MODULATORS OF GROWTH HORMONE RECEPTOR

FIELD

The present embodiments provide methods, compounds, and compositions for treating, preventing, or ameliorating a disease associated with excess growth hormone using antisense compounds or oligonucleotides targeted to growth hormone receptor (GHR).

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 BIOL0226USC3SEQ_ST25.txt, created on May 18, 2022 which is 1028 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND

Growth hormone is produced in the pituitary and secreted into the bloodstream where it binds to growth hormone receptor (GHR) on many cell types, causing production of insulin-like growth factor-1 (IGF-1). IGF-1 is produced mainly in the liver, but also in adipose tissue and the kidney, and secreted into the bloodstream. Several disorders, such as acromegaly and gigantism, are associated with elevated growth hormone levels and/or elevated IGF-I levels in plasma and/or tissues.

Excessive production of growth hormone can lead to diseases such as acromegaly or gigantism. Acromegaly and gigantism are associated with excess growth hormone, often caused by a pituitary tumor, and affects 40-50 per million people worldwide with about 15,000 patients in each of the US and Europe and an annual incidence of about 4-5 per million people. Acromegaly and gigantism are initially characterized by abnormal growth of the hands and feet and bony changes in the facial features. Many of the growth related outcomes are mediated by elevated levels of serum IGF-1.

SUMMARY

Embodiments provided herein relate to methods, compounds, and compositions for treating, preventing, or ameliorating a disease associated with excess growth hormone. Several embodiments provided herein are drawn to antisense compounds or oligonucleotides targeted to growth hormone receptor (GHR). Several embodiments are directed to treatment, prevention, or amelioration of acromegaly with antisense compounds or oligonucleotides targeted to growth hormone receptor (GHR).

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. 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 application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

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

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to an O-methoxy-ethyl modification at 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 2′-MOE modified sugar moiety.

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

“3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular antisense compound.

“5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular antisense compound.

“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 ±10% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of GHR”, it is implied that GHR levels are inhibited within a range of 60% and 80%.

“Administration” or “administering” refers to routes of introducing an antisense compound provided herein to a subject to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.

“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators 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.

“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 in the absence of the antisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with 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 region or 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 moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.

“Bicyclic nucleic acid” or “BNA” or “BNA 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 bicyclic 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.

“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 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 nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.

“Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.

“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.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′ position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.

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

“Effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual'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.

“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 the “gap” and the external regions may be referred to as the “wings.”

“Growth Hormone Receptor (GHR)” means any nucleic acid or protein of GHR. “GHR nucleic acid” means any nucleic acid encoding GHR. For example, in certain embodiments, a GHR nucleic acid includes a DNA sequence encoding GHR, an RNA sequence transcribed from DNA encoding GHR (including genomic DNA comprising introns and exons), including a non-protein encoding (i.e. non-coding) RNA sequence, and an mRNA sequence encoding GHR. “GHR mRNA” means an mRNA encoding a GHR protein.

“GHR specific inhibitor” refers to any agent capable of specifically inhibiting GHR RNA and/or GHR protein expression or activity at the molecular level. For example, GHR specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of GHR RNA and/or GHR protein.

“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 nucleic acid target. 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, or at risk for having, a disease, disorder and/or condition” means identifying an animal having been diagnosed with the disease, disorder and/or condition or identifying an animal predisposed to develop the disease, disorder and/or condition. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.

“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 the expression or activity” refers to a reduction, 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.

“Lengthened” antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.

“Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.

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

“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, or modified nucleobase.

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

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

“Modulating” refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating GHR mRNA can mean to increase or decrease the level of GHR mRNA and/or GHR protein in a cell, tissue, organ or organism. A “modulator” effects the change in the cell, tissue, organ or organism. For example, a GHR antisense compound can be a modulator that decreases the amount of GHR mRNA and/or GHR protein in a cell, tissue, organ or organism.

“Monomer” refers to 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 nucleosides 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, and double-stranded nucleic acids.

“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.

“Oligomeric compound” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.

“Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

“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 or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.

“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.

“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” refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing the risk of developing a disease, disorder, or condition.

“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.

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

“Side effects” means physiological disease and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

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

“Slows progression” means decrease in the development of the said 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” 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,” “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” refers to administering a pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal. In certain embodiments, one or more pharmaceutical compositions can be administered to the animal.

“Unmodified” nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (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).

Certain Embodiments

Certain embodiments provide methods, compounds and compositions for inhibiting growth hormone receptor (GHR) expression.

Certain embodiments provide antisense compounds targeted to a GHR nucleic acid. In certain embodiments, the GHR nucleic acid has the sequence set forth in GENBANK Accession No. NM_000163.4 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No X06562.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. DR006395.1 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No. DB052048.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No. AF230800.1 (incorporated herein as SEQ ID NO: 6), the complement of GENBANK Accession No. AA398260.1 (incorporated herein as SEQ ID NO: 7), GENBANK Accession No. BC136496.1 (incorporated herein as SEQ ID NO: 8), GENBANK Accession No. NM_001242399.2 (incorporated herein as SEQ ID NO: 9), GENBANK Accession No. NM_001242400.2 (incorporated herein as SEQ ID NO: 10), GENBANK Accession No. NM_001242401.3 (incorporated herein as SEQ ID NO: 11), GENBANK Accession No. NM_001242402.2 (incorporated herein as SEQ ID NO: 12), GENBANK Accession No. NM_001242403.2 (incorporated herein as SEQ ID NO: 13), GENBANK Accession No. NM_001242404.2 (incorporated herein as SEQ ID NO: 14), GENBANK Accession No. NM_001242405.2 (incorporated herein as SEQ ID NO: 15), GENBANK Accession No. NM_001242406.2 (incorporated herein as SEQ ID NO: 16), GENBANK Accession No. NM_001242460.1 (incorporated herein as SEQ ID NO: 17), GENBANK Accession NM_001242461.1 (incorporated herein as SEQ ID NO: 18), or GENBANK Accession No. NM_001242462.1 (incorporated herein as SEQ ID NO: 19).

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides complementary within nucleotides 30-51, 63-82, 103-118, 143-159, 164-197, 206-259, 361-388, 554-585, 625-700, 736-776, 862-887, 923-973, 978-996, 1127-1142, 1170-1195, 1317-1347, 1360-1383, 1418-1449, 1492-1507, 1524-1548, 1597-1634, 1641-1660, 1683-1698, 1744-1768, 1827-1860, 1949-2002, 2072-2092, 2095-2110, 2306-2321, 2665-2683, 2685-2719, 2739-2770, 2859-2880, 2941-2960, 2963-2978, 3037-3052, 3205-3252, 3306-3332, 3371-3386, 3518-3542, 3975-3990, 4041-4087, 4418-4446, 4528-4546, 7231-7246, 7570-7585, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 11020-11035, 11793-11808, 12214-12229, 12474-12489, 12905-12920, 13400-13415, 13717-13732, 14149-14164, 14540-14555, 15264-15279, 15849-15864, 16530-16545, 17377-17392, 17581-17596, 17943-17958, 18353-18368, 18636-18651, 19256-19271, 19814-19829, 20365-20380, 20979-20994, 21566-21581, 22150-22165, 22803-22818, 29049-29064, 29554-29569, 30245-30260, 30550-30565, 30915-30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34407-34422, 34846-34861, 35669-35684, 36312-36327, 36812-36827, 37504-37519, 38841-38856, 40250-40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43500-43515, 43947-43962, 44448-44463, 45162-45177, 46010-46025, 46476-46491, 47447-47462, 47752-47767, 48001-48016, 48423-48438, 50195-50210, 50470-50485, 51104-51119, 51756-51771, 52015-52030, 52230-52245, 52588-52603, 53532-53547, or 54645-54660 of SEQ ID NO: 1, wherein said modified oligonucleotide is at least 90% complementary to SEQ ID NO: 1.

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides complementary within nucleotides 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295 of SEQ ID NO: 2, wherein said modified oligonucleotide is at least 90% complementary to SEQ ID NO: 2.

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases 100% complementary to an equal length portion of nucleobases 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295 of SEQ ID NO: 2, wherein the nucleobase sequence of the modified oligonucleotide is complementary to SEQ ID NO: 2. In certain aspects, the compound comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides complementary within nucleotides 155594-155613, 72107-72126, 153921-153940, 159252-159267, 213425-213440, 153004-153019, 155597-155612, 248233-248248 of SEQ ID NO: 2.

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 20-2295.

In certain embodiments, an antisense compound or oligonucleotide targeted to a growth hormone receptor nucleic acid is complementary within the following nucleotide regions of SEQ ID NO: 1: 30-51, 63-82, 103-118, 143-159, 164-197, 206-259, 361-388, 554-585, 625-700, 736-776, 862-887, 923-973, 978-996, 1127-1142, 1170-1195, 1317-1347, 1360-1383, 1418-1449, 1492-1507, 1524-1548, 1597-1634, 1641-1660, 1683-1698, 1744-1768, 1827-1860, 1949-2002, 2072-2092, 2095-2110, 2306-2321, 2665-2683, 2685-2719, 2739-2770, 2859-2880, 2941-2960, 2963-2978, 3037-3052, 3205-3252, 3306-3332, 3371-3386, 3518-3542, 3975-3990, 4041-4087, 4418-4446, 4528-4546, 7231-7246, 7570-7585, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 11020-11035, 11793-11808, 12214-12229, 12474-12489, 12905-12920, 13400-13415, 13717-13732, 14149-14164, 14540-14555, 15264-15279, 15849-15864, 16530-16545, 17377-17392, 17581-17596, 17943-17958, 18353-18368, 18636-18651, 19256-19271, 19814-19829, 20365-20380, 20979-20994, 21566-21581, 22150-22165, 22803-22818, 29049-29064, 29554-29569, 30245-30260, 30550-30565, 30915-30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34407-34422, 34846-34861, 35669-35684, 36312-36327, 36812-36827, 37504-37519, 38841-38856, 40250-40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43500-43515, 43947-43962, 44448-44463, 45162-45177, 46010-46025, 46476-46491, 47447-47462, 47752-47767, 48001-48016, 48423-48438, 50195-50210, 50470-50485, 51104-51119, 51756-51771, 52015-52030, 52230-52245, 52588-52603, 53532-53547, or 54645-54660.

In certain embodiments, an antisense compound or oligonucleotide targeted to a growth hormone receptor nucleic acid target the following nucleotide regions of SEQ ID NO: 1: 30-51, 63-82, 103-118, 143-159, 164-197, 206-259, 361-388, 554-585, 625-700, 736-776, 862-887, 923-973, 978-996, 1127-1142, 1170-1195, 1317-1347, 1360-1383, 1418-1449, 1492-1507, 1524-1548, 1597-1634, 1641-1660, 1683-1698, 1744-1768, 1827-1860, 1949-2002, 2072-2092, 2095-2110, 2306-2321, 2665-2683, 2685-2719, 2739-2770, 2859-2880, 2941-2960, 2963-2978, 3037-3052, 3205-3252, 3306-3332, 3371-3386, 3518-3542, 3975-3990, 4041-4087, 4418-4446, 4528-4546, 7231-7246, 7570-7585, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 11020-11035, 11793-11808, 12214-12229, 12474-12489, 12905-12920, 13400-13415, 13717-13732, 14149-14164, 14540-14555, 15264-15279, 15849-15864, 16530-16545, 17377-17392, 17581-17596, 17943-17958, 18353-18368, 18636-18651, 19256-19271, 19814-19829, 20365-20380, 20979-20994, 21566-21581, 22150-22165, 22803-22818, 29049-29064, 29554-29569, 30245-30260, 30550-30565, 30915-30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34407-34422, 34846-34861, 35669-35684, 36312-36327, 36812-36827, 37504-37519, 38841-38856, 40250-40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43500-43515, 43947-43962, 44448-44463, 45162-45177, 46010-46025, 46476-46491, 47447-47462, 47752-47767, 48001-48016, 48423-48438, 50195-50210, 50470-50485, 51104-51119, 51756-51771, 52015-52030, 52230-52245, 52588-52603, 53532-53547, or 54645-54660.

In certain embodiments, an antisense compound or oligonucleotide targeted to a growth hormone receptor nucleic acid is complementary within the following nucleotide regions of SEQ ID NO: 2: 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295.

In certain embodiments, an antisense compound or oligonucleotide targeted to a growth hormone receptor nucleic acid target the following nucleotide regions of SEQ ID NO: 2: 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295.

In certain embodiments, antisense compounds or oligonucleotides target a region of a growth hormone receptor nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a GHR nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 2: 2571-2586, 2867-3059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 8395-8410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020-11035, 11793-12229, 12469-12920, 13351-13415, 13717-13732, 14149-14164, 14361-14555, 14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17927-17958, 18353-18368, 18636-18773, 19661-19918, 20288-20470, 20979-20994, 21215-21606, 21820-21837, 22150-22165, 22518-22536, 22803-22818, 26494-26522, 29049-29069, 29323-29489, 30550-30565, 30915-31191, 31468-31483, 32363-32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 46476-46532, 48423-48438, 50072-50210, 50470-50485, 50719-51234, 51747-51797, 52015-52143, 52230-52245, 52573-52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727-68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 73350-73369, 73689-73723, 74107-74131, 74317-74557, 74947-75009, 75192-75207, 75979-76066, 76410-77095, 77292-77307, 77638-77869, 78122-78326, 79006-79021, 79478-79505, 80277-80292, 80575-80939, 81207-81222, 81524-81543, 81761-81776, 82233-82248, 82738-83198, 83330-83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 89940-90255, 90473-90528, 91073-91088, 91273-91292, 91647-91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406-100421, 100742-100828, 101080-101103, 101242-101320, 101788-101906, 102549-102568, 103566-103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964-107735, 108514-108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, 111203-111322, 112030-112049, 112499-112514, 112842-112861, 113028-113056, 113646-113665, 113896-113911, 114446-114465, 115087-115106, 119269-119284, 119659-119703, 120376-120497, 120738-120845, 121209-121228, 121823-122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671-124055, 124413-124608, 125178-125197, 125533-125616, 126357-126434, 126736-126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308-130323, 131036-131056, 131286-131305, 131676-131691, 132171-132517, 133168-133241, 133522-133877, 134086-134101, 134240-134259, 134441-134617, 135015-135030, 135431-135519, 135818-135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, 137750-137765, 138048-138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113-142342, 143021-143048, 143185-143486, 143836-144109, 144558-144650, 144990-145078, 145428-145525, 145937-145952, 146235-146386, 147028-147043, 147259-147284, 147671-147686, 148059-148154, 148564-148579, 148904-149084, 149491-149506, 149787-149877, 150236-150251, 150588-151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831-154112, 154171-154186, 154502-154521, 154724-154828, 155283-155304, 155591-155616, 155889-155992, 156233-156612, 156847-156907, 157198-157223, 157330-157349, 157552-157567, 157927-158029, 158542-158631, 159216-159267, 159539-159793, 160352-160429, 160812-160827, 161248-161267, 161461-161607, 161821-161969, 162064-162083, 162132-162147, 162531-162770, 163019-163557, 164839-165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004-168019, 168760-168823, 169062-169092, 169134-169153, 169601-169711, 170081-170291, 170407-170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, 171926-171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514-187644, 187857-189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145-198170, 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258-201861, 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920-211418, 211836-212223, 212606-212816, 213025-213044, 213425-213440, 213825-213933, 214479-214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, 218132-218248, 218526-218541, 218734-21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717-224769, 225133-225148, 225436-225761, 226785-226898, 227025-227040, 227218-227251, 227485-227500, 227914-228837, 229174-229189, 229423-229438, 229615-229640, 230042-230057, 230313-230595, 231218-231345, 231817-232037, 232088-232408, 232823-232848, 232884-232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770-235785, 236071-236213, 236684-237196, 237585-237698, 237949-237557, 244873-244897, 245319-245334, 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214-251233, 251601-251637, 251950-252060, 252665-252680, 252838-252863, 253140-253166, 253594-253819, 254036-254083, 254246-254345, 254641-254660, 254905-254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, 263338-266518, 266861-267131, 267375-268051, 268366-269447, 270038-271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391-278380, 278932-279063, 279303-281001, 281587-281610, 282229-283668, 290035-290474, 290924-292550, 292860-294408, 295475-297012, 297587-298115, 298161-298418, 298489-298738, 299082-299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295.

In certain embodiments, antisense compounds or oligonucleotides target intron 1 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 3058-144965 (intron 1) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, antisense compounds or oligonucleotides target intron 2 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 145047-208139 (intron 2) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, antisense compounds or oligonucleotides target intron 3 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 208206-267991 (intron 3) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, antisense compounds or oligonucleotides target intron 4 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 268122-274018 (intron 4) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, antisense compounds or oligonucleotides target intron 5 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 274192-278925 (intron 5) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, antisense compounds or oligonucleotides target intron 6 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 279105-290308 (intron 6) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, antisense compounds or oligonucleotides target intron 7 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 290475-292530 (intron 7) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, antisense compounds or oligonucleotides target intron 8 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 292622-297153 (intron 8) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, antisense compounds or oligonucleotides target intron 9 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 297224-297554 (intron 9) of a growth hormone receptor nucleic acid having the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises at least one modified sugar. In certain aspects, at least one modified sugar comprises a 2′-O-methoxyethyl group. In certain aspects, at least one modified sugar is a bicyclic sugar, such as a 4′-CH(CH₃)—O-2′ group, a 4′-CH₂—O-2′ group, or a 4′-(CH₂)₂—O-2′-group. In certain aspects, the modified oligonucleotide comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises at least one modified nucleobase, such as 5-methylcytosine.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises:

- a gap segment consisting of linked deoxynucleosides;
- a 5′ wing segment consisting of linked nucleosides; and
- a 3′ wing segment consisting of 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.

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 918, 479, 703, 1800, 1904, 2122, 2127, or 2194.

In certain aspects, the modified oligonucleotide has a nucleobase sequence comprising the sequence recited in SEQ ID NOs: 918, 479 or 703, wherein the modified oligonucleotide comprises

a gap segment consisting of ten linked deoxynucleosides;

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

a 3′ wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine.

In certain aspects, the modified oligonucleotide has a nucleobase sequence comprising the sequence recited in SEQ ID NOs: 1800, 1904, 2122, 2127, or 2194, wherein the modified oligonucleotide comprises:

- a gap segment consisting of ten linked deoxynucleosides;
- a 5′ wing segment consisting of 3 linked nucleosides; and
- a 3′ wing segment consisting of 3 linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar or a constrained ethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.

Certain embodiments provide a compound comprising a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 703. In certain aspects, the modified oligonucleotide comprises at least one modified sugar. In certain aspects, the at least one modified sugar comprises a 2′-O-methoxyethyl group. In certain aspects, the at least one modified sugar is a bicyclic sugar, such as a 4′-CH(CH₃)—O-2′ group, a 4′-CH₂—O-2′ group, or a 4′-(CH₂)₂—O-2′-group. In certain aspects, the modified oligonucleotide comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage. In certain aspects, the modified oligonucleotide comprises at least one modified nucleobase, such as a 5-methylcytosine. In certain aspects, the modified oligonucleotide comprises:

- a gap segment consisting of linked deoxynucleosides;
- a 5′ wing segment consisting of linked nucleosides; and
- a 3′ wing segment consisting of 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.

- a gap segment consisting of ten linked deoxynucleosides;
- a 5′ wing segment consisting of five linked nucleosides; and
- a 3′ wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing embodiments, the compound or oligonucleotide can be at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to a nucleic acid encoding growth hormone receptor.

In any of the foregoing embodiments, the nucleic acid encoding growth hormone receptor can comprise the nucleotide sequence of any one of SEQ ID NOs: 1-19.

In any of the foregoing embodiments, the compound or oligonucleotide can be single-stranded.

Certain embodiments provide a composition comprising the compound of any of the aforementioned embodiments or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent. In certain aspects, the composition has a viscosity less than about 40 centipoise (cP), less than about 30 centipose (cP), less than about 20 centipose (cP), less than about 15 centipose (cP), or less than about 10 centipose (cP). In certain aspects, the composition having any of the aforementioned viscosities comprises a compound provided herein at a concentration of about 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, or about 300 mg/mL. In certain aspects, the composition having any of the aforementioned viscosities and/or compound concentrations has a temperature of room temperature or about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C.

Certain embodiments provide a method of treating a disease associated with excess growth hormone in a human comprising administering to the human a therapeutically effective amount of the compound or composition of any of the aforementioned embodiments, thereby treating the disease associated with excess growth hormone. In certain aspects, the disease associated with excess growth hormone is acromegaly. In certain aspects, the treatment reduces IGF-1 levels.

Certain embodiments provide a method of preventing a disease associated with excess growth hormone in a human comprising administering to the human a therapeutically effective amount of a compound or composition of any of the aforementioned embodiments, thereby preventing the disease associated with excess growth hormone. In certain embodiments, the disease associated with excess growth hormone is acromegaly.

Certain embodiments provide a method of reducing growth hormone receptor (GHR) levels in a human comprising administering to the human a therapeutically effective amount of the compound or composition of any of the aforementioned embodiments, thereby reducing GHR levels in the human. In certain aspects, the human has a disease associated with excess growth hormone. In certain aspects, the disease associated with excess growth hormone is acromegaly.

In certain aspects, the foregoing methods comprise co-administering the compound or composition and a second agent. In certain aspects, the compound or composition and the second agent are administered concomitantly.

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 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 is 10 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 22 subunits in length. In certain embodiments, an antisense compound is 14 to 30 subunits in length. In certain embodiments, an antisense compound is 14 to 20 subunits in length. In certain embodiments, an antisense compound is 15 to 30 subunits in length. In certain embodiments, an antisense compound is 15 to 20 subunits in length. In certain embodiments, an antisense compound is 16 to 30 subunits in length. In certain embodiments, an antisense compound is 16 to 20 subunits in length. In certain embodiments, an antisense compound is 17 to 30 subunits in length. In certain embodiments, an antisense compound is 17 to 20 subunits in length. In certain embodiments, an antisense compound is 18 to 30 subunits in length. In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense compound is 18 to 20 subunits in length. In certain embodiments, an antisense compound is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound is 14 subunits in length. In certain embodiments, an antisense compound is 16 subunits in length. In certain embodiments, an antisense compound is 17 subunits in length. In certain embodiments, an antisense compound is 18 subunits in length. In certain embodiments, an antisense compound is 19 subunits in length. In certain embodiments, an antisense compound is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such 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 nucleotides.

In certain embodiments antisense oligonucleotides 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 GHR 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.

Certain Antisense Compound Motifs and Mechanisms

In certain embodiments, antisense compounds 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 confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect. In certain embodiments, the amount and/or activity of the target nucleic acid is modulated. In certain embodiments, the amount and/or activity of the target nucleic acid is reduced. In certain embodiments, hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation. In certain embodiments, hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation. In certain such embodiments, the presence of the antisense compound hybridized with the target nucleic acid (occupancy) results in a modulation of antisense activity. In certain embodiments, antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms. In certain embodiments, antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.

Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.

RNase H-Mediated Antisense

In certain embodiments, antisense activity results at least in part from degradation of target RNA by RNase H. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid. In certain embodiments, antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides. In certain such embodiments, the modified nucleosides do not support RNase H activity. In certain embodiments, such antisense compounds are gapmers, as described herein. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA-like nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.

Certain 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 constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2′-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X—Y—Z”, where “X” represents the length of the 5′-wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′-wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X—Y—Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′-wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′-wing and gap, or the gap and the 3′-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different. In certain embodiments, “Y” is between 8 and 15 nucleosides. 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 nucleosides.

In certain embodiments, the antisense compound targeted to a GHR nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.

In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J)_m-(B)_n-(J)_p-(B)_r-(A)_t-(D)_g-(A)_v-(B)_w-(J)_x-(B)_y-(J)_z

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

RNAi Compounds

In certain embodiments, antisense compounds are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.

i. ssRNA Compounds

In certain embodiments, antisense compounds including those particularly suited for use as single-stranded RNAi compounds (ssRNA) comprise a modified 5′-terminal end. In certain such embodiments, the 5′-terminal end comprises a modified phosphate moiety. In certain embodiments, such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5′-phosphate). In certain embodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorous moiety. Certain modified 5′-terminal nucleosides may be found in the art, for example in WO/2011/139702.

In certain embodiments, the 5′-nucleoside of an ssRNA compound has Formula IIc:

embedded image

wherein:

T₁is an optionally protected phosphorus moiety;

T₂is an internucleoside linking group linking the compound of Formula IIc to the oligomeric compound;

A has one of the formulas:

embedded image

Q₁and Q₂are each, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy, substituted C₁-C₆alkoxy, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl, substituted C₂-C₆alkynyl or N(R₃)(R₄);

Q₃is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄R₅, R₆and R₇is, independently, H, C₁-C₆alkyl, substituted C₁-C₆alkyl or C₁-C₆alkoxy;

M₃is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇), OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄is H, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy, substituted C₁-C₆alkoxy, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl or substituted C₂-C₆alkynyl;

R₁₅, R₁₆, R₁₇and R₁₈are each, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy, substituted C₁-C₆alkoxy, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl or substituted C₂-C₆alkynyl;

Bx₁is a heterocyclic base moiety;

or if Bx₂is present then Bx₂is a heterocyclic base moiety and Bx₁is H, halogen, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy, substituted C₁-C₆alkoxy, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl or substituted C₂-C₆alkynyl;

J₄, J₅, J₆and J₇are each, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy, substituted C₁-C₆alkoxy, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl or substituted C₂-C₆alkynyl;

or J₄forms a bridge with one of J₅or J₇wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR₁₉, C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other two of J₅, J₆and J₇are each, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy, substituted C₁-C₆alkoxy, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl or substituted C₂-C₆alkynyl;

each R₁₉, R₂₀and R₂₁is, independently, H, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy, substituted C₁-C₆alkoxy, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl or substituted C₂-C₆alkynyl;

G is H, OH, halogen or O—[C(R₈)(R₉)]_n—[(C═O)_m—X₁]_j—Z;

each R₈and R₉is, independently, H, halogen, C₁-C₆alkyl or substituted C₁-C₆alkyl;

X₁is O, S or N(E₁);

Z is H, halogen, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl, substituted C₂-C₆alkynyl or N(E₂)(E₃);

E₁, E₂and E₃are each, independently, H, C₁-C₆alkyl or substituted C₁-C₆alkyl; n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), =NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂is O, S or NJ₃;

each J₁, J₂and J₃is, independently, H or C₁-C₆alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

wherein said oligomeric compound comprises from 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.

In certain embodiments, M₃is O, CH═CH, OCH₂or OC(H)(Bx₂). In certain embodiments, M₃is O.

In certain embodiments, J₄, J₅, J₆and J₇are each H. In certain embodiments, J₄forms a bridge with one of J₅or J₇.

In certain embodiments, A has one of the formulas:

embedded image

wherein:

Q₁and Q₂are each, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy or substituted C₁-C₆alkoxy. In certain embodiments, Q₁and Q₂are each H. In certain embodiments, Q₁and Q₂are each, independently, H or halogen. In certain embodiments, Q₁and Q₂is H and the other of Q₁and Q₂is F, CH₃or OCH₃.

In certain embodiments, T₁has the formula:

embedded image

wherein:

R_aand R_eare each, independently, protected hydroxyl, protected thiol, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy, substituted C₁-C₆alkoxy, protected amino or substituted amino; and

R_bis O or S. In certain embodiments, R_bis O and R_aand R_eare each, independently, OCH₃, OCH₂CH₃or CH(CH₃)₂.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁₀)(R₁₁), O(CH₂)₂—ON(R₁₀)(R₁₁), O(CH₂)₂—O(CH₂)₂—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₂)—(CH₂)₂—N(R₁₀)(R₁₁) or O(CH₂)₂—N(R₁₂)—C(═NR₁₃)[N(R₁₀)(R₁₁)] wherein R₁₀, R₁₁, R₁₂and R₁₃are each, independently, H or C₁-C₆alkyl. In certain embodiments, G is halogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃, OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂or OCH₂—N(H)—C(═NH)NH₂. In certain embodiments, G is F, OCH₃or O(CH₂)₂—OCH₃. In certain embodiments, G is O(CH₂)₂—OCH₃.

In certain embodiments, the 5-terminal nucleoside has Formula IIe:

embedded image

In certain embodiments, antisense compounds, including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2′-F nucleoside. In certain embodiments, each nucleoside of the region is a 2′-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, nucleosides of both types are RNA-like nucleosides. In certain embodiments the alternating nucleosides are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, the alternating modifications are 2′-F and 2′-OMe. Such regions may be contiguous or may be interrupted by differently modified nucleosides or conjugated nucleosides.

In certain embodiments, the alternating region of alternating modifications each consist of a single nucleoside (i.e., the pattern is (AB)_xA_ywherein A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certain embodiments, one or more alternating regions in an alternating motif includes more than a single nucleoside of a type. For example, oligonucleotides may include one or more regions of any of the following nucleoside motifs:

AABBAA;
ABBABB;
AABAAB;
ABBABAABB;
ABABAA;
AABABAB;
ABABAA;
ABBAABBABABAA;
BABBAABBABABAA; or
ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2′-F, 2′-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides having such an alternating motif also comprise a modified 5′ terminal nucleoside, such as those of formula IIc or IIe.

In certain embodiments, oligonucleotides comprise a region having a 2-2-3 motif Such regions comprises the following motif:

-(A)₂-(B)_x-(A)₂-(C)_y-(A)₃-

wherein: A is a first type of modified nucleoside;

B and C, are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certain embodiments, B and C are both 2′-F modified nucleosides. In certain embodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-F modified nucleosides.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(AB)_xA_y-(D)_z

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(A)_x-(D)_z

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

D is a modified nucleoside comprising a modification different from A.

X is 11-30;

Z is 0-4.

In certain embodiments A, B, C, and D in the above motifs are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance). In certain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.

In certain embodiments, antisense compounds, including those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

Oligonucleotides having any of the various sugar motifs described herein, may have any linkage motif. For example, the oligonucleotides, including but not limited to those described above, may have a linkage motif selected from non-limiting the table below:

5′ most linkage
Central region
3′-region

PS
Alternating PO/PS
6 PS

PS
Alternating PO/PS
7 PS

PS
Alternating PO/PS
8 PS

ii. siRNA Compounds

In certain embodiments, antisense compounds are double-stranded RNAi compounds (siRNA). In such embodiments, one or both strands may comprise any modification motif described above for ssRNA. In certain embodiments, ssRNA compounds may be unmodified RNA. In certain embodiments, siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.

Several embodiments relate to double-stranded compositions wherein each strand comprises a motif defined by the location of one or more modified or unmodified nucleosides. In certain embodiments, compositions are provided comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.

The compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. In some embodiments, the target nucleic acid is GHR. In certain embodiment, the degradation of the targeted GHR is facilitated by an activated RISC complex that is formed with compositions of the invention.

Several embodiments are directed to double-stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

Certain embodiments are drawn to double-stranded compositions wherein both the strands comprises a hemimer motif, a fully modified motif, a positionally modified motif or an alternating motif. Each strand of the compositions of the present invention can be modified to fulfil a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modified such that it is enhanced for its particular role. The antisense strand can be modified at the 5′-end to enhance its role in one region of the RISC while the 3′-end can be modified differentially to enhance its role in a different region of the RISC.

The double-stranded oligonucleotide molecules can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double-stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).

The double-stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.

In certain embodiments, the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the double-stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the double-stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.

As used herein, double-stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack 2′-hydroxy (2′-OH) containing nucleotides. In certain embodiments short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such double-stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, double-stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, double-stranded oligonucleotides can be used to epigenetically silence genes at both the post-transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

It is contemplated that compounds and compositions of several embodiments provided herein can target GHR by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., “hairpin” or stem-loop double-stranded RNA effector molecules in which a single RNA strand with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA. In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule. In various embodiments, a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides. Alternatively, the dsRNA may include two different strands that have a region of complementarity to each other.

In various embodiments, both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence. In certain embodiments, the region of the dsRNA that is present in a double-stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA. In some embodiments, the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has one or more single stranded regions or overhangs. In certain embodiments, RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.

In various embodiments, the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell. In yet other embodiments, the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.) Exemplary circular nucleic acids include lariat structures in which the free 5′ phosphoryl group of a nucleotide becomes linked to the 2′ hydroxyl group of another nucleotide in a loop back fashion.

In other embodiments, the dsRNA includes one or more modified nucleotides in which the 2′ position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2′ position contains a hydrogen or an hydroxyl group. In yet other embodiments, the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.

In other embodiments, the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S. Provisional Application 60/419,532, and PCT/US2003/033466, the teaching of which is hereby incorporated by reference. Any of the dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.

Occupancy

In certain embodiments, antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway. In certain such embodiments, antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid. In certain such embodiments, the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode growth hormone receptor (GHR) targetable with the compounds provided herein include, without limitation, the following: GENBANK Accession No. NM_000163.4 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No X06562.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. DR006395.1 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No. DB052048.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No. AF230800.1 (incorporated herein as SEQ ID NO: 6), the complement of GENBANK Accession No. AA398260.1 (incorporated herein as SEQ ID NO: 7), GENBANK Accession No. BC136496.1 (incorporated herein as SEQ ID NO: 8), GENBANK Accession No. NM_001242399.2 (incorporated herein as SEQ ID NO: 9), GENBANK Accession No. NM_001242400.2 (incorporated herein as SEQ ID NO: 10), GENBANK Accession No. NM_001242401.3 (incorporated herein as SEQ ID NO: 11), GENBANK Accession No. NM_001242402.2 (incorporated herein as SEQ ID NO: 12), GENBANK Accession No. NM_001242403.2 (incorporated herein as SEQ ID NO: 13), GENBANK Accession No. NM_001242404.2 (incorporated herein as SEQ ID NO: 14), GENBANK Accession No. NM_001242405.2 (incorporated herein as SEQ ID NO: 15), GENBANK Accession No. NM_001242406.2 (incorporated herein as SEQ ID NO: 16), GENBANK Accession No. NM_001242460.1 (incorporated herein as SEQ ID NO: 17), GENBANK Accession NM_001242461.1 (incorporated herein as SEQ ID NO: 18), GENBANK Accession No. NM_001242462.1 (incorporated herein as SEQ ID NO: 19), or GENBANK Accession No NW_001120958.1 truncated from nucleotides 4410000 to 4720000 (incorporated herein as SEQ ID NO: 2296).

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a GHR 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 GHR 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 GHR nucleic acid).

Non-complementary nucleobases between an antisense compound and a GHR 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 GHR 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 GHR 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 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 GHR 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 11, 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 GHR nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 11, 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 GHR nucleic acid, or specified portion thereof.

The antisense compounds provided 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, or 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 GHR nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

Modified Sugar Moieties

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(R_l)—(CH₂)₂—N(R_m)(R_n), where each R_l, 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 abridge 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′ (also referred to as constrained ethyl or cEt) 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)]_nO—, —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 p-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, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA and (K) vinyl BNA as depicted below:

embedded image

wherein Bx is the base moiety and R is independently H, a protecting group, C₁-C₁₂alkyl or C₁-C₁₂alkoxy.

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_cand 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 comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA'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:

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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, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

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) or fluoro HNA (F-HNA) having a tetrahydropyran ring system as illustrated below:

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In certain embodiments, sugar surrogates are selected 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 methoxyethoxy and R₂is H.

In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following formula:

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In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and 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 bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvith et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.

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wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃and T₄are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T₃and T₄is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T₃and T₄is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈and q₉are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₂-C₆alkenyl, substituted C₂-C₆alkenyl, C₂-C₆alkynyl, substituted C₂-C₆alkynyl or other sugar substituent group.

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)₂O—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_nis, 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 of the sugar ring.

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. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005, and each of which is herein incorporated by reference in its entirety.

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.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a GHR nucleic acid comprise one or more modified nucleobases. In certain embodiments, shortened or gap-widened antisense oligonucleotides targeted to a GHR nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

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.

In certain embodiments, antisense compounds, including, but not limited to those particularly suited for use as ssRNA, are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

For additional conjugates including those useful for ssRNA and their placement within antisense compounds, see e.g., U.S. Application No. 61/583,963.

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.

Cells may be 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 may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 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 may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

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

Yet another technique used to introduce antisense oligonucleotides into cultured cells includes free uptake of the oligonucleotides by the cells.

Cells are treated with antisense oligonucleotides by routine methods. Cells may be 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.

Certain Indications

Certain embodiments provided herein relate to methods of treating, preventing, or ameliorating a disease associated with excess growth hormone in a subject by administering a GHR specific inhibitor, such as an antisense compound or oligonucleotide targeted to GHR. In certain aspects, the disease associated with excess growth hormone is acromegaly. In certain aspects, the disease associated with excess growth hormone is gigantism.

