Compositions for Modulating C9ORF72 Expression

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0235USC1SEQ_ST25.txt created Jan. 15, 2019, which is 444 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

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

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 (Hirtz 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).

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 mutation in the C9ORF72 gene is the most common genetic cause of ALS and FTD. 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).

A founder haplotype, covering the C9ORF72 gene, is present in the majority of cases linked to this region.

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

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.

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, C9ORF72 associated Repeat Associated Non-ATG Translation (RAN translation) products are reduced. In certain embodiments, the C9ORF72 associated RAN translation products are poly-(glycine-proline), poly-(glycine-alanine), and poly-(glycine-arginine). In certain embodiments, certain C9ORF72 mRNA variants are preferentially reduced. In certain embodiments, the C9ORF72 mRNA variants preferentially reduced are variants processed from a pre-mRNA containing intron 1. In certain embodiments, intron 1 contains a hexanucleotide repeat expansion. In certain embodiments, the C9ORF72 mRNA variant preferentially reduced is a C9ORF72 pathogenic associated mRNA variant. In certain embodiments, the C9ORF72 pathogenic associated mRNA variant is NM 001256054.1 (SEQ ID NO: 1). 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 24 GGGGCC repeats. In certain embodiments, the hexanucleotide repeat expansion is associated with nuclear foci. In certain embodiments, C9ORF72 associated RAN translation products are associated with nuclear foci. In certain embodiments, the C9ORF72 associated RAN translation products are poly-(glycine-proline), poly-(glycine-alanine), and poly-(glycine-arginine). In certain embodiments, the compositions and methods described herein are useful for reducing C9ORF72 mRNA levels, C9ORF72 protein levels, C9ORF72 RAN translation products, and nuclear foci. In certain embodiments, the compositions and methods described herein are useful for selectively reducing C9ORF72 pathogenic associated mRNA variants. Such reduction can occur in a time-dependent manner or in a dose-dependent manner.

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).

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.

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

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.

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

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.

Unless otherwise indicated, the following terms have the following meanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-OCH₂CH₂—OCH₃and MOE) refers to an 0-methoxy-ethyl modification of the 2′ position of a furanose ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position of the furanose ring other than H or OH. In certain embodiments, 2′-substituted nucleosides include nucleosides with bicyclic sugar modifications.

“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. A 5-methylcytosine is a modified nucleobase.

“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%.

“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.

“Administering” means providing a pharmaceutical agent to an animal, and includes, but is not limited to administering by a medical professional and self-administering.

“Amelioration” refers to a lessening, slowing, stopping, or reversing of at least one indicator of the severity of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

“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.

“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.

“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.

“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 inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or in the absence of the antisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with a target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding segment of a target nucleic acid.

“Base complementarity” refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.

“Bicyclic sugar” means a furanose ring modified by the bridging of two atoms. A bicyclic sugar is a modified sugar.

“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.

“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.

“C9ORF72 associated RAN translation products” means aberrant peptide or di-peptide polymers translated through RAN translation (i.e., repeat-associated, and non-ATG-dependent translation). In certain embodiments, the C9ORF72 associated RAN translation products are any of poly-(glycine-proline), poly-(glycine-alanine), and poly-(glycine-arginine).

“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 24 times. Such diseases may include a neurodegenerative disease. Such neurodegenerative diseases may include ALS and FTD.

“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 (i.e., pre-mRNA), and an mRNA sequence encoding C9ORF72. “C9ORF72 mRNA” means an mRNA encoding a C9ORF72 protein.

“C9ORF72 pathogenic associated mRNA variant” means the C9ORF72 mRNA variant processed from a C9ORF72 pre-mRNA variant containing the hexanucleotide repeat. A C9ORF72 pre-mRNA contains the hexanucleotide repeat when transcription of the pre-mRNA begins in the region from the start site of exon 1A to the start site of exon 1B, e.g., nucleotides 1107 to 1520 of the genomic sequence (SEQ ID NO: 2, the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, the level of a C9ORF72 pathogenic associated mRNA variant is measured to determine the level of a C9ORF72 pre-mRNA containing the hexanucleotide repeat in a sample.

“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.

“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.

“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(CH₃)—O-2′.

“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

“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.

“Chimeric antisense compound” means an antisense compound that has at least two chemically distinct regions, each position having a plurality of subunits.

“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.

“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

“Comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other.

“Designing” or“designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected, the diluent may be a liquid, e.g. saline solution.

“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.

“Effective amount” in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of pharmaceutical agent to a subject in need of such modulation, treatment, or prophylaxis, either in a single dose or as part of a series, that is effective for modulation of that effect, or for treatment or prophylaxis or improvement of that condition. 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's medical condition, and other relevant factors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.

“Foci” means a nuclear foci comprising a C9ORF72 transcript. In certain embodiments, a foci comprises at least one C9ORF72 transcript. In certain embodiments, C9ORF72 foci comprise transcripts comprising any of the following hexanucleotide repeats: GGGGCC, GGGGGG, GGGGGC, and/or GGGGCG.

“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.

“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.”

“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.

“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.

“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 24 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.

“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a target nucleic acid. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.

“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's medical history and standard clinical tests or assessments, such as genetic testing.

“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment or therapy.

“Inhibiting C9ORF72” means reducing the level or expression of a C9ORF72 mRNA and/or protein. In certain embodiments, C9ORF72 mRNA and/or protein levels are inhibited in the presence of an antisense compound targeting C9ORF72, including an antisense oligonucleotide targeting C9ORF72, as compared to expression of C9ORF72 mRNA and/or protein levels in the absence of a C9ORF72 antisense compound, such as an antisense oligonucleotide.

“Inhibiting the expression or activity” refers to a reduction or blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond between nucleosides.

“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.

“Locked nucleic acid” or “LNA” or “LNA nucleosides” means nucleic acid monomers having a bridge connecting two carbon atoms between the 4′ and 2′ position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugar include, but are not limited to A) α-L-Methyleneoxy (4′-CH₂—O-2′) LNA, (B) β-D-Methyleneoxy (4′-CH₂—O-2′) LNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) LNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH₂—N(R)—O-2′) LNA, as depicted below.

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As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R₁)(R₂)]_n—, —C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—, —C(═S)—, —O—, —Si(R₁)₂—, —S(═O)_x— and —N(R₁)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R₁and R₂is, independently, H, a protecting group, hydroxyl, C₁-C₁₂alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂alkynyl, C₅-C₂₀aryl, substituted C₅-C₂₀aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇alicyclic radical, substituted C₅-C₇alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and each J₁and J₂is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂alkynyl, C₅-C₂₀aryl, substituted C₅-C₂₀aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂aminoalkyl, substituted C₁-C₁₂aminoalkyl or a protecting group.

Examples of 4′-2′ bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: —[C(R₁)(R₂)]_n—, —[C(R₁)(R₂)]_n—O—, —C(R₁R₂)—N(R₁)—O— or —C(R₁R₂)—O—N(R₁)—. Furthermore, other bridging groups encompassed with the definition of LNA are 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R₁)-2′ and 4′-CH₂—N(R₁)—O-2′-bridges, wherein each R₁and R₂is, independently, H, a protecting group or C₁-C₁₂alkyl.

Also included within the definition of LNA according to the invention are LNAs in which the 2′-hydroxyl group of the ribosyl sugar ring is connected to the 4′ carbon atom of the sugar ring, thereby forming a methyleneoxy (4′-CH₂—O-2′) bridge to form the bicyclic sugar moiety. The bridge can also be a methylene (—CH₂—) group connecting the 2′ oxygen atom and the 4′ carbon atom, for which the term methyleneoxy (4′-CH₂—O-2′) LNA is used. Furthermore; in the case of the bicylic sugar moiety having an ethylene bridging group in this position, the term ethyleneoxy (4′-CH₂CH₂—O-2′) LNA is used. α-L-methyleneoxy (4′-CH₂—O-2′), an isomer of methyleneoxy (4′-CH₂—O-2′) LNA is also encompassed within the definition of LNA, as used herein.

“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.

“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e., a phosphodiester internucleoside bond).

“Modified nucleobase” means 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).

“Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, and/or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, modified sugar, and/or modified nucleobase.

“Modified sugar” means substitution and/or any change from a natural sugar moiety.

“Monomer” means a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

“Motif” means the pattern of unmodified and modified nucleoside in an antisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

“Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“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. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

“Off-target effect” refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

“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.

“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection (e.g., bolus 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.

“Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Without limitation, as used herein, peptide refers to polypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to C9ORF72 is a pharmaceutical agent.

“Pharmaceutical composition” means a mixture of substances suitable for administering to as subject. For example, a pharmaceutical composition may comprise an antisense oliognucleotide and a sterile aqueous solution.

“Pharmaceutically acceptable derivative” encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.

“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.

“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 is a modified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. 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.

“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 days, weeks to months, or indefinitely.

“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.

“Prophylactically effective amount” refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.

“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

“Salts” mean a 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.

“Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.

“Shortened” or “truncated” versions of antisense oligonucleotides taught herein have one, two or more nucleosides deleted.

“Side effects” means physiological responses attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies.

“Single-stranded oligonucleotide” means an oligonucleotide which is not hybridized to a complementary strand.

“Sites,” as used herein, are defined as unique nucleobase positions within a target nucleic acid.

“Slows progression” means decrease in the development of the disease.

“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.

“Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

“Subject” means a human or non-human animal selected for treatment or therapy.

“Target” refers to a protein, the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“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.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds.

“Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.

“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.

“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.

“Treat” or “treating” or “treatment” means administering a composition to effect an alteration or improvement of a disease or condition.

“Unmodified nucleobases” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases (T), cytosine (C), and uracil (U).

“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).

“Wing segment” means a plurality of nucleosides modified to impart to an oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Certain Embodiments

Certain embodiments provide compositions and methods for decreasing total C9ORF72 mRNA and protein expression.

Certain embodiments provide compositions and methods for decreasing C9ORF72 pathogenic associated mRNA variants.

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.

Certain embodiments provide compositions and methods for the treatment, prevention, or amelioration of a C9ORF72 hexanucleotide repeat expansion associated disease. In certain embodiments, the hexanucleotide repeat expansion may comprise GGGGCC, GGGGGG, GGGGGC, or GGGGCG.

Provided herein are compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having 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 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: SEQ ID NOs: 20-401 and 441-1545.

Provided herein are compounds, comprising a modified 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 consecutive nucleobases complementary to an equal length portion of nucleobases 1107-1520 of SEQ ID NO: 2.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide is a single-stranded modified oligonucleotide.

In certain embodiments at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.

In certain embodiments, at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, each modified internucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, at least one internucleoside linkage is a phosphodiester internucleoside linkage.

In certain embodiments, at least one internucleoside linkage is a phosphorothioate linkage and at least one internucleoside linkage is a phosphodiester linkage.

In certain embodiments, at least one nucleoside comprises a modified nucleobase.

In certain embodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar.

In certain embodiments, the at least one modified sugar is a bicyclic sugar.

In certain embodiments, the bicyclic sugar comprises a chemical link between the 2′ and 4′ position of the sugar 4′-CH2-N(R)—O-2′ bridge wherein R is, independently, H, C1-C12 alkyl, or a protecting group.

In certain embodiments, the bicyclic sugar comprises a 4′-CH2-N(R)—O-2′ bridge wherein R is, independently, H, C1-C12 alkyl, or a protecting group.

In certain embodiments, at least one modified sugar comprises a 2′-O-methoxyethyl group.

In certain embodiments, the modified sugar comprises a 2′-O(CH₂)₂—OCH₃group.

In certain embodiments, the modified oligonucleotide comprises:

a gap segment consisting of 10 linked deoxynucleosides;

a 5′ wing segment consisting of 5 linked nucleosides; and

a 3′ wing segment consisting of 5 linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

In certain embodiments, the modified oligonucleotide comprises:

a gap segment consisting of 8 linked deoxynucleosides;

a 5′ wing segment consisting of 5 linked nucleosides; and a 3′ wing segment consisting of 5 linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

In certain embodiments, the modified oligonucleotide comprises sugar modifications in any of the following patterns: eeekkdddddddkkeee, eekkddddddddkkeee, ekddddddddekekeee, kekeddddddddekeke, and ekekddddddddkekee; wherein,

e=a 2′-O-methoxyethyl modified nucleoside

d=a 2′-deoxynucleoside, and

k=a cEt nucleoside.

In certain embodiments, the modified oligonucleotide comprises internucleoside linkages in any of the following patterns: soooossssssssssooss, sooosssssssssooss, soosssssssssooss, and sosssssssssoooss; wherein,

s=a phosphorothioate linkage, and

o=a phosphodiester linkage.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 19 linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides.

Provided herein are compositions comprising the compound of any preceding claim or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent.

Provided herein are methods comprising administering to an animal the compound or composition of any preceding claim.

In certain embodiments, the animal is a human.

In certain embodiments, administering the compound prevents, treats, ameliorates, or slows progression of a C9ORF72 associated disease, disorder or condition.

In certain embodiments, administering the compound prevents, treats, ameliorates, or slows progression of a C9ORF72 hexanucleotide repeat expansion associated disease, disorder or condition.

In certain embodiments, the disease, disorder or condition is amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), corticalbasal degeneration syndrome (CBD), atypical Parkinsonian syndrome, and olivopontocerellar degeneration (OPCD).

In certain embodiments, the administering reduces nuclear foci.

In certain embodiments, the administering reduces expression of C9ORF72 associated RAN translation products.

In certain embodiments, the C9ORF72 associated RAN translation products are any of poly-(glycine-proline), poly-(glycine-alanine), and poly-(glycine-arginine).

Provided herein are uses of the compound or composition of any preceding claim for the manufacture of a medicament for treating a neurodegenerative disorder.

Provided herein are methods of selectively inhibiting a C9ORF72 pathogenic associated mRNA variant by administering an antisense compound targeting the region beginning at the start site of exon 1A to the start site of exon 1B of a C9ORF72 pre-mRNA.

Antisense Compounds

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH₂)n-O-2′ bridge, where n=1 or n=2 and 4′-CH₂—O—CH₂-2′). 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.

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.

In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid possess a 5-10-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid possess a 5-8-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid possess sugar modifications in any of the following patterns: eeekkdddddddkkeee, eekkddddddddkkeee, ekddddddddekekeee, kekeddddddddekeke, and ekekddddddddkekee; wherein,

e=a 2′-O-methoxyethyl modified nucleoside

d=a 2′-deoxynucleoside, and

k=a cEt nucleoside.

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′-(CH₂)_n—O-2′ bridge, wherein n is 1 or 2; and 4′-CH₂—O—CH₂-2′. In certain embodiments, the bicyclic sugar is comprises a 4′-CH(CH₃)—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.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

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), and GENBANK Accession No. NW_001101662.1 truncated from nucleosides 8522000 to 8552000 (incorporated herein as SEQ ID NO: 19).

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.

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.

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.

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 embodiments, 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.

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.

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).

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 expression. 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

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.

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.

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

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).

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).

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.

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).

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.

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.

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.

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.

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

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.

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.

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.

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

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.

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.

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

The naturally occurring 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.

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.

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. In certain embodiments, the antisense compounds targeted to a C9ORF72 nucleic acid comprise at least one phosphodiester linkage and at least one phosphorothioate linkage.

In certain embodiments, antisense compounds targeted to a C9ORF72 nucleic acid possess internucleoside linkages in any of the following patterns: soooossssssssssooss, sooosssssssssooss, soosssssssssooss, and sosssssssssoooss; wherein,

s=a phosphorothioate linkage, and

o=a phosphodiester linkage.

Modified Sugar Moieties

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(R₁)(R₂) (R, R₁and R₂are each independently H, C₁-C₁₂alkyl 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).

Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, OCF₃, OCH₂F, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_m)(R_n), O—CH₂—C(═O)—N(R_m)(R_n), and O—CH₂—C(═O)—N(R₁)—(CH₂)₂—N(R_m)(R_n), where each R₁, R_mand R_nis, independently, H or substituted or unsubstituted C₁-C₁₀alkyl.

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′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ and 4′-CH(CH₂OCH₃)—O- 2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof see published International Application WO/2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof see published International Application WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C—(═CH₂)-2′ (and analogs thereof see published International Application WO 2008/154401, published on Dec. 8, 2008).

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).

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)—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_aand R_bis, independently, H, a protecting group, hydroxyl, C₁-C₁₂alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂alkynyl, C₅-C₂₀aryl, substituted C₅-C₂₀aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇alicyclic radical, substituted C₅-C₇alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁and J₂is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂alkynyl, C₅-C₂₀aryl, substituted C₅-C₂₀aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂aminoalkyl, substituted C₁-C₁₂aminoalkyl or a protecting group.

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_aR_b)—N(R)—O— or —C(R_aR_b)—O—N(R)—. In certain embodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)—2′ and 4′-CH₂—N(R)—O-2′- wherein each R is, independently, H, a protecting group or C₁-C₁₂alkyl.

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′-CH₂—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy (4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy (4′-CH₂—O—N(R)—2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O— 2′) BNA, and (F) methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)—2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

embedded image

wherein Bx is the base moiety and R is independently H, a protecting group or C₁-C₁₂alkyl.

In certain embodiments, bicyclic nucleosides are provided having Formula I:

embedded image

wherein:

Bx is a heterocyclic base moiety;

-Q_a-Q_b-Q_c- is —CH₂—N(R_c)—CH₂—, —C(═O)—N(R_c)—CH₂—, —CH₂—O—N(R_c)—, —CH₂—N(R_c)—O— or —N(R_c)—O—CH₂;

R_cis C₁-C₁₂alkyl or an amino protecting group; and

T_aand T_bare 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.

In certain embodiments, bicyclic nucleosides are provided having Formula II:

embedded image

wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare 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;

Z_ais C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆alkenyl, substituted C₂-C₆alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.

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_cJ_d, SJ_c, N₃, OC(═X)J_c, and NJ_eC(═X)NJ_cJ_d, wherein each J_c, J_dand J_eis, independently, H, C₁-C₆alkyl, or substituted C₁-C₆alkyl and X is O or NJ_c.

In certain embodiments, bicyclic nucleosides are provided having Formula III:

embedded image

wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare 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;

Z_bis C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆alkenyl, substituted C₂-C₆alkynyl or substituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides are provided having Formula IV:

embedded image

wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare 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;

R_dis C₁-C₆alkyl, substituted C₁-C₆alkyl, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl or substituted C₂-C₆alkynyl;

each q_a, q_b, q_eand q_dis, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl or substituted C₂-C₆alkynyl, C₁-C₆alkoxyl, substituted C₁-C₆alkoxyl, acyl, substituted acyl, C₁-C₆aminoalkyl or substituted C₁-C₆aminoalkyl;

In certain embodiments, bicyclic nucleosides are provided having Formula V:

embedded image

wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare 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;

q_a, q_b, q_eand q_fare each, independently, hydrogen, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂alkynyl, C₁-C₁₂alkoxy, substituted C₁-C₁₂alkoxy, OJ_j, SJ_j, SOJ_j, SO₂J_j, NJ_jJ_k, N₃, CN, C(═O)OJ_j, C(═O)NJ_jJ_k, C(═O)J_j, O—C(═O)NJ_jJ_k, N(H)C(═NH)NJ_jJ_k, N(H)C(═O)NJ_jJ_kor N(H)C(═S)NJ_jJ_k;

or q_eand q_ftogether are ═C(q_g)(q_h);

q_gand q_hare each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—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.

Analogs of methyleneoxy (4′-CH₂—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 conformationally 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's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

In certain embodiments, bicyclic nucleosides are provided having Formula VI:

embedded image

wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare 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;

each q_i, q_j, q_kand q_lis, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂alkynyl, C₁-C₁₂alkoxyl, substituted C₁-C₁₂alkoxyl, OJ_j, SJ_j, SOJ_j, SO₂J_j, NJ_jJ_k, N₃, CN, C(═O)OJ_j, C(═O)NJ_jJ_k, C(═O)J_j, O—C(═O)NJ_jJ_k, N(H)C(═NH)NJ_jJ_k, N(H)C(═O)NJ_jJ_kor N(H)C(═S)NJ_jJ_k; and

q_iand q_jor q_land q_ktogether are ═C(q_g)(q_h), wherein q_gand q_hare each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and the alkenyl analog bridge 4′-CH═CH—CH₂-2′ 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).

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.

As used herein, “monocyclic 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.

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[(CH₂)_nO]_mCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nF, O(CH₂)_nONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_nON[(CH₂)_nCH₃]2, where n and m are from 1 to about 10. Other 2′- substituent groups can also be selected from: C₁-C₁₂alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, 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, modified 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).

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:

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wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;

T_aand T_bare each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T_aand T_bis an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T_aand T_bis H, a hydroxyl protecting group, a linked conjugate group or a 5′ or 3-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆and q₇are each independently, H, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl or substituted C₂-C₆alkynyl; and each of R₁and R₂is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂and CN, wherein X is O, S or NJ₁and each J₁, J₂and J₃is, independently, H or C₁-C₆alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q₁, q₂, q₃, q₄, q₅, q₆and q₇are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆and q₇is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R₁and R₂is fluoro. In certain embodiments, R₁is fluoro and R₂is H; R₁is methoxy and R₂is H, and R₁is H and R₂is methoxyethoxy.

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—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_m)(R_n), or O—CH₂—C(═O)—N(R_m)(R_n), where each R_mand R_ais, independently, H or substituted or unsubstituted C₁-C₁₀alkyl. 2′-modified nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2′ position.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH₃group at the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or “2′-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a —OCH₂CH₂OCH₃group at the 2′ position of the sugar ring.

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).

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.

Methods for the preparations of modified sugars are well known to those skilled in the art.

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.

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(CH₃)—O-2′) bridging group. In certain embodiments, the (4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wings of a gapmer motif.

Compositions and Methods for Formulating Pharmaceutical Compositions

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.

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.

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.

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

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.

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

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 commercial 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'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

Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.

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.

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.

Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.

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.

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

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's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

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 quantitative 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's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

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's instructions. Methods of quantitative real-time PCR are well known in the art.

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.

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.

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

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

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 (PBS) or artificial cerebrospinal fluid (aCSF). Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous, as well as central routes of administration such as intracerebroventricular or intrathecal. 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

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.

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, the antisense oligonucleotides described herein selectively hybridize to pre-mRNA variants containing the hexanucleotide repeat. In certain embodiments, pre-mRNA 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 24 repeats of any of GGGGCC, GGGGGG, GGGGGC, or GGGGCG.

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 one or more 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, the antisense oligonucleotides described herein selectively inhibit expression of pre-mRNA variants containing the hexanucleotide repeat. In certain embodiments, the antisense oligonucleotides described herein selectively inhibit expression of C9ORF72 pathogenic associated mRNA variants. In certain embodiments, pre-mRNA 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 24 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.

Selective Inhibition of Certain Pathogenic Associated Variants

In certain examples herein, primer probe set RTS3905 detects an mRNA variant (e.g. NM_001256054.1) processed from a pre-mRNA variant containing the hexanucleotide repeat. The mRNA variant processed from a pre-mRNA variant containing the hexanucleotide repeat (i.e., the “C9ORF72 pathogenic associated mRNA variant”). A pre-mRNA contains the hexanucleotide repeat when transcription of the pre-mRNA begins in the region from the start site of exon 1A to the start site of exon 1B, e.g., nucleotides 1107 to 1520 of the genomic sequence (SEQ ID NO: 2, the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). Oligonucleotides were designed in this region to selectively target the pre-mRNA variant containing the hexanucleotide repeat. RTS3905 measures an mRNA product (i.e. the C9ORF72 pathogenic associated mRNA variant) of the pre-mRNA variant containing the hexanucleotide repeat and, therefore, measures the reduction of the pre-mRNA variant containing the hexanucleotide repeat.

C9ORF72 Features

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.

TABLE 1

Functional Segments for NM_001256054.1 (SEQ ID NO: 1)

mRNA
mRNA
Start site
Stop site

Exon
start
stop
in reference to
in reference to

Number
site
site
SEQ ID NO: 2
SEQ 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

TABLE 2

Functional Segments for NM_018325.3 (SEQ ID NO: 4)

mRNA
mRNA
Start site
Stop site

Exon
start
stop
in reference to
in reference to

Number
site
site
SEQ ID NO: 2
SEQ 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

TABLE 3

Functional Segments for NM_145005.5 (SEQ ID NO: 6)

Start site
Stop site

in
in

mRNA
mRNA
reference
reference

start
stop
to SEQ
to SEQ

Exon 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
725
1871
13354
14500

(exon 5 into intron 5)

TABLE 4

Functional Segments for DB079375.1 (SEQ ID NO: 7)

Start site
Stop site

in
in

mRNA
mRNA
reference
reference

start
stop
to SEQ
to SEQ

Exon Number
site
site
ID NO: 2
ID NO: 2

exon 1E
1
35
1135
1169

exon 2
36
524
7839
8326

exon 3
525
562
9413
9450

(EST ends before

end of full exon)

TABLE 5

Functional Segments for BU194591.1 (SEQ ID NO: 8)

Start site
Stop site

in
in

mRNA
mRNA
reference
reference

start
stop
to SEQ
to SEQ

Exon 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
681
798
13354
13465

(exon 5 into intron 5)

Certain Indications

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.

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's response to administration of the antisense compound. An individual's response to administration of the antisense compound may be used by a physician to determine the amount and duration of therapeutic intervention.

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.

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

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.

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.

In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition described herein include Riluzole (Rilutek), Lioresal (Lioresal), and Dexpramipexole.

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.

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.

Certain Amplicon Regions

Certain antisense oligonucleotides described herein may target the amplicon region of the primer probe set. Additional assays may be used to measure the potency and efficacy of these compounds.

Certain Human Therapeutics

The human C9ORF72 antisense oligonucleotides described herein are being evaluated as possible human therapeutics. Various parameters of potency, efficacy, and/or tolerability are being examined. Such parameters include in vitro inhibition of total C9ORF72 RNA expression, in vitro inhibition of C9ORF72 pathogenic associated RNA variant expression, in vitro dose response (IC50), in vivo inhibition of total or pathogenic RNA and/or protein in a transgenic animal containing a human C9ORF72 transgene in relevant tissues (e.g., brain and/or spinal cord), tolerability in mouse, tolerability in rat, and/or tolerability in a primate. Tolerability markers that may be measured include blood and serum chemistry parameters, CSF chemistry parameters, body and organ weights, general observations and/or behavioral tests, and/or biochemical markers such as GFAP and/or AIF1. Acute or long term tolerability may be measured.

Certain Hotspot Regions
1. Nucleobases 1107-1520 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1107-1520 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1107-1520 are a hotspot region. In certain embodiments, nucleobases 1107-1520 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 17, 18, or 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers or 5-8-5 MOE gapmers. In certain embodiments, the antisense oligonucleotides are 17-mer Deoxy, MOE and cEt oligonucleotides. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 1107-1520 are targeted by the following ISIS numbers: 619042-619333, 672581-672714, 672735-672865, 672885-673015, 673035-673165, 673185-673315, and 673335-673465.

In certain embodiments, nucleobases 1107-1520 are targeted by the following SEQ ID NOs: 21-31, 33-50, 52, 54-134, 138-248, 251-319, 325, 744-877, and 898-1028.

In certain embodiments, nucleobases 1107-1520 are targeted by the following ISIS numbers: 619042-619333.

In certain embodiments, nucleobases 1107-1520 are targeted by the following SEQ ID NOs: 21-31, 33-50, 52, 54-134, 138-248, 251-319, and 325.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1107-1520 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1107-1520 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

2. Nucleobases 1111-1200 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1111-1200 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1111-1200 are a hotspot region. In certain embodiments, nucleobases 1111-1200 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 1111-1200 are targeted by the following ISIS numbers: 619042-619095.

In certain embodiments, nucleobases 1111-1200 are targeted by the following SEQ ID NOs: 21, 26-31, 33-50, 52, 54-60, 75, 81, and 87-96.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1111-1200 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1111-1200 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

3. Nucleobases 1211-1318 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1211-1318 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1211-1318 are a hotspot region. In certain embodiments, nucleobases 1211-1318 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 1211-1318 are targeted by the following ISIS numbers: 619096-619172.

In certain embodiments, nucleobases 1211-1318 are targeted by the following SEQ ID NOs: 22-25, 70-74, 76-80, 82-86, 99-134, and 138-159.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1211-1318 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1211-1318 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

4. Nucleobases 1326-1540 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1326-1540 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1326-1540 are a hotspot region. In certain embodiments, nucleobases 1326-1540 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 17, 18, or 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers or 5-8-5 MOE gapmers. In certain embodiments, the antisense oligonucleotides are 17-mer Deoxy, MOE and cEt oligonucleotides. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 1326-1540 are targeted by the following ISIS numbers: 619173-619354, and 672581-673484.

In certain embodiments, nucleobases 1326-1540 are targeted by the following SEQ ID NOs: 97, 98, 160-248, 251-322, 325-343, and 744-1047.

In certain embodiments, nucleobases 1326-1540 are targeted by the following ISIS numbers: 619173-619354.

In certain embodiments, nucleobases 1326-1540 are targeted by the following SEQ ID NOs: 97, 98, 160-248, 251-322, and 325-343.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1326-1540 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1326-1540 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

5. Nucleobases 1331-1375 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1331-1375 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1331-1375 are a hotspot region. In certain embodiments, nucleobases 1331-1375 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”). In certain embodiments, the antisense oligonucleotides comprise the following sugar modification pattern: soooossssssssssooss.

In certain embodiments, nucleobases 1331-1375 are targeted by the following ISIS numbers: 619178-619203.

In certain embodiments, nucleobases 1331-1375 are targeted by the following SEQ ID NOs: 165-190.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1331-1375 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

6. Nucleobases 1368-1391 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1368-1391 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1368-1391 are a hotspot region. In certain embodiments, nucleobases 1368-1391 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”). In certain embodiments, the antisense oligonucleotides comprise the following sugar modification pattern: soooossssssssssooss.

In certain embodiments, nucleobases 1368-1391 are targeted by the following ISIS numbers: 619215-619219.

In certain embodiments, nucleobases 1368-1391 are targeted by the following SEQ ID NOs: 202-206.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1368-1391 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

7. Nucleobases 1398-1424 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1398-1424 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1398-1424 are a hotspot region. In certain embodiments, nucleobases 1398-1424 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”). In certain embodiments, the antisense oligonucleotides comprise the following sugar modification pattern: soooossssssssssooss.

In certain embodiments, nucleobases 1398-1424 are targeted by the following ISIS numbers: 619245-619252.

In certain embodiments, nucleobases 1398-1424 are targeted by the following SEQ ID NOs: 232-239.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1398-1424 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

8. Nucleobases 1411-1440 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1411-1440 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 14l 1-1440 are a hotspot region. In certain embodiments, nucleobases 1411-1440 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”). In certain embodiments, the antisense oligonucleotides comprise the following sugar modification pattern: soooossssssssssooss.

In certain embodiments, nucleobases 1411-1440 are targeted by the following ISIS numbers: 619258-619268.

In certain embodiments, nucleobases 1411-1440 are targeted by the following SEQ ID NOs: 244-248, 251-255, and 325.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1411-1440 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

9. Nucleobases 1429-1481 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1429-1481 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1429-1481 are a hotspot region. In certain embodiments, nucleobases 1429-1481 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”). In certain embodiments, the antisense oligonucleotides comprise the following sugar modification pattern: soooossssssssssooss.

In certain embodiments, nucleobases 1429-1481 are targeted by the following ISIS numbers: 619276-619303.

In certain embodiments, nucleobases 1429-1481 are targeted by the following SEQ ID NOs: 98 and 263-289.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1429-1481 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

10. Nucleobases 1502-1539 of SEO ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1502-1539 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1502-1539 are a hotspot region. In certain embodiments, nucleobases 1502-1539 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”). In certain embodiments, the antisense oligonucleotides comprise the following sugar modification pattern: soooossssssssssooss.

In certain embodiments, nucleobases 1502-1539 are targeted by the following ISIS numbers: 619335-619353.

In certain embodiments, nucleobases 1502-1539 are targeted by the following SEQ ID NOs: 321, 322, and 326-342.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1502-1539 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

11. Nucleobases 1508-1539 of SEO ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 1508-1539 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 1508-1539 are a hotspot region. In certain embodiments, nucleobases 1508-1539 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have “mixed backbones”). In certain embodiments, the antisense oligonucleotides comprise the following sugar modification pattern: soooossssssssssooss.

In certain embodiments, nucleobases 1508-1539 are targeted by the following ISIS numbers: 619341-619353.

In certain embodiments, nucleobases 1508-1539 are targeted by the following SEQ ID NOs: 330-342.

In certain embodiments, antisense oligonucleotides targeting nucleobases 1508-1539 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo

12. Nucleobases 7860-7906 of SEO ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 7860-7906 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 7860-7906 are a hotspot region. In certain embodiments, nucleobases 7860-7906 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleoside linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 7860-7906 are targeted by the following ISIS numbers: 655135-655144.

In certain embodiments, nucleobases 7860-7906 are targeted by the following SEQ ID NOs: 445-454.

In certain embodiments, antisense oligonucleotides targeting nucleobases 7860-7906 achieve at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

13. Nucleobases 7907-7944 of SEO ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 7907-7944 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 7907-7944 are a hotspot region. In certain embodiments, nucleobases 7907-7944 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleoside linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 7907-7944 are targeted by the following ISIS numbers: 655150-655156.

In certain embodiments, nucleobases 7907-7944 are targeted by the following SEQ ID NOs: 460-467.

In certain embodiments, antisense oligonucleotides targeting nucleobases 7907-7944 achieve at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

14. Nucleobases 7989-8038 of SEO ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 7989-8038 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 7989-8038 are a hotspot region. In certain embodiments, nucleobases 7989-8038 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester Internucleoside linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 7989-8038 are targeted by the following ISIS numbers: 619411, 619412, 619420, 625183, 627833, and 655173-655180.

In certain embodiments, nucleobases 7989-8038 are targeted by the following SEQ ID NOs: 20, 51, 53, and 484-493.

In certain embodiments, antisense oligonucleotides targeting nucleobases 7989-8038 achieve at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, or at least 76% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

In certain embodiments, antisense oligonucleotides targeting nucleobases 7989-8038 achieve at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

15. Nucleobases 8020-8135 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 8020-8135 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 8020-8135 are a hotspot region. In certain embodiments, nucleobases 8020-8135 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleoside linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 8020-8135 are targeted by the following ISIS numbers: 619413, 619414, 625255, 627834, 655181-655208.

In certain embodiments, nucleobases 8020-8135 are targeted by the following SEQ ID NOs: 135, 136, 494-511, and 517-528.

In certain embodiments, antisense oligonucleotides targeting nucleobases 8020-8135 achieve at least 7%, 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, or at least 54% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

In certain embodiments, antisense oligonucleotides targeting nucleobases 8020-8135 achieve at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

16. Nucleobases 8136-8161 of SEO ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 8136-8161 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 8136-8161 are a hotspot region. In certain embodiments, nucleobases 8136-8161 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleoside linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 8136-8161 are targeted by the following ISIS numbers: 655215-655217.

In certain embodiments, nucleobases 8136-8161 are targeted by the following SEQ ID NOs: 535-537.

In certain embodiments, antisense oligonucleotides targeting nucleobases 8136-8161 achieve at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, or at least 41% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

In certain embodiments, antisense oligonucleotides targeting nucleobases 8136-8161 achieve at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

17. Nucleobases 8174-8211 of SEO ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 8174-8211 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 8174-8211 are a hotspot region. In certain embodiments, nucleobases 8174-8211 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleoside linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 8174-8211 are targeted by the following ISIS numbers: 655228-655234.

In certain embodiments, nucleobases 8174-8211 are targeted by the following SEQ ID NOs: 548-554.

In certain embodiments, antisense oligonucleotides targeting nucleobases 8174-8211 achieve at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, or at least 63% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

In certain embodiments, antisense oligonucleotides targeting nucleobases 8174-8211 achieve at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

18. Nucleobases 8213-8325 of SEQ ID NO: 2

In certain embodiments, antisense oligonucleotides are designed to target nucleobases 8213-8325 of SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000). In certain embodiments, nucleobases 8213-8325 are a hotspot region. In certain embodiments, nucleobases 8213-8325 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleoside linkages (e.g., the antisense oligonucleotides have “mixed backbones”).

In certain embodiments, nucleobases 8213-8325 are targeted by the following ISIS numbers: 655235-655270.

In certain embodiments, nucleobases 8213-8325 are targeted by the following SEQ ID NOs: 555-590.

In certain embodiments, antisense oligonucleotides targeting nucleobases 8213-8325 achieve at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, or at least 51% reduction of total C9ORF72 mRNA and/or protein levels in vitro and/or in vivo.

In certain embodiments, antisense oligonucleotides targeting nucleobases 8213-8325 achieve at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% reduction of C9ORF72 pathogenic associated mRNA variant levels in vitro and/or in vivo.

EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference

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 a Human C9ORF72 mRNA Variant in HepG2 Cells by MOE Gapmers

Antisense oligonucleotides targeting a C9ORF72 nucleic acid were designed and tested for their effects on C9ORF72 mRNA in vitro. ISIS 576816, previously tested in U.S. Application No. 61/714,132, filed Oct. 15, 2012, was used as a benchmark oligonucleotide. ISIS 576816 is a 5-10-5 MOE gapmer with phosphorothioate linkages throughout. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 4,000 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 RTS3905 (forward primer sequence GGGTCTAGCAAGAGCAGGTG, designated herein as SEQ ID NO: 13; reverse primer sequence GTCTTGGCAACAGCTGGAGAT, designated herein as SEQ ID NO: 14; probe sequence TGATGTCGACTCTTTGCCCACCGC, designated herein as SEQ ID NO: 15—a TAQ-man primer probe set) was used. RTS3905 detects an mRNA variant (e.g. NM_001256054.1) processed from a pre-mRNA variant containing the hexanucleotide repeat. The mRNA variant processed from a pre-mRNA variant containing the hexanucleotide repeat is herein the “C9ORF72 pathogenic associated mRNA variant.” A pre-mRNA contains the hexanucleotide repeat when transcription of the pre-mRNA begins in the region from the start site of exon 1A to the start site of exon 1B (generally nucleotides 1107 to 1520 of the genomic sequence: SEQ ID NO: 2, the complement of GENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000 to 27565000. Therefore, oligonucleotides were designed in this region selectively target the pre-mRNA variant containing the hexanucleotide repeat. RTS3905 measures an mRNA product (i.e. the C9ORF72 pathogenic associated mRNA variant) of the pre-mRNA variant containing the hexanucleotide repeat and, therefore, measures the reduction of the pre-mRNA variant containing the hexanucleotide repeat. The levels of the C9ORF72 pathogenic associated mRNA variant were normalized to the total RNA content of the cell, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells. The oligonucleotide marked with an asterisk (*) targets the amplicon region of the primer probe set. Additional assays may be used to measure the potency and efficacy of these oligonucleotides.

The chimeric antisense oligonucleotides in the Tables below 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 comprises a 2′-MOE group. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. The internucleoside linkages for the gapmers are mixed phosphorothioate and phosphodiester linkages. The internucleoside linkages for each gapmer are presented in the Linkage column, where ‘o’ indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioate linkage.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each antisense oligonucleotide listed in the Table below is targeted to either human C9ORF72 mRNA sequence, designated herein as SEQ TD NO: 1 (GENBANK Accession No. NM_001256054.1) or the human C9ORF72 genomic sequence, designated herein as SEQ TD 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 does not target that particular gene sequence

TABLE 6

Percent inhibition of the C9ORF72 pathogenic associated mRNA variant compared to PBS

control by antisense oligonucleotides targeting SEQ ID NOs: 1 and 2

SEQ
SEQ

ID
ID

NO:
NO:

1
2

SEQ

Start
Start

%
ID

ISIS NO
Site
Site
Sequence
Linkage
inhibition
NO

576816
310
7990
GCCTTACTCTAGGACCAAGA
SSSSSSSSSSSSSSSSSSS
40
20

619060
10
1146
CTTTCCTAGCGGGACACCGT
soooossssssssssooss
34
21

619111
100
1236
AAAAGAGAAGCAACCGGGCA
soooossssssssssooss
34
22

619112
101
1237
CAAAAGAGAAGCAACCGGGC
soooossssssssssooss
38
23

619113
102
1238
CCAAAAGAGAAGCAACCGGG
soooossssssssssooss
44
24

619114
103
1239
CCCAAAAGAGAAGCAACCGG
soooossssssssssooss
100
25

619061
11
1147
TCTTTCCTAGCGGGACACCG
soooossssssssssooss
100
26

619062
12
1148
CTCTTTCCTAGCGGGACACC
soooossssssssssooss
20
27

619063
13
1149
TCTCTTTCCTAGCGGGACAC
soooossssssssssooss
14
28

619064
14
1150
CTCTCTTTCCTAGCGGGACA
soooossssssssssooss
9
29

619065
15
1151
CCTCTCTTTCCTAGCGGGAC
soooossssssssssooss
12
30

619066
16
1152
ACCTCTCTTTCCTAGCGGGA
soooossssssssssooss
0
31

619410*
160
7840
ATCCAAATGCTCCGGAGATA
soooossssssssssooss
96
32

619067
17
1153
CACCTCTCTTTCCTAGCGGG
soooossssssssssooss
0
33

619068
18
1154
GCACCTCTCTTTCCTAGCGG
soooossssssssssooss
24
34

619069
19
1155
CGCACCTCTCTTTCCTAGCG
soooossssssssssooss
39
35

619052
2
1138
GCGGGACACCGTAGGTTACG
soooossssssssssooss
30
36

619070
20
1156
ACGCACCTCTCTTTCCTAGC
soooossssssssssooss
14
37

619071
21
1157
GACGCACCTCTCTTTCCTAG
soooossssssssssooss
7
38

619072
22
1158
TGACGCACCTCTCTTTCCTA
soooossssssssssooss
50
39

619073
23
1159
TTGACGCACCTCTCTTTCCT
soooossssssssssooss
0
40

619074
24
1160
TTTGACGCACCTCTCTTTCC
soooossssssssssooss
55
41

619075
25
1161
GTTTGACGCACCTCTCTTTC
soooossssssssssooss
12
42

619076
26
1162
TGTTTGACGCACCTCTCTTT
soooossssssssssooss
65
43

619077
27
1163
CTGTTTGACGCACCTCTCTT
soooossssssssssooss
28
44

619078
28
1164
GCTGTTTGACGCACCTCTCT
soooossssssssssooss
18
45

619079
29
1165
CGCTGTTTGACGCACCTCTC
soooossssssssssooss
14
46

619053
3
1139
AGCGGGACACCGTAGGTTAC
soooossssssssssooss
23
47

619080
30
1166
TCGCTGTTTGACGCACCTCT
soooossssssssssooss
100
48

619081
31
1167
GTCGCTGTTTGACGCACCTC
soooossssssssssooss
23
49

619082
32
1168
TGTCGCTGTTTGACGCACCT
soooossssssssssooss
0
50

619411
324
8004
TGGAGCCCAAATGTGCCTTA
soooossssssssssooss
99
51

619083
33
1169
TTGTCGCTGTTTGACGCACC
soooossssssssssooss
36
52

619412
332
8012
TCTGTCTTTGGAGCCCAAAT
soooossssssssssooss
100
53

619084
34
1170
CTTGTCGCTGTTTGACGCAC
soooossssssssssooss
28
54

619085
35
1171
ACTTGTCGCTGTTTGACGCA
soooossssssssssooss
55
55

619086
36
1172
AACTTGTCGCTGTTTGACGC
soooossssssssssooss
29
56

619087
37
1173
GAACTTGTCGCTGTTTGACG
soooossssssssssooss
21
57

619088
38
1174
GGAACTTGTCGCTGTTTGAC
soooossssssssssooss
100
58

619089
39
1175
CGGAACTTGTCGCTGTTTGA
soooossssssssssooss
67
59

619054
4
1140
TAGCGGGACACCGTAGGTTA
soooossssssssssooss
59
60

619090
40
1176
GCGGAACTTGTCGCTGTTTG
soooossssssssssooss
8
61

619091
41
1177
GGCGGAACTTGTCGCTGTTT
soooossssssssssooss
38
62

619092
42
1178
GGGCGGAACTTGTCGCTGTT
soooossssssssssooss
16
63

619093
43
1179
TGGGCGGAACTTGTCGCTGT
soooossssssssssooss
22
64

619094
44
1180
GTGGGCGGAACTTGTCGCTG
soooossssssssssooss
24
65

619095
45
1181
CGTGGGCGGAACTTGTCGCT
soooossssssssssooss
100
66

619055
5
1141
CTAGCGGGACACCGTAGGTT
soooossssssssssooss
100
67

619056
6
1142
CCTAGCGGGACACCGTAGGT
soooossssssssssooss
11
68

619057
7
1143
TCCTAGCGGGACACCGTAGG
soooossssssssssooss
22
69

619096
75
1211
GACGGCTGACACACCAAGCG
soooossssssssssooss
100
70

619097
76
1212
GGACGGCTGACACACCAAGC
soooossssssssssooss
88
71

619098
77
1213
GGGACGGCTGACACACCAAG
soooossssssssssooss
29
72

619099
78
1214
AGGGACGGCTGACACACCAA
soooossssssssssooss
83
73

619100
79
1215
CAGGGACGGCTGACACACCA
soooossssssssssooss
23
74

619058
8
1144
TTCCTAGCGGGACACCGTAG
soooossssssssssooss
32
75

619101
80
1216
GCAGGGACGGCTGACACACC
soooossssssssssooss
18
76

619102
81
1217
AGCAGGGACGGCTGACACAC
soooossssssssssooss
39
77

619103
82
1218
CAGCAGGGACGGCTGACACA
soooossssssssssooss
39
78

619104
83
1219
GCAGCAGGGACGGCTGACAC
soooossssssssssooss
22
79

619105
84
1220
GGCAGCAGGGACGGCTGACA
soooossssssssssooss
43
80

619059
9
1145
TTTCCTAGCGGGACACCGTA
soooossssssssssooss
4
81

619106
95
1231
AGAAGCAACCGGGCAGCAGG
soooossssssssssooss
54
82

619107
96
1232
GAGAAGCAACCGGGCAGCAG
soooossssssssssooss
100
83

619108
97
1233
AGAGAAGCAACCGGGCAGCA
soooossssssssssooss
32
84

619109
98
1234
AAGAGAAGCAACCGGGCAGC
soooossssssssssooss
30
85

619110
99
1235
AAAGAGAAGCAACCGGGCAG
soooossssssssssooss
44
86

619042
n/a
1111
GTTTTCTATGTGCGATGACG
soooossssssssssooss
6
87

619043
n/a
1112
TGTTTTCTATGTGCGATGAC
soooossssssssssooss
45
88

619044
n/a
1113
CTGTTTTCTATGTGCGATGA
soooossssssssssooss
23
89

619045
n/a
1114
TCTGTTTTCTATGTGCGATG
soooossssssssssooss
10
90

619046
n/a
1115
GTCTGTTTTCTATGTGCGAT
soooossssssssssooss
11
91

619047
n/a
1116
TGTCTGTTTTCTATGTGCGA
soooossssssssssooss
28
92

619048
n/a
1117
CTGTCTGTTTTCTATGTGCG
soooossssssssssooss
34
93

619049
n/a
1118
TCTGTCTGTTTTCTATGTGC
soooossssssssssooss
72
94

619050
n/a
1119
GTCTGTCTGTTTTCTATGTG
soooossssssssssooss
37
95

619051
n/a
1120
CGTCTGTCTGTTTTCTATGT
soooossssssssssooss
1
96

619253
n/a
1406
TACAGGCTGCGGTTGTTTCC
soooossssssssssooss
100
97

619293
n/a
1446
CCCGGCCCCTAGCGCGCGAC
soooossssssssssooss
98
98

Example 2: Antisense Inhibition of a Human C9ORF72 mRNA Variant in HepG2 Cells by Moe Gapmers

Additional antisense oligonucleotides targeting a C9ORF72 nucleic acid were designed and tested for their effects on C9ORF72 mRNA in vitro. ISIS 576816, previously tested in U.S. Application No. 61/714,132, filed Oct. 15, 2012, was used as a benchmark oligonucleotide. 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 1,000 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 RTS3905 was used to measure the C9ORF72 pathogenic associated mRNA variant, which is the product of a pre-mRNA containing a hexanucleotide repeat. The levels of the C9ORF72 pathogenic associated mRNA variant were normalized to the total RNA content of the cell, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells. The oligonucleotides marked with as asterisk (*) targets the region of the primer probe set. Additional assays may be used to measure the potency and efficacy of these oligonucleotides. ‘n.d.’ indicates that there was no signal reading in the assay for that particular oligonucleotide.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The gapmers of the Tables below also target 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. Some of the gapmers of Table 10 are targeted to GENBANK Accession No. NM_145005.5 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. DB079375.1 (incorporated herein as SEQ ID NO: 4), or GENBANK Accession No. BU194591.1 (incorporated herein as SEQ ID NO: 5). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence.

TABLE 7

Percent inhibition of the C9ORF72 pathogenic associated mRNA variant compared to PBS

control by antisense oligonucleotides targeting SEQ ID NOs: 1 and 2

SEQ
SEQ

ID
ID

NO: 1
NO: 2

Start
Start

%
SEQ ID

ISIS NO
Site
Site
Sequence
Linkage
inhibition
NO

576816
310
7990
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
70
20

619115
104
1240
CCCCAAAAGAGAAGCAACCG
soooossssssssssooss
10
99

619116
105
1241
CCCCCAAAAGAGAAGCAACC
soooossssssssssooss
19
100

619117
106
1242
GCCCCCAAAAGAGAAGCAAC
soooossssssssssooss
0
101

619118*
107
1243
CGCCCCCAAAAGAGAAGCAA
soooossssssssssooss
17
102

619119*
108
1244
CCGCCCCCAAAAGAGAAGCA
soooossssssssssooss
14
103

619120*
109
1245
CCCGCCCCCAAAAGAGAAGC
soooossssssssssooss
4
104

619121*
110
1246
CCCCGCCCCCAAAAGAGAAG
soooossssssssssooss
19
105

619122*
111
1247
ACCCCGCCCCCAAAAGAGAA
soooossssssssssooss
47
106

619123*
112
1248
GACCCCGCCCCCAAAAGAGA
soooossssssssssooss
11
107

619124*
113
1249
AGACCCCGCCCCCAAAAGAG
soooossssssssssooss
16
108

619125*
114
1250
TAGACCCCGCCCCCAAAAGA
soooossssssssssooss
15
109

619126*
115
1251
CTAGACCCCGCCCCCAAAAG
soooossssssssssooss
43
110

619127*
116
1252
GCTAGACCCCGCCCCCAAAA
soooossssssssssooss
67
111

619128*
117
1253
TGCTAGACCCCGCCCCCAAA
soooossssssssssooss
85
112

619129*
118
1254
TTGCTAGACCCCGCCCCCAA
soooossssssssssooss
41
113

619130*
119
1255
CTTGCTAGACCCCGCCCCCA
soooossssssssssooss
62
114

619131*
120
1256
TCTTGCTAGACCCCGCCCCC
soooossssssssssooss
95
115

619132*
121
1257
CTCTTGCTAGACCCCGCCCC
soooossssssssssooss
81
116

619133*
122
1258
GCTCTTGCTAGACCCCGCCC
soooossssssssssooss
90
117

619134*
123
1259
TGCTCTTGCTAGACCCCGCC
soooossssssssssooss
85
118

619135*
124
1260
CTGCTCTTGCTAGACCCCGC
soooossssssssssooss
81
119

619136*
125
1261
CCTGCTCTTGCTAGACCCCG
soooossssssssssooss
78
120

619137*
126
1262
ACCTGCTCTTGCTAGACCCC
soooossssssssssooss
85
121

619138*
127
1263
CACCTGCTCTTGCTAGACCC
soooossssssssssooss
81
122

619139*
128
1264
ACACCTGCTCTTGCTAGACC
soooossssssssssooss
77
123

619140*
129
1265
CACACCTGCTCTTGCTAGAC
soooossssssssssooss
86
124

619141*
130
1266
CCACACCTGCTCTTGCTAGA
soooossssssssssooss
90
125

619142*
131
1267
CCCACACCTGCTCTTGCTAG
soooossssssssssooss
98
126

619143*
132
1268
ACCCACACCTGCTCTTGCTA
soooossssssssssooss
94
127

619144*
133
1269
AACCCACACCTGCTCTTGCT
soooossssssssssooss
93
128

619145*
134
1270
AAACCCACACCTGCTCTTGC
soooossssssssssooss
95
129

619146*
135
1271
TAAACCCACACCTGCTCTTG
soooossssssssssooss
78
130

619147*
136
1272
CTAAACCCACACCTGCTCTT
soooossssssssssooss
64
131

619148*
137
1273
CCTAAACCCACACCTGCTCT
soooossssssssssooss
84
132

619149*
138
1274
TCCTAAACCCACACCTGCTC
soooossssssssssooss
87
133

619150*
139
1275
CTCCTAAACCCACACCTGCT
soooossssssssssooss
89
134

619413
340
8020
GTACCTGTTCTGTCTTTGGA
soooossssssssssooss
70
135

619414
353
8033
CCATCACTGAGAAGTACCTG
soooossssssssssooss
78
136

619415
940
16395
GGCATAATGTTCTGACTATC
soooossssssssssooss
67
137

619151*
n/a
1276
CCTCCTAAACCCACACCTGC
soooossssssssssooss
64
138

619152*
n/a
1277
ACCTCCTAAACCCACACCTG
soooossssssssssooss
45
139

619153*
n/a
1278
CACCTCCTAAACCCACACCT
soooossssssssssooss
36
140

619154*
n/a
1279
ACACCTCCTAAACCCACACC
soooossssssssssooss
26
141

619155*
n/a
1280
CACACCTCCTAAACCCACAC
soooossssssssssooss
50
142

619156*
n/a
1281
ACACACCTCCTAAACCCACA
soooossssssssssooss
53
143

619157*
n/a
1282
CACACACCTCCTAAACCCAC
soooossssssssssooss
44
144

619158*
n/a
1283
ACACACACCTCCTAAACCCA
soooossssssssssooss
65
145

619159*
n/a
1284
AACACACACCTCCTAAACCC
soooossssssssssooss
9
146

619160*
n/a
1285
AAACACACACCTCCTAAACC
soooossssssssssooss
0
147

619161*
n/a
1286
AAAACACACACCTCCTAAAC
soooossssssssssooss
15
148

619162*
n/a
1287
AAAAACACACACCTCCTAAA
soooossssssssssooss
10
149

619163*
n/a
1288
CAAAAACACACACCTCCTAA
soooossssssssssooss
7
150

619164*
n/a
1289
ACAAAAACACACACCTCCTA
soooossssssssssooss
55
151

619165*
n/a
1290
AACAAAAACACACACCTCCT
soooossssssssssooss
24
152

619166*
n/a
1291
AAACAAAAACACACACCTCC
soooossssssssssooss
19
153

619167*
n/a
1292
AAAACAAAAACACACACCTC
soooossssssssssooss
8
154

619168*
n/a
1294
GAAAAACAAAAACACACACC
soooossssssssssooss
17
155

619169*
n/a
1295
GGAAAAACAAAAACACACAC
soooossssssssssooss
26
156

619170
n/a
1297
TGGGAAAAACAAAAACACAC
soooossssssssssooss
30
157

619171
n/a
1298
GTGGGAAAAACAAAAACACA
soooossssssssssooss
23
158

619172
n/a
1299
GGTGGGAAAAACAAAAACAC
soooossssssssssooss
22
159

619173
n/a
1326
CTGTGAGAGCAAGTAGTGGG
soooossssssssssooss
43
160

619174
n/a
1327
ACTGTGAGAGCAAGTAGTGG
soooossssssssssooss
36
161

619175
n/a
1328
TACTGTGAGAGCAAGTAGTG
soooossssssssssooss
24
162

619176
n/a
1329
GTACTGTGAGAGCAAGTAGT
soooossssssssssooss
58
163

619177
n/a
1330
AGTACTGTGAGAGCAAGTAG
soooossssssssssooss
22
164

619178
n/a
1331
GAGTACTGTGAGAGCAAGTA
soooossssssssssooss
100
165

619179
n/a
1332
CGAGTACTGTGAGAGCAAGT
soooossssssssssooss
62
166

619180
n/a
1333
GCGAGTACTGTGAGAGCAAG
soooossssssssssooss
63
167

619181
n/a
1334
AGCGAGTACTGTGAGAGCAA
soooossssssssssooss
36
168

619182
n/a
1335
CAGCGAGTACTGTGAGAGCA
soooossssssssssooss
41
169

619183
n/a
1336
TCAGCGAGTACTGTGAGAGC
soooossssssssssooss
66
170

619184
n/a
1337
CTCAGCGAGTACTGTGAGAG
soooossssssssssooss
28
171

619185
n/a
1338
CCTCAGCGAGTACTGTGAGA
soooossssssssssooss
37
172

619186
n/a
1339
CCCTCAGCGAGTACTGTGAG
soooossssssssssooss
43
173

619187
n/a
1340
ACCCTCAGCGAGTACTGTGA
soooossssssssssooss
84
174

619253
n/a
1406
TACAGGCTGCGGTTGTTTCC
soooossssssssssooss
31
97

619293
n/a
1446
CCCGGCCCCTAGCGCGCGAC
soooossssssssssooss
63
98

TABLE 8

Percent inhibition of the C9ORF72 pathogenic associated

mRNA variant compared to PBS control by

antisense oligonucleotides targeting SEQ ID NOs: 1 and 2

SEQ
SEQ

ID
ID

NO:
NO:

