables 1-5 for the various C9ORF72 variants described below. Antisense oligonucleotides described herein may also target variants not characterized below and such variants are characterized in GENBANK. Moreover, antisense oligonucleotides described herein may also target elements other than exons and such elements are characterized in GENBANK. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Functional Segments for NM_001256054.1 (SEQ ID NO: 1) 
               
             
          
           
               
                   
                   
                   
                   
                 Start site 
                 Stop site 
               
               
                   
                   
                   
                   
                 in 
                 in 
               
               
                   
                   
                 mRNA 
                 mRNA 
                 reference 
                 reference 
               
               
                   
                 Exon 
                 start 
                 stop 
                 to SEQ 
                 to SEQ 
               
               
                   
                 Number 
                 site 
                 site 
                 ID NO: 2 
                 ID NO: 2 
               
               
                   
                   
               
             
          
           
               
                   
                 exon 1C 
                 1 
                 158 
                 1137 
                 1294 
               
               
                   
                 exon 2 
                 159 
                 646 
                 7839 
                 8326 
               
               
                   
                 exon 3 
                 647 
                 706 
                 9413 
                 9472 
               
               
                   
                 exon 4 
                 707 
                 802 
                 12527 
                 12622 
               
               
                   
                 exon 5 
                 803 
                 867 
                 13354 
                 13418 
               
               
                   
                 exon 6 
                 868 
                 940 
                 14704 
                 14776 
               
               
                   
                 exon 7 
                 941 
                 1057 
                 16396 
                 16512 
               
               
                   
                 exon 8 
                 1058 
                 1293 
                 18207 
                 18442 
               
               
                   
                 exon 9 
                 1294 
                 1351 
                 24296 
                 24353 
               
               
                   
                 exon 10 
                 1352 
                 1461 
                 26337 
                 26446 
               
               
                   
                 exon 11 
                 1462 
                 3339 
                 26581 
                 28458 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Functional Segments for NM_018325.3 (SEQ ID NO: 4) 
               
             
          
           
               
                   
                   
                   
                   
                 Start 
                 Stop 
               
               
                   
                   
                   
                   
                 site in 
                 site in 
               
               
                   
                   
                 mRNA 
                 mRNA 
                 reference 
                 reference 
               
               
                   
                 Exon 
                 start 
                 stop 
                 to SEQ 
                 to SEQ 
               
               
                   
                 Number 
                 site 
                 site 
                 ID NO: 2 
                 ID NO: 2 
               
               
                   
                   
               
             
          
           
               
                   
                 exon 1B 
                 1 
                 63 
                 1510 
                 1572 
               
               
                   
                 exon 2 
                 64 
                 551 
                 7839 
                 8326 
               
               
                   
                 exon 3 
                 552 
                 611 
                 9413 
                 9472 
               
               
                   
                 exon 4 
                 612 
                 707 
                 12527 
                 12622 
               
               
                   
                 exon 5 
                 708 
                 772 
                 13354 
                 13418 
               
               
                   
                 exon 6 
                 773 
                 845 
                 14704 
                 14776 
               
               
                   
                 exon 7 
                 846 
                 962 
                 16396 
                 16512 
               
               
                   
                 exon 8 
                 963 
                 1198 
                 18207 
                 18442 
               
               
                   
                 exon 9 
                 1199 
                 1256 
                 24296 
                 24353 
               
               
                   
                 exon 10 
                 1257 
                 1366 
                 26337 
                 26446 
               
               
                   
                 exon 11 
                 1367 
                 3244 
                 26581 
                 28458 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Functional Segments for NM_145005.5 (SEQ ID NO: 6) 
               
             
          
           
               
                   
                   
                   
                 Start 
                 Stop 
               
               
                   
                   
                   
                 site in 
                 site in 
               
               
                   
                 mRNA 
                 mRNA 
                 reference 
                 reference 
               
               
                 Exon 
                 start 
                 stop 
                 to SEQ 
                 to SEQ 
               
               
                 Number 
                 site 
                 site 
                 ID NO: 2 
                 ID NO: 2 
               
               
                   
               
             
          
           
               
                 exon 1A 
                 1 
                 80 
                 1137 
                 1216 
               
               
                 exon 2 
                 81 
                 568 
                 7839 
                 8326 
               
               
                 exon 3 
                 569 
                 628 
                 9413 
                 9472 
               
               
                 exon 4 
                 629 
                 724 
                 12527 
                 12622 
               
               
                 exon 5B (exon 5 
                 725 
                 1871 
                 13354 
                 14500 
               
               
                 into intron 5) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Functional Segments for DB079375.1 (SEQ ID NO: 7) 
               
             
          
           
               
                   
                   
                   
                 Start 
                 Stop 
               
               
                   
                   
                   
                 site in 
                 site in 
               
               
                   
                 mRNA 
                 mRNA 
                 reference 
                 reference 
               
               
                 Exon 
                 start 
                 stop 
                 to SEQ 
                 to SEQ 
               
               
                 Number 
                 site 
                 site 
                 ID NO: 2 
                 ID NO: 2 
               
               
                   
               
             
          
           
               
                 exon 1E 
                 1 
                 35 
                 1135 
                 1169 
               
               
                 exon 2 
                 36 
                 524 
                 7839 
                 8326 
               
               
                 exon 3 (EST ends before 
                 525 
                 562 
                 9413 
                 9450 
               
               
                 end of full exon) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Functional Segments for BU194591.1 (SEQ ID NO: 8) 
               
             
          
           
               
                   
                   
                   
                 Start 
                 Stop 
               
               
                   
                   
                   
                 site in 
                 site in 
               
               
                   
                 mRNA 
                 mRNA 
                 reference 
                 reference 
               
               
                 Exon 
                 start 
                 stop 
                 to SEQ 
                 to SEQ 
               
               
                 Number 
                 site 
                 site 
                 ID NO: 2 
                 ID NO: 2 
               
               
                   
               
             
          
           
               
                 exon 1D 
                 1 
                 36 
                 1241 
                 1279 
               
               
                 exon 2 
                 37 
                 524 
                 7839 
                 8326 
               
               
                 exon 3 
                 525 
                 584 
                 9413 
                 9472 
               
               
                 exon 4 
                 585 
                 680 
                 12527 
                 12622 
               
               
                 exon 5B (exon 5 
                 681 
                 798 
                 13354 
                 13465 
               
               
                 into intron 5) 
               
               
                   
               
             
          
         
       
     
       Certain Indications 
       [0279]    In certain embodiments, provided herein are methods of treating an individual comprising administering one or more pharmaceutical compositions described herein. In certain embodiments, the individual has a neurodegenerative disease. In certain embodiments, the individual is at risk for developing a neurodegenerative disease, including, but not limited to, ALS or FTD. In certain embodiments, the individual has been identified as having a C9ORF72 associated disease. In certain embodiments, the individual has been identified as having a C9ORF72 hexanucleotide repeat expansion associated disease. In certain embodiments, provided herein are methods for prophylactically reducing C9ORF72 expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a therapeutically effective amount of an antisense compound targeted to a C9ORF72 nucleic acid. 
         [0280]    In one embodiment, administration of a therapeutically effective amount of an antisense compound targeted to a C9ORF72 nucleic acid is accompanied by monitoring of C9ORF72 levels in an individual, to determine an individual&#39;s response to administration of the antisense compound. An individual&#39;s response to administration of the antisense compound may be used by a physician to determine the amount and duration of therapeutic intervention. 
         [0281]    In certain embodiments, administration of an antisense compound targeted to a C9ORF72 nucleic acid results in reduction of C9ORF72 expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In certain embodiments, administration of an antisense compound targeted to a C9ORF72 nucleic acid results in improved motor function and respiration in an animal. In certain embodiments, administration of a C9ORF72 antisense compound improves motor function and respiration by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. 
         [0282]    In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to C9ORF72 are used for the preparation of a medicament for treating a patient suffering or susceptible to a neurodegenerative disease including ALS and FTD. 
       Certain Combination Therapies 
       [0283]    In certain embodiments, one or more pharmaceutical compositions described herein are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions described herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions described herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions described herein. In certain embodiments, one or more pharmaceutical compositions described herein are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions described herein are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions described herein are co-administered with another pharmaceutical agent to produce a synergistic effect. 
         [0284]    In certain embodiments, one or more pharmaceutical compositions described herein and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions described herein and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions described herein and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions described herein and one or more other pharmaceutical agents are prepared separately. 
         [0285]    In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition described herein include Riluzole (Rilutek), Lioresal (Lioresal), and Dexpramipexole. 
         [0286]    In certain embodiments, pharmaceutical agents that may be co-administered with a C9ORF72 specific inhibitor described herein include, but are not limited to, an additional C9ORF72 inhibitor. In certain embodiments, the co-adminstered pharmaceutical agent is administered prior to administration of a pharmaceutical composition described herein. In certain embodiments, the co-administered pharmaceutical agent is administered following administration of a pharmaceutical composition described herein. In certain embodiments the co-administered pharmaceutical agent is administered at the same time as a pharmaceutical composition described herein. In certain embodiments the dose of a co-administered pharmaceutical agent is the same as the dose that would be administered if the co-administered pharmaceutical agent was administered alone. In certain embodiments the dose of a co-administered pharmaceutical agent is lower than the dose that would be administered if the co-administered pharmaceutical agent was administered alone. In certain embodiments the dose of a co-administered pharmaceutical agent is greater than the dose that would be administered if the co-administered pharmaceutical agent was administered alone. 
         [0287]    In certain embodiments, the co-administration of a second compound enhances the effect of a first compound, such that co-administration of the compounds results in an effect that is greater than the effect of administering the first compound alone. In other embodiments, the co-administration results in effects that are additive of the effects of the compounds when administered alone. In certain embodiments, the co-administration results in effects that are supra-additive of the effects of the compounds when administered alone. In certain embodiments, the first compound is an antisense compound. In certain embodiments, the second compound is an antisense compound. 
       EXAMPLES 
     Non-Limiting Disclosure and Incorporation by Reference 
       [0288]    While certain compounds, compositions, and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety. 
       Example 1 
     Antisense Inhibition of Human C9ORF72 in HepG2 Cells 
       [0289]    Antisense oligonucleotides were designed targeting a C9ORF72 nucleic acid and were tested for their effects on C9ORF72 mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 7,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and C9ORF72 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3750 (forward sequence TGTGACAGTTGGAATGCAGTGA, designated herein as SEQ ID NO: 15; reverse sequence GCCACTTAAAGCAATCTCTGTCTTG, designated herein as SEQ ID NO: 16; probe sequence TCGACTCTTTGCCCACCGCCA, designated herein as SEQ ID NO: 17) was used to measure mRNA levels. C9ORF72 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells. 
         [0290]    The antisense oligonucleotides in Tables 6-10 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on both the 5′ end and on the 3′ end comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the antisense oligonucleotide is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the antisense oligonucleotide is targeted human gene sequence. Each antisense oligonucleotide listed in Tables 6-9 is targeted to the either human C9ORF72 mRNA sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM — 001256054.1) or the human C9ORF72 genomic sequence, designated herein as SEQ ID NO: 2 (the complement of GENBANK Accession No. NT — 008413.18 truncated from nucleosides 27535000 to 27565000), or both. ‘n/a’ indicates that the antisense oligonucleotide did not target that particular gene sequence. The antisense oligonucleotides of Table 10 are targeted to either SEQ ID NO: 3 (GENBANK Accession No. BQ068108.1) or SEQ ID NO: 4 (GENBANK Accession No. NM — 018325.3). 
         [0291]    As shown in Tables 6-10, below, several of the oligonucleotides targeting SEQ ID NO: 1 exhibit at least 50% inhibition, including those targeted to nucleobases 90-647, 728-1541, 1598-1863, 1935-2146, 2232-2251, 2429-2576, 2632-2743, 2788-2807, 2860-2879, 2949-2968, 3062-3081, 3132-3151, and 3250-3269 of SEQ ID NO 1. These include SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 53, 55, 56, 57, 61, 62, 64, 66, 67, 72, 73, 75, 76, 81, 82, 85, 89, 90, 91, 92, 93, 94, 96, 97, 100, 102, 103, 109, 111, 112, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 130, 131, 132, 133, 137, 139, 140, 141, 145, 146, 149, 150, 151, 152, 153, 154, 165, 166, 168, 169, 170, 171, 174, 179, 181, 182, 183, 185, 186, 187, 188, 190, 192, 195, 197, 199, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, and 332. Several of the oligonucleotides exhibit at least 70% inhibition, including those targeted to nucleobases 90-359, 430-479, 550-569, 617-647, 940-959, 1013-1033, 1446-1465, 1687-1706, 1844-1863, 1935-2007, and 2679-2698 of SEQ ID NO 1. These include SEQ ID NOs: 32, 33, 34, 35, 36, 40, 41, 42, 43, 44, 47, 66, 67, 85, 96, 103, 117, 119, 154, 165, 168, 186, 320, 321, 324, 327, 328, and 331. Several of the oligonucleotides exhibit at least 80% inhibition, including those targeted to nucleobases 90-265 and 310-329. These include SEQ ID NOs: 32, 33, 35, 40, 42, and 321. Several of the oligonucleotides exhibit at least 90% inhibition, including those targeted to nucleobases 190-209 and 310-329 of SEQ ID NO 1. These include SEQ ID NOs: 40 and 321. 
         [0292]    As shown in Tables 6-20, below, several of the oligonucleotides targeting SEQ ID NO: 2 exhibit at least 50% inhibition, including those targeted to nucleobases 1552-1572, 2187-2238, 2728-2779, 3452-2471, 3752-3771, 5025-5044, 5656-5675, 6200-6219, 7594-7613, 7840-8328, 9415-9434, 12526-12545, 13357-13524, 13642-13661, 13790-14130, 14243-14335, 14699-14777, 15587-15606, 16395-16488, 18233-18373, 24306-24340, 24472-24491, 24565-24676, 26400-26424, 26606-26982, 27054-27265, 27351-27370, 27548-27998, 28068-28087, 28181-28270, and 28369-28388 of SEQ ID NO 2. These include SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 53, 55, 56, 57, 64, 67, 72, 73, 75, 76, 81, 82, 85, 89, 90, 91, 92, 93, 94, 96, 97, 100, 102, 103, 111, 112, 115, 117, 118, 119, 121, 122, 123, 124, 125, 126, 130, 131, 132, 133, 137, 139, 140, 141, 145, 146, 149, 150, 151, 152, 153, 154, 165, 166, 168, 169, 170, 171, 174, 179, 181, 182, 183, 185, 186, 187, 188, 190, 192, 195, 197, 199, 205, 206, 208, 211, 212, 224, 226, 230, 231, 250, 251, 252, 256, 300, 301, 304, 306, 307, 310, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, and 332. Several of the oligonucleotides exhibit at least 70% inhibition, including those targeted to nucleobases 3452-2471, 7840-8159, 8230-8249, 12526-12545, 13642-13661, 14075-14094, 14316-14335, 14758-14777, 16395-16414, 16469, 16488, 24655-24674, 26963, 26982, 27054-27126, and 27798-27817 of SEQ ID NO 2. These include SEQ ID NOs: 32, 33, 34, 35, 36, 40, 41, 42, 43, 44, 47, 67, 85, 96, 103, 117, 119, 154, 165, 168, 186, 251, 306, 320, 321, 324, 327, 328, and 331. Several of the oligonucleotides exhibit at least 80% inhibition, including those targeted to nucleobases 7848-8023 of SEQ ID NO 2. These include SEQ ID NOs: 32, 33, 35, 40, 42, and 321. Several of the oligonucleotides exhibit at least 90% inhibition, including those targeted to nucleobases 7870-7889 and 7990-8009 of SEQ ID NO 2. These include SEQ ID NOs: 40 and 321. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Target 
                 Target 
                   