Certain embodiments provide a method of treating, preventing, or ameliorating acromegaly in a subject by administering a GHR specific inhibitor, such as an antisense compound or oligonucleotide targeted to GHR. Acromegaly is a disease associated with excess growth hormone (GH). In over 90 percent of acromegaly patients, the overproduction of growth hormones is caused by a benign tumor of the pituitary gland, called an adenoma, which produces excess growth hormone and compresses surrounding brain tissues. Expansion of the adenoma can cause headaches and visual impairment that often accompany acromegaly. In some instances, acromegaly is caused by tumors of the pancreas, lungs, or adrenal glands that lead to an excess of GH, either by producing GH or by producing Growth Hormone Releasing Hormone (GHRH), the hormone that stimulates the pituitary to make GH.

Acromegaly most commonly affects adults in middle age and can result in severe disfigurement, complicating conditions, and premature death. Because of its pathogenesis and slow progression, acromegaly often goes undiagnosed until changes in external features become noticeable, such as changes in the face. Acromegaly is often associated with gigantism.

Features of acromegaly include soft tissue swelling resulting in enlargement of the hands, feet, nose, lips and ears, and a general thickening of the skin; soft tissue swelling of internal organs, such as the heart and kidney; vocal cord swelling resulting in a low voice and slow speech; expansion of the skull; pronounced eyebrow protrusion, often with ocular distension; pronounced lower jaw protrusion and enlargement of the tongue; teeth gapping; and carpal tunnel syndrome. In certain embodiments, any one or combination of these features of acromegaly can be treated, prevented, or ameliorated by administering a compound or composition targeted to GHR provided herein.

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 Human Growth Hormone Receptor in Hep3B Cells by MOE Gapmers

Antisense oligonucleotides were designed targeting a growth hormone receptor (GHR) nucleic acid and were tested for their effects on GHR mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB (forward sequence CGAGTTCAGTGAGGTGCTCTATGT, designated herein as SEQ ID NO: 2297; reverse sequence AAGAGCCATGGAAAGTAGAAATCTTC, designated herein as SEQ ID NO: 2298; probe sequence TTCCTCAGATGAGCCAATT, designated herein as SEQ ID NO: 2299) was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE or 3-10-4 MOE gapmers. 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. The 3-10-4 MOE gapmers are 17 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 three and four nucleosides respectively. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “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 gapmer listed in the Tables below is targeted to either the human GHR mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000163.4) or the human GHR genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity. In case the sequence alignment for a target gene in a particular table is not shown, it is understood that none of the oligonucleotides presented in that table align with 100% complementarity with that target gene.

TABLE 1

Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting exonic regions of SEQ ID NO: 1 and 2

SEQ

ID
SEQ

SEQ
SEQ

NO:
ID

ID
ID

1
NO: 1

NO: 2
NO: 2
SEQ

Start
Stop
Target

%
Start
Stop
ID

ISIS NO
Site
Site
Region
Sequence
inhibition
Site
Site
NO

523266
164
183
Exon 1
ACCTCCGAGCTTCGCCTCTG
64
3040
3059
20

523267
171
190
Exon-
CTGTAGGACCTCCGAGCTTC
31
n/a
n/a
21

exon

junction

523268
178
197
Exon-
TCCATACCTGTAGGACCTCC
37
n/a
n/a
22

exon

junction

523271
206
225
Exon 2
TGCCAAGGTCAACAGCAGCT
80
144990
145009
23

523272
213
232
Exon 2
CTGCCAGTGCCAAGGTCAAC
53
144997
145016
24

523273
220
239
Exon 2
CTTGATCCTGCCAGTGCCAA
49
145004
145023
25

523274
227
246
Exon 2
AGCATCACTTGATCCTGCCA
67
145011
145030
26

523275
234
253
Exon 2
CAGAAAAAGCATCACTTGAT
0
145018
145037
27

523276
241
260
Exon 2
TCACTTCCAGAAAAAGCATC
1
145025
145044
28

523284
361
380
Exon 4
GTCTCTCGCTCAGGTGAACG
48
268024
268043
29

523285
368
387
Exon 4
TGAAAAAGTCTCTCGCTCAG
15
268031
268050
30

523286
375
394
Exon 4
AGTGGCATGAAAAAGTCTCT
14
268038
268057
31

523287
382
401
Exon 4
TCTGTCCAGTGGCATGAAAA
4
268045
268064
32

523301
625
644
Exon 6
GGATCTGGTTGCACTATTTC
36
n/a
n/a
33

523302
632
651
Exon 6
AATGGGTGGATCTGGTTGCA
28
278926
278945
34

523303
647
666
Exon 6
AGTCCAGTTGAGGGCAATGG
26
278941
278960
35

523304
654
673
Exon 6
TCAGTAAAGTCCAGTTGAGG
0
278948
278967
36

523305
675
694
Exon 6
GAATCCCAGTTAAACTGACG
19
278969
278988
37

523306
682
701
Exon 6
TCTGCATGAATCCCAGTTAA
39
278976
278995
38

523309
736
755
Exon 6
ATCCATCCTTTCTGAATATC
34
279030
279049
39

523310
743
762
Exon 6
CAGAACCATCCATCCTTTCT
31
279037
279056
40

523311
750
769
Exon 6
CATACTCCAGAACCATCCAT
44
279044
279063
41

523312
757
776
Exon 6
TGAAGTTCATACTCCAGAAC
23
279051
279070
42

523313
764
783
Exon 6
TTTGTATTGAAGTTCATACT
6
279058
279077
43

523314
771
790
Exon 6
TTACTTCTTTGTATTGAAGT
0
279065
279084
44

523315
778
797
Exon 6
GTTTCATTTACTTCTTTGTA
3
279072
279091
45

523316
785
804
Exon 6
CCATTTAGTTTCATTTACTT
0
279079
279098
46

523317
792
811
Exon 4-
TCATTTTCCATTTAGTTTCA
19
n/a
n/a
47

exon 5

junction

523323
862
881
Exon 7
ACACGCACTTCATATTCCTT
63
290360
290379
48

523324
869
888
Exon 7
GGATCTCACACGCACTTCAT
80
290367
290386
49

523328
926
945
Exon 7
AAGTGTTACATAGAGCACCT
56
290424
290443
50

523329
933
952
Exon 7
TCTGAGGAAGTGTTACATAG
53
290431
290450
51

523330
957
976
Exon 7
CTTCTTCACATGTAAATTGG
32
290455
290474
52

523331
964
983
Exon 5-
TAGAAATCTTCTTCACATGT
4
n/a
n/a
53

exon 6

junction

523332
971
990
Exon 5-
TGGAAAGTAGAAATCTTCTT
9
n/a
n/a
54

exon 6

junction

523333
978
997
Exon 8
AGAGCCATGGAAAGTAGAAA
46
292532
292551
55

523334
985
1004
Exon 8
ATAATTAAGAGCCATGGAAA
0
292539
292558
56

TABLE 2

Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting exonic regions of SEQ ID NO: 1 and 2

SEQ
SEQ

ID
ID

NO:
NO:

SEQ ID
SEQ ID

1
1

NO: 2
NO: 2
SEQ

ISIS
Start
Stop
Target

%
Start
Stop
ID

NO
Site
Site
Region
Sequence
inhibition
Site
Site
NO

523421
2072
2091
exon 10
CAGTTGGTCTGTGCTCACAT
76
298489
298508
57

533002
207
226
exon 2
GTGCCAAGGTCAACAGCAGC
63
144991
145010
58

533003
208
227
exon 2
AGTGCCAAGGTCAACAGCAG
62
144992
145011
59

533004
225
244
exon 2
CATCACTTGATCCTGCCAGT
53
145009
145028
60

533005
226
245
exon 2
GCATCACTTGATCCTGCCAG
80
145010
145029
61

533006
228
247
exon 2
AAGCATCACTTGATCCTGCC
75
145012
145031
62

533007
229
248
exon 2
AAAGCATCACTTGATCCTGC
61
145013
145032
63

533019
867
886
exon 7
ATCTCACACGCACTTCATAT
35
290365
290384
64

533020
868
887
exon 7
GATCTCACACGCACTTCATA
47
290366
290385
65

533021
870
889
exon 7
TGGATCTCACACGCACTTCA
86
290368
290387
66

533022
871
890
exon 7
TTGGATCTCACACGCACTTC
70
290369
290388
67

533037
1360
1379
exon 10
TCCAGAATGTCAGGTTCACA
59
297777
297796
68

533038
1361
1380
exon 10
CTCCAGAATGTCAGGTTCAC
74
297778
297797
69

533039
1363
1382
exon 10
GTCTCCAGAATGTCAGGTTC
45
297780
297799
70

533040
1364
1383
exon 10
AGTCTCCAGAATGTCAGGTT
51
297781
297800
71

533042
1525
1544
exon 10
GCTTGGATAACACTGGGCTG
41
297942
297961
72

533043
1526
1545
exon 10
TGCTTGGATAACACTGGGCT
46
297943
297962
73

533044
1528
1547
exon 10
TCTGCTTGGATAACACTGGG
55
297945
297964
74

533045
1529
1548
exon 10
CTCTGCTTGGATAACACTGG
47
297946
297965
75

533046
1530
1549
exon 10
TCTCTGCTTGGATAACACTG
54
297947
297966
76

533047
1744
1763
exon 10
CAGAGTGAGACCATTTCCGG
47
298161
298180
77

533048
1745
1764
exon 10
GCAGAGTGAGACCATTTCCG
60
298162
298181
78

533049
1747
1766
exon 10
TGGCAGAGTGAGACCATTTC
65
298164
298183
79

533050
1748
1767
exon 10
TTGGCAGAGTGAGACCATTT
47
298165
298184
80

533051
1749
1768
exon 10
CTTGGCAGAGTGAGACCATT
30
298166
298185
81

533066
2685
2704
exon 10
CAGTGTGTAGTGTAATATAA
53
299102
299121
82

533067
2686
2705
exon 10
ACAGTGTGTAGTGTAATATA
68
299103
299122
83

533068
2688
2707
exon 10
ACACAGTGTGTAGTGTAATA
62
299105
299124
84

533069
2689
2708
exon 10
TACACAGTGTGTAGTGTAAT
55
299106
299125
85

533070
2690
2709
exon 10
GTACACAGTGTGTAGTGTAA
50
299107
299126
86

533071
3205
3224
exon 10
TGTACCTTATTCCCTTCCTG
68
299622
299641
87

533072
3206
3225
exon 10
TTGTACCTTATTCCCTTCCT
61
299623
299642
88

533073
3208
3227
exon 10
TCTTGTACCTTATTCCCTTC
60
299625
299644
89

533074
3209
3228
exon 10
TTCTTGTACCTTATTCCCTT
46
299626
299645
90

TABLE 3

Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intronic and exonic regions of SEQ ID NO: 1

and 2

SEQ

ID
SEQ

SEQ
SEQ

NO:
ID

ID
ID

1
NO: 1

NO: 2
NO: 2

ISIS
Start
Stop
Target

%
Start
Stop
SEQ

NO
Site
Site
Region
Sequence
inhibition
Site
Site
ID NO

532174
n/a
n/a
Intron 1
ACATGTACCCAAACCAACAC
37
18731
18750
91

533086
3210
3229
Exon 10
CTTCTTGTACCTTATTCCCT
72
299627
299646
92

533087
3212
3231
Exon 10
TGCTTCTTGTACCTTATTCC
77
299629
299648
93

533088
3213
3232
Exon 10
ATGCTTCTTGTACCTTATTC
63
299630
299649
94

533089
3215
3234
Exon 10
AAATGCTTCTTGTACCTTAT
67
299632
299651
95

533090
3216
3235
Exon 10
AAAATGCTTCTTGTACCTTA
50
299633
299652
96

533091
3217
3236
Exon 10
CAAAATGCTTCTTGTACCTT
44
299634
299653
97

533092
3518
3537
Exon 10
CTTCTGAATGCTTGCTTTGA
29
299935
299954
98

533093
3519
3538
Exon 10
TCTTCTGAATGCTTGCTTTG
47
299936
299955
99

533094
3521
3540
Exon 10
TTTCTTCTGAATGCTTGCTT
63
299938
299957
100

533095
3522
3541
Exon 10
TTTTCTTCTGAATGCTTGCT
51
299939
299958
101

533096
3523
3542
Exon 10
TTTTTCTTCTGAATGCTTGC
34
299940
299959
102

533097
4041
4060
Exon 10
TGCGATAAATGGGAAATACT
36
300458
300477
103

533098
4042
4061
Exon 10
CTGCGATAAATGGGAAATAC
52
300459
300478
104

533099
4043
4062
Exon 10
TCTGCGATAAATGGGAAATA
41
300460
300479
105

533100
4045
4064
Exon 10
GGTCTGCGATAAATGGGAAA
40
300462
300481
106

533101
4046
4065
Exon 10
AGGTCTGCGATAAATGGGAA
39
300463
300482
107

533102
4048
4067
Exon 10
AAAGGTCTGCGATAAATGGG
34
300465
300484
108

533103
4049
4068
Exon 10
AAAAGGTCTGCGATAAATGG
35
300466
300485
109

533104
4050
4069
Exon 10
AAAAAGGTCTGCGATAAATG
15
300467
300486
110

533115
n/a
n/a
Intron 1
CATGAAGGCCACTCTTCCAA
63
12777
12796
111

533116
n/a
n/a
Intron 1
CCATGAAGGCCACTCTTCCA
78
12778
12797
112

533117
n/a
n/a
Intron 1
CCCATGAAGGCCACTCTTCC
71
12779
12798
113

533118
n/a
n/a
Intron 1
TGCCCATGAAGGCCACTCTT
66
12781
12800
114

533119
n/a
n/a
Intron 1
TTGCCCATGAAGGCCACTCT
60
12782
12801
115

533120
n/a
n/a
Intron 1
GTTGCCCATGAAGGCCACTC
74
12783
12802
116

533121
n/a
n/a
Intron 1
GGTCTTTCATGAATCAAGCT
79
17927
17946
117

533122
n/a
n/a
Intron 1
TGGTCTTTCATGAATCAAGC
83
17928
17947
118

533123
n/a
n/a
Intron 1
ATGGTCTTTCATGAATCAAG
83
17929
17948
119

533124
n/a
n/a
Intron 1
TGATGGTCTTTCATGAATCA
78
17931
17950
120

533125
n/a
n/a
Intron 1
CTGATGGTCTTTCATGAATC
82
17932
17951
121

533126
n/a
n/a
Intron 1
GCTGATGGTCTTTCATGAAT
74
17933
17952
122

533127
n/a
n/a
Intron 1
GTACCCAAACCAACACTAAT
57
18727
18746
123

533128
n/a
n/a
Intron 1
TGTACCCAAACCAACACTAA
65
18728
18747
124

533129
n/a
n/a
Intron 1
ATGTACCCAAACCAACACTA
64
18729
18748
125

533130
n/a
n/a
Intron 1
GACATGTACCCAAACCAACA
63
18732
18751
126

533131
n/a
n/a
Intron 1
AGACATGTACCCAAACCAAC
81
18733
18752
127

533132
n/a
n/a
Intron 1
AGGAATGGAAAACCAAATAT
49
26494
26513
128

533133
n/a
n/a
Intron 1
CAGGAATGGAAAACCAAATA
74
26495
26514
129

121986
122005

533134
n/a
n/a
Intron 1
TCAGGAATGGAAAACCAAAT
73
26496
26515
130

121987
122006

533135
n/a
n/a
Intron 1
ACTCAGGAATGGAAAACCAA
77
26498
26517
131

113032
113051

121989
122008

533136
n/a
n/a
Intron 1
AACTCAGGAATGGAAAACCA
79
26499
26518
132

113033
113052

121990
122009

533137
n/a
n/a
Intron 1
TAACTCAGGAATGGAAAACC
67
26500
26519
133

113034
113053

121991
122010

533138
n/a
n/a
Intron 1
CAAAATTACTGCAGTCACAG
67
39716
39735
134

533139
n/a
n/a
Intron 1
ACAAAATTACTGCAGTCACA
81
39717
39736
135

533140
n/a
n/a
Intron 1
TACAAAATTACTGCAGTCAC
81
39718
39737
136

533141
n/a
n/a
Intron 1
CATACAAAATTACTGCAGTC
67
39720
39739
137

533142
n/a
n/a
Intron 1
ACATACAAAATTACTGCAGT
48
39721
39740
138

533143
n/a
n/a
Intron 1
AACATACAAAATTACTGCAG
53
39722
39741
139

533144
n/a
n/a
Intron 1
TTTTAGTATGAACCTTAAAA
0
42139
42158
140

533145
n/a
n/a
Intron 1
CTTTTAGTATGAACCTTAAA
38
42140
42159
141

533146
n/a
n/a
Intron 1
TCTTTTAGTATGAACCTTAA
57
42141
42160
142

533147
n/a
n/a
Intron 1
AATCTTTTAGTATGAACCTT
60
42143
42162
143

533148
n/a
n/a
Intron 1
CAATCTTTTAGTATGAACCT
70
42144
42163
144

533149
n/a
n/a
Intron 1
ACAATCTTTTAGTATGAACC
60
42145
42164
145

533150
n/a
n/a
Intron 1
AAGTTATGTGACTCTGAGCA
67
43174
43193
146

533151
n/a
n/a
Intron 1
CAAGTTATGTGACTCTGAGC
67
43175
43194
147

533152
n/a
n/a
Intron 1
TCAAGTTATGTGACTCTGAG
63
43176
43195
148

533153
n/a
n/a
Intron 1
AGTTCTCCATTAGGGTTCTG
83
50948
50967
149

533154
n/a
n/a
Intron 1
TAGTTCTCCATTAGGGTTCT
76
50949
50968
150

533155
n/a
n/a
Intron 1
ATAGTTCTCCATTAGGGTTC
51
50950
50969
151

533156
n/a
n/a
Intron 1
AAGCAGGTTGGCAGACAGAC
79
53467
53486
152

533157
n/a
n/a
Intron 1
GAAGCAGGTTGGCAGACAGA
60
53468
53487
153

533158
n/a
n/a
Intron 1
GGAAGCAGGTTGGCAGACAG
67
53469
53488
154

533159
n/a
n/a
Intron 1
TCTTCTTGTGAGCTGGCTTC
61
64882
64901
155

533160
n/a
n/a
Intron 1
GTCTTCTTGTGAGCTGGCTT
83
64883
64902
156

533161
n/a
n/a
Intron 1
AGTCTTCTTGTGAGCTGGCT
81
64884
64903
157

TABLE 4

Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intronic and exonic regions of SEQ ID NO: 1

and 2

SEQ
SEQ

ID
ID

SEQ

NO:
NO:

ID

1
1

SEQ ID
NO: 2
SEQ

ISIS
Start
Stop
Target

%
NO: 2
Stop
ID

NO
Site
Site
Region
Sequence
inhibition
Start Site
Site
NO

533133
n/a
n/a
Intron 1
CAGGAATGGAAAACCAAATA
76
26495
26514
129

121986
122005

533134
n/a
n/a
Intron 1
TCAGGAATGGAAAACCAAAT
83
26496
26515
130

121987
122006

533174
n/a
n/a
Intron 1
TAAGTCTTCTTGTGAGCTGG
73
64886
64905
158

533175
n/a
n/a
Intron 1
TTAAGTCTTCTTGTGAGCTG
58
64887
64906
159

533176
n/a
n/a
Intron 1
ATTAAGTCTTCTTGTGAGCT
51
64888
64907
160

533177
n/a
n/a
Intron 1
TCTCTTCCACTCACATCCAT
72
65989
66008
161

533178
n/a
n/a
Intron 1
GTCTCTTCCACTCACATCCA
86
65990
66009
162

533179
n/a
n/a
Intron 1
AGTCTCTTCCACTCACATCC
80
65991
66010
163

533180
n/a
n/a
Intron 1
TAAGTATTTGTAGCAGTTGC
31
78195
78214
164

533181
n/a
n/a
Intron 1
CTAAGTATTTGTAGCAGTTG
14
78196
78215
165

533182
n/a
n/a
Intron 1
GCTAAGTATTTGTAGCAGTT
59
78197
78216
166

533183
n/a
n/a
Intron 1
TGGCTAAGTATTTGTAGCAG
34
78199
78218
167

533184
n/a
n/a
Intron 1
TTGGCTAAGTATTTGTAGCA
18
78200
78219
168

533185
n/a
n/a
Intron 1
TTTGGCTAAGTATTTGTAGC
21
78201
78220
169

533186
n/a
n/a
Intron 1
AAAATGTCAACAGTGCATAG
61
80636
80655
170

533187
n/a
n/a
Intron 1
CAAAATGTCAACAGTGCATA
78
80637
80656
171

533188
n/a
n/a
Intron 1
CCAAAATGTCAACAGTGCAT
85
80638
80657
172

533189
n/a
n/a
Intron 1
GCCCAAAATGTCAACAGTGC
82
80640
80659
173

533190
n/a
n/a
Intron 1
GGCCCAAAATGTCAACAGTG
60
80641
80660
174

533191
n/a
n/a
Intron 1
TGGCCCAAAATGTCAACAGT
31
80642
80661
175

533192
n/a
n/a
Intron 1
CAGAATCTTCTCTTTGGCCA
66
98624
98643
176

533193
n/a
n/a
Intron 1
GCAGAATCTTCTCTTTGGCC
81
98625
98644
177

533194
n/a
n/a
Intron 1
TGCAGAATCTTCTCTTTGGC
72
98626
98645
178

533195
n/a
n/a
Intron 1
TTTGCAGAATCTTCTCTTTG
33
98628
98647
179

533196
n/a
n/a
Intron 1
ATTTGCAGAATCTTCTCTTT
27
98629
98648
180

533197
n/a
n/a
Intron 1
AATTTGCAGAATCTTCTCTT
38
98630
98649
181

533198
n/a
n/a
Intron 1
ATAAAGCTATGCCATAAAGC
37
99478
99497
182

533199
n/a
n/a
Intron 1
CATAAAGCTATGCCATAAAG
14
99479
99498
183

533200
n/a
n/a
Intron 1
CCATAAAGCTATGCCATAAA
30
99480
99499
184

533201
n/a
n/a
Intron 1
GACCATAAAGCTATGCCATA
54
99482
99501
185

533202
n/a
n/a
Intron 1
TGACCATAAAGCTATGCCAT
64
99483
99502
186

533203
n/a
n/a
Intron 1
CTGACCATAAAGCTATGCCA
61
99484
99503
187

533204
n/a
n/a
Intron 1
CAAAAAGTTGAGCTGAGAAA
0
101078
101097
188

533205
n/a
n/a
Intron 1
CCAAAAAGTTGAGCTGAGAA
28
101079
101098
189

533206
n/a
n/a
Intron 1
CCCAAAAAGTTGAGCTGAGA
52
101080
101099
190

533207
n/a
n/a
Intron 1
CACCCAAAAAGTTGAGCTGA
60
101082
101101
191

533208
n/a
n/a
Intron 1
ACACCCAAAAAGTTGAGCTG
34
101083
101102
192

533209
n/a
n/a
Intron 1
TACACCCAAAAAGTTGAGCT
36
101084
101103
193

533210
n/a
n/a
Intron 1
CTTTTAATGGCACCCAAGCA
41
103566
103585
194

533211
n/a
n/a
Intron 1
GCTTTTAATGGCACCCAAGC
54
103567
103586
195

533212
n/a
n/a
Intron 1
TGCTTTTAATGGCACCCAAG
67
103568
103587
196

533213
n/a
n/a
Intron 1
AATGCTTTTAATGGCACCCA
73
103570
103589
197

533214
n/a
n/a
Intron 1
AAATGCTTTTAATGGCACCC
73
103571
103590
198

533215
n/a
n/a
Intron 1
GAAATGCTTTTAATGGCACC
41
103572
103591
199

533216
n/a
n/a
Intron 1
TAATTCTTAAGGGCCCTCTG
36
106963
106982
200

533217
n/a
n/a
Intron 1
ATAATTCTTAAGGGCCCTCT
45
106964
106983
201

533218
n/a
n/a
Intron 1
CATAATTCTTAAGGGCCCTC
50
106965
106984
202

533219
n/a
n/a
Intron 1
AGCATAATTCTTAAGGGCCC
48
106967
106986
203

533220
n/a
n/a
Intron 1
TAGCATAATTCTTAAGGGCC
52
106968
106987
204

533221
n/a
n/a
Intron 1
TTAGCATAATTCTTAAGGGC
28
106969
106988
205

533222
n/a
n/a
Intron 1
AGGAATGGAAAACCAAACAT
13
113028
113047
206

533223
n/a
n/a
Intron 1
CAGGAATGGAAAACCAAACA
64
113029
113048
207

533224
n/a
n/a
Intron 1
TCAGGAATGGAAAACCAAAC
61
113030
113049
208

533225
n/a
n/a
Intron 1
AGGAATGGAAAACCAAATAC
18
121985
122004
209

533226
n/a
n/a
Intron 1
CATGACTATGTTCTGGCAAG
37
125591
125610
210

533227
n/a
n/a
Intron 1
ACATGACTATGTTCTGGCAA
44
125592
125611
211

533228
n/a
n/a
Intron 1
CACATGACTATGTTCTGGCA
63
125593
125612
212

533229
n/a
n/a
Intron 1
GTCACATGACTATGTTCTGG
47
125595
125614
213

533230
n/a
n/a
Intron 1
GGTCACATGACTATGTTCTG
49
125596
125615
214

533231
n/a
n/a
Intron 1
TGGTCACATGACTATGTTCT
30
125597
125616
215

533232
n/a
n/a
Intron 2
CTGAATTCTGAGCTCTGGAA
73
145428
145447
216

533233
n/a
n/a
Intron 2
CCTGAATTCTGAGCTCTGGA
88
145429
145448
217

533234
n/a
n/a
Intron 2
GCCTGAATTCTGAGCTCTGG
92
145430
145449
218

533235
n/a
n/a
Intron 2
AAGCCTGAATTCTGAGCTCT
83
145432
145451
219

533236
n/a
n/a
Intron 2
CAAGCCTGAATTCTGAGCTC
68
145433
145452
220

533237
n/a
n/a
Intron 2
ACAAGCCTGAATTCTGAGCT
81
145434
145453
221

533238
n/a
n/a
Intron 2
GGATCTCAGCTGCAATTCTT
72
146235
146254
222

533239
n/a
n/a
Intron 2
AGGATCTCAGCTGCAATTCT
53
146236
146255
223

533240
n/a
n/a
Intron 2
GAGGATCTCAGCTGCAATTC
69
146237
146256
224

533241
n/a
n/a
Intron 2
CAGAGGATCTCAGCTGCAAT
69
146239
146258
225

533242
n/a
n/a
Intron 2
GCAGAGGATCTCAGCTGCAA
76
146240
146259
226

533243
230
249
Exon 2
AAAAGCATCACTTGATCCTG
23
145014
145033
227

TABLE 5

Inhibition of GHR mRNA by 3-10-4 MOE gapmers targeting intronic and exonic regions of SEQ ID NO: 1

and 2

SEQ
SEQ

ID
ID

NO:
NO:

SEQ
SEQ ID

1
1

ID NO:
NO: 2

ISIS
Start
Stop
Target

%
2 Start
Stop
SEQ

NO
Site
Site
Region
Sequence
inhibition
Site
Site
ID NO

539284
206
222
Exon 2
CAAGGTCAACAGCAGCT
62
144990
145006
228

539285
207
223
Exon 2
CCAAGGTCAACAGCAGC
74
144991
145007
229

539286
208
224
Exon 2
GCCAAGGTCAACAGCAG
73
144992
145008
230

539290
869
885
Exon 7
TCTCACACGCACTTCAT
29
290367
290383
231

539291
870
886
Exon 7
ATCTCACACGCACTTCA
51
290368
290384
232

539292
871
887
Exon 7
GATCTCACACGCACTTC
56
290369
290385
233

539299
n/a
n/a
Intron 1
CTTTCATGAATCAAGCT
63
17927
17943
234

539300
n/a
n/a
Intron 1
TCTTTCATGAATCAAGC
49
17928
17944
235

539301
n/a
n/a
Intron 1
GTCTTTCATGAATCAAG
61
17929
17945
236

539302
n/a
n/a
Intron 1
GGTCTTTCATGAATCAA
93
17930
17946
237

539303
n/a
n/a
Intron 1
ATGGTCTTTCATGAATC
74
17932
17948
238

539304
n/a
n/a
Intron 1
GATGGTCTTTCATGAAT
56
17933
17949
239

539305
n/a
n/a
Intron 1
TATATCAATATTCTCCC
42
21820
21836
240

539306
n/a
n/a
Intron 1
TTATATCAATATTCTCC
33
21821
21837
241

539307
n/a
n/a
Intron 1
GTTATATCAATATTCTC
12
21822
21838
242

539308
n/a
n/a
Intron 1
TTTCTTTAGCAATAGTT
21
22518
22534
243

539309
n/a
n/a
Intron 1
CTTTCTTTAGCAATAGT
38
22519
22535
244

539310
n/a
n/a
Intron 1
GCTTTCTTTAGCAATAG
39
22520
22536
245

539311
n/a
n/a
Intron 1
AGGAATGGAAAACCAAA
18
26497
26513
246

113031
113047

121988
122004

539312
n/a
n/a
Intron 1
CAGGAATGGAAAACCAA
40
26498
26514
247

113032
113048

121989
122005

539313
n/a
n/a
Intron 1
TCAGGAATGGAAAACCA
49
26499
26515
248

113033
113049

121990
122006

539314
n/a
n/a
Intron 1
TCTCCATTAGGGTTCTG
87
50948
50964
249

539315
n/a
n/a
Intron 1
TTCTCCATTAGGGTTCT
57
50949
50965
250

539316
n/a
n/a
Intron 1
GTTCTCCATTAGGGTTC
73
50950
50966
251

539317
n/a
n/a
Intron 1
AGGTTGGCAGACAGACA
73
53466
53482
252

539318
n/a
n/a
Intron 1
CAGGTTGGCAGACAGAC
84
53467
53483
253

539319
n/a
n/a
Intron 1
GCAGGTTGGCAGACAGA
85
53468
53484
254

539320
n/a
n/a
Intron 1
CTTCTTGTGAGCTGGCT
87
64884
64900
255

539321
n/a
n/a
Intron 1
TCTTCTTGTGAGCTGGC
89
64885
64901
256

539322
n/a
n/a
Intron 1
GTCTTCTTGTGAGCTGG
87
64886
64902
257

539323
n/a
n/a
Intron 1
AGTCTTCTTGTGAGCTG
70
64887
64903
258

539324
n/a
n/a
Intron 1
TCTTCCACTCACATCCA
65
65990
66006
259

539325
n/a
n/a
Intron 1
CTCTTCCACTCACATCC
78
65991
66007
260

539326
n/a
n/a
Intron 1
TCTCTTCCACTCACATC
68
65992
66008
261

539327
n/a
n/a
Intron 1
GTCTCTTCCACTCACAT
74
65993
66009
262

539328
n/a
n/a
Intron 1
ATAGATTTTGACTTCCC
57
72107
72123
263

539329
n/a
n/a
Intron 1
CATAGATTTTGACTTCC
35
72108
72124
264

539330
n/a
n/a
Intron 1
GCATAGATTTTGACTTC
53
72109
72125
265

539331
n/a
n/a
Intron 1
AAAATGTCAACAGTGCA
86
80639
80655
266

539332
n/a
n/a
Intron 1
CAAAATGTCAACAGTGC
73
80640
80656
267

539333
n/a
n/a
Intron 1
CCAAAATGTCAACAGTG
34
80641
80657
268

539334
n/a
n/a
Intron 1
CCCAAAATGTCAACAGT
66
80642
80658
269

539335
n/a
n/a
Intron 1
CATGACTATGTTCTGGC
67
125594
125610
270

539336
n/a
n/a
Intron 1
ACATGACTATGTTCTGG
42
125595
125611
271

539337
n/a
n/a
Intron 1
CACATGACTATGTTCTG
29
125596
125612
272

539338
n/a
n/a
Intron 2
GAATTCTGAGCTCTGGA
77
145429
145445
273

539339
n/a
n/a
Intron 2
TGAATTCTGAGCTCTGG
84
145430
145446
274

539340
n/a
n/a
Intron 2
CTGAATTCTGAGCTCTG
80
145431
145447
275

539341
n/a
n/a
Intron 2
CCTGAATTCTGAGCTCT
84
145432
145448
276

539342
n/a
n/a
Intron 2
GCCTGAATTCTGAGCTC
84
145433
145449
277

539343
n/a
n/a
Intron 2
AGCCTGAATTCTGAGCT
80
145434
145450
278

539344
n/a
n/a
Intron 2
ATATTGTAATTCTTGGT
0
148059
148075
279

539345
n/a
n/a
Intron 2
GATATTGTAATTCTTGG
20
148060
148076
280

539346
n/a
n/a
Intron 2
TGATATTGTAATTCTTG
13
148061
148077
281

539347
n/a
n/a
Intron 2
CTGATATTGTAATTCTT
8
148062
148078
282

539348
n/a
n/a
Intron 2
CCTGATATTGTAATTCT
67
148063
148079
283

539349
n/a
n/a
Intron 2
GCCTGATATTGTAATTC
73
148064
148080
284

539350
n/a
n/a
Intron 2
TGCCTGATATTGTAATT
32
148065
148081
285

539351
n/a
n/a
Intron 2
AATTATGTGCTTTGCCT
58
148907
148923
286

539352
n/a
n/a
Intron 2
CAATTATGTGCTTTGCC
82
148908
148924
287

539353
n/a
n/a
Intron 2
TCAATTATGTGCTTTGC
68
148909
148925
288

539354
n/a
n/a
Intron 2
GTCAATTATGTGCTTTG
80
148910
148926
289

539355
n/a
n/a
Intron 2
GCCATCACCAAACACCA
94
150972
150988
290

539356
n/a
n/a
Intron 2
TGCCATCACCAAACACC
84
150973
150989
291

539357
n/a
n/a
Intron 2
TTGCCATCACCAAACAC
74
150974
150990
292

539358
n/a
n/a
Intron 2
TGGTGACTCTGCCTGAT
85
151387
151403
293

539359
n/a
n/a
Intron 2
CTGGTGACTCTGCCTGA
86
151388
151404
294

TABLE 6

inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting intron 1 of SEQ ID NO: 2