1
2

SEQ

ISIS
Start
Start

%
ID

NO
Site
Site
Sequence
Linkage
inhibition
NO

576816
310
7990
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
82
20

619188
n/a
1341
CACCCTCAGCGAGTACTGTG
soooossssssssssooss
56
175

619189
n/a
1342
TCACCCTCAGCGAGTACTGT
soooossssssssssooss
66
176

619190
n/a
1343
TTCACCCTCAGCGAGTACTG
soooossssssssssooss
57
177

619191
n/a
1344
GTTCACCCTCAGCGAGTACT
soooossssssssssooss
83
178

619192
n/a
1345
TGTTCACCCTCAGCGAGTAC
soooossssssssssooss
66
179

619193
n/a
1346
TTGTTCACCCTCAGCGAGTA
soooossssssssssooss
57
180

619194
n/a
1347
CTTGTTCACCCTCAGCGAGT
soooossssssssssooss
48
181

619195
n/a
1348
TCTTGTTCACCCTCAGCGAG
soooossssssssssooss
46
182

619196
n/a
1349
TTCTTGTTCACCCTCAGCGA
soooossssssssssooss
66
183

619197
n/a
1350
TTTCTTGTTCACCCTCAGCG
soooossssssssssooss
31
184

619198
n/a
1351
TTTTCTTGTTCACCCTCAGC
soooossssssssssooss
47
185

619199
n/a
1352
CTTTTCTTGTTCACCCTCAG
soooossssssssssooss
53
186

619200
n/a
1353
TCTTTTCTTGTTCACCCTCA
soooossssssssssooss
46
187

619201
n/a
1354
GTCTTTTCTTGTTCACCCTC
soooossssssssssooss
71
188

619202
n/a
1355
GGTCTTTTCTTGTTCACCCT
soooossssssssssooss
73
189

619203
n/a
1356
AGGTCTTTTCTTGTTCACCC
soooossssssssssooss
79
190

619204
n/a
1357
CAGGTCTTTTCTTGTTCACC
soooossssssssssooss
0
191

619205
n/a
1358
TCAGGTCTTTTCTTGTTCAC
soooossssssssssooss
68
192

619206
n/a
1359
ATCAGGTCTTTTCTTGTTCA
soooossssssssssooss
52
193

619207
n/a
1360
TATCAGGTCTTTTCTTGTTC
soooossssssssssooss
47
194

619208
n/a
1361
TTATCAGGTCTTTTCTTGTT
soooossssssssssooss
37
195

619209
n/a
1362
TTTATCAGGTCTTTTCTTGT
soooossssssssssooss
31
196

619210
n/a
1363
CTTTATCAGGTCTTTTCTTG
soooossssssssssooss
24
197

619211
n/a
1364
TCTTTATCAGGTCTTTTCTT
soooossssssssssooss
37
198

619212
n/a
1365
ATCTTTATCAGGTCTTTTCT
soooossssssssssooss
34
199

619213
n/a
1366
AATCTTTATCAGGTCTTTTC
soooossssssssssooss
38
200

619214
n/a
1367
TAATCTTTATCAGGTCTTTT
soooossssssssssooss
32
201

619215
n/a
1368
TTAATCTTTATCAGGTCTTT
soooossssssssssooss
55
202

619216
n/a
1369
GTTAATCTTTATCAGGTCTT
soooossssssssssooss
72
203

619217
n/a
1370
GGTTAATCTTTATCAGGTCT
soooossssssssssooss
85
204

619218
n/a
1371
TGGTTAATCTTTATCAGGTC
soooossssssssssooss
82
205

619219
n/a
1372
CTGGTTAATCTTTATCAGGT
soooossssssssssooss
62
206

619220
n/a
1373
TCTGGTTAATCTTTATCAGG
soooossssssssssooss
19
207

619221
n/a
1374
TTCTGGTTAATCTTTATCAG
soooossssssssssooss
31
208

619222
n/a
1375
CTTCTGGTTAATCTTTATCA
soooossssssssssooss
40
209

619223
n/a
1376
TCTTCTGGTTAATCTTTATC
soooossssssssssooss
41
210

619224
n/a
1377
TTCTTCTGGTTAATCTTTAT
soooossssssssssooss
11
211

619225
n/a
1378
TTTCTTCTGGTTAATCTTTA
soooossssssssssooss
46
212

619226
n/a
1379
TTTTCTTCTGGTTAATCTTT
soooossssssssssooss
14
213

619227
n/a
1380
GTTTTCTTCTGGTTAATCTT
soooossssssssssooss
50
214

619228
n/a
1381
TGTTTTCTTCTGGTTAATCT
soooossssssssssooss
49
215

619229
n/a
1382
TTGTTTTCTTCTGGTTAATC
soooossssssssssooss
31
216

619230
n/a
1383
CTTGTTTTCTTCTGGTTAAT
soooossssssssssooss
16
217

619231
n/a
1384
CCTTGTTTTCTTCTGGTTAA
soooossssssssssooss
23
218

619232
n/a
1385
TCCTTGTTTTCTTCTGGTTA
soooossssssssssooss
52
219

619233
n/a
1386
CTCCTTGTTTTCTTCTGGTT
soooossssssssssooss
32
220

619234
n/a
1387
CCTCCTTGTTTTCTTCTGGT
soooossssssssssooss
59
221

619235
n/a
1388
CCCTCCTTGTTTTCTTCTGG
soooossssssssssooss
48
222

619236
n/a
1389
TCCCTCCTTGTTTTCTTCTG
soooossssssssssooss
31
223

619237
n/a
1390
TTCCCTCCTTGTTTTCTTCT
soooossssssssssooss
24
224

619238
n/a
1391
TTTCCCTCCTTGTTTTCTTC
soooossssssssssooss
42
225

619239
n/a
1392
GTTTCCCTCCTTGTTTTCTT
soooossssssssssooss
46
226

619240
n/a
1393
TGTTTCCCTCCTTGTTTTCT
soooossssssssssooss
81
227

619241
n/a
1394
TTGTTTCCCTCCTTGTTTTC
soooossssssssssooss
53
228

619242
n/a
1395
GTTGTTTCCCTCCTTGTTTT
soooossssssssssooss
28
229

619243
n/a
1396
GGTTGTTTCCCTCCTTGTTT
soooossssssssssooss
40
230

619244
n/a
1397
CGGTTGTTTCCCTCCTTGTT
soooossssssssssooss
31
231

619245
n/a
1398
GCGGTTGTTTCCCTCCTTGT
soooossssssssssooss
69
232

619246
n/a
1399
TGCGGTTGTTTCCCTCCTTG
soooossssssssssooss
73
233

619247
n/a
1400
CTGCGGTTGTTTCCCTCCTT
soooossssssssssooss
90
234

619248
n/a
1401
GCTGCGGTTGTTTCCCTCCT
soooossssssssssooss
40
235

619249
n/a
1402
GGCTGCGGTTGTTTCCCTCC
soooossssssssssooss
50
236

619250
n/a
1403
AGGCTGCGGTTGTTTCCCTC
soooossssssssssooss
61
237

619251
n/a
1404
CAGGCTGCGGTTGTTTCCCT
soooossssssssssooss
72
238

619252
n/a
1405
ACAGGCTGCGGTTGTTTCCC
soooossssssssssooss
69
239

619253
n/a
1406
TACAGGCTGCGGTTGTTTCC
soooossssssssssooss
67
97

619254
n/a
1407
CTACAGGCTGCGGTTGTTTC
soooossssssssssooss
43
240

619255
n/a
1408
GCTACAGGCTGCGGTTGTTT
soooossssssssssooss
54
241

619256
n/a
1409
TGCTACAGGCTGCGGTTGTT
soooossssssssssooss
36
242

619257
n/a
1410
TTGCTACAGGCTGCGGTTGT
soooossssssssssooss
29
243

619258
n/a
1411
CTTGCTACAGGCTGCGGTTG
soooossssssssssooss
50
244

619259
n/a
1412
GCTTGCTACAGGCTGCGGTT
soooossssssssssooss
76
245

619260
n/a
1413
AGCTTGCTACAGGCTGCGGT
soooossssssssssooss
80
246

619261
n/a
1414
GAGCTTGCTACAGGCTGCGG
soooossssssssssooss
54
247

619262
n/a
1415
AGAGCTTGCTACAGGCTGCG
soooossssssssssooss
62
248

619293
n/a
1446
CCCGGCCCCTAGCGCGCGAC
soooossssssssssooss
64
98

619416
1937
24657
AAAAAACAGTAGTTGTGGTC
soooossssssssssooss
78
249

27056

619417
1988
27107
GCCAACTCAGATTTCACCTT
soooossssssssssooss
86
250

TABLE 9

Percent inhibition of the C9ORF72 pathogenic associated

mRNA variant compared to PBS control by

antisense oligonucleotides targeting SEQ ID NOs: 1 and 2

SEQ
SEQ

ID
ID

NO:
NO:

1
2

SEQ

ISIS
Start
Start

%
ID

NO
Site
Site
Sequence
Linkage
inhibition
NO

576816
310
7990
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
71
20

619264
n/a
1417
CCAGAGCTTGCTACAGGCTG
soooossssssssssooss
54
251

619265
n/a
1418
TCCAGAGCTTGCTACAGGCT
soooossssssssssooss
69
252

619266
n/a
1419
TTCCAGAGCTTGCTACAGGC
soooossssssssssooss
70
253

619267
n/a
1420
GTTCCAGAGCTTGCTACAGG
soooossssssssssooss
49
254

619268
n/a
1421
AGTTCCAGAGCTTGCTACAG
soooossssssssssooss
95
255

619269
n/a
1422
GAGTTCCAGAGCTTGCTACA
soooossssssssssooss
36
256

619270
n/a
1423
TGAGTTCCAGAGCTTGCTAC
soooossssssssssooss
15
257

619271
n/a
1424
CTGAGTTCCAGAGCTTGCTA
soooossssssssssooss
31
258

619272
n/a
1425
CCTGAGTTCCAGAGCTTGCT
soooossssssssssooss
41
259

619273
n/a
1426
TCCTGAGTTCCAGAGCTTGC
soooossssssssssooss
36
260

619274
n/a
1427
CTCCTGAGTTCCAGAGCTTG
soooossssssssssooss
25
261

619275
n/a
1428
ACTCCTGAGTTCCAGAGCTT
soooossssssssssooss
50
262

619276
n/a
1429
GACTCCTGAGTTCCAGAGCT
soooossssssssssooss
74
263

619277
n/a
1430
CGACTCCTGAGTTCCAGAGC
soooossssssssssooss
69
264

619278
n/a
1431
GCGACTCCTGAGTTCCAGAG
soooossssssssssooss
98
265

619279
n/a
1432
CGCGACTCCTGAGTTCCAGA
soooossssssssssooss
69
266

619280
n/a
1433
GCGCGACTCCTGAGTTCCAG
soooossssssssssooss
75
267

619281
n/a
1434
CGCGCGACTCCTGAGTTCCA
soooossssssssssooss
67
268

619282
n/a
1435
GCGCGCGACTCCTGAGTTCC
soooossssssssssooss
55
269

619283
n/a
1436
AGCGCGCGACTCCTGAGTTC
soooossssssssssooss
62
270

619284
n/a
1437
TAGCGCGCGACTCCTGAGTT
soooossssssssssooss
100
271

619285
n/a
1438
CTAGCGCGCGACTCCTGAGT
soooossssssssssooss
68
272

619286
n/a
1439
CCTAGCGCGCGACTCCTGAG
soooossssssssssooss
41
273

619287
n/a
1440
CCCTAGCGCGCGACTCCTGA
soooossssssssssooss
70
274

619288
n/a
1441
CCCCTAGCGCGCGACTCCTG
soooossssssssssooss
68
275

619289
n/a
1442
GCCCCTAGCGCGCGACTCCT
soooossssssssssooss
52
276

619290
n/a
1443
GGCCCCTAGCGCGCGACTCC
soooossssssssssooss
49
277

619291
n/a
1444
CGGCCCCTAGCGCGCGACTC
soooossssssssssooss
69
278

619292
n/a
1445
CCGGCCCCTAGCGCGCGACT
soooossssssssssooss
76
279

619293
n/a
1446
CCCGGCCCCTAGCGCGCGAC
soooossssssssssooss
52
98

619294
n/a
1447
CCCCGGCCCCTAGCGCGCGA
soooossssssssssooss
62
280

619295
n/a
1448
GCCCCGGCCCCTAGCGCGCG
soooossssssssssooss
68
281

619296
n/a
1449
GGCCCCGGCCCCTAGCGCGC
soooossssssssssooss
56
282

619297
n/a
1450
CGGCCCCGGCCCCTAGCGCG
soooossssssssssooss
71
283

619298
n/a
1451
CCGGCCCCGGCCCCTAGCGC
soooossssssssssooss
73
284

619299
n/a
1452
CCCGGCCCCGGCCCCTAGCG
soooossssssssssooss
51
285

619300
n/a
1453
CCCCGGCCCCGGCCCCTAGC
soooossssssssssooss
49
286

619301
n/a
1454
GCCCCGGCCCCGGCCCCTAG
soooossssssssssooss
60
287

619302
n/a
1455
GGCCCCGGCCCCGGCCCCTA
soooossssssssssooss
48
288

619303
n/a
1462
ACGCCCCGGCCCCGGCCCCG
soooossssssssssooss
49
289

619304
n/a
1463
CACGCCCCGGCCCCGGCCCC
soooossssssssssooss
25
290

619305
n/a
1464
CCACGCCCCGGCCCCGGCCC
soooossssssssssooss
66
291

619306
n/a
1465
ACCACGCCCCGGCCCCGGCC
soooossssssssssooss
70
292

619307
n/a
1466
GACCACGCCCCGGCCCCGGC
soooossssssssssooss
73
293

619308
n/a
1467
CGACCACGCCCCGGCCCCGG
soooossssssssssooss
55
294

619309
n/a
1468
CCGACCACGCCCCGGCCCCG
soooossssssssssooss
70
295

619310
n/a
1469
CCCGACCACGCCCCGGCCCC
soooossssssssssooss
50
296

619311
n/a
1470
CCCCGACCACGCCCCGGCCC
soooossssssssssooss
45
297

619312
n/a
1471
GCCCCGACCACGCCCCGGCC
soooossssssssssooss
36
298

619313
n/a
1472
CGCCCCGACCACGCCCCGGC
soooossssssssssooss
58
299

619314
n/a
1473
CCGCCCCGACCACGCCCCGG
soooossssssssssooss
100
300

619315
n/a
1474
CCCGCCCCGACCACGCCCCG
soooossssssssssooss
44
301

619316
n/a
1475
GCCCGCCCCGACCACGCCCC
soooossssssssssooss
97
302

619317
n/a
1476
GGCCCGCCCCGACCACGCCC
soooossssssssssooss
76
303

619318
n/a
1477
GGGCCCGCCCCGACCACGCC
soooossssssssssooss
44
304

619319
n/a
1478
CGGGCCCGCCCCGACCACGC
soooossssssssssooss
40
305

619320
n/a
1479
CCGGGCCCGCCCCGACCACG
soooossssssssssooss
50
306

619321
n/a
1480
CCCGGGCCCGCCCCGACCAC
soooossssssssssooss
22
307

619322
n/a
1481
CCCCGGGCCCGCCCCGACCA
soooossssssssssooss
56
308

619323
n/a
1482
CCCCCGGGCCCGCCCCGACC
soooossssssssssooss
40
309

619324
n/a
1483
GCCCCCGGGCCCGCCCCGAC
soooossssssssssooss
65
310

619325
n/a
1484
CGCCCCCGGGCCCGCCCCGA
soooossssssssssooss
25
311

619326
n/a
1486
CCCGCCCCCGGGCCCGCCCC
soooossssssssssooss
41
312

619327
n/a
1487
GCCCGCCCCCGGGCCCGCCC
soooossssssssssooss
36
313

619328
n/a
1488
GGCCCGCCCCCGGGCCCGCC
soooossssssssssooss
14
314

619329
n/a
1495
CGCCCCGGGCCCGCCCCCGG
soooossssssssssooss
33
315

619330
n/a
1497
CCCGCCCCGGGCCCGCCCCC
soooossssssssssooss
35
316

619331
n/a
1498
CCCCGCCCCGGGCCCGCCCC
soooossssssssssooss
42
317

619332
n/a
1499
GCCCCGCCCCGGGCCCGCCC
soooossssssssssooss
47
318

619333
n/a
1500
AGCCCCGCCCCGGGCCCGCC
soooossssssssssooss
53
319

619334
n/a
1501
CAGCCCCGCCCCGGGCCCGC
soooossssssssssooss
32
320

619335
n/a
1502
GCAGCCCCGCCCCGGGCCCG
soooossssssssssooss
58
321

619336
n/a
1503
CGCAGCCCCGCCCCGGGCCC
soooossssssssssooss
75
322

619418
n/a
27155
CTACACACCAAAGAATGCCA
soooossssssssssooss
71
323

619419
n/a
15587
GGAATAAGGTCACTAGTTCG
soooossssssssssooss
72
324

619420
n/a
7990
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
100
20

619263
n/a
1416
CAGAGCTTGCTACAGGCTGC
soooossssssssssooss
63
325

619253
n/a
1406
TACAGGCTGCGGTTGTTTCC
soooossssssssssooss
50
97

TABLE 10

Percent inhibition of the C9ORF72 pathogenic associated

mRNA variant compared to PBS control by

antisense oligonucleotides targeting SEQ ID NOs: 1-5

SEQ
SEQ
SEQ
SEQ
SEQ

ID
ID
ID
ID
ID

NO:
NO:
NO:
NO:
NO:

%

1
2
3
4
5

in-
SEQ

ISIS
Start
Start
Start
Start
Start

hibi-
ID

NO
Site
Site
Site
Site
Site
Sequence
Linkage
tion
NO

576816
310
7990
232
188
188
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
69
20

619253
n/a
1406
n/a
n/a
n/a
TACAGGCTGCGGTTGTTTCC
soooossssssssssooss
19
97

619293
n/a
1446
n/a
n/a
n/a
CCCGGCCCCTAGCGCGCGAC
soooossssssssssooss
56
98

619337
n/a
1504
n/a
n/a
n/a
CCGCAGCCCCGCCCCGGGCC
soooossssssssssooss
55
326

619338
n/a
1505
n/a
n/a
n/a
ACCGCAGCCCCGCCCCGGGC
soooossssssssssooss
62
327

619339
n/a
1506
n/a
n/a
n/a
AACCGCAGCCCCGCCCCGGG
soooossssssssssooss
68
328

619340
n/a
1507
n/a
n/a
n/a
CAACCGCAGCCCCGCCCCGG
soooossssssssssooss
14
329

619341
n/a
1508
n/a
n/a
n/a
GCAACCGCAGCCCCGCCCCG
soooossssssssssooss
95
330

619342
n/a
1509
n/a
n/a
n/a
CGCAACCGCAGCCCCGCCCC
soooossssssssssooss
56
331

619343
n/a
1510
n/a
n/a
n/a
CCGCAACCGCAGCCCCGCCC
soooossssssssssooss
58
332

619344
n/a
1511
n/a
n/a
n/a
ACCGCAACCGCAGCCCCGCC
soooossssssssssooss
51
333

619345
n/a
1512
n/a
n/a
n/a
CACCGCAACCGCAGCCCCGC
soooossssssssssooss
97
334

619346
n/a
1513
n/a
n/a
n/a
GCACCGCAACCGCAGCCCCG
soooossssssssssooss
49
335

619347
n/a
1514
n/a
n/a
n/a
GGCACCGCAACCGCAGCCCC
soooossssssssssooss
100
336

619348
n/a
1515
n/a
n/a
n/a
AGGCACCGCAACCGCAGCCC
soooossssssssssooss
46
337

619349
n/a
1516
n/a
n/a
n/a
CAGGCACCGCAACCGCAGCC
soooossssssssssooss
100
338

619350
n/a
1517
n/a
n/a
n/a
GCAGGCACCGCAACCGCAGC
soooossssssssssooss
55
339

619351
n/a
1518
n/a
n/a
n/a
CGCAGGCACCGCAACCGCAG
soooossssssssssooss
63
340

619352
n/a
1519
n/a
n/a
n/a
GCGCAGGCACCGCAACCGCA
soooossssssssssooss
42
341

619353
n/a
1520
n/a
n/a
n/a
GGCGCAGGCACCGCAACCGC
soooossssssssssooss
95
342

619354
n/a
1521
n/a
n/a
n/a
GGGCGCAGGCACCGCAACCG
soooossssssssssooss
23
343

619355*
140
n/a
n/a
n/a
n/a
TCTCCTAAACCCACACCTGC
soooossssssssssooss
94
384

619356*
141
n/a
n/a
n/a
n/a
ATCTCCTAAACCCACACCTG
soooossssssssssooss
69
385

619357
142
n/a
n/a
n/a
n/a
TATCTCCTAAACCCACACCT
soooossssssssssooss
n.d.
386

619358*
143
n/a
n/a
n/a
n/a
ATATCTCCTAAACCCACACC
soooossssssssssooss
53
387

619359
144
n/a
n/a
n/a
n/a
GATATCTCCTAAACCCACAC
soooossssssssssooss
n.d.
388

619360*
145
n/a
n/a
n/a
n/a
AGATATCTCCTAAACCCACA
soooossssssssssooss
30
389

619361*
146
n/a
n/a
n/a
n/a
GAGATATCTCCTAAACCCAC
soooossssssssssooss
48
390

619362*
147
n/a
n/a
n/a
n/a
GGAGATATCTCCTAAACCCA
soooossssssssssooss
60
391

619363*
148
n/a
n/a
n/a
n/a
CGGAGATATCTCCTAAACCC
soooossssssssssooss
26
392

619364*
149
n/a
n/a
n/a
n/a
CCGGAGATATCTCCTAAACC
soooossssssssssooss
97
393

619365*
150
n/a
n/a
n/a
n/a
TCCGGAGATATCTCCTAAAC
soooossssssssssooss
60
394

619366*
151
n/a
n/a
n/a
n/a
CTCCGGAGATATCTCCTAAA
soooossssssssssooss
34
395

619367
152
n/a
n/a
n/a
n/a
GCTCCGGAGATATCTCCTAA
soooossssssssssooss
n.d.
396

619368*
153
n/a
n/a
n/a
n/a
TGCTCCGGAGATATCTCCTA
soooossssssssssooss
95
397

619369
154
n/a
n/a
n/a
n/a
ATGCTCCGGAGATATCTCCT
soooossssssssssooss
n.d.
398

619370*
155
n/a
n/a
n/a
n/a
AATGCTCCGGAGATATCTCC
soooossssssssssooss
59
399

619371*
n/a
n/a
n/a
17
n/a
TCTCTCTTTCCTAGCGGGAC
soooossssssssssooss
0
344

619372*
n/a
n/a
n/a
18
n/a
ATCTCTCTTTCCTAGCGGGA
soooossssssssssooss
8
345

619373*
n/a
n/a
n/a
19
n/a
TATCTCTCTTTCCTAGCGGG
soooossssssssssooss
6
346

619374*
n/a
n/a
n/a
20
n/a
ATATCTCTCTTTCCTAGCGG
soooossssssssssooss
0
347

619375*
n/a
n/a
n/a
21
n/a
GATATCTCTCTTTCCTAGCG
soooossssssssssooss
8
348

619376*
n/a
n/a
n/a
22
n/a
AGATATCTCTCTTTCCTAGC
soooossssssssssooss
0
349

619377*
n/a
n/a
n/a
23
n/a
GAGATATCTCTCTTTCCTAG
soooossssssssssooss
89
350

619378*
n/a
n/a
n/a
24
n/a
GGAGATATCTCTCTTTCCTA
soooossssssssssooss
1
351

619379*
n/a
n/a
n/a
25
n/a
CGGAGATATCTCTCTTTCCT
soooossssssssssooss
0
352

619380
n/a
n/a
n/a
26
n/a
CCGGAGATATCTCTCTTTCC
soooossssssssssooss
n.d.
353

619381*
n/a
n/a
n/a
27
n/a
TCCGGAGATATCTCTCTTTC
soooossssssssssooss
28
354

619382*
n/a
n/a
n/a
28
n/a
CTCCGGAGATATCTCTCTTT
soooossssssssssooss
28
355

619383*
n/a
n/a
n/a
29
n/a
GCTCCGGAGATATCTCTCTT
soooossssssssssooss
91
356

619384*
n/a
n/a
n/a
30
n/a
TGCTCCGGAGATATCTCTCT
soooossssssssssooss
61
357

619385*
n/a
n/a
n/a
31
n/a
ATGCTCCGGAGATATCTCTC
soooossssssssssooss
69
358

619386*
n/a
n/a
n/a
32
n/a
AATGCTCCGGAGATATCTCT
soooossssssssssooss
75
359

619387
156
n/a
n/a
33
n/a
AAATGCTCCGGAGATATCTC
soooossssssssssooss
n.d.
400

619388*
n/a
n/a
n/a
n/a
18
TCTGCTCTTGCTAGACCCCG
soooossssssssssooss
50
360

619389
n/a
n/a
n/a
n/a
19
ATCTGCTCTTGCTAGACCCC
soooossssssssssooss
n.d.
361

619390*
n/a
n/a
n/a
n/a
20
TATCTGCTCTTGCTAGACCC
soooossssssssssooss
5
362

619391*
n/a
n/a
n/a
n/a
21
ATATCTGCTCTTGCTAGACC
soooossssssssssooss
0
363

619392*
n/a
n/a
n/a
n/a
22
GATATCTGCTCTTGCTAGAC
soooossssssssssooss
0
364

619393*
n/a
n/a
n/a
n/a
23
AGATATCTGCTCTTGCTAGA
soooossssssssssooss
6
365

619394*
n/a
n/a
n/a
n/a
24
GAGATATCTGCTCTTGCTAG
soooossssssssssooss
57
366

619395*
n/a
n/a
n/a
n/a
25
GGAGATATCTGCTCTTGCTA
soooossssssssssooss
6
367

619396*
n/a
n/a
n/a
n/a
26
CGGAGATATCTGCTCTTGCT
soooossssssssssooss
0
368

619397*
n/a
n/a
n/a
n/a
27
CCGGAGATATCTGCTCTTGC
soooossssssssssooss
0
369

619398*
n/a
n/a
n/a
n/a
28
TCCGGAGATATCTGCTCTTG
soooossssssssssooss
22
370

619399*
n/a
n/a
n/a
n/a
29
CTCCGGAGATATCTGCTCTT
soooossssssssssooss
14
371

619400*
n/a
n/a
n/a
n/a
30
GCTCCGGAGATATCTGCTCT
soooossssssssssooss
46
372

619401*
n/a
n/a
n/a
n/a
31
TGCTCCGGAGATATCTGCTC
soooossssssssssooss
40
373

619402*
n/a
n/a
n/a
n/a
32
ATGCTCCGGAGATATCTGCT
soooossssssssssooss
79
374

619403*
n/a
n/a
n/a
n/a
33
AATGCTCCGGAGATATCTGC
soooossssssssssooss
65
375

619404*
n/a
n/a
n/a
n/a
34
AAATGCTCCGGAGATATCTC
soooossssssssssooss
22
376

619405*
n/a
n/a
75
n/a
n/a
TGCTCCGGAGATATCAAGCG
soooossssssssssooss
18
377

619406*
n/a
n/a
76
n/a
n/a
ATGCTCCGGAGATATCAAGC
soooossssssssssooss
21
378

619407*
n/a
n/a
77
n/a
n/a
AATGCTCCGGAGATATCAAG
soooossssssssssooss
98
379

619408
n/a
n/a
78
n/a
n/a
AAATGCTCCGGAGATATCAA
soooossssssssssooss
n.d.
380

619409*
n/a
n/a
79
n/a
n/a
CAAATGCTCCGGAGATATCA
soooossssssssssooss
98.
381

619421
3132
28251
n/a
n/a
n/a
GGGACACTACAAGGTAGTAT
soooossssssssssooss
80
401

619422*
n/a
3452
n/a
n/a
n/a
GGTAACTTCAAACTCTTGGG
soooossssssssssooss
85.
382

619423
n/a
13642
1013
n/a
n/a
GCCATGATTTCTTGTCTGGG
soooossssssssssooss
67
383

Example 3: Dose-Dependent Antisense Inhibition of a Human C9ORF72 mRNA Variant in HepG2 Cells

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. ISIS 576816 and ISIS 577061, previously tested in U.S. Application No. 61/714,132, filed Oct. 15, 2012, were used as benchmark oligonucleotides. ISIS 576816 and ISIS 577061 are 5-10-5 MOE gapmers with phosphorothioate linkages throughout. 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 78.1 nM, 312.5 nM, 1,250.0 nM, or 5,000.0 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. Human C9ORF72 primer probe set RTS3905 was used to measure the C9ORF72 pathogenic associated mRNA variant. The levels of the C9ORF72 pathogenic associated mRNA variant were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of variant C9ORF72 levels, relative to untreated control cells. ‘n.d.’ means no data.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in the Tables below. As illustrated, the C9ORF72 pathogenic associated mRNA variant levels were reduced in a dose-dependent manner in some of the antisense oligonucleotide treated cells.

TABLE 11

Dose-dependent inhibition of the C9ORF72

pathogenic associated mRNA variant

ISIS No
78.1 nM
312.5 nM
1250.0 nM
5000.0 nM
IC₅₀(μM)

576816
13
47
92
95
0.4

577061
0
1
23
57
4.2

619049
11
46
34
18
>5.0

619055
14
13
0
0
>5.0

619061
0
2
0
0
>5.0

619080
7
4
0
0
>5.0

619088
0
7
16
16
>5.0

619089
2
0
0
17
>5.0

619095
10
0
0
27
>5.0

619096
16
0
10
27
>5.0

619097
23
55
41
38
>5.0

619099
10
36
46
23
>5.0

619107
15
0
4
33
>5.0

619114
0
0
8
31
>5.0

619253
26
33
66
86
0.5

619293
26
71
n.d.
95
0.2

619410
42
63
86
n.d.
0.1

619411
28
20
96
n.d.
0.3

619412
39
66
93
97
0.1

TABLE 12

Dose-dependent inhibition of the C9ORF72

pathogenic associated mRNA variant

ISIS No
78.1 nM
312.5 nM
1250.0 nM
5000.0 nM
IC₅₀(μM)

576816
39
84
96
90
0.1

577061
29
0
25
71
3.7

619173
18
46
84
98
0.4

619174
21
36
77
n.d.
0.4

619176
16
29
57
95
0.7

619178
5
31
86
97
0.5

619179
0
16
65
96
0.8

619180
4
24
66
96
0.7

619181
10
34
72
95
0.6

619182
23
32
74
92
0.5

619183
0
26
44
96
1.0

619185
14
34
70
93
0.5

619186
8
32
71
98
0.6

619187
18
36
81
95
0.4

619253
17
22
61
97
0.7

619293
0
49
86
99
0.6

619413
8
48
84
95
0.4

619415
26
67
90
n.d.
0.2

TABLE 13

Dose-dependent inhibition of the C9ORF72

pathogenic associated mRNA variant

ISIS No
78.1 nM
312.5 nM
1250.0 nM
5000.0 nM
IC₅₀(μM)

576816
0
71
97
n.d.
0.3

577061
0
12
46
82
1.4

619191
66
84
94
98
<0.07

619201
16
64
95
97
0.3

619202
35
78
95
95
0.1

619203
29
55
92
95
0.2

619216
61
55
92
96
<0.07

619217
44
86
83
n.d.
0.1

619218
35
87
93
n.d.
0.1

619240
0
43
52
78
1.0

619245
0
39
85
n.d.
0.5

619246
34
74
96
99
0.1

619247
0
46
92
93
0.6

619251
40
91
93
96
<0.07

619252
24
67
87
n.d.
0.2

619259
7
76
85
98
0.3

619260
16
80
92
99
0.2

619416
13
63
91
92
0.3

619417
45
88
91
97
<0.07

TABLE 14

Dose-dependent inhibition of the C9ORF72

pathogenic associated mRNA variant

ISIS No
78.1 nM
312.5 nM
1250.0 nM
5000.0 nM
IC₅₀(μM)

576816
38
61
95
98
0.1

577061
0
8
13
77
2.5

619266
0
23
73
98
0.8

619268
5
29
77
97
0.6

619276
31
82
90
91
0.1

619278
35
83
97
99
0.1

619280
66
80
97
96
<0.07

619284
22
55
88
98
0.3

619292
37
79
85
94
0.1

619297
0
68
82
91
0.6

619298
47
89
93
91
<0.07

619307
37
71
96
98
0.1

619314
21
0
61
97
0.8

619316
13
37
71
91
0.5

619317
7
17
68
87
0.8

619336
17
51
64
90
0.5

619418
43
78
89
95
0.1

619419
43
68
94
95
0.1

619420
66
88
100
n.d.
<0.07

TABLE 15

Dose-dependent inhibition of the C9ORF72

pathogenic associated mRNA variant

ISIS No
78.1 nM
312.5 nM
1250.0 nM
5000.0 nM
IC₅₀(μM)

576816
8
48
91
91
0.4

577061
0
13
33
64
2.6

619293
32
77
85
95
0.1

619337
28
42
79
95
0.3

619338
11
55
83
91
0.4

619339
6
42
83
90
0.5

619341
17
14
66
83
0.8

619342
37
57
85
97
0.2

619343
25
51
87
96
0.3

619344
17
27
75
91
0.5

619345
0
18
50
88
1.2

619346
0
22
76
90
0.8

619347
1
8
41
76
1.7

619349
10
12
51
78
1.3

619350
16
46
84
91
0.4

619351
20
21
69
87
0.6

619353
0
13
52
76
1.4

619421
27
78
93
92
0.1

619423
0
53
87
93
0.5

TABLE 16

Gapmers chosen for further analysis

SEQ

ID

NO: 2

SEQ

Isis
IC₅₀

Start

ID

No
(μM)
Sequence
Site
Location
NO

619173
0.4
CTGTGAGAGCAAGTAGTGGG
1326
intron
160

619174
0.4
ACTGTGAGAGCAAGTAGTGG
1327
intron
161

619178
0.5
GAGTACTGTGAGAGCAAGTA
1331
intron
165

619179
0.8
CGAGTACTGTGAGAGCAAGT
1332
intron
166

619181
0.5
AGCGAGTACTGTGAGAGCAA
1334
intron
168

619182
0.5
CAGCGAGTACTGTGAGAGCA
1335
intron
169

619185
0.5
CCTCAGCGAGTACTGTGAGA
1338
intron
172

619186
0.6
CCCTCAGCGAGTACTGTGAG
1339
intron
173

619187
0.4
ACCCTCAGCGAGTACTGTGA
1340
intron
174

619191
<0.07
GTTCACCCTCAGCGAGTACT
1344
intron
178

619201
0.3
GTCTTTTCTTGTTCACCCTC
1354
intron
188

619202
0.1
GGTCTTTTCTTGTTCACCCT
1355
intron
189

619203
0.2
AGGTCTTTTCTTGTTCACCC
1356
intron
190

619216
<0.07
GTTAATCTTTATCAGGTCTT
1369
intron
203

619217
0.1
GGTTAATCTTTATCAGGTCT
1370
intron
204

619218
0.1
TGGTTAATCTTTATCAGGTC
1371
intron
205

619245
0.5
GCGGTTGTTTCCCTCCTTGT
1398
intron
232

619246
0.1
TGCGGTTGTTTCCCTCCTTG
1399
intron
233

619251
<0.07
CAGGCTGCGGTTGTTTCCCT
1404
intron
238

619252
0.2
ACAGGCTGCGGTTGTTTCCC
1405
intron
239

619253
0.5
TACAGGCTGCGGTTGTTTCC
1406
intron
97

619259
0.3
GCTTGCTACAGGCTGCGGTT
1412
intron
245

619260
0.2
AGCTTGCTACAGGCTGCGGT
1413
intron
246

619276
0.1
GACTCCTGAGTTCCAGAGCT
1429
intron
263

619278
0.1
GCGACTCCTGAGTTCCAGAG
1431
intron
265

619280
<0.07
GCGCGACTCCTGAGTTCCAG
1433
intron
267

619284
0.3
TAGCGCGCGACTCCTGAGTT
1437
Intron
271

619292
0.1
CCGGCCCCTAGCGCGCGACT
1445
intron
279

619293
0.2
CCCGGCCCCTAGCGCGCGAC
1446
intron
98

619298
<0.07
CCGGCCCCGGCCCCTAGCGC
1451
intron
284

619307
0.1
GACCACGCCCCGGCCCCGGC
1466
intron
293

619337
0.3
CCGCAGCCCCGCCCCGGGCC
1504
intron:
326

exon 1B

junction

619338
0.4
ACCGCAGCCCCGCCCCGGGC
1505
intron:
327

exon 1B

junction

619339
0.5
AACCGCAGCCCCGCCCCGGG
1506
intron:
328

exon 1B

junction

619342
0.2
CGCAACCGCAGCCCCGCCCC
1509
intron:
331

exon 1B

junction

619343
0.3
CCGCAACCGCAGCCCCGCCC
1510
intron:
332

exon 1B

junction

619344
0.5
ACCGCAACCGCAGCCCCGCC
1511
intron:
333

exon 1B

junction

619350
0.4
GCAGGCACCGCAACCGCAGC
1517
intron:
339

exon 1B

junction

619351
0.6
CGCAGGCACCGCAACCGCAG
1518
intron:
340

exon 1B

junction

Example 4: Antisense Inhibition of C9ORF72 by Human-Rhesus Cross-Reactive Antisense Oligonucleotides in LLC-MK2 Cells

Antisense oligonucleotides targeting a human C9ORF72 nucleic acid and fully cross-reactive with a rhesus C9ORF72 nucleic acid were designed and were tested for their effects on rhesus C9ORF72 mRNA in vitro. ISIS 576816, previously tested in U.S. Application No. 61/714,132, filed Oct. 15, 2012, was used as a benchmark oligonucleotide. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in the tables below. Cultured rhesus LLC-MK2 cells at a density of 20,000 cells per well were transfected using electroporation with 4,000 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: 16; reverse sequence GCCACTTAAAGCAATCTCTGTCTTG, designated herein as SEQ ID NO: 17; probe sequence TCGACTCTTTGCCCACCGCCA, designated herein as SEQ ID NO: 18—a TAQ-man primer probe set) was used to measure total C9ORF72 mRNA levels. RTS3750 targets exon 2 of the mRNA transcripts and, therefore, measures total mRNA transcripts. 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. The oligonucleotides marked with as asterisk (*) target the amplicon region of the primer probe set. Additional assays may be used to measure the potency and efficacy of these oligonucleotides. ‘n.d.’ indicates that there was no signal reading in the assay for that particular oligonucleotide. The antisense oligonucleotides were also tested in HepG2 cells in a series of experiments that had similar culture conditions. The results for each experiment are also presented in tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 4,000 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 RTS3905 was used to measure the C9ORF72 pathogenic associated mRNA variant, which is the product of a pre-mRNA containing a hexanucleotide repeat. The levels of the C9ORF72 pathogenic associated mRNA variant were normalized to the total RNA content of the cell, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells. ‘n.d.’ means no data.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each antisense oligonucleotide listed in the Tables below is targeted to either human C9ORF72 mRNA sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001256054.1), 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), GENBANK Accession No. NM_018325.3 (incorporated herein as SEQ ID NO: 6), or all three. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence.

TABLE 17

Percent inhibition of human C9ORF72 compared

to PBS control by antisense oligonucleotides

targeting SEQ ID NOs: 1, 2, and 6

% inhi-

SEQ
SEQ
SEQ

bition
% inhi-

ID
ID
ID

(LLC-
bition

NO:
NO:
NO:

MK2)
(HepG2)

1
2
6

Primer
Primer
SEQ

ISIS
Start
Start
Start

Probe
Probe
ID

NO
Site
Site
Site
Sequence
Linkage
RTS3750
RTS3905
NO

576816
310
7990
215
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
51
91
20

619343
n/a
1510
1
CCGCAACCGCAGCCCCGCCC
soooossssssssssooss
0
96
332

619420
310
7990
215
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
59
100
20

625183
309
7989
214
CCTTACTCTAGGACCAAGAA
soooossssssssssooss
27
92
484

625249
239
7919
144
AAAGCAATCTCTGTCTTGGC
soooossssssssssooss
78
n.d.
465

625255
364
8044
269
AAGTTATTTCTCCATCACTG
soooossssssssssooss
40
97
502

627833
321
8001
226
AGCCCAAATGTGCCTTACTC
soooossssssssssooss
52
n.d.
487

627834
382
8062
287
GAGTGTGGTTGGCAAGAAAA
soooossssssssssooss
0
61
506

655126
n/a
1548
39
CGCCACCGCCTGCGCCTCCG
soooossssssssssooss
0
96
512

655127
n/a
1551
42
ACTCGCCACCGCCTGCGCCT
soooossssssssssooss
11
93
513

655128
n/a
n/a
45
TCCACTCGCCACCGCCTGCG
soooossssssssssooss
0
60
514

655129
n/a
n/a
48
ATATCCACTCGCCACCGCCT
soooossssssssssooss
1
79
515

655130
n/a
n/a
51
GAGATATCCACTCGCCACCG
soooossssssssssooss
8
45
516

655131*
167
7847
72
TCACATTATCCAAATGCTCC
soooossssssssssooss
36
88
441

655132*
170
7850
75
CTGTCACATTATCCAAATGC
soooossssssssssooss
20
79
442

655133*
173
7853
78
CAACTGTCACATTATCCAAA
soooossssssssssooss
31
82
443

655134*
176
7856
81
TTCCAACTGTCACATTATCC
soooossssssssssooss
43
90
444

655135*
180
7860
85
TGCATTCCAACTGTCACATT
soooossssssssssooss
69
94
445

655136*
183
7863
88
CACTGCATTCCAACTGTCAC
soooossssssssssooss
75
98
446

655137*
186
7866
91
CATCACTGCATTCCAACTGT
soooossssssssssooss
71
95
447

655138
189
7869
94
CGACATCACTGCATTCCAAC
soooossssssssssooss
85
n.d.
448

655139
192
7872
97
AGTCGACATCACTGCATTCC
soooossssssssssooss
89
n.d.
449

655140*
195
7875
100
AAGAGTCGACATCACTGCAT
soooossssssssssooss
81
100
450

655141*
198
7878
103
GCAAAGAGTCGACATCACTG
soooossssssssssooss
68
98
451

655142*
201
7881
106
TGGGCAAAGAGTCGACATCA
soooossssssssssooss
60
98
452

655143*
204
7884
109
CGGTGGGCAAAGAGTCGACA
soooossssssssssooss
69
99
453

655144*
207
7887
112
TGGCGGTGGGCAAAGAGTCG
soooossssssssssooss
60
97
454

655145*
211
7891
116
GAGATGGCGGTGGGCAAAGA
soooossssssssssooss
27
93
455

655146*
214
7894
119
CTGGAGATGGCGGTGGGCAA
soooossssssssssooss
56
99
456

655147*
218
7898
123
ACAGCTGGAGATGGCGGTGG
soooossssssssssooss
17
84
457

655148
221
7901
126
GCAACAGCTGGAGATGGCGG
soooossssssssssooss
34
n.d.
458

655149
224
7904
129
TTGGCAACAGCTGGAGATGG
soooossssssssssooss
27
n.d.
459

655150*
227
7907
132
GTCTTGGCAACAGCTGGAGA
soooossssssssssooss
52
99
460

655151
230
7910
135
TCTGTCTTGGCAACAGCTGG
soooossssssssssooss
60
n.d.
461

655152*
232
7912
137
TCTCTGTCTTGGCAACAGCT
soooossssssssssooss
65
98
462

655153*
236
7916
141
GCAATCTCTGTCTTGGCAAC
soooossssssssssooss
91
99
463

655154*
237
7917
142
AGCAATCTCTGTCTTGGCAA
soooossssssssssooss
87
99
464

655155*
242
7922
147
CTTAAAGCAATCTCTGTCTT
soooossssssssssooss
80
76
466

655156*
245
7925
150
CCACTTAAAGCAATCTCTGT
soooossssssssssooss
74
86
467

655157
267
7947
172
AGCTGCTAATAAAGGTGATT
soooossssssssssooss
27
90
468

655158
270
7950
175
AGTAGCTGCTAATAAAGGTG
soooossssssssssooss
17
87
469

655159
273
7953
178
AAAAGTAGCTGCTAATAAAG
soooossssssssssooss
6
48
470

655160
276
7956
181
AGCAAAAGTAGCTGCTAATA
soooossssssssssooss
3
87
471

655161
279
7959
184
GTAAGCAAAAGTAGCTGCTA
soooossssssssssooss
30
99
472

655162
282
7962
187
CCAGTAAGCAAAAGTAGCTG
soooossssssssssooss
24
85
473

655163
285
7965
190
GTCCCAGTAAGCAAAAGTAG
soooossssssssssooss
15
70
474

655164
286
7966
191
TGTCCCAGTAAGCAAAAGTA
soooossssssssssooss
5
60
475

655165
288
7968
193
ATTGTCCCAGTAAGCAAAAG
soooossssssssssooss
0
46
476

655166
290
7970
195
ATATTGTCCCAGTAAGCAAA
soooossssssssssooss
0
54
477

655167
291
7971
196
AATATTGTCCCAGTAAGCAA
soooossssssssssooss
16
60
478

655168
294
7974
199
AAGAATATTGTCCCAGTAAG
soooossssssssssooss
0
52
479

655169
297
7977
202
ACCAAGAATATTGTCCCAGT
soooossssssssssooss
20
82
480

655170
300
7980
205
AGGACCAAGAATATTGTCCC
soooossssssssssooss
17
54
481

655171
303
7983
208
TCTAGGACCAAGAATATTGT
soooossssssssssooss
0
36
482

655172
306
7986
211
TACTCTAGGACCAAGAATAT
soooossssssssssooss
10
43
483

655173
312
7992
217
GTGCCTTACTCTAGGACCAA
soooossssssssssooss
75
99
485

655174
315
7995
220
AATGTGCCTTACTCTAGGAC
soooossssssssssooss
47
98
486

655175
327
8007
232
CTTTGGAGCCCAAATGTGCC
soooossssssssssooss
21
86
488

655176
330
8010
235
TGTCTTTGGAGCCCAAATGT
soooossssssssssooss
21
88
489

655177
333
8013
238
TTCTGTCTTTGGAGCCCAAA
soooossssssssssooss
42
97
490

655178
334
8014
239
GTTCTGTCTTTGGAGCCCAA
soooossssssssssooss
66
99
491

655179
336
8016
241
CTGTTCTGTCTTTGGAGCCC
soooossssssssssooss
68
95
492

655180
339
8019
244
TACCTGTTCTGTCTTTGGAG
soooossssssssssooss
29
92
493

655181
342
8022
247
AAGTACCTGTTCTGTCTTTG
soooossssssssssooss
26
76
494

655182
345
8025
250
GAGAAGTACCTGTTCTGTCT
soooossssssssssooss
38
94
495

655183
348
8028
253
ACTGAGAAGTACCTGTTCTG
soooossssssssssooss
23
89
496

655184
350
8030
255
TCACTGAGAAGTACCTGTTC
soooossssssssssooss
19
83
497

655185
351
8031
256
ATCACTGAGAAGTACCTGTT
soooossssssssssooss
40
87
498

655186
354
8034
259
TCCATCACTGAGAAGTACCT
soooossssssssssooss
46
97
499

655187
358
8038
263
TTTCTCCATCACTGAGAAGT
soooossssssssssooss
36
94
500

655188
361
8041
266
TTATTTCTCCATCACTGAGA
soooossssssssssooss
9
82
501

655189
367
8047
272
GAAAAGTTATTTCTCCATCA
soooossssssssssooss
37
96
503

655190
376
8056
281
GGTTGGCAAGAAAAGTTATT
soooossssssssssooss
15
74
504

655191
379
8059
284
TGTGGTTGGCAAGAAAAGTT
soooossssssssssooss
15
62
505

655192
385
8065
290
TTAGAGTGTGGTTGGCAAGA
soooossssssssssooss
12
63
507

655193
388
8068
293
CATTTAGAGTGTGGTTGGCA
soooossssssssssooss
7
89
508

655194
389
8069
294
CCATTTAGAGTGTGGTTGGC
soooossssssssssooss
31
93
509

655195
394
8074
299
TTTCTCCATTTAGAGTGTGG
soooossssssssssooss
19
91
510

655196
397
8077
302
GGATTTCTCCATTTAGAGTG
soooossssssssssooss
8
82
511

TABLE 18

Percent inhibition of human C9ORF72

compared to PBS control by antisense oligonucleotides

targeting SEQ ID NOs: 1, 2, and 6

% inhi-

SEQ
SEQ
SEQ

bition
% inhi-

ID
ID
ID

(LLC-
bition

NO:
NO:
NO:

MK2)
(HepG2)

1
2
6

Primer
Primer
SEQ

ISIS
Start
Start
Start

Probe
Probe
ID

NO
Site
Site
Site
Sequence
Linkage
RTS3750
RTS3905
NO

576816
310
7990
215
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
55
97
20

619420
310
7990
215
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
74
97
20

655197
403
8083
308
TTCGAAGGATTTCTCCATTT
soooossssssssssooss
14
87
517

655198
406
8086
311
CATTTCGAAGGATTTCTCCA
soooossssssssssooss
23
83
518

655199
409
8089
314
CTGCATTTCGAAGGATTTCT
soooossssssssssooss
42
97
519

655200
412
8092
317
TCTCTGCATTTCGAAGGATT
soooossssssssssooss
21
87
520

655201
415
8095
320
CACTCTCTGCATTTCGAAGG
soooossssssssssooss
25
89
521

655202
418
8098
323
CACCACTCTCTGCATTTCGA
soooossssssssssooss
54
96
522

655203
420
8100
325
AGCACCACTCTCTGCATTTC
soooossssssssssooss
51
90
523

655204
421
8101
326
TAGCACCACTCTCTGCATTT
soooossssssssssooss
28
94
524

655205
424
8104
329
CTATAGCACCACTCTCTGCA
soooossssssssssooss
17
90
525

655206
427
8107
332
CATCTATAGCACCACTCTCT
soooossssssssssooss
15
83
526

655207
433
8113
338
ACTTTACATCTATAGCACCA
soooossssssssssooss
20
85
527

655208
436
8116
341
AAAACTTTACATCTATAGCA
soooossssssssssooss
25
72
528

655209
443
8123
348
AAGACAAAAAACTTTACATC
soooossssssssssooss
10
45
529

655210
444
8124
349
CAAGACAAAAAACTTTACAT
soooossssssssssooss
0
56
530

655211
446
8126
351
GACAAGACAAAAAACTTTAC
soooossssssssssooss
32
84
531

655212
449
8129
354
TCAGACAAGACAAAAAACTT
soooossssssssssooss
10
74
532

655213
451
8131
356
TTTCAGACAAGACAAAAAAC
soooossssssssssooss
7
54
533

655214
453
8133
358
CTTTTCAGACAAGACAAAAA
soooossssssssssooss
7
57
534

655215
456
8136
361
TCCCTTTTCAGACAAGACAA
soooossssssssssooss
32
91
535

655216
459
8139
364
CACTCCCTTTTCAGACAAGA
soooossssssssssooss
39
91
536

655217
462
8142
367
AATCACTCCCTTTTCAGACA
soooossssssssssooss
41
95
537

655218
465
8145
370
AATAATCACTCCCTTTTCAG
soooossssssssssooss
13
63
538

655219
467
8147
372
ACAATAATCACTCCCTTTTC
soooossssssssssooss
2
60
539

655220
468
8148
373
AACAATAATCACTCCCTTTT
soooossssssssssooss
11
73
540

655221
471
8151
376
TGAAACAATAATCACTCCCT
soooossssssssssooss
17
83
541

655222
474
8154
379
TAATGAAACAATAATCACTC
soooossssssssssooss
1
79
542

655223
477
8157
382
GATTAATGAAACAATAATCA
soooossssssssssooss
12
59
543

655224
482
8162
387
TCAAAGATTAATGAAACAAT
soooossssssssssooss
0
9
544

655225
485
8165
390
CCATCAAAGATTAATGAAAC
soooossssssssssooss
17
81
545

655226
488
8168
393
TTTCCATCAAAGATTAATGA
soooossssssssssooss
14
87
546

655227
491
8171
396
CAGTTTCCATCAAAGATTAA
soooossssssssssooss
7
69
547

655228
494
8174
399
TTCCAGTTTCCATCAAAGAT
soooossssssssssooss
25
88
548

655229
497
8177
402
CCATTCCAGTTTCCATCAAA
soooossssssssssooss
44
90
549

655230
500
8180
405
TCCCCATTCCAGTTTCCATC
soooossssssssssooss
48
94
550

655231
503
8183
408
CGATCCCCATTCCAGTTTCC
soooossssssssssooss
54
96
551

655232
506
8186
411
CTGCGATCCCCATTCCAGTT
soooossssssssssooss
61
96
552

655233
509
8189
414
GTGCTGCGATCCCCATTCCA
soooossssssssssooss
63
100
553

655234
512
8192
417
TATGTGCTGCGATCCCCATT
soooossssssssssooss
34
94
554

655235
533
8213
438
GGAAGTATAATTGATAGTCC
soooossssssssssooss
25
91
555

655236
536
8216
441
TGTGGAAGTATAATTGATAG
soooossssssssssooss
13
66
556

655237
539
8219
444
GTCTGTGGAAGTATAATTGA
soooossssssssssooss
24
83
557

655238
542
8222
447
TCTGTCTGTGGAAGTATAAT
soooossssssssssooss
51
92
558

655239
545
8225
450
AGTTCTGTCTGTGGAAGTAT
soooossssssssssooss
42
96
559

655240
548
8228
453
CTAAGTTCTGTCTGTGGAAG
soooossssssssssooss
16
88
560

655241
551
8231
456
AAACTAAGTTCTGTCTGTGG
soooossssssssssooss
25
93
561

655242
554
8234
459
TAGAAACTAAGTTCTGTCTG
soooossssssssssooss
29
94
562

655243
557
8237
462
AGGTAGAAACTAAGTTCTGT
soooossssssssssooss
43
85
563

655244
560
8240
465
GGGAGGTAGAAACTAAGTTC
soooossssssssssooss
27
77
564

655245
563
8243
468
AGTGGGAGGTAGAAACTAAG
soooossssssssssooss
22
77
565

655246
566
8246
471
TGAAGTGGGAGGTAGAAACT
soooossssssssssooss
16
45
566

655247
569
8249
474
CTATGAAGTGGGAGGTAGAA
soooossssssssssooss
14
60
567

655248
572
8252
477
ACTCTATGAAGTGGGAGGTA
soooossssssssssooss
30
81
568

655249
574
8254
479
ACACTCTATGAAGTGGGAGG
soooossssssssssooss
34
94
569

655250
575
8255
480
CACACTCTATGAAGTGGGAG
soooossssssssssooss
46
98
570

655251
576
8256
481
ACACACTCTATGAAGTGGGA
soooossssssssssooss
20
94
571

655252
578
8258
483
ACACACACTCTATGAAGTGG
soooossssssssssooss
28
97
572

655253
581
8261
486
TCAACACACACTCTATGAAG
soooossssssssssooss
12
62
573

655254
584
8264
489
CTATCAACACACACTCTATG
soooossssssssssooss
6
68
574

655255
587
8267
492
AATCTATCAACACACACTCT
soooossssssssssooss
16
87
575

655256
590
8270
495
GTTAATCTATCAACACACAC
soooossssssssssooss
28
95
576

655257
592
8272
497
GTGTTAATCTATCAACACAC
soooossssssssssooss
15
76
577

655258
593
8273
498
TGTGTTAATCTATCAACACA
soooossssssssssooss
14
53
578

655259
595
8275
500
TATGTGTTAATCTATCAACA
soooossssssssssooss
11
78
579

655260
596
8276
501
ATATGTGTTAATCTATCAAC
soooossssssssssooss
25
79
580

655261
599
8279
504
ATTATATGTGTTAATCTATC
soooossssssssssooss
11
71
581

655262
602
8282
507
CGGATTATATGTGTTAATCT
soooossssssssssooss
21
91
582

655263
605
8285
510
TTCCGGATTATATGTGTTAA
soooossssssssssooss
15
88
583

655264
608
8288
513
CCTTTCCGGATTATATGTGT
soooossssssssssooss
12
83
584

655265
611
8291
516
CTTCCTTTCCGGATTATATG
soooossssssssssooss
1
69
585

655266
614
8294
519
ATTCTTCCTTTCCGGATTAT
soooossssssssssooss
15
76
586

655267
617
8297
522
CATATTCTTCCTTTCCGGAT
soooossssssssssooss
17
69
587

655268
620
8300
525
ATCCATATTCTTCCTTTCCG
soooossssssssssooss
21
67
588

655269
624
8304
529
ATGCATCCATATTCTTCCTT
soooossssssssssooss
5
73
589

655270
626
8306
531
TTATGCATCCATATTCTTCC
soooossssssssssooss
23
71
590

655271
629
n/a
534
TCCTTATGCATCCATATTCT
soooossssssssssooss
15
64
591

655272
632
n/a
537
CTTTCCTTATGCATCCATAT
soooossssssssssooss
24
66
592

655273
635
n/a
540
TGTCTTTCCTTATGCATCCA
soooossssssssssooss
37
76
593

TABLE 19

Percent inhibition of human C9ORF72

compared to PBS control by antisense oligonucleotides

targeting SEQ ID NOs: 1, 2, and 6

% inhi-

SEQ
SEQ
SEQ

bition
% inhi-

ID
ID
ID

(LLC-
bition

NO:
NO:
NO:

MK2)
(HepG2)

1
2
6

Primer
Primer
SEQ

ISIS
Start
Start
Start

Probe
Probe
ID

NO
Site
Site
Site
Sequence
Linkage
RTS3750
RTS3905
NO

576816
310
7990
215
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
53
95
20

619253
n/a
1406
n/a
TACAGGCTGCGGTTGTTTCC
soooossssssssssooss
0
72
97

619293
n/a
1446
n/a
CCCGGCCCCTAGCGCGCGAC
soooossssssssssooss
0
95
98

619422
n/a
3452
n/a
GGTAACTTCAAACTCTTGGG
soooossssssssssooss
0
93
382

655329
n/a
2330
n/a
AGGACCTCCCTCCTGTTTCT
soooossssssssssooss
0
84
594

655330
n/a
2490
n/a
AGAAGTAATGCCAGACAGAT
soooossssssssssooss
0
75
595

655331
n/a
2901
n/a
CTTTGTTTCTCTGAAAGCAA
soooossssssssssooss
16
75
596

655332
n/a
3576
n/a
GTGGTTGGTCCACTGCTATT
soooossssssssssooss
28
94
597

655333
n/a
3801
n/a
TTGAGGGAAGCCAAGATTCA
soooossssssssssooss
10
77
598

655334
n/a
3975
n/a
AGAGCTGTACAATTATTTTA
soooossssssssssooss
13
90
599

655335
n/a
4725
n/a
GGTAATGACACTACTGCTGT
soooossssssssssooss
27
96
600

655336
n/a
5970
n/a
GATCCTAATCCTGTCTATGC
soooossssssssssooss
0
70
601

655337
n/a
7382
n/a
ACTTGTGGGTTGAATTGTGT
soooossssssssssooss
4
66
602

655338
n/a
8310
n/a
TACCTTATGCATCCATATTC
soooossssssssssooss
11
62
603

655339
n/a
8409
n/a
GATGTTCACTGCATATAATT
soooossssssssssooss
31
88
604

655340
n/a
8464
n/a
CCAGATGTATTTGTATCTAA
soooossssssssssooss
41
96
605

655341
n/a
8523
n/a
TAATGTGGAGCTACCATTTC
soooossssssssssooss
6
72
606

655342
n/a
8587
n/a
GCTCCCAAGAAGAATCCAGG
soooossssssssssooss
44
74
607

655343
n/a
8658
n/a
ACTTACACATAGTAGTAAGC
soooossssssssssooss
16
88
608

655344
n/a
8716
n/a
AAAGAGACCAAAGGCTACAT
soooossssssssssooss
4
82
609

655345
n/a
8785
n/a
GGAATTCTCTTGGGAACCAT
soooossssssssssooss
9
80
610

619420
310
7990
215
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
51
98
20