                   
                   
                   
               
               
                 Start 
                 Start 
                   
                   
                 % 
                   
               
               
                 Site at 
                 Site at 
                   
                   
                 inhi- 
                 SEQ 
               
               
                 SEQ ID 
                 SEQ ID 
                   
                 ISIS 
                 bi- 
                 ID 
               
               
                 NO: 1 
                 NO: 2 
                 Sequence 
                 No 
                 tion 
                 NO 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 3 
                 1139 
                 AGCGGGACACCGTAGGTTAC 
                 576883 
                 0 
                 30 
               
               
                   
               
               
                 44 
                 1180 
                 GTGGGCGGAACTTGTCGCTG 
                 576807 
                 1 
                 31 
               
               
                   
               
               
                 90 
                 7848 
                 GTCACATTATCCAAATGCTC 
                 576808 
                 85 
                 32 
               
               
                   
               
               
                 125 
                 7883 
                 GGTGGGCAAAGAGTCGACAT 
                 576809 
                 82 
                 33 
               
               
                   
               
               
                 155 
                 7913 
                 ATCTCTGTCTTGGCAACAGC 
                 576810 
                 78 
                 34 
               
               
                   
               
               
                 160 
                 7918 
                 AAGCAATCTCTGTCTTGGCA 
                 576811 
                 81 
                 35 
               
               
                   
               
               
                 165 
                 7923 
                 ACTTAAAGCAATCTCTGTCT 
                 576812 
                 78 
                 36 
               
               
                   
               
               
                 170 
                 7928 
                 TTGCCACTTAAAGCAATCTC 
                 576813 
                 67 
                 37 
               
               
                   
               
               
                 205 
                 7963 
                 CCCAGTAAGCAAAAGTAGCT 
                 576814 
                 66 
                 38 
               
               
                   
               
               
                 227 
                 7985 
                 ACTCTAGGACCAAGAATATT 
                 576815 
                 11 
                 39 
               
               
                   
               
               
                 232 
                 7990 
                 GCCTTACTCTAGGACCAAGA 
                 576816 
                 78 
                 40 
               
               
                   
               
               
                 240 
                 7998 
                 CCAAATGTGCCTTACTCTAG 
                 576817 
                 73 
                 41 
               
               
                   
               
               
                 246 
                 8004 
                 TGGAGCCCAAATGTGCCTTA 
                 576818 
                 81 
                 42 
               
               
                   
               
               
                 254 
                 8012 
                 TCTGTCTTTGGAGCCCAAAT 
                 576819 
                 76 
                 43 
               
               
                   
               
               
                 275 
                 8033 
                 CCATCACTGAGAAGTACCTG 
                 576820 
                 79 
                 44 
               
               
                   
               
               
                 281 
                 8039 
                 ATTTCTCCATCACTGAGAAG 
                 576821 
                 61 
                 45 
               
               
                   
               
               
                 288 
                 8046 
                 AAAAGTTATTTCTCCATCAC 
                 576822 
                 57 
                 46 
               
               
                   
               
               
                 295 
                 8053 
                 TGGCAAGAAAAGTTATTTCT 
                 576823 
                 70 
                 47 
               
               
                   
               
               
                 302 
                 8060 
                 GTGTGGTTGGCAAGAAAAGT 
                 576824 
                 44 
                 48 
               
               
                   
               
               
                 313 
                 8071 
                 CTCCATTTAGAGTGTGGTTG 
                 576825 
                 39 
                 49 
               
               
                   
               
               
                 330 
                 8088 
                 TGCATTTCGAAGGATTTCTC 
                 576826 
                 65 
                 50 
               
               
                   
               
               
                 338 
                 8096 
                 CCACTCTCTGCATTTCGAAG 
                 576827 
                 67 
                 51 
               
               
                   
               
               
                 362 
                 8120 
                 ACAAAAAACTTTACATCTAT 
                 576828 
                 22 
                 52 
               
               
                   
               
               
                 376 
                 8134 
                 CCTTTTCAGACAAGACAAAA 
                 576829 
                 53 
                 53 
               
               
                   
               
               
                 401 
                 8159 
                 AAGATTAATGAAACAATAAT 
                 576830 
                 0 
                 54 
               
               
                   
               
               
                 411 
                 8169 
                 GTTTCCATCAAAGATTAATG 
                 576831 
                 62 
                 55 
               
               
                   
               
               
                 446 
                 8204 
                 ATTGATAGTCCATATGTGCT 
                 576832 
                 59 
                 56 
               
               
                   
               
               
                 452 
                 8210 
                 AGTATAATTGATAGTCCATA 
                 571818 
                 57 
                 57 
               
               
                   
               
               
                 481 
                 8239 
                 GGAGGTAGAAACTAAGTTCT 
                 576833 
                 45 
                 58 
               
               
                   
               
               
                 516 
                 8274 
                 ATGTGTTAATCTATCAACAC 
                 576834 
                 48 
                 59 
               
               
                   
               
               
                 545 
                 8303 
                 TGCATCCATATTCTTCCTTT 
                 576835 
                 43 
                 60 
               
               
                   
               
               
                 552 
                 n/a 
                 TTCCTTATGCATCCATATTC 
                 576836 
                 64 
                 61 
               
               
                   
               
               
                 559 
                 n/a 
                 CTTGTCTTTCCTTATGCATC 
                 576837 
                 57 
                 62 
               
               
                   
               
               
                 566 
                 n/a 
                 ACATTTTCTTGTCTTTCCTT 
                 576838 
                 43 
                 63 
               
               
                   
               
               
                 571 
                 9415 
                 TCTGGACATTTTCTTGTCTT 
                 576839 
                 61 
                 64 
               
               
                   
               
               
                 578 
                 9422 
                 ATAATCTTCTGGACATTTTC 
                 576840 
                 37 
                 65 
               
               
                   
               
               
                 617 
                 n/a 
                 CTCTGACCCTGATCTTCCAT 
                 576841 
                 79 
                 66 
               
               
                   
               
               
                 628 
                 12526 
                 TTGGAATAATACTCTGACCC 
                 576842 
                 73 
                 67 
               
               
                   
               
               
                 663 
                 12561 
                 CAGTTCCATTACAGGAATCA 
                 576843 
                 45 
                 68 
               
               
                   
               
               
                 697 
                 12595 
                 CTTCAGGAACACTGTGTGAT 
                 576844 
                 20 
                 69 
               
               
                   
               
               
                 705 
                 12603 
                 ATCTATTTCTTCAGGAACAC 
                 576845 
                 46 
                 70 
               
               
                   
               
               
                 722 
                 n/a 
                 AGTACTGTATCAGCTATATC 
                 576846 
                 46 
                 71 
               
               
                   
               
               
                 728 
                 13357 
                 TCATTGAGTACTGTATCAGC 
                 576847 
                 52 
                 72 
               
               
                   
               
               
                 734 
                 13363 
                 TCATCATCATTGAGTACTGT 
                 576848 
                 67 
                 73 
               
               
                   
               
               
                 740 
                 13369 
                 CCAATATCATCATCATTGAG 
                 576849 
                 47 
                 74 
               
               
                   
               
               
                 755 
                 13384 
                 TCATGACAGCTGTCACCAAT 
                 576850 
                 51 
                 75 
               
               
                   
               
               
                 761 
                 13390 
                 AAGCCTTCATGACAGCTGTC 
                 576851 
                 52 
                 76 
               
               
                   
               
               
                 767 
                 13396 
                 AGAAGAAAGCCTTCATGACA 
                 576852 
                 23 
                 77 
               
               
                   
               
               
                 773 
                 13402 
                 TACTTGAGAAGAAAGCCTTC 
                 576853 
                 24 
                 78 
               
               
                   
               
               
                 778 
                 13407 
                 ATTCTTACTTGAGAAGAAAG 
                 576854 
                 12 
                 79 
               
               
                   
               
               
                 782 
                 13411 
                 AAAAATTCTTACTTGAGAAG 
                 576855 
                 0 
                 80 
               
               
                   
               
               
                 817 
                 13446 
                 AGATGGTATCTGCTTCATCC 
                 576856 
                 61 
                 81 
               
               
                   
               
               
                 876 
                 13505 
                 CAATCTAAGTAGACAGTCTG 
                 576857 
                 57 
                 82 
               
               
                   
               
               
                 911 
                 13540 
                 TTAAGCAACAGTTCAAATAC 
                 576858 
                 40 
                 83 
               
               
                   
               
               
                 978 
                 13607 
                 CTTTAAATAGCAAATGGAAT 
                 576859 
                 26 
                 84 
               
               
                   
               
               
                 1013 
                 13642 
                 GCCATGATTTCTTGTCTGGG 
                 576860 
                 79 
                 85 
               
               
                   
               
               
                 1056 
                 13685 
                 GCTTTAATGAGAAGTAAAAC 
                 576861 
                 17 
                 86 
               
               
                   
               
               
                 1091 
                 13720 
                 TCTACAGTACAACTTAATAT 
                 576862 
                 39 
                 87 
               
               
                   
               
               
                 1126 
                 13755 
                 ATAATTTTGTTCTACGCCTA 
                 576863 
                 44 
                 88 
               
               
                   
               
               
                 1161 
                 13790 
                 CACTGCTGGATGGAAAAAGA 
                 576864 
                 65 
                 89 
               
               
                   
               
               
                 1196 
                 13825 
                 TGGTTTAAGGGCACAAACTC 
                 576865 
                 52 
                 90 
               
               
                   
               
               
                 1231 
                 13860 
                 TTGCCCACGGGTACACAGCA 
                 576866 
                 63 
                 91 
               
               
                   
               
               
                 1268 
                 13897 
                 CAGATGAGGAAATAGGTGTA 
                 576867 
                 62 
                 92 
               
               
                   
               
               
                 1303 
                 13932 
                 ACACATTAGGTACTATTACT 
                 576868 
                 63 
                 93 
               
               
                   
               
               
                 1372 
                 14001 
                 TTTTTATGTTCCAGGCACTG 
                 576869 
                 59 
                 94 
               
               
                   
               
               
                 1407 
                 14036 
                 AATAGGAAATGTTAGCTATG 
                 576870 
                 30 
                 95 
               
               
                   
               
               
                 1446 
                 14075 
                 GGCACTCAACAAATACTGGC 
                 576871 
                 72 
                 96 
               
               
                   
               
               
                 1482 
                 14111 
                 TACATGTAAAGCAACTAGTA 
                 576872 
                 55 
                 97 
               
               
                   
               
               
                 1539 
                 14168 
                 TAAAATTTCATGAAAATCTG 
                 576873 
                 0 
                 98 
               
               
                   
               
               
                 1579 
                 14208 
                 AAGTGAATACTTTATACTTT 
                 576874 
                 0 
                 99 
               
               
                   
               
               
                 1614 
                 14243 
                 CATCATGAGCCTAAAGGAAA 
                 576875 
                 51 
                 100 
               
               
                   
               
               
                 1651 
                 14280 
                 GGCTCTTAGGTTAAACACAC 
                 576876 
                 43 
                 101 
               
               
                   
               
               
                 1673 
                 14302 
                 TGCTTCTGATTCAAGCCATT 
                 576877 
                 65 
                 102 
               
               
                   
               
               
                 1687 
                 14316 
                 ATACAGGACTAAAGTGCTTC 
                 576878 
                 74 
                 103 
               
               
                   
               
               
                 1731 
                 14360 
                 CAAATGGGATTTAAAATGAT 
                 576879 
                 0 
                 104 
               
               
                   
               
               
                 1766 
                 14395 
                 TGACATGTAGAGAGATTAAG 
                 576880 
                 26 
                 105 
               
               
                   
               
               
                 1801 
                 14430 
                 TTATTGAAATACCATCATTT 
                 576881 
                 34 
                 106 
               
               
                   
               
               
                 1836 
                 14465 
                 TAGTCAGTATAATATCATTT 
                 576882 
                 18 
                 107 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 Target 
                 Target 
                   
                   
                   
                   
               
               
                 Start 
                 Start 
                   
                   
                 % 
                   
               
               