SEQ
SEQ

ID
ID

NO: 2
NO: 2
SEQ

ISIS

%
Start
Stop
ID

NO
Sequence
inhibition
Site
Site
NO

523561
TATTTCAGAAAGACTTTCTG
11
10373
10392
295

523562
AGGAAAAAATCAAGGAGTTA
8
11173
11192
296

523563
TATTTACTGAACACCTATTC
12
11973
11992
297

523564
GCCCATGAAGGCCACTCTTC
70
12780
12799
298

523565
ACCTATAAATAAAGTGAGGA
0
13581
13600
299

523566
GTTTCATAACCTGCTAATAA
40
14451
14470
300

523567
ATGTGCCTTACAGTTATCAG
36
15251
15270
301

523568
TTCTGAATTTAGAATTATAG
0
16051
16070
302

523569
GTTTATAATCTAGCAGTTAC
26
17130
17149
303

523570
GATGGTCTTTCATGAATCAA
62
17930
17949
304

523571
CATGTACCCAAACCAACACT
65
18730
18749
305

523572
TAAAATACAGCCTACATCAT
0
19637
19656
306

523573
CCATCACTACAACAAACTCA
39
20451
20470
307

523574
ATCTGAAATGATCCCCTTTC
33
21283
21302
308

523575
TGTTGCCCCTCCAAAAAGAC
12
22144
22163
309

523576
ATTAAAATTTTAAATGATGT
0
22944
22963
310

523577
CTCAGGAATGGAAAACCAAA
71
26497
26516
311

113031
113050

121988
122007

523578
AAAATTCTAGAAGATAACAT
0
27838
27857
312

523579
CTAGAAGTCCTAGCCAGAGT
2
28748
28767
313

523580
AACCGATATCACAGAAATAC
0
29548
29567
314

523581
AAGATAGACAGTAACATAAT
0
30348
30367
315

523582
GCACTACAAGAACTGCTTAA
40
31172
31191
316

523583
TTTCCAGACAAAGAATTCAG
6
31978
31997
317

523584
GTAGACAGCCTTTCTGGAAC
20
32827
32846
318

523585
CATCCTACATAGTGGCTGTG
47
33635
33654
319

523586
CAGAACAGTGTGTGGAGACT
8
34452
34471
320

523587
AGCTTTAAAAATACCTCTGC
52
35466
35485
321

523588
CCCAGGTACTTGCTCTCAGA
22
36266
36285
322

523589
TTACACCTGATTCTAGAAAT
30
37066
37085
323

523590
CTTTTCTCTACAACCTCACA
34
38094
38113
324

523591
TAGTAGTTTGAATTTCAAAG
1
38909
38928
325

523592
ATACAAAATTACTGCAGTCA
60
39719
39738
326

523593
GCCACTGCCAAAAAGGAGGA
30
40519
40538
327

523594
TGACAGAAACAGAGCTATGA
33
41342
41361
328

523595
ATCTTTTAGTATGAACCTTA
65
42142
42161
329

523596
AGTTATGTGACTCTGAGCAC
63
43173
43192
330

523597
ACTATGCCCTAGTTACTTCT
29
43973
43992
331

523598
TATAGTGGAAGTGATAGATC
0
44812
44831
332

523599
TGTTTTCTGAAATGGAATGT
0
45733
45752
333

523600
GCTGTAAATGTAATGAGTGT
34
46553
46572
334

523601
GAGAGAAGCCATGGCCCTAG
20
47392
47411
335

523602
CTCTCTTTCCCAGAACAAGA
32
48210
48229
336

523603
TCCAAAATGTCCAGTATAAT
33
50072
50091
337

523604
GTTCTCCATTAGGGTTCTGG
74
50947
50966
338

523605
TTAGTCACCCATCCACCACT
41
51747
51766
339

523606
CATGAATTCACCGAGTTAGG
51
52573
52592
340

523607
AGCAGGTTGGCAGACAGACA
62
53466
53485
341

523608
GAAAGACTTAAATTTTCACA
0
54306
54325
342

523609
TAGTAGAGGAAAAGGAGAAT
0
55730
55749
343

523610
AAACAGGGTCTGGAGTGGAC
3
61243
61262
344

523611
CAAGCTGATAATTAAAAAGA
0
62462
62481
345

523612
ATAAAGATACATTTTCTGGG
8
63277
63296
346

523613
CAGGATTCTTCCTGCCTGGC
47
64085
64104
347

523614
AAGTCTTCTTGTGAGCTGGC
71
64885
64904
348

523615
CTCTTCCACTCACATCCATT
63
65988
66007
349

523616
CCTATATCAGAAGACAAATG
5
66806
66825
350

523617
TCAAAACCCTGCCAAGGTAC
44
67662
67681
351

523618
TCATATTCTACTTCTGTTTA
11
68462
68481
352

523619
CATTCCAGTGTTTCAGTAAG
13
69262
69281
353

523620
GGCCTGGAATTAATCCTCAG
49
70114
70133
354

523621
AATGCCCTCTCCCTGTGCCT
48
70925
70944
355

523622
TTTATAATCAACCTTTGCTA
9
71741
71760
356

523623
ATATAACTACTTAAAATAAT
0
72541
72560
357

523624
TTAGCCAGGATATGGTTGCC
50
73350
73369
358

523625
CTACCTCCATCAAAGAAAAT
0
74190
74209
359

523626
GCATGCATAGATAAGTTTGA
20
74990
75009
360

523627
ATGAGAGTAAATGGATTTTC
10
75790
75809
361

523628
TTGGCAATCCTTGCTTAAAA
34
76598
76617
362

523629
GAATTAAGCCAGACTTATTT
3
77398
77417
363

523630
GGCTAAGTATTTGTAGCAGT
55
78198
78217
364

523631
TTGCCTGTGTGCAACTGGCG
0
79005
79024
365

523632
GTGGCCTTAGTAGGCCAGCT
0
79827
79846
366

523633
CCCAAAATGTCAACAGTGCA
70
80639
80658
367

523634
TTAAGCCTTCAATTTGAAAA
0
81455
81474
368

523635
TGCTCAGAAGGTTGAGCATA
0
82261
82280
369

523636
TTAATGCTTTCCCAAAGCTC
35
83061
83080
370

523637
AAAAGACTTCATACCTTTAC
52
83884
83903
371

TABLE 7

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting intron 1 of SEQ ID NO: 2

SEQ
SEQ

ID
ID

%
NO:
NO:

inhi-
2
2
SEQ

ISIS

bi-
Start
Stop
ID

NO
Sequence
tion
Site
Site
NO

532146
GGCCCCCTGGCACAACAGGA
60
3097
3116
372

532147
TCTAGGGTGATTCAGGTGGA
62
4537
4556
373

532148
CTTAGATTAATGCAAAACAA
25
4875
4894
374

532149
AGGCAGAGGAGGGTGGAACC
34
6246
6265
375

532150
AGTCTAATGAGATCTGATGG
76
6499
6518
376

532151
GCTGAAATGAGTTAAGACTT
89
6737
6756
377

532152
ACTTTGGACTGTGGATTTTT
78
6765
6784
378

532153
GCATATTTACACAATGCCTG
84
6871
6890
379

532154
GGAAATGCCTGGATGTCCAG
27
7241
7260
380

532155
CTGCTGATTTTGGAATGGAG
68
10660
10679
381

532156
ACTGAACACCTATTCTATGG
51
11968
11987
382

532157
TTTACTGAACACCTATTCTA
23
11971
11990
383

532158
CCCTCAAATTATCCACAAAC
89
12053
12072
384

532159
CTTCTAAATGTTTCCAAGGC
63
12186
12205
385

532160
TTACATCCTGTAGGCTAATT
82
12469
12488
386

532161
CCACTAGCCTGGCCAGACTT
73
12487
12506
387

532162
CTGGTAGATGATCTCAAGTT
84
13351
13370
388

532163
AAAGAATTGAGTTATAAATC
23
13670
13689
389

532164
AACTCATCTCTGGCCAGCAG
89
14361
14380
390

532165
CAACATCATTGTATTTTCTG
33
14965
14984
391

532166
TCTTAGCTTACCAATGAGGA
81
15085
15104
392

532167
TTCCCAGAGCCAAAGCTCAA
77
15982
16001
393

532168
TTTGGCCAATCCCAGCTTAT
59
16253
16272
394

532169
GTTTGCAAATCTTCATTCAC
71
16447
16466
395

532170
CAATAGTCCCTGAGGCTTGG
74
16476
16495
396

532171
TTTCCCCAGATTAAATGCCC
85
17650
17669
397

532172
TTCAATAATGCAGTTATTAT
0
18308
18327
398

532173
AAATTCTTGGGCTTAAGCAC
69
18638
18657
399

532174
ACATGTACCCAAACCAACAC
71
18731
18750
91

532175
TGATCCAAATTCAGTACCTA
82
18752
18771
400

532176
GATGATCCAAATTCAGTACC
54
18754
18773
401

532177
CAATATTCATCTTTATATTC
25
19106
19125
402

532178
ATTGCTCTTAAGATAAGTAA
41
19661
19680
403

532179
CAGCTCCCTGAATATCTCTT
74
19783
19802
404

532180
ACTTCACAAATATATTATAA
0
19885
19904
405

532181
GTACAGTCAACTTTACTTCA
89
19899
19918
406

532182
CAATTCCCACTCTTGTCAAC
55
20288
20307
407

532183
TCAACTGCTTTCTGGAGCAG
66
21215
21234
408

532184
ACTGCTGAGCACCTCCAAAA
73
21454
21473
409

532185
CTTAGATTCCTGGTTTATCA
78
21587
21606
410

532186
AGTTATATCAATATTCTCCC
88
21820
21839
411

532187
TATACCATCTTCCCCATAAA
32
22038
22057
412

532188
GGCTTTCTTTAGCAATAGTT
86
22518
22537
413

532189
TACCAGGGATGTAGGTTTAC
82
29050
29069
414

532190
TCACAGCTGAATTCTATCTG
80
29323
29342
415

532191
GGAGATGGACAAATTCCTGC
77
29470
29489
416

532192
CTAGACATGTCATCAAGACA
19
30294
30313
417

532193
CAAATTAATAAAACAATTAC
10
30385
30404
418

532194
TATTCTTATATCAGACAAAA
30
30532
30551
419

532195
TCAAGGGATCCCTGCCATTC
32
32361
32380
420

532196
CGTCAAGGGATCCCTGCCAT
47
32363
32382
421

532197
GGCACTCCCAGTCTCCAGCT
83
34138
34157
422

532198
TTTCTCCAGCAGAAGTGTCA
60
34845
34864
423

532199
AAGTCCTCTTCCGCCTCCCT
82
36023
36042
424

532200
GGAATTTACCAAAAACAGTT
63
36721
36740
425

532201
AGTTAGGTATTGTCCATTTT
74
37032
37051
426

532202
ACATGGGTATCTTCTAGGAA
77
37111
37130
427

532203
TCAGTTTCAGAGAGACAAAA
41
37276
37295
428

532204
TTTGCCAGGTCCTATGTCGA
69
37656
37675
429

532205
ATTCCCTTTTCTCTACAACC
70
38099
38118
430

532206
ATGATAAGAGCCAAGATTTG
13
38994
39013
431

532207
GAAAAAAGGTCCACTGTGGT
49
40356
40375
432

532208
CCTGTCCTGGAATAGTTTCA
49
41164
41183
433

532209
TAGAAAAGTAAATAAGGAAT
15
41501
41520
434

532210
TTATAAAACTATGCAATAGG
0
41889
41908
435

532211
TTATTTCATATTTCCAGAAA
0
42675
42694
436

532212
CATGAATTACAGCTAAAGAT
20
42741
42760
437

532213
TTGCATGTATGTGTTTCTGA
62
43518
43537
438

532214
TCAATCTCTTTATACCCTTA
75
43765
43784
439

532215
TCTTCAATCTCTTTATACCC
58
43768
43787
440

532216
CTATGCCCTAGTTACTTCTA
47
43972
43991
441

532217
AAAGAGAATCTCTTCCTTTT
27
44070
44089
442

532218
TCATTAAAGATTATTATAAC
0
44222
44241
443

532219
TTTGGATGAGTGGAAGGCTA
0
44528
44547
444

532220
GGAAATGGCCTTTTTCCTTA
72
45400
45419
445

532221
GGAGAAGCCCTCTGCCTGTA
60
46477
46496
446

532222
AAACCATATTGTCCACCAGA
84
46510
46529
447

TABLE 8

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting intron 1 of SEQ ID NO: 2

SEQ
SEQ

ID
ID

%
NO:
NO:

inhi-
2
2
SEQ

ISIS

bi-
Start
Stop
ID

NO
Sequence
tion
Site
Site
NO

532223
CTCAAACCATATTGTCCACC
90
46513
46532
448

532224
GTGTAAATAGTGACTTGTAC
76
50123
50142
449

532225
TGAGGCACAGGAAAGTTAAC
52
50719
50738
450

532226
AGCTATAGTTCTCCATTAGG
74
50954
50973
451

532227
TTACTTGCTGACTAAGCCAT
69
51071
51090
452

532228
GTTTGTCAACTCAACATCAA
73
51215
51234
453

532229
GACTATTTGTATATATATAC
33
51491
51510
454

532230
ATGACTATTTGTATATATAT
11
51493
51512
455

532231
ACTCTTCCTTATATTTGCTC
76
51778
51797
456

532232
ATACACTGACTTTTAACATT
67
52039
52058
457

532233
CTTAGAAACAGTAGTTTCAT
42
52124
52143
458

532234
CTGAGCTTTGCCTTAAGAAT
79
52633
52652
459

532235
CACCAGACAGCAGGTAGAGC
81
53540
53559
460

532236
GAGATGGAGTAGAAGGCAAA
43
55926
55945
461

532237
TAGGAAAGGAAGAATACACT
33
63881
63900
462

532238
TAGACCAGGAAGGGTGAGAG
27
64376
64395
463

532239
AAGTTGGATCTGGCATGCAT
64
64574
64593
464

532240
AAAGTTGGATCTGGCATGCA
70
64575
64594
465

532241
CCATAACTCTTCTAACTGGG
84
64643
64662
466

532242
ATATTAAAGTTTGAGAACTA
37
65080
65099
467

532243
CTTAACTACAAAATGCTGGA
71
66164
66183
468

532244
TGAGCAGCTGTCCTCAGTTC
43
67061
67080
469

532245
GAGTTCATAAAAGTTTTACT
26
67251
67270
470

532246
CTATCCACACCATTCCATAA
73
69203
69222
471

532247
AACATCTAAGTAATGCAAAC
58
69223
69242
472

532248
TTTGCATTCAAAGCCCTGGG
91
69565
69584
473

532249
TCCATATTATAGGCTATGAT
73
69889
69908
474

532250
ATTTTATGATAATGTAAAAC
27
69942
69961
475

532251
GAGATCACATTTTCTGAGTA
50
70352
70371
476

532252
ACCTCCCTAGGATTACCTCA
56
71617
71636
477

532253
AAAATCTGATTTATAATCAA
40
71750
71769
478

532254
AGCATAGATTTTGACTTCCC
92
72107
72126
479

532255
AAAGTCATATACACAGGTCT
53
72584
72603
480

532256
CTCATAGCAAATTCCCAGAA
66
73689
73708
481

532257
CAACATGGAGGCTAGCATGT
55
74112
74131
482

532258
AGACTAAGTGGCCTGAATGT
52
74317
74336
483

532259
ACCTACCATGTCACTCTCAA
61
74418
74437
484

532260
AACTTTCTTGTGTTTTATCA
9
75511
75530
485

532261
TTTGCAAGACAAAGAAATGA
31
75915
75934
486

532262
CATGCAAAGTGTTCCTCTTC
63
76024
76043
487

532263
AGTGCTTTGCTTTCTCTTAT
79
76047
76066
488

532264
GAACAAGAAACACTTGGTAA
44
76555
76574
489

532265
AGTGTTCCAATTAAATGGCA
34
76643
76662
490

532266
AAACAATGCCCTTGTAGTGA
57
76703
76722
491

532267
TATTCTAGGTTTTGAGGTGA
60
76752
76771
492

532268
ATATTCTAGGTTTTGAGGTG
24
76753
76772
493

532269
GTTTTCCATTCTTTAAGAAA
41
76896
76915
494

532270
AGCAATCCATTGATTGTATG
59
77044
77063
495

532271
AATTATGGCAAAATGGAAAA
37
77076
77095
496

532272
ACATTTGCTTATGAGACTAT
62
77638
77657
497

532273
GCAGAGATAATCCTATGATG
42
77841
77860
498

532274
TCCATCTGTTACCTCTCTGT
77
78122
78141
499

532275
TTTGCCTGAAGGGCAGAACC
40
79478
79497
500

532276
GAAAAAATCAGATTTTCACA
0
79664
79683
501

532277
AACTTAATTTAATCATTTCT
0
79959
79978
502

532278
TTTGGTTGTCATGAGTTGAG
67
80756
80775
503

532279
TTCCATCTCTAGGGCACTTT
74
80900
80919
504

532280
AGAGCTTATTTTCAAAATTC
36
80920
80939
505

532281
ATAAAGAGCAAACAAACATA
42
81524
81543
506

532282
TATAAATTCCTTGGTCTGAT
33
82835
82854
507

532283
AAAATATAAATTCCTTGGTC
13
82839
82858
508

532284
TTTTATAACAGCCTCTGACA
38
82959
82978
509

532285
AAAAGACCATGTTGCTTATT
72
83179
83198
510

532286
ATAGTCAGTCAGAATGTGGT
72
83330
83349
511

532287
TGCCTTAGCTTGGAAAAGAC
78
83897
83916
512

532288
AGGGCTAGCTGATGCCTCTC
69
84026
84045
513

532289
TTGGACTGGGCTCAAACAGA
72
84381
84400
514

532290
AAAGTCAGGCTAGAGGGACT
49
85713
85732
515

532291
TCCTTGTTTTCTTGTAATGA
50
85945
85964
516

532292
ACACCAGAGGAAGGAAATCA
44
86554
86573
517

532293
GATGTACACCATTTTGAATT
15
86629
86648
518

532294
TGCTCTGGCCTAGCCTATGT
62
86901
86920
519

532295
CAGAGGTGTCTCCCAAGAAA
60
89940
89959
520

532296
AAAGAGAATGGATCAAAGCT
36
91930
91949
521

532297
GATTTGCAGAACAAATCTTG
37
93332
93351
522

532298
TGGTTATGAAGGTTGGACCA
52
94839
94858
523

532299
TGGCTAATTAATGGGCAATT
63
95292
95311
524

TABLE 9

inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting intron 1 of SEQ ID NO: 2

SEQ
SEQ

ID
ID

%
NO:
NO:

inhi-
2
2
SEQ

ISIS

bi-
Start
Stop
ID

NO
Sequence
tion
Site
Site
NO

532300
CTGTGCCATATTGCCTCTAA
87
95471
95490
525

532301
GATTTCAACCAGCTCACCTG
48
95510
95529
526

532302
GCAAAAGGGAACCCTGAAGC
71
95564
95583
527

532303
CTAAGTGTTATAACAAACAC
43
96137
96156
528

532304
GTCCATTGGTATAAAACTCA
84
96282
96301
529

532305
TTTCAATACAATAAGATTTA
34
96793
96812
530

532306
GTCCTTAGACCCCTCAATGG
62
96987
97006
531

532307
GAGGATTTATTCATCTAGGC
68
97806
97825
532

532308
CAGTGGGAGGATCAGATATC
46
97870
97889
533

532309
ATCCCATCCAGCAGCTGGAC
67
98132
98151
534

532310
AACTTGGGATGAGTTACTGA
56
98653
98672
535

532311
GAAGGCTACCTAAAAGAAAT
43
98810
98829
536

532312
AAAGAAATATTCACAACATT
39
99096
99115
537

532313
ATGCTTATACTGCTGCTGTA
69
99791
99810
538

532314
TCCTCACTTCAATCACCTTT
70
99819
99838
539

532315
CTCTTTCTTCATAAATAAGT
33
100809
100828
540

532316
TGGTAATCTGTGTCCCTTTA
96
101242
101261
541

532317
TAATAAAAAAGTTTGAAACA
41
102549
102568
542

532318
GGTGGTGGCAAGAGAAAAAT
56
103015
103034
543

532319
CAAAAGGCCCTTTTTACATG
28
103034
103053
544

532320
ACTCTACTGGTACCAATTTA
31
103173
103192
545

532321
TCTGAACTTTTATGCTCTGT
76
103606
103625
546

532322
AACTTTTGCCTGGGCATCCA
16
104067
104086
547

532323
TGACTCCATGTCTCACATCC
66
104392
104411
548

532324
TTACTTCCTAGATACAACAG
53
104541
104560
549

532325
CTGGCCCCCATGATTCAATT
44
104835
104854
550

532326
AAGACTGGCCCCCATGATTC
49
104839
104858
551

532327
TGTCACTGGTCTGTGTATTT
60
106233
106252
552

532328
ACAGAGTAGATTTAGCATAA
23
106980
106999
553

532329
TAAACAGGTGTACTATTACA
27
107030
107049
554

532330
GCTTTATCAACTAAGTTTAT
22
107716
107735
555

532331
CAGAACTTCTTTTAAAATTG
8
107763
107782
556

532332
GAATACAGACATACCTTGAA
25
108514
108533
557

532333
CCATGACAACAATTTCAGAG
58
109486
109505
558

532334
ACAAATAGCAATGAATGGGT
45
110878
110897
559

532335
CAACAAATAGCAATGAATGG
47
110880
110899
560

532336
GTACACAAATCAGTAGCTCT
72
115087
115106
561

532337
CTATGTCAAAAAGACTGAAA
4
116370
116389
562

532338
ATATACAGAACATTTCATCC
13
116743
116762
563

532339
AGAATAGATAAGAACTCACC
32
117195
117214
564

532340
AGGAAAGATACAGTCATTTT
5
117507
117526
565

532341
GCACAAAGAACACCTGGGAA
43
117781
117800
566

532342
CAAGAAGTCTGGGATTATGT
0
117938
117957
567

532343
GTTAGTTATTAAGCTAATCA
48
118245
118264
568

532344
AACCATTATTTATAGGCTAA
14
119127
119146
569

532345
CCAGAATGCGATCACTTCTT
76
120826
120845
570

532346
CCAGAAATTATCCTCCTCTC
70
121209
121228
571

532347
AGGGAAATGCAAATTAAAAC
20
122479
122498
572

532348
GCATCAAGATACAGAAAAAT
24
122751
122770
573

532349
GAATGTTTATGAGATTTTTC
0
123571
123590
574

532350
GCCAATTATATTGCCACATT
23
124413
124432
575

532351
ATACTTGCTTATGTAGAAAT
45
124589
124608
576

532352
TAATACTTGCTTATGTAGAA
3
124591
124610
577

532353
GAACACATGGCATTCTGATA
36
125178
125197
578

532354
CAGAATTTGCAGTATAAATC
0
126051
126070
579

532355
TATGTTTTGAAATCTTATTT
0
126157
126176
580

532356
ACTCACTGCTACCTCATTAA
11
126998
127017
581

532357
AAGCAGTGATAGGGTATCTG
59
127080
127099
582

532358
ATGAGGCCTATTACAATGGA
14
127170
127189
583

532359
CTGGAGTCTCATGAGGCCTA
53
127180
127199
584

532360
TGACTATCAGCCTTTTAATC
45
127663
127682
585

532361
TTCAGAGAACAACCTTTGAA
0
127959
127978
586

532362
AGCCATGTGTGATCTGATGT
53
128813
128832
587

532363
GAAATTTACTCCAAACTAGC
17
128992
129011
588

532364
AACATCCAGACCACCATCTA
35
130094
130113
589

532365
GTACCAAACCATTCATGCTC
56
131036
131055
590

532366
AGTACCAAACCATTCATGCT
24
131037
131056
591

532367
TTATAGAGCTTGAGATTGAC
7
132165
132184
592

532368
AGTCCATTATAGAGCTTGAG
58
132171
132190
593

532369
AACCATGAGATGCAATGCAG
40
132498
132517
594

532370
AGGATTGAGAATCGCTGATT
42
133168
133187
595

532371
TCTAAAGCATGGCCAGGATT
48
133182
133201
596

532372
GGGACTGAGTATTGATACTT
44
133222
133241
597

532373
AGAAGTAGGGTGTTCCAGAT
29
133523
133542
598

532374
AGAAATAGTCTTCCTACTAA
0
133547
133566
599

532375
GCCTCCTTTAAGCTTCTATG
22
134240
134259
600

532376
GGCCTGCCTTTACTTTCCCA
36
134598
134617
601

TABLE 10

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting introns 1 and 2 of SEQ ID NO: 2

SEQ
SEQ

SEQ
SEQ

ID
ID

ID
ID

NO:
NO:

%
NO:
NO:

1
1

inhi-
2
2
SEQ

ISIS
Start
Stop

Target
bi-
Start
Stop
ID

NO
Site
Site
Sequence
region
tion
Site
Site
NO

523638
n/a
n/a
ACCTCAGTGGACTCTTTCCA
Intron 1
4
84684
84703
602

523639
n/a
n/a
CAAACCTAAGTTCAAGTCCT
Intron 1
62
85523
85542
603

523640
n/a
n/a
AGTTTCACTTCTTGAATCAA
Intron 1
38
86373
86392
604

523641
n/a
n/a
AAGATCAAATGAGGTCAAGG
Intron 1
30
87181
87200
605

523642
n/a
n/a
TAGATACAAATTTCATCACA
Intron 1
23
88063
88082
606

523643
n/a
n/a
ATTCCTAAAATAGGAGCAGG
Intron 1
45
88870
88889
607

523644
n/a
n/a
TTTTTATGTTGTATAAGATA
Intron 1
0
89670
89689
608

523645
n/a
n/a
GTTCAGCCAATACATGAGTA
Intron 1
48
90473
90492
609

523646
n/a
n/a
CCAGAGGGAGTTCATTACCA
Intron 1
62
91273
91292
610

523647
n/a
n/a
TCTCTCTAATTCAACCTTAT
Intron 1
44
92107
92126
611

523648
n/a
n/a
ATAATCCTCAGACCTCTTTA
Intron 1
29
92925
92944
612

523649
n/a
n/a
CACTGTGGCAGAATTCCAAG
Intron 1
28
93762
93781
613

523650
n/a
n/a
ACACCTTGGTGCCTAGAAGC
Intron 1
54
94581
94600
614

523651
n/a
n/a
GTAGCAATGACACCTAAGAA
Intron 1
58
95394
95413
615

523652
n/a
n/a
TTTAAAATAATAAATGCTTA
Intron 1
0
96194
96213
616

523653
n/a
n/a
TCATTTGGTCCTTAGACCCC
Intron 1
27
96994
97013
617

523654
n/a
n/a
TTATTCATCTAGGCCGAGTG
Intron 1
57
97800
97819
618

523655
n/a
n/a
TTGCAGAATCTTCTCTTTGG
Intron 1
65
98627
98646
619

523656
n/a
n/a
ACCATAAAGCTATGCCATAA
Intron 1
63
99481
99500
620

523657
n/a
n/a
GGCAAGGAGCACAATAGGAC
Intron 1
20
100281
100300
621

523658
n/a
n/a
ACCCAAAAAGTTGAGCTGAG
Intron 1
66
101081
101100
622

523659
n/a
n/a
TAGATTTTCAGACTCTTTCT
Intron 1
46
101887
101906
623

523660
n/a
n/a
AATTTCAATATTGTTGTGTT
Intron 1
0
102760
102779
624

523661
n/a
n/a
ATGCTTTTAATGGCACCCAA
Intron 1
69
103569
103588
625

523662
n/a
n/a
CATGTCTCACATCCAGGTCA
Intron 1
37
104386
104405
626

523663
n/a
n/a
TTCACTGGAGTAGACTTTTA
Intron 1
45
105255
105274
627

523664
n/a
n/a
CTTATAAGGGAGGTCTGGTA
Intron 1
41
106147
106166
628

523665
n/a
n/a
GCATAATTCTTAAGGGCCCT
Intron 1
71
106966
106985
629

523666
n/a
n/a
CCACAGAACTTCTTTTAAAA
Intron 1
27
107766
107785
630

523667
n/a
n/a
GGTGACCATGATTTTAACAA
Intron 1
25
108566
108585
631

523668
n/a
n/a
AACAGCTGCATGACAATTTT
Intron 1
50
109382
109401
632

523669
n/a
n/a
AGAAACAGAATCAGTGACTT
Intron 1
44
110403
110422
633

523670
n/a
n/a
CAGATTCCAGAGAAAAGCCA
Intron 1
14
111203
111222
634

523671
n/a
n/a
TGTGAGAAGAACTCTATCAC
Intron 1
12
112030
112049
635

523672
n/a
n/a
CTCACAAATCACCACTAAAG
Intron 1
31
112842
112861
636

523673
n/a
n/a
CAACGAGTGGATAAAGAAAC
Intron 1
28
113646
113665
637

523674
n/a
n/a
ATAAAACTGGATCCTCATCT
Intron 1
13
114446
114465
638

523675
n/a
n/a
ATTAAAACTCTCAGCAAAAT
Intron 1
0
115450
115469
639

523676
n/a
n/a
AAAGACTGAAAGAACACAAA
Intron 1
0
116361
116380
640

523677
n/a
n/a
TATCTGCTGCCTTCAGGAGA
Intron 1
0
117168
117187
641

523678
n/a
n/a
TTTGAATTAACCCAATTCAA
Intron 1
0
117999
118018
642

523679
n/a
n/a
TCTTAATTTACAACAGAGGA
Intron 1
25
118821
118840
643

523680
n/a
n/a
AGAAAAGTGACAGGCTTCCC
Intron 1
31
119659
119678
644

523681
n/a
n/a
ATGTTCCTTGAAGATCCCAA
Intron 1
37
120478
120497
645

523682
n/a
n/a
ATGAATAACACTTGCCACAA
Intron 1
0
121379
121398
646

523683
n/a
n/a
GTATGTTTATCACAGCACAG
Intron 1
56
122180
122199
647

523684
n/a
n/a
AAACACTGCAATATTAGGTT
Intron 1
34
123031
123050
648

523685
n/a
n/a
GATTGGTGCTTTTCAAACTG
Intron 1
39
123936
123955
649

523686
n/a
n/a
ATTTGTAAGACAAACATGAA
Intron 1
9
124764
124783
650

523687
n/a
n/a
TCACATGACTATGTTCTGGC
Intron 1
72
125594
125613
651

523688
n/a
n/a
AGTCCTGTCCACACTATTAA
Intron 1
6
126415
126434
652

523689
n/a
n/a
CTGGGCTCTGCCTGCTGAAC
Intron 1
17
127217
127236
653

523690
n/a
n/a
AAAACCCTTAAGTATTTCCT
Intron 1
12
128054
128073
654

523691
n/a
n/a
CTCTGTTTCAAACCCCCCAG
Intron 1
21
128854
128873
655

523692
n/a
n/a
GGACAGAACACCAATCACAA
Intron 1
18
129654
129673
656

523693
n/a
n/a
ACCTACCCTTCAAAGTCACG
Intron 1
0
130486
130505
657

523694
n/a
n/a
TTCAGTTCCCAGGAGGCTTA
Intron 1
5
131286
131305
658

523695
n/a
n/a
TTTTGCAATGTCTAGCAATT
Intron 1
0
132086
132105
659

523696
n/a
n/a
ATTAAGATCAGAAAATATTA
Intron 1
0
132953
132972
660

523697
n/a
n/a
TTAATGAGATATTTTGCACC
Intron 1
34
133858
133877
661

523698
n/a
n/a
GAGAGGTTAAGTAAATCTCC
Intron 1
0
134678
134697
662

523699
n/a
n/a
CAGACTCAAATTTGAAAATT
Intron 1
14
135500
135519
663

523700
n/a
n/a
GATAAGGCAATAATACAGCC
Intron 1
1
136306
136325
664

523701
n/a
n/a
ATCATTTGCCAATTTCTGTG
Intron 1
28
137133
137152
665

523702
n/a
n/a
CAAGAAGAAAAGATGCAAAA
Intron l
0
138035
138054
666

523703
n/a
n/a
AATTTATTTCCTTCCTATGA
Intron 1
0
138857
138876
667

523704
n/a
n/a
TTTTGGAAATGTGAGAAACG
Intron 1
0
139771
139790
668

523705
n/a
n/a
AAACACATGAGAAAAGATGA
Intron 1
0
140593
140612
669

523706
n/a
n/a
TGTTGGCTCAGTGGGAATGA
Intron 1
0
141412
141431
670

523707
n/a
n/a
TGAACAGGTTTGCATTTCTC
Intron 1
42
142229
142248
671

523708
n/a
n/a
TCCTAGGTGAACAGGCTATG
Intron 1
38
143029
143048
672

523709
n/a
n/a
CCCTAATCAGGCTGAAATAA
Intron 1
0
143829
143848
673

523710
n/a
n/a
AGGGCCAGTAAGGTTTGCTT
Intron 1
12
144631
144650
674

523711
n/a
n/a
AGCCTGAATTCTGAGCTCTG
Intron 2
88
145431
145450
675

523712
n/a
n/a
AGAGGATCTCAGCTGCAATT
Intron 2
71
146238
146257
676

523713
n/a
n/a
GAAAATCCCTGCTCAAGTGC
Intron 2
67
147262
147281
677

523714
n/a
n/a
TGCCTGATATTGTAATTCTT
Intron 2
90
148062
148081
678

TABLE 11

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting introns 1 and 2 of SEQ ID NO: 2

SEQ
SEQ

ID
ID

%
NO:
NO:

inhi-
2
2
SEQ

ISIS

Target
bi-
Start
Stop
ID

NO
Sequence
region
tion
Site
Site
NO

532377
CTCATACAGTGAAGTCTTCA
Intron 1
73
135431
135450
679

532378
CTCACTAAGCTTGATTCACT
Intron 1
67
135818
135837
680

532379
GATACAGAAATCCCAGTGAC
Intron 1
46
136111
136130
681

532380
TGTGCTTGGGTGTACAGGCA
Intron 1
71
136282
136301
682

532381
TCAAGCACTTACATCATATG
Intron 1
42
136377
136396
683

532382
AGGGTTAGTTATTACACTTA
Intron 1
60
136576
136595
684

532383
AGGCTTCATGTGAGGTAACA
Intron 1
58
136996
137015
685

532384
TGAAAGCTTAGTACAAGAAG
Intron 1
51
138048
138067
686

532385
CTCTCCTCTTGGAGATCCAG
Intron 1
58
138782
138801
687

532386
GCTGAGATTTCTCTCCTCTT
Intron 1
78
138792
138811
688

532387
CTTTTGCTGAGATTTCTCTC
Intron 1
58
138797
138816
689

532388
GAACATATGTCCATAGAATG
Intron 1
57
141700
141719
690

532389
GAACAGGCTATGTAATCAAA
Intron 1
68
143021
143040
691

532390
TTTTTATTACTGTGCAAACC
Intron 1
41
143878
143897
692

532391
ACTGAGGGTGGAAATGGAAA
Intron 2
23
145059
145078
693

532392
ATGCCATACTTTTCATTTCA
Intron 2
87
146351
146370
694

532393
TCTTTAAAGATTTCCTATGC
Intron 2
66
146367
146386
695

532394
TCACAATTAAATTATGTTTA
Intron 2
47
149858
149877
696

532395
TTTGCCATCACCAAACACCA
Intron 2
94
150972
150991
697

532396
TCAGAATGCTGAAGGATGGG
Intron 2
70
152208
152227
698

532397
ACAATTGCAGGAGAGAACTG
Intron 2
57
152296
152315
699

532398
GTTCAGTCACCTGGAAAGAG
Intron 2
62
152549
152568
700

532399
CGGAGTTCAGTCACCTGGAA
Intron 2
77
152553
152572
701

532400
AATCTAAAGTTCAATGTCCA
Intron 2
77
152752
152771
702

532401
CCACCTTTGGGTGAATAGCA
Intron 2
95
153921
153940
703

532402
CAACATCAAAAGTTTCCACC
Intron 2
81
153936
153955
704

532403
AAGCTTCTATCAACCAACTG
Intron 2
87
154093
154112
705

532404
ACCATTTTCTAATAATTCAC
Intron 2
46
154502
154521
706

532405
ACCTGCACTTGGACAACTGA
Intron 2
60
154727
154746
707

532406
GTCAGTGCTTTGGTGATGTA
Intron 2
11
155283
155302
708

532407
TAGAAGCACAGGAACTAGAG
Intron 2
68
155889
155908
709

532408
TTTAATTTTATTAGAAGCAC
Intron 2
14
155900
155919
710

532409
GAGCAAGAATTAAGAAAATC
Intron 2
29
155973
155992
711

532410
CTCTGCAGTCATGTACACAA
Intron 2
93
156594
156613
712

532411
GCTTGGTTTGTCAATCCTTT
Intron 2
95
156889
156908
713

532412
GTTCTCAAGCAGGAGCCATT
Intron 2
70
157330
157349
714

532413
AGGGTGATCTTCCAAAACAA
Intron 2
87
158612
158631
715

532414
TCTCCTATGCTTCCTTTAAT
Intron 2
25
158813
158832
716

532415
GACATAAATATGTTCACTGA
Intron 2
81
159216
159235
717

532416
TTACTGAGTGACAGTACAGT
Intron 2
65
161588
161607
718

532417
CCAGGCACCAGCACAGGCAC
Intron 2
47
161950
161969
719

532418
TTAATGTCAGTAGAAAGCTG
Intron 2
0
162349
162368
720

532419
GCAGGTGGAAAGAAGATGTC
Intron 2
50
162531
162550
721

532420
GCCAGGGTCTTTACAAAGTT
Intron 2
93
162751
162770
722

532421
CATTACCTTTGTACATGTAC
Intron 2
83
164839
164858
723

532422
GAAGCAACTTCTCTGAGGTC
Intron 2
68
165040
165059
724

532423
GCCTGGCAAGAAGGGCCCTT
Intron 2
56
165856
165875
725

532424
ACACATGTTTTTAAATTTAT
Intron 2
21
166241
166260
726

532425
TCACAATGCACTAAAAGAAA
Intron 2
53
168760
168779
727

532426
TCCCAATGACTTACTGTAGA
Intron 2
78
169073
169092
728

532427
TAAGCATTTATGGAGGAATG
Intron 2
46
169134
169153
729

532428
TGAGGTGGGTGGCCAACAGG
Intron 2
66
170081
170100
730

532429
GTTTTTCATTTTGATTGCAG
Intron 2
88
170158
170177
731

532430
AGCTCAAGTGTTTTTCATTT
Intron 2
64
170167
170186
732

532431
CAATGTCACAGCTGTTTCCT
Intron 2
62
170272
170291
733

532432
GAACTTTGGAGGCTTTTAGA
Intron 2
55
170703
170722
734

532433
TGTATGCCCCAAACTCCCAT
Intron 2
83
171431
171450
735

532434
ACACAAATAAGGGAATAATA
Intron 2
24
171549
171568
736

532435
TAGTTCAGCCACTATGGAAA
Intron 2
47
171926
171945
737

532436
CTCCAAATTCCAGTCCTAGG
Intron 2
93
172746
172765
738

532437
AGTTGGCACTGCTATATCAG
Intron 2
66
173668
173687
739

532438
GGCCTTAGATTGTAAGTTTT
Intron 2
69
174122
174141
740

532439
TTTTAGTATTATTGTAGGAA
Intron 2
16
174188
174207
741

532440
TTTCATTAATGAAACCTGAT
Intron 2
39
174812
174831
742

532441
CCCTCAGCTGCCTCTTCAAT
Intron 2
51
175014
175033
743

532442
TATTGTATCCTGGCCCCTAA
Intron 2
68
175689
175708
744

532443
AGAACAAGAGCCTAGAAGTA
Intron 2
35
176592
176611
745

532444
GTGACTATGTCACTGAATTT
Intron 2
14
176918
176937
746

532445
GCCCTACCCAGCAGCCTGTG
Intron 2
79
177540
177559
747

532446
CAAACATAAAGAGAGTTCCA
Intron 2
79
177811
177830
748

532447
CTTTAAATGAAGTAGAGCTC
Intron 2
0
178090
178109
749

532448
CTGTTCAAAGAATGCAGGCC
Intron 2
70
178905
178924
750

532449
GTCTAGCCTAACAGAGATAT
Intron 2
47
179137
179156
751

532450
AAAGAGTGATGTCTAGCCTA
Intron 2
55
179147
179166
752

532451
CACTTCTTACTCCTTTGAGG
Intron 2
50
179631
179650
753

532452
TTCCACAAGAAACTCAGTTT
Intron 2
56
181514
181533
754

532453
AGAAATGCCAAAGATAGCTC
Intron 2
56
182105
182124
755

TABLE 12

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting intron 2 of SEQ ID NO: 2