655274
638
n/a
543
TCTTGTCTTTCCTTATGCAT
soooossssssssssooss
0
73
611

655275
641
n/a
546
TTTTCTTGTCTTTCCTTATG
soooossssssssssooss
10
61
612

625344
647
9413
552
TGGACATTTTCTTGTCTTTC
soooossssssssssooss
32
94
613

655276
650
9416
555
TTCTGGACATTTTCTTGTCT
soooossssssssssooss
8
79
614

655277
653
9419
558
ATCTTCTGGACATTTTCTTG
soooossssssssssooss
0
60
615

655278
655
9421
560
TAATCTTCTGGACATTTTCT
soooossssssssssooss
0
66
616

655279
659
9425
564
AAGATAATCTTCTGGACATT
soooossssssssssooss
5
72
617

655280
662
9428
567
TCTAAGATAATCTTCTGGAC
soooossssssssssooss
2
81
618

655281
665
9431
570
CCTTCTAAGATAATCTTCTG
soooossssssssssooss
0
19
619

655282
668
9434
573
GTGCCTTCTAAGATAATCTT
soooossssssssssooss
4
20
620

655283
671
9437
576
TCTGTGCCTTCTAAGATAAT
soooossssssssssooss
9
27
621

655284
674
9440
579
CTCTCTGTGCCTTCTAAGAT
soooossssssssssooss
0
35
622

655285
677
9443
582
ATTCTCTCTGTGCCTTCTAA
soooossssssssssooss
7
40
623

655286
680
9446
585
TCCATTCTCTCTGTGCCTTC
soooossssssssssooss
18
65
624

655287
683
9449
588
TCTTCCATTCTCTCTGTGCC
soooossssssssssooss
14
67
625

655288
686
9452
591
TGATCTTCCATTCTCTCTGT
soooossssssssssooss
7
65
626

655289
691
n/a
596
GACCCTGATCTTCCATTCTC
soooossssssssssooss
12
89
627

655290
694
n/a
599
TCTGACCCTGATCTTCCATT
soooossssssssssooss
13
81
628

655291
697
n/a
602
TACTCTGACCCTGATCTTCC
soooossssssssssooss
0
82
629

655292
700
n/a
605
TAATACTCTGACCCTGATCT
soooossssssssssooss
0
72
630

655293
703
n/a
608
GAATAATACTCTGACCCTGA
soooossssssssssooss
0
77
631

655294
709
12529
614
GCATTGGAATAATACTCTGA
soooossssssssssooss
0
79
632

655295
712
12532
617
TAAGCATTGGAATAATACTC
soooossssssssssooss
8
79
633

655296
715
12535
620
CAGTAAGCATTGGAATAATA
soooossssssssssooss
0
66
634

655297
718
12538
623
CTCCAGTAAGCATTGGAATA
soooossssssssssooss
12
78
635

655298
721
12541
626
CTTCTCCAGTAAGCATTGGA
soooossssssssssooss
0
81
636

655299
724
12544
629
TCACTTCTCCAGTAAGCATT
soooossssssssssooss
0
70
637

655300
727
12547
632
GAATCACTTCTCCAGTAAGC
soooossssssssssooss
0
75
638

655301
730
12550
635
CAGGAATCACTTCTCCAGTA
soooossssssssssooss
20
85
639

655302
733
12553
638
TTACAGGAATCACTTCTCCA
soooossssssssssooss
0
50
640

655303
736
12556
641
CCATTACAGGAATCACTTCT
soooossssssssssooss
0
57
641

655304
744
12564
649
AAGCAGTTCCATTACAGGAA
soooossssssssssooss
0
83
642

655305
747
12567
652
TGAAAGCAGTTCCATTACAG
soooossssssssssooss
0
55
643

655306
750
12570
655
AGATGAAAGCAGTTCCATTA
soooossssssssssooss
0
82
644

655307
753
12573
658
CATAGATGAAAGCAGTTCCA
soooossssssssssooss
2
83
645

655308
756
12576
661
TTTCATAGATGAAAGCAGTT
soooossssssssssooss
0
59
646

655309
762
12582
667
GTGTGATTTCATAGATGAAA
soooossssssssssooss
0
39
647

655310
766
12586
671
CACTGTGTGATTTCATAGAT
soooossssssssssooss
10
31
648

655311
769
12589
674
GAACACTGTGTGATTTCATA
soooossssssssssooss
0
80
649

655312
772
12592
677
CAGGAACACTGTGTGATTTC
soooossssssssssooss
0
53
650

655313
778
12598
683
TTTCTTCAGGAACACTGTGT
soooossssssssssooss
0
45
651

655314
781
12601
686
CTATTTCTTCAGGAACACTG
soooossssssssssooss
0
56
652

655315
784
n/a
689
TATCTATTTCTTCAGGAACA
soooossssssssssooss
0
75
653

655316
787
n/a
692
CTATATCTATTTCTTCAGGA
soooossssssssssooss
0
75
654

655317
790
n/a
695
CAGCTATATCTATTTCTTCA
soooossssssssssooss
23
89
655

655318
793
n/a
698
TATCAGCTATATCTATTTCT
soooossssssssssooss
7
80
656

655319
796
n/a
701
CTGTATCAGCTATATCTATT
soooossssssssssooss
34
86
657

655320
799
n/a
704
GTACTGTATCAGCTATATCT
soooossssssssssooss
42
88
658

655321
802
n/a
707
TGAGTACTGTATCAGCTATA
soooossssssssssooss
31
87
659

655322
805
13356
710
CATTGAGTACTGTATCAGCT
soooossssssssssooss
3
84
660

655323
808
13359
713
CATCATTGAGTACTGTATCA
soooossssssssssooss
5
88
661

655324
811
13362
716
CATCATCATTGAGTACTGTA
soooossssssssssooss
17
90
662

655325
814
13365
719
TATCATCATCATTGAGTACT
soooossssssssssooss
2
84
663

655326
817
13368
722
CAATATCATCATCATTGAGT
soooossssssssssooss
0
74
664

655327
822
13373
727
GTCACCAATATCATCATCAT
soooossssssssssooss
6
78
665

655328
848
13399
753
TTGAGAAGAAAGCCTTCATG
soooossssssssssooss
0
42
666

619421
3132
28251
3037
GGGACACTACAAGGTAGTAT
soooossssssssssooss
0
94
401

TABLE 20

Percent inhibition of human C9ORF72

compared to PBS control by antisense oligonucleotides

targeting SEQ ID NOs: 1, 2, and 6

% inhi-

SEQ
SEQ
SEQ

bition
% inhi-

ID
ID
ID

(LLC-
bition

NO:
NO:
NO:

MK2)
(HepG2)

1
2
6

Primer
Primer
SEQ

ISIS
Start
Start
Start

Probe
Probe
ID

NO
Site
Site
Site
Sequence
Linkage
RTS3750
RTS3905
NO

576816
310
7990
215
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
49
97
20

655346
n/a
8861
n/a
ACTATACTGAAATGTAAATA
soooossssssssssooss
4
7
667

655347
n/a
8915
n/a
TATCAAACTGGAACACAGGA
soooossssssssssooss
16
53
668

655348
n/a
8965
n/a
TGGGCAAAAGCCTTTTAAAA
soooossssssssssooss
13
63
669

655349
n/a
9017
n/a
GCAAACATAGTAAAAAATTA
soooossssssssssooss
6
7
670

655350
n/a
9067
n/a
TTCTCCTGATTTTAAGAGTT
soooossssssssssooss
39
90
671

655351
n/a
9117
n/a
AAGAATGACTTGCACTTTTC
soooossssssssssooss
25
100
672

655352
n/a
9173
n/a
AAAGATAACTTCACAGAAAA
soooossssssssssooss
13
10
673

655353
n/a
9286
n/a
CTTTCTACTTTAGGGAAAAA
soooossssssssssooss
16
64
674

655354
n/a
9336
n/a
TTTTTCAATAGACATGTTCT
soooossssssssssooss
19
81
675

655355
n/a
9403
n/a
CTTGTCTTTCCTGAGCAAGA
soooossssssssssooss
8
54
676

655356
n/a
9455
n/a
ACCTGATCTTCCATTCTCTC
soooossssssssssooss
22
81
677

655357
n/a
9576
n/a
CTCCATAAAAGCTCCATTAA
soooossssssssssooss
34
52
678

655358
n/a
9640
n/a
TGTTTACTGATTTAACTCTT
soooossssssssssooss
34
83
679

655359
n/a
9696
n/a
AACAGAAAAAAAAAGGGAGC
soooossssssssssooss
18
22
680

655360
n/a
9772
n/a
GTACCTTAAAGAACATATCA
soooossssssssssooss
34
100
681

655361
n/a
9920
n/a
AAATGTAAATTGCATGAGTC
soooossssssssssooss
7
100
682

655362
n/a
9970
n/a
GGGTAAGAAATATCACTGAC
soooossssssssssooss
43
63
683

655363
n/a
10055
n/a
AACCATGCTTCTCAAACTCT
soooossssssssssooss
29
75
684

655364
n/a
10122
n/a
AAGAACTTCTCTGCTTTACA
soooossssssssssooss
13
68
685

655365
n/a
10172
n/a
AATGGAAGTAAAAGTGAAGA
soooossssssssssooss
7
13
686

655366
n/a
10233
n/a
AACAGCCATGTTTAAAATAT
soooossssssssssooss
15
37
687

655367
n/a
10283
n/a
TTAAAGTATCATCTGTCTCA
soooossssssssssooss
34
74
688

655368
n/a
10364
n/a
CAATTTGGTAAAGGAGATCA
soooossssssssssooss
6
48
689

655369
n/a
10418
n/a
ACACAGAATAACTGTCTCTG
soooossssssssssooss
16
64
690

655370
n/a
10491
n/a
GCTTATTGACCAGCAAATAA
soooossssssssssooss
22
69
691

655371
n/a
10615
n/a
CCCAGTAAAAGCAGAATTTT
soooossssssssssooss
24
57
692

655372
n/a
10665
n/a
ATTAATAGTAGTCAACTTAA
soooossssssssssooss
20
10
693

655373
n/a
10730
n/a
ACTTGAACTTCTCAGCAGTA
soooossssssssssooss
25
62
694

655374
n/a
10786
n/a
AGAAGAGGCTCTAAAAGAAA
soooossssssssssooss
7
59
695

655375
n/a
10970
n/a
AAAGGCAACTCCTCCTTTTC
soooossssssssssooss
13
78
696

655376
n/a
11020
n/a
ACAGTATTGTTCAAAATAAA
soooossssssssssooss
18
100
697

655377
n/a
11070
n/a
CAGATGACAGCTACAACTGA
soooossssssssssooss
16
49
698

655378
n/a
11121
n/a
GAATAATGACTAGATCCGTG
soooossssssssssooss
26
88
699

655379
n/a
11171
n/a
ATAATTATCATGCCTGTTTA
soooossssssssssooss
22
80
700

655380
n/a
11221
n/a
CTTTAGTAACCTCCACAACT
soooossssssssssooss
19
26
701

655381
n/a
11313
n/a
GAATTTAAATGTGATGCTAC
soooossssssssssooss
15
49
702

655382
n/a
11385
n/a
TGTCAGACCCAGGGCCATTT
soooossssssssssooss
40
83
703

655383
n/a
11445
n/a
ACTTATTTTATGAAATGATT
soooossssssssssooss
7
3
704

655384
n/a
11513
n/a
TTTTAGCTAAACATATTTTT
soooossssssssssooss
2
1
705

655385
n/a
11591
n/a
CAGTCTCATCAGTTTTGTGA
soooossssssssssooss
33
83
706

655386
n/a
11641
n/a
TGTAAAGTGTCTCAAATATG
soooossssssssssooss
21
39
707

655387
n/a
11711
n/a
CTTGAAATTGTAATTTTGAA
soooossssssssssooss
14
42
708

655388
n/a
11798
n/a
AATCAAAATCAGCACATATA
soooossssssssssooss
8
46
709

655389
n/a
11871
n/a
ACCAAATAGGTAAGGAAAAC
soooossssssssssooss
9
32
710

655390
n/a
11978
n/a
AAAGATCTCCTTTAAAATTT
soooossssssssssooss
11
29
711

655391
n/a
12053
n/a
CTTTAGAGAGTATGGAATCA
soooossssssssssooss
22
71
712

655392
n/a
12334
n/a
CAAAGCTCACTTTTATTCTT
soooossssssssssooss
25
70
713

655393
n/a
12384
n/a
ACACAGTATCAAACAAGTCT
soooossssssssssooss
29
45
714

655394
n/a
12459
n/a
AAGCTGGGCAATAAAAAATA
soooossssssssssooss
0
24
715

655395
n/a
12513
n/a
CTGACCCTGCACAATAAAGT
soooossssssssssooss
17
0
716

655396
n/a
12604
n/a
CATCTATTTCTTCAGGAACA
soooossssssssssooss
21
74
717

655397
n/a
12681
n/a
GAATATTAATAATATACATA
soooossssssssssooss
0
16
718

655398
n/a
12765
n/a
AGGATTTTGTGTGTGCTTAT
soooossssssssssooss
30
65
719

655399
n/a
12855
n/a
TTTTAGGAATTATAAAAGTA
soooossssssssssooss
7
61
720

655400
n/a
12924
n/a
ACACAGTTTTGTTTCAAAAG
soooossssssssssooss
13
61
721

655401
n/a
12978
n/a
GGAAACTAAATTTGTGACTA
soooossssssssssooss
21
56
722

655402
n/a
13028
n/a
CTCTTAACACTCATAGTGTG
soooossssssssssooss
13
52
723

655403
n/a
13084
n/a
GAGACTAACCTAAATGACAA
soooossssssssssooss
20
35
724

655404
n/a
13159
n/a
CAAATGTGAAAGCTGGTCAA
soooossssssssssooss
8
100
725

655405
n/a
13237
n/a
TAACACACTGCCTTCATTTC
soooossssssssssooss
17
32
726

655406
n/a
13337
n/a
TATCTAAAATGCATCAAAAA
soooossssssssssooss
2
6
727

655407
n/a
13400
n/a
CTTGAGAAGAAAGCCTTCAT
soooossssssssssooss
23
42
728

655408
n/a
13471
n/a
CCAAATCTTGTCATAGGTGA
soooossssssssssooss
42
95
729

655409
n/a
13550
n/a
TAACACAAATTTAAGCAACA
soooossssssssssooss
21
55
730

655410
n/a
13603
n/a
AAATAGCAAATGGAATAACA
soooossssssssssooss
14
55
731

655411
n/a
13662
n/a
AAACCAGAATCAAGCAAGGG
soooossssssssssooss
27
73
732

655412
n/a
13722
n/a
CATCTACAGTACAACTTAAT
soooossssssssssooss
13
100
733

655413
n/a
13773
n/a
AGATCAGTATAAATATGAAT
soooossssssssssooss
1
43
734

655414
n/a
13823
n/a
GTTTAAGGGCACAAACTCTT
soooossssssssssooss
27
78
735

655415
n/a
13884
n/a
AGGTGTATAGAGAATTCAGG
soooossssssssssooss
43
89
736

655416
n/a
13955
n/a
TACTCAATGCTTATAACAAC
soooossssssssssooss
26
84
737

655417
n/a
14089
n/a
GGAACTAACATGTAGGCACT
soooossssssssssooss
66
100
738

655418
n/a
14213
n/a
CATAAAAGTGAATACTTTAT
soooossssssssssooss
13
0
739

655419
n/a
14281
n/a
AGGCTCTTAGGTTAAACACA
soooossssssssssooss
16
79
740

655420
n/a
14331
n/a
GCTGACACTGAACAGATACA
soooossssssssssooss
52
84
741

655421
n/a
14392
n/a
CATGTAGAGAGATTAAGTGA
soooossssssssssooss
27
42
742

655422
n/a
14452
n/a
ATCATTTAATTAATGTATTT
soooossssssssssooss
13
0
743

619420
310
7990
215
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
76
98
20

Example 5: Dose-Dependent Antisense Inhibition of Human C9ORF72 mRNA in LLC-MK2

Antisense oligonucleotides from the study described above exhibiting significant in vitro inhibition of C9ORF72 mRNA were selected and tested at various doses in LLC-MK2 cells. ISIS 576816, previously tested in U.S. Application No. 61/714,132, filed Oct. 15, 2012, was used as a benchmark oligonucleotide. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.33 μM, 1.00 μM, 3.00 μM, or 9.00 μM 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 total C9ORF72 mRNA levels. C9ORF72 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72 levels, relative to untreated control cells.

As shown in Table 21, total C9ORF72 mRNA levels were reduced in a dose-dependent manner in the antisense oligonucleotide treated cells.

TABLE 21

Dose-dependent inhibition of total C9ORF72

mRNA transcript levels in LLC-MK2 cells

ISIS No
0.33 μM
1.00 μM
3.00 μM
9.00 μM

576816
0
33
55
66

619411
15
43
67
87

619412
9
30
55
84

619413
17
27
58
79

619414
13
49
75
83

619415
15
41
62
57

619416
29
47
70
81

619420
17
49
70
85

619423
25
52
71
82

627833
4
31
63
82

655173
37
72
86
90

655178
9
35
71
86

655179
30
50
52
84

655202
1
12
41
72

655231
0
28
43
71

655232
17
45
64
76

655233
19
30
62
80

655420
24
28
49
78

The antisense oligonucleotides were also selected and tested at various doses in HepG2 cells. ISIS 576816, previously tested in U.S. Application No. 61/714,132, filed Oct. 15, 2012, was used as a benchmark oligonucleotide. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.11 μM, 0.33 μM, 1.00 μM, or 3.00 μM 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 total C9ORF72 mRNA levels. C9ORF72 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72 levels, relative to untreated control cells.

As shown in Table 22, total C9ORF72 mRNA levels were reduced in a dose-dependent manner in the antisense oligonucleotide treated cells.

TABLE 22

Dose-dependent inhibition of total C9ORF72

mRNA transcript levels in HepG2 cells

ISIS No
0.11 μM
0.33 μM
1.00 μM
3.00 μM

576816
16
32
71
90

619411
11
41
71
91

619412
24
44
79
92

619413
0
18
59
84

619414
16
51
76
92

619415
16
38
72
85

619416
16
36
47
80

619420
15
47
75
91

619423
24
50
81
89

627833
0
29
69
90

655173
24
56
88
96

655178
25
48
81
92

655179
16
43
79
87

655202
17
19
61
84

655231
14
45
68
84

655232
13
38
69
79

655233
17
30
65
86

655420
19
35
60
86

Example 6: Design of Mixed Backbone 5-8-5 MOE Gapmers and Deoxy, MOE, and cEt Oligonucleotides Targeting Human C9ORF72

Additional antisense oligonucleotides were designed targeting a C9ORF72 nucleic acid. The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-8-5 MOE gapmers, 5-10-5 MOE gapmers, or deoxy, MOE, and cEt gapmers.

The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The linkages between the nucleosides are described in the Linkage column; ‘o’ indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioate linkage. All cytosine residues throughout each gapmer are 5-methylcytosines.

The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The linkages between the nucleosides are described in the Linkage column; ‘o’ indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioate linkage. All cytosine residues throughout each gapmer are 5-methylcytosines.

The deoxy, MOE, and cEt oligonucleotides are 17 nucleosides in length wherein the nucleoside has either a MOE sugar modification, a cEt sugar modification, or a deoxyribose sugar. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide; ‘k’ indicates a cEt nucleoside; d’ indicates deoxyribonucleosides, the number indicates the number of deoxyribonucleosides; and ‘e’ indicates a 2′-MOE nucleoside. The internucleoside linkages throughout each gapmer are either phosphorothioate linkages or phosphodiester linkages. The linkages between the nucleosides are described in the Linkage column; ‘o’ indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioate linkage. 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 in the human gene sequence. Each antisense oligonucleotide listed in the Tables 23-29 below 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. Table 30 presents a 5-10-5 gapmer that is targeted to C9ORF72 mRNA sequence, SEQ ID NO: 1 (GENBANK Accession No. NM_001256054.1).

TABLE 23

5-8-5 MOE gapmers targeting SEQ ID NO: 2

SEQ ID
SEQ ID

NO: 2
NO: 2
SEQ

ISIS

Start
Stop
ID

NO
Sequence
Linkage
Site
Site
NO

672581
GTGAGAGCAAGTAGTGGG
sooosssssssssooss
1326
1343
744

672582
TGTGAGAGCAAGTAGTGG
sooosssssssssooss
1327
1344
745

672583
CTGTGAGAGCAAGTAGTG
sooosssssssssooss
1328
1345
746

672584
ACTGTGAGAGCAAGTAGT
sooosssssssssooss
1329
1346
747

672585
TACTGTGAGAGCAAGTAG
sooosssssssssooss
1330
1347
748

672586
GTACTGTGAGAGCAAGTA
sooosssssssssooss
1331
1348
749

672587
AGTACTGTGAGAGCAAGT
sooosssssssssooss
1332
1349
750

672588
GAGTACTGTGAGAGCAAG
sooosssssssssooss
1333
1350
751

672589
CGAGTACTGTGAGAGCAA
sooosssssssssooss
1334
1351
752

672590
GCGAGTACTGTGAGAGCA
sooosssssssssooss
1335
1352
753

672591
AGCGAGTACTGTGAGAGC
sooosssssssssooss
1336
1353
754

672592
CAGCGAGTACTGTGAGAG
sooosssssssssooss
1337
1354
755

672593
TCAGCGAGTACTGTGAGA
sooosssssssssooss
1338
1355
756

672594
CTCAGCGAGTACTGTGAG
sooosssssssssooss
1339
1356
757

672595
CCTCAGCGAGTACTGTGA
sooosssssssssooss
1340
1357
758

672596
CCCTCAGCGAGTACTGTG
sooosssssssssooss
1341
1358
759

672597
ACCCTCAGCGAGTACTGT
sooosssssssssooss
1342
1359
760

672598
CACCCTCAGCGAGTACTG
sooosssssssssooss
1343
1360
761

672599
TCACCCTCAGCGAGTACT
sooosssssssssooss
1344
1361
762

672600
TTCACCCTCAGCGAGTAC
sooosssssssssooss
1345
1362
763

672601
GTTCACCCTCAGCGAGTA
sooosssssssssooss
1346
1363
764

672602
TGTTCACCCTCAGCGAGT
sooosssssssssooss
1347
1364
765

672603
TTGTTCACCCTCAGCGAG
sooosssssssssooss
1348
1365
766

672604
CTTGTTCACCCTCAGCGA
sooosssssssssooss
1349
1366
767

672605
TCTTGTTCACCCTCAGCG
sooosssssssssooss
1350
1367
768

672606
TTCTTGTTCACCCTCAGC
sooosssssssssooss
1351
1368
769

672607
TTTCTTGTTCACCCTCAG
sooosssssssssooss
1352
1369
770

672608
TTTTCTTGTTCACCCTCA
sooosssssssssooss
1353
1370
771

672609
CTTTTCTTGTTCACCCTC
sooosssssssssooss
1354
1371
772

672610
TCTTTTCTTGTTCACCCT
sooosssssssssooss
1355
1372
773

672611
GTCTTTTCTTGTTCACCC
sooosssssssssooss
1356
1373
774

672612
GGTCTTTTCTTGTTCACC
sooosssssssssooss
1357
1374
775

672613
AGGTCTTTTCTTGTTCAC
sooosssssssssooss
1358
1375
776

672614
CAGGTCTTTTCTTGTTCA
sooosssssssssooss
1359
1376
777

672615
TCAGGTCTTTTCTTGTTC
sooosssssssssooss
1360
1377
778

672616
ATCAGGTCTTTTCTTGTT
sooosssssssssooss
1361
1378
779

672617
TATCAGGTCTTTTCTTGT
sooosssssssssooss
1362
1379
780

672618
TTATCAGGTCTTTTCTTG
sooosssssssssooss
1363
1380
781

672619
TTTATCAGGTCTTTTCTT
sooosssssssssooss
1364
1381
782

672620
AATCTTTATCAGGTCTTT
sooosssssssssooss
1368
1385
783

672621
TAATCTTTATCAGGTCTT
sooosssssssssooss
1369
1386
784

672622
TTAATCTTTATCAGGTCT
sooosssssssssooss
1370
1387
785

672623
GTTAATCTTTATCAGGTC
sooosssssssssooss
1371
1388
786

672624
GGTTAATCTTTATCAGGT
sooosssssssssooss
1372
1389
787

672625
TGGTTAATCTTTATCAGG
sooosssssssssooss
1373
1390
788

672626
CTGGTTAATCTTTATCAG
sooosssssssssooss
1374
1391
789

672627
TCTGGTTAATCTTTATCA
sooosssssssssooss
1375
1392
790

672628
TTCTGGTTAATCTTTATC
sooosssssssssooss
1376
1393
791

672629
TCCCTCCTTGTTTTCTTC
sooosssssssssooss
1391
1408
792

672630
TTTCCCTCCTTGTTTTCT
sooosssssssssooss
1393
1410
793

672631
GTTTCCCTCCTTGTTTTC
sooosssssssssooss
1394
1411
794

672632
TGTTTCCCTCCTTGTTTT
sooosssssssssooss
1395
1412
795

672633
TTGTTTCCCTCCTTGTTT
sooosssssssssooss
1396
1413
796

672634
GTTGTTTCCCTCCTTGTT
sooosssssssssooss
1397
1414
797

672635
GGTTGTTTCCCTCCTTGT
sooosssssssssooss
1398
1415
798

672636
CGGTTGTTTCCCTCCTTG
sooosssssssssooss
1399
1416
799

672637
GCGGTTGTTTCCCTCCTT
sooosssssssssooss
1400
1417
800

672638
TGCGGTTGTTTCCCTCCT
sooosssssssssooss
1401
1418
801

672639
CTGCGGTTGTTTCCCTCC
sooosssssssssooss
1402
1419
802

672640
GCTGCGGTTGTTTCCCTC
sooosssssssssooss
1403
1420
803

672641
GGCTGCGGTTGTTTCCCT
sooosssssssssooss
1404
1421
804

672642
AGGCTGCGGTTGTTTCCC
sooosssssssssooss
1405
1422
805

672643
CAGGCTGCGGTTGTTTCC
sooosssssssssooss
1406
1423
806

672644
ACAGGCTGCGGTTGTTTC
sooosssssssssooss
1407
1424
807

672645
TACAGGCTGCGGTTGTTT
sooosssssssssooss
1408
1425
808

672646
CTACAGGCTGCGGTTGTT
sooosssssssssooss
1409
1426
809

672647
GCTACAGGCTGCGGTTGT
sooosssssssssooss
1410
1427
810

672648
TGCTACAGGCTGCGGTTG
sooosssssssssooss
1411
1428
811

672649
TTGCTACAGGCTGCGGTT
sooosssssssssooss
1412
1429
812

672650
CTTGCTACAGGCTGCGGT
sooosssssssssooss
1413
1430
813

672651
GCTTGCTACAGGCTGCGG
sooosssssssssooss
1414
1431
814

672652
AGCTTGCTACAGGCTGCG
sooosssssssssooss
1415
1432
815

672653
GAGCTTGCTACAGGCTGC
sooosssssssssooss
1416
1433
816

672654
AGAGCTTGCTACAGGCTG
sooosssssssssooss
1417
1434
817

672655
CAGAGCTTGCTACAGGCT
sooosssssssssooss
1418
1435
818

672656
CCAGAGCTTGCTACAGGC
sooosssssssssooss
1419
1436
819

672657
TCCAGAGCTTGCTACAGG
sooosssssssssooss
1420
1437
820

672658
TTCCAGAGCTTGCTACAG
sooosssssssssooss
1421
1438
821

672659
GTTCCAGAGCTTGCTACA
sooosssssssssooss
1422
1439
822

672660
AGTTCCAGAGCTTGCTAC
sooosssssssssooss
1423
1440
823

672661
GAGTTCCAGAGCTTGCTA
sooosssssssssooss
1424
1441
824

672662
TGAGTTCCAGAGCTTGCT
sooosssssssssooss
1425
1442
825

672663
CTGAGTTCCAGAGCTTGC
sooosssssssssooss
1426
1443
826

672664
CCTGAGTTCCAGAGCTTG
sooosssssssssooss
1427
1444
827

672665
TCCTGAGTTCCAGAGCTT
sooosssssssssooss
1428
1445
828

672666
CTCCTGAGTTCCAGAGCT
sooosssssssssooss
1429
1446
829

672667
ACTCCTGAGTTCCAGAGC
sooosssssssssooss
1430
1447
830

672668
GACTCCTGAGTTCCAGAG
sooosssssssssooss
1431
1448
831

672669
CGACTCCTGAGTTCCAGA
sooosssssssssooss
1432
1449
832

672670
GCGACTCCTGAGTTCCAG
sooosssssssssooss
1433
1450
833

672671
CGCGACTCCTGAGTTCCA
sooosssssssssooss
1434
1451
834

672672
GCGCGACTCCTGAGTTCC
sooosssssssssooss
1435
1452
835

672673
CGCGCGACTCCTGAGTTC
sooosssssssssooss
1436
1453
836

672674
GCGCGCGACTCCTGAGTT
sooosssssssssooss
1437
1454
837

672675
AGCGCGCGACTCCTGAGT
sooosssssssssooss
1438
1455
838

672676
TAGCGCGCGACTCCTGAG
sooosssssssssooss
1439
1456
839

672677
CTAGCGCGCGACTCCTGA
sooosssssssssooss
1440
1457
840

672678
CCTAGCGCGCGACTCCTG
sooosssssssssooss
1441
1458
841

672679
CCCTAGCGCGCGACTCCT
sooosssssssssooss
1442
1459
842

672680
CCCCTAGCGCGCGACTCC
sooosssssssssooss
1443
1460
843

672681
GCCCCTAGCGCGCGACTC
sooosssssssssooss
1444
1461
844

672682
GGCCCCTAGCGCGCGACT
sooosssssssssooss
1445
1462
845

672683
CGGCCCCTAGCGCGCGAC
sooosssssssssooss
1446
1463
846

672684
CCGGCCCCTAGCGCGCGA
sooosssssssssooss
1447
1464
847

672685
CCCGGCCCCTAGCGCGCG
sooosssssssssooss
1448
1465
848

672686
CCCCGGCCCCTAGCGCGC
sooosssssssssooss
1449
1466
849

672687
GCCCCGGCCCCTAGCGCG
sooosssssssssooss
1450
1467
850

672688
GGCCCCGGCCCCTAGCGC
sooosssssssssooss
1451
1468
851

672689
CGGCCCCGGCCCCTAGCG
sooosssssssssooss
1452
1469
852

672690
CCGGCCCCGGCCCCTAGC
sooosssssssssooss
1453
1470
853

672691
CCCGGCCCCGGCCCCTAG
sooosssssssssooss
1454
1471
854

672692
CCCCGGCCCCGGCCCCTA
sooosssssssssooss
1455
1472
855

672693
ACGCCCCGGCCCCGGCCC
sooosssssssssooss
1464
1481
856

672694
CACGCCCCGGCCCCGGCC
sooosssssssssooss
1465
1482
857

672695
CCACGCCCCGGCCCCGGC
sooosssssssssooss
1466
1483
858

672696
ACCACGCCCCGGCCCCGG
sooosssssssssooss
1467
1484
859

672697
GACCACGCCCCGGCCCCG
sooosssssssssooss
1468
1485
860

672698
CGACCACGCCCCGGCCCC
sooosssssssssooss
1469
1486
861

672699
CCGACCACGCCCCGGCCC
sooosssssssssooss
1470
1487
862

672700
CCCGACCACGCCCCGGCC
sooosssssssssooss
1471
1488
863

672701
CCCCGACCACGCCCCGGC
sooosssssssssooss
1472
1489
864

672702
GCCCCGACCACGCCCCGG
sooosssssssssooss
1473
1490
865

672703
CGCCCCGACCACGCCCCG
sooosssssssssooss
1474
1491
866

672704
CCGCCCCGACCACGCCCC
sooosssssssssooss
1475
1492
867

672705
CCCGCCCCGACCACGCCC
sooosssssssssooss
1476
1493
868

672706
GCCCGCCCCGACCACGCC
sooosssssssssooss
1477
1494
869

672707
GGCCCGCCCCGACCACGC
sooosssssssssooss
1478
1495
870

672708
GGGCCCGCCCCGACCACG
sooosssssssssooss
1479
1496
871

672709
CGGGCCCGCCCCGACCAC
sooosssssssssooss
1480
1497
872

672710
CCGGGCCCGCCCCGACCA
sooosssssssssooss
1481
1498
873

672711
CCCGGGCCCGCCCCGACC
sooosssssssssooss
1482
1499
874

672712
CCCCGGGCCCGCCCCGAC
sooosssssssssooss
1483
1500
875

672713
AGCCCCGCCCCGGGCCCG
sooosssssssssooss
1502
1519
876

672714
CAGCCCCGCCCCGGGCCC
sooosssssssssooss
1503
1520
877

672715
GCAGCCCCGCCCCGGGCC
sooosssssssssooss
1504
1521
878

672716
CGCAGCCCCGCCCCGGGC
sooosssssssssooss
1505
1522
879

672717
CCGCAGCCCCGCCCCGGG
sooosssssssssooss
1506
1523
880

672718
ACCGCAGCCCCGCCCCGG
sooosssssssssooss
1507
1524
881

672719
AACCGCAGCCCCGCCCCG
sooosssssssssooss
1508
1525
882

672720
CAACCGCAGCCCCGCCCC
sooosssssssssooss
1509
1526
883

672721
GCAACCGCAGCCCCGCCC
sooosssssssssooss
1510
1527
884

672722
CGCAACCGCAGCCCCGCC
sooosssssssssooss
1511
1528
885

672723
CCGCAACCGCAGCCCCGC
sooosssssssssooss
1512
1529
886

672724
ACCGCAACCGCAGCCCCG
sooosssssssssooss
1513
1530
887

672725
CACCGCAACCGCAGCCCC
sooosssssssssooss
1514
1531
888

672726
GCACCGCAACCGCAGCCC
sooosssssssssooss
1515
1532
889

672727
GGCACCGCAACCGCAGCC
sooosssssssssooss
1516
1533
890

672728
AGGCACCGCAACCGCAGC
sooosssssssssooss
1517
1534
891

672729
CAGGCACCGCAACCGCAG
sooosssssssssooss
1518
1535
892

672730
GCAGGCACCGCAACCGCA
sooosssssssssooss
1519
1536
893

672731
CGCAGGCACCGCAACCGC
sooosssssssssooss
1520
1537
894

672732
GCGCAGGCACCGCAACCG
sooosssssssssooss
1521
1538
895

672733
GGCGCAGGCACCGCAACC
sooosssssssssooss
1522
1539
896

672734
GGGCGCAGGCACCGCAAC
sooosssssssssooss
1523
1540
897

TABLE 24

Deoxy, MOE and cEt oligonucleotides targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO:
NO:

2
2
SEQ

ISIS

Start
Stop
ID

NO
Sequence
Chemistry
Linkage
Site
Site
NO

672735
TGAGAGCAAGTAGTGGG
eeekk-d7-kkeee
soosssssssssooss
1326
1342
898

672736
GTGAGAGCAAGTAGTGG
eeekk-d7-kkeee
soosssssssssooss
1327
1343
899

672737
TGTGAGAGCAAGTAGTG
eeekk-d7-kkeee
soosssssssssooss
1328
1344
900

672738
CTGTGAGAGCAAGTAGT
eeekk-d7-kkeee
soosssssssssooss
1329
1345
901

672739
ACTGTGAGAGCAAGTAG
eeekk-d7-kkeee
soosssssssssooss
1330
1346
902

672740
TACTGTGAGAGCAAGTA
eeekk-d7-kkeee
soosssssssssooss
1331
1347
903

672741
GTACTGTGAGAGCAAGT
eeekk-d7-kkeee
soosssssssssooss
1332
1348
904

672742
AGTACTGTGAGAGCAAG
eeekk-d7-kkeee
soosssssssssooss
1333
1349
905

672743
GAGTACTGTGAGAGCAA
eeekk-d7-kkeee
soosssssssssooss
1334
1350
906

672744
CGAGTACTGTGAGAGCA
eeekk-d7-kkeee
soosssssssssooss
1335
1351
907

672745
GCGAGTACTGTGAGAGC
eeekk-d7-kkeee
soosssssssssooss
1336
1352
908

672746
AGCGAGTACTGTGAGAG
eeekk-d7-kkeee
soosssssssssooss
1337
1353
909

672747
CAGCGAGTACTGTGAGA
eeekk-d7-kkeee
soosssssssssooss
1338
1354
910

672748
TCAGCGAGTACTGTGAG
eeekk-d7-kkeee
soosssssssssooss
1339
1355
911

672749
CTCAGCGAGTACTGTGA
eeekk-d7-kkeee
soosssssssssooss
1340
1356
912

672750
CCTCAGCGAGTACTGTG
eeekk-d7-kkeee
soosssssssssooss
1341
1357
913

672751
CCCTCAGCGAGTACTGT
eeekk-d7-kkeee
soosssssssssooss
1342
1358
914

672752
ACCCTCAGCGAGTACTG
eeekk-d7-kkeee
soosssssssssooss
1343
1359
915

672753
CACCCTCAGCGAGTACT
eeekk-d7-kkeee
soosssssssssooss
1344
1360
916

672754
TCACCCTCAGCGAGTAC
eeekk-d7-kkeee
soosssssssssooss
1345
1361
917

672755
TTCACCCTCAGCGAGTA
eeekk-d7-kkeee
soosssssssssooss
1346
1362
918

672756
GTTCACCCTCAGCGAGT
eeekk-d7-kkeee
soosssssssssooss
1347
1363
919

672757
TGTTCACCCTCAGCGAG
eeekk-d7-kkeee
soosssssssssooss
1348
1364
920

672758
TTGTTCACCCTCAGCGA
eeekk-d7-kkeee
soosssssssssooss
1349
1365
921

672759
CTTGTTCACCCTCAGCG
eeekk-d7-kkeee
soosssssssssooss
1350
1366
922

672760
TCTTGTTCACCCTCAGC
eeekk-d7-kkeee
soosssssssssooss
1351
1367
923

672761
TTCTTGTTCACCCTCAG
eeekk-d7-kkeee
soosssssssssooss
1352
1368
924

672762
TTTCTTGTTCACCCTCA
eeekk-d7-kkeee
soosssssssssooss
1353
1369
925

672763
TTTTCTTGTTCACCCTC
eeekk-d7-kkeee
soosssssssssooss
1354
1370
926

672764
CTTTTCTTGTTCACCCT
eeekk-d7-kkeee
soosssssssssooss
1355
1371
927

672765
TCTTTTCTTGTTCACCC
eeekk-d7-kkeee
soosssssssssooss
1356
1372
928

672766
GTCTTTTCTTGTTCACC
eeekk-d7-kkeee
soosssssssssooss
1357
1373
929

672767
GGTCTTTTCTTGTTCAC
eeekk-d7-kkeee
soosssssssssooss
1358
1374
930

672768
AGGTCTTTTCTTGTTCA
eeekk-d7-kkeee
soosssssssssooss
1359
1375
931

672769
CAGGTCTTTTCTTGTTC
eeekk-d7-kkeee
soosssssssssooss
1360
1376
932

672770
TCAGGTCTTTTCTTGTT
eeekk-d7-kkeee
soosssssssssooss
1361
1377
933

672771
ATCAGGTCTTTTCTTGT
eeekk-d7-kkeee
soosssssssssooss
1362
1378
934

672772
TATCAGGTCTTTTCTTG
eeekk-d7-kkeee
soosssssssssooss
1363
1379
935

672773
TTATCAGGTCTTTTCTT
eeekk-d7-kkeee
soosssssssssooss
1364
1380
936

672774
ATCTTTATCAGGTCTTT
eeekk-d7-kkeee
soosssssssssooss
1368
1384
937

672775
AATCTTTATCAGGTCTT
eeekk-d7-kkeee
soosssssssssooss
1369
1385
938

672776
TAATCTTTATCAGGTCT
eeekk-d7-kkeee
soosssssssssooss
1370
1386
939

672777
TTAATCTTTATCAGGTC
eeekk-d7-kkeee
soosssssssssooss
1371
1387
940

672778
GTTAATCTTTATCAGGT
eeekk-d7-kkeee
soosssssssssooss
1372
1388
941

672779
GGTTAATCTTTATCAGG
eeekk-d7-kkeee
soosssssssssooss
1373
1389
942

672780
TGGTTAATCTTTATCAG
eeekk-d7-kkeee
soosssssssssooss
1374
1390
943

672781
CTGGTTAATCTTTATCA
eeekk-d7-kkeee
soosssssssssooss
1375
1391
944

672782
TCTGGTTAATCTTTATC
eeekk-d7-kkeee
soosssssssssooss
1376
1392
945

672783
CCCTCCTTGTTTTCTTC
eeekk-d7-kkeee
soosssssssssooss
1391
1407
946

672784
TCCCTCCTTGTTTTCTT
eeekk-d7-kkeee
soosssssssssooss
1392
1408
947

672785
TTCCCTCCTTGTTTTCT
eeekk-d7-kkeee
soosssssssssooss
1393
1409
948

672786
TTTCCCTCCTTGTTTTC
eeekk-d7-kkeee
soosssssssssooss
1394
1410
949

672787
GTTTCCCTCCTTGTTTT
eeekk-d7-kkeee
soosssssssssooss
1395
1411
950

672788
TGTTTCCCTCCTTGTTT
eeekk-d7-kkeee
soosssssssssooss
1396
1412
951

672789
TTGTTTCCCTCCTTGTT
eeekk-d7-kkeee
soosssssssssooss
1397
1413
952

672790
GGTTGTTTCCCTCCTTG
eeekk-d7-kkeee
soosssssssssooss
1399
1415
953

672791
CGGTTGTTTCCCTCCTT
eeekk-d7-kkeee
soosssssssssooss
1400
1416
954

672792
GCGGTTGTTTCCCTCCT
eeekk-d7-kkeee
soosssssssssooss
1401
1417
955

672793
TGCGGTTGTTTCCCTCC
eeekk-d7-kkeee
soosssssssssooss
1402
1418
956

672794
CTGCGGTTGTTTCCCTC
eeekk-d7-kkeee
soosssssssssooss
1403
1419
957

672795
GCTGCGGTTGTTTCCCT
eeekk-d7-kkeee
soosssssssssooss
1404
1420
958

672796
GGCTGCGGTTGTTTCCC
eeekk-d7-kkeee
soosssssssssooss
1405
1421
959

672797
AGGCTGCGGTTGTTTCC
eeekk-d7-kkeee
soosssssssssooss
1406
1422
960

672798
CAGGCTGCGGTTGTTTC
eeekk-d7-kkeee
soosssssssssooss
1407
1423
961

672799
ACAGGCTGCGGTTGTTT
eeekk-d7-kkeee
soosssssssssooss
1408
1424
962

672800
TACAGGCTGCGGTTGTT
eeekk-d7-kkeee
soosssssssssooss
1409
1425
963

672801
CTACAGGCTGCGGTTGT
eeekk-d7-kkeee
soosssssssssooss
1410
1426
964

672802
GCTACAGGCTGCGGTTG
eeekk-d7-kkeee
soosssssssssooss
1411
1427
965

672803
TGCTACAGGCTGCGGTT
eeekk-d7-kkeee
soosssssssssooss
1412
1428
966

672804
TTGCTACAGGCTGCGGT
eeekk-d7-kkeee
soosssssssssooss
1413
1429
967

672805
CTTGCTACAGGCTGCGG
eeekk-d7-kkeee
soosssssssssooss
1414
1430
968

672806
GCTTGCTACAGGCTGCG
eeekk-d7-kkeee
soosssssssssooss
1415
1431
969

672807
AGCTTGCTACAGGCTGC
eeekk-d7-kkeee
soosssssssssooss
1416
1432
970

672808
GAGCTTGCTACAGGCTG
eeekk-d7-kkeee
soosssssssssooss
1417
1433
971

672809
AGAGCTTGCTACAGGCT
eeekk-d7-kkeee
soosssssssssooss
1418
1434
972

672810
CAGAGCTTGCTACAGGC
eeekk-d7-kkeee
soosssssssssooss
1419
1435
973

672811
CCAGAGCTTGCTACAGG
eeekk-d7-kkeee
soosssssssssooss
1420
1436
974

672812
TCCAGAGCTTGCTACAG
eeekk-d7-kkeee
soosssssssssooss
1421
1437
975

672813
TTCCAGAGCTTGCTACA
eeekk-d7-kkeee
soosssssssssooss
1422
1438
976

672814
GTTCCAGAGCTTGCTAC
eeekk-d7-kkeee
soosssssssssooss
1423
1439
977

672815
AGTTCCAGAGCTTGCTA
eeekk-d7-kkeee
soosssssssssooss
1424
1440
978

672816
GAGTTCCAGAGCTTGCT
eeekk-d7-kkeee
soosssssssssooss
1425
1441
979

672817
TGAGTTCCAGAGCTTGC
eeekk-d7-kkeee
soosssssssssooss
1426
1442
980

672818
CTGAGTTCCAGAGCTTG
eeekk-d7-kkeee
soosssssssssooss
1427
1443
981

672819
CCTGAGTTCCAGAGCTT
eeekk-d7-kkeee
soosssssssssooss
1428
1444
982

672820
TCCTGAGTTCCAGAGCT
eeekk-d7-kkeee
soosssssssssooss
1429
1445
983

672821
CTCCTGAGTTCCAGAGC
eeekk-d7-kkeee
soosssssssssooss
1430
1446
984

672822
ACTCCTGAGTTCCAGAG
eeekk-d7-kkeee
soosssssssssooss
1431
1447
985

672823
GACTCCTGAGTTCCAGA
eeekk-d7-kkeee
soosssssssssooss
1432
1448
986

672824
CGACTCCTGAGTTCCAG
eeekk-d7-kkeee
soosssssssssooss
1433
1449
987

672825
GCGACTCCTGAGTTCCA
eeekk-d7-kkeee
soosssssssssooss
1434
1450
988

672826
CGCGACTCCTGAGTTCC
eeekk-d7-kkeee
soosssssssssooss
1435
1451
989

672827
GCGCGACTCCTGAGTTC
eeekk-d7-kkeee
soosssssssssooss
1436
1452
990

672828
CGCGCGACTCCTGAGTT
eeekk-d7-kkeee
soosssssssssooss
1437
1453
991

672829
GCGCGCGACTCCTGAGT
eeekk-d7-kkeee
soosssssssssooss
1438
1454
992

672830
AGCGCGCGACTCCTGAG
eeekk-d7-kkeee
soosssssssssooss
1439
1455
993

672831
TAGCGCGCGACTCCTGA
eeekk-d7-kkeee
soosssssssssooss
1440
1456
994

672832
CTAGCGCGCGACTCCTG
eeekk-d7-kkeee
soosssssssssooss
1441
1457
995

672833
CCTAGCGCGCGACTCCT
eeekk-d7-kkeee
soosssssssssooss
1442
1458
996

672834
CCCTAGCGCGCGACTCC
eeekk-d7-kkeee
soosssssssssooss
1443
1459
997

672835
CCCCTAGCGCGCGACTC
eeekk-d7-kkeee
soosssssssssooss
1444
1460
998

672836
GCCCCTAGCGCGCGACT
eeekk-d7-kkeee
soosssssssssooss
1445
1461
999

672837
GGCCCCTAGCGCGCGAC
eeekk-d7-kkeee
soosssssssssooss
1446
1462
1000

672838
CGGCCCCTAGCGCGCGA
eeekk-d7-kkeee
soosssssssssooss
1447
1463
1001

672839
CCGGCCCCTAGCGCGCG
eeekk-d7-kkeee
soosssssssssooss
1448
1464
1002

672840
CCCGGCCCCTAGCGCGC
eeekk-d7-kkeee
soosssssssssooss
1449
1465
1003

672841
CCCCGGCCCCTAGCGCG
eeekk-d7-kkeee
soosssssssssooss
1450
1466
1004

672842
GCCCCGGCCCCTAGCGC
eeekk-d7-kkeee
soosssssssssooss
1451
1467
1005

672843
GGCCCCGGCCCCTAGCG
eeekk-d7-kkeee
soosssssssssooss
1452
1468
1006

672844
CGGCCCCGGCCCCTAGC
eeekk-d7-kkeee
soosssssssssooss
1453
1469
1007

672845
CCGGCCCCGGCCCCTAG
eeekk-d7-kkeee
soosssssssssooss
1454
1470
1008

672846
CCCGGCCCCGGCCCCTA
eeekk-d7-kkeee
soosssssssssooss
1455
1471
1009

672847
ACGCCCCGGCCCCGGCC
eeekk-d7-kkeee
soosssssssssooss
1465
1481
1010

672848
CACGCCCCGGCCCCGGC
eeekk-d7-kkeee
soosssssssssooss
1466
1482
1011

672849
CCACGCCCCGGCCCCGG
eeekk-d7-kkeee
soosssssssssooss
1467
1483
1012

672850
ACCACGCCCCGGCCCCG
eeekk-d7-kkeee
soosssssssssooss
1468
1484
1013

672851
GACCACGCCCCGGCCCC
eeekk-d7-kkeee
soosssssssssooss
1469
1485
1014

672852
CGACCACGCCCCGGCCC
eeekk-d7-kkeee
soosssssssssooss
1470
1486
1015

672853
CCGACCACGCCCCGGCC
eeekk-d7-kkeee
soosssssssssooss
1471
1487
1016

672854
CCCGACCACGCCCCGGC
eeekk-d7-kkeee
soosssssssssooss
1472
1488
1017

672855
CCCCGACCACGCCCCGG
eeekk-d7-kkeee
soosssssssssooss
1473
1489
1018

672856
GCCCCGACCACGCCCCG
eeekk-d7-kkeee
soosssssssssooss
1474
1490
1019

672857
CGCCCCGACCACGCCCC
eeekk-d7-kkeee
soosssssssssooss
1475
1491
1020

672858
CCGCCCCGACCACGCCC
eeekk-d7-kkeee
soosssssssssooss
1476
1492
1021

672859
CCCGCCCCGACCACGCC
eeekk-d7-kkeee
soosssssssssooss
1477
1493
1022

672860
GCCCGCCCCGACCACGC
eeekk-d7-kkeee
soosssssssssooss
1478
1494
1023

672861
GGCCCGCCCCGACCACG
eeekk-d7-kkeee
soosssssssssooss
1479
1495
1024

672862
GGGCCCGCCCCGACCAC
eeekk-d7-kkeee
soosssssssssooss
1480
1496
1025

672863
CGGGCCCGCCCCGACCA
eeekk-d7-kkeee
soosssssssssooss
1481
1497
1026

672864
CCGGGCCCGCCCCGACC
eeekk-d7-kkeee
soosssssssssooss
1482
1498
1027

672865
CCCGGGCCCGCCCCGAC
eeekk-d7-kkeee
soosssssssssooss
1483
1499
1028

672866
GCAGCCCCGCCCCGGGC
eeekk-d7-kkeee
soosssssssssooss
1505
1521
1029

672867
CGCAGCCCCGCCCCGGG
eeekk-d7-kkeee
soosssssssssooss
1506
1522
1030

672868
CCGCAGCCCCGCCCCGG
eeekk-d7-kkeee
soosssssssssooss
1507
1523
1031

672869
ACCGCAGCCCCGCCCCG
eeekk-d7-kkeee
soosssssssssooss
1508
1524
1032

672870
AACCGCAGCCCCGCCCC
eeekk-d7-kkeee
soosssssssssooss
1509
1525
1033

672871
CAACCGCAGCCCCGCCC
eeekk-d7-kkeee
soosssssssssooss
1510
1526
1034

672872
GCAACCGCAGCCCCGCC
eeekk-d7-kkeee
soosssssssssooss
1511
1527
1035

672873
CGCAACCGCAGCCCCGC
eeekk-d7-kkeee
soosssssssssooss
1512
1528
1036

672874
CCGCAACCGCAGCCCCG
eeekk-d7-kkeee
soosssssssssooss
1513
1529
1037

672875
ACCGCAACCGCAGCCCC
eeekk-d7-kkeee
soosssssssssooss
1514
1530
1038

672876
CACCGCAACCGCAGCCC
eeekk-d7-kkeee
soosssssssssooss
1515
1531
1039

672877
GCACCGCAACCGCAGCC
eeekk-d7-kkeee
soosssssssssooss
1516
1532
1040

672878
GGCACCGCAACCGCAGC
eeekk-d7-kkeee
soosssssssssooss
1517
1533
1041

672879
AGGCACCGCAACCGCAG
eeekk-d7-kkeee
soosssssssssooss
1518
1534
1042

672880
CAGGCACCGCAACCGCA
eeekk-d7-kkeee
soosssssssssooss
1519
1535
1043

672881
GCAGGCACCGCAACCGC
eeekk-d7-kkeee
soosssssssssooss
1520
1536
1044

672882
CGCAGGCACCGCAACCG
eeekk-d7-kkeee
soosssssssssooss
1521
1537
1045

672883
GCGCAGGCACCGCAACC
eeekk-d7-kkeee
soosssssssssooss
1522
1538
1046

672884
GGCGCAGGCACCGCAAC
eeekk-d7-kkeee
soosssssssssooss
1523
1539
1047

TABLE 25

Deoxy, MOE and cEt oligonucleotides targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO:
NO:

2
2
SEQ

ISIS

Start
Stop
ID

NO
Sequence
Chemistry
Linkage
Site
Site
NO

672885
TGAGAGCAAGTAGTGGG
eekk-d8-kkeee
soosssssssssooss
1326
1342
898

672886
GTGAGAGCAAGTAGTGG
eekk-d8-kkeee
soosssssssssooss
1327
1343
899

672887
TGTGAGAGCAAGTAGTG
eekk-d8-kkeee
soosssssssssooss
1328
1344
900

672888
CTGTGAGAGCAAGTAGT
eekk-d8-kkeee
soosssssssssooss
1329
1345
901

672889
ACTGTGAGAGCAAGTAG
eekk-d8-kkeee
soosssssssssooss
1330
1346
902

672890
TACTGTGAGAGCAAGTA
eekk-d8-kkeee
soosssssssssooss
1331
1347
903

672891
GTACTGTGAGAGCAAGT
eekk-d8-kkeee
soosssssssssooss
1332
1348
904

672892
AGTACTGTGAGAGCAAG
eekk-d8-kkeee
soosssssssssooss
1333
1349
905

672893
GAGTACTGTGAGAGCAA
eekk-d8-kkeee
soosssssssssooss
1334
1350
906

672894
CGAGTACTGTGAGAGCA
eekk-d8-kkeee
soosssssssssooss
1335
1351
907

672895
GCGAGTACTGTGAGAGC
eekk-d8-kkeee
soosssssssssooss
1336
1352
908

672896
AGCGAGTACTGTGAGAG
eekk-d8-kkeee
soosssssssssooss
1337
1353
909

672897
CAGCGAGTACTGTGAGA
eekk-d8-kkeee
soosssssssssooss
1338
1354
910

672898
TCAGCGAGTACTGTGAG
eekk-d8-kkeee
soosssssssssooss
1339
1355
911

672899
CTCAGCGAGTACTGTGA
eekk-d8-kkeee
soosssssssssooss
1340
1356
912

672900
CCTCAGCGAGTACTGTG
eekk-d8-kkeee
soosssssssssooss
1341
1357
913

672901
CCCTCAGCGAGTACTGT
eekk-d8-kkeee
soosssssssssooss
1342
1358
914

672902
ACCCTCAGCGAGTACTG
eekk-d8-kkeee
soosssssssssooss
1343
1359
915

672903
CACCCTCAGCGAGTACT
eekk-d8-kkeee
soosssssssssooss
1344
1360
916

672904
TCACCCTCAGCGAGTAC
eekk-d8-kkeee
soosssssssssooss
1345
1361
917

672905
TTCACCCTCAGCGAGTA
eekk-d8-kkeee
soosssssssssooss
1346
1362
918

672906
GTTCACCCTCAGCGAGT
eekk-d8-kkeee
soosssssssssooss
1347
1363
919

672907
TGTTCACCCTCAGCGAG
eekk-d8-kkeee
soosssssssssooss
1348
1364
920

672908
TTGTTCACCCTCAGCGA
eekk-d8-kkeee
soosssssssssooss
1349
1365
921

672909
CTTGTTCACCCTCAGCG
eekk-d8-kkeee
soosssssssssooss
1350
1366
922

672910
TCTTGTTCACCCTCAGC
eekk-d8-kkeee
soosssssssssooss
1351
1367
923

672911
TTCTTGTTCACCCTCAG
eekk-d8-kkeee
soosssssssssooss
1352
1368
924

672912
TTTCTTGTTCACCCTCA
eekk-d8-kkeee
soosssssssssooss
1353
1369
925

672913
TTTTCTTGTTCACCCTC
eekk-d8-kkeee
soosssssssssooss
1354
1370
926

672914
CTTTTCTTGTTCACCCT
eekk-d8-kkeee
soosssssssssooss
1355
1371
927

672915
TCTTTTCTTGTTCACCC
eekk-d8-kkeee
soosssssssssooss
1356
1372
928

672916
GTCTTTTCTTGTTCACC
eekk-d8-kkeee
soosssssssssooss
1357
1373
929

672917
GGTCTTTTCTTGTTCAC
eekk-d8-kkeee
soosssssssssooss
1358
1374
930

672918
AGGTCTTTTCTTGTTCA
eekk-d8-kkeee
soosssssssssooss
1359
1375
931

672919
CAGGTCTTTTCTTGTTC
eekk-d8-kkeee
soosssssssssooss
1360
1376
932

672920
TCAGGTCTTTTCTTGTT
eekk-d8-kkeee
soosssssssssooss
1361
1377
933

672921
ATCAGGTCTTTTCTTGT
eekk-d8-kkeee
soosssssssssooss
1362
1378
934

672922
TATCAGGTCTTTTCTTG
eekk-d8-kkeee
soosssssssssooss
1363
1379
935

672923
TTATCAGGTCTTTTCTT
eekk-d8-kkeee
soosssssssssooss
1364
1380
936

672924
ATCTTTATCAGGTCTTT
eekk-d8-kkeee
soosssssssssooss
1368
1384
937

672925
AATCTTTATCAGGTCTT
eekk-d8-kkeee
soosssssssssooss
1369
1385
938

672926
TAATCTTTATCAGGTCT
eekk-d8-kkeee
soosssssssssooss
1370
1386
939

672927
TTAATCTTTATCAGGTC
eekk-d8-kkeee
soosssssssssooss
1371
1387
940

672928
GTTAATCTTTATCAGGT
eekk-d8-kkeee
soosssssssssooss
1372
1388
941

672929
GGTTAATCTTTATCAGG
eekk-d8-kkeee
soosssssssssooss
1373
1389
942

672930
TGGTTAATCTTTATCAG
eekk-d8-kkeee
soosssssssssooss
1374
1390
943

672931
CTGGTTAATCTTTATCA
eekk-d8-kkeee
soosssssssssooss
1375
1391
944

672932
TCTGGTTAATCTTTATC
eekk-d8-kkeee
soosssssssssooss
1376
1392
945

672933
CCCTCCTTGTTTTCTTC
eekk-d8-kkeee
soosssssssssooss
1391
1407
946

672934
TCCCTCCTTGTTTTCTT
eekk-d8-kkeee
soosssssssssooss
1392
1408
947

672935
TTCCCTCCTTGTTTTCT
eekk-d8-kkeee
soosssssssssooss
1393
1409
948

672936
TTTCCCTCCTTGTTTTC
eekk-d8-kkeee
soosssssssssooss
1394
1410
949

672937
GTTTCCCTCCTTGTTTT
eekk-d8-kkeee
soosssssssssooss
1395
1411
950

672938
TGTTTCCCTCCTTGTTT
eekk-d8-kkeee
soosssssssssooss
1396
1412
951

672939
TTGTTTCCCTCCTTGTT
eekk-d8-kkeee
soosssssssssooss
1397
1413
952

672940
GGTTGTTTCCCTCCTTG
eekk-d8-kkeee
soosssssssssooss
1399
1415
953

672941
CGGTTGTTTCCCTCCTT
eekk-d8-kkeee
soosssssssssooss
1400
1416
954

672942
GCGGTTGTTTCCCTCCT
eekk-d8-kkeee
soosssssssssooss
1401
1417
955

672943
TGCGGTTGTTTCCCTCC
eekk-d8-kkeee
soosssssssssooss
1402
1418
956

672944
CTGCGGTTGTTTCCCTC
eekk-d8-kkeee
soosssssssssooss
1403
1419
957

672945
GCTGCGGTTGTTTCCCT
eekk-d8-kkeee
soosssssssssooss
1404
1420
958

672946
GGCTGCGGTTGTTTCCC
eekk-d8-kkeee
soosssssssssooss
1405
1421
959

672947
AGGCTGCGGTTGTTTCC
eekk-d8-kkeee
soosssssssssooss
1406
1422
960

672948
CAGGCTGCGGTTGTTTC
eekk-d8-kkeee
soosssssssssooss
1407
1423
961

672949
ACAGGCTGCGGTTGTTT
eekk-d8-kkeee
soosssssssssooss
1408
1424
962

672950
TACAGGCTGCGGTTGTT
eekk-d8-kkeee
soosssssssssooss
1409
1425
963

672951
CTACAGGCTGCGGTTGT
eekk-d8-kkeee
soosssssssssooss
1410
1426
964

672952
GCTACAGGCTGCGGTTG
eekk-d8-kkeee
soosssssssssooss
1411
1427
965

672953
TGCTACAGGCTGCGGTT
eekk-d8-kkeee
soosssssssssooss
1412
1428
966

672954
TTGCTACAGGCTGCGGT
eekk-d8-kkeee
soosssssssssooss
1413
1429
967

672955
CTTGCTACAGGCTGCGG
eekk-d8-kkeee
soosssssssssooss
1414
1430
968

672956
GCTTGCTACAGGCTGCG
eekk-d8-kkeee
soosssssssssooss
1415
1431
969

672957
AGCTTGCTACAGGCTGC
eekk-d8-kkeee
soosssssssssooss
1416
1432
970

672958
GAGCTTGCTACAGGCTG
eekk-d8-kkeee
soosssssssssooss
1417
1433
971

672959
AGAGCTTGCTACAGGCT
eekk-d8-kkeee
soosssssssssooss
1418
1434
972

672960
CAGAGCTTGCTACAGGC
eekk-d8-kkeee
soosssssssssooss
1419
1435
973

672961
CCAGAGCTTGCTACAGG
eekk-d8-kkeee
soosssssssssooss
1420
1436
974

672962
TCCAGAGCTTGCTACAG
eekk-d8-kkeee
soosssssssssooss
1421
1437
975

672963
TTCCAGAGCTTGCTACA
eekk-d8-kkeee
soosssssssssooss
1422
1438
976

672964
GTTCCAGAGCTTGCTAC
eekk-d8-kkeee
soosssssssssooss
1423
1439
977

672965
AGTTCCAGAGCTTGCTA
eekk-d8-kkeee
soosssssssssooss
1424
1440
978

672966
GAGTTCCAGAGCTTGCT
eekk-d8-kkeee
soosssssssssooss
1425
1441
979

672967
TGAGTTCCAGAGCTTGC
eekk-d8-kkeee
soosssssssssooss
1426
1442
980

672968
CTGAGTTCCAGAGCTTG
eekk-d8-kkeee
soosssssssssooss
1427
1443
981

672969
CCTGAGTTCCAGAGCTT
eekk-d8-kkeee
soosssssssssooss
1428
1444
982

672970
TCCTGAGTTCCAGAGCT
eekk-d8-kkeee
soosssssssssooss
1429
1445
983

672971
CTCCTGAGTTCCAGAGC
eekk-d8-kkeee
soosssssssssooss
1430
1446
984

672972
ACTCCTGAGTTCCAGAG
eekk-d8-kkeee
soosssssssssooss
1431
1447
985

672973
GACTCCTGAGTTCCAGA
eekk-d8-kkeee
soosssssssssooss
1432
1448
986

672974
CGACTCCTGAGTTCCAG
eekk-d8-kkeee
soosssssssssooss
1433
1449
987

672975
GCGACTCCTGAGTTCCA
eekk-d8-kkeee
soosssssssssooss
1434
1450
988

672976
CGCGACTCCTGAGTTCC
eekk-d8-kkeee
soosssssssssooss
1435
1451
989

672977
GCGCGACTCCTGAGTTC
eekk-d8-kkeee
soosssssssssooss
1436
1452
990

672978
CGCGCGACTCCTGAGTT
eekk-d8-kkeee
soosssssssssooss
1437
1453
991

672979
GCGCGCGACTCCTGAGT
eekk-d8-kkeee
soosssssssssooss
1438
1454
992

672980
AGCGCGCGACTCCTGAG
eekk-d8-kkeee
soosssssssssooss
1439
1455
993

672981
TAGCGCGCGACTCCTGA
eekk-d8-kkeee
soosssssssssooss
1440
1456
994

672982
CTAGCGCGCGACTCCTG
eekk-d8-kkeee
soosssssssssooss
1441
1457
995

672983
CCTAGCGCGCGACTCCT
eekk-d8-kkeee
soosssssssssooss
1442
1458
996

672984
CCCTAGCGCGCGACTCC
eekk-d8-kkeee
soosssssssssooss
1443
1459
997

672985
CCCCTAGCGCGCGACTC
eekk-d8-kkeee
soosssssssssooss
1444
1460
998

672986
GCCCCTAGCGCGCGACT
eekk-d8-kkeee
soosssssssssooss
1445
1461
999

672987
GGCCCCTAGCGCGCGAC
eekk-d8-kkeee
soosssssssssooss
1446
1462
1000

672988
CGGCCCCTAGCGCGCGA
eekk-d8-kkeee
soosssssssssooss
1447
1463
1001

672989
CCGGCCCCTAGCGCGCG
eekk-d8-kkeee
soosssssssssooss
1448
1464
1002

672990
CCCGGCCCCTAGCGCGC
eekk-d8-kkeee
soosssssssssooss
1449
1465
1003

672991
CCCCGGCCCCTAGCGCG
eekk-d8-kkeee
soosssssssssooss
1450
1466
1004

672992
GCCCCGGCCCCTAGCGC
eekk-d8-kkeee
soosssssssssooss
1451
1467
1005

672993
GGCCCCGGCCCCTAGCG
eekk-d8-kkeee
soosssssssssooss
1452
1468
1006

672994
CGGCCCCGGCCCCTAGC
eekk-d8-kkeee
soosssssssssooss
1453
1469
1007

672995
CCGGCCCCGGCCCCTAG
eekk-d8-kkeee
soosssssssssooss
1454
1470
1008

672996
CCCGGCCCCGGCCCCTA
eekk-d8-kkeee
soosssssssssooss
1455
1471
1009

672997
ACGCCCCGGCCCCGGCC
eekk-d8-kkeee
soosssssssssooss
1465
1481
1010

672998
CACGCCCCGGCCCCGGC
eekk-d8-kkeee
soosssssssssooss
1466
1482
1011

672999
CCACGCCCCGGCCCCGG
eekk-d8-kkeee
soosssssssssooss
1467
1483
1012

673000
ACCACGCCCCGGCCCCG
eekk-d8-kkeee
soosssssssssooss
1468
1484
1013

673001
GACCACGCCCCGGCCCC
eekk-d8-kkeee
soosssssssssooss
1469
1485
1014

673002
CGACCACGCCCCGGCCC
eekk-d8-kkeee
soosssssssssooss
1470
1486
1015

673003
CCGACCACGCCCCGGCC
eekk-d8-kkeee
soosssssssssooss
1471
1487
1016

673004
CCCGACCACGCCCCGGC
eekk-d8-kkeee
soosssssssssooss
1472
1488
1017

673005
CCCCGACCACGCCCCGG
eekk-d8-kkeee
soosssssssssooss
1473
1489
1018

673006
GCCCCGACCACGCCCCG
eekk-d8-kkeee
soosssssssssooss
1474
1490
1019

673007
CGCCCCGACCACGCCCC
eekk-d8-kkeee
soosssssssssooss
1475
1491
1020

673008
CCGCCCCGACCACGCCC
eekk-d8-kkeee
soosssssssssooss
1476
1492
1021

673009
CCCGCCCCGACCACGCC
eekk-d8-kkeee
soosssssssssooss
1477
1493
1022

673010
GCCCGCCCCGACCACGC
eekk-d8-kkeee
soosssssssssooss
1478
1494
1023

673011
GGCCCGCCCCGACCACG
eekk-d8-kkeee
soosssssssssooss
1479
1495
1024

673012
GGGCCCGCCCCGACCAC
eekk-d8-kkeee
soosssssssssooss
1480
1496
1025

673013
CGGGCCCGCCCCGACCA
eekk-d8-kkeee
soosssssssssooss
1481
1497
1026

673014
CCGGGCCCGCCCCGACC
eekk-d8-kkeee
soosssssssssooss
1482
1498
1027

673015
CCCGGGCCCGCCCCGAC
eekk-d8-kkeee
soosssssssssooss
1483
1499
1028

673016
GCAGCCCCGCCCCGGGC
eekk-d8-kkeee
soosssssssssooss
1505
1521
1029

673017
CGCAGCCCCGCCCCGGG
eekk-d8-kkeee
soosssssssssooss
1506
1522
1030

673018
CCGCAGCCCCGCCCCGG
eekk-d8-kkeee
soosssssssssooss
1507
1523
1031

673019
ACCGCAGCCCCGCCCCG
eekk-d8-kkeee
soosssssssssooss
1508
1524
1032

673020
AACCGCAGCCCCGCCCC
eekk-d8-kkeee
soosssssssssooss
1509
1525
1033

673021
CAACCGCAGCCCCGCCC
eekk-d8-kkeee
soosssssssssooss
1510
1526
1034

673022
GCAACCGCAGCCCCGCC
eekk-d8-kkeee
soosssssssssooss
1511
1527
1035

673023
CGCAACCGCAGCCCCGC
eekk-d8-kkeee
soosssssssssooss
1512
1528
1036

673024
CCGCAACCGCAGCCCCG
eekk-d8-kkeee
soosssssssssooss
1513
1529
1037

673025
ACCGCAACCGCAGCCCC
eekk-d8-kkeee
soosssssssssooss
1514
1530
1038

673026
CACCGCAACCGCAGCCC
eekk-d8-kkeee
soosssssssssooss
1515
1531
1039

673027
GCACCGCAACCGCAGCC
eekk-d8-kkeee
soosssssssssooss
1516
1532
1040

673028
GGCACCGCAACCGCAGC
eekk-d8-kkeee
soosssssssssooss
1517
1533
1041

673029
AGGCACCGCAACCGCAG
eekk-d8-kkeee
soosssssssssooss
1518
1534
1042

673030
CAGGCACCGCAACCGCA
eekk-d8-kkeee
soosssssssssooss
1519
1535
1043

673031
GCAGGCACCGCAACCGC
eekk-d8-kkeee
soosssssssssooss
1520
1536
1044

673032
CGCAGGCACCGCAACCG
eekk-d8-kkeee
soosssssssssooss
1521
1537
1045

673033
GCGCAGGCACCGCAACC
eekk-d8-kkeee
soosssssssssooss
1522
1538
1046

673034
GGCGCAGGCACCGCAAC
eekk-d8-kkeee
soosssssssssooss
1523
1539
1047

TABLE 26

Deoxy, MOE and cEt oligonucleotides targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO:
NO:

2
2
SEQ

ISIS

Start
Stop
ID

NO
Sequence
Chemistry
Linkage
Site
Site
NO

673035
TGAGAGCAAGTAGTGGG
ek-d8-ekekeee
sosssssssssoooss
1326
1342
898

673036
GTGAGAGCAAGTAGTGG
ek-d8-ekekeee
sosssssssssoooss
1327
1343
899

673037
TGTGAGAGCAAGTAGTG
ek-d8-ekekeee
sosssssssssoooss
1328
1344
900

673038
CTGTGAGAGCAAGTAGT
ek-d8-ekekeee
sosssssssssoooss
1329
1345
901

673039
ACTGTGAGAGCAAGTAG
ek-d8-ekekeee
sosssssssssoooss
1330
1346
902

673040
TACTGTGAGAGCAAGTA
ek-d8-ekekeee
sosssssssssoooss
1331
1347
903

673041
GTACTGTGAGAGCAAGT
ek-d8-ekekeee
sosssssssssoooss
1332
1348
904

673042
AGTACTGTGAGAGCAAG
ek-d8-ekekeee
sosssssssssoooss
1333
1349
905

673043
GAGTACTGTGAGAGCAA
ek-d8-ekekeee
sosssssssssoooss
1334
1350
906

673044
CGAGTACTGTGAGAGCA
ek-d8-ekekeee
sosssssssssoooss
1335
1351
907

673045
GCGAGTACTGTGAGAGC
ek-d8-ekekeee
sosssssssssoooss
1336
1352
908

673046
AGCGAGTACTGTGAGAG
ek-d8-ekekeee
sosssssssssoooss
1337
1353
909

673047
CAGCGAGTACTGTGAGA
ek-d8-ekekeee
sosssssssssoooss
1338
1354
910

673048
TCAGCGAGTACTGTGAG
ek-d8-ekekeee
sosssssssssoooss
1339
1355
911

673049
CTCAGCGAGTACTGTGA
ek-d8-ekekeee
sosssssssssoooss
1340
1356
912

673050
CCTCAGCGAGTACTGTG
ek-d8-ekekeee
sosssssssssoooss
1341
1357
913

673051
CCCTCAGCGAGTACTGT
ek-d8-ekekeee
sosssssssssoooss
1342
1358
914

673052
ACCCTCAGCGAGTACTG
ek-d8-ekekeee
sosssssssssoooss
1343
1359
915

673053
CACCCTCAGCGAGTACT
ek-d8-ekekeee
sosssssssssoooss
1344
1360
916

673054
TCACCCTCAGCGAGTAC
ek-d8-ekekeee
sosssssssssoooss
1345
1361
917

673055
TTCACCCTCAGCGAGTA
ek-d8-ekekeee
sosssssssssoooss
1346
1362
918

673056
GTTCACCCTCAGCGAGT
ek-d8-ekekeee
sosssssssssoooss
1347
1363
919

673057
TGTTCACCCTCAGCGAG
ek-d8-ekekeee
sosssssssssoooss
1348
1364
920

673058
TTGTTCACCCTCAGCGA
ek-d8-ekekeee
sosssssssssoooss
1349
1365
921

673059
CTTGTTCACCCTCAGCG
ek-d8-ekekeee
sosssssssssoooss
1350
1366
922

673060
TCTTGTTCACCCTCAGC
ek-d8-ekekeee
sosssssssssoooss
1351
1367
923

673061
TTCTTGTTCACCCTCAG
ek-d8-ekekeee
sosssssssssoooss
1352
1368
924

673062
TTTCTTGTTCACCCTCA
ek-d8-ekekeee
sosssssssssoooss
1353
1369
925

673063
TTTTCTTGTTCACCCTC
ek-d8-ekekeee
sosssssssssoooss
1354
1370
926

673064
CTTTTCTTGTTCACCCT
ek-d8-ekekeee
sosssssssssoooss
1355
1371
927

673065
TCTTTTCTTGTTCACCC
ek-d8-ekekeee
sosssssssssoooss
1356
1372
928

673066
GTCTTTTCTTGTTCACC
ek-d8-ekekeee
sosssssssssoooss
1357
1373
929

673067
GGTCTTTTCTTGTTCAC
ek-d8-ekekeee
sosssssssssoooss
1358
1374
930

673068
AGGTCTTTTCTTGTTCA
ek-d8-ekekeee
sosssssssssoooss
1359
1375
931

673069
CAGGTCTTTTCTTGTTC
ek-d8-ekekeee
sosssssssssoooss
1360
1376
932

673070
TCAGGTCTTTTCTTGTT
ek-d8-ekekeee
sosssssssssoooss
1361
1377
933

673071
ATCAGGTCTTTTCTTGT
ek-d8-ekekeee
sosssssssssoooss
1362
1378
934

673072
TATCAGGTCTTTTCTTG
ek-d8-ekekeee
sosssssssssoooss
1363
1379
935

673073
TTATCAGGTCTTTTCTT
ek-d8-ekekeee
sosssssssssoooss
1364
1380
936

673074
ATCTTTATCAGGTCTTT
ek-d8-ekekeee
sosssssssssoooss
1368
1384
937

673075
AATCTTTATCAGGTCTT
ek-d8-ekekeee
sosssssssssoooss
1369
1385
938

673076
TAATCTTTATCAGGTCT
ek-d8-ekekeee
sosssssssssoooss
1370
1386
939

673077
TTAATCTTTATCAGGTC
ek-d8-ekekeee
sosssssssssoooss
1371
1387
940

673078
GTTAATCTTTATCAGGT
ek-d8-ekekeee
sosssssssssoooss
1372
1388
941

673079
GGTTAATCTTTATCAGG
ek-d8-ekekeee
sosssssssssoooss
1373
1389
942

673080
TGGTTAATCTTTATCAG
ek-d8-ekekeee
sosssssssssoooss
1374
1390
943

673081
CTGGTTAATCTTTATCA
ek-d8-ekekeee
sosssssssssoooss
1375
1391
944

673082
TCTGGTTAATCTTTATC
ek-d8-ekekeee
sosssssssssoooss
1376
1392
945

673083
CCCTCCTTGTTTTCTTC
ek-d8-ekekeee
sosssssssssoooss
1391
1407
946

673084
TCCCTCCTTGTTTTCTT
ek-d8-ekekeee
sosssssssssoooss
1392
1408
947

673085
TTCCCTCCTTGTTTTCT
ek-d8-ekekeee
sosssssssssoooss
1393
1409
948

673086
TTTCCCTCCTTGTTTTC
ek-d8-ekekeee
sosssssssssoooss
1394
1410
949

673087
GTTTCCCTCCTTGTTTT
ek-d8-ekekeee
sosssssssssoooss
1395
1411
950

673088
TGTTTCCCTCCTTGTTT
ek-d8-ekekeee
sosssssssssoooss
1396
1412
951

673089
TTGTTTCCCTCCTTGTT
ek-d8-ekekeee
sosssssssssoooss
1397
1413
952

673090
GGTTGTTTCCCTCCTTG
ek-d8-ekekeee
sosssssssssoooss
1399
1415
953

673091
CGGTTGTTTCCCTCCTT
ek-d8-ekekeee
sosssssssssoooss
1400
1416
954

673092
GCGGTTGTTTCCCTCCT
ek-d8-ekekeee
sosssssssssoooss
1401
1417
955

673093
TGCGGTTGTTTCCCTCC
ek-d8-ekekeee
sosssssssssoooss
1402
1418
956

673094
CTGCGGTTGTTTCCCTC
ek-d8-ekekeee
sosssssssssoooss
1403
1419
957

673095
GCTGCGGTTGTTTCCCT
ek-d8-ekekeee
sosssssssssoooss
1404
1420
958

673096
GGCTGCGGTTGTTTCCC
ek-d8-ekekeee
sosssssssssoooss
1405
1421
959

673097
AGGCTGCGGTTGTTTCC
ek-d8-ekekeee
sosssssssssoooss
1406
1422
960

673098
CAGGCTGCGGTTGTTTC
ek-d8-ekekeee
sosssssssssoooss
1407
1423
961

673099
ACAGGCTGCGGTTGTTT
ek-d8-ekekeee
sosssssssssoooss
1408
1424
962

673100
TACAGGCTGCGGTTGTT
ek-d8-ekekeee
sosssssssssoooss
1409
1425
963

673101
CTACAGGCTGCGGTTGT
ek-d8-ekekeee
sosssssssssoooss
1410
1426
964

673102
GCTACAGGCTGCGGTTG
ek-d8-ekekeee
sosssssssssoooss
1411
1427
965

673103
TGCTACAGGCTGCGGTT
ek-d8-ekekeee
sosssssssssoooss
1412
1428
966

673104
TTGCTACAGGCTGCGGT
ek-d8-ekekeee
sosssssssssoooss
1413
1429
967

673105
CTTGCTACAGGCTGCGG
ek-d8-ekekeee
sosssssssssoooss
1414
1430
968

673106
GCTTGCTACAGGCTGCG
ek-d8-ekekeee
sosssssssssoooss
1415
1431
969

673107
AGCTTGCTACAGGCTGC
ek-d8-ekekeee
sosssssssssoooss
1416
1432
970

673108
GAGCTTGCTACAGGCTG
ek-d8-ekekeee
sosssssssssoooss
1417
1433
971

673109
AGAGCTTGCTACAGGCT
ek-d8-ekekeee
sosssssssssoooss
1418
1434
972

673110
CAGAGCTTGCTACAGGC
ek-d8-ekekeee
sosssssssssoooss
1419
1435
973

673111
CCAGAGCTTGCTACAGG
ek-d8-ekekeee
sosssssssssoooss
1420
1436
974

673112
TCCAGAGCTTGCTACAG
ek-d8-ekekeee
sosssssssssoooss
1421
1437
975

673113
TTCCAGAGCTTGCTACA
ek-d8-ekekeee
sosssssssssoooss
1422
1438
976

673114
GTTCCAGAGCTTGCTAC
ek-d8-ekekeee
sosssssssssoooss
1423
1439
977

673115
AGTTCCAGAGCTTGCTA
ek-d8-ekekeee
sosssssssssoooss
1424
1440
978

673116
GAGTTCCAGAGCTTGCT
ek-d8-ekekeee
sosssssssssoooss
1425
1441
979

673117
TGAGTTCCAGAGCTTGC
ek-d8-ekekeee
sosssssssssoooss
1426
1442
980

673118
CTGAGTTCCAGAGCTTG
ek-d8-ekekeee
sosssssssssoooss
1427
1443
981

673119
CCTGAGTTCCAGAGCTT
ek-d8-ekekeee
sosssssssssoooss
1428
1444
982

673120
TCCTGAGTTCCAGAGCT
ek-d8-ekekeee
sosssssssssoooss
1429
1445
983

673121
CTCCTGAGTTCCAGAGC
ek-d8-ekekeee
sosssssssssoooss
1430
1446
984

673122
ACTCCTGAGTTCCAGAG
ek-d8-ekekeee
sosssssssssoooss
1431
1447
985

673123
GACTCCTGAGTTCCAGA
ek-d8-ekekeee
sosssssssssoooss
1432
1448
986

673124
CGACTCCTGAGTTCCAG
ek-d8-ekekeee
sosssssssssoooss
1433
1449
987

673125
GCGACTCCTGAGTTCCA
ek-d8-ekekeee
sosssssssssoooss
1434
1450
988

673126
CGCGACTCCTGAGTTCC
ek-d8-ekekeee
sosssssssssoooss
1435
1451
989

673127
GCGCGACTCCTGAGTTC
ek-d8-ekekeee
sosssssssssoooss
1436
1452
990

673128
CGCGCGACTCCTGAGTT
ek-d8-ekekeee
sosssssssssoooss
1437
1453
991

673129
GCGCGCGACTCCTGAGT
ek-d8-ekekeee
sosssssssssoooss
1438
1454
992

673130
AGCGCGCGACTCCTGAG
ek-d8-ekekeee
sosssssssssoooss
1439
1455
993

673131
TAGCGCGCGACTCCTGA
ek-d8-ekekeee
sosssssssssoooss
1440
1456
994

673132
CTAGCGCGCGACTCCTG
ek-d8-ekekeee
sosssssssssoooss
1441
1457
995

673133
CCTAGCGCGCGACTCCT
ek-d8-ekekeee
sosssssssssoooss
1442
1458
996

673134
CCCTAGCGCGCGACTCC
ek-d8-ekekeee
sosssssssssoooss
1443
1459
997

673135
CCCCTAGCGCGCGACTC
ek-d8-ekekeee
sosssssssssoooss
1444
1460
998

673136
GCCCCTAGCGCGCGACT
ek-d8-ekekeee
sosssssssssoooss
1445
1461
999

673137
GGCCCCTAGCGCGCGAC
ek-d8-ekekeee
sosssssssssoooss
1446
1462
1000

673138
CGGCCCCTAGCGCGCGA
ek-d8-ekekeee
sosssssssssoooss
1447
1463
1001

673139
CCGGCCCCTAGCGCGCG
ek-d8-ekekeee
sosssssssssoooss
1448
1464
1002

673140
CCCGGCCCCTAGCGCGC
ek-d8-ekekeee
sosssssssssoooss
1449
1465
1003

673141
CCCCGGCCCCTAGCGCG
ek-d8-ekekeee
sosssssssssoooss
1450
1466
1004

673142
GCCCCGGCCCCTAGCGC
ek-d8-ekekeee
sosssssssssoooss
1451
1467
1005

673143
GGCCCCGGCCCCTAGCG
ek-d8-ekekeee
sosssssssssoooss
1452
1468
1006

673144
CGGCCCCGGCCCCTAGC
ek-d8-ekekeee
sosssssssssoooss
1453
1469
1007

673145
CCGGCCCCGGCCCCTAG
ek-d8-ekekeee
sosssssssssoooss
1454
1470
1008

673146
CCCGGCCCCGGCCCCTA
ek-d8-ekekeee
sosssssssssoooss
1455
1471
1009

673147
ACGCCCCGGCCCCGGCC
ek-d8-ekekeee
sosssssssssoooss
1465
1481
1010

673148
CACGCCCCGGCCCCGGC
ek-d8-ekekeee
sosssssssssoooss
1466
1482
1011

673149
CCACGCCCCGGCCCCGG
ek-d8-ekekeee
sosssssssssoooss
1467
1483
1012

673150
ACCACGCCCCGGCCCCG
ek-d8-ekekeee
sosssssssssoooss
1468
1484
1013

673151
GACCACGCCCCGGCCCC
ek-d8-ekekeee
sosssssssssoooss
1469
1485
1014

673152
CGACCACGCCCCGGCCC
ek-d8-ekekeee
sosssssssssoooss
1470
1486
1015

673153
CCGACCACGCCCCGGCC
ek-d8-ekekeee
sosssssssssoooss
1471
1487
1016

673154
CCCGACCACGCCCCGGC
ek-d8-ekekeee
sosssssssssoooss
1472
1488
1017

673155
CCCCGACCACGCCCCGG
ek-d8-ekekeee
sosssssssssoooss
1473
1489
1018

673156
GCCCCGACCACGCCCCG
ek-d8-ekekeee
sosssssssssoooss
1474
1490
1019

673157
CGCCCCGACCACGCCCC
ek-d8-ekekeee
sosssssssssoooss
1475
1491
1020

673158
CCGCCCCGACCACGCCC
ek-d8-ekekeee
sosssssssssoooss
1476
1492
1021

673159
CCCGCCCCGACCACGCC
ek-d8-ekekeee
sosssssssssoooss
1477
1493
1022

673160
GCCCGCCCCGACCACGC
ek-d8-ekekeee
sosssssssssoooss
1478
1494
1023

673161
GGCCCGCCCCGACCACG
ek-d8-ekekeee
sosssssssssoooss
1479
1495
1024

673162
GGGCCCGCCCCGACCAC
ek-d8-ekekeee
sosssssssssoooss
1480
1496
1025

673163
CGGGCCCGCCCCGACCA
ek-d8-ekekeee
sosssssssssoooss
1481
1497
1026

673164
CCGGGCCCGCCCCGACC
ek-d8-ekekeee
sosssssssssoooss
1482
1498
1027

673165
CCCGGGCCCGCCCCGAC
ek-d8-ekekeee
sosssssssssoooss
1483
1499
1028

673166
GCAGCCCCGCCCCGGGC
ek-d8-ekekeee
sosssssssssoooss
1505
1521
1029

673167
CGCAGCCCCGCCCCGGG
ek-d8-ekekeee
sosssssssssoooss
1506
1522
1030

673168
CCGCAGCCCCGCCCCGG
ek-d8-ekekeee
sosssssssssoooss
1507
1523
1031

673169
ACCGCAGCCCCGCCCCG
ek-d8-ekekeee
sosssssssssoooss
1508
1524
1032

673170
AACCGCAGCCCCGCCCC
ek-d8-ekekeee
sosssssssssoooss
1509
1525
1033

673171
CAACCGCAGCCCCGCCC
ek-d8-ekekeee
sosssssssssoooss
1510
1526
1034

673172
GCAACCGCAGCCCCGCC
ek-d8-ekekeee
sosssssssssoooss
1511
1527
1035

673173
CGCAACCGCAGCCCCGC
ek-d8-ekekeee
sosssssssssoooss
1512
1528
1036

673174
CCGCAACCGCAGCCCCG
ek-d8-ekekeee
sosssssssssoooss
1513
1529
1037

673175
ACCGCAACCGCAGCCCC
ek-d8-ekekeee
sosssssssssoooss
1514
1530
1038

673176
CACCGCAACCGCAGCCC
ek-d8-ekekeee
sosssssssssoooss
1515
1531
1039

673177
GCACCGCAACCGCAGCC
ek-d8-ekekeee
sosssssssssoooss
1516
1532
1040

673178
GGCACCGCAACCGCAGC
ek-d8-ekekeee
sosssssssssoooss
1517
1533
1041

673179
AGGCACCGCAACCGCAG
ek-d8-ekekeee
sosssssssssoooss
1518
1534
1042

673180
CAGGCACCGCAACCGCA
ek-d8-ekekeee
sosssssssssoooss
1519
1535
1043

673181
GCAGGCACCGCAACCGC
ek-d8-ekekeee
sosssssssssoooss
1520
1536
1044

673182
CGCAGGCACCGCAACCG
ek-d8-ekekeee
sosssssssssoooss
1521
1537
1045

673183
GCGCAGGCACCGCAACC
ek-d8-ekekeee
sosssssssssoooss
1522
1538
1046

673184
GGCGCAGGCACCGCAAC
ek-d8-ekekeee
sosssssssssoooss
1523
1539
1047

TABLE 27

Deoxy, MOE and cEt oligonucleotides targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO:
NO:

2
2
SEQ

ISIS

Start
Stop
ID

NO
Sequence
Chemistry
Linkage
Site
Site
NO

673185
TGAGAGCAAGTAGTGGG
keke-d8-ekeke
soosssssssssooss
1326
1342
898

673186
GTGAGAGCAAGTAGTGG
keke-d8-ekeke
soosssssssssooss
1327
1343
899

673187
TGTGAGAGCAAGTAGTG
keke-d8-ekeke
soosssssssssooss
1328
1344
900

673188
CTGTGAGAGCAAGTAGT
keke-d8-ekeke
soosssssssssooss
1329
1345
901

673189
ACTGTGAGAGCAAGTAG
keke-d8-ekeke
soosssssssssooss
1330
1346
902

673190
TACTGTGAGAGCAAGTA
keke-d8-ekeke
soosssssssssooss
1331
1347
903

673191
GTACTGTGAGAGCAAGT
keke-d8-ekeke
soosssssssssooss
1332
1348
904

673192
AGTACTGTGAGAGCAAG
keke-d8-ekeke
soosssssssssooss
1333
1349
905

673193
GAGTACTGTGAGAGCAA
keke-d8-ekeke
soosssssssssooss
1334
1350
906

673194
CGAGTACTGTGAGAGCA
keke-d8-ekeke
soosssssssssooss
1335
1351
907

673195
GCGAGTACTGTGAGAGC
keke-d8-ekeke
soosssssssssooss
1336
1352
908

673196
AGCGAGTACTGTGAGAG
keke-d8-ekeke
soosssssssssooss
1337
1353
909

673197
CAGCGAGTACTGTGAGA
keke-d8-ekeke
soosssssssssooss
1338
1354
910

673198
TCAGCGAGTACTGTGAG
keke-d8-ekeke
soosssssssssooss
1339
1355
911

673199
CTCAGCGAGTACTGTGA
keke-d8-ekeke
soosssssssssooss
1340
1356
912

673200
CCTCAGCGAGTACTGTG
keke-d8-ekeke
soosssssssssooss
1341
1357
913

673201
CCCTCAGCGAGTACTGT
keke-d8-ekeke
soosssssssssooss
1342
1358
914

673202
ACCCTCAGCGAGTACTG
keke-d8-ekeke
soosssssssssooss
1343
1359
915

673203
CACCCTCAGCGAGTACT
keke-d8-ekeke
soosssssssssooss
1344
1360
916

673204
TCACCCTCAGCGAGTAC
keke-d8-ekeke
soosssssssssooss
1345
1361
917

673205
TTCACCCTCAGCGAGTA
keke-d8-ekeke
soosssssssssooss
1346
1362
918

673206
GTTCACCCTCAGCGAGT
keke-d8-ekeke
soosssssssssooss
1347
1363
919

673207
TGTTCACCCTCAGCGAG
keke-d8-ekeke
soosssssssssooss
1348
1364
920

673208
TTGTTCACCCTCAGCGA
keke-d8-ekeke
soosssssssssooss
1349
1365
921

673209
CTTGTTCACCCTCAGCG
keke-d8-ekeke
soosssssssssooss
1350
1366
922

673210
TCTTGTTCACCCTCAGC
keke-d8-ekeke
soosssssssssooss
1351
1367
923

673211
TTCTTGTTCACCCTCAG
keke-d8-ekeke
soosssssssssooss
1352
1368
924

673212
TTTCTTGTTCACCCTCA
keke-d8-ekeke
soosssssssssooss
1353
1369
925

673213
TTTTCTTGTTCACCCTC
keke-d8-ekeke
soosssssssssooss
1354
1370
926

673214
CTTTTCTTGTTCACCCT
keke-d8-ekeke
soosssssssssooss
1355
1371
927

673215
TCTTTTCTTGTTCACCC
keke-d8-ekeke
soosssssssssooss
1356
1372
928

673216
GTCTTTTCTTGTTCACC
keke-d8-ekeke
soosssssssssooss
1357
1373
929

673217
GGTCTTTTCTTGTTCAC
keke-d8-ekeke
soosssssssssooss
1358
1374
930

673218
AGGTCTTTTCTTGTTCA
keke-d8-ekeke
soosssssssssooss
1359
1375
931

673219
CAGGTCTTTTCTTGTTC
keke-d8-ekeke
soosssssssssooss
1360
1376
932

673220
TCAGGTCTTTTCTTGTT
keke-d8-ekeke
soosssssssssooss
1361
1377
933

673221
ATCAGGTCTTTTCTTGT
keke-d8-ekeke
soosssssssssooss
1362
1378
934

673222
TATCAGGTCTTTTCTTG
keke-d8-ekeke
soosssssssssooss
1363
1379
935

673223
TTATCAGGTCTTTTCTT
keke-d8-ekeke
soosssssssssooss
1364
1380
936

673224
ATCTTTATCAGGTCTTT
keke-d8-ekeke
soosssssssssooss
1368
1384
937

673225
AATCTTTATCAGGTCTT
keke-d8-ekeke
soosssssssssooss
1369
1385
938

673226
TAATCTTTATCAGGTCT
keke-d8-ekeke
soosssssssssooss
1370
1386
939

673227
TTAATCTTTATCAGGTC
keke-d8-ekeke
soosssssssssooss
1371
1387
940

673228
GTTAATCTTTATCAGGT
keke-d8-ekeke
soosssssssssooss
1372
1388
941

673229
GGTTAATCTTTATCAGG
keke-d8-ekeke
soosssssssssooss
1373
1389
942

673230
TGGTTAATCTTTATCAG
keke-d8-ekeke
soosssssssssooss
1374
1390
943

673231
CTGGTTAATCTTTATCA
keke-d8-ekeke
soosssssssssooss
1375
1391
944

673232
TCTGGTTAATCTTTATC
keke-d8-ekeke
soosssssssssooss
1376
1392
945

673233
CCCTCCTTGTTTTCTTC
keke-d8-ekeke
soosssssssssooss
1391
1407
946

673234
TCCCTCCTTGTTTTCTT
keke-d8-ekeke
soosssssssssooss
1392
1408
947

673235
TTCCCTCCTTGTTTTCT
keke-d8-ekeke
soosssssssssooss
1393
1409
948

673236
TTTCCCTCCTTGTTTTC
keke-d8-ekeke
soosssssssssooss
1394
1410
949

673237
GTTTCCCTCCTTGTTTT
keke-d8-ekeke
soosssssssssooss
1395
1411
950

673238
TGTTTCCCTCCTTGTTT
keke-d8-ekeke
soosssssssssooss
1396
1412
951

673239
TTGTTTCCCTCCTTGTT
keke-d8-ekeke
soosssssssssooss
1397
1413
952

673240
GGTTGTTTCCCTCCTTG
keke-d8-ekeke
soosssssssssooss
1399
1415
953

673241
CGGTTGTTTCCCTCCTT
keke-d8-ekeke
soosssssssssooss
1400
1416
954

673242
GCGGTTGTTTCCCTCCT
keke-d8-ekeke
soosssssssssooss
1401
1417
955

673243
TGCGGTTGTTTCCCTCC
keke-d8-ekeke
soosssssssssooss
1402
1418
956

673244
CTGCGGTTGTTTCCCTC
keke-d8-ekeke
soosssssssssooss
1403
1419
957

673245
GCTGCGGTTGTTTCCCT
keke-d8-ekeke
soosssssssssooss
1404
1420
958

673246
GGCTGCGGTTGTTTCCC
keke-d8-ekeke
soosssssssssooss
1405
1421
959

673247
AGGCTGCGGTTGTTTCC
keke-d8-ekeke
soosssssssssooss
1406
1422
960

673248
CAGGCTGCGGTTGTTTC
keke-d8-ekeke
soosssssssssooss
1407
1423
961

673249
ACAGGCTGCGGTTGTTT
keke-d8-ekeke
soosssssssssooss
1408
1424
962

673250
TACAGGCTGCGGTTGTT
keke-d8-ekeke
soosssssssssooss
1409
1425
963

673251
CTACAGGCTGCGGTTGT
keke-d8-ekeke
soosssssssssooss
1410
1426
964

673252
GCTACAGGCTGCGGTTG
keke-d8-ekeke
soosssssssssooss
1411
1427
965

673253
TGCTACAGGCTGCGGTT
keke-d8-ekeke
soosssssssssooss
1412
1428
966

673254
TTGCTACAGGCTGCGGT
keke-d8-ekeke
soosssssssssooss
1413
1429
967

673255
CTTGCTACAGGCTGCGG
keke-d8-ekeke
soosssssssssooss
1414
1430
968

673256
GCTTGCTACAGGCTGCG
keke-d8-ekeke
soosssssssssooss
1415
1431
969

673257
AGCTTGCTACAGGCTGC
keke-d8-ekeke
soosssssssssooss
1416
1432
970

673258
GAGCTTGCTACAGGCTG
keke-d8-ekeke
soosssssssssooss
1417
1433
971

673259
AGAGCTTGCTACAGGCT
keke-d8-ekeke
soosssssssssooss
1418
1434
972

673260
CAGAGCTTGCTACAGGC
keke-d8-ekeke
soosssssssssooss
1419
1435
973

673261
CCAGAGCTTGCTACAGG
keke-d8-ekeke
soosssssssssooss
1420
1436
974

673262
TCCAGAGCTTGCTACAG
keke-d8-ekeke
soosssssssssooss
1421
1437
975

673263
TTCCAGAGCTTGCTACA
keke-d8-ekeke
soosssssssssooss
1422
1438
976

673264
GTTCCAGAGCTTGCTAC
keke-d8-ekeke
soosssssssssooss
1423
1439
977

673265
AGTTCCAGAGCTTGCTA
keke-d8-ekeke
soosssssssssooss
1424
1440
978

673266
GAGTTCCAGAGCTTGCT
keke-d8-ekeke
soosssssssssooss
1425
1441
979

673267
TGAGTTCCAGAGCTTGC
keke-d8-ekeke
soosssssssssooss
1426
1442
980

673268
CTGAGTTCCAGAGCTTG
keke-d8-ekeke
soosssssssssooss
1427
1443
981

673269
CCTGAGTTCCAGAGCTT
keke-d8-ekeke
soosssssssssooss
1428
1444
982

673270
TCCTGAGTTCCAGAGCT
keke-d8-ekeke
soosssssssssooss
1429
1445
983

673271
CTCCTGAGTTCCAGAGC
keke-d8-ekeke
soosssssssssooss
1430
1446
984

673272
ACTCCTGAGTTCCAGAG
keke-d8-ekeke
soosssssssssooss
1431
1447
985

673273
GACTCCTGAGTTCCAGA
keke-d8-ekeke
soosssssssssooss
1432
1448
986

673274
CGACTCCTGAGTTCCAG
keke-d8-ekeke
soosssssssssooss
1433
1449
987

673275
GCGACTCCTGAGTTCCA
keke-d8-ekeke
soosssssssssooss
1434
1450
988

673276
CGCGACTCCTGAGTTCC
keke-d8-ekeke
soosssssssssooss
1435
1451
989

673277
GCGCGACTCCTGAGTTC
keke-d8-ekeke
soosssssssssooss
1436
1452
990

673278
CGCGCGACTCCTGAGTT
keke-d8-ekeke
soosssssssssooss
1437
1453
991

673279
GCGCGCGACTCCTGAGT
keke-d8-ekeke
soosssssssssooss
1438
1454
992

673280
AGCGCGCGACTCCTGAG
keke-d8-ekeke
soosssssssssooss
1439
1455
993

673281
TAGCGCGCGACTCCTGA
keke-d8-ekeke
soosssssssssooss
1440
1456
994

673282
CTAGCGCGCGACTCCTG
keke-d8-ekeke
soosssssssssooss
1441
1457
995

673283
CCTAGCGCGCGACTCCT
keke-d8-ekeke
soosssssssssooss
1442
1458
996

673284
CCCTAGCGCGCGACTCC
keke-d8-ekeke
soosssssssssooss
1443
1459
997

673285
CCCCTAGCGCGCGACTC
keke-d8-ekeke
soosssssssssooss
1444
1460
998

673286
GCCCCTAGCGCGCGACT
keke-d8-ekeke
soosssssssssooss
1445
1461
999

673287
GGCCCCTAGCGCGCGAC
keke-d8-ekeke
soosssssssssooss
1446
1462
1000

673288
CGGCCCCTAGCGCGCGA
keke-d8-ekeke
soosssssssssooss
1447
1463
1001

673289
CCGGCCCCTAGCGCGCG
keke-d8-ekeke
soosssssssssooss
1448
1464
1002

673290
CCCGGCCCCTAGCGCGC
keke-d8-ekeke
soosssssssssooss
1449
1465
1003

673291
CCCCGGCCCCTAGCGCG
keke-d8-ekeke
soosssssssssooss
1450
1466
1004

673292
GCCCCGGCCCCTAGCGC
keke-d8-ekeke
soosssssssssooss
1451
1467
1005

673293
GGCCCCGGCCCCTAGCG
keke-d8-ekeke
soosssssssssooss
1452
1468
1006

673294
CGGCCCCGGCCCCTAGC
keke-d8-ekeke
soosssssssssooss
1453
1469
1007

673295
CCGGCCCCGGCCCCTAG
keke-d8-ekeke
soosssssssssooss
1454
1470
1008

673296
CCCGGCCCCGGCCCCTA
keke-d8-ekeke
soosssssssssooss
1455
1471
1009

673297
ACGCCCCGGCCCCGGCC
keke-d8-ekeke
soosssssssssooss
1465
1481
1010

673298
CACGCCCCGGCCCCGGC
keke-d8-ekeke
soosssssssssooss
1466
1482
1011

673299
CCACGCCCCGGCCCCGG
keke-d8-ekeke
soosssssssssooss
1467
1483
1012

673300
ACCACGCCCCGGCCCCG
keke-d8-ekeke
soosssssssssooss
1468
1484
1013

673301
GACCACGCCCCGGCCCC
keke-d8-ekeke
soosssssssssooss
1469
1485
1014

673302
CGACCACGCCCCGGCCC
keke-d8-ekeke
soosssssssssooss
1470
1486
1015

673303
CCGACCACGCCCCGGCC
keke-d8-ekeke
soosssssssssooss
1471
1487
1016

673304
CCCGACCACGCCCCGGC
keke-d8-ekeke
soosssssssssooss
1472
1488
1017

673305
CCCCGACCACGCCCCGG
keke-d8-ekeke
soosssssssssooss
1473
1489
1018

673306
GCCCCGACCACGCCCCG
keke-d8-ekeke
soosssssssssooss
1474
1490
1019

673307
CGCCCCGACCACGCCCC
keke-d8-ekeke
soosssssssssooss
1475
1491
1020

673308
CCGCCCCGACCACGCCC
keke-d8-ekeke
soosssssssssooss
1476
1492
1021

673309
CCCGCCCCGACCACGCC
keke-d8-ekeke
soosssssssssooss
1477
1493
1022

673310
GCCCGCCCCGACCACGC
keke-d8-ekeke
soosssssssssooss
1478
1494
1023

673311
GGCCCGCCCCGACCACG
keke-d8-ekeke
soosssssssssooss
1479
1495
1024

673312
GGGCCCGCCCCGACCAC
keke-d8-ekeke
soosssssssssooss
1480
1496
1025

673313
CGGGCCCGCCCCGACCA
keke-d8-ekeke
soosssssssssooss
1481
1497
1026

673314
CCGGGCCCGCCCCGACC
keke-d8-ekeke
soosssssssssooss
1482
1498
1027

673315
CCCGGGCCCGCCCCGAC
keke-d8-ekeke
soosssssssssooss
1483
1499
1028

673316
GCAGCCCCGCCCCGGGC
keke-d8-ekeke
soosssssssssooss
1505
1521
1029

673317
CGCAGCCCCGCCCCGGG
keke-d8-ekeke
soosssssssssooss
1506
1522
1030

673318
CCGCAGCCCCGCCCCGG
keke-d8-ekeke
soosssssssssooss
1507
1523
1031

673319
ACCGCAGCCCCGCCCCG
keke-d8-ekeke
soosssssssssooss
1508
1524
1032

673320
AACCGCAGCCCCGCCCC
keke-d8-ekeke
soosssssssssooss
1509
1525
1033

673321
CAACCGCAGCCCCGCCC
keke-d8-ekeke
soosssssssssooss
1510
1526
1034

673322
GCAACCGCAGCCCCGCC
keke-d8-ekeke
soosssssssssooss
1511
1527
1035

673323
CGCAACCGCAGCCCCGC
keke-d8-ekeke
soosssssssssooss
1512
1528
1036

673324
CCGCAACCGCAGCCCCG
keke-d8-ekeke
soosssssssssooss
1513
1529
1037

673325
ACCGCAACCGCAGCCCC
keke-d8-ekeke
soosssssssssooss
1514
1530
1038

673326
CACCGCAACCGCAGCCC
keke-d8-ekeke
soosssssssssooss
1515
1531
1039

673327
GCACCGCAACCGCAGCC
keke-d8-ekeke
soosssssssssooss
1516
1532
1040

673328
GGCACCGCAACCGCAGC
keke-d8-ekeke
soosssssssssooss
1517
1533
1041

673329
AGGCACCGCAACCGCAG
keke-d8-ekeke
soosssssssssooss
1518
1534
1042

673330
CAGGCACCGCAACCGCA
keke-d8-ekeke
soosssssssssooss
1519
1535
1043

673331
GCAGGCACCGCAACCGC
keke-d8-ekeke
soosssssssssooss
1520
1536
1044

673332
CGCAGGCACCGCAACCG
keke-d8-ekeke
soosssssssssooss
1521
1537
1045

673333
GCGCAGGCACCGCAACC
keke-d8-ekeke
soosssssssssooss
1522
1538
1046

673334
GGCGCAGGCACCGCAAC
keke-d8-ekeke
soosssssssssooss
1523
1539
1047

TABLE 28

Deoxy, MOE and cEt oligonucleotides targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO:
NO:

2
2
SEQ

ISIS

Start
Stop
ID

NO
Sequence
Chemistry
Linkage
Site
Site
NO

673335
TGAGAGCAAGTAGTGGG
ekek-d8-kekee
soosssssssssooss
1326
1342
898

673336
GTGAGAGCAAGTAGTGG
ekek-d8-kekee
soosssssssssooss
1327
1343
899

673337
TGTGAGAGCAAGTAGTG
ekek-d8-kekee
soosssssssssooss
1328
1344
900

673338
CTGTGAGAGCAAGTAGT
ekek-d8-kekee
soosssssssssooss
1329
1345
901

673339
ACTGTGAGAGCAAGTAG
ekek-d8-kekee
soosssssssssooss
1330
1346
902

673340
TACTGTGAGAGCAAGTA
ekek-d8-kekee
soosssssssssooss
1331
1347
903

673341
GTACTGTGAGAGCAAGT
ekek-d8-kekee
soosssssssssooss
1332
1348
904

673342
AGTACTGTGAGAGCAAG
ekek-d8-kekee
soosssssssssooss
1333
1349
905

673343
GAGTACTGTGAGAGCAA
ekek-d8-kekee
soosssssssssooss
1334
1350
906

673344
CGAGTACTGTGAGAGCA
ekek-d8-kekee
soosssssssssooss
1335
1351
907

673345
GCGAGTACTGTGAGAGC
ekek-d8-kekee
soosssssssssooss
1336
1352
908

673346
AGCGAGTACTGTGAGAG
ekek-d8-kekee
soosssssssssooss
1337
1353
909

673347
CAGCGAGTACTGTGAGA
ekek-d8-kekee
soosssssssssooss
1338
1354
910

673348
TCAGCGAGTACTGTGAG
ekek-d8-kekee
soosssssssssooss
1339
1355
911

673349
CTCAGCGAGTACTGTGA
ekek-d8-kekee
soosssssssssooss
1340
1356
912

673350
CCTCAGCGAGTACTGTG
ekek-d8-kekee
soosssssssssooss
1341
1357
913

673351
CCCTCAGCGAGTACTGT
ekek-d8-kekee
soosssssssssooss
1342
1358
914

673352
ACCCTCAGCGAGTACTG
ekek-d8-kekee
soosssssssssooss
1343
1359
915

673353
CACCCTCAGCGAGTACT
ekek-d8-kekee
soosssssssssooss
1344
1360
916

673354
TCACCCTCAGCGAGTAC
ekek-d8-kekee
soosssssssssooss
1345
1361
917

673355
TTCACCCTCAGCGAGTA
ekek-d8-kekee
soosssssssssooss
1346
1362
918

673356
GTTCACCCTCAGCGAGT
ekek-d8-kekee
soosssssssssooss
1347
1363
919

673357
TGTTCACCCTCAGCGAG
ekek-d8-kekee
soosssssssssooss
1348
1364
920

673358
TTGTTCACCCTCAGCGA
ekek-d8-kekee
soosssssssssooss
1349
1365
921

673359
CTTGTTCACCCTCAGCG
ekek-d8-kekee
soosssssssssooss
1350
1366
922

673360
TCTTGTTCACCCTCAGC
ekek-d8-kekee
soosssssssssooss
1351
1367
923

673361
TTCTTGTTCACCCTCAG
ekek-d8-kekee
soosssssssssooss
1352
1368
924

673362
TTTCTTGTTCACCCTCA
ekek-d8-kekee
soosssssssssooss
1353
1369
925

673363
TTTTCTTGTTCACCCTC
ekek-d8-kekee
soosssssssssooss
1354
1370
926

673364
CTTTTCTTGTTCACCCT
ekek-d8-kekee
soosssssssssooss
1355
1371
927

673365
TCTTTTCTTGTTCACCC
ekek-d8-kekee
soosssssssssooss
1356
1372
928

673366
GTCTTTTCTTGTTCACC
ekek-d8-kekee
soosssssssssooss
1357
1373
929

673367
GGTCTTTTCTTGTTCAC
ekek-d8-kekee
soosssssssssooss
1358
1374
930

673368
AGGTCTTTTCTTGTTCA
ekek-d8-kekee
soosssssssssooss
1359
1375
931

673369
CAGGTCTTTTCTTGTTC
ekek-d8-kekee
soosssssssssooss
1360
1376
932

673370
TCAGGTCTTTTCTTGTT
ekek-d8-kekee
soosssssssssooss
1361
1377
933

673371
ATCAGGTCTTTTCTTGT
ekek-d8-kekee
soosssssssssooss
1362
1378
934

673372
TATCAGGTCTTTTCTTG
ekek-d8-kekee
soosssssssssooss
1363
1379
935

673373
TTATCAGGTCTTTTCTT
ekek-d8-kekee
soosssssssssooss
1364
1380
936

673374
ATCTTTATCAGGTCTTT
ekek-d8-kekee
soosssssssssooss
1368
1384
937

673375
AATCTTTATCAGGTCTT
ekek-d8-kekee
soosssssssssooss
1369
1385
938

673376
TAATCTTTATCAGGTCT
ekek-d8-kekee
soosssssssssooss
1370
1386
939

673377
TTAATCTTTATCAGGTC
ekek-d8-kekee
soosssssssssooss
1371
1387
940

673378
GTTAATCTTTATCAGGT
ekek-d8-kekee
soosssssssssooss
1372
1388
941

673379
GGTTAATCTTTATCAGG
ekek-d8-kekee
soosssssssssooss
1373
1389
942

673380
TGGTTAATCTTTATCAG
ekek-d8-kekee
soosssssssssooss
1374
1390
943

673381
CTGGTTAATCTTTATCA
ekek-d8-kekee
soosssssssssooss
1375
1391
944

673382
TCTGGTTAATCTTTATC
ekek-d8-kekee
soosssssssssooss
1376
1392
945

673383
CCCTCCTTGTTTTCTTC
ekek-d8-kekee
soosssssssssooss
1391
1407
946

673384
TCCCTCCTTGTTTTCTT
ekek-d8-kekee
soosssssssssooss
1392
1408
947

673385
TTCCCTCCTTGTTTTCT
ekek-d8-kekee
soosssssssssooss
1393
1409
948

673386
TTTCCCTCCTTGTTTTC
ekek-d8-kekee
soosssssssssooss
1394
1410
949

673387
GTTTCCCTCCTTGTTTT
ekek-d8-kekee
soosssssssssooss
1395
1411
950

673388
TGTTTCCCTCCTTGTTT
ekek-d8-kekee
soosssssssssooss
1396
1412
951

673389
TTGTTTCCCTCCTTGTT
ekek-d8-kekee
soosssssssssooss
1397
1413
952

673390
GGTTGTTTCCCTCCTTG
ekek-d8-kekee
soosssssssssooss
1399
1415
953

673391
CGGTTGTTTCCCTCCTT
ekek-d8-kekee
soosssssssssooss
1400
1416
954

673392
GCGGTTGTTTCCCTCCT
ekek-d8-kekee
soosssssssssooss
1401
1417
955

673393
TGCGGTTGTTTCCCTCC
ekek-d8-kekee
soosssssssssooss
1402
1418
956

673394
CTGCGGTTGTTTCCCTC
ekek-d8-kekee
soosssssssssooss
1403
1419
957

673395
GCTGCGGTTGTTTCCCT
ekek-d8-kekee
soosssssssssooss
1404
1420
958

673396
GGCTGCGGTTGTTTCCC
ekek-d8-kekee
soosssssssssooss
1405
1421
959

673397
AGGCTGCGGTTGTTTCC
ekek-d8-kekee
soosssssssssooss
1406
1422
960

673398
CAGGCTGCGGTTGTTTC
ekek-d8-kekee
soosssssssssooss
1407
1423
961

673399
ACAGGCTGCGGTTGTTT
ekek-d8-kekee
soosssssssssooss
1408
1424
962

673400
TACAGGCTGCGGTTGTT
ekek-d8-kekee
soosssssssssooss
1409
1425
963

673401
CTACAGGCTGCGGTTGT
ekek-d8-kekee
soosssssssssooss
1410
1426
964

673402
GCTACAGGCTGCGGTTG
ekek-d8-kekee
soosssssssssooss
1411
1427
965

673403
TGCTACAGGCTGCGGTT
ekek-d8-kekee
soosssssssssooss
1412
1428
966

673404
TTGCTACAGGCTGCGGT
ekek-d8-kekee
soosssssssssooss
1413
1429
967

673405
CTTGCTACAGGCTGCGG
ekek-d8-kekee
soosssssssssooss
1414
1430
968

673406
GCTTGCTACAGGCTGCG
ekek-d8-kekee
soosssssssssooss
1415
1431
969

673407
AGCTTGCTACAGGCTGC
ekek-d8-kekee
soosssssssssooss
1416
1432
970

673408
GAGCTTGCTACAGGCTG
ekek-d8-kekee
soosssssssssooss
1417
1433
971

673409
AGAGCTTGCTACAGGCT
ekek-d8-kekee
soosssssssssooss
1418
1434
972

673410
CAGAGCTTGCTACAGGC
ekek-d8-kekee
soosssssssssooss
1419
1435
973

673411
CCAGAGCTTGCTACAGG
ekek-d8-kekee
soosssssssssooss
1420
1436
974

673412
TCCAGAGCTTGCTACAG
ekek-d8-kekee
soosssssssssooss
1421
1437
975

673413
TTCCAGAGCTTGCTACA
ekek-d8-kekee
soosssssssssooss
1422
1438
976

673414
GTTCCAGAGCTTGCTAC
ekek-d8-kekee
soosssssssssooss
1423
1439
977

673415
AGTTCCAGAGCTTGCTA
ekek-d8-kekee
soosssssssssooss
1424
1440
978

673416
GAGTTCCAGAGCTTGCT
ekek-d8-kekee
soosssssssssooss
1425
1441
979

673417
TGAGTTCCAGAGCTTGC
ekek-d8-kekee
soosssssssssooss
1426
1442
980

673418
CTGAGTTCCAGAGCTTG
ekek-d8-kekee
soosssssssssooss
1427
1443
981

673419
CCTGAGTTCCAGAGCTT
ekek-d8-kekee
soosssssssssooss
1428
1444
982

673420
TCCTGAGTTCCAGAGCT
ekek-d8-kekee
soosssssssssooss
1429
1445
983

673421
CTCCTGAGTTCCAGAGC
ekek-d8-kekee
soosssssssssooss
1430
1446
984

673422
ACTCCTGAGTTCCAGAG
ekek-d8-kekee
soosssssssssooss
1431
1447
985

673423
GACTCCTGAGTTCCAGA
ekek-d8-kekee
soosssssssssooss
1432
1448
986

673424
CGACTCCTGAGTTCCAG
ekek-d8-kekee
soosssssssssooss
1433
1449
987

673425
GCGACTCCTGAGTTCCA
ekek-d8-kekee
soosssssssssooss
1434
1450
988

673426
CGCGACTCCTGAGTTCC
ekek-d8-kekee
soosssssssssooss
1435
1451
989

673427
GCGCGACTCCTGAGTTC
ekek-d8-kekee
soosssssssssooss
1436
1452
990

673428
CGCGCGACTCCTGAGTT
ekek-d8-kekee
soosssssssssooss
1437
1453
991

673429
GCGCGCGACTCCTGAGT
ekek-d8-kekee
soosssssssssooss
1438
1454
992

673430
AGCGCGCGACTCCTGAG
ekek-d8-kekee
soosssssssssooss
1439
1455
993

673431
TAGCGCGCGACTCCTGA
ekek-d8-kekee
soosssssssssooss
1440
1456
994

673432
CTAGCGCGCGACTCCTG
ekek-d8-kekee
soosssssssssooss
1441
1457
995

673433
CCTAGCGCGCGACTCCT
ekek-d8-kekee
soosssssssssooss
1442
1458
996

673434
CCCTAGCGCGCGACTCC
ekek-d8-kekee
soosssssssssooss
1443
1459
997

673435
CCCCTAGCGCGCGACTC
ekek-d8-kekee
soosssssssssooss
1444
1460
998

673436
GCCCCTAGCGCGCGACT
ekek-d8-kekee
soosssssssssooss
1445
1461
999

673437
GGCCCCTAGCGCGCGAC
ekek-d8-kekee
soosssssssssooss
1446
1462
1000

673438
CGGCCCCTAGCGCGCGA
ekek-d8-kekee
soosssssssssooss
1447
1463
1001

673439
CCGGCCCCTAGCGCGCG
ekek-d8-kekee
soosssssssssooss
1448
1464
1002

673440
CCCGGCCCCTAGCGCGC
ekek-d8-kekee
soosssssssssooss
1449
1465
1003

673441
CCCCGGCCCCTAGCGCG
ekek-d8-kekee
soosssssssssooss
1450
1466
1004

673442
GCCCCGGCCCCTAGCGC
ekek-d8-kekee
soosssssssssooss
1451
1467
1005

673443
GGCCCCGGCCCCTAGCG
ekek-d8-kekee
soosssssssssooss
1452
1468
1006

673444
CGGCCCCGGCCCCTAGC
ekek-d8-kekee
soosssssssssooss
1453
1469
1007

673445
CCGGCCCCGGCCCCTAG
ekek-d8-kekee
soosssssssssooss
1454
1470
1008

673446
CCCGGCCCCGGCCCCTA
ekek-d8-kekee
soosssssssssooss
1455
1471
1009

673447
ACGCCCCGGCCCCGGCC
ekek-d8-kekee
soosssssssssooss
1465
1481
1010

673448
CACGCCCCGGCCCCGGC
ekek-d8-kekee
soosssssssssooss
1466
1482
1011

673449
CCACGCCCCGGCCCCGG
ekek-d8-kekee
soosssssssssooss
1467
1483
1012

673450
ACCACGCCCCGGCCCCG
ekek-d8-kekee
soosssssssssooss
1468
1484
1013

673451
GACCACGCCCCGGCCCC
ekek-d8-kekee
soosssssssssooss
1469
1485
1014

673452
CGACCACGCCCCGGCCC
ekek-d8-kekee
soosssssssssooss
1470
1486
1015

673453
CCGACCACGCCCCGGCC
ekek-d8-kekee
soosssssssssooss
1471
1487
1016

673454
CCCGACCACGCCCCGGC
ekek-d8-kekee
soosssssssssooss
1472
1488
1017

673455
CCCCGACCACGCCCCGG
ekek-d8-kekee
soosssssssssooss
1473
1489
1018

673456
GCCCCGACCACGCCCCG
ekek-d8-kekee
soosssssssssooss
1474
1490
1019

673457
CGCCCCGACCACGCCCC
ekek-d8-kekee
soosssssssssooss
1475
1491
1020

673458
CCGCCCCGACCACGCCC
ekek-d8-kekee
soosssssssssooss
1476
1492
1021

673459
CCCGCCCCGACCACGCC
ekek-d8-kekee
soosssssssssooss
1477
1493
1022

673460
GCCCGCCCCGACCACGC
ekek-d8-kekee
soosssssssssooss
1478
1494
1023

673461
GGCCCGCCCCGACCACG
ekek-d8-kekee
soosssssssssooss
1479
1495
1024

673462
GGGCCCGCCCCGACCAC
ekek-d8-kekee
soosssssssssooss
1480
1496
1025

673463
CGGGCCCGCCCCGACCA
ekek-d8-kekee
soosssssssssooss
1481
1497
1026

673464
CCGGGCCCGCCCCGACC
ekek-d8-kekee
soosssssssssooss
1482
1498
1027

673465
CCCGGGCCCGCCCCGAC
ekek-d8-kekee
soosssssssssooss
1483
1499
1028

673466
GCAGCCCCGCCCCGGGC
ekek-d8-kekee
soosssssssssooss
1505
1521
1029

673467
CGCAGCCCCGCCCCGGG
ekek-d8-kekee
soosssssssssooss
1506
1522
1030

673468
CCGCAGCCCCGCCCCGG
ekek-d8-kekee
soosssssssssooss
1507
1523
1031

673469
ACCGCAGCCCCGCCCCG
ekek-d8-kekee
soosssssssssooss
1508
1524
1032

673470
AACCGCAGCCCCGCCCC
ekek-d8-kekee
soosssssssssooss
1509
1525
1033

673471
CAACCGCAGCCCCGCCC
ekek-d8-kekee
soosssssssssooss
1510
1526
1034

673472
GCAACCGCAGCCCCGCC
ekek-d8-kekee
soosssssssssooss
1511
1527
1035

673473
CGCAACCGCAGCCCCGC
ekek-d8-kekee
soosssssssssooss
1512
1528
1036

673474
CCGCAACCGCAGCCCCG
ekek-d8-kekee
soosssssssssooss
1513
1529
1037

673475
ACCGCAACCGCAGCCCC
ekek-d8-kekee
soosssssssssooss
1514
1530
1038

673476
CACCGCAACCGCAGCCC
ekek-d8-kekee
soosssssssssooss
1515
1531
1039

673477
GCACCGCAACCGCAGCC
ekek-d8-kekee
soosssssssssooss
1516
1532
1040

673478
GGCACCGCAACCGCAGC
ekek-d8-kekee
soosssssssssooss
1517
1533
1041

673479
AGGCACCGCAACCGCAG
ekek-d8-kekee
soosssssssssooss
1518
1534
1042

673480
CAGGCACCGCAACCGCA
ekek-d8-kekee
soosssssssssooss
1519
1535
1043

673481
GCAGGCACCGCAACCGC
ekek-d8-kekee
soosssssssssooss
1520
1536
1044

673482
CGCAGGCACCGCAACCG
ekek-d8-kekee
soosssssssssooss
1521
1537
1045

673483
GCGCAGGCACCGCAACC
ekek-d8-kekee
soosssssssssooss
1522
1538
1046

673484
GGCGCAGGCACCGCAAC
ekek-d8-kekee
soosssssssssooss
1523
1539
1047

TABLE 29

5-10-5 MOE gapmers targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO:
NO:

2
2
SEQ

ISIS

Start
Stop
ID

NO
Sequence
Linkage
Site
Site
NO

653222
CCACTCGCCA
soooossssssssssooss
1553
1572
1048

CCGCCTGCGC

653223
TGCATTCCTA
soooossssssssssooss
5325
5344
1049

AGCAATGTGT

655016
CCCGGCCCCG
soooossssssssssooss
1458
1477
1050

GCCCCGGCCC

655017
CCCCGGCCCC
soooossssssssssooss
1459
1478
1051

GGCCCCGGCC

671081
TTACATCTAT
soooossssssssssooss
8110
8129
1052

AGCACCACTC

671082
TCACTCCCTT
soooossssssssssooss
8140
8159
1053

TTCAGACAAG

671083
AACTAAGTTC
soooossssssssssooss
8230
8249
1054

TGTCTGTGGA

671084
ATACAGGACT
soooossssssssssooss
14316
14335
1055

AAAGTGCTTC

TABLE 30

5-10-5 MOE gapmers targeting SEQ ID NO: 1

SEQ
SEQ

ID
ID

NO:
NO:

1
1
SEQ

ISIS

Start
Stop
ID

NO
Sequence
Linkage
Site
Site
NO

672561
CTCTGACCCT
soooossssssssssooss
695
714
1056

GATCTTCCAT

Example 7: In Vivo Rodent Inhibition and Tolerability with Treatment of C9ORF72 Antisense Oligonucleotides

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.