                 Site at 
                 Site at 
                   
                   
                 inhi- 
                 SEQ 
               
               
                 SEQ ID 
                 SEQ ID 
                   
                 ISIS 
                 bi- 
                 ID 
               
               
                 NO: 1 
                 NO: 2 
                 Sequence 
                 No 
                 tion 
                 NO 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 851 
                 n/a 
                 GCATTGAGAAGAAAGCCTTC 
                 571824 
                 25 
                 108 
               
               
                   
               
               
                 1337 
                 n/a 
                 AAGACCTGATCCAGGAAGGC 
                 571836 
                 53 
                 109 
               
               
                   
               
               
                 861 
                 n/a 
                 TGAGCTGATGGCATTGAGAA 
                 571981 
                 41 
                 110 
               
               
                   
               
               
                 890 
                 14726 
                 ACAACGGAACAGCCACAGGT 
                 571983 
                 66 
                 111 
               
               
                   
               
               
                 1420 
                 26405 
                 TTAGTGTCAAGGCTTTTCTG 
                 572007 
                 60 
                 112 
               
               
                   
               
               
                 75 
                 1211 
                 GACGGCTGACACACCAAGCG 
                 576884 
                 8 
                 113 
               
               
                   
               
               
                 856 
                 n/a 
                 TGATGGCATTGAGAAGAAAG 
                 576891 
                 6 
                 114 
               
               
                   
               
               
                 917 
                 14753 
                 TTTACTTTCTCTGCACTGCT 
                 576892 
                 68 
                 115 
               
               
                   
               
               
                 922 
                 n/a 
                 TCTTATTTACTTTCTCTGCA 
                 576893 
                 63 
                 116 
               
               
                   
               
               
                 940 
                 16395 
                 GGCATAATGTTCTGACTATC 
                 576894 
                 71 
                 117 
               
               
                   
               
               
                 979 
                 16434 
                 ATAACCTGGAGCATTTTCTC 
                 576895 
                 65 
                 118 
               
               
                   
               
               
                 1014 
                 16469 
                 CCCTGACTCATATTTAAATG 
                 576896 
                 70 
                 119 
               
               
                   
               
               
                 1049 
                 n/a 
                 CCAGTTGAATCCTTTAGCAG 
                 576897 
                 51 
                 120 
               
               
                   
               
               
                 1084 
                 18233 
                 CATACATGACTTGCCGGAAA 
                 576898 
                 66 
                 121 
               
               
                   
               
               
                 1119 
                 18268 
                 GACATCCACATCTATGTGTG 
                 576899 
                 63 
                 122 
               
               
                   
               
               
                 1154 
                 18303 
                 TGTTCATGACAGGGTGGCAT 
                 576900 
                 66 
                 123 
               
               
                   
               
               
                 1163 
                 18312 
                 TTATAAATATGTTCATGACA 
                 576901 
                 51 
                 124 
               
               
                   
               
               
                 1191 
                 18340 
                 CAGCTCGGATCTCATGTATC 
                 576902 
                 52 
                 125 
               
               
                   
               
               
                 1205 
                 18354 
                 CTCCAGAAGGCTGTCAGCTC 
                 576903 
                 59 
                 126 
               
               
                   
               
               
                 1238 
                 18387 
                 GTATCCTGAGCCATGTCTTC 
                 576904 
                 33 
                 127 
               
               
                   
               
               
                 1273 
                 18422 
                 AATCAGGAGTAAAGCTTTCG 
                 576905 
                 48 
                 128 
               
               
                   
               
               
                 1283 
                 n/a 
                 AAAATATTCAAATCAGGAGT 
                 576906 
                 23 
                 129 
               
               
                   
               
               
                 1304 
                 24306 
                 TCTCTGTGTAAGACATCTTG 
                 576907 
                 51 
                 130 
               
               
                   
               
               
                 1309 
                 24311 
                 GAGTGTCTCTGTGTAAGACA 
                 576908 
                 54 
                 131 
               
               
                   
               
               
                 1314 
                 24316 
                 CACTAGAGTGTCTCTGTGTA 
                 576909 
                 50 
                 132 
               
               
                   
               
               
                 1319 
                 24321 
                 GCTTTCACTAGAGTGTCTCT 
                 576910 
                 60 
                 133 
               
               
                   
               
               
                 1330 
                 24332 
                 GATCCAGGAAGGCTTTCACT 
                 576911 
                 35 
                 134 
               
               
                   
               
               
                 1373 
                 26358 
                 AAAGTACTTCTGAGAGATAA 
                 576912 
                 38 
                 135 
               
               
                   
               
               
                 1385 
                 26370 
                 AACTGTGCAAGGAAAGTACT 
                 576913 
                 43 
                 136 
               
               
                   
               
               
                 1415 
                 26400 
                 GTCAAGGCTTTTCTGTGAAG 
                 576914 
                 65 
                 137 
               
               
                   
               
               
                 1472 
                 26591 
                 AGAGATTTAAAGGGCTTTTT 
                 576915 
                 46 
                 138 
               
               
                   
               
               
                 1487 
                 26606 
                 ATCTTCAGGTTCCGAAGAGA 
                 576916 
                 53 
                 139 
               
               
                   
               
               
                 1511 
                 26630 
                 CCCTCTGCTGTTAAATCAAG 
                 576917 
                 51 
                 140 
               
               
                   
               
               
                 1522 
                 26641 
                 TGTTAAGATCGCCCTCTGCT 
                 576918 
                 64 
                 141 
               
               
                   
               
               
                 1529 
                 26648 
                 ATTATTATGTTAAGATCGCC 
                 576919 
                 46 
                 142 
               
               
                   
               
               
                 1535 
                 26654 
                 AGAGCCATTATTATGTTAAG 
                 576920 
                 36 
                 143 
               
               
                   
               
               
                 1571 
                 26690 
                 ATAAAAGAGTGTAGGCCTGG 
                 576921 
                 46 
                 144 
               
               
                   
               
               
                 1598 
                 26717 
                 ACACTAGTGTAGAAAGGTCT 
                 576922 
                 55 
                 145 
               
               
                   
               
               
                 1606 
                 26725 
                 GTTCTTGCACACTAGTGTAG 
                 576923 
                 62 
                 146 
               
               
                   
               
               
                 1628 
                 26747 
                 TAAAAAGTCATTAGAACATC 
                 576924 
                 10 
                 147 
               
               
                   
               
               
                 1644 
                 26763 
                 TATTAAGTTACACATTTAAA 
                 576925 
                 20 
                 148 
               
               
                   
               
               
                 1679 
                 26798 
                 CTTTACCAGCGATCATGATT 
                 576926 
                 57 
                 149 
               
               
                   
               
               
                 1725 
                 26844 
                 TTCTGGAGTATGATCCAGGG 
                 576927 
                 64 
                 150 
               
               
                   
               
               
                 1752 
                 24472 
                 ACTTAACTGCAATTGCTGAG 
                 576928 
                 66 
                 151 
               
               
                   
                 26871 
                   
                   
                   
                   
               
               
                   
               
               
                 1765 
                 26884 
                 TGTAGTGTAACTTACTTAAC 
                 576929 
                 60 
                 152 
               
               
                   
               
               
                 1802 
                 26921 
                 ATGCACCTGACATCCCCTCA 
                 576930 
                 56 
                 153 
               
               
                   
               
               
                 1844 
                 26963 
                 CCCAAAAGCATAAATCTAGG 
                 576931 
                 71 
                 154 
               
               
                   
               
               
                 1876 
                 24596 
                 ATATTTATTATATTGTAAAC 
                 576932 
                 0 
                 155 
               
               
                   
                 26995 
                   
                   
                   
                   
               
               
                   
               
               
                 1883 
                 24603 
                 AGCAATAATATTTATTATAT 
                 576933 
                 1 
                 156 
               
               
                   
                 27002 
                   
                   
                   
                   
               
               
                   
               
               
                 1887 
                 24607 
                 AGATAGCAATAATATTTATT 
                 576934 
                 0 
                 157 
               
               
                   
                 27006 
                   
                   
                   
                   
               
               
                   
               
               
                 1889 
                 24609 
                 AAAGATAGCAATAATATTTA 
                 576935 
                 0 
                 158 
               
               
                   
                 27008 
                   
                   
                   
                   
               
               
                   
               
               
                 1892 
                 24612 
                 TTAAAAGATAGCAATAATAT 
                 576936 
                 3 
                 159 
               
               
                   
                 27011 
                   
                   
                   
                   
               
               
                   
               
               
                 1896 
                 24616 
                 ATCTTTAAAAGATAGCAATA 
                 576937 
                 14 
                 160 
               
               
                   
                 27015 
                   
                   
                   
                   
               
               
                   
               
               
                 1898 
                 24618 
                 ATATCTTTAAAAGATAGCAA 
                 576938 
                 15 
                 161 
               
               
                   
                 27017 
                   
                   
                   
                   
               
               
                   
               
               
                 1901 
                 24621 
                 ATTATATCTTTAAAAGATAG 
                 576939 
                 12 
                 162 
               
               
                   
                 27020 
                   
                   
                   
                   
               
               
                   
               
               
                 1905 
                 24625 
                 TATTATTATATCTTTAAAAG 
                 576940 
                 6 
                 163 
               
               
                   
                 27024 
                   
                   
                   
                   
               
               
                   
               
               
                 1918 
                 27037 
                 CAAGTTTACATCCTATTATT 
                 576941 
                 48 
                 164 
               
               
                   
               
               
                 1935 
                 24655 
                 AAAACAGTAGTTGTGGTCAA 
                 576942 
                 77 
                 165 
               
               
                   
                 27054 
                   
                   
                   
                   
               
               
                   
               
               
                 1937 
                 24657 
                 AAAAAACAGTAGTTGTGGTC 
                 576943 
                 69 
                 166 
               
               
                   
                 27056 
                   
                   
                   
                   
               
               
                   
               
               
                 1953 
                 27072 
                 TGAATCATGTATTTCAAAAA 
                 576944 
                 17 
                 167 
               
               
                   
               
               
                 1988 
                 27107 
                 GCCAACTCAGATTTCACCTT 
                 576945 
                 71 
                 168 
               
               
                   
               
               
                 2036 
                 27155 
                 CTACACACCAAAGAATGCCA 
                 576946 
                 69 
                 169 
               
               
                   
               
               
                 2071 
                 27190 
                 AGTTTTCAGTTGATTGCAGA 
                 576947 
                 58 
                 170 
               
               
                   
               
               
                 2127 
                 27246 
                 CATCCTATGTTCAAGCTCAC 
                 576948 
                 51 
                 171 
               
               
                   
               
               
                 2162 
                 27281 
                 TAAACATCTGCTTGATCAAT 
                 576949 
                 44 
                 172 
               
               
                   
               
               
                 2197 
                 27316 
                 AATCCACAAAGTAGGATCTA 
                 576950 
                 42 
                 173 
               
               
                   
               
               
                 2232 
                 27351 
                 ATTAGACATTTCTACAGACT 
                 576951 
                 56 
                 174 
               
               
                   
               
               
                 2325 
                 27444 
                 CTCAACTACATAGAATATCA 
                 576952 
                 45 
                 175 
               
               
                   
               
               
                 2371 
                 27490 
                 TTGGCAACAATTACTAAAAC 
                 576953 
                 48 
                 176 
               
               
                   
               
               
                 2400 
                 27519 
                 TCAAAAATAATGAAAATTAA 
                 576954 
                 0 
                 177 
               
               
                   
               
               
                 2409 
                 27528 
                 CAATTTGGCTCAAAAATAAT 
                 576955 
                 3 
                 178 
               
               
                   
               
               
                 2429 
                 27548 
                 GGCACAGGAGGTGCACATTT 
                 576956 
                 60 
                 179 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 Target 
                 Target 
                   
                   
                   
                   
               
               
                 Start 
                 Start 
                   
                   
                   
                   
               
               
                 Site at 
                 Site at 
                   
                   
                 % 
                 SEQ 
               
               
                 SEQ ID 
                 SEQ ID 
                   
                 ISIS 
                 inhi- 
                 ID 
               
               
                 NO: 1 
                 NO: 2 
                 Sequence 
                 No 
                 bition 
                 NO 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 2451 
                 27570 
                 TAGATTTTCTAAGGAGAAAA 
                 576957 
                 8 
                 180 
               
               
                   
               
               
                 2486 
                 27605 
                 ACTGACCAGTGAAATCTGAA 
                 576958 
                 50 
                 181 
               
               
                   
               
               
                 2522 
                 27641 
                 GGTAAGACTTAGCAAGAAGA 
                 576959 
                 59 
                 182 
               
               
                   
               
               
                 2557 
                 27676 
                 TCTCAGAGTTGCAATGATTG 
                 576960 
                 63 
                 183 
               
               
                   
               
               
                 2597 
                 27716 
                 AGATCTTATTAGTTAGTATA 
                 576961 
                 18 
                 184 
               
               
                   
               
               
                 2632 
                 27751 
                 AGTACTCAAGGAACTATTTT 
                 576962 
                 57 
                 185 
               
               
                   
               
               
                 2679 
                 27798 
                 GGCAAACAGCAACAACTTCA 
                 576963 
                 71 
                 186 
               
               
                   
               
               
                 2724 
                 27843 
                 GCACTTCAGTAAAATTTCTC 
                 576964 
                 69 
                 187 
               
               
                   
               
               
                 2788 
                 27907 
                 GGTCCAAACGCATTAAGAAA 
                 576965 
                 58 
                 188 
               
               
                   
               
               
                 2825 
                 27944 
                 GAATTATATTAATCAGTTAT 
                 576966 
                 0 
                 189 
               
               
                   
               
               
                 2860 
                 27979 
                 TGTGTTTGTGTAACTACAAT 
                 576967 
                 67 
                 190 
               
               
                   
               
               
                 2895 
                 28014 
                 ATATTACTTCCAGAATTTTA 
                 576968 
                 19 
                 191 
               
               
                   
               
               
                 2949 
                 28068 
                 GGCAGAAGGGCTCTATTACC 
                 576969 
                 59 
                 192 
               