SEQ
SEQ

ID
ID

%
NO:
NO:

inhi-
2
2
SEQ

ISIS

bi-
Start
Stop
ID

NO
Sequence
tion
Site
Site
NO

533249
AGCAGAGGATCTCAGCTGCA
84
146241
146260
756

533250
AATCCCTGCTCAAGTGCTAC
75
147259
147278
757

533251
AAATCCCTGCTCAAGTGCTA
71
147260
147279
758

533252
AAAATCCCTGCTCAAGTGCT
73
147261
147280
759

533253
AGAAAATCCCTGCTCAAGTG
56
147263
147282
760

533254
AAGAAAATCCCTGCTCAAGT
58
147264
147283
761

533255
CAAGAAAATCCCTGCTCAAG
46
147265
147284
762

533256
CTGATATTGTAATTCTTGGT
91
148059
148078
763

533257
CCTGATATTGTAATTCTTGG
90
148060
148079
764

533258
GCCTGATATTGTAATTCTTG
94
148061
148080
765

533259
ATGCCTGATATTGTAATTCT
91
148063
148082
766

533260
AATGCCTGATATTGTAATTC
74
148064
148083
767

533261
CAATGCCTGATATTGTAATT
76
148065
148084
768

533262
AATTATGTGCTTTGCCTGCA
92
148904
148923
769

533263
CAATTATGTGCTTTGCCTGC
83
148905
148924
770

533264
TCAATTATGTGCTTTGCCTG
83
148906
148925
771

533265
TGTCAATTATGTGCTTTGCC
91
148908
148927
772

533266
ATGTCAATTATGTGCTTTGC
83
148909
148928
773

533267
GATGTCAATTATGTGCTTTG
74
148910
148929
774

533268
CTGGTGACTCTGCCTGATGA
77
151385
151404
775

533269
GCTGGTGACTCTGCCTGATG
87
151386
151405
776

533270
TGCTGGTGACTCTGCCTGAT
89
151387
151406
777

533271
GCTGCTGGTGACTCTGCCTG
94
151389
151408
778

533272
GGCTGCTGGTGACTCTGCCT
77
151390
151409
779

533273
TGGCTGCTGGTGACTCTGCC
82
151391
151410
780

533274
GCTGAAGGATGGGCATCCAG
85
152201
152220
781

533275
TGCTGAAGGATGGGCATCCA
85
152202
152221
782

533276
ATGCTGAAGGATGGGCATCC
78
152203
152222
783

533277
GAATGCTGAAGGATGGGCAT
66
152205
152224
784

533278
AGAATGCTGAAGGATGGGCA
81
152206
152225
785

533279
CAGAATGCTGAAGGATGGGC
85
152207
152226
786

533280
TCCAGTAGTCAATATTATTT
87
153001
153020
787

533281
ATCCAGTAGTCAATATTATT
85
153002
153021
788

533282
TATCCAGTAGTCAATATTAT
69
153003
153022
789

533283
GTTATCCAGTAGTCAATATT
77
153005
153024
790

533284
GGTTATCCAGTAGTCAATAT
85
153006
153025
791

533285
TGGTTATCCAGTAGTCAATA
86
153007
153026
792

533286
CAACTTGAGGACAATAAGAG
35
155591
155610
793

533287
TCAACTTGAGGACAATAAGA
62
155592
155611
794

533288
CTCAACTTGAGGACAATAAG
86
155593
155612
795

533289
AACTCAACTTGAGGACAATA
82
155595
155614
796

533290
TAACTCAACTTGAGGACAAT
66
155596
155615
797

533291
ATAACTCAACTTGAGGACAA
87
155597
155616
798

533292
CAGGAAGAAAGGAACCTTAG
77
156391
156410
799

533293
CCAGGAAGAAAGGAACCTTA
84
156392
156411
800

533294
ACCAGGAAGAAAGGAACCTT
86
156393
156412
801

533295
AGACCAGGAAGAAAGGAACC
74
156395
156414
802

533296
TAGACCAGGAAGAAAGGAAC
59
156396
156415
803

533297
ATAGACCAGGAAGAAAGGAA
65
156397
156416
804

533298
TACAATGCACAGGACACGCC
73
157198
157217
805

533299
CTACAATGCACAGGACACGC
85
157199
157218
806

533300
GCTACAATGCACAGGACACG
83
157200
157219
807

533301
ATGCTACAATGCACAGGACA
89
157202
157221
808

533302
TATGCTACAATGCACAGGAC
82
157203
157222
809

533303
ATATGCTACAATGCACAGGA
84
157204
157223
810

533304
CTGATATTTATTGCTGTACG
76
158006
158025
811

533305
CTCTGATATTTATTGCTGTA
80
158008
158027
812

533306
TCTCTGATATTTATTGCTGT
86
158009
158028
813

533307
GTCTCTGATATTTATTGCTG
80
158010
158029
814

533308
CCAGAAGAATTACCCATGCA
85
165550
165569
815

533309
TCCAGAAGAATTACCCATGC
84
165551
165570
816

533310
TTCCAGAAGAATTACCCATG
81
165552
165571
817

533311
TCTTCCAGAAGAATTACCCA
58
165554
165573
818

533312
ATCTTCCAGAAGAATTACCC
64
165555
165574
819

533313
CATCTTCCAGAAGAATTACC
58
165556
165575
820

533314
TTTCTGCAGTATCCTAGCCT
78
166350
166369
821

533315
GTTTCTGCAGTATCCTAGCC
88
166351
166370
822

533316
AGTTTCTGCAGTATCCTAGC
86
166352
166371
823

533317
TCAGTTTCTGCAGTATCCTA
88
166354
166373
824

533318
TTCAGTTTCTGCAGTATCCT
87
166355
166374
825

533319
TTTCAGTTTCTGCAGTATCC
80
166356
166375
826

533320
GTTTCCATTTTCTTGATTCC
70
169601
169620
827

533321
TGTTTCCATTTTCTTGATTC
54
169602
169621
828

533322
GTGTTTCCATTTTCTTGATT
55
169603
169622
829

533323
TGGTGTTTCCATTTTCTTGA
73
169605
169624
830

533324
ATGGTGTTTCCATTTTCTTG
76
169606
169625
831

533325
AATGGTGTTTCCATTTTCTT
78
169607
169626
832

TABLE 13

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting introns 2 and 3 of SEQ ID NO: 2

SEQ
SEQ

ID
ID

%
NO:
NO:

inhi-
2
2
SEQ

ISIS

Target
bi-
Start
Stop
ID

NO
Sequence
region
tion
Site
Site
NO

533326
AACCCATTTCATCCATTTAA
Intron 2
93
175369
175388
833

533327
GAACCCATTTCATCCATTTA
Intron 2
83
175370
175389
834

533328
GGAACCCATTTCATCCATTT
Intron 2
92
175371
175390
835

533329
TAGGAACCCATTTCATCCAT
Intron 2
91
175373
175392
836

533330
GTAGGAACCCATTTCATCCA
Intron 2
95
175374
175393
837

533331
GGTAGGAACCCATTTCATCC
Intron 2
92
175375
175394
838

533332
TGAGGGATTGCCTCAGTAGC
Intron 2
66
179616
179635
839

533333
TTGAGGGATTGCCTCAGTAG
Intron 2
72
179617
179636
840

533334
TTTGAGGGATTGCCTCAGTA
Intron 2
67
179618
179637
841

533335
CCTTTGAGGGATTGCCTCAG
Intron 2
74
179620
179639
842

533336
TCCTTTGAGGGATTGCCTCA
Intron 2
66
179621
179640
843

533337
CTCCTTTGAGGGATTGCCTC
Intron 2
76
179622
179641
844

533338
AACTTAGGACTTGGGACATT
Intron 2
64
184575
184594
845

533339
TAACTTAGGACTTGGGACAT
Intron 2
54
184576
184595
846

533340
CTAACTTAGGACTTGGGACA
Intron 2
63
184577
184596
847

533341
CACTAACTTAGGACTTGGGA
Intron 2
82
184579
184598
848

533342
TCACTAACTTAGGACTTGGG
Intron 2
77
184580
184599
849

533343
GTCACTAACTTAGGACTTGG
Intron 2
83
184581
184600
850

533344
TGGGCTAGATCAGGATTGGT
Intron 2
81
188617
188636
851

533345
ATGGGCTAGATCAGGATTGG
Intron 2
70
188618
188637
852

533346
CATGGGCTAGATCAGGATTG
Intron 2
64
188619
188638
853

533347
ACCATGGGCTAGATCAGGAT
Intron 2
82
188621
188640
854

533348
TACCATGGGCTAGATCAGGA
Intron 2
88
188622
188641
855

533349
CTACCATGGGCTAGATCAGG
Intron 2
87
188623
188642
856

533350
ATGAGCTTAGCAGTCACTTA
Intron 2
83
189482
189501
857

533351
CATGAGCTTAGCAGTCACTT
Intron 2
87
189483
189502
858

533352
CCATGAGCTTAGCAGTCACT
Intron 2
92
189484
189503
859

533353
GTCTCAGCAAACCTGGGATA
Intron 2
84
190283
190302
860

533354
TGTCTCAGCAAACCTGGGAT
Intron 2
82
190284
190303
861

533355
ATGTCTCAGCAAACCTGGGA
Intron 2
81
190285
190304
862

533356
GAATGTCTCAGCAAACCTGG
Intron 2
76
190287
190306
863

533357
GGAATGTCTCAGCAAACCTG
Intron 2
82
190288
190307
864

533358
AGGAATGTCTCAGCAAACCT
Intron 2
85
190289
190308
865

533359
TACAGACATAGCTCTAACCT
Intron 2
79
191139
191158
866

533360
ATACAGACATAGCTCTAACC
Intron 2
79
191140
191159
867

533361
GATACAGACATAGCTCTAAC
Intron 2
71
191141
191160
868

533362
TGGATACAGACATAGCTCTA
Intron 2
79
191143
191162
869

533363
CTGGATACAGACATAGCTCT
Intron 2
82
191144
191163
870

533364
GCTGGATACAGACATAGCTC
Intron 2
95
191145
191164
871

533365
ACACTGTTTGTGAGGGTCAA
Intron 2
87
191939
191958
872

533366
AACACTGTTTGTGAGGGTCA
Intron 2
81
191940
191959
873

533367
CAACACTGTTTGTGAGGGTC
Intron 2
85
191941
191960
874

533368
AACAACACTGTTTGTGAGGG
Intron 2
65
191943
191962
875

533369
AAACAACACTGTTTGTGAGG
Intron 2
76
191944
191963
876

533370
CAAACAACACTGTTTGTGAG
Intron 2
67
191945
191964
877

533371
TTCAAGTTTAGGATCTGCAG
Intron 2
73
196536
196555
878

533372
CTTCAAGTTTAGGATCTGCA
Intron 2
88
196537
196556
879

533373
GCTTCAAGTTTAGGATCTGC
Intron 2
86
196538
196557
880

533374
GGGCTTCAAGTTTAGGATCT
Intron 2
67
196540
196559
881

533375
AGGGCTTCAAGTTTAGGATC
Intron 2
66
196541
196560
882

533376
CAGGGCTTCAAGTTTAGGAT
Intron 2
74
196542
196561
883

533377
TGTGGCTTTAATTCACTAAT
Intron 2
84
198145
198164
884

533378
ATGTGGCTTTAATTCACTAA
Intron 2
86
198146
198165
885

533379
TATGTGGCTTTAATTCACTA
Intron 2
79
198147
198166
886

533380
GGTATGTGGCTTTAATTCAC
Intron 2
83
198149
198168
887

533381
TGGTATGTGGCTTTAATTCA
Intron 2
81
198150
198169
888

533382
GTGGTATGTGGCTTTAATTC
Intron 2
86
198151
198170
889

533383
TCTGTGTTCAGTTGCATCAC
Intron 2
75
199817
199836
890

533384
TTCTGTGTTCAGTTGCATCA
Intron 2
82
199818
199837
891

533385
GTTCTGTGTTCAGTTGCATC
Intron 2
86
199819
199838
892

533386
GTACTCATGAGGAGGCACTT
Intron 2
81
201413
201432
893

533387
GGTACTCATGAGGAGGCACT
Intron 2
82
201414
201433
894

533388
TGGTACTCATGAGGAGGCAC
Intron 2
78
201415
201434
895

533389
ATTGGTACTCATGAGGAGGC
Intron 2
64
201417
201436
896

533390
AATTGGTACTCATGAGGAGG
Intron 2
47
201418
201437
897

533391
CAATTGGTACTCATGAGGAG
Intron 2
54
201419
201438
898

533392
AAACTCTGCAACTCCAACCC
Intron 2
69
205549
205568
899

533393
GAAACTCTGCAACTCCAACC
Intron 2
64
205550
205569
900

533394
GGAAACTCTGCAACTCCAAC
Intron 2
83
205551
205570
901

533395
ATGGAAACTCTGCAACTCCA
Intron 2
88
205553
205572
902

533396
CATGGAAACTCTGCAACTCC
Intron 2
70
205554
205573
903

533397
TCATGGAAACTCTGCAACTC
Intron 2
69
205555
205574
904

533398
ACATCTGGATGTGAGGCTCG
Intron 3
64
210559
210578
905

533399
CACATCTGGATGTGAGGCTC
Intron 3
84
210560
210579
906

533400
GTCACATCTGGATGTGAGGC
Intron 3
75
210562
210581
907

533401
TGTCACATCTGGATGTGAGG
Intron 3
51
210563
210582
908

533402
CTGTCACATCTGGATGTGAG
Intron 3
30
210564
210583
909

TABLE 14

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting introns 2 and 3 of SEQ ID NO: 2

SEQ
SEQ

ID
ID

%
NO:
NO:

inhi-
2
2
SEQ

ISIS

Target
bi-
Start
Stop
ID

NO
Sequence
region
tion
Site
Site
NO

523715
GTCAATTATGTGCTTTGCCT
Intron 2
91
148907
148926
910

523716
ACATTCAAAATTCTTCCTTG
Intron 2
50
149787
149806
911

523717
ATCCTGCATATATTTTATTG
Intron 2
20
150588
150607
912

523718
CTGCTGGTGACTCTGCCTGA
Intron 2
77
151388
151407
913

523719
AATGCTGAAGGATGGGCATC
Intron 2
66
152204
152223
914

523720
TTATCCAGTAGTCAATATTA
Intron 2
71
153004
153023
915

523721
TCTCATGTTAAAGTTCTTAA
Intron 2
48
153831
153850
916

523722
TGCACTTGGACAACTGATAG
Intron 2
29
154724
154743
917

523723
ACTCAACTTGAGGACAATAA
Intron 2
88
155594
155613
918

523724
GACCAGGAAGAAAGGAACCT
Intron 2
72
156394
156413
919

523725
TGCTACAATGCACAGGACAC
Intron 2
80
157201
157220
920

523726
TCTGATATTTATTGCTGTAC
Intron 2
73
158007
158026
921

523727
ATGCTTCCTTTAATAAATGT
Intron 2
0
158807
158826
922

523728
AACATTTAGAACCTAGGAGA
Intron 2
20
159610
159629
923

523729
CAAGCTTGCAAGTAGGAAAA
Intron 2
51
160410
160429
924

523730
CCAGGCTGTTCATGCCAAGG
Intron 2
26
161248
161267
925

523731
CCTGCCAAGGGCAAGCCAGG
Intron 2
17
162064
162083
926

523732
TTTCACCTGGTGACTGGAAG
Intron 2
51
163019
163038
927

523733
ATTTTCTACCATCAAAGAGA
Intron 2
4
163943
163962
928

523734
GATTAAGTTTTCTTTAAAAA
Intron 2
0
164746
164765
929

523735
CTTCCAGAAGAATTACCCAT
Intron 2
56
165553
165572
930

523736
CAGTTTCTGCAGTATCCTAG
Intron 2
77
166353
166372
931

523737
TATTTTGAAAATGAGATTCA
Intron 2
0
167195
167214
932

523738
GTGGCCCGAGTAAAGATAAA
Intron 2
21
167995
168014
933

523739
CCTGTCAATCCTCTTATATG
Intron 2
37
168804
168823
934

523740
GGTGTTTCCATTTTCTTGAT
Intron 2
65
169604
169623
935

523741
ACAGGGTCAAAAGTTCACTT
Intron 2
44
170407
170426
936

523742
TAGGAAAGCTGAGAGAATCC
Intron 2
35
171207
171226
937

523743
AGCATATGAAAAAATACTCA
Intron 2
0
172101
172120
938

523744
CTTCAGAAATCAGCATCTGA
Intron 2
45
172937
172956
939

523745
TTACAAGTGACAGTGTTTGT
Intron 2
28
173737
173756
940

523746
ATCAGACCCTGAAGAATTTA
Intron 2
29
174560
174579
941

523747
AGGAACCCATTTCATCCATT
Intron 2
83
175372
175391
942

523748
CACATTGGTAACTTAAAGTT
Intron 2
18
176263
176282
943

523749
TATTATCTGACTCATTTCTG
Intron 2
16
177072
177091
944

523750
AAATAAGACAAAGAAAATTC
Intron 2
0
177872
177891
945

523751
TTTTAAAAATAACCAATTCA
Intron 2
0
178788
178807
946

523752
CTTTGAGGGATTGCCTCAGT
Intron 2
66
179619
179638
947

523753
ACAGTCCTCATGAACAGATT
Intron 2
37
180513
180532
948

523754
ACTATCATTAATAATATTGT
Intron 2
0
181323
181342
949

523755
ATCTAGATTTGCCTTATAAG
Intron 2
27
182123
182142
950

523756
TGGTTGAGGAAGACAGTCTC
Intron 2
16
182962
182981
951

523757
TGGCTCATAACTTCCTTAGC
Intron 2
43
183762
183781
952

523758
ACTAACTTAGGACTTGGGAC
Intron 2
72
184578
184597
953

523759
CTTATAGCATTACTAAGTGG
Intron 2
49
185403
185422
954

523760
TGGTGGCAGGAGAGAGGGAA
Intron 2
48
186203
186222
955

523761
TTTGCCAGGAAATCTTGAAA
Intron 2
35
187003
187022
956

523762
ATAACTTTTCTCTGAAATTT
Intron 2
8
187803
187822
957

523763
CCATGGGCTAGATCAGGATT
Intron 2
59
188620
188639
958

523764
TGAGCTTAGCAGTCACTTAG
Intron 2
62
189481
189500
959

523765
AATGTCTCAGCAAACCTGGG
Intron 2
62
190286
190305
960

523766
GGATACAGACATAGCTCTAA
Intron 2
75
191142
191161
961

523767
ACAACACTGTTTGTGAGGGT
Intron 2
66
191942
191961
962

523768
TCTATTTTCTAATAGCTGTT
Intron 2
49
192742
192761
963

523769
GGCCCCACCTCTGACCTTCA
Intron 2
7
193542
193561
964

523770
TGGTAAAGCTAGAAAAAAAA
Intron 2
0
194346
194365
965

523771
AAGTGGTAAATATGATCACA
Intron 2
23
195159
195178
966

523772
GGCTTCAAGTTTAGGATCTG
Intron 2
52
196539
196558
967

523773
TTGTTGACACTCTCTTTTGG
Intron 2
18
197348
197367
968

523774
GTATGTGGCTTTAATTCACT
Intron 2
71
198148
198167
969

523775
AATTAGTTGTTTTGGCAAAT
Intron 2
14
198988
199007
970

523776
CTGTGTTCAGTTGCATCACG
Intron 2
75
199816
199835
971

523777
AATGTGGAAGTTTCCTAACA
Intron 2
15
200616
200635
972

523778
TTGGTACTCATGAGGAGGCA
Intron 2
58
201416
201435
973

523779
TTTCTCTGTGTTTAAAATTG
Intron 2
13
202308
202327
974

523780
GTAAAGCACAATGAACAAAA
Intron 2
21
203115
203134
975

523781
ATCACAGATCTTTGCTACAA
Intron 2
51
203915
203934
976

523782
TCCTGCCTTTCTGAACCAAA
Intron 2
50
204721
204740
977

523783
TGGAAACTCTGCAACTCCAA
Intron 2
58
205552
205571
978

523784
ACACAGTAGGGAACAATTTT
Intron 2
8
206412
206431
979

523785
AGACAGATGGTGAAATGATG
Intron 2
0
207219
207238
980

523786
AAACAGAAAGAGAAGAAAAC
Intron 2
0
208117
208136
981

523787
CTTAGATAAATACTTCAAGA
Intron 3
0
208938
208957
982

523788
AGCCACTTCTTTTACAACCT
Intron 3
0
209742
209761
983

523789
TCACATCTGGATGTGAGGCT
Intron 3
80
210561
210580
984

523790
GACTGAAACTTAAAGGTGGG
Intron 3
7
211399
211418
985

523791
AAAGATGTGCAATCATCTAA
Intron 3
44
212204
212223
986

TABLE 15

Inhibition of GHR mRNA by 3-10-4 MOE gapmers

targeting introns 2 and 3 of SEQ ID NO: 2

SEQ
SEQ

ID
ID

%
NO:
NO:

inhi-
2
2
SEQ

ISIS

Target
bi-
Start
Stop
ID

NO
Sequence
region
tion
Site
Site
NO

539360
GCTGGTGACTCTGCCTG
Intron 2
95
151389
151405
987

539361
TGCTGGTGACTCTGCCT
Intron 2
95
151390
151406
988

539362
CTGCTGGTGACTCTGCC
Intron 2
93
151391
151407
989

539363
AGTAGTCAATATTATTT
Intron 2
31
153001
153017
990

539364
CAGTAGTCAATATTATT
Intron 2
13
153002
153018
991

539365
CCAGTAGTCAATATTAT
Intron 2
34
153003
153019
992

539366
CCTTTGGGTGAATAGCA
Intron 2
64
153921
153937
993

539367
ACCTTTGGGTGAATAGC
Intron 2
78
153922
153938
994

539368
CACCTTTGGGTGAATAG
Intron 2
40
153923
153939
995

539369
CAACTTGAGGACAATAA
Intron 2
38
155594
155610
996

539370
TCAACTTGAGGACAATA
Intron 2
63
155595
155611
997

539371
CTCAACTTGAGGACAAT
Intron 2
81
155596
155612
998

539372
CAGGAAGAAAGGAACCT
Intron 2
70
156394
156410
999

539373
CCAGGAAGAAAGGAACC
Intron 2
59
156395
156411
1000

539374
ACCAGGAAGAAAGGAAC
Intron 2
43
156396
156412
1001

539375
TGCAGTCATGTACACAA
Intron 2
93
156594
156610
1002

539376
CTGCAGTCATGTACACA
Intron 2
91
156595
156611
1003

539377
TCTGCAGTCATGTACAC
Intron 2
87
156596
156612
1004

539378
TGGTTTGTCAATCCTTT
Intron 2
95
156889
156905
1005

539379
TTGGTTTGTCAATCCTT
Intron 2
97
156890
156906
1006

539380
CTTGGTTTGTCAATCCT
Intron 2
97
156891
156907
1007

539381
TACAATGCACAGGACAC
Intron 2
65
157201
157217
1008

539382
CTACAATGCACAGGACA
Intron 2
85
157202
157218
1009

539383
GCTACAATGCACAGGAC
Intron 2
96
157203
157219
1010

539384
GATATTTATTGCTGTAC
Intron 2
43
158007
158023
1011

539385
TGATATTTATTGCTGTA
Intron 2
35
158008
158024
1012

539386
CTGATATTTATTGCTGT
Intron 2
38
158009
158025
1013

539387
AGGGTCTTTACAAAGTT
Intron 2
61
162751
162767
1014

539388
CAGGGTCTTTACAAAGT
Intron 2
65
162752
162768
1015

539389
CCAGGGTCTTTACAAAG
Intron 2
88
162753
162769
1016

539390
TTCTGCAGTATCCTAGC
Intron 2
72
166352
166368
1017

539391
TTTCTGCAGTATCCTAG
Intron 2
53
166353
166369
1018

539392
GTTTCTGCAGTATCCTA
Intron 2
84
166354
166370
1019

539393
AGTTTCTGCAGTATCCT
Intron 2
78
166355
166371
1020

539394
CAGTTTCTGCAGTATCC
Intron 2
77
166356
166372
1021

539395
CAAATTCCAGTCCTAGG
Intron 2
60
172746
172762
1022

539396
CCAAATTCCAGTCCTAG
Intron 2
75
172747
172763
1023

539397
TCCAAATTCCAGTCCTA
Intron 2
62
172748
172764
1024

539398
AACCCATTTCATCCATT
Intron 2
82
175372
175388
1025

539399
GAACCCATTTCATCCAT
Intron 2
86
175373
175389
1026

539400
GGAACCCATTTCATCCA
Intron 2
84
175374
175390
1027

539401
GCTTCATGTCTTTCTAG
Intron 2
88
189119
189135
1028

539402
TGCTTCATGTCTTTCTA
Intron 2
77
189120
189136
1029

539403
GTGCTTCATGTCTTTCT
Intron 2
95
189121
189137
1030

539404
TGAGCTTAGCAGTCACT
Intron 2
92
189484
189500
1031

539405
CATGAGCTTAGCAGTCA
Intron 2
82
189486
189502
1032

539406
TACAGACATAGCTCTAA
Intron 2
45
191142
191158
1033

539407
ATACAGACATAGCTCTA
Intron 2
53
191143
191159
1034

539408
GATACAGACATAGCTCT
Intron 2
67
191144
191160
1035

539409
TGTGGCTTTAATTCACT
Intron 2
70
198148
198164
1036

539410
ATGTGGCTTTAATTCAC
Intron 2
40
198149
198165
1037

539411
TATGTGGCTTTAATTCA
Intron 2
35
198150
198166
1038

539412
TGTTCAGTTGCATCACG
Intron 2
84
199816
199832
1039

539413
GTGTTCAGTTGCATCAC
Intron 2
80
199817
199833
1040

539414
TGTGTTCAGTTGCATCA
Intron 2
74
199818
199834
1041

539415
CATCTGGATGTGAGGCT
Intron 3
82
210561
210577
1042

539416
ACATCTGGATGTGAGGC
Intron 3
86
210562
210578
1043

539417
CACATCTGGATGTGAGG
Intron 3
55
210563
210579
1044

539418
TCAGGTAATTTCTGGAA
Intron 3
35
219019
219035
1045

539419
CTCAGGTAATTTCTGGA
Intron 3
44
219020
219036
1046

539420
TCTCAGGTAATTTCTGG
Intron 3
31
219021
219037
1047

539421
TTGCTTATTTACCTGGG
Intron 3
0
225568
225584
1048

539422
TTTGCTTATTTACCTGG
Intron 3
38
225569
225585
1049

539423
TTTTGCTTATTTACCTG
Intron 3
33
225570
225586
1050

539424
ATGATGTTACTACTACT
Intron 3
29
229618
229634
1051

539425
AATGATGTTACTACTAC
Intron 3
10
229619
229635
1052

539426
CAATGATGTTACTACTA
Intron 3
0
229620
229636
1053

539427
CCCCTAGAGCAATGGTC
Intron 3
67
232826
232842
1054

539428
CCCCCTAGAGCAATGGT
Intron 3
65
232827
232843
1055

539429
TCCCCCTAGAGCAATGG
Intron 3
45
232828
232844
1056

539430
TCAATTGCAGATGCTCT
Intron 3
78
237675
237691
1057

539431
CTCAATTGCAGATGCTC
Intron 3
82
237676
237692
1058

539432
GCTCAATTGCAGATGCT
Intron 3
92
237677
237693
1059

539433
AGCTCAATTGCAGATGC
Intron 3
85
237678
237694
1060

539434
GTATATTCAGTCCAAGG
Intron 3
73
248231
248247
1061

539435
AGTATATTCAGTCCAAG
Intron 3
70
248232
248248
1062

539436
CAGTATATTCAGTCCAA
Intron 3
40
248233
248249
1063

TABLE 16

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting introns 1 and 3 of SEQ ID NO: 2

ISIS

Target
%
SEQ ID NO: 2
SEQ ID NO: 2
SEQ

NO
Sequence
region
inhibition
Start Site
Stop Site
ID NO

532502
GAGTATTTCAGGCTGGAAAA
Intron 3
43
214623
214642
1064

533404
GTAACTCAGGAATGGAAAAC
Intron 1
56
26501
26520
1065

113035
113054

121992
122011

533405
AGTAACTCAGGAATGGAAAA
Intron 1
41
26502
26521
1066

113036
113055

121993
122012

533406
AAGTAACTCAGGAATGGAAA
Intron 1
43
26503
26522
1067

113037
113056

121994
122013

533407
GAGATTTCAAATAAATCTCA
Intron 1
0
143207
143226
1068

143235
143254

143263
143282

143291
143310

143319
143338

143347
143366

143375
143394

143403
143422

143431
143450

143459
143478

533408
TGAGATTTCAAATAAATCTC
Intron 1
11
143208
143227
1069

143236
143255

143264
143283

143292
143311

143320
143339

143348
143367

143376
143395

143404
143423

143432
143451

143460
143479

533409
GTGAGATTTCAAATAAATCT
Intron 1
0
143209
143228
1070

143237
143256

143265
143284

143293
143312

143321
143340

143349
143368

143377
143396

143405
143424

143433
143452

143461
143480

533410
TGTGAGATTTCAAATAAATC
Intron 1
0
143210
143229
1071

143238
143257

143266
143285

143294
143313

143322
143341

143350
143369

143378
143397

143406
143425

143434
143453

143462
143481

533411
TTGTGAGATTTCAAATAAAT
Intron 1
10
143183
143202
1072

143211
143230

143239
143258

143267
143286

143295
143314

143323
143342

143351
143370

143379
143398

143407
143426

143435
143454

143463
143482

533412
TTTGTGAGATTTCAAATAAA
Intron 1
0
143184
143203
1073

143212
143231

143240
143259

143296
143315

143324
143343

143352
143371

143380
143399

143464
143483

533413
CTTTGTGAGATTTCAAATAA
Intron 1
20
143185
143204
1074

143213
143232

143241
143260

143297
143316

143325
143344

143353
143372

143381
143400

143465
143484

533414
ACTTTGTGAGATTTCAAATA
Intron l
57
143186
143205
1075

143214
143233

143242
143261

143298
143317

143326
143345

143354
143373

143382
143401

143466
143485

533415
CACTTTGTGAGATTTCAAAT
Intron 1
69
143187
143206
1076

143215
143234

143243
143262

143299
143318

143327
143346

143355
143374

143383
143402

143467
143486

533895
AGTATTTCAGGCTGGAAAAA
Intron 3
35
214622
214641
1077

533896
TGAGTATTTCAGGCTGGAAA
Intron 3
55
214624
214643
1078

533897
TCTGAGTATTTCAGGCTGGA
Intron 3
71
214626
214645
1079

533898
ATCTGAGTATTTCAGGCTGG
Intron 3
77
214627
214646
1080

533899
TATCTGAGTATTTCAGGCTG
Intron 3
58
214628
214647
1081

533900
TTTTGTGTTATGCCTTGAGG
Intron 3
51
221483
221502
1082

533901
TTTTTGTGTTATGCCTTGAG
Intron 3
55
221484
221503
1083

533902
ATTTTTGTGTTATGCCTTGA
Intron 3
57
221485
221504
1084

533903
ATATTTTTGTGTTATGCCTT
Intron 3
56
221487
221506
1085

533904
AATATTTTTGTGTTATGCCT
Intron 3
61
221488
221507
1086

533905
AAATATTTTTGTGTTATGCC
Intron 3
18
221489
221508
1087

533906
TTGCTTATTTACCTGGGTAA
Intron 3
58
225565
225584
1088

533907
TTTGCTTATTTACCTGGGTA
Intron 3
64
225566
225585
1089

533908
TTTTGCTTATTTACCTGGGT
Intron 3
77
225567
225586
1090

533909
CCTTTTGCTTATTTACCTGG
Intron 3
69
225569
225588
1091

533910
GCCTTTTGCTTATTTACCTG
Intron 3
69
225570
225589
1092

533911
TGCCTTTTGCTTATTTACCT
Intron 3
55
225571
225590
1093

533912
ATGATGTTACTACTACTCAA
Intron 3
60
229615
229634
1094

533913
AATGATGTTACTACTACTCA
Intron 3
48
229616
229635
1095

533914
CAATGATGTTACTACTACTC
Intron 3
57
229617
229636
1096

533915
TCCAATGATGTTACTACTAC
Intron 3
69
229619
229638
1097

533916
TTCCAATGATGTTACTACTA
Intron 3
74
229620
229639
1098

533917
ATTCCAATGATGTTACTACT
Intron 3
74
229621
229640
1099

533918
CCCCTAGAGCAATGGTCTAG
Intron 3
71
232823
232842
1100

533919
CCCCCTAGAGCAATGGTCTA
Intron 3
44
232824
232843
1101

533920
TCCCCCTAGAGCAATGGTCT
Intron 3
54
232825
232844
1102

533921
TATCCCCCTAGAGCAATGGT
Intron 3
62
232827
232846
1103

533922
ATATCCCCCTAGAGCAATGG
Intron 3
50
232828
232847
1104

533923
AATATCCCCCTAGAGCAATG
Intron 3
61
232829
232848
1105

533924
GCTCACATTTGGAAGACAGT
Intron 3
68
233623
233642
1106

533925
GGCTCACATTTGGAAGACAG
Intron 3
74
233624
233643
1107

533926
AGGCTCACATTTGGAAGACA
Intron 3
56
233625
233644
1108

533927
AGAGGCTCACATTTGGAAGA
Intron 3
34
233627
233646
1109

533928
TAGAGGCTCACATTTGGAAG
Intron 3
18
233628
233647
1110

533929
TTAGAGGCTCACATTTGGAA
Intron 3
19
233629
233648
1111

533930
CTCAATTGCAGATGCTCTGA
Intron 3
66
237673
237692
1112

533931
GCTCAATTGCAGATGCTCTG
Intron 3
72
237674
237693
1113

533932
AGCTCAATTGCAGATGCTCT
Intron 3
74
237675
237694
1114

533933
AAAGCTCAATTGCAGATGCT
Intron 3
66
237677
237696
1115

533934
TAAAGCTCAATTGCAGATGC
Intron 3
59
237678
237697
1116

533935
ATAAAGCTCAATTGCAGATG
Intron 3
23
237679
237698
1117

533936
GTGAGTCCATTAAACCTCTT
Intron 3
73
244873
244892
1118

533937
TGTGAGTCCATTAAACCTCT
Intron 3
73
244874
244893
1119

533938
ACTGTGAGTCCATTAAACCT
Intron 3
17
244876
244895
1120

533939
AACTGTGAGTCCATTAAACC
Intron 3
19
244877
244896
1121

533940
GAACTGTGAGTCCATTAAAC
Intron 3
28
244878
244897
1122

533941
ATATTGAAAGGCCCATCAAA
Intron 3
13
246498
246517
1123

533942
AATATTGAAAGGCCCATCAA
Intron 3
31
246499
246518
1124

533943
AAATATTGAAAGGCCCATCA
Intron 3
51
246500
246519
1125

533944
GAAAATATTGAAAGGCCCAT
Intron 3
22
246502
246521
1126

533945
GGAAAATATTGAAAGGCCCA
Intron 3
42
246503
246522
1127

533946
AGGAAAATATTGAAAGGCCC
Intron 3
28
246504
246523
1128

533947
GTATATTCAGTCCAAGGATC
Intron 3
65
248228
248247
1129

533948
AGTATATTCAGTCCAAGGAT
Intron 3
63
248229
248248
1130

533949
CAGTATATTCAGTCCAAGGA
Intron 3
67
248230
248249
1131

533950
AACAGTATATTCAGTCCAAG
Intron 3
56
248232
248251
1132

533951
AAACAGTATATTCAGTCCAA
Intron 3
60
248233
248252
1133

533952
AAAACAGTATATTCAGTCCA
Intron 3
59
248234
248253
1134

533953
TCTATTGTTGCCACCTTTAT
Intron 3
45
252838
252857
1135

533954
TTCTATTGTTGCCACCTTTA
Intron 3
52
252839
252858
1136

533955
TTTCTATTGTTGCCACCTTT
Intron 3
46
252840
252859
1137

533956
AGTTTCTATTGTTGCCACCT
Intron 3
59
252842
252861
1138

533957
CAGTTTCTATTGTTGCCACC
Intron 3
41
252843
252862
1139

533958
CCAGTTTCTATTGTTGCCAC
Intron 3
48
252844
252863
1140

TABLE 17

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting intron 3 of SEQ ID NO: 2