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).

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

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 anterior-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

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 31. The results indicate that treatment with increasing doses of ISIS 571883 resulted in dose-dependent inhibition of C9ORF72 mRNA expression.

The induction of the microglial marker AIF-1 as a measure of CNS toxicity was also assessed. The data is presented in Table 32. 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.

TABLE 31

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

TABLE 32

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

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

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 33 and 34. 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.

TABLE 33

Latency to fall (sec) in the rotarod assay

Weeks after

ISIS

injection
PBS
571883

0
66
66

4
91
70

8
94
84

TABLE 34

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

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

RNA was extracted from a 2-3 mm brain section posterior to the injection site, 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 35. The results indicate that treatment with increasing doses of ISIS 603538 resulted in dose-dependent inhibition of C9ORF72 mRNA expression.

The induction of the microglial marker AIF-1 as a measure of CNS toxicity was also assessed. The data is presented in Table 36. 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.

TABLE 35

Percentage inhibition of C9ORF72 mRNA

expression compared to the PBS control

Dose
Brain

Spinal cord
Spinal cord

(μg)
(1 mm section)
Cortex
(lumbar)
(cervical)

700
21
4
86
74

1000
53
49
88
82

3000
64
62
88
80

TABLE 36

Percentage expression of AIF-1

mRNA expression compared to the PBS control

Dose
Brain (1 mm

Spinal cord
Spinal cord

(hg)
section)
Cortex
(lumbar)
(cervical)

700
97
119
98
89

1000
105
113
122
96

3000
109
141
156
115

Body Weight Analysis

Body weights of the rats were measured at regular time point intervals. The data is presented in Table 37. The results indicate that treatment with increasing doses of ISIS 603538 did not have any significant changes in the body weights of the rats.

TABLE 37

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 8: Antisense Inhibition of C9ORF72 by 5-8-5 MOE Gapmers with Mixed Backbones and Deoxy, MOE and cEt Antisense Oligonucleotides with Mixed Backbones

Antisense oligonucleotides described in Example 6 hereinabove (see Table 23 and Table 24 hereinabove) were tested in HepG2 cells in a series of experiments that had similar culture conditions. ISIS 576816, previously tested in PCT/US2013/065073 (claiming priority to U.S. Application No. 61/714,132, filed Oct. 15, 2012, was used as a benchmark oligonucleotide for study with deoxy, MOE, and cEt antisense oligonucleotides. The results for each experiment are presented in tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 500 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 RTS3905 was used to measure the C9ORF72 pathogenic associated mRNA variant, which is the product of a pre-mRNA containing a hexanucleotide repeat. The levels of the C9ORF72 pathogenic associated mRNA variant were normalized to the total RNA content of the cell, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells.

TABLE 38

Percent inhibition of the C9ORF72 pathogenic associated mRNA

variant compared to PBS control by 5-8-5 MOE gapmers with mixed

backbones targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO:
NO:

2
2

SEQ

ISIS

Start
Stop
%
ID

NO
Sequence
Linkage
Site
Site
inhibition
NO

672581
GTGAGAGCAAGTAGTGGG
sooosssssssssooss
1326
1343
11
744

672582
TGTGAGAGCAAGTAGTGG
sooosssssssssooss
1327
1344
32
745

672583
CTGTGAGAGCAAGTAGTG
sooosssssssssooss
1328
1345
14
746

672584
ACTGTGAGAGCAAGTAGT
sooosssssssssooss
1329
1346
1
747

672585
TACTGTGAGAGCAAGTAG
sooosssssssssooss
1330
1347
14
748

672586
GTACTGTGAGAGCAAGTA
sooosssssssssooss
1331
1348
22
749

672587
AGTACTGTGAGAGCAAGT
sooosssssssssooss
1332
1349
0
750

672588
GAGTACTGTGAGAGCAAG
sooosssssssssooss
1333
1350
8
751

672589
CGAGTACTGTGAGAGCAA
sooosssssssssooss
1334
1351
15
752

672590
GCGAGTACTGTGAGAGCA
sooosssssssssooss
1335
1352
13
753

672591
AGCGAGTACTGTGAGAGC
sooosssssssssooss
1336
1353
32
754

672592
CAGCGAGTACTGTGAGAG
sooosssssssssooss
1337
1354
39
755

672593
TCAGCGAGTACTGTGAGA
sooosssssssssooss
1338
1355
15
756

672594
CTCAGCGAGTACTGTGAG
sooosssssssssooss
1339
1356
14
757

672595
CCTCAGCGAGTACTGTGA
sooosssssssssooss
1340
1357
40
758

672596
CCCTCAGCGAGTACTGTG
sooosssssssssooss
1341
1358
28
759

672597
ACCCTCAGCGAGTACTGT
sooosssssssssooss
1342
1359
30
760

672598
CACCCTCAGCGAGTACTG
sooosssssssssooss
1343
1360
46
761

672599
TCACCCTCAGCGAGTACT
sooosssssssssooss
1344
1361
40
762

672600
TTCACCCTCAGCGAGTAC
sooosssssssssooss
1345
1362
25
763

672601
GTTCACCCTCAGCGAGTA
sooosssssssssooss
1346
1363
15
764

672602
TGTTCACCCTCAGCGAGT
sooosssssssssooss
1347
1364
35
765

672603
TTGTTCACCCTCAGCGAG
sooosssssssssooss
1348
1365
22
766

672604
CTTGTTCACCCTCAGCGA
sooosssssssssooss
1349
1366
6
767

672605
TCTTGTTCACCCTCAGCG
sooosssssssssooss
1350
1367
26
768

672606
TTCTTGTTCACCCTCAGC
sooosssssssssooss
1351
1368
11
769

672607
TTTCTTGTTCACCCTCAG
sooosssssssssooss
1352
1369
9
770

672608
TTTTCTTGTTCACCCTCA
sooosssssssssooss
1353
1370
36
771

672609
CTTTTCTTGTTCACCCTC
sooosssssssssooss
1354
1371
25
772

672610
TCTTTTCTTGTTCACCCT
sooosssssssssooss
1355
1372
16
773

672611
GTCTTTTCTTGTTCACCC
sooosssssssssooss
1356
1373
45
774

672612
GGTCTTTTCTTGTTCACC
sooosssssssssooss
1357
1374
15
775

672613
AGGTCTTTTCTTGTTCAC
sooosssssssssooss
1358
1375
10
776

672614
CAGGTCTTTTCTTGTTCA
sooosssssssssooss
1359
1376
25
777

672615
TCAGGTCTTTTCTTGTTC
sooosssssssssooss
1360
1377
21
778

672616
ATCAGGTCTTTTCTTGTT
sooosssssssssooss
1361
1378
15
779

672617
TATCAGGTCTTTTCTTGT
sooosssssssssooss
1362
1379
27
780

672618
TTATCAGGTCTTTTCTTG
sooosssssssssooss
1363
1380
14
781

672619
TTTATCAGGTCTTTTCTT
sooosssssssssooss
1364
1381
46
782

672620
AATCTTTATCAGGTCTTT
sooosssssssssooss
1368
1385
31
783

672621
TAATCTTTATCAGGTCTT
sooosssssssssooss
1369
1386
12
784

672622
TTAATCTTTATCAGGTCT
sooosssssssssooss
1370
1387
27
785

672623
GTTAATCTTTATCAGGTC
sooosssssssssooss
1371
1388
9
786

672624
GGTTAATCTTTATCAGGT
sooosssssssssooss
1372
1389
53
787

672625
TGGTTAATCTTTATCAGG
sooosssssssssooss
1373
1390
17
788

672626
CTGGTTAATCTTTATCAG
sooosssssssssooss
1374
1391
11
789

672627
TCTGGTTAATCTTTATCA
sooosssssssssooss
1375
1392
16
790

672628
TTCTGGTTAATCTTTATC
sooosssssssssooss
1376
1393
22
791

672629
TCCCTCCTTGTTTTCTTC
sooosssssssssooss
1391
1408
0
792

672630
TTTCCCTCCTTGTTTTCT
sooosssssssssooss
1393
1410
8
793

672631
GTTTCCCTCCTTGTTTTC
sooosssssssssooss
1394
1411
0
794

672632
TGTTTCCCTCCTTGTTTT
sooosssssssssooss
1395
1412
25
795

672633
TTGTTTCCCTCCTTGTTT
sooosssssssssooss
1396
1413
0
796

672634
GTTGTTTCCCTCCTTGTT
sooosssssssssooss
1397
1414
10
797

672635
GGTTGTTTCCCTCCTTGT
sooosssssssssooss
1398
1415
15
798

672636
CGGTTGTTTCCCTCCTTG
sooosssssssssooss
1399
1416
49
799

672637
GCGGTTGTTTCCCTCCTT
sooosssssssssooss
1400
1417
49
800

672638
TGCGGTTGTTTCCCTCCT
sooosssssssssooss
1401
1418
23
801

672639
CTGCGGTTGTTTCCCTCC
sooosssssssssooss
1402
1419
21
802

672640
GCTGCGGTTGTTTCCCTC
sooosssssssssooss
1403
1420
52
803

672641
GGCTGCGGTTGTTTCCCT
sooosssssssssooss
1404
1421
23
804

672642
AGGCTGCGGTTGTTTCCC
sooosssssssssooss
1405
1422
35
805

672643
CAGGCTGCGGTTGTTTCC
sooosssssssssooss
1406
1423
22
806

672644
ACAGGCTGCGGTTGTTTC
sooosssssssssooss
1407
1424
27
807

672645
TACAGGCTGCGGTTGTTT
sooosssssssssooss
1408
1425
21
808

672646
CTACAGGCTGCGGTTGTT
sooosssssssssooss
1409
1426
18
809

672647
GCTACAGGCTGCGGTTGT
sooosssssssssooss
1410
1427
16
810

672648
TGCTACAGGCTGCGGTTG
sooosssssssssooss
1411
1428
10
811

672649
TTGCTACAGGCTGCGGTT
sooosssssssssooss
1412
1429
0
812

672650
CTTGCTACAGGCTGCGGT
sooosssssssssooss
1413
1430
27
813

672651
GCTTGCTACAGGCTGCGG
sooosssssssssooss
1414
1431
53
814

672652
AGCTTGCTACAGGCTGCG
sooosssssssssooss
1415
1432
38
815

672653
GAGCTTGCTACAGGCTGC
sooosssssssssooss
1416
1433
7
816

672654
AGAGCTTGCTACAGGCTG
sooosssssssssooss
1417
1434
7
817

672655
CAGAGCTTGCTACAGGCT
sooosssssssssooss
1418
1435
15
818

672656
CCAGAGCTTGCTACAGGC
sooosssssssssooss
1419
1436
22
819

672657
TCCAGAGCTTGCTACAGG
sooosssssssssooss
1420
1437
43
820

672658
TTCCAGAGCTTGCTACAG
sooosssssssssooss
1421
1438
20
821

672659
GTTCCAGAGCTTGCTACA
sooosssssssssooss
1422
1439
12
822

672660
AGTTCCAGAGCTTGCTAC
sooosssssssssooss
1423
1440
11
823

672661
GAGTTCCAGAGCTTGCTA
sooosssssssssooss
1424
1441
39
824

672662
TGAGTTCCAGAGCTTGCT
sooosssssssssooss
1425
1442
18
825

672663
CTGAGTTCCAGAGCTTGC
sooosssssssssooss
1426
1443
26
826

672664
CCTGAGTTCCAGAGCTTG
sooosssssssssooss
1427
1444
69
827

672665
TCCTGAGTTCCAGAGCTT
sooosssssssssooss
1428
1445
56
828

672666
CTCCTGAGTTCCAGAGCT
sooosssssssssooss
1429
1446
28
829

672667
ACTCCTGAGTTCCAGAGC
sooosssssssssooss
1430
1447
51
830

672668
GACTCCTGAGTTCCAGAG
sooosssssssssooss
1431
1448
39
831

672669
CGACTCCTGAGTTCCAGA
sooosssssssssooss
1432
1449
32
832

672670
GCGACTCCTGAGTTCCAG
sooosssssssssooss
1433
1450
66
833

672671
CGCGACTCCTGAGTTCCA
sooosssssssssooss
1434
1451
67
834

672672
GCGCGACTCCTGAGTTCC
sooosssssssssooss
1435
1452
48
835

672673
CGCGCGACTCCTGAGTTC
sooosssssssssooss
1436
1453
38
836

672674
GCGCGCGACTCCTGAGTT
sooosssssssssooss
1437
1454
53
837

672675
AGCGCGCGACTCCTGAGT
sooosssssssssooss
1438
1455
58
838

672676
TAGCGCGCGACTCCTGAG
sooosssssssssooss
1439
1456
55
839

672677
CTAGCGCGCGACTCCTGA
sooosssssssssooss
1440
1457
47
840

672678
CCTAGCGCGCGACTCCTG
sooosssssssssooss
1441
1458
56
841

672679
CCCTAGCGCGCGACTCCT
sooosssssssssooss
1442
1459
74
842

672680
CCCCTAGCGCGCGACTCC
sooosssssssssooss
1443
1460
43
843

672681
GCCCCTAGCGCGCGACTC
sooosssssssssooss
1444
1461
53
844

672682
GGCCCCTAGCGCGCGACT
sooosssssssssooss
1445
1462
42
845

672683
CGGCCCCTAGCGCGCGAC
sooosssssssssooss
1446
1463
69
846

672684
CCGGCCCCTAGCGCGCGA
sooosssssssssooss
1447
1464
29
847

672685
CCCGGCCCCTAGCGCGCG
sooosssssssssooss
1448
1465
21
848

672686
CCCCGGCCCCTAGCGCGC
sooosssssssssooss
1449
1466
35
849

672687
GCCCCGGCCCCTAGCGCG
sooosssssssssooss
1450
1467
41
850

672688
GGCCCCGGCCCCTAGCGC
sooosssssssssooss
1451
1468
46
851

672689
CGGCCCCGGCCCCTAGCG
sooosssssssssooss
1452
1469
28
852

672690
CCGGCCCCGGCCCCTAGC
sooosssssssssooss
1453
1470
33
853

672691
CCCGGCCCCGGCCCCTAG
sooosssssssssooss
1454
1471
10
854

672692
CCCCGGCCCCGGCCCCTA
sooosssssssssooss
1455
1472
35
855

672693
ACGCCCCGGCCCCGGCCC
sooosssssssssooss
1464
1481
57
856

672694
CACGCCCCGGCCCCGGCC
sooosssssssssooss
1465
1482
39
857

672695
CCACGCCCCGGCCCCGGC
sooosssssssssooss
1466
1483
48
858

672696
ACCACGCCCCGGCCCCGG
sooosssssssssooss
1467
1484
39
859

672697
GACCACGCCCCGGCCCCG
sooosssssssssooss
1468
1485
54
860

672698
CGACCACGCCCCGGCCCC
sooosssssssssooss
1469
1486
48
861

672699
CCGACCACGCCCCGGCCC
sooosssssssssooss
1470
1487
52
862

672700
CCCGACCACGCCCCGGCC
sooosssssssssooss
1471
1488
67
863

672701
CCCCGACCACGCCCCGGC
sooosssssssssooss
1472
1489
42
864

672702
GCCCCGACCACGCCCCGG
sooosssssssssooss
1473
1490
11
865

672703
CGCCCCGACCACGCCCCG
sooosssssssssooss
1474
1491
23
866

672704
CCGCCCCGACCACGCCCC
sooosssssssssooss
1475
1492
50
867

672705
CCCGCCCCGACCACGCCC
sooosssssssssooss
1476
1493
23
868

672706
GCCCGCCCCGACCACGCC
sooosssssssssooss
1477
1494
24
869

672707
GGCCCGCCCCGACCACGC
sooosssssssssooss
1478
1495
44
870

672708
GGGCCCGCCCCGACCACG
sooosssssssssooss
1479
1496
29
871

672709
CGGGCCCGCCCCGACCAC
sooosssssssssooss
1480
1497
7
872

672710
CCGGGCCCGCCCCGACCA
sooosssssssssooss
1481
1498
30
873

672711
CCCGGGCCCGCCCCGACC
sooosssssssssooss
1482
1499
16
874

672712
CCCCGGGCCCGCCCCGAC
sooosssssssssooss
1483
1500
14
875

672713
AGCCCCGCCCCGGGCCCG
sooosssssssssooss
1502
1519
32
876

672714
CAGCCCCGCCCCGGGCCC
sooosssssssssooss
1503
1520
22
877

672715
GCAGCCCCGCCCCGGGCC
sooosssssssssooss
1504
1521
1
878

672716
CGCAGCCCCGCCCCGGGC
sooosssssssssooss
1505
1522
29
879

672717
CCGCAGCCCCGCCCCGGG
sooosssssssssooss
1506
1523
51
880

672718
ACCGCAGCCCCGCCCCGG
sooosssssssssooss
1507
1524
45
881

672719
AACCGCAGCCCCGCCCCG
sooosssssssssooss
1508
1525
12
882

672720
CAACCGCAGCCCCGCCCC
sooosssssssssooss
1509
1526
7
883

672721
GCAACCGCAGCCCCGCCC
sooosssssssssooss
1510
1527
38
884

672722
CGCAACCGCAGCCCCGCC
sooosssssssssooss
1511
1528
34
885

672723
CCGCAACCGCAGCCCCGC
sooosssssssssooss
1512
1529
58
886

672724
ACCGCAACCGCAGCCCCG
sooosssssssssooss
1513
1530
39
887

672725
CACCGCAACCGCAGCCCC
sooosssssssssooss
1514
1531
43
888

672726
GCACCGCAACCGCAGCCC
sooosssssssssooss
1515
1532
41
889

672727
GGCACCGCAACCGCAGCC
sooosssssssssooss
1516
1533
18
890

672728
AGGCACCGCAACCGCAGC
sooosssssssssooss
1517
1534
53
891

672729
CAGGCACCGCAACCGCAG
sooosssssssssooss
1518
1535
26
892

672730
GCAGGCACCGCAACCGCA
sooosssssssssooss
1519
1536
54
893

672731
CGCAGGCACCGCAACCGC
sooosssssssssooss
1520
1537
41
894

672732
GCGCAGGCACCGCAACCG
sooosssssssssooss
1521
1538
46
895

672733
GGCGCAGGCACCGCAACC
sooosssssssssooss
1522
1539
7
896

672734
GGGCGCAGGCACCGCAAC
sooosssssssssooss
1523
1540
26
897

TABLE 39

Percent inhibition of the C9ORF72 pathogenic associated mRNA variant compared to PBS control by

Deoxy, MOE and cEt antisense oligonucleotides with mixed backbones targeting SEQ ID NO: 2

SEQ ID
SEQ ID

NO: 2
NO: 2
%
SEQ

ISIS

Start
Stop
inhib-
ID

NO
Sequence
Chemistry
Linkage
Site
Site
ition
NO

576816
GCCTTACTCTAGGACCAAGA
eeeee-d10-eeeee
sssssssssssssssssss
7990
8009
71
20

672735
TGAGAGCAAGTAGTGGG
eeekk-d7-kkeee
soosssssssssooss
1326
1342
5
898

672736
GTGAGAGCAAGTAGTGG
eeekk-d7-kkeee
soosssssssssooss
1327
1343
35
899

672737
TGTGAGAGCAAGTAGTG
eeekk-d7-kkeee
soosssssssssooss
1328
1344
0
900

672738
CTGTGAGAGCAAGTAGT
eeekk-d7-kkeee
soosssssssssooss
1329
1345
63
901

672739
ACTGTGAGAGCAAGTAG
eeekk-d7-kkeee
soosssssssssooss
1330
1346
0
902

672740
TACTGTGAGAGCAAGTA
eeekk-d7-kkeee
soosssssssssooss
1331
1347
65
903

672741
GTACTGTGAGAGCAAGT
eeekk-d7-kkeee
soosssssssssooss
1332
1348
13
904

672742
AGTACTGTGAGAGCAAG
eeekk-d7-kkeee
soosssssssssooss
1333
1349
46
905

672743
GAGTACTGTGAGAGCAA
eeekk-d7-kkeee
soosssssssssooss
1334
1350
55
906

672744
CGAGTACTGTGAGAGCA
eeekk-d7-kkeee
soosssssssssooss
1335
1351
76
907

672745
GCGAGTACTGTGAGAGC
eeekk-d7-kkeee
soosssssssssooss
1336
1352
11
908

672746
AGCGAGTACTGTGAGAG
eeekk-d7-kkeee
soosssssssssooss
1337
1353
11
909

672747
CAGCGAGTACTGTGAGA
eeekk-d7-kkeee
soosssssssssooss
1338
1354
51
910

672748
TCAGCGAGTACTGTGAG
eeekk-d7-kkeee
soosssssssssooss
1339
1355
20
911

672749
CTCAGCGAGTACTGTGA
eeekk-d7-kkeee
soosssssssssooss
1340
1356
12
912

672750
CCTCAGCGAGTACTGTG
eeekk-d7-kkeee
soosssssssssooss
1341
1357
2
913

672751
CCCTCAGCGAGTACTGT
eeekk-d7-kkeee
soosssssssssooss
1342
1358
39
914

672752
ACCCTCAGCGAGTACTG
eeekk-d7-kkeee
soosssssssssooss
1343
1359
27
915

672753
CACCCTCAGCGAGTACT
eeekk-d7-kkeee
soosssssssssooss
1344
1360
33
916

672754
TCACCCTCAGCGAGTAC
eeekk-d7-kkeee
soosssssssssooss
1345
1361
0
917

672755
TTCACCCTCAGCGAGTA
eeekk-d7-kkeee
soosssssssssooss
1346
1362
13
918

672756
GTTCACCCTCAGCGAGT
eeekk-d7-kkeee
soosssssssssooss
1347
1363
45
919

672757
TGTTCACCCTCAGCGAG
eeekk-d7-kkeee
soosssssssssooss
1348
1364
11
920

672758
TTGTTCACCCTCAGCGA
eeekk-d7-kkeee
soosssssssssooss
1349
1365
0
921

672759
CTTGTTCACCCTCAGCG
eeekk-d7-kkeee
soosssssssssooss
1350
1366
12
922

672760
TCTTGTTCACCCTCAGC
eeekk-d7-kkeee
soosssssssssooss
1351
1367
21
923

672761
TTCTTGTTCACCCTCAG
eeekk-d7-kkeee
soosssssssssooss
1352
1368
0
924

672762
TTTCTTGTTCACCCTCA
eeekk-d7-kkeee
soosssssssssooss
1353
1369
34
925

672763
TTTTCTTGTTCACCCTC
eeekk-d7-kkeee
soosssssssssooss
1354
1370
16
926

672764
CTTTTCTTGTTCACCCT
eeekk-d7-kkeee
soosssssssssooss
1355
1371
2
927

672765
TCTTTTCTTGTTCACCC
eeekk-d7-kkeee
soosssssssssooss
1356
1372
24
928

672766
GTCTTTTCTTGTTCACC
eeekk-d7-kkeee
soosssssssssooss
1357
1373
28
929

672767
GGTCTTTTCTTGTTCAC
eeekk-d7-kkeee
soosssssssssooss
1358
1374
30
930

672768
AGGTCTTTTCTTGTTCA
eeekk-d7-kkeee
soosssssssssooss
1359
1375
14
931

672769
CAGGTCTTTTCTTGTTC
eeekk-d7-kkeee
soosssssssssooss
1360
1376
20
932

672770
TCAGGTCTTTTCTTGTT
eeekk-d7-kkeee
soosssssssssooss
1361
1377
2
933

672771
ATCAGGTCTTTTCTTGT
eeekk-d7-kkeee
soosssssssssooss
1362
1378
0
934

672772
TATCAGGTCTTTTCTTG
eeekk-d7-kkeee
soosssssssssooss
1363
1379
23
935

672773
TTATCAGGTCTTTTCTT
eeekk-d7-kkeee
soosssssssssooss
1364
1380
39
936

672774
ATCTTTATCAGGTCTTT
eeekk-d7-kkeee
soosssssssssooss
1368
1384
92
937

672775
AATCTTTATCAGGTCTT
eeekk-d7-kkeee
soosssssssssooss
1369
1385
61
938

672776
TAATCTTTATCAGGTCT
eeekk-d7-kkeee
soosssssssssooss
1370
1386
2
939

672777
TTAATCTTTATCAGGTC
eeekk-d7-kkeee
soosssssssssooss
1371
1387
33
940

672778
GTTAATCTTTATCAGGT
eeekk-d7-kkeee
soosssssssssooss
1372
1388
53
941

672779
GGTTAATCTTTATCAGG
eeekk-d7-kkeee
soosssssssssooss
1373
1389
40
942

672780
TGGTTAATCTTTATCAG
eeekk-d7-kkeee
soosssssssssooss
1374
1390
26
943

672781
CTGGTTAATCTTTATCA
eeekk-d7-kkeee
soosssssssssooss
1375
1391
25
944

672782
TCTGGTTAATCTTTATC
eeekk-d7-kkeee
soosssssssssooss
1376
1392
44
945

672783
CCCTCCTTGTTTTCTTC
eeekk-d7-kkeee
soosssssssssooss
1391
1407
37
946

672784
TCCCTCCTTGTTTTCTT
eeekk-d7-kkeee
soosssssssssooss
1392
1408
10
947

672785
TTCCCTCCTTGTTTTCT
eeekk-d7-kkeee
soosssssssssooss
1393
1409
0
948

672786
TTTCCCTCCTTGTTTTC
eeekk-d7-kkeee
soosssssssssooss
1394
1410
0
949

672787
GTTTCCCTCCTTGTTTT
eeekk-d7-kkeee
soosssssssssooss
1395
1411
0
950

672788
TGTTTCCCTCCTTGTTT
eeekk-d7-kkeee
soosssssssssooss
1396
1412
0
951

672789
TTGTTTCCCTCCTTGTT
eeekk-d7-kkeee
soosssssssssooss
1397
1413
0
952

672790
GGTTGTTTCCCTCCTTG
eeekk-d7-kkeee
soosssssssssooss
1399
1415
33
953

672791
CGGTTGTTTCCCTCCTT
eeekk-d7-kkeee
soosssssssssooss
1400
1416
0
954

672792
GCGGTTGTTTCCCTCCT
eeekk-d7-kkeee
soosssssssssooss
1401
1417
23
955

672793
TGCGGTTGTTTCCCTCC
eeekk-d7-kkeee
soosssssssssooss
1402
1418
0
956

672794
CTGCGGTTGTTTCCCTC
eeekk-d7-kkeee
soosssssssssooss
1403
1419
25
957

672795
GCTGCGGTTGTTTCCCT
eeekk-d7-kkeee
soosssssssssooss
1404
1420
5
958

672796
GGCTGCGGTTGTTTCCC
eeekk-d7-kkeee
soosssssssssooss
1405
1421
16
959

672797
AGGCTGCGGTTGTTTCC
eeekk-d7-kkeee
soosssssssssooss
1406
1422
7
960

672798
CAGGCTGCGGTTGTTTC
eeekk-d7-kkeee
soosssssssssooss
1407
1423
0
961

672799
ACAGGCTGCGGTTGTTT
eeekk-d7-kkeee
soosssssssssooss
1408
1424
28
962

672800
TACAGGCTGCGGTTGTT
eeekk-d7-kkeee
soosssssssssooss
1409
1425
33
963

672801
CTACAGGCTGCGGTTGT
eeekk-d7-kkeee
soosssssssssooss
1410
1426
53
964

672802
GCTACAGGCTGCGGTTG
eeekk-d7-kkeee
soosssssssssooss
1411
1427
0
965

672803
TGCTACAGGCTGCGGTT
eeekk-d7-kkeee
soosssssssssooss
1412
1428
0
966

672804
TTGCTACAGGCTGCGGT
eeekk-d7-kkeee
soosssssssssooss
1413
1429
0
967

672805
CTTGCTACAGGCTGCGG
eeekk-d7-kkeee
soosssssssssooss
1414
1430
1
968

672806
GCTTGCTACAGGCTGCG
eeekk-d7-kkeee
soosssssssssooss
1415
1431
58
969

672807
AGCTTGCTACAGGCTGC
eeekk-d7-kkeee
soosssssssssooss
1416
1432
0
970

672808
GAGCTTGCTACAGGCTG
eeekk-d7-kkeee
soosssssssssooss
1417
1433
0
971

672809
AGAGCTTGCTACAGGCT
eeekk-d7-kkeee
soosssssssssooss
1418
1434
71
972

672810
CAGAGCTTGCTACAGGC
eeekk-d7-kkeee
soosssssssssooss
1419
1435
18
973

672811
CCAGAGCTTGCTACAGG
eeekk-d7-kkeee
soosssssssssooss
1420
1436
0
974

672812
TCCAGAGCTTGCTACAG
eeekk-d7-kkeee
soosssssssssooss
1421
1437
19
975

672813
TTCCAGAGCTTGCTACA
eeekk-d7-kkeee
soosssssssssooss
1422
1438
19
976

672814
GTTCCAGAGCTTGCTAC
eeekk-d7-kkeee
soosssssssssooss
1423
1439
0
977

672815
AGTTCCAGAGCTTGCTA
eeekk-d7-kkeee
soosssssssssooss
1424
1440
0
978

672816
GAGTTCCAGAGCTTGCT
eeekk-d7-kkeee
soosssssssssooss
1425
1441
14
979

672817
TGAGTTCCAGAGCTTGC
eeekk-d7-kkeee
soosssssssssooss
1426
1442
31
980

672818
CTGAGTTCCAGAGCTTG
eeekk-d7-kkeee
soosssssssssooss
1427
1443
21
981

672819
CCTGAGTTCCAGAGCTT
eeekk-d7-kkeee
soosssssssssooss
1428
1444
19
982

672820
TCCTGAGTTCCAGAGCT
eeekk-d7-kkeee
soosssssssssooss
1429
1445
46
983

672821
CTCCTGAGTTCCAGAGC
eeekk-d7-kkeee
soosssssssssooss
1430
1446
7
984

672822
ACTCCTGAGTTCCAGAG
eeekk-d7-kkeee
soosssssssssooss
1431
1447
13
985

672823
GACTCCTGAGTTCCAGA
eeekk-d7-kkeee
soosssssssssooss
1432
1448
19
986

672824
CGACTCCTGAGTTCCAG
eeekk-d7-kkeee
soosssssssssooss
1433
1449
23
987

672825
GCGACTCCTGAGTTCCA
eeekk-d7-kkeee
soosssssssssooss
1434
1450
0
988

672826
CGCGACTCCTGAGTTCC
eeekk-d7-kkeee
soosssssssssooss
1435
1451
19
989

672827
GCGCGACTCCTGAGTTC
eeekk-d7-kkeee
soosssssssssooss
1436
1452
31
990

672828
CGCGCGACTCCTGAGTT
eeekk-d7-kkeee
soosssssssssooss
1437
1453
63
991

672829
GCGCGCGACTCCTGAGT
eeekk-d7-kkeee
soosssssssssooss
1438
1454
28
992

672830
AGCGCGCGACTCCTGAG
eeekk-d7-kkeee
soosssssssssooss
1439
1455
58
993

672831
TAGCGCGCGACTCCTGA
eeekk-d7-kkeee
soosssssssssooss
1440
1456
42
994

672832
CTAGCGCGCGACTCCTG
eeekk-d7-kkeee
soosssssssssooss
1441
1457
42
995

672833
CCTAGCGCGCGACTCCT
eeekk-d7-kkeee
soosssssssssooss
1442
1458
24
996

672834
CCCTAGCGCGCGACTCC
eeekk-d7-kkeee
soosssssssssooss
1443
1459
58
997

672835
CCCCTAGCGCGCGACTC
eeekk-d7-kkeee
soosssssssssooss
1444
1460
40
998

672836
GCCCCTAGCGCGCGACT
eeekk-d7-kkeee
soosssssssssooss
1445
1461
0
999

672837
GGCCCCTAGCGCGCGAC
eeekk-d7-kkeee
soosssssssssooss
1446
1462
2
1000

672838
CGGCCCCTAGCGCGCGA
eeekk-d7-kkeee
soosssssssssooss
1447
1463
72
1001

672839
CCGGCCCCTAGCGCGCG
eeekk-d7-kkeee
soosssssssssooss
1448
1464
0
1002

672840
CCCGGCCCCTAGCGCGC
eeekk-d7-kkeee
soosssssssssooss
1449
1465
28
1003

672841
CCCCGGCCCCTAGCGCG
eeekk-d7-kkeee
soosssssssssooss
1450
1466
28
1004

672842
GCCCCGGCCCCTAGCGC
eeekk-d7-kkeee
soosssssssssooss
1451
1467
0
1005

672843
GGCCCCGGCCCCTAGCG
eeekk-d7-kkeee
soosssssssssooss
1452
1468
23
1006

672844
CGGCCCCGGCCCCTAGC
eeekk-d7-kkeee
soosssssssssooss
1453
1469
26
1007

672845
CCGGCCCCGGCCCCTAG
eeekk-d7-kkeee
soosssssssssooss
1454
1470
24
1008

672846
CCCGGCCCCGGCCCCTA
eeekk-d7-kkeee
soosssssssssooss
1455
1471
9
1009

672847
ACGCCCCGGCCCCGGCC
eeekk-d7-kkeee
soosssssssssooss
1465
1481
3
1010

672848
CACGCCCCGGCCCCGGC
eeekk-d7-kkeee
soosssssssssooss
1466
1482
19
1011

672849
CCACGCCCCGGCCCCGG
eeekk-d7-kkeee
soosssssssssooss
1467
1483
50
1012

672850
ACCACGCCCCGGCCCCG
eeekk-d7-kkeee
soosssssssssooss
1468
1484
0
1013

672851
GACCACGCCCCGGCCCC
eeekk-d7-kkeee
soosssssssssooss
1469
1485
3
1014

672852
CGACCACGCCCCGGCCC
eeekk-d7-kkeee
soosssssssssooss
1470
1486
9
1015

672853
CCGACCACGCCCCGGCC
eeekk-d7-kkeee
soosssssssssooss
1471
1487
24
1016

672854
CCCGACCACGCCCCGGC
eeekk-d7-kkeee
soosssssssssooss
1472
1488
9
1017

672855
CCCCGACCACGCCCCGG
eeekk-d7-kkeee
soosssssssssooss
1473
1489
18
1018

672856
GCCCCGACCACGCCCCG
eeekk-d7-kkeee
soosssssssssooss
1474
1490
8
1019

672857
CGCCCCGACCACGCCCC
eeekk-d7-kkeee
soosssssssssooss
1475
1491
0
1020

672858
CCGCCCCGACCACGCCC
eeekk-d7-kkeee
soosssssssssooss
1476
1492
48
1021

672859
CCCGCCCCGACCACGCC
eeekk-d7-kkeee
soosssssssssooss
1477
1493
28
1022

672860
GCCCGCCCCGACCACGC
eeekk-d7-kkeee
soosssssssssooss
1478
1494
0
1023

672861
GGCCCGCCCCGACCACG
eeekk-d7-kkeee
soosssssssssooss
1479
1495
33
1024

672862
GGGCCCGCCCCGACCAC
eeekk-d7-kkeee
soosssssssssooss
1480
1496
32
1025

672863
CGGGCCCGCCCCGACCA
eeekk-d7-kkeee
soosssssssssooss
1481
1497
0
1026

672864
CCGGGCCCGCCCCGACC
eeekk-d7-kkeee
soosssssssssooss
1482
1498
0
1027

672865
CCCGGGCCCGCCCCGAC
eeekk-d7-kkeee
soosssssssssooss
1483
1499
11
1028

672866
GCAGCCCCGCCCCGGGC
eeekk-d7-kkeee
soosssssssssooss
1505
1521
23
1029

672867
CGCAGCCCCGCCCCGGG
eeekk-d7-kkeee
soosssssssssooss
1506
1522
26
1030

672868
CCGCAGCCCCGCCCCGG
eeekk-d7-kkeee
soosssssssssooss
1507
1523
2
1031

672869
ACCGCAGCCCCGCCCCG
eeekk-d7-kkeee
soosssssssssooss
1508
1524
8
1032

672870
AACCGCAGCCCCGCCCC
eeekk-d7-kkeee
soosssssssssooss
1509
1525
7
1033

672871
CAACCGCAGCCCCGCCC
eeekk-d7-kkeee
soosssssssssooss
1510
1526
1
1034

672872
GCAACCGCAGCCCCGCC
eeekk-d7-kkeee
soosssssssssooss
1511
1527
37
1035

672873
CGCAACCGCAGCCCCGC
eeekk-d7-kkeee
soosssssssssooss
1512
1528
20
1036

672874
CCGCAACCGCAGCCCCG
eeekk-d7-kkeee
soosssssssssooss
1513
1529
23
1037

672875
ACCGCAACCGCAGCCCC
eeekk-d7-kkeee
soosssssssssooss
1514
1530
8
1038

672876
CACCGCAACCGCAGCCC
eeekk-d7-kkeee
soosssssssssooss
1515
1531
22
1039

672877
GCACCGCAACCGCAGCC
eeekk-d7-kkeee
soosssssssssooss
1516
1532
19
1040

672878
GGCACCGCAACCGCAGC
eeekk-d7-kkeee
soosssssssssooss
1517
1533
25
1041

672879
AGGCACCGCAACCGCAG
eeekk-d7-kkeee
soosssssssssooss
1518
1534
21
1042

672880
CAGGCACCGCAACCGCA
eeekk-d7-kkeee
soosssssssssooss
1519
1535
12
1043

672881
GCAGGCACCGCAACCGC
eeekk-d7-kkeee
soosssssssssooss
1520
1536
18
1044

672882
CGCAGGCACCGCAACCG
eeekk-d7-kkeee
soosssssssssooss
1521
1537
15
1045

672883
GCGCAGGCACCGCAACC
eeekk-d7-kkeee
soosssssssssooss
1522
1538
0
1046

672884
GGCGCAGGCACCGCAAC
eeekk-d7-kkeee
soosssssssssooss
1523
1539
0
1047

Example 9: Dose-Dependent Antisense Inhibition of Human C9ORF72 mRNA in HepG2 Cells

Antisense oligonucleotides from the study described in Example 8 hereinabove exhibiting significant in vitro inhibition of C9ORF72 mRNA were selected and tested at various doses in HepG2 cells. ISIS 576816, previously tested in PCT/US2013/065073 (claiming priority to U.S. Application No. 61/714,132, filed Oct. 15, 2012, was used as a benchmark oligonucleotide. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.11 μM, 0.33 μM, 1.00 μM, or 3.00 μM 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 primer probe set RTS3905 was used to measure the C9ORF72 pathogenic associated mRNA variant, which is the product of a pre-mRNA containing a hexanucleotide repeat. C9ORF72 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72 levels, relative to untreated control cells.

As shown in Tables 39 and 40, total C9ORF72 mRNA levels were reduced in a dose-dependent manner in some of the antisense oligonucleotide treated cells.

TABLE 39

Dose-dependent inhibition of the C9ORF72 pathogenic

associated mRNA variant transcript levels in HepG2 cells

0.11
0.33
1.00
3.00

ISIS No
μM
μM
μM
μM

672651
12
39
63
88

672611
13
50
59
79

672602
16
36
62
79

672624
25
54
80
95

672657
19
30
53
75

672582
0
0
20
57

672683
33
57
73
84

672595
39
39
66
88

576816
23
53
78
87

672640
17
26
56
84

672599
28
59
69
87

672637
36
50
66
88

672592
16
38
37
65

672636
24
39
69
88

672652
26
48
63
94

672619
8
12
6
0

672608
12
45
37
59

672598
21
9
55
69

672642
28
35
59
72

TABLE 40

Dose-dependent inhibition of the C9ORF72 pathogenic

associated mRNA variant transcript levels in HepG2 cells

0.11
0.33
1.00
3.00

ISIS No
μM
μM
μM
μM

672679
2
52
81
95

672693
14
41
53
83

672681
27
42
63
87

672683
10
35
64
82

672678
24
56
77
91

672699
17
31
46
83

672664
7
28
58
87

672665
35
46
62
75

576816
15
55
71
84

672671
36
66
79
87

672676
33
53
71
77

672700
25
45
68
81

672730
24
41
59
80

672670
25
40
60
75

672697
11
41
64
80

672723
30
48
68
88

672728
11
44
49
68

672675
41
48
74
88

672674
19
34
55
61

Example 10: Antisense Inhibition of C9ORF72 by Deoxy, MOE and cEt Antisense Oligonucleotides with Mixed Backbones

Antisense oligonucleotides described in Example 6 hereinabove (see Tables 25-28 hereinabove) were tested in HepG2 cells in a series of experiments that had similar culture conditions. ISIS 576816, which was previously tested in PCT/US2013/065073 (claiming priority to U.S. Application No. 61/714,132, filed Oct. 15, 2012) was used as a benchmark oligonucleotide for study with deoxy, MOE, and cEt antisense oligonucleotides. The results for each experiment are presented in tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 700 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 RTS3905 was used to measure the C9ORF72 pathogenic associated mRNA variant, which is the product of a pre-mRNA containing a hexanucleotide repeat. The levels of the C9ORF72 pathogenic associated mRNA variant were normalized to the total RNA content of the cell, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72, relative to untreated control cells.

TABLE 41

Percent inhibition of the C9ORF72 pathogenic associated mRNA variant compared to PBS control by

Deoxy, MOE and cEt antisense oligonucleotides with mixed backbones targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO:
NO:

2
2
%
SEQ

ISIS

Start
Stop
inhib-
ID

NO
Sequence
Chemistry
Linkage
Site
Site
ition
NO

576816
GCCTTACTCTAGGACCAAGA
eeeee-d10-eeeee
sssssssssssssssssss
7990
8009
52
20

672885
TGAGAGCAAGTAGTGGG
eekk-d8-kkeee
soosssssssssooss
1326
1342
10
898

672886
GTGAGAGCAAGTAGTGG
eekk-d8-kkeee
soosssssssssooss
1327
1343
24
899

672887
TGTGAGAGCAAGTAGTG
eekk-d8-kkeee
soosssssssssooss
1328
1344
38
900

672888
CTGTGAGAGCAAGTAGT
eekk-d8-kkeee
soosssssssssooss
1329
1345
37
901

672889
ACTGTGAGAGCAAGTAG
eekk-d8-kkeee
soosssssssssooss
1330
1346
16
902

672890
TACTGTGAGAGCAAGTA
eekk-d8-kkeee
soosssssssssooss
1331
1347
26
903

672891
GTACTGTGAGAGCAAGT
eekk-d8-kkeee
soosssssssssooss
1332
1348
38
904

672892
AGTACTGTGAGAGCAAG
eekk-d8-kkeee
soosssssssssooss
1333
1349
44
905

672893
GAGTACTGTGAGAGCAA
eekk-d8-kkeee
soosssssssssooss
1334
1350
57
906

672894
CGAGTACTGTGAGAGCA
eekk-d8-kkeee
soosssssssssooss
1335
1351
62
907

672895
GCGAGTACTGTGAGAGC
eekk-d8-kkeee
soosssssssssooss
1336
1352
39
908

672896
AGCGAGTACTGTGAGAG
eekk-d8-kkeee
soosssssssssooss
1337
1353
54
909

672897
CAGCGAGTACTGTGAGA
eekk-d8-kkeee
soosssssssssooss
1338
1354
57
910

672898
TCAGCGAGTACTGTGAG
eekk-d8-kkeee
soosssssssssooss
1339
1355
23
911

672899
CTCAGCGAGTACTGTGA
eekk-d8-kkeee
soosssssssssooss
1340
1356
34
912

672900
CCTCAGCGAGTACTGTG
eekk-d8-kkeee
soosssssssssooss
1341
1357
33
913

672901
CCCTCAGCGAGTACTGT
eekk-d8-kkeee
soosssssssssooss
1342
1358
32
914

672902
ACCCTCAGCGAGTACTG
eekk-d8-kkeee
soosssssssssooss
1343
1359
62
915

672903
CACCCTCAGCGAGTACT
eekk-d8-kkeee
soosssssssssooss
1344
1360
59
916

672904
TCACCCTCAGCGAGTAC
eekk-d8-kkeee
soosssssssssooss
1345
1361
52
917

672905
TTCACCCTCAGCGAGTA
eekk-d8-kkeee
soosssssssssooss
1346
1362
50
918

672906
GTTCACCCTCAGCGAGT
eekk-d8-kkeee
soosssssssssooss
1347
1363
38
919

672907
TGTTCACCCTCAGCGAG
eekk-d8-kkeee
soosssssssssooss
1348
1364
20
920

672908
TTGTTCACCCTCAGCGA
eekk-d8-kkeee
soosssssssssooss
1349
1365
57
921

672909
CTTGTTCACCCTCAGCG
eekk-d8-kkeee
soosssssssssooss
1350
1366
66
922

672910
TCTTGTTCACCCTCAGC
eekk-d8-kkeee
soosssssssssooss
1351
1367
47
923

672911
TTCTTGTTCACCCTCAG
eekk-d8-kkeee
soosssssssssooss
1352
1368
37
924

672912
TTTCTTGTTCACCCTCA
eekk-d8-kkeee
soosssssssssooss
1353
1369
36
925

672913
TTTTCTTGTTCACCCTC
eekk-d8-kkeee
soosssssssssooss
1354
1370
34
926

672914
CTTTTCTTGTTCACCCT
eekk-d8-kkeee
soosssssssssooss
1355
1371
35
927

672915
TCTTTTCTTGTTCACCC
eekk-d8-kkeee
soosssssssssooss
1356
1372
41
928

672916
GTCTTTTCTTGTTCACC
eekk-d8-kkeee
soosssssssssooss
1357
1373
34
929

672917
GGTCTTTTCTTGTTCAC
eekk-d8-kkeee
soosssssssssooss
1358
1374
23
930

672918
AGGTCTTTTCTTGTTCA
eekk-d8-kkeee
soosssssssssooss
1359
1375
31
931

672919
CAGGTCTTTTCTTGTTC
eekk-d8-kkeee
soosssssssssooss
1360
1376
51
932

672920
TCAGGTCTTTTCTTGTT
eekk-d8-kkeee
soosssssssssooss
1361
1377
15
933

672921
ATCAGGTCTTTTCTTGT
eekk-d8-kkeee
soosssssssssooss
1362
1378
0
934

672922
TATCAGGTCTTTTCTTG
eekk-d8-kkeee
soosssssssssooss
1363
1379
31
935

672923
TTATCAGGTCTTTTCTT
eekk-d8-kkeee
soosssssssssooss
1364
1380
14
936

672924
ATCTTTATCAGGTCTTT
eekk-d8-kkeee
soosssssssssooss
1368
1384
71
937

672925
AATCTTTATCAGGTCTT
eekk-d8-kkeee
soosssssssssooss
1369
1385
72
938

672926
TAATCTTTATCAGGTCT
eekk-d8-kkeee
soosssssssssooss
1370
1386
40
939

672927
TTAATCTTTATCAGGTC
eekk-d8-kkeee
soosssssssssooss
1371
1387
66
940

672928
GTTAATCTTTATCAGGT
eekk-d8-kkeee
soosssssssssooss
1372
1388
56
941

672929
GGTTAATCTTTATCAGG
eekk-d8-kkeee
soosssssssssooss
1373
1389
80
942

672930
TGGTTAATCTTTATCAG
eekk-d8-kkeee
soosssssssssooss
1374
1390
48
943

672931
CTGGTTAATCTTTATCA
eekk-d8-kkeee
soosssssssssooss
1375
1391
48
944

672932
TCTGGTTAATCTTTATC
eekk-d8-kkeee
soosssssssssooss
1376
1392
54
945

672933
CCCTCCTTGTTTTCTTC
eekk-d8-kkeee
soosssssssssooss
1391
1407
18
946

672934
TCCCTCCTTGTTTTCTT
eekk-d8-kkeee
soosssssssssooss
1392
1408
7
947

672935
TTCCCTCCTTGTTTTCT
eekk-d8-kkeee
soosssssssssooss
1393
1409
10
948

672936
TTTCCCTCCTTGTTTTC
eekk-d8-kkeee
soosssssssssooss
1394
1410
0
949

672937
GTTTCCCTCCTTGTTTT
eekk-d8-kkeee
soosssssssssooss
1395
1411
0
950

672938
TGTTTCCCTCCTTGTTT
eekk-d8-kkeee
soosssssssssooss
1396
1412
9
951

672939
TTGTTTCCCTCCTTGTT
eekk-d8-kkeee
soosssssssssooss
1397
1413
27
952

672940
GGTTGTTTCCCTCCTTG
eekk-d8-kkeee
soosssssssssooss
1399
1415
49
953

672941
CGGTTGTTTCCCTCCTT
eekk-d8-kkeee
soosssssssssooss
1400
1416
17
954

672942
GCGGTTGTTTCCCTCCT
eekk-d8-kkeee
soosssssssssooss
1401
1417
10
955

672943
TGCGGTTGTTTCCCTCC
eekk-d8-kkeee
soosssssssssooss
1402
1418
33
956

672944
CTGCGGTTGTTTCCCTC
eekk-d8-kkeee
soosssssssssooss
1403
1419
29
957

672945
GCTGCGGTTGTTTCCCT
eekk-d8-kkeee
soosssssssssooss
1404
1420
23
958

672946
GGCTGCGGTTGTTTCCC
eekk-d8-kkeee
soosssssssssooss
1405
1421
15
959

672947
AGGCTGCGGTTGTTTCC
eekk-d8-kkeee
soosssssssssooss
1406
1422
24
960

672948
CAGGCTGCGGTTGTTTC
eekk-d8-kkeee
soosssssssssooss
1407
1423
49
961

672949
ACAGGCTGCGGTTGTTT
eekk-d8-kkeee
soosssssssssooss
1408
1424
35
962

672950
TACAGGCTGCGGTTGTT
eekk-d8-kkeee
soosssssssssooss
1409
1425
37
963

672951
CTACAGGCTGCGGTTGT
eekk-d8-kkeee
soosssssssssooss
1410
1426
4
964

672952
GCTACAGGCTGCGGTTG
eekk-d8-kkeee
soosssssssssooss
1411
1427
4
965

672953
TGCTACAGGCTGCGGTT
eekk-d8-kkeee
soosssssssssooss
1412
1428
24
966

672954
TTGCTACAGGCTGCGGT
eekk-d8-kkeee
soosssssssssooss
1413
1429
8
967

672955
CTTGCTACAGGCTGCGG
eekk-d8-kkeee
soosssssssssooss
1414
1430
28
968

672956
GCTTGCTACAGGCTGCG
eekk-d8-kkeee
soosssssssssooss
1415
1431
8
969

672957
AGCTTGCTACAGGCTGC
eekk-d8-kkeee
soosssssssssooss
1416
1432
5
970

672958
GAGCTTGCTACAGGCTG
eekk-d8-kkeee
soosssssssssooss
1417
1433
4
971

672959
AGAGCTTGCTACAGGCT
eekk-d8-kkeee
soosssssssssooss
1418
1434
0
972

672960
CAGAGCTTGCTACAGGC
eekk-d8-kkeee
soosssssssssooss
1419
1435
12
973

672961
CCAGAGCTTGCTACAGG
eekk-d8-kkeee
soosssssssssooss
1420
1436
36
974

672962
TCCAGAGCTTGCTACAG
eekk-d8-kkeee
soosssssssssooss
1421
1437
0
975

672963
TTCCAGAGCTTGCTACA
eekk-d8-kkeee
soosssssssssooss
1422
1438
11
976

672964
GTTCCAGAGCTTGCTAC
eekk-d8-kkeee
soosssssssssooss
1423
1439
8
977

672965
AGTTCCAGAGCTTGCTA
eekk-d8-kkeee
soosssssssssooss
1424
1440
19
978

672966
GAGTTCCAGAGCTTGCT
eekk-d8-kkeee
soosssssssssooss
1425
1441
48
979

672967
TGAGTTCCAGAGCTTGC
eekk-d8-kkeee
soosssssssssooss
1426
1442
41
980

672968
CTGAGTTCCAGAGCTTG
eekk-d8-kkeee
soosssssssssooss
1427
1443
54
981

672969
CCTGAGTTCCAGAGCTT
eekk-d8-kkeee
soosssssssssooss
1428
1444
58
982

672970
TCCTGAGTTCCAGAGCT
eekk-d8-kkeee
soosssssssssooss
1429
1445
12
983

672971
CTCCTGAGTTCCAGAGC
eekk-d8-kkeee
soosssssssssooss
1430
1446
23
984

672972
ACTCCTGAGTTCCAGAG
eekk-d8-kkeee
soosssssssssooss
1431
1447
30
985

672973
GACTCCTGAGTTCCAGA
eekk-d8-kkeee
soosssssssssooss
1432
1448
39
986

672974
CGACTCCTGAGTTCCAG
eekk-d8-kkeee
soosssssssssooss
1433
1449
41
987

672975
GCGACTCCTGAGTTCCA
eekk-d8-kkeee
soosssssssssooss
1434
1450
31
988

672976
CGCGACTCCTGAGTTCC
eekk-d8-kkeee
soosssssssssooss
1435
1451
56
989

672977
GCGCGACTCCTGAGTTC
eekk-d8-kkeee
soosssssssssooss
1436
1452
31
990

672978
CGCGCGACTCCTGAGTT
eekk-d8-kkeee
soosssssssssooss
1437
1453
45
991

672979
GCGCGCGACTCCTGAGT
eekk-d8-kkeee
soosssssssssooss
1438
1454
29
992

672980
AGCGCGCGACTCCTGAG
eekk-d8-kkeee
soosssssssssooss
1439
1455
48
993

672981
TAGCGCGCGACTCCTGA
eekk-d8-kkeee
soosssssssssooss
1440
1456
68
994

672982
CTAGCGCGCGACTCCTG
eekk-d8-kkeee
soosssssssssooss
1441
1457
59
995

672983
CCTAGCGCGCGACTCCT
eekk-d8-kkeee
soosssssssssooss
1442
1458
62
996

672984
CCCTAGCGCGCGACTCC
eekk-d8-kkeee
soosssssssssooss
1443
1459
69
997

672985
CCCCTAGCGCGCGACTC
eekk-d8-kkeee
soosssssssssooss
1444
1460
65
998

672986
GCCCCTAGCGCGCGACT
eekk-d8-kkeee
soosssssssssooss
1445
1461
34
999

672987
GGCCCCTAGCGCGCGAC
eekk-d8-kkeee
soosssssssssooss
1446
1462
22
1000

672988
CGGCCCCTAGCGCGCGA
eekk-d8-kkeee
soosssssssssooss
1447
1463
16
1001

672989
CCGGCCCCTAGCGCGCG
eekk-d8-kkeee
soosssssssssooss
1448
1464
24
1002

672990
CCCGGCCCCTAGCGCGC
eekk-d8-kkeee
soosssssssssooss
1449
1465
10
1003

672991
CCCCGGCCCCTAGCGCG
eekk-d8-kkeee
soosssssssssooss
1450
1466
24
1004

672992
GCCCCGGCCCCTAGCGC
eekk-d8-kkeee
soosssssssssooss
1451
1467
29
1005

672993
GGCCCCGGCCCCTAGCG
eekk-d8-kkeee
soosssssssssooss
1452
1468
24
1006

672994
CGGCCCCGGCCCCTAGC
eekk-d8-kkeee
soosssssssssooss
1453
1469
25
1007

672995
CCGGCCCCGGCCCCTAG
eekk-d8-kkeee
soosssssssssooss
1454
1470
28
1008

672996
CCCGGCCCCGGCCCCTA
eekk-d8-kkeee
soosssssssssooss
1455
1471
25
1009

672997
ACGCCCCGGCCCCGGCC
eekk-d8-kkeee
soosssssssssooss
1465
1481
21
1010

672998
CACGCCCCGGCCCCGGC
eekk-d8-kkeee
soosssssssssooss
1466
1482
17
1011

672999
CCACGCCCCGGCCCCGG
eekk-d8-kkeee
soosssssssssooss
1467
1483
30
1012

673000
ACCACGCCCCGGCCCCG
eekk-d8-kkeee
soosssssssssooss
1468
1484
29
1013

673001
GACCACGCCCCGGCCCC
eekk-d8-kkeee
soosssssssssooss
1469
1485
25
1014

673002
CGACCACGCCCCGGCCC
eekk-d8-kkeee
soosssssssssooss
1470
1486
37
1015

673003
CCGACCACGCCCCGGCC
eekk-d8-kkeee
soosssssssssooss
1471
1487
23
1016

673004
CCCGACCACGCCCCGGC
eekk-d8-kkeee
soosssssssssooss
1472
1488
21
1017

673005
CCCCGACCACGCCCCGG
eekk-d8-kkeee
soosssssssssooss
1473
1489
9
1018

673006
GCCCCGACCACGCCCCG
eekk-d8-kkeee
soosssssssssooss
1474
1490
13
1019

673007
CGCCCCGACCACGCCCC
eekk-d8-kkeee
soosssssssssooss
1475
1491
17
1020

673008
CCGCCCCGACCACGCCC
eekk-d8-kkeee
soosssssssssooss
1476
1492
20
1021

673009
CCCGCCCCGACCACGCC
eekk-d8-kkeee
soosssssssssooss
1477
1493
36
1022

673010
GCCCGCCCCGACCACGC
eekk-d8-kkeee
soosssssssssooss
1478
1494
16
1023

673011
GGCCCGCCCCGACCACG
eekk-d8-kkeee
soosssssssssooss
1479
1495
3
1024

673012
GGGCCCGCCCCGACCAC
eekk-d8-kkeee
soosssssssssooss
1480
1496
21
1025

673013
CGGGCCCGCCCCGACCA
eekk-d8-kkeee
soosssssssssooss
1481
1497
4
1026

673014
CCGGGCCCGCCCCGACC
eekk-d8-kkeee
soosssssssssooss
1482
1498
21
1027

673015
CCCGGGCCCGCCCCGAC
eekk-d8-kkeee
soosssssssssooss
1483
1499
15
1028

673016
GCAGCCCCGCCCCGGGC
eekk-d8-kkeee
soosssssssssooss
1505
1521
3
1029

673017
CGCAGCCCCGCCCCGGG
eekk-d8-kkeee
soosssssssssooss
1506
1522
7
1030

673018
CCGCAGCCCCGCCCCGG
eekk-d8-kkeee
soosssssssssooss
1507
1523
7
1031

673019
ACCGCAGCCCCGCCCCG
eekk-d8-kkeee
soosssssssssooss
1508
1524
8
1032

673020
AACCGCAGCCCCGCCCC
eekk-d8-kkeee
soosssssssssooss
1509
1525
42
1033

673021
CAACCGCAGCCCCGCCC
eekk-d8-kkeee
soosssssssssooss
1510
1526
58
1034

673022
GCAACCGCAGCCCCGCC
eekk-d8-kkeee
soosssssssssooss
1511
1527
44
1035

673023
CGCAACCGCAGCCCCGC
eekk-d8-kkeee
soosssssssssooss
1512
1528
46
1036

673024
CCGCAACCGCAGCCCCG
eekk-d8-kkeee
soosssssssssooss
1513
1529
26
1037

673025
ACCGCAACCGCAGCCCC
eekk-d8-kkeee
soosssssssssooss
1514
1530
20
1038

673026
CACCGCAACCGCAGCCC
eekk-d8-kkeee
soosssssssssooss
1515
1531
52
1039

673027
GCACCGCAACCGCAGCC
eekk-d8-kkeee
soosssssssssooss
1516
1532
22
1040

673028
GGCACCGCAACCGCAGC
eekk-d8-kkeee
soosssssssssooss
1517
1533
32
1041

673029
AGGCACCGCAACCGCAG
eekk-d8-kkeee
soosssssssssooss
1518
1534
27
1042

673030
CAGGCACCGCAACCGCA
eekk-d8-kkeee
soosssssssssooss
1519
1535
32
1043

673031
GCAGGCACCGCAACCGC
eekk-d8-kkeee
soosssssssssooss
1520
1536
38
1044

673032
CGCAGGCACCGCAACCG
eekk-d8-kkeee
soosssssssssooss
1521
1537
54
1045

673033
GCGCAGGCACCGCAACC
eekk-d8-kkeee
soosssssssssooss
1522
1538
24
1046

673034
GGCGCAGGCACCGCAAC
eekk-d8-kkeee
soosssssssssooss
1523
1539
17
1047

TABLE 42

Percent inhibition of the C9ORF72 pathogenic associated mRNA variant compared to PBS control by