               
                   
               
               
                 2992 
                 28111 
                 CATTCGAACATGTCATTTTG 
                 576970 
                 40 
                 193 
               
               
                   
               
               
                 3027 
                 28146 
                 CTGATTCATGATGGGAAAGC 
                 576971 
                 34 
                 194 
               
               
                   
               
               
                 3062 
                 28181 
                 GTGGTTGTCTAAAACATCAA 
                 576972 
                 58 
                 195 
               
               
                   
               
               
                 3097 
                 28216 
                 ATGACTGAGCTACAGTACAA 
                 576973 
                 47 
                 196 
               
               
                   
               
               
                 3132 
                 28251 
                 GGGACACTACAAGGTAGTAT 
                 576974 
                 56 
                 197 
               
               
                   
               
               
                 3167 
                 28286 
                 TTAAATAAGAATCTACCATG 
                 576975 
                 12 
                 198 
               
               
                   
               
               
                 3250 
                 28369 
                 GCTTTAATAACTTATTTCAC 
                 576976 
                 54 
                 199 
               
               
                   
               
               
                 3282 
                 28401 
                 AGGAGAAAAGATATATAACA 
                 576977 
                 0 
                 200 
               
               
                   
               
               
                 3288 
                 28407 
                 CCATTTAGGAGAAAAGATAT 
                 576978 
                 0 
                 201 
               
               
                   
               
               
                 n/a 
                 1343 
                 TTCACCCTCAGCGAGTACTG 
                 576979 
                 0 
                 202 
               
               
                   
               
               
                 n/a 
                 1403 
                 AGGCTGCGGTTGTTTCCCTC 
                 576980 
                 0 
                 203 
               
               
                   
               
               
                 n/a 
                 1800 
                 GCCAGATCCCCATCCCTTGT 
                 576981 
                 11 
                 204 
               
               
                   
               
               
                 n/a 
                 2187 
                 TCACTTCCTTTAAGCAAGTC 
                 576982 
                 52 
                 205 
               
               
                   
               
               
                 n/a 
                 2209 
                 AGTGATGCCCAAGTCACAAT 
                 576983 
                 53 
                 206 
               
               
                   
               
               
                 n/a 
                 2214 
                 AGTCAAGTGATGCCCAAGTC 
                 576984 
                 47 
                 207 
               
               
                   
               
               
                 n/a 
                 2219 
                 CCATCAGTCAAGTGATGCCC 
                 576985 
                 60 
                 208 
               
               
                   
               
               
                 n/a 
                 2224 
                 GATTACCATCAGTCAAGTGA 
                 576986 
                 29 
                 209 
               
               
                   
               
               
                 n/a 
                 2229 
                 CAACTGATTACCATCAGTCA 
                 576987 
                 42 
                 210 
               
               
                   
               
               
                 n/a 
                 2728 
                 GCAGTTTCCAACTGATTCAG 
                 576988 
                 58 
                 211 
               
               
                   
               
               
                 n/a 
                 2760 
                 CGTTCTTGTTTCAGATGTAC 
                 576989 
                 57 
                 212 
               
               
                   
               
               
                 n/a 
                 2862 
                 GCCAAACAAAATATTTTATC 
                 576990 
                 22 
                 213 
               
               
                   
               
               
                 n/a 
                 2995 
                 TAGGTAGGCTAACCTAGTCC 
                 576991 
                 47 
                 214 
               
               
                   
               
               
                 n/a 
                 3196 
                 TCCCAGCCCAAAGAGAAGCA 
                 576992 
                 41 
                 215 
               
               
                   
               
               
                 n/a 
                 3466 
                 GGATCATAGCTCTCGGTAAC 
                 576993 
                 26 
                 216 
               
               
                   
               
               
                 n/a 
                 3540 
                 AATCATAAAGCCCTCACTTC 
                 576994 
                 7 
                 217 
               
               
                   
               
               
                 n/a 
                 3595 
                 CTGATTGGTATTTAGAAAGG 
                 576995 
                 3 
                 218 
               
               
                   
               
               
                 n/a 
                 3705 
                 ATGCAGACATGATTACATTA 
                 576996 
                 48 
                 219 
               
               
                   
               
               
                 n/a 
                 4560 
                 TTCATCATTAAACTGAAAAT 
                 576997 
                 0 
                 220 
               
               
                   
               
               
                 n/a 
                 4613 
                 CTTTTAGGTTAAAAAGGTGG 
                 576998 
                 35 
                 221 
               
               
                   
               
               
                 n/a 
                 4986 
                 ATACAGAGCCTGGCAAAACA 
                 576999 
                 30 
                 222 
               
               
                   
               
               
                 n/a 
                 5036 
                 TTCTATTTACAGAGCATTAG 
                 577000 
                 29 
                 223 
               
               
                   
               
               
                 n/a 
                 5656 
                 GCCTTCACATTAATTCACCA 
                 577001 
                 62 
                 224 
               
               
                   
               
               
                 n/a 
                 6051 
                 TGTGTTATTGCCCCTAAAAA 
                 577002 
                 24 
                 225 
               
               
                   
               
               
                 n/a 
                 6200 
                 TGTATTCACTATACTATGCC 
                 577003 
                 52 
                 226 
               
               
                   
               
               
                 n/a 
                 6276 
                 AAGTTATTTAAAGTATAGCA 
                 577004 
                 0 
                 227 
               
               
                   
               
               
                 n/a 
                 6762 
                 GACATTGAAGTATCAAGACA 
                 577005 
                 34 
                 228 
               
               
                   
               
               
                 n/a 
                 6965 
                 TGTTAAGTAATCTTAGAAAA 
                 577006 
                 0 
                 229 
               
               
                   
               
               
                 n/a 
                 7594 
                 GGCATACATTTAGAAATTCA 
                 577007 
                 60 
                 230 
               
               
                   
               
               
                 n/a 
                 8309 
                 ACCTTATGCATCCATATTCT 
                 577008 
                 59 
                 231 
               
               
                   
               
               
                 n/a 
                 8784 
                 GAATTCTCTTGGGAACCATT 
                 577009 
                 42 
                 232 
               
               
                   
               
               
                 n/a 
                 8834 
                 ATATTCAACTACAGGATTTA 
                 577010 
                 13 
                 233 
               
               
                   
               
               
                 n/a 
                 8884 
                 ATGTGTTCTTTAGATACATC 
                 577011 
                 42 
                 234 
               
               
                   
               
               
                 n/a 
                 9510 
                 CCTTATACAGATACATGCTG 
                 577012 
                 37 
                 235 
               
               
                   
               
               
                 n/a 
                 9663 
                 TAGATGCAATTACTATTTTC 
                 577013 
                 34 
                 236 
               
               
                   
               
               
                 n/a 
                 10742 
                 TGTACTTCCCAAACTTGAAC 
                 577014 
                 24 
                 237 
               
               
                   
               
               
                 n/a 
                 10845 
                 CTGAAGCTCAACAACACCAA 
                 577015 
                 49 
                 238 
               
               
                   
               
               
                 n/a 
                 11684 
                 GTCTATAGAATCAAACTGAA 
                 577016 
                 38 
                 239 
               
               
                   
               
               
                 n/a 
                 11851 
                 TTGAATCAATACCTAACCTC 
                 577017 
                 23 
                 240 
               
               
                   
               
               
                 n/a 
                 11991 
                 TGCCTCTTTTAGAAAAGATC 
                 577018 
                 44 
                 241 
               
               
                   
               
               
                 n/a 
                 12042 
                 ATGGAATCATTGGTTTATCG 
                 577019 
                 43 
                 242 
               
               
                   
               
               
                 n/a 
                 12069 
                 AAAGCTCACTTTTATTCTTT 
                 577020 
                 37 
                 243 
               
               
                 n/a 
                 12333 
                   
                   
                   
                   
               
               
                   
               
               
                 n/a 
                 12170 
                 GGTGCCGCCACCATGCCCGG 
                 577021 
                 0 
                 244 
               
               
                   
               
               
                 n/a 
                 12464 
                 GAGAGAAGCTGGGCAATAAA 
                 577022 
                 2 
                 245 
               
               
                   
               
               
                 n/a 
                 12514 
                 TCTGACCCTGCACAATAAAG 
                 577023 
                 0 
                 246 
               
               
                   
               
               
                 n/a 
                 13016 
                 ATAGTGTGTGATTCAAAACG 
                 577024 
                 17 
                 247 
               
               
                   
               
               
                 n/a 
                 13348 
                 ACTGTATCAGCTATCTAAAA 
                 577025 
                 22 
                 248 
               
               
                   
               
               
                 n/a 
                 14540 
                 TTATTTGTATAGGAACCTAC 
                 577026 
                 44 
                 249 
               
               
                   
               
               
                 n/a 
                 14699 
                 TGTGAGCTGATGGCACTGTA 
                 577027 
                 61 
                 250 
               
               
                   
               
               
                 n/a 
                 14758 
                 CCTTATTTACTTTCTCTGCA 
                 577028 
                 71 
                 251 
               
               
                   
               
               
                 n/a 
                 15587 
                 GGAATAAGGTCACTAGTTCG 
                 577029 
                 69 
                 252 
               
               
                   
               
               
                 n/a 
                 17187 
                 ATTTGCAACAATTTTTAAAT 
                 577030 
                 8 
                 253 
               
               
                   
               
               
                 n/a 
                 21808 
                 ATAAACTACCAATGATATCC 
                 577031 
                 13 
                 254 
               
               
                   
               
               
                 n/a 
                 24337 
                 TACCTGATCCAGGAAGGCTT 
                 577032 
                 40 
                 255 
               
               
                   
               
               
                 n/a 
                 24565 
                 TTCCCGAAGCATAAATCTAG 
                 577033 
                 53 
                 256 
               
               
                   
               
               
                 n/a 
                 25549 
                 TTGAGAAGCATGAAATTCCA 
                 577034 
                 48 
                 257 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 Target 
                 Target 
                   
                   
                   
                   
               
               
                 Start 
                 Start 
                   
                   
                   
                   
               
               
                 Site at 
                 Site at 
                   
                   
                 % 
                 SEQ 
               
               
                 SEQ ID 
                 SEQ ID 
                   
                 ISIS 
                 inhi- 
                 ID 
               
               
                 NO: 1 
                 NO: 2 
                 Sequence 
                 No 
                 bition 
                 NO 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 310 
                 7990 
                 GCCTTACTCTAGGACCAAGA 
                 576816 
                 90 
                 40 
               
               
                   
               
               
                 75 
                 1211 
                 GACGGCTGACACACCAAGCG 
                 576884 
                 0 
                 113 
               
               
                   
               
               
                 2 
                 1138 
                 GCGGGACACCGTAGGTTACG 
                 577035 
                 0 
                 258 
               
               
                   
               
               
                 10 
                 1146 
                 CTTTCCTAGCGGGACACCGT 
                 577036 
                 1 
                 259 
               
               
                   
               
               
                 18 
                 1154 
                 GCACCTCTCTTTCCTAGCGG 
                 577037 
                 0 
                 260 
               
               
                   
               
               
                 26 
                 1162 
                 TGTTTGACGCACCTCTCTTT 
                 577038 
                 0 
                 261 
               
               
                   
               
               
                 34 
                 1170 
                 CTTGTCGCTGTTTGACGCAC 
                 577039 
                 0 
                 262 
               
               
                   
               
               
                 42 
                 1178 
                 GGGCGGAACTTGTCGCTGTT 
                 577040 
                 0 
                 263 
               
               
                   
               
               
                 83 
                 1219 
                 GCAGCAGGGACGGCTGACAC 
                 577041 
                 0 
                 264 
               
               
                   
               
               
                 95 
                 1231 
                 AGAAGCAACCGGGCAGCAGG 
                 577042 
                 0 
                 265 
               
               
                   
               
               
                 103 
                 1239 
                 CCCAAAAGAGAAGCAACCGG 
                 577043 
                 0 
                 266 
               
               
                   
               
               
                 111 
                 1247 
                 ACCCCGCCCCCAAAAGAGAA 
                 577044 
                 1 
                 267 
               
               
                   
               
               
                 119 
                 1255 
                 CTTGCTAGACCCCGCCCCCA 
                 577045 
                 0 
                 268 
               
               
                   
               
               
                 127 
                 1263 
                 CACCTGCTCTTGCTAGACCC 
                 577046 
                 0 
                 269 
               
               
                   
               
               
                 135 
                 1271 
                 TAAACCCACACCTGCTCTTG 
                 577047 
                 0 
                 270 
               
               
                   
               
               
                 139 
                 1275 
                 CTCCTAAACCCACACCTGCT 
                 577048 
                 0 
                 271 
               
               
                   
               
               
                 n/a 
                 1283 
                 ACACACACCTCCTAAACCCA 
                 577049 
                 0 
                 272 
               
               
                   
               
               
                 n/a 
                 1291 
                 AAACAAAAACACACACCTCC 
                 577050 
                 5 
                 273 
               
               
                   
               
               
                 n/a 
                 1299 
                 GGTGGGAAAAACAAAAACAC 
                 577051 
                 1 
                 274 
               
               
                   
               
               
                 n/a 
                 1326 
                 CTGTGAGAGCAAGTAGTGGG 
                 577052 
                 3 
                 275 
               
               
                   
               
               
                 n/a 
                 1334 
                 AGCGAGTACTGTGAGAGCAA 
                 577053 
                 0 
                 276 
               
               
                   
               
               
                 n/a 
                 1342 
                 TCACCCTCAGCGAGTACTGT 
                 577054 
                 0 
                 277 
               
               
                   
               
               
                 n/a 
                 1358 
                 TCAGGTCTTTTCTTGTTCAC 
                 577055 
                 0 
                 278 
               
               
                   
               
               
                 n/a 
                 1366 
                 AATCTTTATCAGGTCTTTTC 
                 577056 
                 16 
                 279 
               
               
                   
               
               
                 n/a 
                 1374 
                 TTCTGGTTAATCTTTATCAG 
                 577057 
                 22 
                 280 
               