ISIS

%
SEQ ID NO: 2
SEQ ID NO: 2
SEQ

NO
Sequence
inhibition
Start Site
Stop Site
ID NO

532454
GCAGAACTGATTGCTTACTT
78
182862
182881
1141

532455
AGGTCATAAGATTTTCATTT
48
183533
183552
1142

532456
GCCTCTGGCCATAAAGAAAT
54
183578
183597
1143

532457
AAAGTTTAAGAGGCACCCCA
31
184508
184527
1144

532458
GAATAAGCACAAAAGTTTAA
28
184519
184538
1145

532459
GAACCAAATAAACCTCTCTT
52
185452
185471
1146

532460
ATGTTGAAATTTGATCCCCA
79
185763
185782
1147

532461
TGTGAGAGCTCACTCACTAT
42
186134
186153
1148

532462
CTTGTGAGAGCTCACTCACT
72
186136
186155
1149

532463
ACATGGTGGCAGGAGAGAGG
42
186206
186225
1150

532464
CTAGAAAGAAACTACCTGAG
12
186341
186360
1151

532465
AACTTCAGTTGTAAAATAAT
27
187044
187063
1152

532466
GAAAAGGATTTTGAGATTTC
43
188897
188916
1153

532467
CTTAGCTGTCAAGGCCCTTT
80
189084
189103
1154

532468
TGTGCTTCATGTCTTTCTAG
88
189119
189138
1155

532469
CCCTTGAACATGCTATCCTT
85
189256
189275
1156

532470
CTTGCAGGGATGCATCTCAG
87
189625
189644
1157

532471
TCTCTTGCACATCTAATTTC
82
189656
189675
1158

532472
CTTCCAGCACAACCCATCAC
77
190109
190128
1159

532473
GTAACTACATTCCCTTTATC
52
190860
190879
1160

532474
AGTAACTACATTCCCTTTAT
58
190861
190880
1161

532475
CAGATAGCACAGGGCTAAAA
84
190979
190998
1162

532476
AGAATCAGGAATGTTTGCCT
86
192904
192923
1163

532477
TGACTCAATCATTTAGACTT
45
192990
193009
1164

532478
TCAACAGTCAATGGACTTGT
71
193042
193061
1165

532479
AATTTCTACTGCTATGATGC
75
194806
194825
1166

532480
ATGGTTCCAAATTTCTATCT
86
195704
195723
1167

532481
CTGTATGGCTTTAAGTATTC
63
196756
196775
1168

532482
AACTTATGAACTGTTCACCA
86
198307
198326
1169

532483
AATAAGCTTGAAGTCTGAAG
63
199520
199539
1170

532484
TAGTTATCTAACTGCCCAAT
77
199544
199563
1171

532485
TTCTGCAAAGCTTCCCAGTA
72
200314
200333
1172

532486
ACAACTTCAAGCTTCACATA
65
200599
200618
1173

532487
GAATCAATGTTCTGGCAAGA
52
201842
201861
1174

532488
CAGCCTTTCAGCTGTGAAAG
52
204181
204200
1175

532489
AACAATGCCAAGAAATCTAT
74
204369
204388
1176

532490
CCCACAGTAACAATGCCAAG
90
204377
204396
1177

532491
TTTTACCTCCCAGTGAAACT
34
205896
205915
1178

532492
TAATTGTTGATCCATGATGT
5
208856
208875
1179

532493
GTTGGAGAGACAAGTTTAAC
29
208975
208994
1180

532494
AGTCATAAAATTCAAATTAT
39
209537
209556
1181

532495
GGCCTTGGGCACACTTTCTC
82
207510
207529
1182

210189
210208

532496
AAGTTTTTATTGAAGTTAAT
0
212551
212570
1183

532497
AAGAAAAATTAGGAAGCTAG
31
212649
212668
1184

532498
CAGGGAGATAAGTTTATTCA
61
212797
212816
1185

532499
ATTTAATACACATTGGAATA
15
213390
213409
1186

532500
GTAGGACTATTTATGATTCC
86
213914
213933
1187

532501
CACTCTCTTGGGCTGTTAAG
82
214479
214498
1188

532502
GAGTATTTCAGGCTGGAAAA
66
214623
214642
1064

532503
TTGTTTGAGTTCCAAAAGAA
39
214932
214951
1189

532504
TTTGCCATGAGACACACAAT
77
215932
215951
1190

532505
CACCAAACCTCAGAGACATG
80
216468
216487
1191

532506
CCACTGTTAAGTGATGCATG
83
217480
217499
1192

532507
CTCTCAGGTAATTTCTGGAA
86
219019
219038
1193

532508
GCTCCTCACAATGACCCTTT
84
219452
219471
1194

532509
GGGACTGGCACTGGTAATTT
56
220062
220081
1195

532510
CTAACCATTAGTTACTGTAT
69
220558
220577
1196

532511
GGATTTTAGGTTCTTGCTGT
51
221588
221607
1197

532512
TGAATCATATACTGATATCA
63
222914
222933
1198

532513
TTGAGGTATTAAATTTTAAA
0
223001
223020
1199

532514
AGTTTGTAATGTAGTGATTT
19
223156
223175
1200

532515
AAATATTTGATAGCTCACAT
18
224409
224428
1201

532516
AGAAATATTTGATAGCTCAC
57
224411
224430
1202

532517
CCACATTTCAAATGTTCTCT
80
224717
224736
1203

532518
GCAGGAAGAGTGGCATGGAC
59
224750
224769
1204

532519
CACTTATCCAAATGCAGAGA
82
225742
225761
1205

532520
CAAGGTAATGGGAGGCTAGC
47
225903
225922
1206

532521
ATAGTCAAAGCTAAGGATAT
4
226177
226196
1207

532522
GTAATTTCATTCATGCTTCC
67
226804
226823
1208

532523
GTCCACATTCAGCTGTGTGT
72
231912
231931
1209

532524
TCATTCAGGAAATTCTGCTA
62
232286
232305
1210

532525
AACATGTCTCATTCAGGAAA
71
232294
232313
1211

532526
TAACATGTCTCATTCAGGAA
85
232295
232314
1212

532527
AGATTCCTCAAATTCAGTGA
66
232389
232408
1213

532528
TAAGCGGAAAAGGAGAAAAG
0
233684
233703
1214

532529
AAAGCAAGAGAATTCCTAAA
32
234203
234222
1215

532530
AATGAACCTTTAACTTAGTA
40
234876
234895
1216

TABLE 18

Inhibition of GHR mRNA by 5-10-5 MOE gapmers targeting

introns 3-8 and intron-exonic regions of SEQ ID NO: 2

ISIS

%
SEQ ID NO: 2
SEQ ID NO: 2
SEQ

NO
Sequence
Target region
inhibition
Start Site
Stop Site
ID NO

523792
AAAGCTTTGTGGATAAAGTT
Intron 3
44
213025
213044
1217

523793
GAAGGAAAGGTTCTGTGGAA
Intron 3
38
213825
213844
1218

523794
CTGAGTATTTCAGGCTGGAA
Intron 3
84
214625
214644
1219

523795
TTGAATTATCCCTTTAAAAA
Intron 3
38
215446
215465
1220

523796
TTTAGAATGGTTTGGCATAC
Intron 3
66
216365
216384
1221

523797
GATATGTCCACATTGATTAG
Intron 3
65
218132
218151
1222

523798
ATTATTTAAGCTTCTACTTT
Intron 3
44
218973
218992
1223

523799
ATACATGGCAATTAAAAGAT
Intron 3
26
219886
219905
1224

523800
TGAGATAGTGTGGGAAATAT
Intron 3
18
220686
220705
1225

523801
TATTTTTGTGTTATGCCTTG
Intron 3
73
221486
221505
1226

523802
TTATTAACTAGAATATGCCT
Intron 3
16
223110
223129
1227

523803
GATTATTCTATTTTTATTTT
Intron 3
33
223948
223967
1228

523804
AGGAAGAGTGGCATGGACAT
Intron 3
43
224748
224767
1229

523805
CTTTTGCTTATTTACCTGGG
Intron 3
84
225568
225587
1230

523806
TTTATATTATTAATATCATT
Intron 3
31
226371
226390
1231

523807
GGTACATGGCTTTTAAGTGG
Intron 3
53
227218
227237
1232

523808
AATATTGGTCAGGTTTAAGA
Intron 3
28
228018
228037
1233

523809
ATTTCATCTCTTTCTTAGTT
Intron 3
45
228818
228837
1234

523810
CCAATGATGTTACTACTACT
Intron 3
89
229618
229637
1235

523811
GTTCCCCCAACCCCTTGGAA
Intron 3
28
230418
230437
1236

523812
TATAGGAAGTGAGATGTATG
Intron 3
46
231218
231237
1237

523813
ATTATTCTAGAAGAAGATTT
Intron 3
12
232018
232037
1238

523814
ATCCCCCTAGAGCAATGGTC
Intron 3
79
232826
232845
1239

523815
GAGGCTCACATTTGGAAGAC
Intron 3
69
233626
233645
1240

523816
TACACAAATCCAAGGCAGAG
Intron 3
57
234447
234466
1241

523817
AGGAAGAGTGGGAGTGTTAC
Intron 3
35
235258
235277
1242

523818
GTCCCTGACTAGGCATTTTG
Intron 3
43
236071
236090
1243

523819
AAGCTCAATTGCAGATGCTC
Intron 3
80
237676
237695
1244

523820
CTGTGAGTCCATTAAACCTC
Intron 3
81
244875
244894
1245

523821
TGAAATGTGGCTAGTGTGAC
Intron 3
51
245701
245720
1246

523822
AAAATATTGAAAGGCCCATC
Intron 3
68
246501
246520
1247

523823
AATGTCAATAGTGCCCTATT
Intron 3
48
247431
247450
1248

523824
ACAGTATATTCAGTCCAAGG
Intron 3
82
248231
248250
1249

523825
TGTCTATTTAAGTTTGTTGC
Intron 3
45
250001
250020
1250

523826
TTCAAGTACTGTCATGAATA
Intron 3
47
251214
251233
1251

523827
TTTCTTTTTCTTAAACTAAG
Intron 3
11
252041
252060
1252

523828
GTTTCTATTGTTGCCACCTT
Intron 3
70
252841
252860
1253

523829
AAGGCCACATATTATAGTAT
Intron 3
29
253698
253717
1254

523830
ACCTGAACTATTAATTTCTT
Intron 3
19
255397
255416
1255

523831
GAATGGGCTGAGTAGTTGAA
Intron 3
47
256197
256216
1256

523832
TGATGAACATTGCTAATTTG
Intron 3
26
257018
257037
1257

523833
ATCTTGCCTCGATGAAAGTT
Intron 3
17
257818
257837
1258

523834
TTAAGTGGCACAGCCATGAT
Intron 3
9
258774
258793
1259

523835
AATGAGTTAAGTTGGAACAC
Intron 3
25
261294
261313
1260

523836
TCCTTAGTAGAATGCCTGGA
Intron 3
57
263338
263357
1261

523837
TATGTAGAAAAATAAGCTGG
Intron 3
0
266514
266533
1262

523838
GCCGAGGCAGGCACCTGAGT
Intron 3
43
267375
267394
1263

523839
TGGTACCTATATTGAGAGGT
Intron 4
46
269052
269071
1264

523840
TTAAGGAAAAATATAGTATA
Intron 4
7
269854
269873
1265

523841
TTATTTATGTGTCAGGGATG
Intron 4
28
270668
270687
1266

523842
CAAAAGTTAAGTGCTTTAGG
Intron 4
10
271468
271487
1267

523843
TTCATAGATGTCTAAGGAAT
Intron 4
32
273341
273360
1268

523844
ACCTGTGATTTACCTATTTC
Exon 5-intron 5
18
274185
274204
1269

junction

523845
TGCCTAGAAAACCACATAAA
Intron 5
38
274985
275004
1270

523846
AAACATCCTCAAAGGTACCT
Intron 5
64
275808
275827
1271

523847
CTTCCCTGAGACACACACAT
Intron 5
35
276617
276636
1272

523848
CTTCTTCAATCTTCTCATAC
Intron 5
33
278288
278307
1273

523849
TACCATTTTCCATTTAGTTT
Exon 6-intron 6
7
279088
279107
1274

junction

523850
ATTGGCATCTTTTTCAGTGG
Intron 6
34
279902
279921
1275

523851
TCAAGCTCACGGTTGGAGAC
Intron 6
36
280799
280818
1276

523852
AAATGAAATCAGTATGTTGA
Intron 6
0
281622
281641
1277

523853
TGATTTATCACAAAGGTGCT
Intron 6
29
282437
282456
1278

523854
AAAACAGTAGAAAAGATTAA
Intron 6
14
284073
284092
1279

523855
CTACATCACAGCAGTCAGAA
Intron 6
23
285187
285206
1280

523856
AAAAGATGTAAGTGTGACAT
Intron 6
28
286349
286368
1281

286919
286938

523857
TTACAAGAACTGCTAAAGGG
Intron 6
15
287151
287170
1282

523858
ATAAAGAAAAAGTTAACTGA
Intron 6
9
287982
288001
1283

523859
AGATAATATACTTCTTCTAT
Intron 6
4
288809
288828
1284

523860
CCTTCTTCACATGTAAATTG
Exon 7-intron 7
19
290456
290475
1285

junction

523861
TTTCTATGTAGCTTGTGGTT
Intron 7
30
291258
291277
1286

523862
AGGCAGAGTTTTTATTGATA
Intron 7
19
292058
292077
1287

523863
ATAGTCACCAGCCTAAGCCT
Intron 8
28
292858
292877
1288

523864
AGACTTTTAGCATGCTTGAC
Intron 8
56
293658
293677
1289

523865
TTTACAGCCCTACAGTTCTA
Intron 8
7
294464
294483
1290

523866
CCAGAGAACCTGACTCCAAA
Intron 8
6
295330
295349
1291

523867
CAGAAGAAAATATTAGACAG
Intron 8
10
296993
297012
1292

TABLE 19

Inhibition of GHR mRNA by 5-10-5 MOE gapmers

targeting introns 3-8 of SEQ ID NO: 2

ISIS

Target
%
SEQ ID NO: 2
SEQ ID NO: 2
SEQ

NO
Sequence
Region
inhibition
Start Site
Stop Site
ID NO

532531
TATTATACTTCTAAATTCCC
Intron 3
70
236716
236735
1293

532532
TAAAAGCAAGAAAAAGGAAC
Intron 3
52
236889
236908
1294

532533
CCTAATTTATATGAACAAAC
Intron 3
56
237177
237196
1295

532534
TGCAATGCCTTAGCCTAAAA
Intron 3
86
238087
238106
1296

532535
CACCACCATTATTACACTAC
Intron 3
75
238186
238205
1297

532536
AAATAAATCAGATTATTATA
Intron 3
52
238242
238261
1298

532537
CTTAGATCTGTGCTGTCCAA
Intron 3
81
245758
245777
1299

532538
GTTAGTGTTAGATTCTTTGA
Intron 3
67
246152
246171
1300

532539
CATGCTCACGGCTGTGTTAC
Intron 3
66
246248
246267
1301

532540
CCCATCAAATACTGAGTTCT
Intron 3
86
246487
246506
1302

532541
GAAAGTAGTGATTAATGAGA
Intron 3
38
247012
247031
1303

532542
ATTAATCAACAAGTGGCATT
Intron 3
72
247203
247222
1304

532543
TTTAATTTTAGGGTTTAGAG
Intron 3
48
248344
248363
1305

532544
CTTGCTACCACTAGAGCCTT
Intron 3
69
248694
248713
1306

532545
ACCACTGACTTATATCATTT
Intron 3
58
248743
248762
1307

532546
TTCCCCATTGCTAATTTTGT
Intron 3
48
251601
251620
1308

532547
TCCTGAAACTTAGTAGCTGG
Intron 3
83
253147
253166
1309

532548
TGTCTTAAAAAGGAATAAAA
Intron 3
52
253785
253804
1310

532549
CCTATAATAAAGTATTGTCT
Intron 3
70
253800
253819
1311

532550
ATGTAAAATGGTATAGCTAC
Intron 3
50
254040
254059
1312

532551
AACCCTCACACACTTCTGTT
Intron 3
71
254064
254083
1313

532552
ATTCTGCATAAGCAGTGTTT
Intron 3
53
254246
254265
1314

532553
TTACTACCCTGAAGAAGAAC
Intron 3
35
254314
254333
1315

532554
AAGACCTATAACTTACTACC
Intron 3
49
254326
254345
1316

532555
TTTCACAAGATTTACTTGGT
Intron 3
77
254641
254660
1317

532556
CAGTTGTGATTGTCAACCTA
Intron 3
77
257073
257092
1318

532557
AATCTTGCCTCGATGAAAGT
Intron 3
57
257819
257838
1319

532558
TGGCCTAAATGTATCAGTTA
Intron 3
66
259157
259176
1320

532559
AGGCTTTGGGTAAAATCTTT
Intron 3
67
259184
259203
1321

532560
TATGATTTTTAAAGATTAAA
Intron 3
20
261419
261438
1322

532561
GTACAGTGAAAAAGATGTGT
Intron 3
56
263666
263685
1323

532562
GACAGGTATGAAGCAAAACA
Intron 3
64
267033
267052
1324

532563
TGAGCTGAGGGTCTTTGCCG
Intron 3
61
267391
267410
1325

532564
AGGCTGAGTTGTACACAAAC
Intron 4
52
269422
269441
1326

532565
ATGAGGAGGCTGAGTTGTAC
Intron 4
43
269428
269447
1327

532566
TCATAAAGTGGGCCCAGCTT
Intron 4
70
270044
270063
1328

532567
ACTCCTAATCCCTCAGTTTT
Intron 4
62
270492
270511
1329

532568
TTTACATGCAAGGAGCTGAG
Intron 4
61
271047
271066
1330

532569
TAATGCCCTTTCTCCCTACT
Intron 4
60
271215
271234
1331

532570
CCTGTTTAGATTATCCCAAA
Intron 4
62
271763
271782
1332

532571
CATGATTCACAGAATTTCTC
Intron 4
56
271831
271850
1333

532572
AGTTAGAAAACTCAAAGTAT
Intron 4
2
271915
271934
1334

532573
TCAAATGTACTTAGCATAAG
Intron 4
9
271947
271966
1335

532574
ATATCAAATGTACTTAGCAT
Intron 4
59
271950
271969
1336

532575
AAAGTTCAGAAGAGGGAATG
Intron 4
51
273233
273252
1337

532576
AATTCCCATCTGAGTAGTTT
Intron 4
56
273440
273459
1338

532577
GTCCCCTAATTTCAGGCTAA
Intron 4
31
273471
273490
1339

532578
CTATGTCAAATGAAACAAAA
Intron 5
38
274205
274224
1340

532579
TGATTATGCTTTGTGATAAA
Intron 5
42
274624
274643
1341

532580
TCCAGCTGACTAGGAGGGCT
Intron 5
7
275732
275751
1342

532581
CATACCAGTCTCCTCGCTCA
Intron 5
0
276738
276757
1343

532582
ATATAACAGAATCCAACCAT
Intron 5
47
277045
277064
1344

278361
278380

532583
TGCAAAATGGCCAAACTACA
Intron 5
56
277577
277596
1345

532584
TCTTCCTAGCCACATGTGAT
Intron 5
32
278227
278246
1346

532585
TACCATGCTCTCTAATTGCC
Intron 6
47
279624
279643
1347

532586
AGTGATCTGTGCCAGGCTGC
Intron 6
65
279848
279867
1348

532587
AAGTTACAGAACAGATATCT
Intron 6
61
280012
280031
1349

532588
GTATTGTGAAAATAGTACTG
Intron 6
45
280226
280245
1350

532589
AAACACTATCAAGCTCACGG
Intron 6
54
280807
280826
1351

532590
TTCAAGAAAAGTCTTCAAAT
Intron 6
24
280831
280850
1352

532591
GGATCATTTCCCCATGCATG
Intron 6
52
280982
281001
1353

532592
ATATTATATTAAGAAAAATG
Intron 6
4
281422
281441
1354

532593
CTCCCATGTTCATTACTTAT
Intron 6
49
281587
281606
1355

532594
CATGACATTGGTTTGGGCAA
Intron 6
43
282229
282248
1356

532595
AATGTTGTTGGGAAAATTGG
Intron 6
42
282383
282402
1357

532596
AGCTGCAGGATACAAAGTCA
Intron 6
49
282986
283005
1358

532597
ATATCCTTTCATGATAAAAA
Intron 6
31
283354
283373
1359

532598
ATGGGCTAATATCTCTGATA
Intron 6
50
283590
283609
1360

532599
ACATTACTAATAATTAGAGA
Intron 6
0
285236
285255
1361

532600
ATAAAAACATATGAAAGTAT
Intron 6
12
287093
287112
1362

532601
TTCTGAATTAAATCTATTAG
Intron 6
16
287408
287427
1363

532602
TTACATTTTTGCAAATTTAT
Intron 6
31
287472
287491
1364

532603
TGAACAGTTGATTAACAAAG
Intron 6
15
287887
287906
1365

532604
AAGTTATTGGTTTACTAGAT
Intron 6
0
288598
288617
1366

532605
TTGGAAAAGGTCCTAGAAAA
Intron 6
24
289808
289827
1367

532606
CATGACAGAAACTTCTTAGA
Intron 7
25
292035
292054
1368

532607
CCATACTTGCTGACAAATAT
Intron 8
39
294389
294408
1369

Example 2: Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by MOE Gapmers

Gapmers from Example 1 exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN©. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 20

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523271
41
61
73
86
92
0.8

523274
20
36
64
80
92
1.8

523324
35
45
68
91
90
1.2

TABLE 21

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523604
21
42
68
58
86
2.0

523577
6
22
56
66
91
2.7

523614
14
44
61
84
87
1.9

523564
4
26
48
67
86
2.8

523633
30
43
71
82
84
1.4

523571
2
9
38
55
82
3.9

TABLE 22

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523570
25
50
64
77
88
1.5

523592
27
42
59
79
88
1.7

523595
21
50
62
76
90
1.6

523596
36
47
62
75
77
1.4

523607
49
62
71
82
84
0.5

523615
20
49
63
83
91
1.6

523630
4
28
54
79
78
2.6

523661
4
34
48
73
79
2.7

523665
4
28
54
73
79
2.7

523687
30
56
61
78
81
1.4

523711
42
66
78
94
95
0.7

523712
6
37
60
72
89
2.3

523713
4
32
55
72
85
2.5

523714
59
75
88
95
97
0.2

TABLE 23

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523655
26
33
60
67
78
2.1

523656
19
33
45
69
87
2.4

523658
0
42
62
67
79
3.1

523715
78
90
92
93
95
<0.6

523718
30
46
67
84
92
1.4

523723
56
69
83
92
94
0.3

523725
45
64
79
89
95
0.6

523726
32
48
77
88
89
1.2

523736
0
64
75
90
96
1.5

523747
48
64
80
91
92
0.6

523758
25
39
61
74
84
1.9

523766
7
37
66
81
93
2.0

523776
26
54
72
78
83
1.3

523789
62
68
81
85
90
0.2

TABLE 24

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523719
24
46
65
84
93
1.5

523720
18
49
72
85
93
1.5

523724
43
61
77
91
91
0.7

523735
8
42
63
81
93
2.0

523740
37
58
72
83
88
1.0

523752
9
29
52
72
86
2.5

523763
8
32
57
70
80
2.6

523764
43
52
67
77
79
0.9

523765
24
48
62
88
4
1.5

523767
49
62
67
72
82
0.6

523772
29
39
54
62
61
2.7

523774
28
59
63
88
91
1.2

523778
25
32
63
78
84
1.9

523783
0
22
53
72
88
2.8

TABLE 25

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

532151
57
69
76
85
88
<0.6

532153
23
43
54
80
86
1.8

532158
46
58
81
87
87
0.6

532160
17
26
55
76
92
2.2

532162
14
46
71
83
93
1.7

532164
37
76
82
90
93
0.6

532171
41
81
67
81
83
<0.6

532181
56
81
84
89
93
0.2

532186
26
65
75
83
91
1.1

532188
51
68
80
89
93
<0.6

532189
24
31
52
75
86
2.1

532197
0
40
66
85
93
2.1

532199
24
37
50
73
87
2.1

532222
12
41
67
84
94
1.8

TABLE 26

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

532175
41
54
76
84
89
0.9

532223
53
69
75
88
94
<0.6

532235
43
58
67
77
82
0.8

532241
39
53
62
73
87
1.2

532248
49
65
72
85
93
0.6

532254
52
62
85
87
92
<0.6

532300
20
29
49
66
78
2.7

532304
26
39
66
78
90
1.7

532316
41
66
76
86
94
0.7

532395
32
56
84
93
97
1.0

532401
47
80
92
96
98
<0.6

532411
73
90
94
97
98
<0.6

532420
38
49
82
85
97
1.0

532436
37
58
75
90
96
0.9

TABLE 27

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

532410
66
83
92
94
97
<0.6

532468
45
68
78
93
94
0.6

532469
0
17
56
76
92
2.8

532470
10
34
62
84
94
2.0

532475
13
36
52
64
87
2.5

532476
34
64
73
79
93
0.9

532480
28
54
67
78
87
1.4

532482
21
39
69
83
92
1.7

532490
42
60
68
84
93
0.9

532500
37
50
63
81
87
1.2

532506
13
41
66
75
89
1.9

532507
47
59
71
86
89
0.7

532508
0
31
73
83
89
2.2

532526
31
56
78
79
88
1.1

TABLE 28

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

532495
59
74
81
87
95
<0.6

532501
49
53
71
83
84
0.7

532534
53
75
85
91
97
<0.6

532535
0
34
61
84
92
2.6

532537
49
67
80
90
94
<0.6

532540
59
70
87
93
95
<0.6

532547
57
71
81
91
92
<0.6

532555
48
36
61
72
85
1.3

532556
33
57
67
86
90
1.1

TABLE 29

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523421
32
57
81
82
88
1.0

533006
46
43
69
83
91
1.0

533121
53
75
75
88
93
<0.6

533122
65
77
82
90
93
<0.6

533123
39
71
84
91
95
0.6

533125
49
61
81
85
91
0.6

533131
3
57
59
82
90
1.9

533136
32
65
62
81
88
1.1

533139
13
51
72
90
94
1.5

533140
36
66
39
87
92
1.2

533153
50
65
83
89
90
<0.6

533156
43
64
74
85
90
0.7

533160
57
80
87
91
95
<0.6

533161
54
62
81
89
92
<0.6

TABLE 30

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

533234
50
70
86
93
95
<0.6

533237
5
45
63
84
93
1.9

533233
43
55
76
90
95
0.8

533179
31
63
75
87
87
1.0

533178
53
67
76
89
94
<0.6

533187
5
15
53
79
86
2.7

533188
49
68
83
89
94
<0.6

533271
45
66
85
92
94
0.6

533134
22
45
64
81
89
1.6

533258
52
72
88
93
95
<0.6

533235
50
54
75
82
90
0.7

533262
23
54
78
91
96
1.2

533189
48
66
78
82
88
<0.6

533193
38
53
72
77
91
1.0

TABLE 31

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

533259
63
78
84
90
92
<0.6

533291
25
57
75
86
96
1.2

533256
67
76
90
95
95
<0.6

533269
42
75
82
94
97
0.6

533265
67
78
91
95
97
<0.6

533318
16
45
77
87
95
1.5

533257
55
84
91
96
96
<0.6

533280
34
62
80
91
91
0.9

533301
52
77
84
93
96
<0.6

533316
41
50
79
93
94
0.9

533270
62
71
88
94
97
<0.6

533330
46
76
93
97
98
<0.6

533317
55
60
82
87
96
<0.6

533315
39
56
82
87
93
0.9

TABLE 32

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

533364
71
77
92
90
94
<0.6

533925
26
55
61
85
91
1.4

533326
54
77
80
93
95
<0.6

533916
18
62
69
83
93
1.4

533328
52
68
89
94
98
<0.6

533932
42
49
80
86
92
0.9

533352
42
82
88
93
94
<0.6

533917
20
37
57
78
84
2.0

533331
54
83
89
93
96
<0.6

533936
21
46
73
84
88
1.5

533329
56
73
84
92
98
<0.6

533937
26
32
79
86
94
1.5

533908
58
66
81
88
94
<0.6

533898
61
64
84
90
92
<0.6

TABLE 33

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

539371
32
41
82
92
98
1.2

539382
18
58
74
91
97
1.3

539392
34
59
79
94
96
0.9

539398
31
53
89
94
98
1.0

539399
31
72
87
95
97
0.8

539400
36
60
79
93
97
0.9

539405
33
58
74
91
94
1.0

539412
23
61
80
93
95
1.1

539413
53
75
86
92
96
<0.6

539415
47
62
84
91
96
0.6

539416
61
85
94
97
96
<0.6

539430
24
48
68
80
93
1.5

539431
14
40
71
89
95
1.7

539433
46
67
74
92
95
0.6

Example 3: Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by MOE Gapmers

Gapmers from the studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.3125 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

TABLE 34

0.3125
0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
μM
(μM)

523814
0
24
48
52
68
82
2.2

523805
13
29
55
0
79
85
1.5

523822
0
19
26
41
65
85
2.8

523820
0
19
29
58
74
86
2.3

523815
3
6
19
37
45
71
4.8

523828
12
19
32
51
64
74
2.7

523801
3
9
31
43
59
76
3.3

523824
12
28
44
63
77
85
1.7

523794
13
21
30
51
66
78
2.5

523810
15
34
55
72
78
86
1.3

523819
0
24
40
60
66
75
2.4

TABLE 35

0.3125
0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
μM
(μM)

539302
31
56
80
92
97
98
0.5

539314
16
28
49
69
85
95
1.3

539319
8
30
45
71
90
94
1.4

539320
11
42
64
83
92
95
1.0

539321
25
48
64
82
95
97
0.8

539322
19
34
58
72
90
96
1.1

539331
7
14
46
69
88
96
1.6

539355
28
35
67
89
96
98
0.8

539358
12
39
56
80
93
98
1.1

539359
15
23
58
77
93
98
1.2

TABLE 36

0.3125
0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
μM
(μM)

539318
23
21
56
73
88
94
1.2

539325
14
26
38
74
92
98
1.4

539339
18
23
58
83
92
98
1.1

539341
17
29
62
84
94
95
1.0

539342
20
31
43
71
90
95
1.2

539352
15
23
41
61
89
95
1.5

539356
24
46
62
83
90
97
0.8

539361
37
42
73
88
96
98
0.6

539379
53
66
83
96
96
98
0.2

539380
52
77
91
97
97
99
0.1

539383
34
61
71
89
98
98
0.5

TABLE 37

0.3125
0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
μM
(μM)

539360
45
60
81
94
97
98
0.3

539362
21
36
72
90
98
99
0.8

539375
23
36
66
85
95
99
0.9

539376
26
35
58
82
95
99
0.9

539377
29
31
43
64
85
89
1.3

539378
37
59
81
93
97
98
0.4

539389
34
61
61
87
95
97
0.5

539401
34
52
63
84
92
95
0.6

539403
52
73
83
94
97
98
0.1

539404
22
55
74
88
94
96
0.6

539432
32
50
75
86
94
96
0.6

Example 4: Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by MOE Gapmers

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

TABLE 38

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523271
26
41
80
89
94
1.4

523274
13
35
63
85
95
1.9

523324
26
40
64
88
95
1.6

523577
27
50
72
87
95
1.3

523604
49
66
74
81
87
0.5

523614
43
54
82
92
89
0.8

TABLE 39

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523564
16
48
69
75
91
1.7

523570
24
52
65
71
88
1.6

523592
6
31
52
65
81
2.8

523595
13
49
60
79
92
1.8

523596
20
49
62
71
75
1.9

523607
38
63
66
74
76
0.8

523615
17
48
60
80
92
1.8

523630
19
42
42
67
80
2.5

523633
41
69
78
79
80
0.6

523665
16
45
56
71
80
2.1

523687
37
59
73
75
78
0.9

523711
33
63
78
91
93
0.9

523712
13
36
61
78
87
2.1

523714
63
85
91
96
96
<0.6

TABLE 40

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523655
28
42
57
74
76
1.9

523656
33
43
53
74
88
1.7

523661
29
29
66
79
82
1.9

523713
35
45
64
83
87
1.3

523715
83
86
92
93
94
<0.6

523718
27
52
69
84
95
1.3

523723
65
74
86
85
94
<0.6

523725
37
63
78
78
92
0.8

523726
43
57
72
86
89
0.8

523736
39
65
80
88
95
0.8

523747
51
71
83
86
93
<0.6

523766
30
50
70
82
89
1.3

523776
45
59
67
79
84
0.7

523789
63
75
76
83
83
<0.6

TABLE 41

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523719
18
40
56
73
83
2.1

523720
36
46
59
64
89
1.5

523724
44
60
75
81
87
0.7

523735
11
40
60
78
84
2.1

523740
17
47
61
80
81
1.8

523752
25
31
38
70
84
2.5

523758
23
48
58
72
80
1.8

523763
2
24
48
64
75
3.3

523764
22
49
45
73
75
2.1

523765
42
40
57
79
87
1.4

523767
43
53
56
69
79
1.2

523774
36
52
71
81
89
1.1

523778
15
45
59
75
79
2.0

523783
5
30
48
66
83
2.9

TABLE 42

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

532151
40
45
64
71
82
1.3

532158
28
47
63
70
87
1.6

532164
36
47
64
75
89
1.3

532171
35
47
50
69
89
1.6

532175
27
38
43
75
87
2.1

532181
21
56
63
69
80
1.7

532186
28
49
62
73
91
1.5

532188
40
52
73
75
90
1.0

532223
22
34
53
71
90
2.2

532235
35
31
48
68
73
2.3

532241
6
24
29
51
72
4.5

532248
19
37
47
73
84
2.3

532254
56
56
72
85
90
0.5

532316
32
55
50
78
90
1.5

TABLE 43

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

532304
44
57
68
78
73
0.7

532395
47
62
82
91
96
0.6

532401
70
83
91
94
96
<0.6

532410
56
71
85
90
96
<0.6

532411
88
93
96
97
98
<0.6

532420
61
67
82
85
96
<0.6

532436
48
49
77
90
97
0.8

532468
42
67
82
89
94
0.6

532476
32
58
75
84
90
1.1

532482
5
26
56
71
87
2.6

532490
18
47
55
69
86
2.0

532501
4
22
43
59
77
3.5

532507
39
63
66
83
89
0.9

532526
30
48
67
82
88
1.4

TABLE 44

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

533121
59
67
78
83
87
0.2

533122
48
73
78
84
90
0.4

533125
47
61
74
89
89
0.6

533136
5
25
58
79
90
2.4

533156
37
48
69
77
87
1.2

533161
28
67
77
89
90
1.0

533178
30
60
72
90
92
1.1

533179
37
66
76
76
87
0.8

533188
32
64
74
80
90
1.0

533189
49
66
77
81
81
0.4

533193
26
48
69
75
85
1.5

533233
39
60
59
84
93
1.0

533234
45
69
84
91
94
0.5

533235
28
49
69
82
90
1.4

TABLE 45

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

533256
47
72
86
90
94
<0.6

533257
63
77
88
91
96
<0.6

533258
66
81
88
95
95
<0.6

533259
48
70
84
90
93
<0.6

533262
44
66
79
90
96
0.7

533265
59
74
85
93
96
<0.6

533269
25
55
74
86
87
1.2

533270
34
59
73
86
95
1.0

533271
63
82
88
92
92
<0.6

533291
14
46
64
84
89
1.8

533301
49
61
75
83
91
0.6

533315
22
39
73
76
91
1.7

533317
26
53
68
85
94
1.3

533318
29
40
46
77
91
1.9

TABLE 46

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

533280
58
64
77
82
87
0.3

533316
35
55
68
87
91
1.1

533326
34
68
76
89
96
0.8

533328
54
55
79
83
92
0.5

533329
46
62
72
83
95
0.7

533330
56
75
83
91
94
0.3

533331
54
61
80
86
89
0.4

533352
54
62
79
83
89
0.4

533364
52
73
83
91
94
0.4

533898
17
47
63
78
87
1.8

533908
35
58
74
82
87
1

533916
22
46
72
78
88
1.6

533932
51
62
70
79
80
0.5

533937
20
40
61
79
85
1.9

Example 5: Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by MOE Gapmers

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.3125 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

TABLE 47

0.3125
0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
μM
(μM)

523577
0
16
33
59
72
94
2.2

523633
15
33
66
73
82
86
1.1

523764
11
33
50
68
78
83
1.5

523794
12
30
33
56
76
82
1.9

523805
21
48
66
78
85
92
0.8

523810
18
36
61
80
89
90
1.0

523814
13
35
52
67
81
88
1.3

523819
11
30
57
72
81
89
1.3

523820
0
15
43
61
84
92
1.8

523824
21
27
59
72
84
90
1.2

TABLE 48

0.3125
0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
μM
(μM)

539302
34
41
56
83
83
96
0.8

539321
30
32
76
73
80
94
0.8

539322
22
36
57
72
78
94
1.1

539355
23
42
48
72
71
88
1.2

539359
21
38
48
73
78
92
1.2

539320
14
32
53
72
82
91
1.3

539341
3
19
35
56
78
89
2.0

539342
6
18
33
51
70
83
2.3

539356
0
0
21
45
73
94
2.7

539358
0
15
23
50
52
91
2.9

TABLE 49

0.3125
0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
μM
(μM)

539339
22
37
52
77
90
92
1.0

539360
28
49
72
82
95
97
0.7

539361
36
56
75
86
95
98
0.5

539362
24
26
63
77
91
97
1.0

539375
21
29
39
63
77
91
1.5

539378
8
42
64
85
92
97
1.0

539379
43
59
80
89
96
98
0.3

539380
61
73
90
95
98
98
0.1

539383
30
49
75
87
97
98
0.6

539403
48
55
75
85
94
96
0.3

539432
36
42
69
79
88
95
0.7

TABLE 50

0.3125
0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
μM
(μM)

539376
34
46
62
82
94
98
0.7

539389
53
58
78
86
94
97
0.2

539392
1
19
26
68
81
94
1.9

539399
27
52
65
78
92
98
0.7

539400
7
26
43
59
88
95
1.6

539401
32
39
77
90
92
95
0.6

539404
22
59
77
87
93
95
0.6

539413
16
33
53
82
86
96
1.1

539415
4
44
56
74
81
94
1.2

539416
37
61
70
85
92
95
0.4

539433
31
52
70
85
87
94
0.6

Example 6: Antisense Inhibition of Human Growth Hormone Receptor in Hep3B Cells by Deoxy, MOE and cEt Gapmers

Additional antisense oligonucleotides were designed targeting a growth hormone receptor (GHR) nucleic acid and were tested for their effects on GHR mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 5,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as deoxy, MOE, and cEt gapmers. The deoxy, MOE and cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates a cEt sugar modification; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “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 gapmer listed in the Tables below is targeted to either the human GHR mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000163.4) or the human GHR genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity. In case the sequence alignment for a target gene in a particular table is not shown, it is understood that none of the oligonucleotides presented in that table align with 100% complementarity with that target gene.