Deoxy, MOE and cEt antisense oligonucleotides with mixed backbones targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO: 2
NO: 2
%
SEQ

ISIS

Start
Stop
inhib-
ID

NO
Sequence
Chemistry
Linkage
Site
Site
ition
NO

576816
GCCTTACTCTAGGACCAAGA
eeeee-d10-eeeee
sssssssssssssssssss
7990
8009
75
20

673035
TGAGAGCAAGTAGTGGG
ek-d8-ekekeee
sosssssssssoooss
1326
1342
47
898

673036
GTGAGAGCAAGTAGTGG
ek-d8-ekekeee
sosssssssssoooss
1327
1343
57
899

673037
TGTGAGAGCAAGTAGTG
ek-d8-ekekeee
sosssssssssoooss
1328
1344
39
900

673038
CTGTGAGAGCAAGTAGT
ek-d8-ekekeee
sosssssssssoooss
1329
1345
48
901

673039
ACTGTGAGAGCAAGTAG
ek-d8-ekekeee
sosssssssssoooss
1330
1346
35
902

673040
TACTGTGAGAGCAAGTA
ek-d8-ekekeee
sosssssssssoooss
1331
1347
40
903

673041
GTACTGTGAGAGCAAGT
ek-d8-ekekeee
sosssssssssoooss
1332
1348
35
904

673042
AGTACTGTGAGAGCAAG
ek-d8-ekekeee
sosssssssssoooss
1333
1349
26
905

673043
GAGTACTGTGAGAGCAA
ek-d8-ekekeee
sosssssssssoooss
1334
1350
44
906

673044
CGAGTACTGTGAGAGCA
ek-d8-ekekeee
sosssssssssoooss
1335
1351
44
907

673045
GCGAGTACTGTGAGAGC
ek-d8-ekekeee
sosssssssssoooss
1336
1352
36
908

673046
AGCGAGTACTGTGAGAG
ek-d8-ekekeee
sosssssssssoooss
1337
1353
37
909

673047
CAGCGAGTACTGTGAGA
ek-d8-ekekeee
sosssssssssoooss
1338
1354
66
910

673048
TCAGCGAGTACTGTGAG
ek-d8-ekekeee
sosssssssssoooss
1339
1355
22
911

673049
CTCAGCGAGTACTGTGA
ek-d8-ekekeee
sosssssssssoooss
1340
1356
49
912

673050
CCTCAGCGAGTACTGTG
ek-d8-ekekeee
sosssssssssoooss
1341
1357
53
913

673051
CCCTCAGCGAGTACTGT
ek-d8-ekekeee
sosssssssssoooss
1342
1358
52
914

673052
ACCCTCAGCGAGTACTG
ek-d8-ekekeee
sosssssssssoooss
1343
1359
20
915

673053
CACCCTCAGCGAGTACT
ek-d8-ekekeee
sosssssssssoooss
1344
1360
66
916

673054
TCACCCTCAGCGAGTAC
ek-d8-ekekeee
sosssssssssoooss
1345
1361
50
917

673055
TTCACCCTCAGCGAGTA
ek-d8-ekekeee
sosssssssssoooss
1346
1362
35
918

673056
GTTCACCCTCAGCGAGT
ek-d8-ekekeee
sosssssssssoooss
1347
1363
46
919

673057
TGTTCACCCTCAGCGAG
ek-d8-ekekeee
sosssssssssoooss
1348
1364
51
920

673058
TTGTTCACCCTCAGCGA
ek-d8-ekekeee
sosssssssssoooss
1349
1365
57
921

673059
CTTGTTCACCCTCAGCG
ek-d8-ekekeee
sosssssssssoooss
1350
1366
50
922

673060
TCTTGTTCACCCTCAGC
ek-d8-ekekeee
sosssssssssoooss
1351
1367
41
923

673061
TTCTTGTTCACCCTCAG
ek-d8-ekekeee
sosssssssssoooss
1352
1368
14
924

673062
TTTCTTGTTCACCCTCA
ek-d8-ekekeee
sosssssssssoooss
1353
1369
28
925

673063
TTTTCTTGTTCACCCTC
ek-d8-ekekeee
sosssssssssoooss
1354
1370
38
926

673064
CTTTTCTTGTTCACCCT
ek-d8-ekekeee
sosssssssssoooss
1355
1371
38
927

673065
TCTTTTCTTGTTCACCC
ek-d8-ekekeee
sosssssssssoooss
1356
1372
30
928

673066
GTCTTTTCTTGTTCACC
ek-d8-ekekeee
sosssssssssoooss
1357
1373
47
929

673067
GGTCTTTTCTTGTTCAC
ek-d8-ekekeee
sosssssssssoooss
1358
1374
63
930

673068
AGGTCTTTTCTTGTTCA
ek-d8-ekekeee
sosssssssssoooss
1359
1375
71
931

673069
CAGGTCTTTTCTTGTTC
ek-d8-ekekeee
sosssssssssoooss
1360
1376
41
932

673070
TCAGGTCTTTTCTTGTT
ek-d8-ekekeee
sosssssssssoooss
1361
1377
24
933

673071
ATCAGGTCTTTTCTTGT
ek-d8-ekekeee
sosssssssssoooss
1362
1378
53
934

673072
TATCAGGTCTTTTCTTG
ek-d8-ekekeee
sosssssssssoooss
1363
1379
44
935

673073
TTATCAGGTCTTTTCTT
ek-d8-ekekeee
sosssssssssoooss
1364
1380
28
936

673074
ATCTTTATCAGGTCTTT
ek-d8-ekekeee
sosssssssssoooss
1368
1384
57
937

673075
AATCTTTATCAGGTCTT
ek-d8-ekekeee
sosssssssssoooss
1369
1385
49
938

673076
TAATCTTTATCAGGTCT
ek-d8-ekekeee
sosssssssssoooss
1370
1386
40
939

673077
TTAATCTTTATCAGGTC
ek-d8-ekekeee
sosssssssssoooss
1371
1387
33
940

673078
GTTAATCTTTATCAGGT
ek-d8-ekekeee
sosssssssssoooss
1372
1388
46
941

673079
GGTTAATCTTTATCAGG
ek-d8-ekekeee
sosssssssssoooss
1373
1389
87
942

673080
TGGTTAATCTTTATCAG
ek-d8-ekekeee
sosssssssssoooss
1374
1390
35
943

673081
CTGGTTAATCTTTATCA
ek-d8-ekekeee
sosssssssssoooss
1375
1391
56
944

673082
TCTGGTTAATCTTTATC
ek-d8-ekekeee
sosssssssssoooss
1376
1392
59
945

673083
CCCTCCTTGTTTTCTTC
ek-d8-ekekeee
sosssssssssoooss
1391
1407
11
946

673084
TCCCTCCTTGTTTTCTT
ek-d8-ekekeee
sosssssssssoooss
1392
1408
10
947

673085
TTCCCTCCTTGTTTTCT
ek-d8-ekekeee
sosssssssssoooss
1393
1409
8
948

673086
TTTCCCTCCTTGTTTTC
ek-d8-ekekeee
sosssssssssoooss
1394
1410
26
949

673087
GTTTCCCTCCTTGTTTT
ek-d8-ekekeee
sosssssssssoooss
1395
1411
25
950

673088
TGTTTCCCTCCTTGTTT
ek-d8-ekekeee
sosssssssssoooss
1396
1412
62
951

673089
TTGTTTCCCTCCTTGTT
ek-d8-ekekeee
sosssssssssoooss
1397
1413
51
952

673090
GGTTGTTTCCCTCCTTG
ek-d8-ekekeee
sosssssssssoooss
1399
1415
34
953

673091
CGGTTGTTTCCCTCCTT
ek-d8-ekekeee
sosssssssssoooss
1400
1416
13
954

673092
GCGGTTGTTTCCCTCCT
ek-d8-ekekeee
sosssssssssoooss
1401
1417
37
955

673093
TGCGGTTGTTTCCCTCC
ek-d8-ekekeee
sosssssssssoooss
1402
1418
49
956

673094
CTGCGGTTGTTTCCCTC
ek-d8-ekekeee
sosssssssssoooss
1403
1419
15
957

673095
GCTGCGGTTGTTTCCCT
ek-d8-ekekeee
sosssssssssoooss
1404
1420
17
958

673096
GGCTGCGGTTGTTTCCC
ek-d8-ekekeee
sosssssssssoooss
1405
1421
33
959

673097
AGGCTGCGGTTGTTTCC
ek-d8-ekekeee
sosssssssssoooss
1406
1422
43
960

673098
CAGGCTGCGGTTGTTTC
ek-d8-ekekeee
sosssssssssoooss
1407
1423
53
961

673099
ACAGGCTGCGGTTGTTT
ek-d8-ekekeee
sosssssssssoooss
1408
1424
21
962

673100
TACAGGCTGCGGTTGTT
ek-d8-ekekeee
sosssssssssoooss
1409
1425
23
963

673101
CTACAGGCTGCGGTTGT
ek-d8-ekekeee
sosssssssssoooss
1410
1426
16
964

673102
GCTACAGGCTGCGGTTG
ek-d8-ekekeee
sosssssssssoooss
1411
1427
24
965

673103
TGCTACAGGCTGCGGTT
ek-d8-ekekeee
sosssssssssoooss
1412
1428
41
966

673104
TTGCTACAGGCTGCGGT
ek-d8-ekekeee
sosssssssssoooss
1413
1429
30
967

673105
CTTGCTACAGGCTGCGG
ek-d8-ekekeee
sosssssssssoooss
1414
1430
13
968

673106
GCTTGCTACAGGCTGCG
ek-d8-ekekeee
sosssssssssoooss
1415
1431
7
969

673107
AGCTTGCTACAGGCTGC
ek-d8-ekekeee
sosssssssssoooss
1416
1432
7
970

673108
GAGCTTGCTACAGGCTG
ek-d8-ekekeee
sosssssssssoooss
1417
1433
23
971

673109
AGAGCTTGCTACAGGCT
ek-d8-ekekeee
sosssssssssoooss
1418
1434
38
972

673110
CAGAGCTTGCTACAGGC
ek-d8-ekekeee
sosssssssssoooss
1419
1435
22
973

673111
CCAGAGCTTGCTACAGG
ek-d8-ekekeee
sosssssssssoooss
1420
1436
14
974

673112
TCCAGAGCTTGCTACAG
ek-d8-ekekeee
sosssssssssoooss
1421
1437
11
975

673113
TTCCAGAGCTTGCTACA
ek-d8-ekekeee
sosssssssssoooss
1422
1438
24
976

673114
GTTCCAGAGCTTGCTAC
ek-d8-ekekeee
sosssssssssoooss
1423
1439
37
977

673115
AGTTCCAGAGCTTGCTA
ek-d8-ekekeee
sosssssssssoooss
1424
1440
34
978

673116
GAGTTCCAGAGCTTGCT
ek-d8-ekekeee
sosssssssssoooss
1425
1441
21
979

673117
TGAGTTCCAGAGCTTGC
ek-d8-ekekeee
sosssssssssoooss
1426
1442
47
980

673118
CTGAGTTCCAGAGCTTG
ek-d8-ekekeee
sosssssssssoooss
1427
1443
27
981

673119
CCTGAGTTCCAGAGCTT
ek-d8-ekekeee
sosssssssssoooss
1428
1444
44
982

673120
TCCTGAGTTCCAGAGCT
ek-d8-ekekeee
sosssssssssoooss
1429
1445
39
983

673121
CTCCTGAGTTCCAGAGC
ek-d8-ekekeee
sosssssssssoooss
1430
1446
37
984

673122
ACTCCTGAGTTCCAGAG
ek-d8-ekekeee
sosssssssssoooss
1431
1447
40
985

673123
GACTCCTGAGTTCCAGA
ek-d8-ekekeee
sosssssssssoooss
1432
1448
26
986

673124
CGACTCCTGAGTTCCAG
ek-d8-ekekeee
sosssssssssoooss
1433
1449
36
987

673125
GCGACTCCTGAGTTCCA
ek-d8-ekekeee
sosssssssssoooss
1434
1450
55
988

673126
CGCGACTCCTGAGTTCC
ek-d8-ekekeee
sosssssssssoooss
1435
1451
55
989

673127
GCGCGACTCCTGAGTTC
ek-d8-ekekeee
sosssssssssoooss
1436
1452
64
990

673128
CGCGCGACTCCTGAGTT
ek-d8-ekekeee
sosssssssssoooss
1437
1453
59
991

673129
GCGCGCGACTCCTGAGT
ek-d8-ekekeee
sosssssssssoooss
1438
1454
42
992

673130
AGCGCGCGACTCCTGAG
ek-d8-ekekeee
sosssssssssoooss
1439
1455
60
993

673131
TAGCGCGCGACTCCTGA
ek-d8-ekekeee
sosssssssssoooss
1440
1456
59
994

673132
CTAGCGCGCGACTCCTG
ek-d8-ekekeee
sosssssssssoooss
1441
1457
49
995

673133
CCTAGCGCGCGACTCCT
ek-d8-ekekeee
sosssssssssoooss
1442
1458
62
996

673134
CCCTAGCGCGCGACTCC
ek-d8-ekekeee
sosssssssssoooss
1443
1459
62
997

673135
CCCCTAGCGCGCGACTC
ek-d8-ekekeee
sosssssssssoooss
1444
1460
65
998

673136
GCCCCTAGCGCGCGACT
ek-d8-ekekeee
sosssssssssoooss
1445
1461
27
999

673137
GGCCCCTAGCGCGCGAC
ek-d8-ekekeee
sosssssssssoooss
1446
1462
6
1000

673138
CGGCCCCTAGCGCGCGA
ek-d8-ekekeee
sosssssssssoooss
1447
1463
26
1001

673139
CCGGCCCCTAGCGCGCG
ek-d8-ekekeee
sosssssssssoooss
1448
1464
15
1002

673140
CCCGGCCCCTAGCGCGC
ek-d8-ekekeee
sosssssssssoooss
1449
1465
24
1003

673141
CCCCGGCCCCTAGCGCG
ek-d8-ekekeee
sosssssssssoooss
1450
1466
27
1004

673142
GCCCCGGCCCCTAGCGC
ek-d8-ekekeee
sosssssssssoooss
1451
1467
28
1005

673143
GGCCCCGGCCCCTAGCG
ek-d8-ekekeee
sosssssssssoooss
1452
1468
28
1006

673144
CGGCCCCGGCCCCTAGC
ek-d8-ekekeee
sosssssssssoooss
1453
1469
49
1007

673145
CCGGCCCCGGCCCCTAG
ek-d8-ekekeee
sosssssssssoooss
1454
1470
24
1008

673146
CCCGGCCCCGGCCCCTA
ek-d8-ekekeee
sosssssssssoooss
1455
1471
32
1009

673147
ACGCCCCGGCCCCGGCC
ek-d8-ekekeee
sosssssssssoooss
1465
1481
12
1010

673148
CACGCCCCGGCCCCGGC
ek-d8-ekekeee
sosssssssssoooss
1466
1482
4
1011

673149
CCACGCCCCGGCCCCGG
ek-d8-ekekeee
sosssssssssoooss
1467
1483
18
1012

673150
ACCACGCCCCGGCCCCG
ek-d8-ekekeee
sosssssssssoooss
1468
1484
5
1013

673151
GACCACGCCCCGGCCCC
ek-d8-ekekeee
sosssssssssoooss
1469
1485
20
1014

673152
CGACCACGCCCCGGCCC
ek-d8-ekekeee
sosssssssssoooss
1470
1486
52
1015

673153
CCGACCACGCCCCGGCC
ek-d8-ekekeee
sosssssssssoooss
1471
1487
4
1016

673154
CCCGACCACGCCCCGGC
ek-d8-ekekeee
sosssssssssoooss
1472
1488
9
1017

673155
CCCCGACCACGCCCCGG
ek-d8-ekekeee
sosssssssssoooss
1473
1489
23
1018

673156
GCCCCGACCACGCCCCG
ek-d8-ekekeee
sosssssssssoooss
1474
1490
9
1019

673157
CGCCCCGACCACGCCCC
ek-d8-ekekeee
sosssssssssoooss
1475
1491
22
1020

673158
CCGCCCCGACCACGCCC
ek-d8-ekekeee
sosssssssssoooss
1476
1492
27
1021

673159
CCCGCCCCGACCACGCC
ek-d8-ekekeee
sosssssssssoooss
1477
1493
49
1022

673160
GCCCGCCCCGACCACGC
ek-d8-ekekeee
sosssssssssoooss
1478
1494
33
1023

673161
GGCCCGCCCCGACCACG
ek-d8-ekekeee
sosssssssssoooss
1479
1495
7
1024

673162
GGGCCCGCCCCGACCAC
ek-d8-ekekeee
sosssssssssoooss
1480
1496
8
1025

673163
CGGGCCCGCCCCGACCA
ek-d8-ekekeee
sosssssssssoooss
1481
1497
12
1026

673164
CCGGGCCCGCCCCGACC
ek-d8-ekekeee
sosssssssssoooss
1482
1498
2
1027

673165
CCCGGGCCCGCCCCGAC
ek-d8-ekekeee
sosssssssssoooss
1483
1499
13
1028

673166
GCAGCCCCGCCCCGGGC
ek-d8-ekekeee
sosssssssssoooss
1505
1521
7
1029

673167
CGCAGCCCCGCCCCGGG
ek-d8-ekekeee
sosssssssssoooss
1506
1522
14
1030

673168
CCGCAGCCCCGCCCCGG
ek-d8-ekekeee
sosssssssssoooss
1507
1523
17
1031

673169
ACCGCAGCCCCGCCCCG
ek-d8-ekekeee
sosssssssssoooss
1508
1524
44
1032

673170
AACCGCAGCCCCGCCCC
ek-d8-ekekeee
sosssssssssoooss
1509
1525
40
1033

673171
CAACCGCAGCCCCGCCC
ek-d8-ekekeee
sosssssssssoooss
1510
1526
45
1034

673172
GCAACCGCAGCCCCGCC
ek-d8-ekekeee
sosssssssssoooss
1511
1527
28
1035

673173
CGCAACCGCAGCCCCGC
ek-d8-ekekeee
sosssssssssoooss
1512
1528
31
1036

673174
CCGCAACCGCAGCCCCG
ek-d8-ekekeee
sosssssssssoooss
1513
1529
38
1037

673175
ACCGCAACCGCAGCCCC
ek-d8-ekekeee
sosssssssssoooss
1514
1530
47
1038

673176
CACCGCAACCGCAGCCC
ek-d8-ekekeee
sosssssssssoooss
1515
1531
37
1039

673177
GCACCGCAACCGCAGCC
ek-d8-ekekeee
sosssssssssoooss
1516
1532
41
1040

673178
GGCACCGCAACCGCAGC
ek-d8-ekekeee
sosssssssssoooss
1517
1533
34
1041

673179
AGGCACCGCAACCGCAG
ek-d8-ekekeee
sosssssssssoooss
1518
1534
19
1042

673180
CAGGCACCGCAACCGCA
ek-d8-ekekeee
sosssssssssoooss
1519
1535
36
1043

673181
GCAGGCACCGCAACCGC
ek-d8-ekekeee
sosssssssssoooss
1520
1536
33
1044

673182
CGCAGGCACCGCAACCG
ek-d8-ekekeee
sosssssssssoooss
1521
1537
37
1045

673183
GCGCAGGCACCGCAACC
ek-d8-ekekeee
sosssssssssoooss
1522
1538
6
1046

673184
GGCGCAGGCACCGCAAC
ek-d8-ekekeee
sosssssssssoooss
1523
1539
11
1047

TABLE 43

Percent inhibition of the C9ORF72 pathogenic associated mRNA variant compared to PBS control by

Deoxy, MOE and cEt antisense oligonucleotides with mixed backbones targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO: 2
NO: 2
%
SEQ

ISIS

Start
Stop
inhib-
ID

NO
Sequence
Chemistry
Linkage
Site
Site
ition
NO

576816
GCCTTACTCTAGGACCAAGA
eeeee-d10-eeeee
sssssssssssssssssss
7990
8009
79
20

673185
TGAGAGCAAGTAGTGGG
keke-d8-ekeke
soosssssssssooss
1326
1342
37
898

673186
GTGAGAGCAAGTAGTGG
keke-d8-ekeke
soosssssssssooss
1327
1343
39
899

673187
TGTGAGAGCAAGTAGTG
keke-d8-ekeke
soosssssssssooss
1328
1344
33
900

673188
CTGTGAGAGCAAGTAGT
keke-d8-ekeke
soosssssssssooss
1329
1345
40
901

673189
ACTGTGAGAGCAAGTAG
keke-d8-ekeke
soosssssssssooss
1330
1346
26
902

673190
TACTGTGAGAGCAAGTA
keke-d8-ekeke
soosssssssssooss
1331
1347
23
903

673191
GTACTGTGAGAGCAAGT
keke-d8-ekeke
soosssssssssooss
1332
1348
50
904

673192
AGTACTGTGAGAGCAAG
keke-d8-ekeke
soosssssssssooss
1333
1349
39
905

673193
GAGTACTGTGAGAGCAA
keke-d8-ekeke
soosssssssssooss
1334
1350
69
906

673194
CGAGTACTGTGAGAGCA
keke-d8-ekeke
soosssssssssooss
1335
1351
72
907

673195
GCGAGTACTGTGAGAGC
keke-d8-ekeke
soosssssssssooss
1336
1352
51
908

673196
AGCGAGTACTGTGAGAG
keke-d8-ekeke
soosssssssssooss
1337
1353
51
909

673197
CAGCGAGTACTGTGAGA
keke-d8-ekeke
soosssssssssooss
1338
1354
59
910

673198
TCAGCGAGTACTGTGAG
keke-d8-ekeke
soosssssssssooss
1339
1355
33
911

673199
CTCAGCGAGTACTGTGA
keke-d8-ekeke
soosssssssssooss
1340
1356
32
912

673200
CCTCAGCGAGTACTGTG
keke-d8-ekeke
soosssssssssooss
1341
1357
46
913

673201
CCCTCAGCGAGTACTGT
keke-d8-ekeke
soosssssssssooss
1342
1358
53
914

673202
ACCCTCAGCGAGTACTG
keke-d8-ekeke
soosssssssssooss
1343
1359
58
915

673203
CACCCTCAGCGAGTACT
keke-d8-ekeke
soosssssssssooss
1344
1360
68
916

673204
TCACCCTCAGCGAGTAC
keke-d8-ekeke
soosssssssssooss
1345
1361
70
917

673205
TTCACCCTCAGCGAGTA
keke-d8-ekeke
soosssssssssooss
1346
1362
47
918

673206
GTTCACCCTCAGCGAGT
keke-d8-ekeke
soosssssssssooss
1347
1363
65
919

673207
TGTTCACCCTCAGCGAG
keke-d8-ekeke
soosssssssssooss
1348
1364
31
920

673208
TTGTTCACCCTCAGCGA
keke-d8-ekeke
soosssssssssooss
1349
1365
51
921

673209
CTTGTTCACCCTCAGCG
keke-d8-ekeke
soosssssssssooss
1350
1366
49
922

673210
TCTTGTTCACCCTCAGC
keke-d8-ekeke
soosssssssssooss
1351
1367
61
923

673211
TTCTTGTTCACCCTCAG
keke-d8-ekeke
soosssssssssooss
1352
1368
49
924

673212
TTTCTTGTTCACCCTCA
keke-d8-ekeke
soosssssssssooss
1353
1369
42
925

673213
TTTTCTTGTTCACCCTC
keke-d8-ekeke
soosssssssssooss
1354
1370
36
926

673214
CTTTTCTTGTTCACCCT
keke-d8-ekeke
soosssssssssooss
1355
1371
41
927

673215
TCTTTTCTTGTTCACCC
keke-d8-ekeke
soosssssssssooss
1356
1372
41
928

673216
GTCTTTTCTTGTTCACC
keke-d8-ekeke
soosssssssssooss
1357
1373
36
929

673217
GGTCTTTTCTTGTTCAC
keke-d8-ekeke
soosssssssssooss
1358
1374
29
930

673218
AGGTCTTTTCTTGTTCA
keke-d8-ekeke
soosssssssssooss
1359
1375
50
931

673219
CAGGTCTTTTCTTGTTC
keke-d8-ekeke
soosssssssssooss
1360
1376
60
932

673220
TCAGGTCTTTTCTTGTT
keke-d8-ekeke
soosssssssssooss
1361
1377
34
933

673221
ATCAGGTCTTTTCTTGT
keke-d8-ekeke
soosssssssssooss
1362
1378
33
934

673222
TATCAGGTCTTTTCTTG
keke-d8-ekeke
soosssssssssooss
1363
1379
31
935

673223
TTATCAGGTCTTTTCTT
keke-d8-ekeke
soosssssssssooss
1364
1380
10
936

673224
ATCTTTATCAGGTCTTT
keke-d8-ekeke
soosssssssssooss
1368
1384
61
937

673225
AATCTTTATCAGGTCTT
keke-d8-ekeke
soosssssssssooss
1369
1385
74
938

673226
TAATCTTTATCAGGTCT
keke-d8-ekeke
soosssssssssooss
1370
1386
62
939

673227
TTAATCTTTATCAGGTC
keke-d8-ekeke
soosssssssssooss
1371
1387
51
940

673228
GTTAATCTTTATCAGGT
keke-d8-ekeke
soosssssssssooss
1372
1388
73
941

673229
GGTTAATCTTTATCAGG
keke-d8-ekeke
soosssssssssooss
1373
1389
66
942

673230
TGGTTAATCTTTATCAG
keke-d8-ekeke
soosssssssssooss
1374
1390
38
943

673231
CTGGTTAATCTTTATCA
keke-d8-ekeke
soosssssssssooss
1375
1391
41
944

673232
TCTGGTTAATCTTTATC
keke-d8-ekeke
soosssssssssooss
1376
1392
37
945

673233
CCCTCCTTGTTTTCTTC
keke-d8-ekeke
soosssssssssooss
1391
1407
26
946

673234
TCCCTCCTTGTTTTCTT
keke-d8-ekeke
soosssssssssooss
1392
1408
10
947

673235
TTCCCTCCTTGTTTTCT
keke-d8-ekeke
soosssssssssooss
1393
1409
20
948

673236
TTTCCCTCCTTGTTTTC
keke-d8-ekeke
soosssssssssooss
1394
1410
1
949

673237
GTTTCCCTCCTTGTTTT
keke-d8-ekeke
soosssssssssooss
1395
1411
2
950

673238
TGTTTCCCTCCTTGTTT
keke-d8-ekeke
soosssssssssooss
1396
1412
26
951

673239
TTGTTTCCCTCCTTGTT
keke-d8-ekeke
soosssssssssooss
1397
1413
28
952

673240
GGTTGTTTCCCTCCTTG
keke-d8-ekeke
soosssssssssooss
1399
1415
70
953

673241
CGGTTGTTTCCCTCCTT
keke-d8-ekeke
soosssssssssooss
1400
1416
36
954

673242
GCGGTTGTTTCCCTCCT
keke-d8-ekeke
soosssssssssooss
1401
1417
21
955

673243
TGCGGTTGTTTCCCTCC
keke-d8-ekeke
soosssssssssooss
1402
1418
24
956

673244
CTGCGGTTGTTTCCCTC
keke-d8-ekeke
soosssssssssooss
1403
1419
41
957

673245
GCTGCGGTTGTTTCCCT
keke-d8-ekeke
soosssssssssooss
1404
1420
23
958

673246
GGCTGCGGTTGTTTCCC
keke-d8-ekeke
soosssssssssooss
1405
1421
39
959

673247
AGGCTGCGGTTGTTTCC
keke-d8-ekeke
soosssssssssooss
1406
1422
50
960

673248
CAGGCTGCGGTTGTTTC
keke-d8-ekeke
soosssssssssooss
1407
1423
32
961

673249
ACAGGCTGCGGTTGTTT
keke-d8-ekeke
soosssssssssooss
1408
1424
28
962

673250
TACAGGCTGCGGTTGTT
keke-d8-ekeke
soosssssssssooss
1409
1425
41
963

673251
CTACAGGCTGCGGTTGT
keke-d8-ekeke
soosssssssssooss
1410
1426
18
964

673252
GCTACAGGCTGCGGTTG
keke-d8-ekeke
soosssssssssooss
1411
1427
28
965

673253
TGCTACAGGCTGCGGTT
keke-d8-ekeke
soosssssssssooss
1412
1428
28
966

673254
TTGCTACAGGCTGCGGT
keke-d8-ekeke
soosssssssssooss
1413
1429
9
967

673255
CTTGCTACAGGCTGCGG
keke-d8-ekeke
soosssssssssooss
1414
1430
34
968

673256
GCTTGCTACAGGCTGCG
keke-d8-ekeke
soosssssssssooss
1415
1431
28
969

673257
AGCTTGCTACAGGCTGC
keke-d8-ekeke
soosssssssssooss
1416
1432
25
970

673258
GAGCTTGCTACAGGCTG
keke-d8-ekeke
soosssssssssooss
1417
1433
23
971

673259
AGAGCTTGCTACAGGCT
keke-d8-ekeke
soosssssssssooss
1418
1434
0
972

673260
CAGAGCTTGCTACAGGC
keke-d8-ekeke
soosssssssssooss
1419
1435
4
973

673261
CCAGAGCTTGCTACAGG
keke-d8-ekeke
soosssssssssooss
1420
1436
40
974

673262
TCCAGAGCTTGCTACAG
keke-d8-ekeke
soosssssssssooss
1421
1437
35
975

673263
TTCCAGAGCTTGCTACA
keke-d8-ekeke
soosssssssssooss
1422
1438
23
976

673264
GTTCCAGAGCTTGCTAC
keke-d8-ekeke
soosssssssssooss
1423
1439
36
977

673265
AGTTCCAGAGCTTGCTA
keke-d8-ekeke
soosssssssssooss
1424
1440
54
978

673266
GAGTTCCAGAGCTTGCT
keke-d8-ekeke
soosssssssssooss
1425
1441
32
979

673267
TGAGTTCCAGAGCTTGC
keke-d8-ekeke
soosssssssssooss
1426
1442
32
980

673268
CTGAGTTCCAGAGCTTG
keke-d8-ekeke
soosssssssssooss
1427
1443
44
981

673269
CCTGAGTTCCAGAGCTT
keke-d8-ekeke
soosssssssssooss
1428
1444
70
982

673270
TCCTGAGTTCCAGAGCT
keke-d8-ekeke
soosssssssssooss
1429
1445
43
983

673271
CTCCTGAGTTCCAGAGC
keke-d8-ekeke
soosssssssssooss
1430
1446
41
984

673272
ACTCCTGAGTTCCAGAG
keke-d8-ekeke
soosssssssssooss
1431
1447
23
985

673273
GACTCCTGAGTTCCAGA
keke-d8-ekeke
soosssssssssooss
1432
1448
48
986

673274
CGACTCCTGAGTTCCAG
keke-d8-ekeke
soosssssssssooss
1433
1449
34
987

673275
GCGACTCCTGAGTTCCA
keke-d8-ekeke
soosssssssssooss
1434
1450
64
988

673276
CGCGACTCCTGAGTTCC
keke-d8-ekeke
soosssssssssooss
1435
1451
65
989

673277
GCGCGACTCCTGAGTTC
keke-d8-ekeke
soosssssssssooss
1436
1452
62
990

673278
CGCGCGACTCCTGAGTT
keke-d8-ekeke
soosssssssssooss
1437
1453
45
991

673279
GCGCGCGACTCCTGAGT
keke-d8-ekeke
soosssssssssooss
1438
1454
52
992

673280
AGCGCGCGACTCCTGAG
keke-d8-ekeke
soosssssssssooss
1439
1455
65
993

673281
TAGCGCGCGACTCCTGA
keke-d8-ekeke
soosssssssssooss
1440
1456
89
994

673282
CTAGCGCGCGACTCCTG
keke-d8-ekeke
soosssssssssooss
1441
1457
69
995

673283
CCTAGCGCGCGACTCCT
keke-d8-ekeke
soosssssssssooss
1442
1458
68
996

673284
CCCTAGCGCGCGACTCC
keke-d8-ekeke
soosssssssssooss
1443
1459
73
997

673285
CCCCTAGCGCGCGACTC
keke-d8-ekeke
soosssssssssooss
1444
1460
70
998

673286
GCCCCTAGCGCGCGACT
keke-d8-ekeke
soosssssssssooss
1445
1461
45
999

673287
GGCCCCTAGCGCGCGAC
keke-d8-ekeke
soosssssssssooss
1446
1462
33
1000

673288
CGGCCCCTAGCGCGCGA
keke-d8-ekeke
soosssssssssooss
1447
1463
29
1001

673289
CCGGCCCCTAGCGCGCG
keke-d8-ekeke
soosssssssssooss
1448
1464
0
1002

673290
CCCGGCCCCTAGCGCGC
keke-d8-ekeke
soosssssssssooss
1449
1465
31
1003

673291
CCCCGGCCCCTAGCGCG
keke-d8-ekeke
soosssssssssooss
1450
1466
28
1004

673292
GCCCCGGCCCCTAGCGC
keke-d8-ekeke
soosssssssssooss
1451
1467
12
1005

673293
GGCCCCGGCCCCTAGCG
keke-d8-ekeke
soosssssssssooss
1452
1468
29
1006

673294
CGGCCCCGGCCCCTAGC
keke-d8-ekeke
soosssssssssooss
1453
1469
39
1007

673295
CCGGCCCCGGCCCCTAG
keke-d8-ekeke
soosssssssssooss
1454
1470
28
1008

673296
CCCGGCCCCGGCCCCTA
keke-d8-ekeke
soosssssssssooss
1455
1471
4
1009

673297
ACGCCCCGGCCCCGGCC
keke-d8-ekeke
soosssssssssooss
1465
1481
17
1010

673298
CACGCCCCGGCCCCGGC
keke-d8-ekeke
soosssssssssooss
1466
1482
35
1011

673299
CCACGCCCCGGCCCCGG
keke-d8-ekeke
soosssssssssooss
1467
1483
28
1012

673300
ACCACGCCCCGGCCCCG
keke-d8-ekeke
soosssssssssooss
1468
1484
21
1013

673301
GACCACGCCCCGGCCCC
keke-d8-ekeke
soosssssssssooss
1469
1485
28
1014

673302
CGACCACGCCCCGGCCC
keke-d8-ekeke
soosssssssssooss
1470
1486
46
1015

673303
CCGACCACGCCCCGGCC
keke-d8-ekeke
soosssssssssooss
1471
1487
40
1016

673304
CCCGACCACGCCCCGGC
keke-d8-ekeke
soosssssssssooss
1472
1488
16
1017

673305
CCCCGACCACGCCCCGG
keke-d8-ekeke
soosssssssssooss
1473
1489
11
1018

673306
GCCCCGACCACGCCCCG
keke-d8-ekeke
soosssssssssooss
1474
1490
13
1019

673307
CGCCCCGACCACGCCCC
keke-d8-ekeke
soosssssssssooss
1475
1491
43
1020

673308
CCGCCCCGACCACGCCC
keke-d8-ekeke
soosssssssssooss
1476
1492
20
1021

673309
CCCGCCCCGACCACGCC
keke-d8-ekeke
soosssssssssooss
1477
1493
16
1022

673310
GCCCGCCCCGACCACGC
keke-d8-ekeke
soosssssssssooss
1478
1494
44
1023

673311
GGCCCGCCCCGACCACG
keke-d8-ekeke
soosssssssssooss
1479
1495
33
1024

673312
GGGCCCGCCCCGACCAC
keke-d8-ekeke
soosssssssssooss
1480
1496
1
1025

673313
CGGGCCCGCCCCGACCA
keke-d8-ekeke
soosssssssssooss
1481
1497
0
1026

673314
CCGGGCCCGCCCCGACC
keke-d8-ekeke
soosssssssssooss
1482
1498
0
1027

673315
CCCGGGCCCGCCCCGAC
keke-d8-ekeke
soosssssssssooss
1483
1499
8
1028

673316
GCAGCCCCGCCCCGGGC
keke-d8-ekeke
soosssssssssooss
1505
1521
31
1029

673317
CGCAGCCCCGCCCCGGG
keke-d8-ekeke
soosssssssssooss
1506
1522
4
1030

673318
CCGCAGCCCCGCCCCGG
keke-d8-ekeke
soosssssssssooss
1507
1523
18
1031

673319
ACCGCAGCCCCGCCCCG
keke-d8-ekeke
soosssssssssooss
1508
1524
16
1032

673320
AACCGCAGCCCCGCCCC
keke-d8-ekeke
soosssssssssooss
1509
1525
39
1033

673321
CAACCGCAGCCCCGCCC
keke-d8-ekeke
soosssssssssooss
1510
1526
50
1034

673322
GCAACCGCAGCCCCGCC
keke-d8-ekeke
soosssssssssooss
1511
1527
45
1035

673323
CGCAACCGCAGCCCCGC
keke-d8-ekeke
soosssssssssooss
1512
1528
56
1036

673324
CCGCAACCGCAGCCCCG
keke-d8-ekeke
soosssssssssooss
1513
1529
10
1037

673325
ACCGCAACCGCAGCCCC
keke-d8-ekeke
soosssssssssooss
1514
1530
43
1038

673326
CACCGCAACCGCAGCCC
keke-d8-ekeke
soosssssssssooss
1515
1531
49
1039

673327
GCACCGCAACCGCAGCC
keke-d8-ekeke
soosssssssssooss
1516
1532
35
1040

673328
GGCACCGCAACCGCAGC
keke-d8-ekeke
soosssssssssooss
1517
1533
24
1041

673329
AGGCACCGCAACCGCAG
keke-d8-ekeke
soosssssssssooss
1518
1534
52
1042

673330
CAGGCACCGCAACCGCA
keke-d8-ekeke
soosssssssssooss
1519
1535
38
1043

673331
GCAGGCACCGCAACCGC
keke-d8-ekeke
soosssssssssooss
1520
1536
51
1044

673332
CGCAGGCACCGCAACCG
keke-d8-ekeke
soosssssssssooss
1521
1537
59
1045

673333
GCGCAGGCACCGCAACC
keke-d8-ekeke
soosssssssssooss
1522
1538
24
1046

673334
GGCGCAGGCACCGCAAC
keke-d8-ekeke
soosssssssssooss
1523
1539
18
1047

TABLE 44

Percent inhibition of the C9ORF72 pathogenic associated mRNA variant compared to PBS control by

Deoxy, MOE and cEt antisense oligonucleotides with mixed backbones targeting SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO:
NO:

2
2
%
SEQ

ISIS

Start
Stop
inhib-
ID

NO
Sequence
Chemistry
Linkage
Site
Site
ition
NO

576816
GCCTTACTCTAGGACCAAGA
eeeee-d10-eeeee
SSSSSSSSSSSSSSSSSSS
7990
8009
60
20

673335
TGAGAGCAAGTAGTGGG
ekek-d8-kekee
soosssssssssooss
1326
1342
28
898

673336
GTGAGAGCAAGTAGTGG
ekek-d8-kekee
soosssssssssooss
1327
1343
27
899

673337
TGTGAGAGCAAGTAGTG
ekek-d8-kekee
soosssssssssooss
1328
1344
32
900

673338
CTGTGAGAGCAAGTAGT
ekek-d8-kekee
soosssssssssooss
1329
1345
43
901

673339
ACTGTGAGAGCAAGTAG
ekek-d8-kekee
soosssssssssooss
1330
1346
22
902

673340
TACTGTGAGAGCAAGTA
ekek-d8-kekee
soosssssssssooss
1331
1347
20
903

673341
GTACTGTGAGAGCAAGT
ekek-d8-kekee
soosssssssssooss
1332
1348
53
904

673342
AGTACTGTGAGAGCAAG
ekek-d8-kekee
soosssssssssooss
1333
1349
20
905

673343
GAGTACTGTGAGAGCAA
ekek-d8-kekee
soosssssssssooss
1334
1350
50
906

673344
CGAGTACTGTGAGAGCA
ekek-d8-kekee
soosssssssssooss
1335
1351
45
907

673345
GCGAGTACTGTGAGAGC
ekek-d8-kekee
soosssssssssooss
1336
1352
45
908

673346
AGCGAGTACTGTGAGAG
ekek-d8-kekee
soosssssssssooss
1337
1353
53
909

673347
CAGCGAGTACTGTGAGA
ekek-d8-kekee
soosssssssssooss
1338
1354
35
910

673348
TCAGCGAGTACTGTGAG
ekek-d8-kekee
soosssssssssooss
1339
1355
36
911

673349
CTCAGCGAGTACTGTGA
ekek-d8-kekee
soosssssssssooss
1340
1356
19
912

673350
CCTCAGCGAGTACTGTG
ekek-d8-kekee
soosssssssssooss
1341
1357
21
913

673351
CCCTCAGCGAGTACTGT
ekek-d8-kekee
soosssssssssooss
1342
1358
46
914

673352
ACCCTCAGCGAGTACTG
ekek-d8-kekee
soosssssssssooss
1343
1359
43
915

673353
CACCCTCAGCGAGTACT
ekek-d8-kekee
soosssssssssooss
1344
1360
46
916

673354
TCACCCTCAGCGAGTAC
ekek-d8-kekee
soosssssssssooss
1345
1361
40
917

673355
TTCACCCTCAGCGAGTA
ekek-d8-kekee
soosssssssssooss
1346
1362
33
918

673356
GTTCACCCTCAGCGAGT
ekek-d8-kekee
soosssssssssooss
1347
1363
11
919

673357
TGTTCACCCTCAGCGAG
ekek-d8-kekee
soosssssssssooss
1348
1364
34
920

673358
TTGTTCACCCTCAGCGA
ekek-d8-kekee
soosssssssssooss
1349
1365
47
921

673359
CTTGTTCACCCTCAGCG
ekek-d8-kekee
soosssssssssooss
1350
1366
54
922

673360
TCTTGTTCACCCTCAGC
ekek-d8-kekee
soosssssssssooss
1351
1367
26
923

673361
TTCTTGTTCACCCTCAG
ekek-d8-kekee
soosssssssssooss
1352
1368
36
924

673362
TTTCTTGTTCACCCTCA
ekek-d8-kekee
soosssssssssooss
1353
1369
29
925

673363
TTTTCTTGTTCACCCTC
ekek-d8-kekee
soosssssssssooss
1354
1370
29
926

673364
CTTTTCTTGTTCACCCT
ekek-d8-kekee
soosssssssssooss
1355
1371
23
927

673365
TCTTTTCTTGTTCACCC
ekek-d8-kekee
soosssssssssooss
1356
1372
36
928

673366
GTCTTTTCTTGTTCACC
ekek-d8-kekee
soosssssssssooss
1357
1373
27
929

673367
GGTCTTTTCTTGTTCAC
ekek-d8-kekee
soosssssssssooss
1358
1374
21
930

673368
AGGTCTTTTCTTGTTCA
ekek-d8-kekee
soosssssssssooss
1359
1375
29
931

673369
CAGGTCTTTTCTTGTTC
ekek-d8-kekee
soosssssssssooss
1360
1376
65
932

673370
TCAGGTCTTTTCTTGTT
ekek-d8-kekee
soosssssssssooss
1361
1377
2
933

673371
ATCAGGTCTTTTCTTGT
ekek-d8-kekee
soosssssssssooss
1362
1378
23
934

673372
TATCAGGTCTTTTCTTG
ekek-d8-kekee
soosssssssssooss
1363
1379
40
935

673373
TTATCAGGTCTTTTCTT
ekek-d8-kekee
soosssssssssooss
1364
1380
13
936

673374
ATCTTTATCAGGTCTTT
ekek-d8-kekee
soosssssssssooss
1368
1384
76
937

673375
AATCTTTATCAGGTCTT
ekek-d8-kekee
soosssssssssooss
1369
1385
62
938

673376
TAATCTTTATCAGGTCT
ekek-d8-kekee
soosssssssssooss
1370
1386
39
939

673377
TTAATCTTTATCAGGTC
ekek-d8-kekee
soosssssssssooss
1371
1387
71
940

673378
GTTAATCTTTATCAGGT
ekek-d8-kekee
soosssssssssooss
1372
1388
61
941

673379
GGTTAATCTTTATCAGG
ekek-d8-kekee
soosssssssssooss
1373
1389
74
942

673380
TGGTTAATCTTTATCAG
ekek-d8-kekee
soosssssssssooss
1374
1390
24
943

673381
CTGGTTAATCTTTATCA
ekek-d8-kekee
soosssssssssooss
1375
1391
32
944

673382
TCTGGTTAATCTTTATC
ekek-d8-kekee
soosssssssssooss
1376
1392
38
945

673383
CCCTCCTTGTTTTCTTC
ekek-d8-kekee
soosssssssssooss
1391
1407
21
946

673384
TCCCTCCTTGTTTTCTT
ekek-d8-kekee
soosssssssssooss
1392
1408
0
947

673385
TTCCCTCCTTGTTTTCT
ekek-d8-kekee
soosssssssssooss
1393
1409
0
948

673386
TTTCCCTCCTTGTTTTC
ekek-d8-kekee
soosssssssssooss
1394
1410
5
949

673387
GTTTCCCTCCTTGTTTT
ekek-d8-kekee
soosssssssssooss
1395
1411
0
950

673388
TGTTTCCCTCCTTGTTT
ekek-d8-kekee
soosssssssssooss
1396
1412
0
951

673389
TTGTTTCCCTCCTTGTT
ekek-d8-kekee
soosssssssssooss
1397
1413
22
952

673390
GGTTGTTTCCCTCCTTG
ekek-d8-kekee
soosssssssssooss
1399
1415
55
953

673391
CGGTTGTTTCCCTCCTT
ekek-d8-kekee
soosssssssssooss
1400
1416
25
954

673392
GCGGTTGTTTCCCTCCT
ekek-d8-kekee
soosssssssssooss
1401
1417
19
955

673393
TGCGGTTGTTTCCCTCC
ekek-d8-kekee
soosssssssssooss
1402
1418
0
956

673394
CTGCGGTTGTTTCCCTC
ekek-d8-kekee
soosssssssssooss
1403
1419
13
957

673395
GCTGCGGTTGTTTCCCT
ekek-d8-kekee
soosssssssssooss
1404
1420
19
958

673396
GGCTGCGGTTGTTTCCC
ekek-d8-kekee
soosssssssssooss
1405
1421
27
959

673397
AGGCTGCGGTTGTTTCC
ekek-d8-kekee
soosssssssssooss
1406
1422
13
960

673398
CAGGCTGCGGTTGTTTC
ekek-d8-kekee
soosssssssssooss
1407
1423
22
961

673399
ACAGGCTGCGGTTGTTT
ekek-d8-kekee
soosssssssssooss
1408
1424
5
962

673400
TACAGGCTGCGGTTGTT
ekek-d8-kekee
soosssssssssooss
1409
1425
0
963

673401
CTACAGGCTGCGGTTGT
ekek-d8-kekee
soosssssssssooss
1410
1426
0
964

673402
GCTACAGGCTGCGGTTG
ekek-d8-kekee
soosssssssssooss
1411
1427
39
965

673403
TGCTACAGGCTGCGGTT
ekek-d8-kekee
soosssssssssooss
1412
1428
20
966

673404
TTGCTACAGGCTGCGGT
ekek-d8-kekee
soosssssssssooss
1413
1429
24
967

673405
CTTGCTACAGGCTGCGG
ekek-d8-kekee
soosssssssssooss
1414
1430
0
968

673406
GCTTGCTACAGGCTGCG
ekek-d8-kekee
soosssssssssooss
1415
1431
18
969

673407
AGCTTGCTACAGGCTGC
ekek-d8-kekee
soosssssssssooss
1416
1432
3
970

673408
GAGCTTGCTACAGGCTG
ekek-d8-kekee
soosssssssssooss
1417
1433
13
971

673409
AGAGCTTGCTACAGGCT
ekek-d8-kekee
soosssssssssooss
1418
1434
29
972

673410
CAGAGCTTGCTACAGGC
ekek-d8-kekee
soosssssssssooss
1419
1435
22
973

673411
CCAGAGCTTGCTACAGG
ekek-d8-kekee
soosssssssssooss
1420
1436
24
974

673412
TCCAGAGCTTGCTACAG
ekek-d8-kekee
soosssssssssooss
1421
1437
4
975

673413
TTCCAGAGCTTGCTACA
ekek-d8-kekee
soosssssssssooss
1422
1438
0
976

673414
GTTCCAGAGCTTGCTAC
ekek-d8-kekee
soosssssssssooss
1423
1439
19
977

673415
AGTTCCAGAGCTTGCTA
ekek-d8-kekee
soosssssssssooss
1424
1440
0
978

673416
GAGTTCCAGAGCTTGCT
ekek-d8-kekee
soosssssssssooss
1425
1441
48
979

673417
TGAGTTCCAGAGCTTGC
ekek-d8-kekee
soosssssssssooss
1426
1442
14
980

673418
CTGAGTTCCAGAGCTTG
ekek-d8-kekee
soosssssssssooss
1427
1443
37
981

673419
CCTGAGTTCCAGAGCTT
ekek-d8-kekee
soosssssssssooss
1428
1444
80
982

673420
TCCTGAGTTCCAGAGCT
ekek-d8-kekee
soosssssssssooss
1429
1445
26
983

673421
CTCCTGAGTTCCAGAGC
ekek-d8-kekee
soosssssssssooss
1430
1446
5
984

673422
ACTCCTGAGTTCCAGAG
ekek-d8-kekee
soosssssssssooss
1431
1447
23
985

673423
GACTCCTGAGTTCCAGA
ekek-d8-kekee
soosssssssssooss
1432
1448
37
986

673424
CGACTCCTGAGTTCCAG
ekek-d8-kekee
soosssssssssooss
1433
1449
5
987

673425
GCGACTCCTGAGTTCCA
ekek-d8-kekee
soosssssssssooss
1434
1450
39
988

673426
CGCGACTCCTGAGTTCC
ekek-d8-kekee
soosssssssssooss
1435
1451
46
989

673427
GCGCGACTCCTGAGTTC
ekek-d8-kekee
soosssssssssooss
1436
1452
50
990

673428
CGCGCGACTCCTGAGTT
ekek-d8-kekee
soosssssssssooss
1437
1453
19
991

673429
GCGCGCGACTCCTGAGT
ekek-d8-kekee
soosssssssssooss
1438
1454
13
992

673430
AGCGCGCGACTCCTGAG
ekek-d8-kekee
soosssssssssooss
1439
1455
51
993

673431
TAGCGCGCGACTCCTGA
ekek-d8-kekee
soosssssssssooss
1440
1456
83
994

673432
CTAGCGCGCGACTCCTG
ekek-d8-kekee
soosssssssssooss
1441
1457
60
995

673433
CCTAGCGCGCGACTCCT
ekek-d8-kekee
soosssssssssooss
1442
1458
37
996

673434
CCCTAGCGCGCGACTCC
ekek-d8-kekee
soosssssssssooss
1443
1459
60
997

673435
CCCCTAGCGCGCGACTC
ekek-d8-kekee
soosssssssssooss
1444
1460
62
998

673436
GCCCCTAGCGCGCGACT
ekek-d8-kekee
soosssssssssooss
1445
1461
41
999

673437
GGCCCCTAGCGCGCGAC
ekek-d8-kekee
soosssssssssooss
1446
1462
8
1000

673438
CGGCCCCTAGCGCGCGA
ekek-d8-kekee
soosssssssssooss
1447
1463
31
1001

673439
CCGGCCCCTAGCGCGCG
ekek-d8-kekee
soosssssssssooss
1448
1464
18
1002

673440
CCCGGCCCCTAGCGCGC
ekek-d8-kekee
soosssssssssooss
1449
1465
6
1003

673441
CCCCGGCCCCTAGCGCG
ekek-d8-kekee
soosssssssssooss
1450
1466
23
1004

673442
GCCCCGGCCCCTAGCGC
ekek-d8-kekee
soosssssssssooss
1451
1467
8
1005

673443
GGCCCCGGCCCCTAGCG
ekek-d8-kekee
soosssssssssooss
1452
1468
18
1006

673444
CGGCCCCGGCCCCTAGC
ekek-d8-kekee
soosssssssssooss
1453
1469
28
1007

673445
CCGGCCCCGGCCCCTAG
ekek-d8-kekee
soosssssssssooss
1454
1470
9
1008

673446
CCCGGCCCCGGCCCCTA
ekek-d8-kekee
soosssssssssooss
1455
1471
5
1009

673447
ACGCCCCGGCCCCGGCC
ekek-d8-kekee
soosssssssssooss
1465
1481
23
1010

673448
CACGCCCCGGCCCCGGC
ekek-d8-kekee
soosssssssssooss
1466
1482
14
1011

673449
CCACGCCCCGGCCCCGG
ekek-d8-kekee
soosssssssssooss
1467
1483
35
1012

673450
ACCACGCCCCGGCCCCG
ekek-d8-kekee
soosssssssssooss
1468
1484
30
1013

673451
GACCACGCCCCGGCCCC
ekek-d8-kekee
soosssssssssooss
1469
1485
0
1014

673452
CGACCACGCCCCGGCCC
ekek-d8-kekee
soosssssssssooss
1470
1486
15
1015

673453
CCGACCACGCCCCGGCC
ekek-d8-kekee
soosssssssssooss
1471
1487
42
1016

673454
CCCGACCACGCCCCGGC
ekek-d8-kekee
soosssssssssooss
1472
1488
19
1017

673455
CCCCGACCACGCCCCGG
ekek-d8-kekee
soosssssssssooss
1473
1489
21
1018

673456
GCCCCGACCACGCCCCG
ekek-d8-kekee
soosssssssssooss
1474
1490
9
1019

673457
CGCCCCGACCACGCCCC
ekek-d8-kekee
soosssssssssooss
1475
1491
45
1020

673458
CCGCCCCGACCACGCCC
ekek-d8-kekee
soosssssssssooss
1476
1492
14
1021

673459
CCCGCCCCGACCACGCC
ekek-d8-kekee
soosssssssssooss
1477
1493
2
1022

673460
GCCCGCCCCGACCACGC
ekek-d8-kekee
soosssssssssooss
1478
1494
28
1023

673461
GGCCCGCCCCGACCACG
ekek-d8-kekee
soosssssssssooss
1479
1495
19
1024

673462
GGGCCCGCCCCGACCAC
ekek-d8-kekee
soosssssssssooss
1480
1496
26
1025

673463
CGGGCCCGCCCCGACCA
ekek-d8-kekee
soosssssssssooss
1481
1497
12
1026

673464
CCGGGCCCGCCCCGACC
ekek-d8-kekee
soosssssssssooss
1482
1498
18
1027

673465
CCCGGGCCCGCCCCGAC
ekek-d8-kekee
soosssssssssooss
1483
1499
19
1028

673466
GCAGCCCCGCCCCGGGC
ekek-d8-kekee
soosssssssssooss
1505
1521
11
1029

673467
CGCAGCCCCGCCCCGGG
ekek-d8-kekee
soosssssssssooss
1506
1522
40
1030

673468
CCGCAGCCCCGCCCCGG
ekek-d8-kekee
soosssssssssooss
1507
1523
12
1031

673469
ACCGCAGCCCCGCCCCG
ekek-d8-kekee
soosssssssssooss
1508
1524
26
1032

673470
AACCGCAGCCCCGCCCC
ekek-d8-kekee
soosssssssssooss
1509
1525
36
1033

673471
CAACCGCAGCCCCGCCC
ekek-d8-kekee
soosssssssssooss
1510
1526
63
1034

673472
GCAACCGCAGCCCCGCC
ekek-d8-kekee
soosssssssssooss
1511
1527
35
1035

673473
CGCAACCGCAGCCCCGC
ekek-d8-kekee
soosssssssssooss
1512
1528
51
1036

673474
CCGCAACCGCAGCCCCG
ekek-d8-kekee
soosssssssssooss
1513
1529
27
1037

673475
ACCGCAACCGCAGCCCC
ekek-d8-kekee
soosssssssssooss
1514
1530
49
1038

673476
CACCGCAACCGCAGCCC
ekek-d8-kekee
soosssssssssooss
1515
1531
34
1039

673477
GCACCGCAACCGCAGCC
ekek-d8-kekee
soosssssssssooss
1516
1532
36
1040

673478
GGCACCGCAACCGCAGC
ekek-d8-kekee
soosssssssssooss
1517
1533
22
1041

673479
AGGCACCGCAACCGCAG
ekek-d8-kekee
soosssssssssooss
1518
1534
23
1042

673480
CAGGCACCGCAACCGCA
ekek-d8-kekee
soosssssssssooss
1519
1535
27
1043

673481
GCAGGCACCGCAACCGC
ekek-d8-kekee
soosssssssssooss
1520
1536
41
1044

673482
CGCAGGCACCGCAACCG
ekek-d8-kekee
soosssssssssooss
1521
1537
60
1045

673483
GCGCAGGCACCGCAACC
ekek-d8-kekee
soosssssssssooss
1522
1538
22
1046

673484
GGCGCAGGCACCGCAAC
ekek-d8-kekee
soosssssssssooss
1523
1539
11
1047

Example 11: Dose-Dependent Antisense Inhibition of Human C9ORF72 mRNA in HepG2 Cells

Antisense oligonucleotides from the study described in Example 10 hereinabove exhibiting significant in vitro inhibition of C9ORF72 mRNA were selected and tested at various doses in HepG2 cells. ISIS 576816, which was previously tested in PCT/US2013/065073 (claiming priority to U.S. Application No. 61/714,132, filed Oct. 15, 2012) was used as a benchmark oligonucleotide. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.185 μM, 0.56 μM, 1.67 μM, or 5.00 μM 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 primer probe set RTS3905 was used to measure the C9ORF72 pathogenic associated mRNA variant, which is the product of a pre-mRNA containing a hexanucleotide repeat. C9ORF72 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72 levels, relative to untreated control cells.