               
                   
               
               
                 n/a 
                 1382 
                 TTGTTTTCTTCTGGTTAATC 
                 577058 
                 19 
                 281 
               
               
                   
               
               
                 n/a 
                 1390  
                 TTCCCTCCTTGTTTTCTTCT 
                 577059 
                 28 
                 282 
               
               
                   
               
               
                 n/a 
                 1398  
                 GCGGTTGTTTCCCTCCTTGT 
                 577060 
                 17 
                 283 
               
               
                   
               
               
                 n/a 
                 1406  
                 TACAGGCTGCGGTTGTTTCC 
                 577061 
                 28 
                 284 
               
               
                   
               
               
                 n/a 
                 1414  
                 GAGCTTGCTACAGGCTGCGG 
                 577062 
                 23 
                 285 
               
               
                   
               
               
                 n/a 
                 1422  
                 GAGTTCCAGAGCTTGCTACA 
                 577063 
                 14 
                 286 
               
               
                   
               
               
                 n/a 
                 1430  
                 CGACTCCTGAGTTCCAGAGC 
                 577064 
                 0 
                 287 
               
               
                   
               
               
                 n/a 
                 1446  
                 CCCGGCCCCTAGCGCGCGAC 
                 577065 
                 0 
                 288 
               
               
                   
               
               
                 n/a 
                 1454  
                 GCCCCGGCCCCGGCCCCTAG 
                 577066 
                 0 
                 289 
               
               
                   
               
               
                 n/a 
                 1465  
                 ACCACGCCCCGGCCCCGGCC 
                 577067 
                 0 
                 290 
               
               
                   
               
               
                 n/a 
                 1473  
                 CCGCCCCGACCACGCCCCGG 
                 577068 
                 0 
                 291 
               
               
                   
               
               
                 n/a 
                 1481  
                 CCCCGGGCCCGCCCCGACCA 
                 577069 
                 0 
                 292 
               
               
                   
               
               
                 n/a 
                 1495  
                 CGCCCCGGGCCCGCCCCCGG 
                 577070 
                 0 
                 293 
               
               
                   
               
               
                 n/a 
                 1503  
                 CGCAGCCCCGCCCCGGGCCC 
                 577071 
                 0 
                 294 
               
               
                   
               
               
                 n/a 
                 1511  
                 ACCGCAACCGCAGCCCCGCC 
                 577072 
                 0 
                 295 
               
               
                   
               
               
                 n/a 
                 1519  
                 GCGCAGGCACCGCAACCGCA 
                 577073 
                 18 
                 296 
               
               
                   
               
               
                 n/a 
                 1520  
                 GGCGCAGGCACCGCAACCGC 
                 577074 
                 17 
                 297 
               
               
                   
               
               
                 n/a 
                 1536  
                 CGCCTCCGCCGCCGCGGGCG 
                 577075 
                 32 
                 298 
               
               
                   
               
               
                 n/a 
                 1544  
                 ACCGCCTGCGCCTCCGCCGC 
                 577076 
                 43 
                 299 
               
               
                   
               
               
                 n/a 
                 1552  
                 CACTCGCCACCGCCTGCGCC 
                 577077 
                 52 
                 300 
               
               
                   
               
               
                 n/a 
                 1553 
                 CCACTCGCCACCGCCTGCGC 
                 577078 
                 52 
                 301 
               
               
                   
               
               
                 n/a 
                 1853 
                 GGTCCCCGGGAAGGAGACAG 
                 577079 
                 41 
                 302 
               
               
                   
               
               
                 n/a 
                 2453 
                 AACAACTGGTGCATGGCAAC 
                 577080 
                 42 
                 303 
               
               
                   
               
               
                 n/a 
                 2753 
                 GTTTCAGATGTACTATCAGC 
                 577081 
                 63 
                 304 
               
               
                   
               
               
                 n/a 
                 3053 
                 AAGGTGAAGTTCATATCACT 
                 577082 
                 10 
                 305 
               
               
                   
               
               
                 n/a 
                 3452 
                 GGTAACTTCAAACTCTTGGG 
                 577083 
                 70 
                 306 
               
               
                   
               
               
                 n/a 
                 3752 
                 GGTTCATGAGAGGTTTCCCA 
                 577084 
                 53 
                 307 
               
               
                   
               
               
                 n/a 
                 4052 
                 TACTGAATTGCTTAGTTTTA 
                 577085 
                 25 
                 308 
               
               
                   
               
               
                 n/a 
                 4425 
                 CTAACAGAATAAGAAAAAAA 
                 577086 
                 0 
                 309 
               
               
                   
               
               
                 n/a 
                 5025 
                 GAGCATTAGATGAGTGCTTT 
                 577087 
                 52 
                 310 
               
               
                   
               
               
                 n/a 
                 5325  
                 TGCATTCCTAAGCAATGTGT 
                 577088 
                 28 
                 311 
               
               
                   
               
               
                 n/a 
                 5661 
                 TCTAGGCCTTCACATTAATT 
                 577089 
                 37 
                 312 
               
               
                   
               
               
                 n/a 
                 5961 
                 CCTGTCTATGCCTAGGTGAA 
                 577090 
                 19 
                 313 
               
               
                   
               
               
                 n/a 
                 6261 
                 TAGCACATACAATTATTACA 
                 577091 
                 38 
                 314 
               
               
                   
               
               
                 n/a 
                 6566 
                 GAGGAGAAGAACATAAACGC 
                 577092 
                 20 
                 315 
               
               
                   
               
               
                 n/a 
                 6866 
                 TACCACAAGTCTGGAGCCAT 
                 577093 
                 27 
                 316 
               
               
                   
               
               
                 n/a 
                 7166 
                 GATACTGGATTGTTGAAACT 
                 577094 
                 1 
                 317 
               
               
                   
               
               
                 n/a 
                 7466 
                 TAGTATGACTGGAGATTTGG 
                 577095 
                 1 
                 318 
               
               
                   
               
               
                 n/a 
                 7766  
                 ATCAAAACCCCAAATGATTT 
                 577096 
                 13 
                 319 
               
               
                   
               
               
                 160 
                 7840 
                 ATCCAAATGCTCCGGAGATA 
                 577097 
                 78 
                 320 
               
               
                   
               
               
                 190 
                 7870 
                 TCGACATCACTGCATTCCAA 
                 577098 
                 95 
                 321 
               
               
                   
               
               
                 220 
                 7900 
                 CAACAGCTGGAGATGGCGGT 
                 577099 
                 56 
                 322 
               
               
                   
               
               
                 250 
                 7930 
                 ATTTGCCACTTAAAGCAATC 
                 577100 
                 62 
                 323 
               
               
                   
               
               
                 340 
                 8020 
                 GTACCTGTTCTGTCTTTGGA 
                 577101 
                 76 
                 324 
               
               
                   
               
               
                 370 
                 8050 
                 CAAGAAAAGTTATTTCTCCA 
                 577102 
                 65 
                 325 
               
               
                   
               
               
                 400 
                 8080 
                 GAAGGATTTCTCCATTTAGA 
                 577103 
                 50 
                 326 
               
               
                   
               
               
                 430 
                 8110 
                 TTACATCTATAGCACCACTC 
                 577104 
                 73 
                 327 
               
               
                   
               
               
                 460 
                 8140 
                 TCACTCCCTTTTCAGACAAG 
                 577105 
                 73 
                 328 
               
               
                   
               
               
                 490 
                 8170 
                 AGTTTCCATCAAAGATTAAT 
                 577106 
                 55 
                 329 
               
               
                   
               
               
                 520 
                 8200 
                 ATAGTCCATATGTGCTGCGA 
                 577107 
                 57 
                 330 
               
               
                   
               
               
                 550 
                 8230 
                 AACTAAGTTCTGTCTGTGGA 
                 577108 
                 71 
                 331 
               
               
                   
               
               
                 580 
                 8260 
                 CAACACACACTCTATGAAGT 
                 577109 
                 54 
                 332 
               
               
                   
               
               
                 610 
                 8290 
                 TTCCTTTCCGGATTATATGT 
                 577110 
                 0 
                 333 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                 Target 
                 Target 
                   
                   
                 % 
                 SEQ 
               
               
                 SEQ ID 
                 Start 
                 ISIS 
                   
                 inhibi- 
                 ID 
               
               
                 NO 
                 Site 
                 No 
                 Sequence 
                 tion 
                 NO 
               
               
                   
               
             
             
               
                 3 
                 751 
                 576885 
                 TTTCCATTACAGGAATCACT 
                 63 
                 334 
               
               
                   
               
               
                 3 
                 807 
                 576886 
                 ATCAGCCTATATCTATTTCC 
                 15 
                 335 
               
               
                   
               
               
                 3 
                 855 
                 576887 
                 TCAATGACCAGGCGGTCCCC 
                  0 
                 336 
               
               
                   
               
               
                 3 
                 905 
                 576888 
                 CTTTTTATGGAAAAGGAAAA 
                  0 
                 337 
               
               
                   
               
               
                 3 
                 984 
                 576889 
                 TGTTTCCCCAAAAATTTCTG 
                  0 
                 338 
               
               
                   
               
               
                 4 
                  50 
                 576890 
                 AGATATCCACTCGCCACCGC 
                 42 
                 339 
               
               
                   
               
             
          
         
       
     
       Example 2 
     Dose-Dependent Antisense Inhibition of Human C9ORF72 in HepG2 Cells 
       [0293]    Antisense oligonucleotides from the study described above exhibiting significant in vitro inhibition of C9ORF72 mRNA were selected and tested at various doses in HepG2 cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 82.3 nM, 246.9 nM, 740.7 nM, 2,222.2 nM, 6,666.7 nM, or 20,000 nM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and C9ORF72 mRNA levels were measured by quantitative real-time PCR. Human C9ORF72 primer probe set RTS3750 was used to measure mRNA levels. C9ORF72 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells. 
         [0294]    The half maximal inhibitory concentration (IC 50 ) of each oligonucleotide is also presented in Tables 11-13. As illustrated, C9ORF72 mRNA levels were reduced in a dose-dependent manner in the antisense oligonucleotide treated cells. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                 ISIS No 
                 82.3 nM 
                 246.9 nM 
                 740.7 nM 
                 2222.2 nM 
                 6666.7 nM 
                 20000.0 nM 
                 IC 50  (μM) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 576816 
                 5 
                 23 
                 49 
                 76 
                 91 
                 96 
                 0.9 
               
               
                 576817 
                 8 
                 2 
                 6 
                 29 
                 58 
                 83 
                 4.7 
               
               
                 576818 
                 0 
                 22 
                 31 
                 68 
                 87 
                 90 
                 1.4 
               
               
                 576819 
                 0 
                 12 
                 44 
                 72 
                 81 
                 86 
                 1.4 
               
               
                 576820 
                 18 
                 24 
                 52 
                 78 
                 91 
                 93 
                 0.7 
               
               
                 576841 
                 23 
                 19 
                 29 
                 52 
                 75 
                 85 
                 1.6 
               
               
                 576842 
                 6 
                 12 
                 13 
                 37 
                 53 
                 83 
                 4.1 
               
               
                 576860 
                 9 
                 24 
                 54 
                 70 
                 83 
                 87 
                 1.0 
               
               
                 576878 
                 1 
                 9 
                 26 
                 61 
                 77 
                 83 
                 2.0 
               
               
                 576931 
                 16 
                 21 
                 24 
                 49 
                 77 
                 83 
                 1.8 
               
               
                 576942 
                 6 
                 16 
                 26 
                 57 
                 78 
                 85 
                 1.8 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                 ISIS No 
                 82.3 nM 
                 246.9 nM 
                 740.7 nM 
                 2222.2 nM 
                 6666.7 nM 
                 20000.0 nM 
                 IC 50  (μM) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 576894 
                 9 
                 30 
                 38 
                 61 
                 75 
                 84 
                 1.3 
               
               
                 576896 
                 17 
                 17 
                 28 
                 47 
                 66 
                 76 
                 2.5 
               
               
                 576927 
                 3 
                 26 
                 40 
                 60 
                 79 
                 81 
                 1.5 
               
               
                 576943 
                 37 
                 37 
                 55 
                 77 
                 84 
                 82 
                 0.4 
               
               
                 576945 
                 20 
                 41 
                 56 
                 73 
                 83 
                 84 
                 0.6 
               
               
                 576946 
                 8 
                 28 
                 46 
                 69 
                 81 
                 88 
                 1.0 
               
               
                 576963 
                 0 
                 0 
                 25 
                 51 
                 63 
                 83 
                 2.9 
               
               
                 576964 
                 11 
                 18 
                 37 
                 58 
                 73 
                 77 
                 1.8 
               
               
                 576967 
                 19 
                 31 
                 48 
                 68 
                 77 
                 85 
                 0.9 
               
               
                 577028 
                 6 
                 19 
                 25 
                 59 
                 79 
                 88 
                 1.6 
               
               
                 577029 
                 7 
                 22 
                 44 
                 67 
                 77 
                 85 
                 1.3 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
               
                   
               
               
                 ISIS No 
                 82.3 nM 
                 246.9 nM 
                 740.7 nM 
                 2222.2 nM 
                 6666.7 nM 
                 20000.0 nM 
                 IC 50  (μM) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 576960 
                 0 
                 12 
                 28 
                 49 
                 58 
                 78 
                 3.2 
               
               
                 576974 
                 25 
                 45 
                 65 
                 70 
                 65 
                 78 
                 0.5 
               
               
                 576816 
                 18 
                 36 
                 53 
                 82 
                 91 
                 95 
                 0.6 
               
               
                 577097 
                 22 
                 20 
                 31 
                 63 
                 82 
                 94 
                 1.1 
               
               
                 577101 
                 16 
                 23 
                 39 
                 62 
                 80 
                 89 
                 1.2 
               
               
                 577105 
                 0 
                 4 
                 30 
                 48 
                 78 
                 92 
                 2.0 
               
               
                 577104 
                 4 
                 1 
                 16 
                 56 
                 80 
                 92 
                 2.0 
               