TABLE 51

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting

intronic and exonic regions of SEQ ID NO: 1 and 2

ISIS

%

SEQ

NO
Start Site
Target Region
Sequence
Chemistry
inhibition
Start Site
ID NO

541262
n/a
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
84
156891
1370

541263
164
Intron 1
CCGAGCTTCGCCTCTG
eekddddddddddkke
89
3040
1371

541264
167
Intron 1
CCTCCGAGCTTCGCCT
eekddddddddddkke
90
3043
1372

541265
170
Junction
GGACCTCCGAGCTTCG
eekddddddddddkke
89
n/a
1373

spanning two

exons

541266
176
Junction
CCTGTAGGACCTCCGA
eekddddddddddkke
83
n/a
1374

spanning two

exons

541268
214
Exon 2
CCAGTGCCAAGGTCAA
eekddddddddddkke
87
144998
1375

541269
226
Exon 2
CACTTGATCCTGCCAG
eekddddddddddkke
67
145010
1376

541270
244
Exon 2
CACTTCCAGAAAAAGC
eekddddddddddkke
34
145028
1377

541278
365
Exon 4/Intron 3
GTCTCTCGCTCAGGTG
eekddddddddddkke
77
268028
1378

541279
368
Exon 4/Intron 3
AAAGTCTCTCGCTCAG
eekddddddddddkke
76
268031
1379

541280
373
Exon 4/Intron 3
ATGAAAAAGTCTCTCG
eekddddddddddkke
66
268036
1380

541283
445
exon 2-exon 3
TCCTTCTGGTATAGAA
eekddddddddddkke
37
n/a
1381

junction

541288
554
Exon 5
CAATAAGGTATCCAGA
eekddddddddddkke
49
274114
1382

541289
561
Exon 5
CTTGATACAATAAGGT
eekddddddddddkke
66
274121
1383

541290
569
Exon 5
CTAGTTAGCTTGATAC
eekddddddddddkke
61
274129
1384

541293
628
exon 3-exon 4
GATCTGGTTGCACTAT
eekddddddddddkke
57
n/a
1385

junction

541294
639
Exon 6
GGCAATGGGTGGATCT
eekddddddddddkke
38
278933
1386

541295
648
Exon 6
CCAGTTGAGGGCAATG
eekddddddddddkke
67
278942
1387

541296
654
Exon 6
TAAAGTCCAGTTGAGG
eekddddddddddkke
43
278948
1388

541301
924
Exon 7
TACATAGAGCACCTCA
eekddddddddddkke
86
290422
1389

541302
927
Exon 7
TGTTACATAGAGCACC
eekddddddddddkke
78
290425
1390

541303
930
Exon 7
AAGTGTTACATAGAGC
eekddddddddddkke
59
290428
1391

541304
958
Exon 7
CTTCACATGTAAATTG
eekddddddddddkke
26
290456
1392

541305
981
Exon 8
GAGCCATGGAAAGTAG
eekddddddddddkke
66
292535
1393

541310
1127
Exon 7-exon 8
CCTTCCTTGAGGAGAT
eekddddddddddkke
26
n/a
1394

junction

541320
1317
Exon 10
CTTCACCCCTAGGTTA
eekddddddddddkke
38
297734
1395

541321
1322
Exon 10
CCATCCTTCACCCCTA
eekddddddddddkke
81
297739
1396

541322
1326
Exon 10
GTCGCCATCCTTCACC
eekddddddddddkke
79
297743
1397

541323
1331
Exon 10
CCAGAGTCGCCATCCT
eekddddddddddkke
64
297748
1398

541325
1420
Exon 10
GTGGCTGAGCAACCTC
eekddddddddddkke
79
297837
1399

541326
1434
Exon 10
CCCTTTTAACCTCTGT
eekddddddddddkke
67
297851
1400

541331
1492
Exon 10
CATCATGATAAGGTGA
eekddddddddddkke
16
297909
1401

541332
1526
Exon 10
TGGATAACACTGGGCT
eekddddddddddkke
30
297943
1402

541333
1532
Exon 10
TCTGCTTGGATAACAC
eekddddddddddkke
63
297949
1403

541335
1597
Exon 10
GAATATGGGCAGCTTG
eekddddddddddkke
33
298014
1404

541336
1601
Exon 10
AGCTGAATATGGGCAG
eekddddddddddkke
34
298018
1405

541337
1607
Exon 10
TTGCTTAGCTGAATAT
eekddddddddddkke
39
298024
1406

541338
1611
Exon 10
TGGATTGCTTAGCTGA
eekddddddddddkke
79
298028
1407

541339
1614
Exon 10
ACTTGGATTGCTTAGC
eekddddddddddkke
73
298031
1408

Example 7: Antisense Inhibition of Human Growth Hormone Receptor in Hep3B Cells by Deoxy, MOE and cEt Gapmers

Additional antisense oligonucleotides were designed targeting a growth hormone receptor (GHR) nucleic acid and were tested for their effects on GHR mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as deoxy, MOE, and cEt gapmers. The deoxy, MOE and cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, a cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates a cEt sugar modification; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “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 gapmer listed in the Tables below is targeted to either the human GHR mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000163.4) or the human GHR genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity. In case the sequence alignment for a target gene in a particular table is not shown, it is understood that none of the oligonucleotides presented in that table align with 100% complementarity with that target gene. The oligonucleotides of Table 54 do not target SEQ ID NOs: 1 or 2, but instead target variant gene sequences SEQ ID NO: 4 (GENBANK Accession No. DR006395.1) or SEQ ID NO: 7 (the complement of GENBANK Accession No. AA398260.1).

TABLE 52

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting

intronic and exonic regions of SEQ ID NO: 1 and 2

ISIS
Start
Target

%
Start
SEQ

NO
Site
Region
Sequence
Chemistry
inhibition
Site
ID NO

541262
n/a
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
86
156891
1370

541340
1619
Exon 10
AGTGAACTTGGATTGC
eekddddddddddkke
73
298036
1409

541341
1641
Exon 10
GGCATAAAAGTCGATG
eekddddddddddkke
41
298058
1410

541342
1644
Exon 10
CTGGGCATAAAAGTCG
eekddddddddddkke
33
298061
1411

541343
1683
Exon 10
GGAAAGGACCACACTA
eekddddddddddkke
34
298100
1412

541344
1746
Exon 10
GAGTGAGACCATTTCC
eekddddddddddkke
65
298163
1413

541345
1827
Exon 10
GATGTGAGGAGCCACA
eekddddddddddkke
54
298244
1414

541346
1830
Exon 10
CTTGATGTGAGGAGCC
eekddddddddddkke
70
298247
1415

541347
1835
Exon 10
TCAACCTTGATGTGAG
eekddddddddddkke
38
298252
1416

541348
1839
Exon 10
TGATTCAACCTTGATG
eekddddddddddkke
39
298256
1417

541349
1842
Exon 10
GTGTGATTCAACCTTG
eekddddddddddkke
74
298259
1418

541350
1845
Exon 10
TATGTGTGATTCAACC
eekddddddddddkke
58
298262
1419

541351
1949
Exon 10
GGCATCTCAGAACCTG
eekddddddddddkke
41
298366
1420

541352
1965
Exon 10
GGTATAGTCTGGGACA
eekddddddddddkke
18
298382
1421

541353
1969
Exon 10
TGGAGGTATAGTCTGG
eekddddddddddkke
17
298386
1422

541354
1972
Exon 10
GAATGGAGGTATAGTC
eekddddddddddkke
0
298389
1423

541355
1975
Exon 10
TATGAATGGAGGTATA
eekddddddddddkke
0
298392
1424

541356
1978
Exon 10
CTATATGAATGGAGGT
eekddddddddddkke
30
298395
1425

541357
1981
Exon 10
GTACTATATGAATGGA
eekddddddddddkke
43
298398
1426

541358
1987
Exon 10
GGGACTGTACTATATG
eekddddddddddkke
12
298404
1427

541369
2306
Exon 10
TTACATTGCACAATAG
eekddddddddddkke
21
298723
1428

541373
2667
Exon 10
TAGCCATGCTTGAAGT
eekddddddddddkke
34
299084
1429

541374
2686
Exon 10
TGTGTAGTGTAATATA
eekddddddddddkke
10
299103
1430

541375
2690
Exon 10
ACAGTGTGTAGTGTAA
eekddddddddddkke
82
299107
1431

541376
2697
Exon 10
GCAGTACACAGTGTGT
eekddddddddddkke
46
299114
1432

541377
2700
Exon 10
ACTGCAGTACACAGTG
eekddddddddddkke
32
299117
1433

541378
2740
Exon 10
TTAGACTGTAGTTGCT
eekddddddddddkke
25
299157
1434

541379
2746
Exon 10
CCAGCTTTAGACTGTA
eekddddddddddkke
69
299163
1435

541380
2750
Exon 10
TAAACCAGCTTTAGAC
eekddddddddddkke
20
299167
1436

541381
2755
Exon 10
AACATTAAACCAGCTT
eekddddddddddkke
64
299172
1437

541382
2849
Exon 10
ACTACAATCATTTTAG
eekddddddddddkke
0
299266
1438

541383
2853
Exon 10
GATTACTACAATCATT
eekddddddddddkke
0
299270
1439

541384
2859
Exon 10
AATGCAGATTACTACA
eekddddddddddkke
46
299276
1440

541385
2865
Exon 10
TCCAATAATGCAGATT
eekddddddddddkke
52
299282
1441

541386
2941
Exon 10
GTTGATCTGTGCAAAC
eekddddddddddkke
74
299358
1442

541389
3037
Exon 10
TCTACTTCTCTTAGCA
eekddddddddddkke
50
299454
1443

541393
3215
Exon 10
GCTTCTTGTACCTTAT
eekddddddddddkke
84
299632
1444

541394
3237
Exon 10
GATTTGCTTCAACTTA
eekddddddddddkke
47
299654
1445

541395
3305
Exon 10
GGTTATAGGCTGTGAA
eekddddddddddkke
0
299722
1446

541396
3308
Exon 10
TCTGGTTATAGGCTGT
eekddddddddddkke
88
299725
1447

541397
3311
Exon 10
GTGTCTGGTTATAGGC
eekddddddddddkke
56
299728
1448

541398
3316
Exon 10
AGTATGTGTCTGGTTA
eekddddddddddkke
76
299733
1449

541399
3371
Exon 10
GGGACTGAAAACCTTG
eekddddddddddkke
50
299788
1450

541400
3975
Exon 10
AGTATTCTTCACTGAG
eekddddddddddkke
36
300392
1451

541401
4044
Exon 10
GCGATAAATGGGAAAT
eekddddddddddkke
36
300461
1452

541402
4048
Exon 10
GTCTGCGATAAATGGG
eekddddddddddkke
52
300465
1453

541403
4058
Exon 10
CCTAAAAAAGGTCTGC
eekddddddddddkke
51
300475
1454

541404
4072
Exon 10
CATTAAGCTTGCTTCC
eekddddddddddkke
53
300489
1455

TABLE 53

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting

intronic and exonic regions of SEQ ID NO: 1 and 2

ISIS
Start

%
Start
SEQ

NO
Site
Target Region
Sequence
Chemistry
inhibition
Site
ID NO

541262
n/a
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
85
156891
1370

541421
4418
Exon 10
CACAACTAGTCATACT
eekddddddddddkke
42
300835
1456

541422
4428
Exon 10
AACTGCCAGACACAAC
eekddddddddddkke
68
300845
1457

541423
4431
Exon 10
ATAAACTGCCAGACAC
eekddddddddddkke
86
300848
1458

541424
4503
Exon 10
TATCAGGAATCCAAGA
eekddddddddddkke
11
300920
1459

541425
4521
Exon 10
TTGATAACAGAAGCAC
eekddddddddddkke
16
300938
1460

541426
4528
Exon 10
TTGGTGTTTGATAACA
eekddddddddddkke
31
300945
1461

541427
4531
Exon 10
ATGTTGGTGTTTGATA
eekddddddddddkke
32
300948
1462

541429
30
Exon 1
CCGCCACTGTAGCAGC
eekddddddddddkke
77
2906
1463

541430
35
Exon 1
CGCCACCGCCACTGTA
eekddddddddddkke
88
2911
1464

541431
63
Exon 1
GCCGCCCGGGCTCAGC
eekddddddddddkke
86
2939
1465

541432
67
Exon 1
CGCCGCCGCCCGGGCT
eekddddddddddkke
61
2943
1466

541433
144
Exon 1
GAGAGCGCGGGTTCGC
eekddddddddddkke
57
3020
1467

541434
n/a
Exon 1/Intron 1
CTACTGACCCCAGTTC
eekddddddddddkke
80
3655
1468

541435
n/a
Exon 1/Intron 1
TCACTCTACTGACCCC
eekddddddddddkke
90
3660
1469

541436
n/a
Exon 1/Intron 1
TCATGCGGACTGGTGG
eekddddddddddkke
56
3679
1470

541437
n/a
Exon 3/Intron 3
ATGTGAGCATGGACCC
eekddddddddddkke
82
225438
1471

541438
n/a
Exon 3/Intron 3
TCTTGATATGTGAGCA
eekddddddddddkke
93
225445
1472

541439
n/a
Exon 3/Intron 3
TTCAAGTTGGTGAGCT
eekddddddddddkke
72
226788
1473

541440
n/a
Exon 3/Intron 3
TGCTTCCTTCAAGTTG
eekddddddddddkke
68
226795
1474

541441
n/a
Exon 3/Intron 3
TGTAATTTCATTCATG
eekddddddddddkke
62
226809
1475

541442
n/a
Exon 3/Intron 3
CCTTTTGCCAAGAGCA
eekddddddddddkke
85
226876
1476

541443
n/a
Exon 3/Intron 3
GATCCTTTTGCCAAGA
eekddddddddddkke
77
226879
1477

541444
n/a
Exon 3/Intron 3
GCTAGTAATGTTACAT
eekddddddddddkke
68
238331
1478

541445
n/a
Exon 3/Intron 3
GCAACTTGCTAGTAAT
eekddddddddddkke
65
238338
1479

541446
n/a
Exon 3/Intron 3
TGTGCAACTTGCTAGT
eekddddddddddkke
44
238341
1480

541447
n/a
Exon 3/Intron 3
GGATTTCAGTTTGAAT
eekddddddddddkke
0
238363
1481

541448
n/a
Exon 3/Intron 3
CTCAGAGCCTTGGTAG
eekddddddddddkke
65
238428
1482

541449
n/a
Exon 1/Intron 1
CAAACGCGCAAAAGAC
eekddddddddddkke
1
3608
1483

541450
n/a
Exon 1/Intron 1
GCCCGCACAAACGCGC
eekddddddddddkke
11
3615
1484

541451
n/a
Exon 1/Intron 1
GGTTAAAGAAGTTGCT
eekddddddddddkke
60
93190
1485

541452
n/a
Exon 1/Intron 1
CCCAGTGAATTCAGCA
eekddddddddddkke
85
93245
1486

541453
n/a
Exon 1/Intron 1
GCGCCCAGTGAATTCA
eekddddddddddkke
74
93248
1487

541454
n/a
Exon 1/Intron 1
AAGATGCGCCCAGTGA
eekddddddddddkke
71
93253
1488

541455
n/a
Exon 1/Intron 1
TGTAAGATGCGCCCAG
eekddddddddddkke
75
93256
1489

541456
n/a
Exon 1/Intron 1
AATTACTTGTAAGATG
eekddddddddddkke
15
93263
1490

541457
n/a
Exon 1/Intron 1
CCCAGAAGGCACTTGT
eekddddddddddkke
61
93302
1491

541458
n/a
Exon 1/Intron 1
TTGCAGAACAAATCTT
eekddddddddddkke
3
93333
1492

541459
n/a
Exon 1/Intron 1
CATGGAAGATTTGCAG
eekddddddddddkke
17
93343
1493

541460
n/a
Exon 1/Intron 1
GGTCATGGAAGATTTG
eekddddddddddkke
57
93346
1494

541461
n/a
Exon 1/Intron 1
GACCTTGGTCATGGAA
eekddddddddddkke
51
93352
1495

541462
n/a
Exon 1/Intron 1
TGCCAATCCAAAGAGG
eekddddddddddkke
34
93369
1496

541463
n/a
Exon 1/Intron 1
GGGTCTGCCAATCCAA
eekddddddddddkke
67
93374
1497

541464
n/a
Exon 1/Intron 1
TCCCTGGGTCTGCCAA
eekddddddddddkke
82
93379
1498

541465
n/a
Exon 1/Intron 1
AAGTGTGAATTTATCT
eekddddddddddkke
16
93408
1499

541466
n/a
Exon 1/Intron 1
GGAGATCTCAACAAGG
eekddddddddddkke
38
93428
1500

541468
n/a
Exon 1/Intron 1
TCGCCCATCACTCTTC
eekddddddddddkke
43
93989
1501

541469
n/a
Exon 1/Intron 1
CCTGTCGCCCATCACT
eekddddddddddkke
61
93993
1502

541470
n/a
Exon 1/Intron 1
TCACCTGTCGCCCATC
eekddddddddddkke
70
93996
1503

541471
n/a
Exon 1/Intron 1
CCATCACCTGTCGCCC
eekddddddddddkke
89
93999
1504

541472
n/a
Exon 1/Intron 1
TCACCATCACCTGTCG
eekddddddddddkke
72
94002
1505

541473
n/a
Exon 1/Intron 1
TAATAGTTGTCACCAT
eekddddddddddkke
42
94011
1506

541474
n/a
Exon 1/Intron 1
TTCAGATCTTATTAAT
eekddddddddddkke
0
94023
1507

541475
n/a
Exon 1/Intron 1
TTGCAAATTCAGTCTG
eekddddddddddkke
32
94096
1508

541477
n/a
Exon 2/Intron 2
CGTTCTCTTGGAAGTA
eekddddddddddkke
78
198766
1509

541478
n/a
Exon 2/Intron 2
TCTTGAATAAATTTCG
eekddddddddddkke
25
198780
1510

541479
n/a
Exon 2/Intron 2
AAGCTCACTCTTCAAT
eekddddddddddkke
60
198810
1511

541480
n/a
Exon 2/Intron 2
TCCAAGCTCACTCTTC
eekddddddddddkke
49
198813
1512

541481
n/a
Exon 2/Intron 2
GCTCCTGCCACTCTGT
eekddddddddddkke
75
198837
1513

541482
n/a
Exon 2/Intron 2
ATGGGCAAAGGCATCT
eekddddddddddkke
60
198874
1514

541483
n/a
5′UTR
AGTCTTCCCGGCGAGG
eekddddddddddkke
32
2571
1515

541484
n/a
5′ and overlappig
CCGCCGCTCCCTAGCC
eekddddddddddkke
73
2867
1516

with exon 1

541485
n/a
Intron 1
GCCCGCAACTCCCTGC
eekddddddddddkke
37
3341
1517

541486
n/a
Intron 1
CGCCTCCCCAGGCGCA
eekddddddddddkke
34
4024
1518

541487
n/a
Intron 1
GAGTGTCTTCCCAGGC
eekddddddddddkke
86
4446
1519

541488
n/a
Intron 1
CTGAAGACTCCTTGAA
eekddddddddddkke
39
4721
1520

541489
n/a
Intron 1
GGCTAGCCAAGTTGGA
eekddddddddddkke
54
5392
1521

541490
n/a
Intron 1
TGACTCCAGTCTTACC
eekddddddddddkke
76
5802
1522

541491
n/a
Intron 1
ATTCATTGTGGTCAGC
eekddddddddddkke
91
6128
1523

541492
n/a
Intron 1
GAAGTGGGTTTTTCCC
eekddddddddddkke
86
6543
1524

541493
n/a
Intron 1
GCCTTGGTTCAGGTGA
eekddddddddddkke
79
6786
1525

TABLE 54

Inhibition of GHR mRNA by deoxy, MOE and cEt

gapmers targeting SEQ ID NO: 3 and 4

ISIS
Target
Targe

%
SEQ

NO
Start Site
SEQ ID NO
Sequence
Chemistry
inhibition
ID NO

541428
66
3
CCACTGTAGCAGCCGC
eekddddddddddkke
92
1526

541476
263
4
TAGGTATTTCAGAGCC
eekddddddddddkke
80
1527

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting intronic regions of SEQ ID NO: 2

SEQ ID
SEQ ID

ISIS
NO: 1
NO: 1
Target

%
SEQ

NO
Start Site
Target Region
Region
Sequence
Chemistry
inhibition
ID NO

541262
156891
541277
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
80
1370

541494
7231
541509
Intron 1
GTCCAGGCAGAGTTGT
eekddddddddddkke
30
1528

541495
7570
541510
Intron 1
AGCCAAATGTTGGTCA
eekddddddddddkke
19
1529

541496
8395
541511
Intron 1
GAGGGCGAGTTTTTCC
eekddddddddddkke
71
1530

541497
9153
541512
Intron 1
GTGGCATTGGCAAGCC
eekddddddddddkke
81
1531

541498
9554
541513
Intron 1
ACCCCACTGCACCAAG
eekddddddddddkke
67
1532

541499
9931
541514
Intron 1
TCCAAGTACTTGCCAA
eekddddddddddkke
83
1533

541500
10549
541515
Intron 1
AGTGCCTGGCCTAAGG
eekddddddddddkke
75
1534

541501
11020
541516
Intron 1
GCGCTTCTTCCCTAGG
eekddddddddddkke
71
1535

541502
11793
541517
Intron 1
CATCTTGCCCAGGGAT
eekddddddddddkke
84
1536

541503
12214
541518
Intron 1
CCATCTTGCTCCAAGT
eekddddddddddkke
93
1537

541504
12474
541519
Intron 1
CTTACATCCTGTAGGC
eekddddddddddkke
71
1538

541505
12905
541520
Intron 1
CGCCTCCTGGTCCTCA
eekddddddddddkke
97
1539

541506
13400
541521
Intron 1
CCCTATGCACTACCTA
eekddddddddddkke
49
1540

541507
13717
541522
Intron 1
GAGGGACTGTGGTGCT
eekddddddddddkke
65
1541

541508
14149
541523
Intron 1
GCCCAATATGTGCCAG
eekddddddddddkke
60
1542

541509
14540
541524
Intron 1
GCTCTCTCATCGCTGG
eekddddddddddkke
90
1543

541510
15264
541525
Intron 1
CTCAAGGCTATGTGCC
eekddddddddddkke
67
1544

541511
15849
541526
Intron 1
TCCACATCCCTCATGT
eekddddddddddkke
68
1545

541512
16530
541527
Intron 1
AGGACTGAAGGCCCAT
eekddddddddddkke
49
1546

541513
17377
541528
Intron 1
GTGCGACTTACCAGCT
eekddddddddddkke
85
1547

541514
17581
541529
Intron 1
TCGCTAAAGCCACACA
eekddddddddddkke
89
1548

541515
17943
541530
Intron 1
GCTCTGGCTGATGGTC
eekddddddddddkke
92
1549

541516
18353
541531
Intron 1
TTCCCATGAGGATTTC
eekddddddddddkke
70
1550

541517
18636
541532
Intron 1
TTGGGCTTAAGCACTA
eekddddddddddkke
71
1551

541518
19256
541533
Intron 1
GCTAGCACCTAGTCCA
eekddddddddddkke
71
1552

541519
19814
541534
Intron 1
CCTCTGGCCTACAACA
eekddddddddddkke
64
1553

541520
20365
541535
Intron 1
ACCCCTCATCAGCACC
eekddddddddddkke
93
1554

541521
20979
541536
Intron 1
GGCCACCCCTGATCCT
eekddddddddddkke
66
1555

541522
21566
541537
Intron 1
GAAGCTCCCTTGCCCA
eekddddddddddkke
96
1556

541523
22150
541538
Intron 1
AGTGTTGCCCCTCCAA
eekddddddddddkke
83
1557

541524
22803
541539
Intron 1
GGGTCTCCAACCTACT
eekddddddddddkke
70
1558

541525
29049
541540
Intron 1
GGGATGTAGGTTTACC
eekddddddddddkke
74
1559

541526
29554
541541
Intron 1
GCAACCGATATCACAG
eekddddddddddkke
60
1560

541527
30245
541542
Intron 1
TGCCCTGGAACAAATT
eekddddddddddkke
13
1561

541528
30550
541543
Intron 1
AGTCTAGGAGTAGCTA
eekddddddddddkke
50
1562

541529
30915
541544
Intron 1
GCTGTTGTCAAGAGAC
eekddddddddddkke
55
1563

541530
31468
541545
Intron 1
CACCTAGACACTCAGT
eekddddddddddkke
47
1564

541531
32366
541546
Intron 1
GTCAAGGGATCCCTGC
eekddddddddddkke
34
1565

541532
32897
541547
Intron 1
TCCCCCTGGCACTCCA
eekddddddddddkke
79
1566

541533
33187
541548
Intron 1
GCCTGGTAACTCCATT
eekddddddddddkke
56
1567

541534
33780
541549
Intron 1
GGGCTCACCAACTGTG
eekddddddddddkke
39
1568

541535
34407
541550
Intron 1
CCACAGGATCATATCA
eekddddddddddkke
37
1569

541536
34846
541551
Intron 1
CTCCAGCAGAAGTGTC
eekddddddddddkke
10
1570

541537
35669
541552
Intron 1
AGCCCAACTGTTGCCT
eekddddddddddkke
79
1571

541538
36312
541553
Intron 1
TGCCAGGCAGTTGCCA
eekddddddddddkke
75
1572

541539
36812
541554
Intron 1
GCCAGTAAGCACCTTG
eekddddddddddkke
93
1573

541540
37504
541555
Intron 1
CTAGCTTCCCAGCCCC
eekddddddddddkke
46
1574

541541
38841
541556
Intron 1
TCAAGCCCAGCTAGCA
eekddddddddddkke
39
1575

541542
39108
541557
Intron 1
CCTCACAGGCCCTAAT
eekddddddddddkke
4
1576

541543
39408
541558
Intron 1
ACCTGCTTACATGGTA
eekddddddddddkke
21
1577

541544
40250
541559
Intron 1
CCTTTGCTAGGACCCA
eekddddddddddkke
52
1578

541545
40706
541560
Intron 1
GGGACTGCCACCAAGG
eekddddddddddkke
27
1579

541546
40922
541561
Intron 1
GCTAGATGTTCAGGCC
eekddddddddddkke
34
1580

541547
41424
541562
Intron 1
CCTATGGCCATGCTGA
eekddddddddddkke
32
1581

541548
41999
541563
Intron 1
GTATGCTAGTTCCCAT
eekddddddddddkke
83
1582

541549
42481
541564
Intron 1
CCCTCATAATCTTGGG
eekddddddddddkke
13
1583

541550
42700
541565
Intron 1
GTCCAACCACTACCAC
eekddddddddddkke
74
1584

541551
43291
541566
Intron 1
ACTTGCAGATAGCTGA
eekddddddddddkke
73
1585

541552
43500
541567
Intron 1
GCATGACCCCACTGCC
eekddddddddddkke
72
1586

541553
43947
541568
Intron 1
GAGGGTCACATTCCCT
eekddddddddddkke
23
1587

541554
44448
541569
Intron 1
TCTCTTACTGGTGGGT
eekddddddddddkke
90
1588

541555
45162
541570
Intron 1
GCCCCCTTCCTGGATA
eekddddddddddkke
28
1589

541556
46010
541571
Intron 1
CCTCATGCGACACCAC
eekddddddddddkke
71
1590

541557
46476
541572
Intron 1
AGCCCTCTGCCTGTAA
eekddddddddddkke
67
1591

541558
47447
541573
Intron 1
CTCCCAGCTATAGGCG
eekddddddddddkke
38
1592

541559
47752
541574
Intron 1
GCTAGCTGCGCAAGGA
eekddddddddddkke
5
1593

541560
48001
541575
Intron 1
GCGCAGCCCGCTGCAA
eekddddddddddkke
18
1594

541561
48423
541576
Intron 1
TGCATGATCCACCCCA
eekddddddddddkke
65
1595

541562
50195
541577
Intron 1
GCTTAGTGCTGGCCCA
eekddddddddddkke
72
1596

541563
50470
541578
Intron 1
CCTTCCAGTCCTCATA
eekddddddddddkke
81
1597

541564
51104
541579
Intron 1
ATAGTGTCAAGGCCCA
eekddddddddddkke
91
1598

541565
51756
541580
Intron 1
AGGCCTTAGTCACCCA
eekddddddddddkke
88
1599

541566
52015
541581
Intron 1
TAACCAACCTAAGGGA
eekddddddddddkke
11
1600

541567
52230
541582
Intron 1
ATTCTGGTGATGCCCT
eekddddddddddkke
66
1601

541568
52588
541583
Intron 1
GTGTTCACTGCCATGA
eekddddddddddkke
67
1602

541569
53532
541584
Intron 1
GGTAGAGCACACTGCC
eekddddddddddkke
47
1603

541570
54645
541585
Intron 1
CCACTTTAATGCCACC
eekddddddddddkke
76
1604

TABLE 56

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting intronic regions of SEQ ID NO: 2

SEQ ID
SEQ ID

ISIS
NO: 2
NO: 2
Target

%
SEQ

NO
Start Site
Target Region
Region
Sequence
Chemistry
inhibition
ID NO

541262
156891
156906
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
88
1370