As shown in Tables 45-52, total C9ORF72 mRNA levels were reduced in a dose-dependent manner in some of the antisense oligonucleotide treated cells.

TABLE 45

Dose-dependent inhibition of the C9ORF72 pathogenic

associated mRNA variant transcript levels in HepG2 cells

0.185
0.56
1.67
5.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

576816
21
66
82
91
0.5

672893
4
50
83
82
0.8

672894
13
55
70
88
0.7

672896
13
57
81
89
0.6

672897
2
38
72
79
1.1

672902
20
40
83
88
0.7

672903
19
44
73
80
0.8

672904
16
35
49
85
1.2

672905
15
30
67
82
1.0

672908
41
49
79
83
0.4

672909
20
54
72
90
0.6

672919
31
58
69
92
0.5

672924
34
60
89
97
0.4

672925
41
58
88
94
0.3

672927
31
78
81
92
0.3

672928
30
62
79
92
0.4

672929
51
71
89
94
0.1

672932
10
54
83
88
0.7

672940
14
36
58
87
1.0

TABLE 46

Dose-dependent inhibition of the C9ORF72 pathogenic

associated mRNA variant transcript levels in HepG2 cells

0.185
0.56
1.67
5.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

576816
24
51
78
88
0.6

672948
17
27
52
65
1.8

672966
1
36
77
73
1.1

672967
24
36
44
75
1.4

672968
15
46
69
83
0.8

672969
1
39
66
93
1.0

672976
47
65
74
80
0.2

672978
36
32
52
76
1.0

672980
24
45
77
86
0.6

672981
48
74
86
93
0.1

672982
42
63
91
90
0.2

672983
38
56
83
92
0.4

672984
33
53
72
88
0.5

672985
38
46
66
78
0.6

673021
43
48
76
79
0.4

673022
2
52
58
89
1.0

673023
44
36
76
78
0.5

673026
22
77
70
76
0.4

673032
19
37
55
80
1.1

TABLE 47

Dose-dependent inhibition of the C9ORF72 pathogenic associated

mRNA variant transcript levels in HepG2 cells

0.185

5.00
IC₅₀

ISIS No
μM
0.56 μM
1.67 μM
μM
(μM)

576816
48
49
80
95
0.3

673036
38
54
73
88
0.4

673047
27
75
93
87
0.3

673050
6
66
83
82
0.6

673051
31
57
69
85
0.5

673053
17
59
76
92
0.6

673054
15
45
76
90
0.7

673057
37
67
64
81
0.3

673058
35
62
79
87
0.4

673067
59
74
98
97
<0.2

673068
37
71
85
95
0.3

673071
43
5
59
64
1.9

673074
37
44
89
89
0.4

673079
41
71
89
95
0.2

673081
21
37
80
76
0.8

673082
27
58
87
92
0.4

673088
63
79
96
97
<0.2

673089
11
41
71
83
0.9

673098
15
61
68
93
0.6

TABLE 48

Dose-dependent inhibition of the C9ORF72 pathogenic associated

mRNA variant transcript levels in HepG2 cells

0.185
0.56
1.67
5.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

576816
43
71
80
93
0.2

673098
18
53
81
88
0.6

673117
22
45
76
80
0.7

673119
17
47
65
90
0.8

673125
33
64
79
79
0.4

673126
41
56
70
82
0.4

673127
46
85
92
96
<0.2

673128
32
71
88
99
0.3

673130
42
69
91
91
0.2

673131
34
62
74
99
0.4

673132
47
44
75
89
0.4

673133
54
61
78
84
<0.2

673134
41
62
77
77
0.3

673135
28
60
77
82
0.5

673144
24
58
64
92
0.6

673152
18
59
70
72
0.7

673159
4
50
75
80
0.9

673171
17
43
58
86
0.9

673175
30
45
76
78
0.6

TABLE 49

Dose-dependent inhibition of the C9ORF72 pathogenic associated

mRNA variant transcript levels in HepG2 cells

0.185
0.56
1.67
5.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

576816
22
69
78
93
0.4

673193
60
74
90
89
<0.2

673194
15
54
75
77
0.7

673196
36
38
71
73
0.7

673197
28
39
68
78
0.8

673201
0
40
69
91
1.0

673202
9
50
77
89
0.7

673203
40
52
84
98
0.4

673204
44
67
91
92
0.2

673206
27
40
70
90
0.7

673210
22
43
79
94
0.6

673211
0
45
53
85
1.2

673219
27
36
67
88
0.8

673224
41
65
86
95
0.3

673225
34
73
78
97
0.3

673226
19
59
83
94
0.5

673228
8
67
79
94
0.6

673229
46
76
89
86
<0.2

673240
18
58
75
93
0.6

TABLE 50

Dose-dependent inhibition of the C9ORF72 pathogenic associated

mRNA variant transcript levels in HepG2 cells

0.185
0.56
1.67
5.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

576816
31
65
81
89
0.4

673265
24
43
73
83
0.7

673269
12
58
81
89
0.6

673275
31
57
63
94
0.5

673276
33
56
69
91
0.5

673277
37
51
65
66
0.6

673279
11
57
68
86
0.8

673280
38
60
80
95
0.3

673281
51
83
92
86
<0.2

673282
60
73
93
95
<0.2

673283
59
66
94
96
<0.2

673284
45
59
78
91
0.3

673285
30
59
78
86
0.4

673321
10
44
72
79
0.9

673323
43
54
76
86
0.3

673326
0
46
72
81
0.8

673329
15
30
64
76
1.2

673331
47
40
66
79
0.5

673332
58
49
71
78
<0.2

TABLE 51

Dose-dependent inhibition of the C9ORF72 pathogenic associated

mRNA variant transcript levels in HepG2 cells

0.185
0.56
1.67
5.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

576816
38
61
75
92
0.3

673338
0
13
48
61
2.6

673341
29
39
66
82
0.8

673343
38
33
58
81
0.8

673344
29
40
69
72
0.8

673345
24
11
51
66
2.2

673346
19
27
52
74
1.4

673351
17
40
61
82
1.0

673352
18
36
62
71
1.2

673353
29
38
47
74
1.2

673358
11
39
63
71
1.2

673359
15
46
51
65
1.4

673369
17
33
55
70
1.4

673374
42
62
77
87
0.3

673375
28
66
79
94
0.4

673377
32
51
77
87
0.5

673378
32
47
76
89
0.5

673379
33
58
76
83
0.4

673390
21
40
57
74
1.1

TABLE 52

Dose-dependent inhibition of the C9ORF72 pathogenic associated

mRNA variant transcript levels in HepG2 cells

0.185
0.56
1.67
5.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

576816
6
54
75
88
0.7

673416
31
51
61
67
0.7

673419
16
34
41
67
2.0

673426
31
62
53
68
0.7

673427
41
52
52
59
0.8

673430
27
46
76
83
0.6

673431
49
68
83
96
0.2

673432
43
72
72
86
0.2

673434
41
70
80
90
0.2

673435
8
48
71
69
1.0

673436
15
20
65
66
1.6

673453
18
49
57
72
1.0

673457
0
19
43
63
2.5

673467
12
25
35
42
>5.0

673471
13
45
57
79
1.0

673473
13
48
62
92
0.8

673475
23
30
65
61
1.4

673481
26
33
52
41
>5.0

673482
14
45
56
75
1.1

Example 12: Antisense Inhibition of C9ORF72 by Human-Rhesus Cross-Reactive Antisense Oligonucleotides in LLC-MK2 Cells by Mixed Backbone 5-8-5 MOE and 5-10-5 MOE Gapmers

Antisense oligonucleotides targeting a human C9ORF72 nucleic acid and cross-reactive with a rhesus C9ORF72 nucleic acid were designed and tested for their effects on rhesus C9ORF72 mRNA expression in vitro. ISIS 576816, previously tested in U.S. Application No. 61/714,132, filed Oct. 15, 2012, was used as a benchmark oligonucleotide. ISIS 619420, which is the mixed backbone version of ISIS 576816, described in Example 2 hereinabove was also tested. 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 LLC-MK2 cells at a density of 20,000 cells per well were transfected using electroporation with 3,500 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 (a TAQ-man primer probe set) was used to measure total C9ORF72 mRNA levels. RTS3750 targets exon 2 of the mRNA transcripts and, therefore, measures total mRNA transcripts. In cases where the oligonucleotide overlapped the amplicon of the primer probe set RTS3750 (see, e.g., Table 53), an alternative primer probe set, RTS3760_MGB (forward sequence TTCCAATGCTTACTGGAGAAGTGA, designated herein as SEQ ID NO: 1546; reverse sequence GGAACACTGTGTGATTTCATAGATGA, designated herein as SEQ ID NO: 1547; probe sequence TCCTGTAATGGAACTGC, designated herein as SEQ ID NO: 1548—a TAQ-man primer probe set) was used to measure total mRNA transcripts. The levels of the C9ORF72 mRNA were normalized to the total RNA content of the cell, as measured by RIBOGREEN®. Results are presented as percent inhibition of rhesus C9ORF72 mRNA expression, relative to untreated control cells. The oligonucleotides marked with as asterisk (*) target the amplicon region of the primer probe set. Additional assays may be used to measure the potency and efficacy of these oligonucleotides. ‘n.d.’ indicates that there was no signal reading in the assay for that particular oligonucleotide.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-8-5 MOE gapmers and 5-10-5 MOE gapmers.

The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises eight 2′-deoxynucleosides and is flanked by wing segments on boht the 5′ end and the 3′ end comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE group. All cytosine residues throughout each gapmer are 5-methylcytosines. The internucleoside linkages for the gapmers are mixed phosphorothioate and phosphodiester linkages. The internucleoside linkages for each gapmer are presented in the Linkage column, where ‘o’ indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioate linkage.

The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on boht the 5′ end and the 3′ end comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE group. All cytosine residues throughout each gapmer are 5-methylcytosines. The internucleoside linkages for the gapmers are mixed phosphorothioate and phosphodiester linkages. The internucleoside linkages for each gapmer are presented in the Linkage column, where ‘o’ indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioate linkage.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted in the gene sequence. Each gapmer listed in the Tables below is targeted to one or more of human C9ORF72 mRNA sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001256054.1), 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), and rhesus C9ORF72 genomic sequence designated herein as SEQ ID NO: 19 (GENBANK Accession No. NW_001101662.1 truncated from nucleosides 8522000 to 8552000). The ‘Mismatches’ column indicates the number of mismatches the human antisense oligonucleotide has with the rhesus genomic sequence. ‘n/a’ in the rhesus sequence columns indicates that the human oligonucleotide has more than 3 mismatches with the rhesus genomic sequence. Where the ‘Mismtaches’ column is not provided in a Table, it is understood that the human oligonucleotides of the Table are fully cross-reactive with the rhesus genomic sequence.

TABLE 53

Percent inhibition of the C9ORF72 mRNA levels compared to PBS control by antisense

oligonucleotides targeting SEQ ID NOs: 1, 2 and 19

SEQ

SEQ
SEQ
ID

%

ID
ID
NO:

%
inhib-

NO: 1
NO: 2
19

inhib-
ition
SEQ

ISIS
Start
Start
Start

ition
(RTS3760_
ID

NO
Site
Site
Site
Sequence
Linkage
Motif
(RTS3750)
MGB)
NO

576816
310
7990
8043
GCCTTACTCTAGGACCAA
sssssssssssssssss
5-10-5
60
54
20

GA
ss

619420
310
7990
8043
GCCTTACTCTAGGACCAA
soooossssssssssoo
5-10-5
69
68
20

GA
ss

688005*
221
7901
7954
AACAGCTGGAGATGGCGG
sooosssssssssooss
5-8-5
12
36
1057

688006*
222
7902
7955
CAACAGCTGGAGATGGCG
sooosssssssssooss
5-8-5
29
20
1058

688007*
223
7903
7956
GCAACAGCTGGAGATGGC
sooosssssssssooss
5-8-5
46
33
1059

688008*
224
7904
7957
GGCAACAGCTGGAGATGG
sooosssssssssooss
5-8-5
59
41
1060

688009*
225
7905
7958
TGGCAACAGCTGGAGATG
sooosssssssssooss
5-8-5
43
24
1061

688010*
226
7906
7959
TTGGCAACAGCTGGAGAT
sooosssssssssooss
5-8-5
31
0
1062

688011*
227
7907
7960
CTTGGCAACAGCTGGAGA
sooosssssssssooss
5-8-5
45
18
1063

688012*
228
7908
7961
TCTTGGCAACAGCTGGAG
sooosssssssssooss
5-8-5
67
0
1064

688013*
229
7909
7962
GTCTTGGCAACAGCTGGA
sooosssssssssooss
5-8-5
48
13
1065

688014*
230
7910
7963
TGTCTTGGCAACAGCTGG
sooosssssssssooss
5-8-5
16
16
1066

688015*
231
7911
7964
CTGTCTTGGCAACAGCTG
sooosssssssssooss
5-8-5
75
33
1067

688016*
232
7912
7965
TCTGTCTTGGCAACAGCT
sooosssssssssooss
5-8-5
37
36
1068

688017*
233
7913
7966
CTCTGTCTTGGCAACAGC
sooosssssssssooss
5-8-5
59
28
1069

688018*
234
7914
7967
TCTCTGTCTTGGCAACAG
sooosssssssssooss
5-8-5
40
36
1070

688019*
235
7915
7968
ATCTCTGTCTTGGCAACA
sooosssssssssooss
5-8-5
34
31
1071

688020*
236
7916
7969
AATCTCTGTCTTGGCAAC
sooosssssssssooss
5-8-5
30
26
1072

688021*
237
7917
7970
CAATCTCTGTCTTGGCAA
sooosssssssssooss
5-8-5
58
0
1073

688022*
238
7918
7971
GCAATCTCTGTCTTGGCA
sooosssssssssooss
5-8-5
90
52
1074

688023*
239
7919
7972
AGCAATCTCTGTCTTGGC
sooosssssssssooss
5-8-5
92
73
1075

688024*
240
7920
7973
AAGCAATCTCTGTCTTGG
sooosssssssssooss
5-8-5
74
24
1076

688025*
241
7921
7974
AAAGCAATCTCTGTCTTG
sooosssssssssooss
5-8-5
76
0
1077

688026*
242
7922
7975
TAAAGCAATCTCTGTCTT
sooosssssssssooss
5-8-5
28
6
1078

688027*
243
7923
7976
TTAAAGCAATCTCTGTCT
sooosssssssssooss
5-8-5
32
18
1079

688028*
244
7924
7977
CTTAAAGCAATCTCTGTC
sooosssssssssooss
5-8-5
41
4
1080

688029*
245
7925
7978
ACTTAAAGCAATCTCTGT
sooosssssssssooss
5-8-5
63
0
1081

688030*
246
7926
7979
CACTTAAAGCAATCTCTG
sooosssssssssooss
5-8-5
26
31
1082

688031*
247
7927
7980
CCACTTAAAGCAATCTCT
sooosssssssssooss
5-8-5
73
33
1083

688032
267
7947
8000
CTGCTAATAAAGGTGATT
sooosssssssssooss
5-8-5
24
34
1084

688033
268
7948
8001
GCTGCTAATAAAGGTGAT
sooosssssssssooss
5-8-5
11
18
1085

688034
269
7949
8002
AGCTGCTAATAAAGGTGA
sooosssssssssooss
5-8-5
40
34
1086

688035
270
7950
8003
TAGCTGCTAATAAAGGTG
sooosssssssssooss
5-8-5
46
1
1087

688036
271
7951
8004
GTAGCTGCTAATAAAGGT
sooosssssssssooss
5-8-5
13
17
1088

688037
272
7952
8005
AGTAGCTGCTAATAAAGG
sooosssssssssooss
5-8-5
0
0
1089

688038
273
7953
8006
AAGTAGCTGCTAATAAAG
sooosssssssssooss
5-8-5
0
0
1090

688039
274
7954
8007
AAAGTAGCTGCTAATAAA
sooosssssssssooss
5-8-5
0
3
1091

688040
275
7955
8008
AAAAGTAGCTGCTAATAA
sooosssssssssooss
5-8-5
0
0
1092

688041
276
7956
8009
CAAAAGTAGCTGCTAATA
sooosssssssssooss
5-8-5
0
0
1093

688042
277
7957
8010
GCAAAAGTAGCTGCTAAT
sooosssssssssooss
5-8-5
32
31
1094

688043
278
7958
8011
AGCAAAAGTAGCTGCTAA
sooosssssssssooss
5-8-5
18
14
1095

688044
279
7959
8012
AAGCAAAAGTAGCTGCTA
sooosssssssssooss
5-8-5
17
24
1096

688045
280
7960
8013
TAAGCAAAAGTAGCTGCT
sooosssssssssooss
5-8-5
38
7
1097

688046
281
7961
8014
GTAAGCAAAAGTAGCTGC
sooosssssssssooss
5-8-5
37
38
1098

688047
282
7962
8015
AGTAAGCAAAAGTAGCTG
sooosssssssssooss
5-8-5
12
33
1099

688048
283
7963
8016
CAGTAAGCAAAAGTAGCT
sooosssssssssooss
5-8-5
17
29
1100

688049
284
7964
8017
CCAGTAAGCAAAAGTAGC
sooosssssssssooss
5-8-5
17
0
1101

688050
285
7965
8018
CCCAGTAAGCAAAAGTAG
sooosssssssssooss
5-8-5
7
27
1102

688051
286
7966
8019
TCCCAGTAAGCAAAAGTA
sooosssssssssooss
5-8-5
0
0
1103

688052
287
7967
8020
GTCCCAGTAAGCAAAAGT
sooosssssssssooss
5-8-5
16
24
1104

688053
288
7968
8021
TGTCCCAGTAAGCAAAAG
sooosssssssssooss
5-8-5
0
0
1105

688054
289
7969
8022
TTGTCCCAGTAAGCAAAA
sooosssssssssooss
5-8-5
9
10
1106

688055
290
7970
8023
ATTGTCCCAGTAAGCAAA
sooosssssssssooss
5-8-5
6
15
1107

688056
291
7971
8024
TATTGTCCCAGTAAGCAA
sooosssssssssooss
5-8-5
21
0
1108

688057
292
7972
8025
ATATTGTCCCAGTAAGCA
sooosssssssssooss
5-8-5
11
4
1109

688058
293
7973
8026
AATATTGTCCCAGTAAGC
sooosssssssssooss
5-8-5
23
29
1110

688059
294
7974
8027
GAATATTGTCCCAGTAAG
sooosssssssssooss
5-8-5
5
13
1111

688060
295
7975
8028
AGAATATTGTCCCAGTAA
sooosssssssssooss
5-8-5
0
31
1112

688061
296
7976
8029
AAGAATATTGTCCCAGTA
sooosssssssssooss
5-8-5
0
16
1113

688062
297
7977
8030
CAAGAATATTGTCCCAGT
sooosssssssssooss
5-8-5
29
0
1114

688063
298
7978
8031
CCAAGAATATTGTCCCAG
sooosssssssssooss
5-8-5
26
42
1115

688064
299
7979
8032
ACCAAGAATATTGTCCCA
sooosssssssssooss
5-8-5
19
11
1116

688065
300
7980
8033
GACCAAGAATATTGTCCC
sooosssssssssooss
5-8-5
31
26
1117

688066
301
7981
8034
GGACCAAGAATATTGTCC
sooosssssssssooss
5-8-5
0
0
1118

688067
302
7982
8035
AGGACCAAGAATATTGTC
sooosssssssssooss
5-8-5
5
10
1119

688068
303
7983
8036
TAGGACCAAGAATATTGT
sooosssssssssooss
5-8-5
20
13
1120

688069
304
7984
8037
CTAGGACCAAGAATATTG
sooosssssssssooss
5-8-5
0
22
1121

688070
305
7985
8038
TCTAGGACCAAGAATATT
sooosssssssssooss
5-8-5
0
0
1122

688071
306
7986
8039
CTCTAGGACCAAGAATAT
sooosssssssssooss
5-8-5
5
18
1123

688072
307
7987
8040
ACTCTAGGACCAAGAATA
sooosssssssssooss
5-8-5
17
27
1124

688073
308
7988
8041
TACTCTAGGACCAAGAAT
sooosssssssssooss
5-8-5
13
8
1125

688074
309
7989
8042
TTACTCTAGGACCAAGAA
sooosssssssssooss
5-8-5
0
0
1126

688075
310
7990
8043
CTTACTCTAGGACCAAGA
sooosssssssssooss
5-8-5
0
0
1127

688076
311
7991
8044
CCTTACTCTAGGACCAAG
sooosssssssssooss
5-8-5
45
71
1128

688077
312
7992
8045
GCCTTACTCTAGGACCAA
sooosssssssssooss
5-8-5
62
67
1129

688078
313
7993
8046
TGCCTTACTCTAGGACCA
sooosssssssssooss
5-8-5
33
30
1130

688079
314
7994
8047
GTGCCTTACTCTAGGACC
sooosssssssssooss
5-8-5
59
49
1131

688080
315
7995
8048
TGTGCCTTACTCTAGGAC
sooosssssssssooss
5-8-5
47
41
1132

688081
316
7996
8049
ATGTGCCTTACTCTAGGA
sooosssssssssooss
5-8-5
44
13
1133

688082
317
7997
8050
AATGTGCCTTACTCTAGG
sooosssssssssooss
5-8-5
6
0
1134

TABLE 54

Percent inhibition of the C9ORF72 mRNA levels compared to PBS control by antisense

oligonucleotides targeting SEQ ID NOs: 1, 2 and 19

SEQ
SEQ
SEQ

ID
ID
ID

NO:
NO:
NO:

%

1
2
19

inhib-
SEQ

ISIS
Start
Start
Start

ition
ID

NO
Site
Site
Site
Sequence
Linkage
Motif
(RTS3750)
NO

576816
310
7990
8043
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
5-10-5
58
20

619420
310
7990
8043
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
5-10-5
70
20

688078
313
7993
8046
TGCCTTACTCTAGGACCA
sooosssssssssooss
5-8-5
50
1130

688083
318
7998
8051
AAATGTGCCTTACTCTAG
sooosssssssssooss
5-8-5
7
1135

688084
319
7999
8052
CAAATGTGCCTTACTCTA
sooosssssssssooss
5-8-5
11
1136

688085
320
8000
8053
CCAAATGTGCCTTACTCT
sooosssssssssooss
5-8-5
29
1137

688086
321
8001
8054
CCCAAATGTGCCTTACTC
sooosssssssssooss
5-8-5
43
1138

688087
322
8002
8055
GCCCAAATGTGCCTTACT
sooosssssssssooss
5-8-5
58
1139

688088
323
8003
8056
AGCCCAAATGTGCCTTAC
sooosssssssssooss
5-8-5
66
1140

688089
324
8004
8057
GAGCCCAAATGTGCCTTA
sooosssssssssooss
5-8-5
61
1141

688090
325
8005
8058
GGAGCCCAAATGTGCCTT
sooosssssssssooss
5-8-5
52
1142

688091
326
8006
8059
TGGAGCCCAAATGTGCCT
sooosssssssssooss
5-8-5
34
1143

688092
327
8007
8060
TTGGAGCCCAAATGTGCC
sooosssssssssooss
5-8-5
15
1144

688093
328
8008
8061
TTTGGAGCCCAAATGTGC
sooosssssssssooss
5-8-5
31
1145

688094
329
8009
8062
CTTTGGAGCCCAAATGTG
sooosssssssssooss
5-8-5
23
1146

688095
330
8010
8063
TCTTTGGAGCCCAAATGT
sooosssssssssooss
5-8-5
13
1147

688096
331
8011
8064
GTCTTTGGAGCCCAAATG
sooosssssssssooss
5-8-5
30
1148

688097
332
8012
8065
TGTCTTTGGAGCCCAAAT
sooosssssssssooss
5-8-5
32
1149

688098
333
8013
8066
CTGTCTTTGGAGCCCAAA
sooosssssssssooss
5-8-5
48
1150

688099
334
8014
8067
TCTGTCTTTGGAGCCCAA
sooosssssssssooss
5-8-5
62
1151

688100
335
8015
8068
TTCTGTCTTTGGAGCCCA
sooosssssssssooss
5-8-5
61
1152

688101
336
8016
8069
GTTCTGTCTTTGGAGCCC
sooosssssssssooss
5-8-5
77
1153

688102
337
8017
8070
TGTTCTGTCTTTGGAGCC
sooosssssssssooss
5-8-5
58
1154

688103
338
8018
8071
CTGTTCTGTCTTTGGAGC
sooosssssssssooss
5-8-5
57
1155

688104
339
8019
8072
CCTGTTCTGTCTTTGGAG
sooosssssssssooss
5-8-5
55
1156

688105
340
8020
8073
ACCTGTTCTGTCTTTGGA
sooosssssssssooss
5-8-5
51
1157

688106
341
8021
8074
TACCTGTTCTGTCTTTGG
sooosssssssssooss
5-8-5
42
1158

688107
342
8022
8075
GTACCTGTTCTGTCTTTG
sooosssssssssooss
5-8-5
49
1159

688108
343
8023
8076
AGTACCTGTTCTGTCTTT
sooosssssssssooss
5-8-5
25
1160

688109
344
8024
8077
AAGTACCTGTTCTGTCTT
sooosssssssssooss
5-8-5
22
1161

688110
345
8025
8078
GAAGTACCTGTTCTGTCT
sooosssssssssooss
5-8-5
42
1162

688111
346
8026
8079
AGAAGTACCTGTTCTGTC
sooosssssssssooss
5-8-5
21
1163

688112
347
8027
8080
GAGAAGTACCTGTTCTGT
sooosssssssssooss
5-8-5
22
1164

688113
348
8028
8081
TGAGAAGTACCTGTTCTG
sooosssssssssooss
5-8-5
13
1165

688114
349
8029
8082
CTGAGAAGTACCTGTTCT
sooosssssssssooss
5-8-5
25
1166

688115
350
8030
8083
ACTGAGAAGTACCTGTTC
sooosssssssssooss
5-8-5
14
1167

688116
351
8031
8084
CACTGAGAAGTACCTGTT
sooosssssssssooss
5-8-5
36
1168

688117
352
8032
8085
TCACTGAGAAGTACCTGT
sooosssssssssooss
5-8-5
28
1169

688118
353
8033
8086
ATCACTGAGAAGTACCTG
sooosssssssssooss
5-8-5
36
1170

688119
354
8034
8087
CATCACTGAGAAGTACCT
sooosssssssssooss
5-8-5
32
1171

688120
355
8035
8088
CCATCACTGAGAAGTACC
sooosssssssssooss
5-8-5
44
1172

688121
356
8036
8089
TCCATCACTGAGAAGTAC
sooosssssssssooss
5-8-5
31
1173

688122
357
8037
8090
CTCCATCACTGAGAAGTA
sooosssssssssooss
5-8-5
56
1174

688123
358
8038
8091
TCTCCATCACTGAGAAGT
sooosssssssssooss
5-8-5
53
1175

688124
359
8039
8092
TTCTCCATCACTGAGAAG
sooosssssssssooss
5-8-5
14
1176

688125
360
8040
8093
TTTCTCCATCACTGAGAA
sooosssssssssooss
5-8-5
12
1177

688126
361
8041
8094
ATTTCTCCATCACTGAGA
sooosssssssssooss
5-8-5
18
1178

688127
362
8042
8095
TATTTCTCCATCACTGAG
sooosssssssssooss
5-8-5
11
1179

688128
364
8044
8097
GTTATTTCTCCATCACTG
sooosssssssssooss
5-8-5
40
1180

688129
365
8045
8098
AGTTATTTCTCCATCACT
sooosssssssssooss
5-8-5
37
1181

688130
366
8046
8099
AAGTTATTTCTCCATCAC
sooosssssssssooss
5-8-5
20
1182

688131
367
8047
8100
AAAGTTATTTCTCCATCA
sooosssssssssooss
5-8-5
22
1183

688132
368
8048
8101
AAAAGTTATTTCTCCATC
sooosssssssssooss
5-8-5
31
1184

688133
369
8049
8102
GAAAAGTTATTTCTCCAT
sooosssssssssooss
5-8-5
19
1185

688134
371
8051
8104
AAGAAAAGTTATTTCTCC
sooosssssssssooss
5-8-5
34
1186

688135
372
8052
8105
CAAGAAAAGTTATTTCTC
sooosssssssssooss
5-8-5
0
1187

688136
373
8053
8106
GCAAGAAAAGTTATTTCT
sooosssssssssooss
5-8-5
21
1188

688137
374
8054
8107
GGCAAGAAAAGTTATTTC
sooosssssssssooss
5-8-5
51
1189

688138
375
8055
8108
TGGCAAGAAAAGTTATTT
sooosssssssssooss
5-8-5
42
1190

688139
376
8056
8109
TTGGCAAGAAAAGTTATT
sooosssssssssooss
5-8-5
13
1191

688140
377
8057
8110
GTTGGCAAGAAAAGTTAT
sooosssssssssooss
5-8-5
16
1192

688141
378
8058
8111
GGTTGGCAAGAAAAGTTA
sooosssssssssooss
5-8-5
23
1193

688142
379
8059
8112
TGGTTGGCAAGAAAAGTT
sooosssssssssooss
5-8-5
7
1194

688143
380
8060
8113
GTGGTTGGCAAGAAAAGT
sooosssssssssooss
5-8-5
30
1195

688144
381
8061
8114
TGTGGTTGGCAAGAAAAG
sooosssssssssooss
5-8-5
12
1196

688145
382
8062
8115
GTGTGGTTGGCAAGAAAA
sooosssssssssooss
5-8-5
7
1197

688146
383
8063
8116
AGTGTGGTTGGCAAGAAA
sooosssssssssooss
5-8-5
0
1198

688147
384
8064
8117
GAGTGTGGTTGGCAAGAA
sooosssssssssooss
5-8-5
27
1199

688148
385
8065
8118
AGAGTGTGGTTGGCAAGA
sooosssssssssooss
5-8-5
17
1200

688149
386
8066
8119
TAGAGTGTGGTTGGCAAG
sooosssssssssooss
5-8-5
17
1201

688150
387
8067
8120
TTAGAGTGTGGTTGGCAA
sooosssssssssooss
5-8-5
20
1202

688151
388
8068
8121
TTTAGAGTGTGGTTGGCA
sooosssssssssooss
5-8-5
22
1203

688152
389
8069
8122
ATTTAGAGTGTGGTTGGC
sooosssssssssooss
5-8-5
47
1204

688153
390
8070
8123
CATTTAGAGTGTGGTTGG
sooosssssssssooss
5-8-5
21
1205

688154
391
8071
8124
CCATTTAGAGTGTGGTTG
sooosssssssssooss
5-8-5
23
1206

688155
392
8072
8125
TCCATTTAGAGTGTGGTT
sooosssssssssooss
5-8-5
9
1207

688156
393
8073
8126
CTCCATTTAGAGTGTGGT
sooosssssssssooss
5-8-5
25
1208

688157
394
8074
8127
TCTCCATTTAGAGTGTGG
sooosssssssssooss
5-8-5
41
1209

688158
395
8075
8128
TTCTCCATTTAGAGTGTG
sooosssssssssooss
5-8-5
19
1210

688159
396
8076
8129
TTTCTCCATTTAGAGTGT
sooosssssssssooss
5-8-5
0
1211

TABLE 55

Percent inhibition of the C9ORF72 mRNA levels compared to PBS control by antisense

oligonucleotides targeting SEQ ID NOs: 1, 2 and 19

SEQ
SEQ
SEQ

ID
ID
ID

NO:
NO:
NO:

%

1
2
19

inhib-
SEQ

ISIS
Start
Start
Start

ition
ID

NO
Site
Site
Site
Sequence
Linkage
Motif
(RTS3750)
NO

576816
310
7990
8043
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
5-10-5
57
20

619420
310
7990
8043
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
5-10-5
64
20

688078
313
7993
8046
TGCCTTACTCTAGGACCA
sooosssssssssooss
5-8-5
37
1130

688160
397
8077
8130
ATTTCTCCATTTAGAGTG
sooosssssssssooss
5-8-5
0
1212

688161
398
8078
8131
GATTTCTCCATTTAGAGT
sooosssssssssooss
5-8-5
9
1213

688162
399
8079
8132
GGATTTCTCCATTTAGAG
sooosssssssssooss
5-8-5
2
1214

688163
400
8080
8133
AGGATTTCTCCATTTAGA
sooosssssssssooss
5-8-5
0
1215

688164
401
8081
8134
AAGGATTTCTCCATTTAG
sooosssssssssooss
5-8-5
12
1216

688165
402
8082
8135
GAAGGATTTCTCCATTTA
sooosssssssssooss
5-8-5
0
1217

688166
403
8083
8136
CGAAGGATTTCTCCATTT
sooosssssssssooss
5-8-5
18
1218

688167
404
8084
8137
TCGAAGGATTTCTCCATT
sooosssssssssooss
5-8-5
12
1219

688168
405
8085
8138
TTCGAAGGATTTCTCCAT
sooosssssssssooss
5-8-5
6
1220

688169
406
8086
8139
TTTCGAAGGATTTCTCCA
sooosssssssssooss
5-8-5
0
1221

688170
407
8087
8140
ATTTCGAAGGATTTCTCC
sooosssssssssooss
5-8-5
8
1222

688171
408
8088
8141
CATTTCGAAGGATTTCTC
sooosssssssssooss
5-8-5
16
1223

688172
409
8089
8142
GCATTTCGAAGGATTTCT
sooosssssssssooss
5-8-5
55
1224

688173
410
8090
8143
TGCATTTCGAAGGATTTC
sooosssssssssooss
5-8-5
0
1225

688174
411
8091
8144
CTGCATTTCGAAGGATTT
sooosssssssssooss
5-8-5
25
1226

688175
412
8092
8145
TCTGCATTTCGAAGGATT
sooosssssssssooss
5-8-5
33
1227

688176
413
8093
8146
CTCTGCATTTCGAAGGAT
sooosssssssssooss
5-8-5
12
1228

688177
414
8094
8147
TCTCTGCATTTCGAAGGA
sooosssssssssooss
5-8-5
14
1229

688178
415
8095
8148
CTCTCTGCATTTCGAAGG
sooosssssssssooss
5-8-5
5
1230

688179
416
8096
8149
ACTCTCTGCATTTCGAAG
sooosssssssssooss
5-8-5
0
1231

688180
417
8097
8150
CACTCTCTGCATTTCGAA
sooosssssssssooss
5-8-5
12
1232

688181
418
8098
8151
CCACTCTCTGCATTTCGA
sooosssssssssooss
5-8-5
40
1233

688182
419
8099
8152
ACCACTCTCTGCATTTCG
sooosssssssssooss
5-8-5
40
1234

688183
420
8100
8153
CACCACTCTCTGCATTTC
sooosssssssssooss
5-8-5
45
1235

688184
421
8101
8154
GCACCACTCTCTGCATTT
sooosssssssssooss
5-8-5
24
1236

688185
422
8102
8155
AGCACCACTCTCTGCATT
sooosssssssssooss
5-8-5
12
1237

688186
423
8103
8156
TAGCACCACTCTCTGCAT
sooosssssssssooss
5-8-5
21
1238

688187
424
8104
8157
ATAGCACCACTCTCTGCA
sooosssssssssooss
5-8-5
25
1239

688188
425
8105
8158
TATAGCACCACTCTCTGC
sooosssssssssooss
5-8-5
12
1240

688189
426
8106
8159
CTATAGCACCACTCTCTG
sooosssssssssooss
5-8-5
0
1241

688190
427
8107
8160
TCTATAGCACCACTCTCT
sooosssssssssooss
5-8-5
0
1242

688191
428
8108
8161
ATCTATAGCACCACTCTC
sooosssssssssooss
5-8-5
20
1243

688192
429
8109
8162
CATCTATAGCACCACTCT
sooosssssssssooss
5-8-5
0
1244

688193
430
8110
8163
ACATCTATAGCACCACTC
sooosssssssssooss
5-8-5
31
1245

688194
431
8111
8164
TACATCTATAGCACCACT
sooosssssssssooss
5-8-5
4
1246

688195
432
8112
8165
TTACATCTATAGCACCAC
sooosssssssssooss
5-8-5
32
1247

688196
433
8113
8166
TTTACATCTATAGCACCA
sooosssssssssooss
5-8-5
37
1248

688197
434
8114
8167
CTTTACATCTATAGCACC
sooosssssssssooss
5-8-5
25
1249

688198
435
8115
8168
ACTTTACATCTATAGCAC
sooosssssssssooss
5-8-5
0
1250

688199
436
8116
8169
AACTTTACATCTATAGCA
sooosssssssssooss
5-8-5
9
1251

688200
437
8117
8170
AAACTTTACATCTATAGC
sooosssssssssooss
5-8-5
8
1252

688201
438
8118
8171
AAAACTTTACATCTATAG
sooosssssssssooss
5-8-5
4
1253

688202
440
8120
8173
AAAAAACTTTACATCTAT
sooosssssssssooss
5-8-5
8
1254

688203
441
8121
8174
CAAAAAACTTTACATCTA
sooosssssssssooss
5-8-5
4
1255

688204
442
8122
8175
ACAAAAAACTTTACATCT
sooosssssssssooss
5-8-5
0
1256

688205
443
8123
8176
GACAAAAAACTTTACATC
sooosssssssssooss
5-8-5
0
1257

688206
446
8126
8179
CAAGACAAAAAACTTTAC
sooosssssssssooss
5-8-5
5
1258

688207
448
8128
8181
GACAAGACAAAAAACTTT
sooosssssssssooss
5-8-5
27
1259

688208
449
8129
8182
AGACAAGACAAAAAACTT
sooosssssssssooss
5-8-5
9
1260

688209
450
8130
8183
CAGACAAGACAAAAAACT
sooosssssssssooss
5-8-5
0
1261

688210
451
8131
8184
TCAGACAAGACAAAAAAC
sooosssssssssooss
5-8-5
11
1262

688211
452
8132
8185
TTCAGACAAGACAAAAAA
sooosssssssssooss
5-8-5
0
1263

688212
454
8134
8187
TTTTCAGACAAGACAAAA
sooosssssssssooss
5-8-5
0
1264

688213
455
8135
8188
CTTTTCAGACAAGACAAA
sooosssssssssooss
5-8-5
0
1265

688214
456
8136
8189
CCTTTTCAGACAAGACAA
sooosssssssssooss
5-8-5
30
1266

688215
457
8137
8190
CCCTTTTCAGACAAGACA
sooosssssssssooss
5-8-5
31
1267

688216
458
8138
8191
TCCCTTTTCAGACAAGAC
sooosssssssssooss
5-8-5
24
1268

688217
459
8139
8192
CTCCCTTTTCAGACAAGA
sooosssssssssooss
5-8-5
47
1269

688218
460
8140
8193
ACTCCCTTTTCAGACAAG
sooosssssssssooss
5-8-5
34
1270

688219
461
8141
8194
CACTCCCTTTTCAGACAA
sooosssssssssooss
5-8-5
5
1271

688220
462
8142
8195
TCACTCCCTTTTCAGACA
sooosssssssssooss
5-8-5
15
1272

688221
463
8143
8196
ATCACTCCCTTTTCAGAC
sooosssssssssooss
5-8-5
14
1273

688222
464
8144
8197
AATCACTCCCTTTTCAGA
sooosssssssssooss
5-8-5
18
1274

688223
465
8145
8198
TAATCACTCCCTTTTCAG
sooosssssssssooss
5-8-5
14
1275

688224
466
8146
8199
ATAATCACTCCCTTTTCA
sooosssssssssooss
5-8-5
15
1276

688225
467
8147
8200
AATAATCACTCCCTTTTC
sooosssssssssooss
5-8-5
8
1277

688226
468
8148
8201
CAATAATCACTCCCTTTT
sooosssssssssooss
5-8-5
9
1278

688227
469
8149
8202
ACAATAATCACTCCCTTT
sooosssssssssooss
5-8-5
24
1279

688228
470
8150
8203
AACAATAATCACTCCCTT
sooosssssssssooss
5-8-5
21
1280

688229
471
8151
8204
AAACAATAATCACTCCCT
sooosssssssssooss
5-8-5
21
1281

688230
472
8152
8205
GAAACAATAATCACTCCC
sooosssssssssooss
5-8-5
36
1282

688231
473
8153
8206
TGAAACAATAATCACTCC
sooosssssssssooss
5-8-5
7
1283

688232
474
8154
8207
ATGAAACAATAATCACTC
sooosssssssssooss
5-8-5
20
1284

688233
475
8155
8208
AATGAAACAATAATCACT
sooosssssssssooss
5-8-5
0
1285

688234
476
8156
8209
TAATGAAACAATAATCAC
sooosssssssssooss
5-8-5
16
1286

688235
477
8157
8210
TTAATGAAACAATAATCA
sooosssssssssooss
5-8-5
0
1287

688236
478
8158
8211
ATTAATGAAACAATAATC
sooosssssssssooss
5-8-5
0
1288

TABLE 56

Percent inhibition of the C9ORF72 mRNA levels compared to PBS control by antisense

oligonucleotides targeting SEQ ID NOs: 1, 2 and 19

SEQ
SEQ
SEQ

ID
ID
ID

NO:
NO:
NO:

%

1
2
19

inhib-
SEQ

ISIS
Start
Start
Start

ition
ID

NO
Site
Site
Site
Sequence
Linkage
Motif
(RTS3750)
NO

576816
310
7990
8043
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
5-10-5
61
20

619420
310
7990
8043
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
5-10-5
75
20

688078
313
7993
8046
TGCCTTACTCTAGGACCA
sooosssssssssooss
5-8-5
67
1130

688237
485
8165
8218
ATCAAAGATTAATGAAAC
sooosssssssssooss
5-8-5
43
1289

688238
486
8166
8219
CATCAAAGATTAATGAAA
sooosssssssssooss
5-8-5
0
1290

688239
487
8167
8220
CCATCAAAGATTAATGAA
sooosssssssssooss
5-8-5
42
1291

688240
488
8168
8221
TCCATCAAAGATTAATGA
sooosssssssssooss
5-8-5
46
1292

688241
489
8169
8222
TTCCATCAAAGATTAATG
sooosssssssssooss
5-8-5
0
1293

688242
490
8170
8223
TTTCCATCAAAGATTAAT
sooosssssssssooss
5-8-5
55
1294

688243
491
8171
8224
GTTTCCATCAAAGATTAA
sooosssssssssooss
5-8-5
49
1295

688244
492
8172
8225
AGTTTCCATCAAAGATTA
sooosssssssssooss
5-8-5
44
1296

688245
493
8173
8226
CAGTTTCCATCAAAGATT
sooosssssssssooss
5-8-5
0
1297

688246
494
8174
8227
CCAGTTTCCATCAAAGAT
sooosssssssssooss
5-8-5
46
1298

688247
495
8175
8228
TCCAGTTTCCATCAAAGA
sooosssssssssooss
5-8-5
46
1299

688248
496
8176
8229
TTCCAGTTTCCATCAAAG
sooosssssssssooss
5-8-5
59
1300

688249
497
8177
8230
ATTCCAGTTTCCATCAAA
sooosssssssssooss
5-8-5
48
1301

688250
498
8178
8231
CATTCCAGTTTCCATCAA
sooosssssssssooss
5-8-5
54
1302

688251
499
8179
8232
CCATTCCAGTTTCCATCA
sooosssssssssooss
5-8-5
59
1303

688252
500
8180
8233
CCCATTCCAGTTTCCATC
sooosssssssssooss
5-8-5
67
1304

688253
501
8181
8234
CCCCATTCCAGTTTCCAT
sooosssssssssooss
5-8-5
58
1305

688254
502
8182
8235
TCCCCATTCCAGTTTCCA
sooosssssssssooss
5-8-5
55
1306

688255
503
8183
8236
ATCCCCATTCCAGTTTCC
sooosssssssssooss
5-8-5
61
1307

688256
504
8184
8237
GATCCCCATTCCAGTTTC
sooosssssssssooss
5-8-5
51
1308

688257
505
8185
8238
CGATCCCCATTCCAGTTT
sooosssssssssooss
5-8-5
49
1309

688258
506
8186
8239
GCGATCCCCATTCCAGTT
sooosssssssssooss
5-8-5
43
1310

688259
507
8187
8240
TGCGATCCCCATTCCAGT
sooosssssssssooss
5-8-5
51
1311

688260
508
8188
8241
CTGCGATCCCCATTCCAG
sooosssssssssooss
5-8-5
70
1312

688261
509
8189
8242
GCTGCGATCCCCATTCCA
sooosssssssssooss
5-8-5
72
1313

688262
510
8190
8243
TGCTGCGATCCCCATTCC
sooosssssssssooss
5-8-5
49
1314

688263
511
8191
8244
GTGCTGCGATCCCCATTC
sooosssssssssooss
5-8-5
0
1315

688264
512
8192
8245
TGTGCTGCGATCCCCATT
sooosssssssssooss
5-8-5
58
1316

688265
513
8193
8246
ATGTGCTGCGATCCCCAT
sooosssssssssooss
5-8-5
66
1317

688266
514
8194
8247
TATGTGCTGCGATCCCCA
sooosssssssssooss
5-8-5
63
1318

688267
533
8213
8266
AAGTATAATTGATAGTCC
sooosssssssssooss
5-8-5
49
1319

688268
534
8214
8267
GAAGTATAATTGATAGTC
sooosssssssssooss
5-8-5
50
1320

688269
535
8215
8268
GGAAGTATAATTGATAGT
sooosssssssssooss
5-8-5
39
1321

688270
536
8216
8269
TGGAAGTATAATTGATAG
sooosssssssssooss
5-8-5
43
1322

688271
537
8217
8270
GTGGAAGTATAATTGATA
sooosssssssssooss
5-8-5
49
1323

688272
538
8218
8271
TGTGGAAGTATAATTGAT
sooosssssssssooss
5-8-5
40
1324

688273
539
8219
8272
CTGTGGAAGTATAATTGA
sooosssssssssooss
5-8-5
34
1325

688274
540
8220
8273
TCTGTGGAAGTATAATTG
sooosssssssssooss
5-8-5
0
1326

688275
541
8221
8274
GTCTGTGGAAGTATAATT
sooosssssssssooss
5-8-5
0
1327

688276
542
8222
8275
TGTCTGTGGAAGTATAAT
sooosssssssssooss
5-8-5
66
1328

688277
543
8223
8276
CTGTCTGTGGAAGTATAA
sooosssssssssooss
5-8-5
0
1329

688278
544
8224
8277
TCTGTCTGTGGAAGTATA
sooosssssssssooss
5-8-5
63
1330

688279
545
8225
8278
TTCTGTCTGTGGAAGTAT
sooosssssssssooss
5-8-5
55
1331

688280
546
8226
8279
GTTCTGTCTGTGGAAGTA
sooosssssssssooss
5-8-5
78
1332

688281
547
8227
8280
AGTTCTGTCTGTGGAAGT
sooosssssssssooss
5-8-5
63
1333

688282
548
8228
8281
AAGTTCTGTCTGTGGAAG
sooosssssssssooss
5-8-5
50
1334

688283
549
8229
8282
TAAGTTCTGTCTGTGGAA
sooosssssssssooss
5-8-5
0
1335

688284
550
8230
8283
CTAAGTTCTGTCTGTGGA
sooosssssssssooss
5-8-5
55
1336

688285
551
8231
8284
ACTAAGTTCTGTCTGTGG
sooosssssssssooss
5-8-5
69
1337

688286
552
8232
8285
AACTAAGTTCTGTCTGTG
sooosssssssssooss
5-8-5
66
1338

688287
553
8233
8286
AAACTAAGTTCTGTCTGT
sooosssssssssooss
5-8-5
43
1339

688288
554
8234
8287
GAAACTAAGTTCTGTCTG
sooosssssssssooss
5-8-5
37
1340

688289
555
8235
8288
AGAAACTAAGTTCTGTCT
sooosssssssssooss
5-8-5
47
1341

688290
556
8236
8289
TAGAAACTAAGTTCTGTC
sooosssssssssooss
5-8-5
50
1342

688291
557
8237
8290
GTAGAAACTAAGTTCTGT
sooosssssssssooss
5-8-5
47
1343

688292
558
8238
8291
GGTAGAAACTAAGTTCTG
sooosssssssssooss
5-8-5
46
1344

688293
559
8239
8292
AGGTAGAAACTAAGTTCT
sooosssssssssooss
5-8-5
59
1345

688294
560
8240
8293
GAGGTAGAAACTAAGTTC
sooosssssssssooss
5-8-5
48
1346

688295
561
8241
8294
GGAGGTAGAAACTAAGTT
sooosssssssssooss
5-8-5
47
1347

688296
562
8242
8295
GGGAGGTAGAAACTAAGT
sooosssssssssooss
5-8-5
43
1348

688297
563
8243
8296
TGGGAGGTAGAAACTAAG
sooosssssssssooss
5-8-5
49
1349

688298
564
8244
8297
GTGGGAGGTAGAAACTAA
sooosssssssssooss
5-8-5
51
1350

688299
565
8245
8298
AGTGGGAGGTAGAAACTA
sooosssssssssooss
5-8-5
45
1351

688300
566
8246
8299
AAGTGGGAGGTAGAAACT
sooosssssssssooss
5-8-5
40
1352

688301
567
8247
8300
GAAGTGGGAGGTAGAAAC
sooosssssssssooss
5-8-5
39
1353

688302
568
8248
8301
TGAAGTGGGAGGTAGAAA
sooosssssssssooss
5-8-5
0
1354

688303
569
8249
8302
ATGAAGTGGGAGGTAGAA
sooosssssssssooss
5-8-5
42
1355

688304
570
8250
8303
TATGAAGTGGGAGGTAGA
sooosssssssssooss
5-8-5
32
1356

688305
571
8251
8304
CTATGAAGTGGGAGGTAG
sooosssssssssooss
5-8-5
47
1357

688306
572
8252
8305
TCTATGAAGTGGGAGGTA
sooosssssssssooss
5-8-5
33
1358

688307
573
8253
8306
CTCTATGAAGTGGGAGGT
sooosssssssssooss
5-8-5
55
1359

688308
574
8254
8307
ACTCTATGAAGTGGGAGG
sooosssssssssooss
5-8-5
51
1360

688309
575
8255
8308
CACTCTATGAAGTGGGAG
sooosssssssssooss
5-8-5
59
1361

688310
576
8256
8309
ACACTCTATGAAGTGGGA
sooosssssssssooss
5-8-5
58
1362

688311
577
8257
8310
CACACTCTATGAAGTGGG
sooosssssssssooss
5-8-5
59
1363

688312
578
8258
8311
ACACACTCTATGAAGTGG
sooosssssssssooss
5-8-5
42
1364

688313
579
8259
8312
CACACACTCTATGAAGTG
sooosssssssssooss
5-8-5
40
1365

TABLE 57

Percent inhibition of the C9ORF72 mRNA levels compared to PBS control by antisense

oligonucleotides targeting SEQ ID NOs: 1, 2 and 19

SEQ
SEQ
SEQ

ID
ID
ID
Mis-

NO:
NO:
NO:
matches

%

1
2
19
with SEQ

inhib-
SEQ

ISIS
Start
Start
Start
ID NO:

ition
ID

NO
Site
Site
Site
19
Sequence
Linkage
Motif
(RTS3750)
NO

576816
310
7990
8043
0
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
5-10-5
47
20

619420
310
7990
8043
0
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
5-10-5
51
20

688078
313
7993
8046
0
TGCCTTACTCTAGGACCA
sooosssssssssooss
5-8-5
32
1130

688314
580
8260
8313
0
ACACACACTCTATGAAGT
sooosssssssssooss
5-8-5
9
1366

688315
691
n/a
9505
1
CCCTGATCTTCCATTCTC
sooosssssssssooss
5-8-5
21
1367

688316
692
n/a
9506
2
ACCCTGATCTTCCATTCT
sooosssssssssooss
5-8-5
2
1368

688317
693
n/a
9507
3
GACCCTGATCTTCCATTC
sooosssssssssooss
5-8-5
16
1369

688318
694
n/a
9508
3
TGACCCTGATCTTCCATT
sooosssssssssooss
5-8-5
18
1370

688319
695
n/a
n/a
n/a
CTGACCCTGATCTTCCAT
sooosssssssssooss
5-8-5
5
1371

688320
696
n/a
n/a
n/a
TCTGACCCTGATCTTCCA
sooosssssssssooss
5-8-5
11
1372

688321
697
n/a
n/a
n/a
CTCTGACCCTGATCTTCC
sooosssssssssooss
5-8-5
21
1373

688322
698
n/a
n/a
n/a
ACTCTGACCCTGATCTTC
sooosssssssssooss
5-8-5
26
1374

688323
699
n/a
n/a
n/a
TACTCTGACCCTGATCTT
sooosssssssssooss
5-8-5
6
1375

688324
700
n/a
12554
3
ATACTCTGACCCTGATCT
sooosssssssssooss
5-8-5
7
1376

688325
701
n/a
12555
2
AATACTCTGACCCTGATC
sooosssssssssooss
5-8-5
0
1377

687955
n/a
13641
13680
0
ATGATTTCTTGTCTGGGA
sooosssssssssooss
5-8-5
31
1378

687956
n/a
13642
13681
0
CATGATTTCTTGTCTGGG
sooosssssssssooss
5-8-5
38
1379

687957
n/a
13643
13682
0
CCATGATTTCTTGTCTGG
sooosssssssssooss
5-8-5
12
1380

687958
n/a
13644
13683
0
GCCATGATTTCTTGTCTG
sooosssssssssooss
5-8-5
27
1381

687959
n/a
13645
13684
0
GGCCATGATTTCTTGTCT
sooosssssssssooss
5-8-5
26
1382

687960
n/a
13646
13685
0
GGGCCATGATTTCTTGTC
sooosssssssssooss
5-8-5
18
1383

687961
n/a
14089
14136
0
AACTAACATGTAGGCACT
sooosssssssssooss
5-8-5
38
1384

687962
n/a
14090
14137
0
GAACTAACATGTAGGCAC
sooosssssssssooss
5-8-5
57
1385

687963
n/a
14091
14138
0
GGAACTAACATGTAGGCA
sooosssssssssooss
5-8-5
63
1386

687964
n/a
14092
14139
0
AGGAACTAACATGTAGGC
sooosssssssssooss
5-8-5
29
1387

687965
n/a
14302
14349
0
CTTCTGATTCAAGCCATT
sooosssssssssooss
5-8-5
25
1388

687966
n/a
14303
14350
0
GCTTCTGATTCAAGCCAT
sooosssssssssooss
5-8-5
51
1389

687967
n/a
14304
14351
0
TGCTTCTGATTCAAGCCA
sooosssssssssooss
5-8-5
46
1390

687968
n/a
14305
14352
0
GTGCTTCTGATTCAAGCC
sooosssssssssooss
5-8-5
21
1391

687969
n/a
14306
14353
0
AGTGCTTCTGATTCAAGC
sooosssssssssooss
5-8-5
36
1392

687970
n/a
14307
14354
0
AAGTGCTTCTGATTCAAG
sooosssssssssooss
5-8-5
25
1393

687971
n/a
14308
14355
0
AAAGTGCTTCTGATTCAA
sooosssssssssooss
5-8-5
28
1394

687972
n/a
14309
14356
0
TAAAGTGCTTCTGATTCA
sooosssssssssooss
5-8-5
0
1395

687973
n/a
14310
14357
0
CTAAAGTGCTTCTGATTC
sooosssssssssooss
5-8-5
25
1396

687974
n/a
14311
14358
0
ACTAAAGTGCTTCTGATT
sooosssssssssooss
5-8-5
17
1397

687975
n/a
14312
14359
0
GACTAAAGTGCTTCTGAT
sooosssssssssooss
5-8-5
33
1398

687976
n/a
14313
14360
0
GGACTAAAGTGCTTCTGA
sooosssssssssooss
5-8-5
47
1399

687977
n/a
14314
14361
0
AGGACTAAAGTGCTTCTG
sooosssssssssooss
5-8-5
44
1400

687978
n/a
14315
14362
0
CAGGACTAAAGTGCTTCT
sooosssssssssooss
5-8-5
57
1401

687979
n/a
14316
14363
0
ACAGGACTAAAGTGCTTC
sooosssssssssooss
5-8-5
31
1402

687980
n/a
14317
14364
0
TACAGGACTAAAGTGCTT
sooosssssssssooss
5-8-5
24
1403

687981
n/a
14318
14365
0
ATACAGGACTAAAGTGCT
sooosssssssssooss
5-8-5
21
1404

687982
n/a
14319
14366
0
GATACAGGACTAAAGTGC
sooosssssssssooss
5-8-5
15
1405

687983
n/a
14320
14367
0
AGATACAGGACTAAAGTG
sooosssssssssooss
5-8-5
8
1406

687984
n/a
14321
14368
0
CAGATACAGGACTAAAGT
sooosssssssssooss
5-8-5
1
1407

687985
n/a
14322
14369
0
ACAGATACAGGACTAAAG
sooosssssssssooss
5-8-5
10
1408

687986
n/a
14323
14370
0
AACAGATACAGGACTAAA
sooosssssssssooss
5-8-5
11
1409

687987
n/a
14324
14371
0
GAACAGATACAGGACTAA
sooosssssssssooss
5-8-5
20
1410

687988
n/a
14325
14372
0
TGAACAGATACAGGACTA
sooosssssssssooss
5-8-5
25
1411

687989
n/a
14326
14373
0
CTGAACAGATACAGGACT
sooosssssssssooss
5-8-5
12
1412

687990
n/a
14327
14374
0
ACTGAACAGATACAGGAC
sooosssssssssooss
5-8-5
25
1413

687991
n/a
14328
14375
0
CACTGAACAGATACAGGA
sooosssssssssooss
5-8-5
8
1414

687992
n/a
14329
14376
0
ACACTGAACAGATACAGG
sooosssssssssooss
5-8-5
10
1415

687993
n/a
14330
14377
0
GACACTGAACAGATACAG
sooosssssssssooss
5-8-5
13
1416

687994
n/a
14331
14378
0
TGACACTGAACAGATACA
sooosssssssssooss
5-8-5
25
1417

687995
n/a
14332
14379
0
CTGACACTGAACAGATAC
sooosssssssssooss
5-8-5
35
1418

687996
n/a
14333
14380
0
GCTGACACTGAACAGATA
sooosssssssssooss
5-8-5
24
1419

687997
n/a
14334
14381
0
GGCTGACACTGAACAGAT
sooosssssssssooss
5-8-5
40
1420

687998
n/a
14335
14382
0
AGGCTGACACTGAACAGA
sooosssssssssooss
5-8-5
10
1421

687999
n/a
14336
14383
0
AAGGCTGACACTGAACAG
sooosssssssssooss
5-8-5
3
1422

688000
n/a
14337
14384
0
AAAGGCTGACACTGAACA
sooosssssssssooss
5-8-5
18
1423

688001
n/a
14338
14385
0
GAAAGGCTGACACTGAAC
sooosssssssssooss
5-8-5
17
1424

688002
n/a
14339
14386
0
TGAAAGGCTGACACTGAA
sooosssssssssooss
5-8-5
12
1425

688003
n/a
14358
14405
0
TGGGATTTAAAATGATGT
sooosssssssssooss
5-8-5
17
1426

688004
n/a
14359
14406
0
ATGGGATTTAAAATGATG
sooosssssssssooss
5-8-5
11
1427

688326
n/a
13402
13443
0
CTTGAGAAGAAAGCCTTC
sooosssssssssooss
5-8-5
6
1428

688327
n/a
14287
14334
0
ATTAAGGCTCTTAGGTTA
sooosssssssssooss
5-8-5
0
1429

688328
n/a
13499
13530
0
GTAGACAGTCTGTTATTT
sooosssssssssooss
5-8-5
27
1430

688329
n/a
14397
14444
0
TGACATGTAGAGAGATTA
sooosssssssssooss
5-8-5
43
1431

688330
n/a
13827
13866
0
TGGTTTAAGGGCACAAAC
sooosssssssssooss
5-8-5
0
1432

688331
n/a
13403
13444
0
ACTTGAGAAGAAAGCCTT
sooosssssssssooss
5-8-5
27
1433

688332
n/a
14257
14304
0
CCTCTGATACTCCATCAT
sooosssssssssooss
5-8-5
28
1434

688333
n/a
13471
13502
0
AAATCTTGTCATAGGTGA
sooosssssssssooss
5-8-5
21
1435

688334
n/a
13410
13451
0
AATTCTTACTTGAGAAGA
sooosssssssssooss
5-8-5
7
1436

688335
n/a
13885
13924
0
GGTGTATAGAGAATTCAG
sooosssssssssooss
5-8-5
41
1437

688336
n/a
14250
14297
0
TACTCCATCATGAGCCTA
sooosssssssssooss
5-8-5
35
1438

688337
n/a
13788
13827
0
GCTGGATGGAAAAAGATC
sooosssssssssooss
5-8-5
12
1439

688338
n/a
13517
13548
0
GTCCCTAGAACAATCTAA
sooosssssssssooss
5-8-5
28
1440

688339
n/a
14405
14452
0
GAAGAAATTGACATGTAG
sooosssssssssooss
5-8-5
12
1441

688340
n/a
13724
13763
0
CATCTACAGTACAACTTA
sooosssssssssooss
5-8-5
4
1442

TABLE 58

Percent inhibition of the C9ORF72 mRNA levels compared to PBS control by antisense

oligonucleotides targeting SEQ ID NOs: 1, 2 and 19

SEQ
SEQ
SEQ

ID
ID
ID

NO:
NO:
NO:

%

1
2
19

inhib-
SEQ

ISIS
Start
Start
Start

ition
ID

NO
Site
Site
Site
Sequence
Linkage
Motif
(RTS3750)
NO

688341
233
7913
7966
ATCTCTGTCTTGGCAACAGC
soooossssssssssooss
5-10-5
57
1443

688342
234
7914
7967
AATCTCTGTCTTGGCAACAG
soooossssssssssooss
5-10-5
35
1444

688343
235
7915
7968
CAATCTCTGTCTTGGCAACA
soooossssssssssooss
5-10-5
36
1445

655153
236
7916
7969
GCAATCTCTGTCTTGGCAAC
soooossssssssssooss
5-10-5
89
463

655154
237
7917
7970
AGCAATCTCTGTCTTGGCAA
soooossssssssssooss
5-10-5
81
464

688344
238
7918
7971
AAGCAATCTCTGTCTTGGCA
soooossssssssssooss
5-10-5
83
1446

655172
306
7986
8039
TACTCTAGGACCAAGAATAT
soooossssssssssooss
5-10-5
10
483

688345
307
7987
8040
TTACTCTAGGACCAAGAATA
soooossssssssssooss
5-10-5
1
1447

688346
308
7988
8041
CTTACTCTAGGACCAAGAAT
soooossssssssssooss
5-10-5
10
1448

625183
309
7989
8042
CCTTACTCTAGGACCAAGAA
soooossssssssssooss
5-10-5
44
484

576816
310
7990
8043
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
5-10-5
56
20

619420
310
7990
8043
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
5-10-5
73
20

688347
311
7991
8044
TGCCTTACTCTAGGACCAAG
soooossssssssssooss
5-10-5
62
1449

655173
312
7992
8045
GTGCCTTACTCTAGGACCAA
soooossssssssssooss
5-10-5
59
485

688348
313
7993
8046
TGTGCCTTACTCTAGGACCA
soooossssssssssooss
5-10-5
62
1450

688349
314
7994
8047
ATGTGCCTTACTCTAGGACC
soooossssssssssooss
5-10-5
62
1451

655174
315
7995
8048
AATGTGCCTTACTCTAGGAC
soooossssssssssooss
5-10-5
60
486

688350
319
7999
8052
CCCAAATGTGCCTTACTCTA
soooossssssssssooss
5-10-5
41
1452

688351
320
8000
8053
GCCCAAATGTGCCTTACTCT
soooossssssssssooss
5-10-5
62
1453

627833
321
8001
8054
AGCCCAAATGTGCCTTACTC
soooossssssssssooss
5-10-5
51
487

688352
322
8002
8055
GAGCCCAAATGTGCCTTACT
soooossssssssssooss
5-10-5
66
1454

688353
323
8003
8056
GGAGCCCAAATGTGCCTTAC
soooossssssssssooss
5-10-5
48
1455

619411
324
8004
8057
TGGAGCCCAAATGTGCCTTA
soooossssssssssooss
5-10-5
67
51

688354
325
8005
8058
TTGGAGCCCAAATGTGCCTT
soooossssssssssooss
5-10-5
53
1456

627608
326
8006
8059
TTTGGAGCCCAAATGTGCCT
soooossssssssssooss
5-10-5
42
1457

655175
327
8007
8060
CTTTGGAGCCCAAATGTGCC
soooossssssssssooss
5-10-5
26
488

655176
330
8010
8063
TGTCTTTGGAGCCCAAATGT
soooossssssssssooss
5-10-5
33
489

688355
331
8011
8064
CTGTCTTTGGAGCCCAAATG
soooossssssssssooss
5-10-5
56
1458

619412
332
8012
8065
TCTGTCTTTGGAGCCCAAAT
soooossssssssssooss
5-10-5
60
53

655177
333
8013
8066
TTCTGTCTTTGGAGCCCAAA
soooossssssssssooss
5-10-5
47
490

655178
334
8014
8067
GTTCTGTCTTTGGAGCCCAA
soooossssssssssooss
5-10-5
67
491

688356
335
8015
8068
TGTTCTGTCTTTGGAGCCCA
soooossssssssssooss
5-10-5
60
1459

655179
336
8016
8069
CTGTTCTGTCTTTGGAGCCC
soooossssssssssooss
5-10-5
59
492

688357
337
8017
8070
CCTGTTCTGTCTTTGGAGCC
soooossssssssssooss
5-10-5
50
1460

688358
338
8018
8071
ACCTGTTCTGTCTTTGGAGC
soooossssssssssooss
5-10-5
44
1461

655180
339
8019
8072
TACCTGTTCTGTCTTTGGAG
soooossssssssssooss
5-10-5
42
493

619413
340
8020
8073
GTACCTGTTCTGTCTTTGGA
soooossssssssssooss
5-10-5
56
135

688359
341
8021
8074
AGTACCTGTTCTGTCTTTGG
soooossssssssssooss
5-10-5
38
1462

655181
342
8022
8075
AAGTACCTGTTCTGTCTTTG
soooossssssssssooss
5-10-5
31
494

655185
351
8031
8084
ATCACTGAGAAGTACCTGTT
soooossssssssssooss
5-10-5
32
498

688360
352
8032
8085
CATCACTGAGAAGTACCTGT
soooossssssssssooss
5-10-5
53
1463

619414
353
8033
8086
CCATCACTGAGAAGTACCTG
soooossssssssssooss
5-10-5
48
136

655186
354
8034
8087
TCCATCACTGAGAAGTACCT
soooossssssssssooss
5-10-5
56
499

688361
355
8035
8088
CTCCATCACTGAGAAGTACC
soooossssssssssooss
5-10-5
54
1464

655201
415
8095
8148
CACTCTCTGCATTTCGAAGG
soooossssssssssooss
5-10-5
32
521

688362
416
8096
8149
CCACTCTCTGCATTTCGAAG
soooossssssssssooss
5-10-5
39
1465

688363
417
8097
8150
ACCACTCTCTGCATTTCGAA
soooossssssssssooss
5-10-5
37
1466

655202
418
8098
8151
CACCACTCTCTGCATTTCGA
soooossssssssssooss
5-10-5
50
522

688364
419
8099
8152
GCACCACTCTCTGCATTTCG
soooossssssssssooss
5-10-5
56
1467

655203
420
8100
8153
AGCACCACTCTCTGCATTTC
soooossssssssssooss
5-10-5
56
523

655204
421
8101
8154
TAGCACCACTCTCTGCATTT
soooossssssssssooss
5-10-5
25
524

688365
422
8102
8155
ATAGCACCACTCTCTGCATT
soooossssssssssooss
5-10-5
29
1468

688366
423
8103
8156
TATAGCACCACTCTCTGCAT
soooossssssssssooss
5-10-5
31
1469

655206
427
8107
8160
CATCTATAGCACCACTCTCT
soooossssssssssooss
5-10-5
20
526

688367
428
8108
8161
ACATCTATAGCACCACTCTC
soooossssssssssooss
5-10-5
28
1470

688368
429
8109
8162
TACATCTATAGCACCACTCT
soooossssssssssooss
5-10-5
24
1471

671081
430
8110
8163
TTACATCTATAGCACCACTC
soooossssssssssooss
5-10-5
26
1052

688369
431
8111
8164
TTTACATCTATAGCACCACT
soooossssssssssooss
5-10-5
40
1472

688370
432
8112
8165
CTTTACATCTATAGCACCAC
soooossssssssssooss
5-10-5
43
1473

655207
433
8113
8166
ACTTTACATCTATAGCACCA
soooossssssssssooss
5-10-5
33
527

688371
434
8114
8167
AACTTTACATCTATAGCACC
soooossssssssssooss
5-10-5
15
1474

688372
435
8115
8168
AAACTTTACATCTATAGCAC
soooossssssssssooss
5-10-5
24
1475

655208
436
8116
8169
AAAACTTTACATCTATAGCA
soooossssssssssooss
5-10-5
28
528

655215
456
8136
8189
TCCCTTTTCAGACAAGACAA
soooossssssssssooss
5-10-5
35
535

688373
457
8137
8190
CTCCCTTTTCAGACAAGACA
soooossssssssssooss
5-10-5
42
1476

688374
458
8138
8191
ACTCCCTTTTCAGACAAGAC
soooossssssssssooss
5-10-5
57
1477

655216
459
8139
8192
CACTCCCTTTTCAGACAAGA
soooossssssssssooss
5-10-5
51
536

671082
460
8140
8193
TCACTCCCTTTTCAGACAAG
soooossssssssssooss
5-10-5
45
1053

688375
461
8141
8194
ATCACTCCCTTTTCAGACAA
soooossssssssssooss
5-10-5
39
1478

655217
462
8142
8195
AATCACTCCCTTTTCAGACA
soooossssssssssooss
5-10-5
45
537

688376
463
8143
8196
TAATCACTCCCTTTTCAGAC
soooossssssssssooss
5-10-5
7
1479

688377
464
8144
8197
ATAATCACTCCCTTTTCAGA
soooossssssssssooss
5-10-5
1
1480

655218
465
8145
8198
AATAATCACTCCCTTTTCAG
soooossssssssssooss
5-10-5
23
538

655230
500
8180
8233
TCCCCATTCCAGTTTCCATC
soooossssssssssooss
5-10-5
57
550

688378
501
8181
8234
ATCCCCATTCCAGTTTCCAT
soooossssssssssooss
5-10-5
60
1481

688379
502
8182
8235
GATCCCCATTCCAGTTTCCA
soooossssssssssooss
5-10-5
55
1482

655231
503
8183
8236
CGATCCCCATTCCAGTTTCC
soooossssssssssooss
5-10-5
58
551

688380
504
8184
8237
GCGATCCCCATTCCAGTTTC
soooossssssssssooss
5-10-5
56
1483

688381
505
8185
8238
TGCGATCCCCATTCCAGTTT
soooossssssssssooss
5-10-5
46
1484

TABLE 59

Percent inhibition of the C9ORF72 mRNA levels compared to PBS control by antisense

oligonucleotides targeting SEQ ID NOs: 1, 2 and 19

SEQ
SEQ
SEQ

ID
ID
ID
Mis-

NO:
NO:
NO:
matches

%

1
2
19
with SEQ

inhib-
SEQ

ISIS
Start
Start
Start
ID NO:

ition
ID

NO
Site
Site
Site
19
Sequence
Linkage
Motif
(RTS3750)
NO

576816
310
7990
8043
0
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
5-10-5
53
20

619420
310
7990
8043
0
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
5-10-5
54
20

655173
312
7992
8045
0
GTGCCTTACTCTAGGACCAA
soooossssssssssooss
5-10-5
77
485

655232
506
8186
8239
0
CTGCGATCCCCATTCCAGTT
soooossssssssssooss
5-10-5
46
552

688382
507
8187
8240
0
GCTGCGATCCCCATTCCAGT
soooossssssssssooss
5-10-5
65
1485

688383
508
8188
8241
0
TGCTGCGATCCCCATTCCAG
soooossssssssssooss
5-10-5
60
1486

655233
509
8189
8242
0
GTGCTGCGATCCCCATTCCA
soooossssssssssooss
5-10-5
57
553

688384
510
8190
8243
0
TGTGCTGCGATCCCCATTCC
soooossssssssssooss
5-10-5
45
1487

688385
511
8191
8244
0
ATGTGCTGCGATCCCCATTC
soooossssssssssooss
5-10-5
68
1488

655234
512
8192
8245
0
TATGTGCTGCGATCCCCATT
soooossssssssssooss
5-10-5
32
554

655240
548
8228
8281
0
CTAAGTTCTGTCTGTGGAAG
soooossssssssssooss
5-10-5
23
560

688386
549
8229
8282
0
ACTAAGTTCTGTCTGTGGAA
soooossssssssssooss
5-10-5
45
1489

671083
550
8230
8283
0
AACTAAGTTCTGTCTGTGGA
soooossssssssssooss
5-10-5
41
1054

655241
551
8231
8284
0
AAACTAAGTTCTGTCTGTGG
soooossssssssssooss
5-10-5
28
561

688387
552
8232
8285
0
GAAACTAAGTTCTGTCTGTG
soooossssssssssooss
5-10-5
49
1490

688388
553
8233
8286
0
AGAAACTAAGTTCTGTCTGT
soooossssssssssooss
5-10-5
46
1491

655242
554
8234
8287
0
TAGAAACTAAGTTCTGTCTG
soooossssssssssooss
5-10-5
25
562

655289
691
n/a
9505
3
GACCCTGATCTTCCATTCTC
soooossssssssssooss
5-10-5
34
627

688389
692
n/a
9506
3
TGACCCTGATCTTCCATTCT
soooossssssssssooss
5-10-5
19
1492

688390
693
n/a
n/a
n/a
CTGACCCTGATCTTCCATTC
soooossssssssssooss
5-10-5
21
1493

655290
694
n/a
n/a
n/a
TCTGACCCTGATCTTCCATT
soooossssssssssooss
5-10-5
67
628

672561
695
n/a
n/a
n/a
CTCTGACCCTGATCTTCCAT
soooossssssssssooss
5-10-5
25
1056

625345
696
n/a
n/a
n/a
ACTCTGACCCTGATCTTCCA
soooossssssssssooss
5-10-5
15
1494

655291
697
n/a
n/a
n/a
TACTCTGACCCTGATCTTCC
soooossssssssssooss
5-10-5
18
629

688391
698
n/a
n/a
n/a
ATACTCTGACCCTGATCTTC
soooossssssssssooss
5-10-5
40
1495

688392
699
n/a
n/a
n/a
AATACTCTGACCCTGATCTT
soooossssssssssooss
5-10-5
8
1496

619423
n/a
13642
13681
0
GCCATGATTTCTTGTCTGGG
soooossssssssssooss
5-10-5
77
383

655417
n/a
14089
14136
0
GGAACTAACATGTAGGCACT
soooossssssssssooss
5-10-5
83
738

655420
n/a
14331
14378
0
GCTGACACTGAACAGATACA
soooossssssssssooss
5-10-5
51
741

655422
n/a
14452
14499
0
ATCATTTAATTAATGTATTT
soooossssssssssooss
5-10-5
33
743

671084
n/a
14316
14363
0
ATACAGGACTAAAGTGCTTC
soooossssssssssooss
5-10-5
74
1055

688393
n/a
13641
13680
0
CCATGATTTCTTGTCTGGGA
soooossssssssssooss
5-10-5
54
1497

688394
n/a
13643
13682
0
GGCCATGATTTCTTGTCTGG
soooossssssssssooss
5-10-5
52
1498

688395
n/a
13644
13683
0
GGGCCATGATTTCTTGTCTG
soooossssssssssooss
5-10-5
46
1499

688396
n/a
13731
13770
0
ACTTAAGTTCATCTACAGTA
soooossssssssssooss
5-10-5
18
1500

688397
n/a
13792
13831
0
TCCACTGCTGGATGGAAAAA
soooossssssssssooss
5-10-5
20
1501

688398
n/a
13968
14007
0
ATATTATTTATCTTACTCAA
soooossssssssssooss
5-10-5
32
1502

688399
n/a
13982
14021
0
GTTCTAAGTGCTTTATATTA
soooossssssssssooss
5-10-5
30
1503

688400
n/a
14090
14137
0
AGGAACTAACATGTAGGCAC
soooossssssssssooss
5-10-5
87
1504

688401
n/a
14122
14169
0
ATATAAGATAATACATGTAA
soooossssssssssooss
5-10-5
6
1505

688402
n/a
14243
14290
0
CATCATGAGCCTAAAGGAAA
soooossssssssssooss
5-10-5
22
1506

688403
n/a
14244
14291
0
CCATCATGAGCCTAAAGGAA
soooossssssssssooss
5-10-5
37
1507

688404
n/a
14300
14347
0
CTTCTGATTCAAGCCATTAA
soooossssssssssooss
5-10-5
76
1508

688405
n/a
14302
14349
0
TGCTTCTGATTCAAGCCATT
soooossssssssssooss
5-10-5
55
1509

688406
n/a
14303
14350
0
GTGCTTCTGATTCAAGCCAT
soooossssssssssooss
5-10-5
50
1510

688407
n/a
14304
14351
0
AGTGCTTCTGATTCAAGCCA
soooossssssssssooss
5-10-5
68
1511

688408
n/a
14305
14352
0
AAGTGCTTCTGATTCAAGCC
soooossssssssssooss
5-10-5
41
1512

688409
n/a
14306
14353
0
AAAGTGCTTCTGATTCAAGC
soooossssssssssooss
5-10-5
18
1513

688410
n/a
14307
14354
0
TAAAGTGCTTCTGATTCAAG
soooossssssssssooss
5-10-5
29
1514

688411
n/a
14308
14355
0
CTAAAGTGCTTCTGATTCAA
soooossssssssssooss
5-10-5
43
1515

688412
n/a
14309
14356
0
ACTAAAGTGCTTCTGATTCA
soooossssssssssooss
5-10-5
33
1516

688413
n/a
14310
14357
0
GACTAAAGTGCTTCTGATTC
soooossssssssssooss
5-10-5
49
1517

688414
n/a
14311
14358
0
GGACTAAAGTGCTTCTGATT
soooossssssssssooss
5-10-5
49
1518

688415
n/a
14312
14359
0
AGGACTAAAGTGCTTCTGAT
soooossssssssssooss
5-10-5
55
1519

688416
n/a
14313
14360
0
CAGGACTAAAGTGCTTCTGA
soooossssssssssooss
5-10-5
71
1520

688417
n/a
14314
14361
0
ACAGGACTAAAGTGCTTCTG
soooossssssssssooss
5-10-5
66
1521

688418
n/a
14315
14362
0
TACAGGACTAAAGTGCTTCT
soooossssssssssooss
5-10-5
61
1522

688419
n/a
14317
14364
0
GATACAGGACTAAAGTGCTT
soooossssssssssooss
5-10-5
76
1523

688420
n/a
14318
14365
0
AGATACAGGACTAAAGTGCT
soooossssssssssooss
5-10-5
46
1524

688421
n/a
14319
14366
0
CAGATACAGGACTAAAGTGC
soooossssssssssooss
5-10-5
53
1525

688422
n/a
14320
14367
0
ACAGATACAGGACTAAAGTG
soooossssssssssooss
5-10-5
23
1526

688423
n/a
14321
14368
0
AACAGATACAGGACTAAAGT
soooossssssssssooss
5-10-5
28
1527

688424
n/a
14322
14369
0
GAACAGATACAGGACTAAAG
soooossssssssssooss
5-10-5
26
1528

688425
n/a
14323
14370
0
TGAACAGATACAGGACTAAA
soooossssssssssooss
5-10-5
13
1529

688426
n/a
14324
14371
0
CTGAACAGATACAGGACTAA
soooossssssssssooss
5-10-5
27
1530

688427
n/a
14325
14372
0
ACTGAACAGATACAGGACTA
soooossssssssssooss
5-10-5
37
1531

688428
n/a
14326
14373
0
CACTGAACAGATACAGGACT
soooossssssssssooss
5-10-5
35
1532

688429
n/a
14327
14374
0
ACACTGAACAGATACAGGAC
soooossssssssssooss
5-10-5
28
1533

688430
n/a
14328
14375
0
GACACTGAACAGATACAGGA
soooossssssssssooss
5-10-5
39
1534

688431
n/a
14329
14376
0
TGACACTGAACAGATACAGG
soooossssssssssooss
5-10-5
38
1535

688432
n/a
14330
14377
0
CTGACACTGAACAGATACAG
soooossssssssssooss
5-10-5
64
1536

688433
n/a
14332
14379
0
GGCTGACACTGAACAGATAC
soooossssssssssooss
5-10-5
50
1537

688434
n/a
14333
14380
0
AGGCTGACACTGAACAGATA
soooossssssssssooss
5-10-5
18
1538

688435
n/a
14334
14381
0
AAGGCTGACACTGAACAGAT
soooossssssssssooss
5-10-5
45
1539

688436
n/a
14335
14382
0
AAAGGCTGACACTGAACAGA
soooossssssssssooss
5-10-5
28
1540

688437
n/a
14336
14383
0
GAAAGGCTGACACTGAACAG
soooossssssssssooss
5-10-5
18
1541

688438
n/a
14337
14384
0
TGAAAGGCTGACACTGAACA
soooossssssssssooss
5-10-5
23
1542

688439
n/a
14358
14405
0
AATGGGATTTAAAATGATGT
soooossssssssssooss
5-10-5
26
1543

688440
n/a
14359
14406
0
AAATGGGATTTAAAATGATG
soooossssssssssooss
5-10-5
30
1544

688441
n/a
14360
14407
0
CAAATGGGATTTAAAATGAT
soooossssssssssooss
5-10-5
16
1545

TABLE 60

Percent inhibition of the C9ORF72 mRNA levels compared to PBS control by antisense

oligonucleotides targeting SEQ ID NOs: 1, 2 and 19

SEQ
SEQ
SEQ

ID
ID
ID

NO:
NO:
NO:

%

1
2
19

inhib-
SEQ

ISIS
Start
Start
Start

ition
ID

NO
Site
Site
Site
Sequence
Linkage
Motif
(RTS3750)
NO

576816
310
7990
8043
GCCTTACTCTAGGACCAAGA
sssssssssssssssssss
5-10-5
62
20

619420
310
7990
8043
GCCTTACTCTAGGACCAAGA
soooossssssssssooss
5-10-5
75
20

688078
313
7993
8046
TGCCTTACTCTAGGACCA
sooosssssssssooss
5-8-5
67
1130

688237
485
8165
8218
ATCAAAGATTAATGAAAC
sooosssssssssooss
5-8-5
42
1289

688238
486
8166
8219
CATCAAAGATTAATGAAA
sooosssssssssooss
5-8-5
0
1290

688239
487
8167
8220
CCATCAAAGATTAATGAA
sooosssssssssooss
5-8-5
39
1291

688240
488
8168
8221
TCCATCAAAGATTAATGA
sooosssssssssooss
5-8-5
45
1292

688241
489
8169
8222
TTCCATCAAAGATTAATG
sooosssssssssooss
5-8-5
0
1293

688242
490
8170
8223
TTTCCATCAAAGATTAAT
sooosssssssssooss
5-8-5
53
1294

688243
491
8171
8224
GTTTCCATCAAAGATTAA
sooosssssssssooss
5-8-5
46
1295

688244
492
8172
8225
AGTTTCCATCAAAGATTA
sooosssssssssooss
5-8-5
43
1296

688245
493
8173
8226
CAGTTTCCATCAAAGATT
sooosssssssssooss
5-8-5
0
1297

688246
494
8174
8227
CCAGTTTCCATCAAAGAT
sooosssssssssooss
5-8-5
44
1298

688247
495
8175
8228
TCCAGTTTCCATCAAAGA
sooosssssssssooss
5-8-5
46
1299

688248
496
8176
8229
TTCCAGTTTCCATCAAAG
sooosssssssssooss
5-8-5
58
1300

688249
497
8177
8230
ATTCCAGTTTCCATCAAA
sooosssssssssooss
5-8-5
46
1301

688250
498
8178
8231
CATTCCAGTTTCCATCAA
sooosssssssssooss
5-8-5
50
1302

688251
499
8179
8232
CCATTCCAGTTTCCATCA
sooosssssssssooss
5-8-5
58
1303

688252
500
8180
8233
CCCATTCCAGTTTCCATC
sooosssssssssooss
5-8-5
66
1304

688253
501
8181
8234
CCCCATTCCAGTTTCCAT
sooosssssssssooss
5-8-5
59
1305

688254
502
8182
8235
TCCCCATTCCAGTTTCCA
sooosssssssssooss
5-8-5
52
1306

688255
503
8183
8236
ATCCCCATTCCAGTTTCC
sooosssssssssooss
5-8-5
61
1307

688256
504
8184
8237
GATCCCCATTCCAGTTTC
sooosssssssssooss
5-8-5
50
1308

688257
505
8185
8238
CGATCCCCATTCCAGTTT
sooosssssssssooss
5-8-5
48
1309

688258
506
8186
8239
GCGATCCCCATTCCAGTT
sooosssssssssooss
5-8-5
44
1310

688259
507
8187
8240
TGCGATCCCCATTCCAGT
sooosssssssssooss
5-8-5
49
1311

688260
508
8188
8241
CTGCGATCCCCATTCCAG
sooosssssssssooss
5-8-5
70
1312

688261
509
8189
8242
GCTGCGATCCCCATTCCA
sooosssssssssooss
5-8-5
72
1313

688262
510
8190
8243
TGCTGCGATCCCCATTCC
sooosssssssssooss
5-8-5
47
1314

688263
511
8191
8244
GTGCTGCGATCCCCATTC
sooosssssssssooss
5-8-5
0
1315

688264
512
8192
8245
TGTGCTGCGATCCCCATT
sooosssssssssooss
5-8-5
56
1316

688265
513
8193
8246
ATGTGCTGCGATCCCCAT
sooosssssssssooss
5-8-5
65
1317

688266
514
8194
8247
TATGTGCTGCGATCCCCA
sooosssssssssooss
5-8-5
61
1318

688267
533
8213
8266
AAGTATAATTGATAGTCC
sooosssssssssooss
5-8-5
47
1319

688268
534
8214
8267
GAAGTATAATTGATAGTC
sooosssssssssooss
5-8-5
48
1320

688269
535
8215
8268
GGAAGTATAATTGATAGT
sooosssssssssooss
5-8-5
35
1321

688270
536
8216
8269
TGGAAGTATAATTGATAG
sooosssssssssooss
5-8-5
41
1322

688271
537
8217
8270
GTGGAAGTATAATTGATA
sooosssssssssooss
5-8-5
48
1323

688272
538
8218
8271
TGTGGAAGTATAATTGAT
sooosssssssssooss
5-8-5
42
1324

688273
539
8219
8272
CTGTGGAAGTATAATTGA
sooosssssssssooss
5-8-5
32
1325

688274
540
8220
8273
TCTGTGGAAGTATAATTG
sooosssssssssooss
5-8-5
0
1326

688275
541
8221
8274
GTCTGTGGAAGTATAATT
sooosssssssssooss
5-8-5
0
1327

688276
542
8222
8275
TGTCTGTGGAAGTATAAT
sooosssssssssooss
5-8-5
65
1328

688277
543
8223
8276
CTGTCTGTGGAAGTATAA
sooosssssssssooss
5-8-5
0
1329

688278
544
8224
8277
TCTGTCTGTGGAAGTATA
sooosssssssssooss
5-8-5
62
1330

688279
545
8225
8278
TTCTGTCTGTGGAAGTAT
sooosssssssssooss
5-8-5
55
1331

688280
546
8226
8279
GTTCTGTCTGTGGAAGTA
sooosssssssssooss
5-8-5
77
1332

688281
547
8227
8280
AGTTCTGTCTGTGGAAGT
sooosssssssssooss
5-8-5
63
1333

688282
548
8228
8281
AAGTTCTGTCTGTGGAAG
sooosssssssssooss
5-8-5
49
1334

688283
549
8229
8282
TAAGTTCTGTCTGTGGAA
sooosssssssssooss
5-8-5
0
1335

688284
550
8230
8283
CTAAGTTCTGTCTGTGGA
sooosssssssssooss
5-8-5
54
1336

688285
551
8231
8284
ACTAAGTTCTGTCTGTGG
sooosssssssssooss
5-8-5
69
1337

688286
552
8232
8285
AACTAAGTTCTGTCTGTG
sooosssssssssooss
5-8-5
65
1338

688287
553
8233
8286
AAACTAAGTTCTGTCTGT
sooosssssssssooss
5-8-5
40
1339

688288
554
8234
8287
GAAACTAAGTTCTGTCTG
sooosssssssssooss
5-8-5
36
1340

688289
555
8235
8288
AGAAACTAAGTTCTGTCT
sooosssssssssooss
5-8-5
47
1341

688290
556
8236
8289
TAGAAACTAAGTTCTGTC
sooosssssssssooss
5-8-5
48
1342

688291
557
8237
8290
GTAGAAACTAAGTTCTGT
sooosssssssssooss
5-8-5
45
1343

688292
558
8238
8291
GGTAGAAACTAAGTTCTG
sooosssssssssooss
5-8-5
44
1344

688293
559
8239
8292
AGGTAGAAACTAAGTTCT
sooosssssssssooss
5-8-5
58
1345

688294
560
8240
8293
GAGGTAGAAACTAAGTTC
sooosssssssssooss
5-8-5
45
1346

688295
561
8241
8294
GGAGGTAGAAACTAAGTT
sooosssssssssooss
5-8-5
47
1347

688296
562
8242
8295
GGGAGGTAGAAACTAAGT
sooosssssssssooss
5-8-5
44
1348

688297
563
8243
8296
TGGGAGGTAGAAACTAAG
sooosssssssssooss
5-8-5
47
1349

688298
564
8244
8297
GTGGGAGGTAGAAACTAA
sooosssssssssooss
5-8-5
53
1350

688299
565
8245
8298
AGTGGGAGGTAGAAACTA
sooosssssssssooss
5-8-5
43
1351

688300
566
8246
8299
AAGTGGGAGGTAGAAACT
sooosssssssssooss
5-8-5
36
1352

688301
567
8247
8300
GAAGTGGGAGGTAGAAAC
sooosssssssssooss
5-8-5
41
1353

688302
568
8248
8301
TGAAGTGGGAGGTAGAAA
sooosssssssssooss
5-8-5
0
1354

688303
569
8249
8302
ATGAAGTGGGAGGTAGAA
sooosssssssssooss
5-8-5
41
1355

688304
570
8250
8303
TATGAAGTGGGAGGTAGA
sooosssssssssooss
5-8-5
31
1356

688305
571
8251
8304
CTATGAAGTGGGAGGTAG
sooosssssssssooss
5-8-5
46
1357

688306
572
8252
8305
TCTATGAAGTGGGAGGTA
sooosssssssssooss
5-8-5
30
1358

688307
573
8253
8306
CTCTATGAAGTGGGAGGT
sooosssssssssooss
5-8-5
54
1359

688308
574
8254
8307
ACTCTATGAAGTGGGAGG
sooosssssssssooss
5-8-5
50
1360

688309
575
8255
8308
CACTCTATGAAGTGGGAG
sooosssssssssooss
5-8-5
60
1361

688310
576
8256
8309
ACACTCTATGAAGTGGGA
sooosssssssssooss
5-8-5
56
1362

688311
577
8257
8310
CACACTCTATGAAGTGGG
sooosssssssssooss
5-8-5
57
1363

688312
578
8258
8311
ACACACTCTATGAAGTGG
sooosssssssssooss
5-8-5
41
1364

688313
579
8259
8312
CACACACTCTATGAAGTG
sooosssssssssooss
5-8-5
38
1365

Example 13: Dose-Dependent Antisense Inhibition of Human C9ORF72 mRNA in HepG2 Cells

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. ISIS 619420 described in Example 2 hereinabove was also tested. 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 0.33 μM, 1.00 μM, 3.00 μM, or 9.00 μM 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 primer probe set RTS3750 was used to measure the total C9ORF72 mRNA levels. C9ORF72 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of C9ORF72 levels, relative to untreated control cells.

The half maximal inhibitory concentration (IC⁵⁰) of each oligonucleotide is also presented in the Tables below. As shown in the Tables below, total C9ORF72 mRNA levels were reduced in a dose-dependent manner in some of the antisense oligonucleotide treated cells.

TABLE 61

Dose-dependent inhibition of

total C9ORF72 mRNA levels in HepG2 cells

0.33
1.00
3.00
9.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

619411
55
68
89
90
<0.3

619420
41
74
87
95
0.4

655178
45
63
84
92
0.4

687962
48
61
83
86
0.4

687963
34
53
77
76
0.8

687978
50
60
70
65
<0.3

688077
49
63
70
86
0.3

688088
51
72
86
92
<0.3

688089
51
66
76
85
<0.3

688099
41
66
73
85
0.5

688100
44
64
79
86
0.4

688101
53
64
77
74
<0.3

688102
29
43
52
62
2.4

688172
31
47
77
88
1.0

688261
35
41
50
49
6.7

688347
41
60
79
89
0.5

688348
57
61
85
92
<0.3

688352
51
64
71
75
<0.3

TABLE 62

Dose-dependent inhibition of

total C9ORF72 mRNA levels in HepG2 cells

0.33
1.00
3.00
9.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

619412
48
69
83
87
0.3

619413
34
33
47
85
1.8

619420
53
72
80
84
<0.3

655173
48
62
76
89
0.4

655174
38
59
82
79
0.6

655179
47
68
83
80
0.3

655186
40
53
82
85
0.6

655203
28
61
87
79
0.7

655230
36
59
77
89
0.7

655231
49
69
78
81
<0.3

688349
40
63
84
79
0.5

688351
47
74
81
88
0.3

688355
50
56
79
83
0.4

688356
47
64
78
85
0.3

688364
29
44
57
78
1.5

688374
38
50
79
70
0.8

688378
41
67
85
87
0.4

688379
39
42
83
83
0.8

688380
50
62
68
82
0.3

TABLE 63

Dose-dependent inhibition of

total C9ORF72 mRNA levels in HepG2 cells

0.33
1.00
3.00
9.00
IC₅₀

ISIS No
μM
μM
μM
μM
(μM)

619420
37
39
87
92
0.3

619423
55
73
84
82
<0.3

655173
53
83
82
94
<0.3

655233
34
28
41
79
2.4

655290
16
27
68
78
2.0

655417
73
42
87
84
<0.3

688022
51
71
80
76
<0.3

688360
32
63
85
72
0.6

688361
46
70
34
85
0.5

688382
42
58
79
81
0.5

688383
43
63
80
87
0.5

688385
41
60
82
84
0.5

688400
61
81
85
85
<0.3

688404
38
62
78
81
0.6

688407
37
53
71
85
0.8

688415
43
18
79
73
1.3

688416
48
74
73
81
<0.3

688417
33
61
67
74
0.8

688418
34
55
77
82
0.8

Example 14: Tolerability of Antisense Oligonucleotides Targeting Human C9ORF72 in Mice

Antisense oligonucleotides from the Examples above were tested in a standard mouse model to assess tolerability of the oligonucleotides. The rodents were assessed by standard FOB assays and measurement of GFAP and/or AIF expression levels.

Groups of mice were administered a single ICV dose of 700 μg of ISIS oligonucleotides.

Mouse FOB Assay

At 3 hours, one week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks post injection, each mouse was evaluated according to 7 different criteria. The 7 criteria are (1) the mouse was bright, alert, and responsive; (2) the mouse was standing or hunched without stimuli; (3) the mouse showed any movement without stimuli (4) the mouse demonstrated forward movement after it was lifted; (5) the mouse demonstrated any movement after it was lifted; (6) the mouse responded to a tail pinch; (7) regular breathing. For each of the 7 different criteria, each mouse was given a sub-score of 0 if it met the criteria or 1 if it did not. After all of the 7 criteria were evaluated, the sub-scores were summed for each mouse and then averaged for each group. For example, if a mouse was bright, alert, and responsive 3 hours after the 700 μg ICV dose, and met all other criteria, it would get a summed score of 0. If another mouse was not bright, alert, and responsive 3 hours after the 700 μg ICV dose but met all other criteria, it would receive a score of 1.

The results are presented as individual scores for each mouse in each group. The expression levels of GFAP and AIF1 in the thoracic spinal cord of the mice were assessed by qRT-PCR after 8 weeks.

Study 1 with cEt Oligonucleotides

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 64

Antisense oligonucleotides targeted to SEQ ID NO: 2

ISIS No
Start Site

672744
1335

672747
1338

672774
1368

672775
1369

672778
1372

672779
1373

672831
1440

672908
1349

672909
1350

672919
1360

672924
1368

672925
1369

672927
1371

672928
1372

672929
1373

672976
1435

672980
1439

672981
1440

TABLE 65

FOB scores in C57/Bl6 mice

3 hr

PBS
0, 0, 0, 0

672744
7, 7, 7, 7

672747
7, 7, 7, 7

672774
5, 5, 5, 5

672775
5, 7, 5, 7

672778
1,7, 1, 1

672779
7, 2, 5, 7

672831
7, 7, 7, 7

672908
6, 1, 1, 1

672909
6, 6, 6, 4

672919
5, 5, 5, 5

672924
4, 6, 7, 6

672925
4, 4, 4, 4

672927
0, 0, 0, 0

672928
0, 0, 0, 0

672929
7, 7, 7, 7

672976
5, 7, 7, 7

672980
2, 2, 2, 2

672981
6, 6, 6, 6

Study 2 with cEt Oligonucleotides

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 66

Antisense oligonucleotides

targeted to SEQ ID NO: 2

Start Site of

ISIS No
SEQ ID NO: 2

672982
1441

672983
1442

672984
1443

672985
1444

673021
1510

673023
1512

673026
1515

673036
1327

673047
1338

673057
1348

673058
1349

673067
1358

673068
1359

673074
1368

673079
1373

673082
1376

673088
1396

673125
1434

673126
1435

673127
1436

672832
1441

TABLE 67

FOB scores in C57/Bl6 mice

3 hr

PBS
0, 0, 0, 0

672982
7, 7, 7, 7

672983
5, 7, 5, 7

672984
4, 6, 6, 6

672985
5, 4, 4, 4

673021
5, 5, 5, 5

673023
7, 7, 7, 7

673026
0, 0, 0, 0

673036
7, 7, 7, 7

673047
5, 5, 5, 6

673057
5, 0, 5, 7

673058
3, 3, 3, 3

673067
7, 0, 0, 6

673068
6, 6, 6, 6

673074
1, 1, 1, 1

673079
6, 6, 7, 6

673082
1, 3, 0, 3

673088
6, 6, 6, 6

673125
0, 0, 0, 0

673126
0, 0, 0, 0

673127
7, 7, 7, 7

672832
6, 6, 6, 7

Study 3 with cEt Oligonucleotides

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 68

Antisense oligonucleotides

targeted to SEQ ID NO: 2

Start Site of

ISIS No
SEQ ID NO: 2

673131
1440

673132
1441

673133
1442

673134
1443

673135
1444

673193
1334

673203
1344

673204
1345

673224
1368

673225
1369

673226
1370

673228
1372

673229
1373

673275
1434

673276
1435

673280
1439

TABLE 69

FOB scores in C57/Bl6 mice

3 hr

PBS
0, 0, 0, 0

673131
4, 4, 4, 5

673132
4, 4, 4, 4

673133
4, 3, 4, 3

673134
5, 6, 3, 6

673135
5, 3, 4, 5

673193
6, 6, 6, 6

673203
4, 3, 4, 4

673204
2, 3, 2, 2

673224
5, 5, 5, 5

673225
1, 2, 2, 2

673226
1, 1, 1, 1

673228
2, 2, 2, 2

673229
6, 6, 6, 6

673275
1, 1, 1, 1

673276
3, 3, 3, 3

673280
1, 1, 1, 1

Study 4 with cEt Oligonucleotides

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 70

Antisense oligonucleotides

targeted to SEQ ID NO: 2

Start Site of

ISIS No
SEQ ID NO: 2

673128
1437

673130
1439

673281
1440

673282
1441

673283
1442

673284
1443

673285
1444

673323
1512

673331
1520

673332
1521

673374
1368

673375
1369

673377
1371

673378
1372

673379
1373

673430
1439

673431
1440

673432
1441

673434
1443

TABLE 71

FOB scores in C57/Bl6 mice

3 hr

PBS
0, 0, 0, 0

673128
0, 0, 0, 0

673130
0, 0, 0, 0

673281
7, 7, 7, 7

673282
7, 7, 7, 7

673283
5, 6, 6, 7

673284
5, 0, 0, 0

673285
3, 3, 3, 3

673323
7, 7, 7, 7

673331
7, 7, 7, 7

673332
7, 7, 7, 7

673374
7, 7, 7, 7

673375
2, 3, 2, 3

673377
0, 0, 0, 0

673378
1, 1, 1, 1

673379
4, 6, 6, 7

673430
7, 7, 7, 7

673431
7, 7, 7, 7

673432
7, 6, 6, 7

673434
7, 7, 7, 7

Study 1 with MOE Oligonucleotides

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 72

Antisense oligonucleotides

targeted to SEQ ID NO: 2

Start Site of

ISIS No
SEQ ID NO: 2

619253
1406

619293
1446

619322
1481

619352
1519

619353
1520

619410
7840

619414
8033

619420
7990

619421
28251

619422
3452

619423
13642

653222
1553

653223
5325

655016
1458

655017
1459

TABLE 73

FOB scores in C57/Bl6 mice

3 hrs

PBS
0, 0, 0, 0

619253
3, 3, 7, 7

619293
6, 4, 2, 4

619322
2, 2, 2, 4

619352
0, 0, 7, 0

619353
6, 4, 7, 6

619410
7, 3, 3, 3

619414
6, 6, 6, 6

619420
7, 7, 7, 7

619421
1, 1, 1, 1

619422
5, 5, 5, 5

619423
6, 6, 6, 6

653222
3, 3, 3, 3

653223
1, 1, 7, 1

655016
5, 5, 3, 3

655017
0, 0, 0, 0

Study 2 with MOE Oligonucleotides

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 74

Antisense oligonucleotides

targeted to SEQ ID NO: 2

Start Site of

ISIS No
SEQ ID NO: 2

619173
1326

619174
1327

619178
1331

619179
1332

619180
1333

619181
1334

619182
1335

619185
1338

619186
1339

619187
1340

619191
1344

619201
1354

619202
1355

619203
1356

619216
1369

619217
1370

TABLE 75

FOB scores in C57/Bl6 mice

3 hrs

PBS
0, 0, 0, 0

619173
7, 7, 7, 7

619174
7, 7, 7, 7

619178
7, 7, 7, 7

619179
7, 7, 7, 7

619180
7, 7, 7, 7

619181
7, 7, 7, 7

619182
7, 7, 7, 7

619185
7, 6, 6, 7

619186
6, 6, 6, 7

619187
5, 7, 5, 7

619191
5, 5, 3, 5

619201
3, 3, 3, 1

619202
6, 6, 6, 6

619203
5, 1, 5, 5

619216
4, 4, 4, 4

619217
2, 2, 2, 2

Study 3 with MOE Oligonucleotides

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 76

Antisense oligonucleotides

targeted to SEQ ID NO: 2

Start Site of

ISIS No
SEQ ID NO: 2

619218
1371

619245
1398

619246
1399

619247
1400

619251
1404

619252
1405

619259
1412

619260
1413

619276
1429

619278
1431

619280
1433

619284
1437

619292
1445

619297
1450

619298
1451

619307
1466

TABLE 77

FOB scores in C57/Bl6 mice

3 hrs

PBS
0, 0, 0, 0

619218
4, 2, 7, 7

619245
4, 4, 4, 4

619246
4, 5, 5, 4

619247
1, 1, 1, 1

619251
4, 7, 4, 4

619252
6, 6, 6, 0

619259
6, 6, 4, 5

619260
4, 4, 4, 4

619276
3, 3, 4, 4

619278
4, 7, 2, 7

619280
7, 7, 7, 7

619284
0, 0, 0, 0

619292
6, 5, 5, 5

619297
7, 4, 4, 4

619298
0, 0, 0, 0

619307
0, 0, 0, 0

Study 4 with MOE Oligonucleotides

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 78

Antisense oligonucleotides

targeted to SEQ ID NO: 2

Start Site of

ISIS No
SEQ ID NO: 2

619266
1419

619268
1421

619316
1475

619317
1476

619336
1503

619337
1504

619338
1505

619339
1506

619341
1508

619342
1509

619343
1510

619344
1511

619346
1513

619350
1517

619351
1518

619413
8020

TABLE 79

FOB scores in C57/Bl6 mice

3 hrs

PBS
0, 0, 0, 0

619266
2, 2, 2, 2

619268
6, 6, 7, 7

619316
5, 5, 5, 7

619317
2, 3, 3, 4

619336
1, 1, 1, 7

619338
0, 0, 7, 7

619339
0, 5, 7, 7

619341
7, 7, 7, 7

619342
6, 6, 6, 6

619343
6, 6, 6, 6

619344
7, 7, 7, 7

619346
5, 5, 7, 7

619350
6, 6, 7, 7

619351
6, 6, 7, 7

619413
7, 6, 7, 6

Example 15: Tolerability of Antisense Oligonucleotides Targeting Human C9ORF72 in Rats

Antisense oligonucleotides from the Examples above were tested in a standard rat model to assess tolerability of the oligonucleotides. The rodents were assessed by standard FOB assays and measurement of GFAP and/or AIF expression levels. Groups of Sprague-Dawley rats were administered intrathecally with 2,000 μg or 3,000 μg of ISIS oligonucleotide as specified in the Tables below.

Rat FOB Assay

At 3 hours, one week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks post injection the movement of 7 different parts of the body was evaluated for each rat. The 7 body parts were (1) the rat's tail; (2) the rat's posterior posture; (3) the rat's hind limbs; (4) the rat's hind paws; (5) the rat's forepaws; (6) the rat's anterior posture; and (7) the rat's head. For each of the 7 different body parts, each rat was given a sub-score of 0 if the body part was moving or 1 if the body part was paralyzed. After each of the 7 body parts was evaluated, the sub-scores were summed for each rat and then averaged for each group. Saline treated rats generally receive a score of 0.

Study 1 with 5-10-5 MOE Oligonucleotides at 3,000 μg

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 80

Antisense oligonucleotides targeted to SEQ ID NO: 2

Start Site of

ISIS No
SEQ ID NO: 2

619185
1338

619186
1339

619187
1340

619191
1344

619201
1354

619203
1356

619217
1370

619218
1371

619251
1404

619252
1405

619259
1412

619266
1419

619268
1421

619278
1431

619284
1437

619346
1513

619350
1517

619351
1518

TABLE 81

FOB scores in Sprague-Dawley rats

3 hrs

PBS
0, 0, 0

619185
7, 7, 6

619186
0, 6, 6

619187
7, 0, 6

619191
6, 0, 0

619201
1, 4, 4

619203
5, 1, 5

619217
4, 4, 4

619218
4, 2, 4

619251
6, 6, 6

619252
6, 6, 6

619259
0, 6, 6

619266
4, 4, 3

619268
6, 6, 6

619278
6, 6, 6

619284
1

619346
0, 0, 6

619350
4, 0, 3

619351
3, 3, 3

Study 2 with 5-10-5 MOE Oligonucleotides at 3,000 μg

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 82

Antisense oligonucleotides targeted to SEQ ID NO: 2

Start

Site of

ISIS
SEQ ID

No
NO: 2

619176
1329

619183
1336

619253
1406

619260
1413

619276
1429

619292
1445

619293
1446

619307
1466

619317
1476

619338
1505

619339
1506

619342
1509

TABLE 83

FOB scores in Sprague-Dawley rats

3 hrs

PBS
0, 1, 1

619176
7, 7, 6

619183
6, 6, 6

619253
7, 6, 6

619260
6, 5, 5

619276
4, 5, 5

619292
6, 6, 0

619293
ND

619307
ND

619317
6, 6, 7

619338
7, 7, 7

619339
7, 6, 3

619342
6, 6, 6

Study 3 with 5-8-5 MOE Oligonucleotides at 3,000 μg

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 84

Antisense oligonucleotides targeted to SEQ ID NO: 2

Start Site of

ISIS No
SEQ ID NO: 2

672595
1340

672599
1344

672602
1347

672624
1372

672636
1399

672637
1400

672640
1403

672642
1405

672651
1414

672652
1415

TABLE 85

FOB scores in Sprague-Dawley rats

3 hr

PBS
0, 0, 0, 0

672595
4, 0, 3, 0, 4, 4, 4

672599
3, 0, 3, 0, 1, 3

672602
3, 3, 1, 0, 0, 3

672624
3, 0, 0, 7, 3, 3, 3

672636
5, 5, 5, 5

672637
1, 1, 2, 1

672640
6, 6, 5, 1, 6, 0

672642
6, 7, 0, 7, 6, 6

672651
6, 1, 1, 6, 3, 2, 3

672652
6, 1, 1, 5, 4, 4

Study 4 with 5-8-5 MOE Oligonucleotides at 3,000 μg

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below.

TABLE 86

Antisense oligonucleotides targeted to SEQ ID NO: 2

Start

Site of

ISIS
SEQ ID

No
NO: 2

672664
1427

672665
1428

672670
1433

672671
1434

672675
1438

672676
1439

672678
1441

672679
1442

672681
1444

672683
1446

672693
1464

672697
1468

672699
1470

672700
1471

672723
1512

672730
1519

TABLE 87

FOB scores in Sprague-Dawley rats

ISIS

No
3 hr

PBS
1, 1, 1, 0

672664
6, 6, 6, 5

672665
4, 0, 4, 0, 4, 1

672670
4, 2, 4, 4

672671
4, 0, 0, 2, 5, 3

672675
3, 3, 0, 2, 1, 1

672676
1, 5, 0, 1, 1, 4

672678
0, 6, 6, 4, 6, 6

672679
3, 0, 0, 2, 3, 4

672681
1, 2, 2, 2

672683
0, 2, 5, 5, 0, 4

672693
ND

672697
6, 6, 6, 6

672699
0, 0, 3, 4, 4, 1

672700
4, 7, 4, 4

672723
4, 4, 4, 4

672730
2, 2, 3, 3

Study 5 with 5-10-5 MOE Oligonucleotides at 3,000 μg

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below and indicate that several oligonucleotides were tolerable in this model.

TABLE 88

Antisense oligonucleotides targeted to SEQ ID NO: 2

Start Site of SEQ

ISIS No
ID NO: 2

619411
8004

619412
8012

627833
8001

655153
7916

655173
7992

655178
8014

655179
8016

655202
8098

655231
8183

655232
8186

655233
8189

655417
14089

655420
14331

671081
8110

671082
8140

671083
8230

671084
14316

672561
mRNA

TABLE 89

FOB scores in Sprague-Dawley rats

3 hr

PBS
0, 0, 0, 0

619411
6, 6, 6, 6

619412
4, 6, 6, 0

627833
6, 6, 6, 1

655153
6, 6, 6, 1

655173
6, 6, 6, 0

655178
5, 5, 6, 6

655179
6, 6, 6, 6

655202
6, 0, 6, 6

655231
4, 4, 4, 4

655232
0, 0, 0, 0

655233
6, 6, 6, 6

655417
1, 6, 6, 7

655420
5, 7, 5, 5

671081
5, 5, 5, 5

671082
5, 6, 5, 7

671083
7, 6, 7, 6

671084
6, 0, 4, 3

672561
4, 4, 4, 1

Study 6 with 5-8-5 MOE Oligonucleotides at 2,000 μg

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below and indicate that several oligonucleotides were tolerable in this model.

TABLE 90

Antisense oligonucleotides targeted to SEQ ID NO: 2

Start

Site of

ISIS
SEQ ID

No
NO: 2

687978
14315

688022
7918

688077
7992

688088
8003

688089
8004

688099
8014

688100
8015

TABLE 91

FOB scores in Sprague-Dawley rats

3 hr

PBS
0, 0, 0, 0

687978
3, 3, 3, 3

688022
3, 3, 3, 3

688077
0, 3, 1, 3,

3, 3

688088
3, 0, 3, 3, 3

688089
1, 3, 3, 3, 3

688099
3, 4, 0, 3, 3

688100
0, 3, 4, 4

Study 7 with 5-8-5 and 5-10-5 MOE Oligonucleotides at 2,000 μg

The oligonucleotides tested in this study are presented in the Table below. The FOB scores are given below and indicate that several oligonucleotides were tolerable in this model.

TABLE 92

Antisense oligonucleotides targeted to SEQ ID NO: 2

Start

Site of

ISIS
SEQ ID

No
NO: 2

688101
8016

688348
7993

688356
8015

688380
8184

688382
8187

688400
14090

688407
14304

688415
14312

688416
14313

688417
14314

TABLE 93

FOB scores in Sprague-Dawley rats

3 hr

PBS
0, 0, 0, 0

688101
3, 2, 2, 2

688348
2, 1, 2, 2

688356
3, 3, 3, 3

688380
0, 0, 1, 0

688382
2, 2, 2, 2

688400
0, 3, 3, 3

688407
2, 2, 2, 2

688415
3, 0, 3, 3

688416
3, 3, 3, 3

688417
4, 4, 3, 3

Example 16: Acute Tolerability of Oligonucleotides from WO 2014/062691

Oligonucleotides described in WO 2014/062691 were tested in an acute tolerability study in mice. The tested oligonucleotides include ISIS 576816, ISIS 576974, ISIS 577061, ISIS 577065, and ISIS 577083, which are 5-10-5 MOE gapmers with a full phosphorothioate backbone and each cytosine is a 5-methylcytosine. The sequences are provided in the Table below. Mice were separated into groups of 3 or 4 mice. Each mouse in each group of mice was administered a single ICV dose of either 700 μg of the oligonucleotides in the table below. At 3 hours post injection, each mouse was evaluated according to 7 different criteria. The 7 criteria are (1) the mouse was bright, alert, and responsive; (2) the mouse was standing or hunched without stimuli; (3) the mouse shows any movement without stimuli (4) the mouse demonstrates forward movement after its lifted; (5) the mouse demonstrates any movement after its lifted; (6) the mouse responds to a tail pinch; (7) regular breathing. For each of the 7 different criteria, each mouse was given a sub-score of 0 if it met the criteria or 1 if it did not. After all of the 7 criteria were evaluated, the sub-scores were summed for each mouse and then averaged for each group. For example, if a mouse was bright, alert, and responsive 3 hours after the ICV dose, and met all other other criteria, it would get a summed score of 0. If another mouse was not bright, alert, and responsive 3 hours after the dose but met all other criteria, it would receive a score of 1. Saline treated mice generally receive a score of 0. Results are presented as the average score for each treatment group in the Table below. These results demonstrate that ISIS 576816, ISIS 576974, ISIS 577061, ISIS 577065, and ISIS 577083 were poorly tolerated.

TABLE 94

Antisense oligonucleotides from WO 2014/062691

ISIS No
Sequence
SEQ ID NO

576816
GCCTTACTCTAGGACCAAGA
20

576974
GGGACACTACAAGGTAGTAT
401

577061
TACAGGCTGCGGTTGTTTCC
97

577065
CCCGGCCCCTAGCGCGCGAC
98

577083
GGTAACTTCAAACTCTTGGG
382

TABLE 95

FOB scores in mice

3 hrs
5 hrs

576816
7, 7, 7
7, 7, 7

576974
6, 5, 6
7, 5, 7

577061
7, 7, 7
7, 7, 7

577065
6, 6, 6
7, 7, 7

577083
7, 7, 7
7, 7, 7

Gapmers from the studies described above, including compounds ISIS 576816, ISIS 576974, ISIS 577061, ISIS 577065, and ISIS 577083 which were previously disclosed in WO 2014/062691, are tested for tolerability in Sprague-Dawley rats. Rats are injected intrathecally with 3 mg of a single dose of ISIS oligonucleotide. A control group of rats is injected intrathecally with PBS. Acute tolerability is assessed 3 hours post-dose using a functional observational battery (FOB). This score is used to evaluate the acute tolerability of a compound with lower scores denoting better tolerated compounds. Control animals usually have a score of ‘0’ or ‘1’. At 3 hours post injection, the rats are observed by placing each rat on the cage top and evaluating certain functions, assigning a number of ‘0’ or ‘1’ depending on whether the rat exhibits normal function in the region of interest (0) or does not (1) for each function, and then adding the total scores. Seven regions are assessed, including tail, hind paws, hind legs, hind end, front posture, fore paws, and head.

Poor acute tolerability in mice is generally predictive of poor acute tolerability in rats. For example, ISIS 619185 (see Example 14 hereinabove) had an acute tolerability score of 7,6,6,6 (4 animals) in mouse, and an acute tolerability score of 7,7,6 (3 animals) in rats (See Example 15 hereinabove). ISIS 619342 (see Example 14 hereinabove) had an acute tolerability score of 6,6,6,6 (4 animals) in mouse, and an acute tolerability score of 6,6,6 (3 animals) in rats (see Example 15 hereinabove). Both compounds were deemed to be poorly tolerated acutely. It is therefore expected that the compounds ISIS 576816, ISIS 576974, ISIS 577061, ISIS 577065, and ISIS 577083, which were previously disclosed in WO 2014/062691, will show similarly high FOB scores in rats as they did in mice.

Number	Date	Country
61980502	Apr 2014	US
61927903	Jan 2014	US
61919540	Dec 2013	US
61891313	Oct 2013	US
61890108	Oct 2013	US

	Number	Date	Country
Parent	15028626	Apr 2016	US
Child	16248612		US

	Number	Date	Country
Parent	16248612	Jan 2019	US
Child	17721765		US

Compositions for Modulating C9ORF72 Expression

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