               
                 577108 
                 0 
                 0 
                 24 
                 52 
                 76 
                 83 
                 2.9 
               
               
                 577083 
                 0 
                 0 
                 24 
                 50 
                 73 
                 74 
                 3.0 
               
               
                 577078 
                 0 
                 0 
                 10 
                 15 
                 30 
                 75 
                 10.8 
               
               
                 577077 
                 0 
                 0 
                 22 
                 22 
                 51 
                 83 
                 5.0 
               
               
                   
               
             
          
         
       
     
       Example 3 
     Dose-Dependent Antisense Inhibition of Human C9ORF72 in HepG2 Cells 
       [0295]    Antisense oligonucleotides from the study described above exhibiting significant in vitro inhibition of C9ORF72 mRNA were selected and tested at various doses in HepG2 cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 246.9 nM, 740.7 nM, 2,222.2 nM, 6,666.7 nM, or 20,000 nM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and C9ORF72 total mRNA levels, as well as mRNA levels of the exon 1 transcript, were measured by quantitative real-time PCR. Human C9ORF72 primer probe set RTS3750 was used to measure total C9ORF72 mRNA levels. Primer probe set RTS3905 (forward sequence GGGTCTAGCAAGAGCAGGTG, designated herein as SEQ ID NO: 18; reverse sequence GTCTTGGCAACAGCTGGAGAT, designated herein as SEQ ID NO: 19; probe sequence TGATGTCGACTCTTTGCCCACCGC, designated herein as SEQ ID NO: 20) was used to measure exon 1 message transcript. C9ORF72 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells. 
         [0296]    The half maximal inhibitory concentration (IC 50 ) of each oligonucleotide is also presented in Tables 14 and 15. As illustrated, C9ORF72 mRNA levels were reduced in a dose-dependent manner in the antisense oligonucleotide treated cells. ‘n.d.’ indicates that there is no data for that particular dose. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 % inhibition of total C9ORF72 mRNA levels 
               
             
          
           
               
                 ISIS No 
                 246.9 nM 
                 740.7 nM 
                 2222.2 nM 
                 6666.7 nM 
                 20000.0 nM 
                 IC 50  (μM) 
               
               
                   
               
             
          
           
               
                 576816 
                 29 
                 53 
                 84 
                 90 
                 92 
                 0.60 
               
               
                 576820 
                 20 
                 42 
                 70 
                 87 
                 75 
                 1.19 
               
               
                 576860 
                 25 
                 53 
                 72 
                 86 
                 85 
                 0.80 
               
               
                 576974 
                 36 
                 49 
                 64 
                 65 
                 68 
                 0.95 
               
               
                 577041 
                 3 
                 0 
                 0 
                 0 
                 0 
                 &gt;20.00 
               
               
                 577042 
                 0 
                 2 
                 0 
                 3 
                 0 
                 &gt;20.00 
               
               
                 577061 
                 0 
                 3 
                 0 
                 4 
                 0 
                 &gt;20.00 
               
               
                 577065 
                 7 
                 0 
                 1 
                 6 
                 0 
                 &gt;20.00 
               
               
                 577069 
                 3 
                 0 
                 3 
                 0 
                 0 
                 &gt;20.00 
               
               
                 577073 
                 7 
                 0 
                 8 
                 11 
                 0 
                 &gt;20.00 
               
               
                 577074 
                 0 
                 7 
                 11 
                 15 
                 0 
                 &gt;20.00 
               
               
                 577078 
                 0 
                 2 
                 20 
                 65 
                 81 
                 5.22 
               
               
                 577083 
                 0 
                 19 
                 55 
                 71 
                 75 
                 3.35 
               
               
                 577088 
                 6 
                 11 
                 49 
                 61 
                 74 
                 3.93 
               
               
                 577097 
                 3 
                 38 
                 62 
                 78 
                 82 
                 1.94 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 % inhibition of C9ORF72 exon 1 mRNA levels 
               
             
          
           
               
                 ISIS No 
                 246.9 nM 
                 740.7 nM 
                 2222.2 nM 
                 6666.7 nM 
                 20000.0 nM 
                 IC 50  (μM) 
               
               
                   
               
             
          
           
               
                 576794 
                 42 
                 67 
                 n.d. 
                 93 
                 87 
                 0.27 
               
               
                 576816 
                 45 
                 78 
                 93 
                 n.d. 
                 n.d. 
                 0.26 
               
               
                 576820 
                 54 
                 65 
                 92 
                 98 
                 94 
                 &lt;0.247 
               
               
                 576860 
                 43 
                 36 
                 71 
                 95 
                 91 
                 0.66 
               
               
                 577041 
                 0 
                 0 
                 49 
                 4 
                 31 
                 &gt;20.00 
               
               
                 577042 
                 9 
                 15 
                 0 
                 33 
                 12 
                 &gt;20.00 
               
               
                 577061 
                 8 
                 36 
                 70 
                 67 
                 76 
                 2.03 
               
               
                 577065 
                 20 
                 55 
                 67 
                 82 
                 62 
                 1.06 
               
               
                 577069 
                 22 
                 24 
                 61 
                 74 
                 70 
                 2.16 
               
               
                 577073 
                 4 
                 62 
                 69 
                 82 
                 81 
                 1.21 
               
               
                 577074 
                 8 
                 49 
                 69 
                 85 
                 85 
                 1.29 
               
               
                 577078 
                 0 
                 21 
                 59 
                 81 
                 n.d. 
                 1.90 
               
               
                 577083 
                 30 
                 43 
                 85 
                 88 
                 92 
                 0.71 
               
               
                 577088 
                 38 
                 44 
                 79 
                 87 
                 91 
                 0.61 
               
               
                 577097 
                 17 
                 47 
                 52 
                 94 
                 89 
                 1.27 
               
               
                   
               
             
          
         
       
     
       Example 4 
     Antisense Inhibition of Human C9ORF72 in HepG2 Cells 
       [0297]    Antisense oligonucleotides were designed targeting the hexanucleotide repeat expansion of a C9ORF72 nucleic acid and were tested for their effects on C9ORF72 mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. ISIS 576816 and ISIS 577065 were included in these assays for comparison. Cultured C9ORF72 fibroblasts at a density of 35,000 cells per well were transfected using electroporation with 7,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and C9ORF72 mRNA levels were measured by quantitative real-time PCR. Human primer probe sets RTS3750, RTS 3905, or RTS4097 (forward sequence CAAGCCACCGTCTCACTCAA, designated herein as SEQ ID NO: 24; reverse sequence GTAGTGCTGTCTACTCCAGAGAGTTACC, designated herein as SEQ ID NO: 25; probe sequence CTTGGCTTCCCTCAAAAGACTGGCTAATGT, designated herein as SEQ ID NO: 26) were used to measure mRNA levels. RTS3750 targets exon 2 of the mRNA transcripts and, therefore, measures total mRNA transcripts. RTS3905 targets the hexanucleotide repeat expansion containing transcript and, therefore, measures only mRNA transcripts that contain the hexanucleotide repeat expansion. RTS4097 targets the gene sequence at a site 3′ of the hexanucleotide repeat expansion. mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells. ‘n.d.’ indicates that there is no data for that particular antisense oligonucleotide. 
         [0298]    The antisense oligonucleotides in Table 16 were designed as uniform MOE oligonucleotides, or 3-10-3 MOE, 4-10-3 MOE, 4-10-4 MOE, 5-10-4 MOE, or 5-10-5 MOE gapmers. The uniform MOE oligonucleotides are 20 nucleosides in length, wherein each nucleoside comprises a 2′-MOE group. The 3-10-3 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on both the 5′ end and on the 3′ end comprising three nucleosides each. The 4-10-3 gapmers are 17 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on both the 5′ end and on the 3′ end comprising four and three nucleosides, respectively. The 4-10-4 gapmers are 18 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on both the 5′ end and on the 3′ end comprising four nucleosides each. The 5-10-4 gapmers are 19 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on both the 5′ end and on the 3′ end comprising five and four nucleosides, respectively. The 5-10-5 gapmers are 20 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on both the 5′ end and on the 3′ end comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment comprises a 2′-MOE group. The internucleoside linkages throughout each oligonucleotide are phosphorothioate linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the antisense oligonucleotide is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the antisense oligonucleotide is targeted human gene sequence. Each antisense oligonucleotide listed in Table 16 is targeted to the human C9ORF72 genomic sequence, designated herein as SEQ ID NO: 2 (the complement of GENBANK Accession No. NT — 008413.18 truncated from nucleosides 27535000 to 27565000) or SEQ ID NO: 13, which is an expanded version of the hexanucleotide repeat from intron 1 of the C9ORF72 gene. 
         [0299]    The data indicates that certain antisense oligonucleotides preferentially inhibit levels of C9ORF72 mRNA transcript levels that contain the hexanucleotide repeat. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 16 
               
               
                   
               
               
                 Target 
                 Target 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Start 
                 Start 
                   
                   
                   
                 % 
                 % 
                 % 
                   
               
               
                 Site 
                 Site on 
                   
                   
                   
                 inhi- 
                 inhi- 
                 inhi- 
                 SEQ 
               
               
                 on SEQ 
                 SEQ ID 
                   
                   
                 ISIS 
                 bition 
                 bition 
                 bition 
                 ID 
               
               
                 ID NO: 2 
                 NO: 13 
                 Motif 
                 Sequence 
                 NO 
                 (RTS3750) 
                 (RTS3905) 
                 (RTS4097) 
                 NO 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1457 
                 1 
                 Uniform 
                 CCGGCCCCGGCCC 
                 573674 
                 0 
                 34 
                 0 
                 340 
               
               
                   
                 7 
                 MOE 
                 CGGCCCC 
                   
                   
                   
                   
                   
               
               
                   
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 Uniform 
                 CCCGGCCCCGGCC 
                 573675 
                 0 
                 28 
                 0 
                 341 
               
               
                   
                 8 
                 MOE 
                 CCGGCCC 
                   
                   
                   
                   
                   
               
               
                   
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 Uniform 
                 CCCCGGCCCCGGC 
                 573676 
                 0 
                 34 
                 0 
                 342 
               
               
                   
                 9 
                 MOE 
                 CCCGGCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 Uniform 
                 GCCCCGGCCCCGG 
                 573677 
                 4 
                 41 
                 0 
                 343 
               
               
                   
                 10 
                 MOE 
                 CCCCGGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 n/a 
                 5 
                 Uniform 
                 GGCCCCGGCCCCG 
                 573678 
                 12 
                 11 
                 6 
                 344 
               
               
                   
                 11 
                 MOE 
                 GCCCCGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 n/a 
                 6 
                 Uniform 
                 CGGCCCCGGCCCC 
                 573679 
                 0 
                 0 
                 0 
                 345 
               
               
                   
                 12 
                 MOE 
                 GGCCCCG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1457 
                 1 
                 Uniform 
                 CGGCCCCGGCCCC 
                 573680 
                 10 
                 6 
                 0 
                 346 
               
               
                   
                 7 
                 MOE 
                 GGCCCC 
                   
                   
                   
                   
                   
               
               
                   
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 Uniform 
                 CCGGCCCCGGCCC 
                 573681 
                 13 
                 23 
                 0 
                 347 
               
               
                   
                 8 
                 MOE 
                 CGGCCC 
                   
                   
                   
                   
                   
               
               
                   
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 Uniform 
                 CCCGGCCCCGGCC 
                 573682 
                 2 
                 48 
                 0 
                 348 
               
               
                   
                 9 
                 MOE 
                 CCGGCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 Uniform 
                 CCCCGGCCCCGGC 
                 573683 
                 0 
                 38 
                 0 
                 349 
               
               
                   
                 10 
                 MOE 
                 CCCGGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1461 
                 5 
                 Uniform 
                 GCCCCGGCCCCGG 
                 573684 
                 0 
                 0 
                 0 
                 350 
               
               
                   
                 11 
                 MOE 
                 CCCCGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 n/a 
                 6 
                 Uniform 
                 GGCCCCGGCCCCG 
                 573685 
                 0 
                 27 
                 0 
                 351 
               
               
                   
                 12 
                 MOE 
                 GCCCCG 
                   
                   
                   
                   
                   
               
               
                   
                 18 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1457 
                 1 
                 Uniform 
                 GGCCCCGGCCCCG 
                 573686 
                 0 
                 40 
                 0 
                 352 
               
               
                   
                 7 
                 MOE 
                 GCCCC 
                   
                   
                   
                   
                   
               
               
                   
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 19 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 Uniform 
                 CGGCCCCGGCCCC 
                 573687 
                 0 
                 0 
                 0 
                 353 
               
               
                   
                 8 
                 MOE 
                 GGCCC 
                   
                   
                   
                   
                   
               
               
                   
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 Uniform 
                 CCGGCCCCGGCCC 
                 573688 
                 22 
                 0 
                 0 
                 354 
               
               
                   
                 9 
                 MOE 
                 CGGCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 Uniform 
                 CCCGGCCCCGGCC 
                 573689 
                 0 
                 22 
                 0 
                 355 
               
               
                   
                 10 
                 MOE 
                 CCGGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1461 
                 5 
                 Uniform 
                 CCCCGGCCCCGGC 
                 573690 
                 15 
                 43 
                 0 
                 356 
               
               
                   
                 11 
                 MOE 
                 CCCGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1462 
                 6 
                 Uniform 
                 GCCCCGGCCCCGG 
                 573691 
                 10 
                 16 
                 0 
                 357 
               
               
                   
                 12 
                 MOE 
                 CCCCG 
                   
                   
                   
                   
                   
               
               
                   
                 18 
                   
                   
                   
                   
                   
                   
                   
               
               
                 1463 
                 13 
                   
                 GCCCCGGCCCCGG 
                 573692 
                 6 
                 65 
                 0 
                 358 
               
               
                   
                 19 
                   
                 CCCC 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 Uniform 
                 GGCCCCGGCCCCG 
                 573693 
                 9 
                 0 
                 0 
                 359 
               
               
                   
                 8 
                 MOE 
                 GCCC 
                   
                   
                   
                   
                   
               
               
                   
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 20 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 Uniform 
                 CGGCCCCGGCCCC 
                 573694 
                 10 
                 0 
                 0 
                 360 
               
               
                   
                 9 
                 MOE 
                 GGCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 Uniform 
                 CCGGCCCCGGCCC 
                 573695 
                 3 
                 42 
                 0 
                 361 
               
               
                   
                 10 
                 MOE 
                 CGGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1461 
                 5 
                 Uniform 
                 CCCGGCCCCGGCC 
                 573696 
                 0 
                 23 
                 0 
                 362 
               
               
                   
                 11 
                 MOE 
                 CCGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1462 
                 6 
                 Uniform 
                 CCCCGGCCCCGGC 
                 573697 
                 0 
                 28 
                 0 
                 363 
               
               
                   
                 12 
                 MOE 
                 CCCG 
                   
                   
                   
                   
                   
               
               
                   
                 18 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1457 
                 1 
                 Uniform 
                 CCCCGGCCCCGGC 
                 573698 
                 1 
                 68 
                 0 
                 364 
               
               
                   
                 7 
                 MOE 
                 CCC 
                   
                   
                   
                   
                   
               
               
                 1463 
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 19 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 Uniform 
                 GCCCCGGCCCCGG 
                 573699 
                 0 
                 31 
                 0 
                 365 
               
               
                   
                 8 
                 MOE 
                 CCC 
                   
                   
                   
                   
                   
               
               
                   
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 20 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 Uniform 
                 GGCCCCGGCCCCG 
                 573700 
                 7 
                 2 
                 2 
                 366 
               
               
                   
                 9 
                 MOE 
                 GCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 21 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 Uniform 
                 CGGCCCCGGCCCC 
                 573701 
                 15 
                 1 
                 8 
                 367 
               
               
                   
                 10 
                 MOE 
                 GGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1461 
                 5 
                 Uniform 
                 CCGGCCCCGGCCC 
                 573702 
                 26 
                 0 
                 0 
                 368 
               
               
                   
                 11 
                 MOE 
                 CGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1462 
                 6 
                 Uniform 
                 CCCGGCCCCGGCC 
                 573703 
                 12 
                 52 
                 10 
                 369 
               
               
                   
                 12 
                 MOE 
                   
                   
                   
                   
                   
                   
               
               
                   
                 18 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1457 
                 1 
                 5-10-5 
                 CCGGCCCCGGCCC 
                 573716 
                 0 
                 93 
                 46 
                 340 
               
               
                   
                 7 
                 MOE 
                 CGGCCCC 
                   
                   
                   
                   
                   
               
               
                   
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 5-10-5 
                 CCCGGCCCCGGCC 
                 573717 
                 0 
                 98 
                 0 
                 341 
               
               
                   
                 8 
                 MOE 
                 CCGGCCC 
                   
                   
                   
                   
                   
               
               
                   
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 5-10-5 
                 CCCCGGCCCCGGC 
                 573718 
                 0 
                 98 
                 2 
                 342 
               
               
                   
                 9 
                 MOE 
                 CCCGGCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 5-10-5 
                 GCCCCGGCCCCGG 
                 573719 
                 0 
                 68 
                 19 
                 343 
               
               
                   
                 10 
                 MOE 
                 CCCCGGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 n/a 
                 5 
                 5-10-5 
                 GGCCCCGGCCCCG 
                 573720 
                 13 
                 90 
                 18 
                 344 
               
               
                   
                 11 
                 MOE 
                 GCCCCGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 n/a 
                 6 
                 5-10-5 
                 CGGCCCCGGCCCC 
                 573721 
                 0 
                 98 
                 18 
                 345 
               
               
                   
                 12 
                 MOE 
                 GGCCCCG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1457 
                 1 
                 5-10-4 
                 CGGCCCCGGCCCC 
                 573722 
                 0 
                 97 
                 0 
                 346 
               
               
                   
                 7 
                 MOE 
                 GGCCCC 
                   
                   
                   
                   
                   
               
               
                   
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 5-10-4 
                 CCGGCCCCGGCCC 
                 573723 
                 0 
                 n.d. 
                 8 
                 347 
               
               
                   
                 8 
                 MOE 
                 CGGCCC 
                   
                   
                   
                   
                   
               
               
                   
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 5-10-4 
                 CCCGGCCCCGGCC 
                 573724 
                 0 
                 94 
                 28 
                 348 
               
               
                   
                 9 
                 MOE 
                 CCGGCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 5-10-4 
                 CCCCGGCCCCGGC 
                 573725 
                 0 
                 94 
                 7 
                 349 
               
               
                   
                 10 
                 MOE 
                 CCCGGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1461 
                 5 
                 5-10-4 
                 GCCCCGGCCCCGG 
                 573726 
                 0 
                 n.d. 
                 28 
                 350 
               
               
                   
                 11 
                 MOE 
                 CCCCGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 n/a 
                 6 
                 5-10-4 
                 GGCCCCGGCCCCG 
                 573727 
                 0 
                 98 
                 40 
                 351 
               
               
                   
                 12 
                 MOE 
                 GCCCCG 
                   
                   
                   
                   
                   
               
               
                   
                 18 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1457 
                 1 
                 4-10-4 
                 GGCCCCGGCCCCG 
                 573728 
                 0 
                 97 
                 19 
                 352 
               
               
                   
                 7 
                 MOE 
                 GCCCC 
                   
                   
                   
                   
                   
               
               
                   
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 19 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 4-10-4 
                 CGGCCCCGGCCCC 
                 573729 
                 0 
                 n.d. 
                 36 
                 353 
               
               
                   
                 8 
                 MOE 
                 GGCCC 
                   
                   
                   
                   
                   
               
               
                   
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 4-10-4 
                 CCGGCCCCGGCCC 
                 573730 
                 0 
                 94 
                 24 
                 354 
               
               
                   
                 9 
                 MOE 
                 CGGCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 4-10-4 
                 CCCGGCCCCGGCC 
                 573731 
                 0 
                 97 
                 13 
                 355 
               
               
                   
                 10 
                 MOE 
                 CCGGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1461 
                 5 
                 4-10-4 
                 CCCCGGCCCCGGC 
                 573732 
                 0 
                 97 
                 1 
                 356 
               
               
                   
                 11 
                 MOE 
                 CCCGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1462 
                 6 
                 4-10-4 
                 GCCCCGGCCCCGG 
                 573733 
                 0 
                 n.d. 
                 0 
                 357 
               
               
                   
                 12 
                 MOE 
                 CCCCG 
                   
                   
                   
                   
                   
               
               
                   
                 18 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1457 
                 1 
                 4-10-3 
                 GCCCCGGCCCCGG 
                 573734 
                 0 
                 96 
                 0 
                 358 
               
               
                   
                 7 
                 MOE 
                 CCCC 
                   
                   
                   
                   
                   
               
               
                 1463 
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 19 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 4-10-3 
                 GGCCCCGGCCCCG 
                 573735 
                 0 
                 94 
                 21 
                 359 
               
               
                   
                 8 
                 MOE 
                 GCCC 
                   
                   
                   
                   
                   
               
               
                   
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 20 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 4-10-3 
                 CGGCCCCGGCCCC 
                 573736 
                 0 
                 93 
                 43 
                 360 
               
               
                   
                 9 
                 MOE 
                 GGCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 4-10-3 
                 CCGGCCCCGGCCC 
                 573737 
                 0 
                 96 
                 19 
                 361 
               
               
                   
                 10 
                 MOE 
                 CGGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1461 
                 5 
                 4-10-3 
                 CCCGGCCCCGGCC 
                 573738 
                 0 
                 n.d. 
                 24 
                 362 
               
               
                   
                 11 
                 MOE 
                 CCGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1462 
                 6 
                 4-10-3 
                 CCCCGGCCCCGGC 
                 573739 
                 0 
                 n.d. 
                 34 
                 363 
               
               
                   
                 12 
                 MOE 
                 CCCG 
                   
                   
                   
                   
                   
               
               
                   
                 18 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1457 
                 1 
                 3-10-3 
                 CCCCGGCCCCGGC 
                 573740 
                 0 
                 n.d. 
                 4 
                 364 
               
               
                   
                 7 
                 MOE 
                 CCC 
                   
                   
                   
                   
                   
               
               
                 1463 
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 19 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1458 
                 2 
                 3-10-3 
                 GCCCCGGCCCCGG 
                 573741 
                 0 
                 95 
                 6 
                 365 
               
               
                   
                 8 
                 MOE 
                 CCC 
                   
                   
                   
                   
                   
               
               
                 1464 
                 14 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 20 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 3 
                 3-10-3 
                 GGCCCCGGCCCCG 
                 573742 
                 23 
                 97 
                 49 
                 366 
               
               
                   
                 9 
                 MOE 
                 GCC 
                   
                   
                   
                   
                   
               
               
                   
                 15 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 21 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1460 
                 4 
                 3-10-3 
                 CGGCCCCGGCCCC 
                 573743 
                 0 
                 96 
                 0 
                 367 
               
               
                   
                 10 
                 MOE 
                 GGC 
                   
                   
                   
                   
                   
               
               
                   
                 16 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1461 
                 5 
                 3-10-3 
                 CCGGCCCCGGCCC 
                 573744 
                 0 
                 94 
                 34 
                 368 
               
               
                   
                 11 
                 MOE 
                 CGG 
                   
                   
                   
                   
                   
               
               
                   
                 17 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1462 
                 6 
                 3-10-3 
                 CCCGGCCCCGGCC 
                 573745 
                 0 
                 n.d. 
                 8 
                 369 
               
               
                   
                 12 
                 MOE 
                 CCG 
                   
                   
                   
                   
                   
               
               
                   
                 18 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 7990 
                 n/a 
                 5-10-5 
                 GCCTTACTCTAGG 
                 576816 
                 83 
                 91 
                 29 
                 40 
               
               
                   
                   
                 MOE 
                 ACCAAGA 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1446 
                 n/a 
                 5-10-5 
                 CCCGGCCCCTAGC 
                 577065 
                 0 
                 87 
                 34 
                 288 
               
               
                   
                   
                 MOE 
                 GCGCGAC 
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
       Example 5 
     In Vivo Rodent Inhibition and Tolerability with Treatment of C9ORF72 Antisense Oligonucleotides 
       [0300]    In order to assess the tolerability of inhibition of C9ORF72 expression in vivo, antisense oligonucleotides targeting a murine C9ORF72 nucleic acid were designed and assessed in mouse and rat models. 
         [0301]    ISIS 571883 was designed as a 5-10-5 MOE gapmer, 20 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on both the 5′ end and on the 3′ end comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The internucleoside linkages are phosphorothioate linkages. All cytosine residues throughout the gapmer are 5-methylcytosines. ISIS 571883 has a target start site of nucleoside 33704 on the murine C9ORF72 genomic sequence, designated herein as SEQ ID NO: 11 (the complement of GENBANK Accession No. NT — 166289.1 truncated from nucleosides 3587000 to 3625000). 
         [0302]    ISIS 603538 was designed as a 5-10-5 MOE gapmer, 20 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on both the 5′ end and on the 3′ end comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The internucleoside linkages are either phosphorothioate linkages or phosphate ester linkages (Gs Ao Co Co Gs Cs Ts Ts Gs As Gs Ts Ts Ts Gs Co Co Ao Cs A; wherein ‘s’ denotes a phosphorothioate internucleoside linkage, ‘o’ denotes a phosphate ester linkage; and A, G, C, T denote the relevant nucleosides). All cytosine residues throughout the gapmer are 5-methylcytosines. ISIS 603538 has a target start site of nucleoside 2872 on the rat C9ORF72 mRNA sequence, designated herein as SEQ ID NO: 12 (GENBANK Accession No. NM —  001007702.1). 
       Mouse Experiment 1 
       [0303]    Groups of 4 C57BL/6 mice each were injected with 50 μg, 100 μg, 300 μg, 500 μg, or 700 μg of ISIS 571883 administered via an intracerebroventricular bolus injection. A control group of four C57/BL6 mice were similarly treated with PBS. Animals were anesthetized with 3% isofluorane and placed in a stereotactic frame. After sterilizing the surgical site, each mouse was injected −0.2 mm anterio-posterior from the bregma na d 3 mm dorsoventral to the bregma with the above-mentioned doses of ISIS 571883 using a Hamilton syringe. The incision was closed with sutures. The mice were allowed to recover for 14 days, after which animals were euthanized according to a humane protocol approved by the Institutional Animal Care and Use Committee. Brain and spinal cord tissue were harvested and snap frozen in liquid nitrogen. Prior to freezing, brain tissue was cut transversely five sections using a mouse brain matrix. 
       RNA Analysis 
       [0304]    RNA was extracted from a 2-3 mm brain section posterior to the injection site, from brain frontal cortex and from the lumbar section of the spinal cord tissue for analysis of C9ORF72 mRNA expression. C9ORF72 mRNA expression was measured by RT-PCR. The data is presented in Table 17. The results indicate that treatment with increasing doses of ISIS 571883 resulted in dose-dependent inhibition of C9ORF72 mRNA expression. 
         [0305]    The induction of the microglial marker AIF-1 as a measure of CNS toxicity was also assessed. The data is presented in Table 18. The results indicate that treatment with increasing doses of ISIS 571883 did not result in significant increases in AIF-1 mRNA expression. Hence, the injection of ISIS 571883 was deemed tolerable in this model. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 17 
               
             
             
               
                   
               
               
                 Percentage inhibition of C9ORF72 mRNA 
               
               
                 expression compared to the PBS control 
               
             
          
           
               
                   
                   
                 Posterior 
                   
                 Spinal 
               
               
                   
                 Dose (μg) 
                 brain 
                 Cortex 
                 cord 
               
               
                   
                   
               
             
          
           
               
                   
                 50 
                 22 
                 8 
                 46 
               
               
                   
                 100 
                 22 
                 12 
                 47 
               
               
                   
                 300 
                 55 
                 47 
                 67 
               
               
                   
                 500 
                 61 
                 56 
                 78 
               
               
                   
                 700 
                 65 
                 65 
                 79 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 18 
               
             
             
               
                   
               
               
                 Percentage expression of AIF-1 mRNA expression 
               
               
                 compared to the PBS control 
               
             
          
           
               
                   
                 Posterior 
                 Spinal 
               
               
                 Dose (μg) 
                 brain 
                 cord 
               
               
                   
               
             
          
           
               
                 50 
                 102 
                 89 
               
               
                 100 
                 105 
                 111 
               
               
                 300 
                 107 
                 98 
               
               
                 500 
                 131 
                 124 
               
               
                 700 
                 122 
                 116 
               
               
                   
               
             
          
         
       
     
       Mouse Experiment 2 
       [0306]    Groups of 4 C57BL/6 mice each were injected with 500 μg of ISIS 571883 administered via an intracerebroventricular bolus injection in a procedure similar to that described above. A control group of four C57/BL6 mice were similarly treated with PBS. The mice were tested at regular time points after ICV administration. 
       Behavior Analysis 
       [0307]    Two standard assays to assess motor behavior were employed; the rotarod assay and grip strength assay. In case of the rotarod assays, the time of latency to fall was measured. The data for the assays is presented in Tables 19 and 20. The results indicate that there were no significant changes in the motor behavior of the mice as a result of antisense inhibition of ISIS 571883 or due to the ICV injection. Hence, antisense inhibition of C9ORF72 was deemed tolerable in this model. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 19 
               
             
             
               
                   
               
               
                 Latency to fall (sec) in the rotarod assay 
               
             
          
           
               
                 Weeks after 
                   
                 ISIS 
               
               
                 injection 
                 PBS 
                 571883 
               
               
                   
               
               
                 0 
                 66 
                 66 
               
               
                 4 
                 91 
                 70 
               
               
                 8 
                 94 
                 84 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 20 
               
             
             
               
                   
               
               
                 Mean hindlimb grip strength (g) in the grip strength assay 
               
             
          
           
               
                 Weeks after 
                   
                 ISIS 
               
               
                 injection 
                 PBS 
                 571883 
               
               
                   
               
             
          
           
               
                 0 
                 57 
                 63 
               
               
                 1 
                 65 
                 51 
               
               
                 2 
                 51 
                 52 
               
               
                 3 
                 51 
                 51 
               
               
                 4 
                 59 
                 72 
               
               
                 5 
                 60 
                 64 
               
               
                 6 
                 61 
                 72 
               
               
                 7 
                 67 
                 68 
               
               
                 8 
                 66 
                 70 
               
               
                 9 
                 63 
                 61 
               
               
                 10 
                 48 
                 46 
               
               
                   
               
             
          
         
       
     
       Rat Experiment 
       [0308]    Groups of 4 Sprague-Dawley rats each were injected with 700 μg, 1,000 μg, or 3,000 μg of ISIS 603538 administered via an intrathecal bolus injection. A control group of four Sprague-Dawley rats were similarly treated with PBS. Animals were anesthetized with 3% isofluorane and placed in a stereotactic frame. After sterilizing the surgical site, each rat was injected with 30 μL of ASO solution administered via 8 cm intrathecal catheter 2 cm into the spinal canal with a 50 μL flush. The rats were allowed to recover for 4 weeks, after which animals were euthanized according to a humane protocol approved by the Institutional Animal Care and Use Committee. 
       RNA Analysis 
       [0309]    RNA was extracted from a 2-3 mm brain section posterior to the injection stie, from brain frontal cortex, and from the cervical and lumbar sections of the spinal cord tissue for analysis of C9ORF72 mRNA expression. C9ORF72 mRNA expression was measured by RT-PCR. The data is presented in Table 21. The results indicate that treatment with increasing doses of ISIS 603538 resulted in dose-dependent inhibition of C9ORF72 mRNA expression. 
         [0310]    The induction of the microglial marker AIF-1 as a measure of CNS toxicity was also assessed. The data is presented in Table 22. The results indicate that treatment with increasing doses of ISIS 603538 did not result in significant increases in AIF-1 mRNA expression. Hence, the injection of ISIS 603538 was deemed tolerable in this model. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 21 
               
             
             
               
                   
               
               
                 Percentage inhibition of C9ORF72 mRNA 
               
               
                 expression compared to the PBS control 
               
             
          
           
               
                 Dose 
                 Brain (1 mm 
                   
                 Spinal cord 
                 Spinal cord 
               
               
                 (μg) 
                 section) 
                 Cortex 
                 (lumbar) 
                 (cervical) 
               
               
                   
               
             
          
           
               
                 700 
                 21 
                 4 
                 86 
                 74 
               
               
                 1000 
                 53 
                 49 
                 88 
                 82 
               
               
                 3000 
                 64 
                 62 
                 88 
                 80 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 22 
               
             
             
               
                   
               
               
                 Percentage expression of AIF-1 mRNA expression 
               
               
                 compared to the PBS control 
               
             
          
           
               
                 Dose 
                 Brain (1 mm 
                   
                 Spinal cord 
                 Spinal cord 
               
               
                 (μg) 
                 section) 
                 Cortex 
                 (lumbar) 
                 (cervical) 
               
               
                   
               
             
          
           
               
                 700 
                 97 
                 119 
                 98 
                 89 
               
               
                 1000 
                 105 
                 113 
                 122 
                 96 
               
               
                 3000 
                 109 
                 141 
                 156 
                 115 
               
               
                   
               
             
          
         
       
     
       Body Weight Analysis 
       [0311]    Body weights of the rats were measured at regular time point intervals. The data is presented in Table 23. The results indicate that treatment with increasing doses of ISIS 603538 did not have any significant changes in the body weights of the rats. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 23 
               
             
             
               
                   
               
               
                 Body weights of the rats (% initial body weight) 
               
             
          
           
               
                   
                 Dose 
                   
                   
                   
                   
                   
               
               
                   
                 (μg) 
                 Week 1 
                 Week 2 
                 Week 3 
                 Week 4 
                 Week 5 
               
               
                   
                   
               
             
          
           
               
                 PBS 
                   
                 100 
                 94 
                 103 
                 105 
                 109 
               
               
                 ISIS 
                 700 
                 100 
                 94 
                 98 
                 103 
                 107 
               
               
                 603538 
                 1000 
                 100 
                 95 
                 97 
                 101 
                 103 
               
               
                   
                 3000 
                 100 
                 92 
                 98 
                 102 
                 105 
               
               
                   
               
             
          
         
       
     
       Example 6 
     Preferential Inhibition of Human C9ORF72 Expression in Two Patient Fibroblast Lines 
       [0312]    Two different fibroblast cell lines from human patients (F09-152 and F09-229) were analyzed with antisense oligonucleotides that target the C9ORF72 pre-mRNA sequence before exon 1B; i.e. antisense oligonucleotides that target the hexanucleotide repeat expansion containing transcript and antisense oligonucleotides that target downstream of exon 1. The target start and stop sites and the target regions with respect to SEQ ID NOs: 1 and 2 for each oligonucleotide are provided in Table 24. ISIS 577061 and ISIS 577065 target C9ORF72 upstream of exon 1B and just upstream of the hexanucleotide repeat. The rest of the ISIS oligonucleotides of Table 24 target C9ORF72 downstream of exon 1B and the hexanucleotide repeat. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 24 
               
             
             
               
                   
               
               
                 Target Start and Stop sites of ISIS oligonucleotides used 
               
               
                 in a dose response assay in C9ORF72 patient fibroblasts 
               
             
          
           
               
                   
                   
                 Target 
                 Target 
                   
               
               
                   
                   
                 Start Site 
                 Start Site 
               
               
                   
                 ISIS 
                 at SEQ ID 
                 at SEQ ID 
                 Target 
               
               
                   
                 No 
                 NO: 1 
                 NO: 2 
                 Region 
               
               
                   
                   
               
             
          
           
               
                   
                 577061 
                 n/a 
                 1406 
                 Upstream of 
               
               
                   
                   
                   
                   
                 exon 1B 
               
               
                   
                 577065 
                 n/a 
                 1446 
                 Upstream of 
               
               
                   
                   
                   
                   
                 exon 1B 
               
               
                   
                 577083 
                 n/a 
                 3452 
                 Downstream 
               
               
                   
                   
                   
                   
                 of exon 1B 
               
               
                   
                 576816 
                  232 
                 7990 
                 Exon 2 
               
               
                   
                 576974 
                 3132 
                 28251 
                 Exon 11 
               
               
                   
                   
               
             
          
         
       
     
         [0313]    Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 246.9 nM, 740.7 nM, 2,222.2 nM, 6,666.7 nM, and 20,000.0 nM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and C9ORF72 mRNA levels were measured by quantitative real-time PCR. Two primer probe sets were used: (1) human C9ORF72 primer probe set RTS3750, which measures total mRNA levels, and (2) RTS3905, which targets the hexanucleotide repeat expansion containing transcript, which measures only mRNA transcripts that contain the hexanucleotide repeat expansion. C9ORF72 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells. 
         [0314]    As illustrated in Table 25, below, the two oligonucleotides that target upstream of exon 1B and, therefore, target mRNA transcripts containing the hexanucleotide repeat expansion (ISIS 577061 and ISIS 577065), do not inhibit total mRNA levels of C9ORF72 (as measured by RTS3750) as well as ISIS 576974, 576816, and 577083, which target downstream of exon 1B and, therefore, do not target the mRNA transcript containing the hexanucleotide repeat expansion. Expression levels of the C9ORF72 mRNA transcript containing the hexanucleotide repeat expansion are low (about 10% of the total C9ORF72 expression products), therefore, oligonucleotides targeting the mRNA transcript containing the hexanucleotide repeat expansion do not robustly inhibit total C9ORF72 mRNA (as measured by RTS3905), as suggested by Table 25 below. Thus, ISIS 577061 and ISIS 577065 preferentially inhibit expression of mRNA transcripts containing the hexanucleotide repeat expansion. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 25 
               
             
             
               
                   
               
               
                 Percent inhibition of C9ORF72 total mRNA in F09-152 patient 
               
               
                 fibroblasts in a dose response assay as measured with RTS3750 
               
             
          
           
               
                   
                 ISIS 
                 246.9 
                 740.7 
                 2222.2 
                 6666.7 
                 20000.0 
               
               
                   
                 No 
                 nM 
                 nM 
                 nM 
                 nM 
                 nM 
               
               
                   
                   
               
             
          
           
               
                   
                 577061 
                 6 
                 11 
                 0 
                 18 
                 10 
               
               
                   
                 577065 
                 10 
                 11 
                 30 
                 29 
                 0 
               
               
                   
                 576974 
                 61 
                 69 
                 72 
                 83 
                 83 
               
               
                   
                 576816 
                 35 
                 76 
                 82 
                 91 
                 93 
               
               
                   
                 577083 
                 28 
                 38 
                 52 
                 75 
                 80 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 26 
               
             
             
               
                   
               
               
                 Percent inhibition of C9ORF72 mRNA transcripts containing 
               
               
                 the hexanucleotide repeat expansion in F09-152 patient fibroblasts 
               
               
                 in a dose response assay as measured with RTS3905 
               
             
          
           
               
                   
                 ISIS 
                 246.9 
                 740.7 
                 2222.2 
                 6666.7 
                 20000.0 
               
               
                   
                 No 
                 nM 
                 nM 
                 nM 
                 nM 
                 nM 
               
               
                   
                   
               
             
          
           
               
                   
                 577061 
                 4 
                 28 
                 58 
                 81 
                 87 
               
               
                   
                 577065 
                 25 
                 54 
                 70 
                 90 
                 94 
               
               
                   
                 576974 
                 57 
                 77 
                 81 
                 93 
                 92 
               
               
                   
                 576816 
                 37 
                 77 
                 91 
                 97 
                 98 
               
               
                   
                 577083 
                 37 
                 53 
                 74 
                 93 
                 94 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 27 
               
             
             
               
                   
               
               
                 Percent inhibition of C9ORF72 total mRNA in F09-229 patient 
               
               
                 fibroblasts in a dose response assay as measured with RTS3750 
               
             
          
           
               
                   
                 ISIS 
                 246.9 
                 740.7 
                 2222.2 
                 6666.7 
                 20000.0 
               
               
                   
                 No 
                 nM 
                 nM 
                 nM 
                 nM 
                 nM 
               
               
                   
                   
               
             
          
           
               
                   
                 577061 
                 0 
                 0 
                 0 
                 17 
                 7 
               
               
                   
                 577065 
                 8 
                 17 
                 17 
                 16 
                 3 
               
               
                   
                 576974 
                 43 
                 58 
                 85 
                 85 
                 74 
               
               
                   
                 576816 
                 45 
                 70 
                 85 
                 81 
                 89 
               
               
                   
                 577083 
                 22 
                 45 
                 56 
                 76 
                 78 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 28 
               
             
             
               
                   
               
               
                 Percent inhibition of C9ORF72 mRNA transcripts containing 
               
               
                 the hexanucleotide repeat expansion in F09-229 patient fibroblasts 
               
               
                 in a dose response assay as measured with RTS3905 
               
             
          
           
               
                   
                 ISIS 
                 246.9 
                 740.7 
                 2222.2 
                 6666.7 
                 20000.0 
               
               
                   
                 No 
                 nM 
                 nM 
                 nM 
                 nM 
                 nM 
               
               
                   
                   
               
             
          
           
               
                   
                 577061 
                 14 
                 36 
                 70 
                 87 
                 89 
               
               
                   
                 577065 
                 26 
                 48 
                 92 
                 91 
                 98 
               
               
                   
                 576974 
                 63 
                 87 
                 91 
                 92 
                 91 
               
               
                   
                 576816 
                 62 
                 81 
                 96 
                 98 
                 100 
               
               
                   
                 577083 
                 36 
                 64 
                 82 
                 98 
                 96