541571
54886
54901
Intron 1
GTCAAATGCTGTTGGG
eekddddddddddkke
91
1605

541572
55900
55915
Intron 1
CATCCCCTATCAGGGT
eekddddddddddkke
53
1606

541573
62266
62281
Intron 1
CTCGAATCCCTTGAGC
eekddddddddddkke
73
1607

541574
62733
62748
Intron 1
GATTCCCTCCCCTAAC
eekddddddddddkke
27
1608

541575
63173
63188
Intron 1
ATCCATCCATGTGCTG
eekddddddddddkke
92
1609

541576
63751
63766
Intron 1
GAGCATGCCTCAGTGG
eekddddddddddkke
81
1610

541577
63964
63979
Intron 1
CAGAAGGACTGCCTCT
eekddddddddddkke
50
1611

541578
64213
64228
Intron 1
ACAATGCTCAACAGCC
eekddddddddddkke
75
1612

541579
64576
64591
Intron 1
GTTGGATCTGGCATGC
eekddddddddddkke
80
1613

541580
65027
65042
Intron 1
CGGCTGAGAGCAAGGG
eekddddddddddkke
88
1614

541581
65363
65378
Intron 1
GAGAGGGTTCAGCCTG
eekddddddddddkke
62
1615

541582
65600
65615
Intron 1
ACTTAGTTCCTAGCCA
eekddddddddddkke
91
1616

541583
66087
66102
Intron 1
GTGAACCAGATGTGCT
eekddddddddddkke
86
1617

541584
66566
66581
Intron 1
GGAGTGACAGCTAAGT
eekddddddddddkke
98
1618

541585
66978
66993
Intron 1
AAGTGTTCAGAGCCAC
eekddddddddddkke
97
1619

541586
67662
67677
Intron 1
AACCCTGCCAAGGTAC
eekddddddddddkke
45
1620

541587
67914
67929
Intron 1
GATGGTGAGCACTACC
eekddddddddddkke
78
1621

541588
68278
68293
Intron 1
GGCAGGATAGGACAGA
eekddddddddddkke
11
1622

541589
68727
68742
Intron 1
GCAAAGTGATGAGCCT
eekddddddddddkke
81
1623

541590
69207
69222
Intron 1
CTATCCACACCATTCC
eekddddddddddkke
93
1624

541591
69605
69620
Intron 1
GGATCATGGGCCCCTA
eekddddddddddkke
70
1625

541592
70130
70145
Intron 1
GTGAATTTGCTGGGCC
eekddddddddddkke
94
1626

541593
70569
70584
Intron 1
GTGATGGGCCCAAGGC
eekddddddddddkke
67
1627

541594
71056
71071
Intron 1
TCCTCAGTCGGCTTGC
eekddddddddddkke
69
1628

541595
71314
71329
Intron 1
CAGCCTTTTGCCAGAT
eekddddddddddkke
93
1629

541596
71620
71635
Intron 1
CCTCCCTAGGATTACC
eekddddddddddkke
42
1630

541597
72226
72241
Intron 1
ACGCCCCAATCACTCA
eekddddddddddkke
79
1631

541598
72655
72670
Intron 1
GCATGACCCATTATGT
eekddddddddddkke
94
1632

541599
73061
73076
Intron 1
TCCCTCCAAGAGCTCA
eekddddddddddkke
83
1633

541600
73708
73723
Intron 1
GATGCCTGTGGCTGAC
eekddddddddddkke
84
1634

541601
74107
74122
Intron 1
GGCTAGCATGTTGCCT
eekddddddddddkke
19
1635

541602
74542
74557
Intron 1
TAACCCACTAGGCTGG
eekddddddddddkke
84
1636

541603
74947
74962
Intron 1
TGGCCCAAAACTAATC
eekddddddddddkke
34
1637

541604
75192
75207
Intron 1
GGAGCAGTCTGGCACC
eekddddddddddkke
85
1638

541605
75699
75714
Intron 1
TATTCTGTGGGACAAG
eekddddddddddkke
51
1639

541606
75979
75994
Intron 1
GTGTCTAGTTCCAGCC
eekddddddddddkke
86
1640

541607
76410
76425
Intron 1
TACTATCATGTAGCGC
eekddddddddddkke
87
1641

541608
76701
76716
Intron 1
TGCCCTTGTAGTGAGA
eekddddddddddkke
31
1642

541609
76980
76995
Intron 1
TCCCCAACCTACAAGC
eekddddddddddkke
41
1643

541610
77292
77307
Intron 1
GCTCTAGGCATATGAA
eekddddddddddkke
63
1644

541611
77555
77570
Intron 1
TACCTCCCTTGTAGGG
eekddddddddddkke
27
1645

541612
77854
77869
Intron 1
GGTTCCCTTGCAGAGA
eekddddddddddkke
62
1646

541613
78311
78326
Intron 1
GTGCCCTCTTCATGCC
eekddddddddddkke
68
1647

541614
79006
79021
Intron 1
CCTGTGTGCAACTGGC
eekddddddddddkke
85
1648

541615
79490
79505
Intron 1
CTGAGTCATTTGCCTG
eekddddddddddkke
93
1649

541616
79829
79844
Intron 1
GGCCTTAGTAGGCCAG
eekddddddddddkke
0
1650

541617
80277
80292
Intron 1
GTCCTTGCAGTCAACC
eekddddddddddkke
77
1651

541618
80575
80590
Intron 1
GCTGGGCCAAGTCCAT
eekddddddddddkke
77
1652

541619
80895
80910
Intron 1
TAGGGCACTTTTTGCC
eekddddddddddkke
31
1653

541620
81207
81222
Intron 1
GCTGAGGTCCCTCTCT
eekddddddddddkke
34
1654

541621
81761
81776
Intron 1
CTTTGGTCCCATTGCC
eekddddddddddkke
83
1655

541622
82233
82248
Intron 1
GGAACATGCCAAGGGC
eekddddddddddkke
91
1656

541623
82738
82753
Intron 1
AGGTGGTCTCCCTTCA
eekddddddddddkke
74
1657

541624
83056
83071
Intron 1
TCCCAAAGCTCCCCTC
eekddddddddddkke
53
1658

541625
83401
83416
Intron 1
CCTGGCCTAGCAAGCT
eekddddddddddkke
47
1659

541626
84048
84063
Intron 1
TCTTAGCCCTGGGCTA
eekddddddddddkke
12
1660

541627
84388
84403
Intron 1
GACTTGGACTGGGCTC
eekddddddddddkke
81
1661

541628
85261
85276
Intron 1
GGCCTAGGATCTAGGA
eekddddddddddkke
0
1662

541629
85714
85729
Intron 1
GTCAGGCTAGAGGGAC
eekddddddddddkke
41
1663

541630
86220
86235
Intron 1
GGAAGTTCTCCCAGCC
eekddddddddddkke
47
1664

541631
86640
86655
Intron 1
CCTGACTGATGTACAC
eekddddddddddkke
35
1665

541632
86903
86918
Intron 1
CTCTGGCCTAGCCTAT
eekddddddddddkke
54
1666

541633
87247
87262
Intron 1
GGCTGCTGTCAGATGC
eekddddddddddkke
79
1667

541634
88293
88308
Intron 1
TCTCAGGTGTAGGCAG
eekddddddddddkke
59
1668

541635
88605
88620
Intron 1
GGTCACTGAGACTGGG
eekddddddddddkke
88
1669

541636
88952
88967
Intron 1
ACCCACTAGCAGCTAG
eekddddddddddkke
61
1670

541637
89160
89175
Intron 1
CGGATGAGGCAGTTAG
eekddddddddddkke
42
1671

541638
89855
89870
Intron 1
TGGTAGGCCCTCTGGC
eekddddddddddkke
28
1672

541639
90240
90255
Intron 1
GTCACAAGGTGGGTGC
eekddddddddddkke
28
1673

541640
90513
90528
Intron 1
GTCTTGCCCTCACGGA
eekddddddddddkke
73
1674

541641
91073
91088
Intron 1
GCAGTCTGTGGACTTA
eekddddddddddkke
93
1675

541642
91647
91662
Intron 1
TGCTCTCTGGTCACAC
eekddddddddddkke
75
1676

541643
92069
92084
Intron 1
TATCCCCCAGAGCCAT
eekddddddddddkke
68
1677

541644
92356
92371
Intron 1
AAGGTGAGAGGGCACT
eekddddddddddkke
75
1678

541645
92904
92919
Intron 1
GTTTTAACCTCACCCT
eekddddddddddkke
0
1679

541646
93846
93861
Intron 1
CCTTCCACTGACCTTC
eekddddddddddkke
56
1680

541647
94374
94389
Intron 1
GACACTAGCCTAAGCC
eekddddddddddkke
37
1681

TABLE 57

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting intronic regions of SEQ ID NO: 2

SEQ ID
SEQ ID

NO: 2
NO: 2

ISIS
Start
Stop
Target

%
SEQ

NO
Site
Site
Region
Sequence
Chemistry
inhibition
ID NO

541262
156891
156906
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
94
1370

541648
94638
94653
Intron 1
GGTTAGCCCTCAGCCT
eekddddddddddkke
61
1682

541649
94839
94854
Intron 1
TATGAAGGTTGGACCA
eekddddddddddkke
69
1683

541650
95509
95524
Intron 1
CAACCAGCTCACCTGA
eekddddddddddkke
37
1684

541651
95829
95844
Intron 1
GGGCTCCAAGGCTCTC
eekddddddddddkke
75
1685

541652
96158
96173
Intron 1
AGCTGTTACATGCCAA
eekddddddddddkke
93
1686

541653
96488
96503
Intron 1
GGCCCAGAGGTTATAG
eekddddddddddkke
30
1687

541654
96991
97006
Intron 1
GTCCTTAGACCCCTCA
eekddddddddddkke
70
1688

541655
97539
97554
Intron 1
GCCCTGGCTAGAGACA
eekddddddddddkke
39
1689

541656
98132
98147
Intron 1
CATCCAGCAGCTGGAC
eekddddddddddkke
35
1690

541657
98833
98848
Intron 1
GACTGAGGTCATCACA
eekddddddddddkke
60
1691

541658
99258
99273
Intron 1
GGCCAGGCACATCATG
eekddddddddddkke
45
1692

541659
99843
99858
Intron 1
GGAGCTCATTGAGCCA
eekddddddddddkke
36
1693

541660
100406
100421
Intron 1
GTGCCCATTGCTGTGT
eekddddddddddkke
70
1694

541661
100742
100757
Intron 1
CCAAGTGTGGCTTCAG
eekddddddddddkke
54
1695

541662
101305
101320
Intron 1
CCACCCTTTATACGCA
eekddddddddddkke
87
1696

541663
101788
101803
Intron 1
CAGTAACCCCAAGGGA
eekddddddddddkke
12
1697

541664
102649
102664
Intron 1
CCCCACCTTATATGGG
eekddddddddddkke
9
1698

541665
103034
103049
Intron 1
AGGCCCTTTTTACATG
eekddddddddddkke
9
1699

541666
103316
103331
Intron 1
TCAATAAGTCCCTAGG
eekddddddddddkke
20
1700

541667
104277
104292
Intron 1
GGCATTGAGTGACTGC
eekddddddddddkke
51
1701

541668
104679
104694
Intron 1
ATAATGCCTTCTCAGC
eekddddddddddkke
62
1702

541669
106349
106364
Intron 1
GTGAGGCATTTAGCCC
eekddddddddddkke
35
1703

541670
106632
106647
Intron 1
GCTCTTGTGTTGGGTA
eekddddddddddkke
89
1704

541671
107084
107099
Intron 1
TGTGCAGGAGGTCTCA
eekddddddddddkke
60
1705

541672
107949
107964
Intron 1
TGGAGAGTCTTGTCTC
eekddddddddddkke
17
1706

541673
108773
108788
Intron 1
GTGACCCACCCAAGAG
eekddddddddddkke
34
1707

541674
109336
109351
Intron 1
GTTGTAGCTAGTGTTC
eekddddddddddkke
74
1708

541675
109849
109864
Intron 1
GCCTTAGTTTGTGCCA
eekddddddddddkke
78
1709

541676
110427
110442
Intron 1
GCCCCAGCTGAGAATT
eekddddddddddkke
29
1710

541677
110701
110716
Intron 1
ACAACAATCCAGGGTG
eekddddddddddkke
61
1711

541678
110959
110974
Intron 1
CTCCCCTGGAAGTCAC
eekddddddddddkke
59
1712

541679
111307
111322
Intron 1
GCCCTCATGGCTCAAG
eekddddddddddkke
60
1713

541680
112499
112514
Intron 1
TCAGCAGATAGGGAGC
eekddddddddddkke
61
1714

541681
113896
113911
Intron 1
GAATGCGGTGATCAGG
eekddddddddddkke
29
1715

541682
117477
117492
Intron 1
CTGAGAGAATTGGCCC
eekddddddddddkke
5
1716

541683
117740
117755
Intron 1
AGGCACATTGTTACCA
eekddddddddddkke
26
1717

541684
118229
118244
Intron 1
GGGAGGCACTAGAGAA
eekddddddddddkke
13
1718

541685
119269
119284
Intron 1
TACAGTAACACATCCC
eekddddddddddkke
78
1719

541686
119688
119703
Intron 1
GAAGCTCAGCCTGATC
eekddddddddddkke
45
1720

541687
120376
120391
Intron 1
CTTGCCTGACAACCTA
eekddddddddddkke
53
1721

541688
120738
120753
Intron 1
GCCTACCTGCTTTTGC
eekddddddddddkke
10
1722

541689
121242
121257
Intron 1
TTTCCCAACCACTTAG
eekddddddddddkke
7
1723

541690
121615
121630
Intron 1
TCTCCTATTTCAGTTA
eekddddddddddkke
23
1724

541691
121823
121838
Intron 1
GGGTGATGGATGAACT
eekddddddddddkke
40
1725

541692
122345
122360
Intron 1
ACACTGCTGGTAGTGA
eekddddddddddkke
0
1726

541693
122588
122603
Intron 1
ACCCAACTAGCCTGTC
eekddddddddddkke
35
1727

541694
123152
123167
Intron 1
GAGACCTGCTGCCTGA
eekddddddddddkke
80
1728

541695
123671
123686
Intron 1
ACATCTCTTGGGAGGT
eekddddddddddkke
78
1729

541696
124040
124055
Intron 1
ACATAGTACCCCTCCA
eekddddddddddkke
35
1730

541697
124430
124445
Intron 1
CTCTCAAGTACCTGCC
eekddddddddddkke
72
1731

541698
124824
124839
Intron 1
TTTGTACCCAACCCCC
eekddddddddddkke
15
1732

541699
125032
125047
Intron 1
AGGCCCACATAAATGC
eekddddddddddkke
21
1733

541700
125533
125548
Intron 1
GAGCATCCCCTACACT
eekddddddddddkke
12
1734

541701
126357
126372
Intron 1
GCTGGGCCTTTAGCTG
eekddddddddddkke
66
1735

541702
126736
126751
Intron 1
TTGGTCAATTGGGCAG
eekddddddddddkke
79
1736

541703
127179
127194
Intron 1
GTCTCATGAGGCCTAT
eekddddddddddkke
60
1737

541704
127454
127469
Intron 1
GGAGGTGGGATCCCAC
eekddddddddddkke
35
1738

541705
128467
128482
Intron 1
GCCCACTACCTAGCAC
eekddddddddddkke
30
1739

541706
129096
129111
Intron 1
CCCAGCTGGCTGGTCG
eekddddddddddkke
50
1740

541707
129312
129327
Intron 1
GCACCAGGTCTCCTGT
eekddddddddddkke
7
1741

541708
129516
129531
Intron 1
GTCTAGAAGCCTAGGG
eekddddddddddkke
23
1742

541709
129976
129991
Intron 1
GCCGGGTGTTGGTGCA
eekddddddddddkke
50
1743

541710
130308
130323
Intron 1
TTGGTGCCTGTGTTGC
eekddddddddddkke
49
1744

541711
130767
130782
Intron 1
TGCTTCTGATCCCTAC
eekddddddddddkke
18
1745

541712
131286
131301
Intron 1
GTTCCCAGGAGGCTTA
eekddddddddddkke
56
1746

541713
131676
131691
Intron 1
AGGCCCCTAGAGTCTA
eekddddddddddkke
41
1747

541714
132292
132307
Intron 1
TGGTGTGCCCAGACTT
eekddddddddddkke
60
1748

541715
132730
132745
Intron 1
GATGGCTAACCCACTG
eekddddddddddkke
14
1749

541716
133101
133116
Intron 1
CCCCCAAAAGTTGCCC
eekddddddddddkke
12
1750

541717
133522
133537
Intron 1
TAGGGTGTTCCAGATC
eekddddddddddkke
44
1751

541718
133724
133739
Intron 1
GTACCATGAAGCTCTG
eekddddddddddkke
67
1752

541719
134086
134101
Intron 1
CTTGGACTTGGACCAT
eekddddddddddkke
42
1753

541720
134441
134456
Intron 1
GTGCATAGGGCCTGTC
eekddddddddddkke
42
1754

541721
135015
135030
Intron 1
CCTCACCTGAACACCC
eekddddddddddkke
23
1755

541722
135859
135874
Intron 1
ATGCCTCCCCGCAACT
eekddddddddddkke
27
1756

541723
136287
136302
Intron 1
TTGTGCTTGGGTGTAC
eekddddddddddkke
39
1757

541724
137000
137015
Intron 1
AGGCTTCATGTGAGGT
eekddddddddddkke
86
1758

TABLE 58

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting introns 1 and 2 of SEQ ID NO: 2

SEQ
SEQ

ID NO:
ID NO:

ISIS
2 Start
2 Stop
Target

%
SEQ

NO
Site
Site
Region
Sequence
Chemistry
inhibition
ID NO

541262
156891
156906
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
95
1370

541725
137372
137387
Intron 1
TGTAAAAGGTCCTCCC
eekddddddddddkke
53
1759

541726
137750
137765
Intron 1
GACCTGTGCAGCAGGT
eekddddddddddkke
32
1760

541727
138783
138798
Intron 1
TCCTCTTGGAGATCCA
eekddddddddddkke
44
1761

541728
139825
139840
Intron 1
AGGTCATAGGACTGCT
eekddddddddddkke
73
1762

541729
140343
140358
Intron 1
GAAGGTCAGACTAGGG
eekddddddddddkke
53
1763

541730
140686
140701
Intron 1
TCTGTAGACTGCCCAG
eekddddddddddkke
87
1764

541731
141116
141131
Intron 1
GTCCCTCTATTCCCCT
eekddddddddddkke
57
1765

541732
141591
141606
Intron 1
AATTGCCATGCTCCCA
eekddddddddddkke
56
1766

541733
142113
142128
Intron 1
GATGACCTTCCTCCAA
eekddddddddddkke
15
1767

541734
142327
142342
Intron 1
GTTTCCAGTAGCACCT
eekddddddddddkke
82
1768

541735
143118
143133
Intron 1
GGCCTTGAGCTGATGG
eekddddddddddkke
11
1769

541736
143836
143851
Intron 1
TATCCCTAATCAGGCT
eekddddddddddkke
40
1770

541737
144094
144109
Intron 1
GGTGTCCACATCCCGG
eekddddddddddkke
58
1771

541738
144558
144573
Intron 1
AGCTGGACAGGCCATA
eekddddddddddkke
27
1772

541740
145510
145525
Intron 2
GGTAATCACCCAGAGA
eekddddddddddkke
90
1773

541741
145937
145952
Intron 2
GCGCTAAGTCTGCTGT
eekddddddddddkke
92
1774

541742
146320
146335
Intron 2
CCTCAAATCTTGCCCA
eekddddddddddkke
96
1775

541743
147028
147043
Intron 2
ATCCAGACCTGGCAGA
eekddddddddddkke
84
1776

541744
147262
147277
Intron 2
ATCCCTGCTCAAGTGC
eekddddddddddkke
89
1777

541745
147671
147686
Intron 2
CAGGCACTCCTTGGAA
eekddddddddddkke
93
1778

541746
148139
148154
Intron 2
AGCTGAGGTATCCCTC
eekddddddddddkke
94
1779

541747
148564
148579
Intron 2
GGGCCCAGCAAGTCTT
eekddddddddddkke
33
1780

541748
149069
149084
Intron 2
GTTTTGTCAGTGTGCA
eekddddddddddkke
98
1781

541749
149491
149506
Intron 2
GTGACCTGCTGAACTC
eekddddddddddkke
95
1782

541750
150236
150251
Intron 2
GGCTGAACTGTGCACC
eekddddddddddkke
95
1783

541751
150748
150763
Intron 2
GGGTGGTCCCACTCCT
eekddddddddddkke
91
1784

541752
151124
151139
Intron 2
GAGGAATCCTGGGCCC
eekddddddddddkke
94
1785

541753
151373
151388
Intron 2
ATGACAAGCTAGGTGC
eekddddddddddkke
81
1786

541754
151644
151659
Intron 2
TTGCCAGACAGGGCAC
eekddddddddddkke
18
1787

541755
152373
152388
Intron 2
AGACCCCTCCCACTAT
eekddddddddddkke
43
1788

541756
152617
152632
Intron 2
GGTGCTGGGTGACCGG
eekddddddddddkke
91
1789

541757
153349
153364
Intron 2
GGCCAAACGGTGCCCT
eekddddddddddkke
23
1790

541758
153918
153933
Intron 2
TGGGTGAATAGCAACC
eekddddddddddkke
85
1791

541759
154171
154186
Intron 2
GCCCCCAAGGAAGTGA
eekddddddddddkke
76
1792

541760
154813
154828
Intron 2
CAGGCTTCATGTGTGG
eekddddddddddkke
92
1793

541761
155289
155304
Intron 2
CTGTCAGTGCTTTGGT
eekddddddddddkke
52
1794

541762
156233
156248
Intron 2
GAGTACCCTGGCAGGT
eekddddddddddkke
58
1795

541763
156847
156862
Intron 2
TAGCTAGCACCTGGGT
eekddddddddddkke
90
1796

541764
157552
157567
Intron 2
GGCAAACCTTTGAGCC
eekddddddddddkke
27
1797

541765
157927
157942
Intron 2
GCTATCATTGGAGCAG
eekddddddddddkke
94
1798

541766
158542
158557
Intron 2
CCTCTGAGTACTCCCT
eekddddddddddkke
96
1799

541767
159252
159267
Intron 2
AGCTGAAGGCAACCAG
eekddddddddddkke
97
1800

541768
159539
159554
Intron 2
GGGCAGTTTTCCATAG
eekddddddddddkke
89
1801

541769
159778
159793
Intron 2
GGTCCTACCTCTGACA
eekddddddddddkke
82
1802

541770
160352
160367
Intron 2
GGCTGCCTTAGGGTGG
eekddddddddddkke
90
1803

541771
160812
160827
Intron 2
CGCACCTCCCCCACTA
eekddddddddddkke
15
1804

541772
161461
161476
Intron 2
GCTTATTGGTCCATGG
eekddddddddddkke
93
1805

541773
161821
161836
Intron 2
AACCGCAGAGCCCCCA
eekddddddddddkke
76
1806

541774
162132
162147
Intron 2
GGGCTTGTTCTGCCAA
eekddddddddddkke
33
1807

541775
162639
162654
Intron 2
GGGACCTGCGCTGACT
eekddddddddddkke
86
1808

541776
163024
163039
Intron 2
CTTTCACCTGGTGACT
eekddddddddddkke
83
1809

541777
163542
163557
Intron 2
AGCTTGAGGGAGTATA
eekddddddddddkke
52
1810

541778
164144
164159
Intron 2
GCCTGCTCAATTGAGG
eekddddddddddkke
32
1811

541779
164570
164585
Intron 2
ATAGCAGCTGGCTGCC
eekddddddddddkke
24
1812

541780
165419
165434
Intron 2
AAAAGCTTGGCACCCC
eekddddddddddkke
91
1813

541781
165859
165874
Intron 2
CCTGGCAAGAAGGGCC
eekddddddddddkke
65
1814

541782
166435
166450
Intron 2
TTAGCCCATCTATCCC
eekddddddddddkke
82
1815

541783
166837
166852
Intron 2
GTGGTCTCCCTGTGCC
eekddddddddddkke
90
1816

541784
167107
167122
Intron 2
AGCCCTCTCTGGCAAA
eekddddddddddkke
38
1817

541785
168004
168019
Intron 2
TTACTGTGGCCCGAGT
eekddddddddddkke
94
1818

541786
169062
169077
Intron 2
GTAGACTCCTAGGGTC
eekddddddddddkke
90
1819

541787
169696
169711
Intron 2
CCTCCAGTTAGTGTGC
eekddddddddddkke
91
1820

541788
170081
170096
Intron 2
GTGGGTGGCCAACAGG
eekddddddddddkke
91
1821

541789
170799
170814
Intron 2
GGGATTCCCTGGTAGC
eekddddddddddkke
77
1822

541790
171021
171036
Intron 2
GTGAGACCGGCCTTTG
eekddddddddddkke
23
1823

541791
171530
171545
Intron 2
ACTGGCACCCACTTGG
eekddddddddddkke
54
1824

541792
172447
172462
Intron 2
ATTGGCCTAATGCCCC
eekddddddddddkke
76
1825

541793
172733
172748
Intron 2
AGGCTATACATTCCAG
eekddddddddddkke
94
1826

541794
173045
173060
Intron 2
GGTGGCAGCTAGGTGG
eekddddddddddkke
80
1827

541795
173677
173692
Intron 2
TCCACAGTTGGCACTG
eekddddddddddkke
77
1828

541796
174128
174143
Intron 2
TGGGCCTTAGATTGTA
eekddddddddddkke
69
1829

541797
174521
174536
Intron 2
TGTCTTCCTGGTGGCC
eekddddddddddkke
97
1830

541798
174870
174885
Intron 2
CCCGCCTCTCCAGCAA
eekddddddddddkke
89
1831

541799
175275
175290
Intron 2
GCAGCAGCCAATAAGT
eekddddddddddkke
76
1832

541800
175691
175706
Intron 2
TTGTATCCTGGCCCCT
eekddddddddddkke
80
1833

541801
176038
176053
Intron 2
GCCTCATGGGCCTTAC
eekddddddddddkke
66
1834

TABLE 59

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting introns 2 and 3 of SEQ ID NO: 2

SEQ
SEQ ID

ID NO:
NO: 2

SEQ

ISIS
2 Start
Stop
Target

%
ID

NO
Site
Site
Region
Sequence
Chemistry
inhibition
NO

541262
156891
156906
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
97
1370

541802
176619
176634
Intron 2
GGATGCCAGTCTTGGC
eekddddddddddkke
48
1835

541803
176835
176850
Intron 2
CTGCTCTCAGTACCTC
eekddddddddddkke
87
1836

541804
177300
177315
Intron 2
ACCCAAGAAGTCACCT
eekddddddddddkke
93
1837

541805
177551
177566
Intron 2
GCCTCAAGCCCTACCC
eekddddddddddkke
73
1838

541806
178066
178081
Intron 2
AGCTCCAGCCTATAGA
eekddddddddddkke
81
1839

541807
178361
178376
Intron 2
GGTCCACATGGCCCTA
eekddddddddddkke
90
1840

541808
178895
178910
Intron 2
CAGGCCCAGGATTGTC
eekddddddddddkke
81
1841

541809
179444
179459
Intron 2
GGGCCTGCTTTGCAGC
eekddddddddddkke
81
1842

541810
179863
179878
Intron 2
ACTCCTCTCTTTAGGC
eekddddddddddkke
87
1843

541811
180524
180539
Intron 2
CTGGGTAACAGTCCTC
eekddddddddddkke
98
1844

541812
181528
181543
Intron 2
ACTGTATGGTTTCCAC
eekddddddddddkke
83
1845

541813
182103
182118
Intron 2
GCCAAAGATAGCTCTT
eekddddddddddkke
94
1846

541814
182978
182993
Intron 2
GGCATTGGAAGTTGGT
eekddddddddddkke
87
1847

541815
183193
183208
Intron 2
CCCTTCCTGACCTTAC
eekddddddddddkke
55
1848

541816
183658
183673
Intron 2
TTACCCTCTATTCACC
eekddddddddddkke
65
1849

541818
184501
184516
Intron 2
GGCACCCCAGGCCGGG
eekddddddddddkke
25
1850

541819
185080
185095
Intron 2
CAGCAGCTAGTTCCCC
eekddddddddddkke
96
1851

541820
185327
185342
Intron 2
GTGGGCACTAGTGTGT
eekddddddddddkke
75
1852

541821
185682
185697
Intron 2
TGCCCTTGTCAGGGCA
eekddddddddddkke
20
1853

541822
186025
186040
Intron 2
GCAGATAGGCTCAGCA
eekddddddddddkke
98
1854

541823
186570
186585
Intron 2
CCCTAGCCCTTAGCAC
eekddddddddddkke
44
1855

541824
186841
186856
Intron 2
ACTGGAATGGCCCTCT
eekddddddddddkke
86
1856

541825
187176
187191
Intron 2
TTTGCTCATGCTCACA
eekddddddddddkke
96
1857

541826
187629
187644
Intron 2
GCCTTTGTGTGTCACT
eekddddddddddkke
99
1858

541827
187857
187872
Intron 2
TATGTGGTAGCATGTC
eekddddddddddkke
96
1859

541828
188442
188457
Intron 2
CCCCAGGAAGTTGGCC
eekddddddddddkke
68
1860

541829
189086
189101
Intron 2
TAGCTGTCAAGGCCCT
eekddddddddddkke
90
1861

541830
189534
189549
Intron 2
CCTAGTCAGCCACTAG
eekddddddddddkke
20
1862

541831
189889
189904
Intron 2
AGACTCCCCATCAGCC
eekddddddddddkke
74
1863

541832
190172
190187
Intron 2
GTGAAGGGCCTTCATC
eekddddddddddkke
68
1864

541833
190961
190976
Intron 2
GGTTGAGAGTCCAATG
eekddddddddddkke
95
1865

541834
191404
191419
Intron 2
CAGCTAATTCCCTCAT
eekddddddddddkke
79
1866

541835
191614
191629
Intron 2
TTGTGTCTCAACCCAC
eekddddddddddkke
95
1867

541836
191999
192014
Intron 2
GGCTATGCTGCATGCT
eekddddddddddkke
91
1868

541837
192860
192875
Intron 2
CCCCATACCCAGTGGA
eekddddddddddkke
71
1869

541838
193460
193475
Intron 2
GGTGGTTTTCCTCCCT
eekddddddddddkke
95
1870

541839
194144
194159
Intron 2
GAGCCTGCCCAACTTT
eekddddddddddkke
90
1871

541840
194425
194440
Intron 2
TGATGCCCAAGAGTGA
eekddddddddddkke
85
1872

541841
194953
194968
Intron 2
TTCCCTCTGCGAACAT
eekddddddddddkke
96
1873

541842
195428
195443
Intron 2
GTTCCATCTCAATCCA
eekddddddddddkke
94
1874

541843
196858
196873
Intron 2
ACGGCCACTCCACTGG
eekddddddddddkke
44
1875

541844
197326
197341
Intron 2
TGGAAGTGGTTCCAGA
eekddddddddddkke
90
1876

541845
197946
197961
Intron 2
TTGCCCCAGACCAACA
eekddddddddddkke
47
1877

541846
198366
198381
Intron 2
GAGGTTGTGGAGGTGC
eekddddddddddkke
26
1878

541847
198715
198730
Intron 2
GAGTTGCTGTGTGTGA
eekddddddddddkke
83
1879

541848
198939
198954
Intron 2
CATGTCAGAGGTGTCC
eekddddddddddkke
93
1880

541849
199506
199521
Intron 2
AGGTAAGGATCATGGC
eekddddddddddkke
87
1881

541850
199816
199831
Intron 2
GTTCAGTTGCATCACG
eekddddddddddkke
90
1882

541851
200249
200264
Intron 2
GCCCAGCTAGCCACCC
eekddddddddddkke
68
1883

541852
201258
201273
Intron 2
CCTTAGCAGCCAGGCC
eekddddddddddkke
86
1884

541853
202079
202094
Intron 2
GCACTTAGGGTTTTGC
eekddddddddddkke
94
1885

541854
202382
202397
Intron 2
GTTGAACTTTCCCTAC
eekddddddddddkke
53
1886

541855
202702
202717
Intron 2
TGACTCCTTGAGACAG
eekddddddddddkke
83
1887

541856
203098
203113
Intron 2
TGCGCTGGCTTAGCAA
eekddddddddddkke
59
1888

541857
203464
203479
Intron 2
GGCCTAACATCAGCAG
eekddddddddddkke
88
1889

541858
204212
204227
Intron 2
ACTCCTCCCAGTTAGC
eekddddddddddkke
70
1890

541859
205630
205645
Intron 2
ACCAGTGGCCAATGTC
eekddddddddddkke
92
1891

541861
206422
206437
Intron 2
GCCTAGACACAGTAGG
eekddddddddddkke
70
1892

541862
206749
206764
Intron 2
TATTCTCCCCCTAGGG
eekddddddddddkke
42
1893

541863
207517
207532
Intron 2
GACGGCCTTGGGCACA
eekddddddddddkke
96
1894

210196
210211

541865
208659
208674
Intron 3
GCAGGCTGTATTAGCA
eekddddddddddkke
15
1895

541867
209999
210014
Intron 3
ACCCCCTATCCTGCAC
eekddddddddddkke
58
1896

541868
210281
210296
Intron 3
TCCTCCATACCTAGAG
eekddddddddddkke
61
1897

211033
211048

541869
210502
210517
Intron 3
GATAGGTGCCCACTGT
eekddddddddddkke
80
1898

541870
210920
210935
Intron 3
GTCAGTTCTGGCTAGG
eekddddddddddkke
97
1899

541871
211269
211284
Intron 3
GCCTGAACTTACAAGC
eekddddddddddkke
68
1900

541872
211836
211851
Intron 3
ACCCTGGGCTGACCTT
eekddddddddddkke
92
1901

541873
212606
212621
Intron 3
GGACCTGGACAAGCAA
eekddddddddddkke
97
1902

541874
213099
213114
Intron 3
CTCCTTGCGAGAGAGG
eekddddddddddkke
7
1903

541875
213425
213440
Intron 3
AGAGTTGACATGGGCA
eekddddddddddkke
96
1904

541876
213846
213861
Intron 3
CACTAGGTCCCTGACC
eekddddddddddkke
37
1905

541877
214483
214498
Intron 3
CACTCTCTTGGGCTGT
eekddddddddddkke
94
1906

541878
214884
214899
Intron 3
AGGGACCTGCATTCCA
eekddddddddddkke
72
1907

TABLE 60

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting introns 2 and 3 of SEQ ID NO: 2

SEQ ID
SEQ

NO: 2
ID NO:

ISIS
Start
2 Stop
Target

%
SEQ ID

NO
Site
Site
Region
Sequence
Chemistry
inhibition
NO

541262
156891
156906
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
91
1370

541879
215493
215508
Intron 3
TTCACCACCCATTGGG
eekddddddddddkke
63
1908

541880
216192
216207
Intron 3
ATCTGGTCTGAGGGCC
eekddddddddddkke
92
1909

541881
216458
216473
Intron 3
GACATGCAATTGACCC
eekddddddddddkke
98
1910

541882
217580
217595
Intron 3
GTGTGCAGCAGACTGT
eekddddddddddkke
92
1911

541883
218233
218248
Intron 3
GACAGTCCAGCTGCAA
eekddddddddddkke
84
1912

541884
218526
218541
Intron 3
CCTGCGGCAGTGAAGA
eekddddddddddkke
85
1913

541885
218734
218749
Intron 3
CTCTGAGGATAACCCT
eekddddddddddkke
76
1914

541886
219342
219357
Intron 3
GTTCCCAGCTCCCCAA
eekddddddddddkke
68
1915

541887
219618
219633
Intron 3
TAGGGTCAGTGTCCCA
eekddddddddddkke
79
1916

541888
220039
220054
Intron 3
GGCGAGCCTCTCAGCC
eekddddddddddkke
52
1917

541889
220393
220408
Intron 3
GACTCATCCAGGCAGT
eekddddddddddkke
91
1918

541890
220665
220680
Intron 3
TCCCTCCCTTAGGCAC
eekddddddddddkke
71
1919

541891
221044
221059
Intron 3
GAGGAGCCAGGCATAT
eekddddddddddkke
80
1920

541892
221562
221577
Intron 3
CACCAACGAAGTCCCC
eekddddddddddkke
89
1921

541893
221947
221962
Intron 3
GCTGGCAGTCACCAAA
eekddddddddddkke
90
1922

541894
222569
222584
Intron 3
GCCCACACCATTGAGC
eekddddddddddkke
70
1923

541895
222983
222998
Intron 3
AGTGAGATGCCCTGGT
eekddddddddddkke
92
1924

541896
223436
223451
Intron 3
CACTGGCAGTTAGACC
eekddddddddddkke
88
1925

541897
224107
224122
Intron 3
ACTCTGGCCACTAGTA
eekddddddddddkke
80
1926

541898
224731
224746
Intron 3
GGTAGGGTGGCCACAT
eekddddddddddkke
78
1927

541899
225133
225148
Intron 3
GAGCCATGTCTAGGCA
eekddddddddddkke
18
1928

541900
225465
225480
Intron 3
CAGACTGAAACCCACC
eekddddddddddkke
86
1929

541901
225671
225686
Intron 3
TATGGTCCAGCCACCA
eekddddddddddkke
76
1930

541902
226110
226125
Intron 3
TACCTCCTCTGTTGGT
eekddddddddddkke
36
1931

541903
227025
227040
Intron 3
ACACCTCAGTCATGAT
eekddddddddddkke
92
1932

541904
227236
227251
Intron 3
AACAGGCTTCAAGAGG
eekddddddddddkke
91
1933

541905
227485
227500
Intron 3
GTACTACTGGCCATGT
eekddddddddddkke
73
1934

541906
227914
227929
Intron 3
CTGCAGGCGGTTGCTA
eekddddddddddkke
60
1935

541907
228718
228733
Intron 3
GTCTGTTGCCAAGAGC
eekddddddddddkke
95
1936

541908
229174
229189
Intron 3
CCCTGGGTCACTTAAG
eekddddddddddkke
44
1937

541909
229423
229438
Intron 3
CCTGTCCTTGCTTGCA
eekddddddddddkke
96
1938

541910
230042
230057
Intron 3
GCCCAGCTTATCCTAA
eekddddddddddkke
78
1939

541911
230313
230328
Intron 3
AGTAGAGCCTTTGCCT
eekddddddddddkke
75
1940

541912
230580
230595
Intron 3
CTGTCTCTTGGCCCAT
eekddddddddddkke
80
1941

541913
231330
231345
Intron 3
GGCCCAAATCTTGAGT
eekddddddddddkke
67
1942

541914
231817
231832
Intron 3
GCTTGTTACAGCACTA
eekddddddddddkke
92
1943

541915
232088
232103
Intron 3
ACTTTGGCCCAGAGAT
eekddddddddddkke
51
1944

541916
232884
232899
Intron 3
GCAGTCAGGTCAGCTG
eekddddddddddkke
75
1945

541917
233210
233225
Intron 3
GCCTTGTCCTACTACC
eekddddddddddkke
65
1946

541918
233657
233672
Intron 3
GGCTCTGCTATTGGCC
eekddddddddddkke
59
1947

541919
233998
234013
Intron 3
CTTATAGAGCCTTGCC
eekddddddddddkke
59
1948

541920
234296
234311
Intron 3
GGAAGGGCCCAAATAT
eekddddddddddkke
15
1949

541921
234903
234918
Intron 3
GATCTACTCCTACTGC
eekddddddddddkke
65
1950

541922
235313
235328
Intron 3
GTCAGCCTGTGTCTGA
eekddddddddddkke
45
1951

541923
235770
235785
Intron 3
AGCTTCCTCCTTACAC
eekddddddddddkke
54
1952

541924
236198
236213
Intron 3
CTGCTAAGCCCCTACC
eekddddddddddkke
59
1953

541925
236684
236699
Intron 3
AGAGGTCAGGTGCATA
eekddddddddddkke
77
1954

541926
237055
237070
Intron 3
TTCAGCCTGGTTGGGA
eekddddddddddkke
71
1955

541927
237585
237600
Intron 3
GATTGATTGAGCTCCT
eekddddddddddkke
86
1956

541928
237949
237964
Intron 3
ATGGACTCCCTAGGCT
eekddddddddddkke
61
1957

541929
238542
238557
Intron 3
TACTCAAGGGCCCCTC
eekddddddddddkke
67
1958

541930
245319
245334
Intron 3
GGCATATGTAGCTTGC
eekddddddddddkke
91
1959

541931
245765
245780
Intron 3
GAGCTTAGATCTGTGC
eekddddddddddkke
73
1960

541932
246251
246266
Intron 3
ATGCTCACGGCTGTGT
eekddddddddddkke
81
1961

541933
246500
246515
Intron 3
ATTGAAAGGCCCATCA
eekddddddddddkke
45
1962

541934
246936
246951
Intron 3
CAACCCAGTTTGCCGG
eekddddddddddkke
71
1963

541935
247225
247240
Intron 3
CAGCTATTCCCTGTTT
eekddddddddddkke
53
1964

541936
247644
247659
Intron 3
GCTGTGTCACACTTCC
eekddddddddddkke
98
1965

541937
248223
248238
Intron 3
GTCCAAGGATCACAGC
eekddddddddddkke
86
1966

541938
248695
248710
Intron 3
GCTACCACTAGAGCCT
eekddddddddddkke
81
1967

541939
249494
249509
Intron 3
GTTTCAGGGCTTATGT
eekddddddddddkke
63
1968

541940
250693
250708
Intron 3
TCCCACACCTATTGAA
eekddddddddddkke
51
1969

541941
251622
251637
Intron 3
ACTGACTAGAGAGTCC
eekddddddddddkke
81
1970

541942
251950
251965
Intron 3
TCCAAGGCTGATGTCC
eekddddddddddkke
85
1971

541943
252665
252680
Intron 3
TCCCATGGTGGACATG
eekddddddddddkke
39
1972

541944
253140
253155
Intron 3
AGTAGCTGGCAGAAGG
eekddddddddddkke
85
1973

541945
253594
253609
Intron 3
CTGGGAGTGACTACTA
eekddddddddddkke
77
1974

541946
254036
254051
Intron 3
TGGTATAGCTACTGGG
eekddddddddddkke
84
1975

541947
254905
254920
Intron 3
CTGTGGTTTGGCAGGT
eekddddddddddkke
90
1976

541948
255407
255422
Intron 3
GTTCTCACCTGAACTA
eekddddddddddkke
65
1977

541949
255618
255633
Intron 3
ATAGGCTACTGGCAGG
eekddddddddddkke
89
1978

541950
255992
256007
Intron 3
CCCAGCTAGCTGGAGT
eekddddddddddkke
50
1979

541951
256428
256443
Intron 3
GGCTGGCTCTCAAAGG
eekddddddddddkke
61
1980

541952
256689
256704
Intron 3
TGGTGATACTGTGGCA
eekddddddddddkke
94
1981

541953
257317
257332
Intron 3
GCTGATTTTGGTGCCA
eekddddddddddkke
92
1982

541954
257826
257841
Intron 3
GCTAATCTTGCCTCGA
eekddddddddddkke
52
1983

541955
258407
258422
Intron 3
CACTGGTGGCTTTCAA
eekddddddddddkke
31
1984

TABLE 61

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting intronic and exonic regions

of SEQ ID NOs: 1 and 2

SEQ

SEQ ID

ID NO:

NO: 2
SEQ

ISIS
1 Start
Target

%
Start
ID

NO
Site
Region
Sequence
Chemistry
inhibition
Site
NO

541262
n/a
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
93
156891
1370

541956
n/a
Intron 3
GTCCCCTTCTTAAGCA
eekddddddddddkke
56
258980
1985

541957
n/a
Intron 3
GCCAGGCCAACTGTGG
eekddddddddddkke
53
259290
1986

541958
n/a
Intron 3
GGCCCGTTATGGTGGA
eekddddddddddkke
72
259500
1987

541959
n/a
Intron 3
CCTAAAGTCCAACTCC
eekddddddddddkke
76
261641
1988

541960
n/a
Intron 3
CCCTATCCAGCCTTCA
eekddddddddddkke
77
262021
1989

541961
n/a
Intron 3
AAGCATGGCCTCTGGC
eekddddddddddkke
23
262453
1990

541962
n/a
Intron 3
TACCCTGCACCCTCCT
eekddddddddddkke
71
262764
1991

541963
n/a
Intron 3
TCCTTAGTAGAATGCC
eekddddddddddkke
82
263342
1992

541964
n/a
Intron 3
TTAGCCCTGGGAGCAC
eekddddddddddkke
78
263913
1993

541965
n/a
Intron 3
GCTGGGTCAGGTAGCG
eekddddddddddkke
71
266503
1994

541966
n/a
Intron 3
GGGAGGCTCTCAATCT
eekddddddddddkke
75
266861
1995

541967
n/a
Intron 3
GTAAGTGCAGAATGCC
eekddddddddddkke
87
267116
1996

541968
n/a
Intron 3
TGCCGAGGCAGGCACC
eekddddddddddkke
33
267380
1997

541969
n/a
Intron 3
TCCGTGTCTAGGAGGT
eekddddddddddkke
84
267865
1998

541970
n/a
Intron 4
GTCTCCCTGCATTGGA
eekddddddddddkke
31
268366
1999

541971
n/a
Intron 4
CCATATCACTCTCCTC
eekddddddddddkke
79
268786
2000

541972
n/a
Intron 4
CGAACACCTTGAGCCA
eekddddddddddkke
90
269252
2001

541973
n/a
Intron 4
GGCCCAGCTTAAGAGG
eekddddddddddkke
59
270038
2002

541974
n/a
Intron 4
CTGATACTCCTAATCC
eekddddddddddkke
70
270501
2003

541975
n/a
Intron 4
GCCTGTAGGGCTGTGC
eekddddddddddkke
82
270817
2004

541976
n/a
Intron 4
TGCCCTTTCTCCCTAC
eekddddddddddkke
87
271216
2005

541977
n/a
Intron 4
AGTGCATGTCAGTACC
eekddddddddddkke
75
271812
2006

541978
n/a
Intron 4
TGCTCCTCAGCTGTTG
eekddddddddddkke
44
272631
2007

541979
n/a
Intron 4
GTTTGGGACCATCCCT
eekddddddddddkke
41
272834
2008

541980
n/a
Intron 4
AGTGCTCTCTAGGGTC
eekddddddddddkke
87
273257
2009

541981
n/a
Intron 4
TACAGAGAATCACCCC
eekddddddddddkke
82
273651
2010

541982
n/a
Intron 4
GTCCAAGTAAGGTGCT
eekddddddddddkke
57
273947
2011

541983
n/a
Intron 5
GACCTTGCAGGCTTCC
eekddddddddddkke
87
274244
2012

541984
n/a
Intron 5
GGGCAAAGGATCCTCT
eekddddddddddkke
71
274758
2013

541985
n/a
Intron 5
CCCATTCTGCTATCCC
eekddddddddddkke
92
275198
2014

541986
n/a
Intron 5
GCTGACTAGGAGGGCT
eekddddddddddkke
62
275732
2015

541987
n/a
Intron 5
CCTGTGAGGTAGTACC
eekddddddddddkke
83
276309
2016

541988
n/a
Intron 5
GTCCCCCTCCAGTCTA
eekddddddddddkke
50
276932
2017

541989
n/a
Intron 5
GAGGACTCAATTCCTC
eekddddddddddkke
0
277149
2018

541990
n/a
Intron 5
GACAAGGTCCTTTTGG
eekddddddddddkke
43
277391
2019

541991
n/a
Intron 5
GCTCTTGTGTGCACCC
eekddddddddddkke
90
277730
2020

541992
n/a
Intron 5
TCACCGCCTGCACCAC
eekddddddddddkke
75
278342
2021

541993
n/a
Intron 5
GGTTGCACTGTGCAAT
eekddddddddddkke
26
278917
2022

541994
n/a
Intron 6
TTCCACAGGCCTCCAT
eekddddddddddkke
64
279303
2023

541995
n/a
Intron 6
GCTGAGTTCCATATGC
eekddddddddddkke
72
279679
2024

541996
n/a
Intron 6
GAACCGCCACCTCAGG
eekddddddddddkke
38
280157
2025

541997
n/a
Intron 6
GCTCACGGTTGGAGAC
eekddddddddddkke
42
280799
2026

541998
n/a
Intron 6
TGGGCTCCCATGTTCA
eekddddddddddkke
45
281595
2027

541999
n/a
Intron 6
TCACTCTACCAACCTC
eekddddddddddkke
33
282572
2028

542000
n/a
Intron 6
TCCTTGCTTACAGATG
eekddddddddddkke
33
283079
2029

542001
n/a
Intron 6
TGATGCTAGCATTACC
eekddddddddddkke
37
283653
2030

542002
n/a
Intron 6
TGGGTAACTGGCTAGT
eekddddddddddkke
47
285711
2031

542003
n/a
Intron 6
AACCATTCCTCACCAA
eekddddddddddkke
53
287181
2032

542004
n/a
Intron 6
GCCCTGAACAGTTGAT
eekddddddddddkke
37
287895
2033

542005
n/a
Intron 6
GGCTCCTATCATACCT
eekddddddddddkke
38
288943
2034

542006
n/a
Intron 6
TAGGTCTCACAACCCT
eekddddddddddkke
10
289638
2035

542007
n/a
Intron 6
GTGCATTAGTCTTCCA
eekddddddddddkke
74
290035
2036

542008
n/a
Intron 7
CAAAAGCCAGGTTAGC
eekddddddddddkke
13
290503
2037

542009
n/a
Intron 7
CTGCTGTTGACTACCT
eekddddddddddkke
50
290924
2038

542010
n/a
Intron 7
GTACCTGCCAGCTACT
eekddddddddddkke
35
291807
2039

542011
n/a
Exon 8-
CCTACCTTTGCTGTTT
eekddddddddddkke
12
292611
2040

intron 8

junction

542012
n/a
Intron 8
AGTCACCAGCCTAAGC
eekddddddddddkke
47
292860
2041

542013
n/a
Intron 8
AGGCAACCTGGGAGTG
eekddddddddddkke
52
293377
2042

542014
n/a
Intron 8
TGGCCTTCACAATGGC
eekddddddddddkke
33
294052
2043

542015
n/a
Intron 8
GGTGAAGTGGGTTGGA
eekddddddddddkke
27
294536
2044

542016
n/a
Intron 8
GCTGGTTGTCTGCTGC
eekddddddddddkke
60
294931
2045

542017
n/a
Intron 8
AGTTTGTGACCCCTGC
eekddddddddddkke
81
295475
2046

542018
n/a
Intron 8
CCACTCAGTGTGAATG
eekddddddddddkke
85
295955
2047

542019
n/a
Intron 8
CTGGCCTCAGGGCAAT
eekddddddddddkke
51
296186
2048

542020
n/a
Intron 8
GTAGACTTGGGTAGGT
eekddddddddddkke
53
296680
2049

542022
n/a
3'UTR
TGGTGCTAAGCTCTCC
eekddddddddddkke
67
301009
2050

542023
n/a
3'UTR
CATGCTCAAGCTGGAA
eekddddddddddkke
47
301280
2051

542024
206
Exon 2
AAGGTCAACAGCAGCT
eekddddddddddkke
93
144990
2052

542025
207
Exon 2
CAAGGTCAACAGCAGC
eekddddddddddkke
85
144991
2053

542026
208
Exon 2
CCAAGGTCAACAGCAG
eekddddddddddkke
82
144992
2054

542027
209
Exon 2
GCCAAGGTCAACAGCA
eekddddddddddkke
84
144993
2055

TABLE 62

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting intronic and exonic regions

of SEQ ID NOs: 1 and 2

SEQ

SEQ ID

ID NO:

NO: 2

ISIS
1 Start
Target

%
Start
SEQ

NO
Site
Region
Sequence
Chemistry
inhibition
Site
ID NO

541262
n/a
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
86
156891
1370

542034
870
Exon 7
TCTCACACGCACTTCA
eekddddddddddkke
49
290368
2056

542035
871
Exon 7
ATCTCACACGCACTTC
eekddddddddddkke
39
290369
2057

542036
872
Exon 7
GATCTCACACGCACTT
eekddddddddddkke
50
290370
2058

542049
n/a
Intron 1
CTTTCATGAATCAAGC
eekddddddddddkke
85
17928
2059

542050
n/a
Intron 1
TCTTTCATGAATCAAG
eekddddddddddkke
54
17929
2060

542051
n/a
Intron 1
GTCTTTCATGAATCAA
eekddddddddddkke
96
17930
2061

542052
n/a
Intron 1
GGTCTTTCATGAATCA
eekddddddddddkke
98
17931
2062

542053
n/a
Intron 1
ATGGTCTTTCATGAAT
eekddddddddddkke
94
17933
2063

542054
n/a
Intron 1
GATGGTCTTTCATGAA
eekddddddddddkke
73
17934
2064

542055
n/a
Intron 1
TGATGGTCTTTCATGA
eekddddddddddkke
83
17935
2065

542056
n/a
Intron 1
TATATCAATATTCTCC
eekddddddddddkke
75
21821
2066

542057
n/a
Intron 1
TTATATCAATATTCTC
eekddddddddddkke
23
21822
2067

542058
n/a
Intron 1
GTTATATCAATATTCT
eekddddddddddkke
87
21823
2068

542059
n/a
Intron 1
TTTCTTTAGCAATAGT
eekddddddddddkke
85
22519
2069

542060
n/a
Intron 1
CTTTCTTTAGCAATAG
eekddddddddddkke
81
22520
2070

542061
n/a
Intron 1
GCTTTCTTTAGCAATA
eekddddddddddkke
68
22521
2071

542062
n/a
Intron 1
CTCCATTAGGGTTCTG
eekddddddddddkke
91
50948
2072

542063
n/a
Intron 1
TCTCCATTAGGGTTCT
eekddddddddddkke
88
50949
2073

542064
n/a
Intron 1
TTCTCCATTAGGGTTC
eekddddddddddkke
85
50950
2074

542065
n/a
Intron 1
GTTCTCCATTAGGGTT
eekddddddddddkke
84
50951
2075

542066
n/a
Intron 1
AGGTTGGCAGACAGAC
eekddddddddddkke
92
53467
2076

542067
n/a
Intron 1
CAGGTTGGCAGACAGA
eekddddddddddkke
93
53468
2077

542068
n/a
Intron 1
GCAGGTTGGCAGACAG
eekddddddddddkke
91
53469
2078

542069
n/a
Intron 1
CTTCTTGTGAGCTGGC
eekddddddddddkke
95
64885
2079

542070
n/a
Intron 1
TCTTCTTGTGAGCTGG
eekddddddddddkke
89
64886
2080

542071
n/a
Intron 1
GTCTTCTTGTGAGCTG
eekddddddddddkke
96
64887
2081

542072
n/a
Intron 1
AGTCTTCTTGTGAGCT
eekddddddddddkke
81
64888
2082

542073
n/a
Intron 1
TCTTCCACTCACATCC
eekddddddddddkke
89
65991
2083

542074
n/a
Intron 1
CTCTTCCACTCACATC
eekddddddddddkke
79
65992
2084

542075
n/a
Intron 1
TCTCTTCCACTCACAT
eekddddddddddkke
86
65993
2085

542076
n/a
Intron 1
GTCTCTTCCACTCACA
eekddddddddddkke
92
65994
2086

542077
n/a
Intron 1
ATAGATTTTGACTTCC
eekddddddddddkke
86
72108
2087

542078
n/a
Intron 1
CATAGATTTTGACTTC
eekddddddddddkke
42
72109
2088

542079
n/a
Intron 1
GCATAGATTTTGACTT
eekddddddddddkke
66
72110
2089

542080
n/a
Intron 1
AAATGTCAACAGTGCA
eekddddddddddkke
97
80639
2090

542081
n/a
Intron 1
CATGACTATGTTCTGG
eekddddddddddkke
68
125595
2091

542082
n/a
Intron 1
ACATGACTATGTTCTG
eekddddddddddkke
66
125596
2092

542083
n/a
Intron 1
CACATGACTATGTTCT
eekddddddddddkke
74
125597
2093

542084
n/a
Intron 2
GAATTCTGAGCTCTGG
eekddddddddddkke
91
145430
2094

542085
n/a
Intron 2
TGAATTCTGAGCTCTG
eekddddddddddkke
94
145431
2095

542086
n/a
Intron 2
CTGAATTCTGAGCTCT
eekddddddddddkke
94
145432
2096

542087
n/a
Intron 2
CCTGAATTCTGAGCTC
eekddddddddddkke
93
145433
2097

542088
n/a
Intron 2
GCCTGAATTCTGAGCT
eekddddddddddkke
87
145434
2098

542089
n/a
Intron 2
AGCCTGAATTCTGAGC
eekddddddddddkke
84
145435
2099

542090
n/a
Intron 2
ATATTGTAATTCTTGG
eekddddddddddkke
47
148060
2100

542091
n/a
Intron 2
GATATTGTAATTCTTG
eekddddddddddkke
61
148061
2101

542092
n/a
Intron 2
TGATATTGTAATTCTT
eekddddddddddkke
0
148062
2102

542093
n/a
Intron 2
CTGATATTGTAATTCT
eekddddddddddkke
58
148063
2103

542094
n/a
Intron 2
CCTGATATTGTAATTC
eekddddddddddkke
95
148064
2104

542095
n/a
Intron 2
GCCTGATATTGTAATT
eekddddddddddkke
85
148065
2105

542096
n/a
Intron 2
TGCCTGATATTGTAAT
eekddddddddddkke
86
148066
2106

542097
n/a
Intron 2
ATTATGTGCTTTGCCT
eekddddddddddkke
86
148907
2107

542098
n/a
Intron 2
AATTATGTGCTTTGCC
eekddddddddddkke
75
148908
2108

542099
n/a
Intron 2
CAATTATGTGCTTTGC
eekddddddddddkke
88
148909
2109

542100
n/a
Intron 2
TCAATTATGTGCTTTG
eekddddddddddkke
78
148910
2110

542101
n/a
Intron 2
GTCAATTATGTGCTTT
eekddddddddddkke
97
148911
2111

542102
n/a
Intron 2
GCCATCACCAAACACC
eekddddddddddkke
97
150973
2112

542103
n/a
Intron 2
TGCCATCACCAAACAC
eekddddddddddkke
90
150974
2113

542104
n/a
Intron 2
TTGCCATCACCAAACA
eekddddddddddkke
89
150975
2114

542105
n/a
Intron 2
TGGTGACTCTGCCTGA
eekddddddddddkke
98
151388
2115

542106
n/a
Intron 2
CTGGTGACTCTGCCTG
eekddddddddddkke
96
151389
2116

542107
n/a
Intron 2
GCTGGTGACTCTGCCT
eekddddddddddkke
98
151390
2117

542108
n/a
Intron 2
TGCTGGTGACTCTGCC
eekddddddddddkke
97
151391
2118

542109
n/a
Intron 2
CTGCTGGTGACTCTGC
eekddddddddddkke
93
151392
2119

TABLE 63

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting introns 2 and 3 of SEQ ID NO: 2

SEQ

ID
SEQ ID

NO: 2
NO: 2

ISIS
Start
Stop
Target

%
SEQ

NO
Site
Site
Region
Sequence
Chemistry
inhibition
ID NO

541262
156891
156906
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
95
1370

542110
153002
153017
Intron 2
AGTAGTCAATATTATT
eekddddddddddkke
74
2120

542111
153003
153018
Intron 2
CAGTAGTCAATATTAT
eekddddddddddkke
55
2121

542112
153004
153019
Intron 2
CCAGTAGTCAATATTA
eekddddddddddkke
97
2122

542113
153922
153937
Intron 2
CCTTTGGGTGAATAGC
eekddddddddddkke
90
2123

542114
153923
153938
Intron 2
ACCTTTGGGTGAATAG
eekddddddddddkke
71
2124

542115
153924
153939
Intron 2
CACCTTTGGGTGAATA
eekddddddddddkke
78
2125

542116
155595
155610
Intron 2
CAACTTGAGGACAATA
eekddddddddddkke
89
2126

542118
155597
155612
Intron 2
CTCAACTTGAGGACAA
eekddddddddddkke
98
2127

542119
156395
156410
Intron 2
CAGGAAGAAAGGAACC
eekddddddddddkke
95
2128

542120
156396
156411
Intron 2
CCAGGAAGAAAGGAAC
eekddddddddddkke
83
2129

542121
156397
156412
Intron 2
ACCAGGAAGAAAGGAA
eekddddddddddkke
90
2130

542122
156595
156610
Intron 2
TGCAGTCATGTACACA
eekddddddddddkke
97
2131

542123
156596
156611
Intron 2
CTGCAGTCATGTACAC
eekddddddddddkke
90
2132

542124
156597
156612
Intron 2
TCTGCAGTCATGTACA
eekddddddddddkke
81
2133

542125
156890
156905
Intron 2
TGGTTTGTCAATCCTT
eekddddddddddkke
97
2134

542126
156892
156907
Intron 2
CTTGGTTTGTCAATCC
eekddddddddddkke
99
2135

542127
157204
157219
Intron 2
GCTACAATGCACAGGA
eekddddddddddkke
98
2136

542128
157205
157220
Intron 2
TGCTACAATGCACAGG
eekddddddddddkke
98
2137

542129
158008
158023
Intron 2
GATATTTATTGCTGTA
eekddddddddddkke
61
2138

542130
158009
158024
Intron 2
TGATATTTATTGCTGT
eekddddddddddkke
41
2139

542131
158010
158025
Intron 2
CTGATATTTATTGCTG
eekddddddddddkke
86
2140

542132
162752
162767
Intron 2
AGGGTCTTTACAAAGT
eekddddddddddkke
69
2141

542133
162753
162768
Intron 2
CAGGGTCTTTACAAAG
eekddddddddddkke
71
2142

542134
162754
162769
Intron 2
CCAGGGTCTTTACAAA
eekddddddddddkke
93
2143

542135
166353
166368
Intron 2
TTCTGCAGTATCCTAG
eekddddddddddkke
84
2144

542136
166354
166369
Intron 2
TTTCTGCAGTATCCTA
eekddddddddddkke
88
2145

542137
166355
166370
Intron 2
GTTTCTGCAGTATCCT
eekddddddddddkke
95
2146

542138
166356
166371
Intron 2
AGTTTCTGCAGTATCC
eekddddddddddkke
92
2147

542139
166357
166372
Intron 2
CAGTTTCTGCAGTATC
eekddddddddddkke
93
2148

542140
172747
172762
Intron 2
CAAATTCCAGTCCTAG
eekddddddddddkke
73
2149

542141
172748
172763
Intron 2
CCAAATTCCAGTCCTA
eekddddddddddkke
91
2150

542142
172749
172764
Intron 2
TCCAAATTCCAGTCCT
eekddddddddddkke
90
2151

542143
175372
175387
Intron 2
ACCCATTTCATCCATT
eekddddddddddkke
94
2152

542144
175373
175388
Intron 2
AACCCATTTCATCCAT
eekddddddddddkke
93
2153

542145
175374
175389
Intron 2
GAACCCATTTCATCCA
eekddddddddddkke
97
2154

542146
175375
175390
Intron 2
GGAACCCATTTCATCC
eekddddddddddkke
96
2155

542147
175376
175391
Intron 2
AGGAACCCATTTCATC
eekddddddddddkke
68
2156

542148
189120
189135
Intron 2
GCTTCATGTCTTTCTA
eekddddddddddkke
90
2157

542149
189121
189136
Intron 2
TGCTTCATGTCTTTCT
eekddddddddddkke
96
2158

542150
189122
189137
Intron 2
GTGCTTCATGTCTTTC
eekddddddddddkke
97
2159

542151
189485
189500
Intron 2
TGAGCTTAGCAGTCAC
eekddddddddddkke
92
2160

542152
189486
189501
Intron 2
ATGAGCTTAGCAGTCA
eekddddddddddkke
95
2161

542153
189487
189502
Intron 2
CATGAGCTTAGCAGTC
eekddddddddddkke
95
2162

542154
191143
191158
Intron 2
TACAGACATAGCTCTA
eekddddddddddkke
91
2163

542155
191144
191159
Intron 2
ATACAGACATAGCTCT
eekddddddddddkke
74
2164

542156
191145
191160
Intron 2
GATACAGACATAGCTC
eekddddddddddkke
91
2165

542157
191146
191161
Intron 2
GGATACAGACATAGCT
eekddddddddddkke
94
2166

542158
198149
198164
Intron 2
TGTGGCTTTAATTCAC
eekddddddddddkke
71
2167

542159
198150
198165
Intron 2
ATGTGGCTTTAATTCA
eekddddddddddkke
81
2168

542160
198151
198166
Intron 2
TATGTGGCTTTAATTC
eekddddddddddkke
78
2169

542161
199817
199832
Intron 2
TGTTCAGTTGCATCAC
eekddddddddddkke
91
2170

542162
199818
199833
Intron 2
GTGTTCAGTTGCATCA
eekddddddddddkke
89
2171

542163
199819
199834
Intron 2
TGTGTTCAGTTGCATC
eekddddddddddkke
90
2172

542164
210562
210577
Intron 3
CATCTGGATGTGAGGC
eekddddddddddkke
90
2173

542165
210563
210578
Intron 3
ACATCTGGATGTGAGG
eekddddddddddkke
78
2174

542166
210564
210579
Intron 3
CACATCTGGATGTGAG
eekddddddddddkke
55
2175

542167
219020
219035
Intron 3
TCAGGTAATTTCTGGA
eekddddddddddkke
82
2176

542168
219021
219036
Intron 3
CTCAGGTAATTTCTGG
eekddddddddddkke
73
2177

542169
219022
219037
Intron 3
TCTCAGGTAATTTCTG
eekddddddddddkke
40
2178

542170
225568
225583
Intron 3
TGCTTATTTACCTGGG
eekddddddddddkke
90
2179

542171
225569
225584
Intron 3
TTGCTTATTTACCTGG
eekddddddddddkke
90
2180

542172
225570
225585
Intron 3
TTTGCTTATTTACCTG
eekddddddddddkke
79
2181

542173
225571
225586
Intron 3
TTTTGCTTATTTACCT
eekddddddddddkke
32
2182

542174
229619
229634
Intron 3
ATGATGTTACTACTAC
eekddddddddddkke
63
2183

542175
229620
229635
Intron 3
AATGATGTTACTACTA
eekddddddddddkke
53
2184

542176
229621
229636
Intron 3
CAATGATGTTACTACT
eekddddddddddkke
12
2185

542177
232827
232842
Intron 3
CCCCTAGAGCAATGGT
eekddddddddddkke
76
2186

542178
232828
232843
Intron 3
CCCCCTAGAGCAATGG
eekddddddddddkke
83
2187

542179
232829
232844
Intron 3
TCCCCCTAGAGCAATG
eekddddddddddkke
49
2188

542180
237676
237691
Intron 3
TCAATTGCAGATGCTC
eekddddddddddkke
88
2189

542181
237677
237692
Intron 3
CTCAATTGCAGATGCT
eekddddddddddkke
90
2190

542182
237678
237693
Intron 3
GCTCAATTGCAGATGC
eekddddddddddkke
81
2191

542183
237679
237694
Intron 3
AGCTCAATTGCAGATG
eekddddddddddkke
85
2192

542184
248232
248247
Intron 3
GTATATTCAGTCCAAG
eekddddddddddkke
90
2193

542185
248233
248248
Intron 3
AGTATATTCAGTCCAA
eekddddddddddkke
94
2194

542186
248234
248249
Intron 3
CAGTATATTCAGTCCA
eekddddddddddkke
97
2195

TABLE 64

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting intronic and exonic regions

of SEQ ID NOs: 1 and 2

SEQ

ID

SEQ ID

NO: 1

NO: 2

ISIS
Start

%
Start
SEQ ID

NO
Site
Target Region
Sequence
Chemistry
inhibition
Site
NO

541262
n/a
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
93
156891
1370

545316
168
exon 1-intron 1
ACCTCCGAGCTTCGCC
eekddddddddddkke
80
3044
2196

junction

545317
173
exon-exon
GTAGGACCTCCGAGCT
eekddddddddddkke
74
n/a
2197

junction

545318
177
exon-exon
ACCTGTAGGACCTCCG
eekddddddddddkke
70
n/a
2198

junction

545321
213
Exon 2
CAGTGCCAAGGTCAAC
eekddddddddddkke
77
144997
2199

545322
225
Exon 2
ACTTGATCCTGCCAGT
eekddddddddddkke
36
145009
2200

545332
361
Exon 4/Intron 3
CTCGCTCAGGTGAACG
eekddddddddddkke
57
268024
2201

545333
366
Exon 4/Intron 3
AGTCTCTCGCTCAGGT
eekddddddddddkke
88
268029
2202

545337
444
Exon 4-intron 4
CCTTCTGGTATAGAAC
eekddddddddddkke
21
268107
2203

junction

545340
570
Exon 5
GCTAGTTAGCTTGATA
eekddddddddddkke
39
274130
2204

545343
626
exon 3-exon 4
TCTGGTTGCACTATTT
eekddddddddddkke
34
n/a
2205

junction

545344
629
exon 3-exon 4
GGATCTGGTTGCACTA
eekddddddddddkke
30
n/a
2206

junction

545345
632
Exon 6
GGTGGATCTGGTTGCA
eekddddddddddkke
18
278926
2207

545346
638
Exon 6
GCAATGGGTGGATCTG
eekddddddddddkke
50
278932
2208

545347
647
Exon 6
CAGTTGAGGGCAATGG
eekddddddddddkke
71
278941
2209

545348
651
Exon 6
AGTCCAGTTGAGGGCA
eekddddddddddkke
58
278945
2210

545349
655
Exon 6
GTAAAGTCCAGTTGAG
eekddddddddddkke
34
278949
2211

545350
660
Exon 6
GTTCAGTAAAGTCCAG
eekddddddddddkke
52
278954
2212

545351
685
Exon 6
CTGCATGAATCCCAGT
eekddddddddddkke
77
278979
2213

545355
923
Exon 7
ACATAGAGCACCTCAC
eekddddddddddkke
38
290421
2214

545356
926
Exon 7
GTTACATAGAGCACCT
eekddddddddddkke
79
290424
2215

545357
929
Exon 7
AGTGTTACATAGAGCA
eekddddddddddkke
70
290427
2216

545362
1124
Exon 7-exon 8
TCCTTGAGGAGATCTG
eekddddddddddkke
3
n/a
2217

junction

545363
1170
Exon 10
GCTATCATGAATGGCT
eekddddddddddkke
69
297587
2218

545364
1180
Exon 10
CGGGTTTATAGCTATC
eekddddddddddkke
58
297597
2219

545369
1320
Exon 10
ATCCTTCACCCCTAGG
eekddddddddddkke
46
297737
2220

545370
1328
Exon 10
GAGTCGCCATCCTTCA
eekddddddddddkke
60
297745
2221

545371
1332
Exon 10
TCCAGAGTCGCCATCC
eekddddddddddkke
51
297749
2222

545373
1418
Exon 10
GGCTGAGCAACCTCTG
eekddddddddddkke
80
297835
2223

545374
1422
Exon 10
CTGTGGCTGAGCAACC
eekddddddddddkke
63
297839
2224

545380
1524
Exon 10
GATAACACTGGGCTGC
eekddddddddddkke
60
297941
2225

545381
1530
Exon 10
TGCTTGGATAACACTG
eekddddddddddkke
76
297947
2226

545382
1533
Exon 10
CTCTGCTTGGATAACA
eekddddddddddkke
60
297950
2227

545386
1600
Exon 10
GCTGAATATGGGCAGC
eekddddddddddkke
29
298017
2228

545387
1613
Exon 10
CTTGGATTGCTTAGCT
eekddddddddddkke
59
298030
2229

545388
1645
Exon 10
CCTGGGCATAAAAGTC
eekddddddddddkke
47
298062
2230

545392
1832
Exon 10
ACCTTGATGTGAGGAG
eekddddddddddkke
44
298249
2231

TABLE 65

Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting intronic and exonic regions

of SEQ ID NOs: 1 and 2

SEQ

SEQ

ID

ID

NO: 1

NO: 2

ISIS
Start
Target

%
Start
SEQ ID

NO
Site
Region
Sequence
Chemistry
inhibition
Site
NO

541262
n/a
Intron 2
TTGGTTTGTCAATCCT
eekddddddddddkke
89
156891
1370

545393
1838
Exon 10
GATTCAACCTTGATGT
eekddddddddddkke
40
298255
2232

545394
1844
Exon 10
ATGTGTGATTCAACCT
eekddddddddddkke
80
298261
2233

545395
1956
Exon 10
TGGGACAGGCATCTCA
eekddddddddddkke
29
298373
2234

545396
1961
Exon 10
TAGTCTGGGACAGGCA
eekddddddddddkke
48
298378
2235

545397
1968
Exon 10
GGAGGTATAGTCTGGG
eekddddddddddkke
61
298385
2236

545398
1986
Exon 10
GGACTGTACTATATGA
eekddddddddddkke
48
298403
2237

545401
2077
Exon 10
TCAGTTGGTCTGTGCT
eekddddddddddkke
60
298494
2238

545402
2095
Exon 10
GCTAAGGCATGATTTT
eekddddddddddkke
53
298512
2239

545406
2665
Exon 10
GCCATGCTTGAAGTCT
eekddddddddddkke
87
299082
2240

545407
2668
Exon 10
ATAGCCATGCTTGAAG
eekddddddddddkke
70
299085
2241

545408
2692
Exon 10
ACACAGTGTGTAGTGT
eekddddddddddkke
60
299109
2242

545409
2699
Exon 10
CTGCAGTACACAGTGT
eekddddddddddkke
31
299116
2243

545410
2704
Exon 10
ACCAACTGCAGTACAC
eekddddddddddkke
57
299121
2244

545411
2739
Exon 10
TAGACTGTAGTTGCTA
eekddddddddddkke
53
299156
2245

545412
2747
Exon 10
ACCAGCTTTAGACTGT
eekddddddddddkke
56
299164
2246

545413
2945
Exon 10
GTAAGTTGATCTGTGC
eekddddddddddkke
79
299362
2247

545414
2963
Exon 10
TACTTCTTTTGGTGCC
eekddddddddddkke
82
299380
2248

545416
3212
Exon 10
TCTTGTACCTTATTCC
eekddddddddddkke
73
299629
2249

545417
3306
Exon 10
TGGTTATAGGCTGTGA
eekddddddddddkke
90
299723
2250

545418
3309
Exon 10
GTCTGGTTATAGGCTG
eekddddddddddkke
88
299726
2251

545419
3313
Exon 10
ATGTGTCTGGTTATAG
eekddddddddddkke
68
299730
2252

545420
3317
Exon 10
GAGTATGTGTCTGGTT
eekddddddddddkke
84
299734
2253

545421
4049
Exon 10
GGTCTGCGATAAATGG
eekddddddddddkke
69
300466
2254

545429
4424
Exon 10
GCCAGACACAACTAGT
eekddddddddddkke
59
300841
2255

545430
31
Exon 1
ACCGCCACTGTAGCAG
eekddddddddddkke
76
2907
2256

545431
36
Exon 1
CCGCCACCGCCACTGT
eekddddddddddkke
94
2912
2257

545432
103
Exon 1
GGGCCTCCGGCCCGCG
eekddddddddddkke
22
2979
2258

545433
143
Exon 1
AGAGCGCGGGTTCGCG
eekddddddddddkke
61
3019
2259

545434
n/a
Intron
TACTGACCCCAGTTCC
eekddddddddddkke
68
3654
2260

1/Exon 1

545435
n/a
Intron
ACTCTACTGACCCCAG
eekddddddddddkke
70
3658
2261

1/Exon 1

545436
n/a
Intron
GTCACTCTACTGACCC
eekddddddddddkke
83
3661
2262

1/Exon 1

545437
n/a
Intron
TTCATGCGGACTGGTG
eekddddddddddkke
68
3680
2263

1/Exon 1

545438
n/a
Intron
GTGAGCATGGACCCCA
eekddddddddddkke
94
225436
2264

3/Exon 3

545439
n/a
Intron
TGATATGTGAGCATGG
eekddddddddddkke
88
225442
2265

3/Exon 3

545440
n/a
Intron
AAGTTGGTGAGCTTCT
eekddddddddddkke
85
226785
2266

3/Exon 3

545441
n/a
Intron
CCTTCAAGTTGGTGAG
eekddddddddddkke
88
226790
2267

3/Exon 3

545442
n/a
Intron
GTAAGATCCTTTTGCC
eekddddddddddkke
70
226883
2268

3/Exon 3

545443
n/a
Intron
CAGCTGTGCAACTTGC
eekddddddddddkke
50
238345
2269

3/Exon 3

545444
n/a
Intron
GCCTTGGTAGGTAGGG
eekddddddddddkke
68
238422
2270

3/Exon 3

545445
n/a
Intron
AGAGCCTTGGTAGGTA
eekddddddddddkke
85
238425
2271

3/Exon 3

545446
n/a
Intron
CCCGCACAAACGCGCA
eekddddddddddkke
10
3614
2272

1/Exon 1

545447
n/a
Intron
GTCTTCAAGGTCAGTT
eekddddddddddkke
92
93208
2273

1/Exon 1

545448
n/a
Intron
GCCCAGTGAATTCAGC
eekddddddddddkke
76
93246
2274

1/Exon 1

545449
n/a
Intron
AGATGCGCCCAGTGAA
eekddddddddddkke
60
93252
2275

1/Exon 1

545450
n/a
Intron
GTAAGATGCGCCCAGT
eekddddddddddkke
78
93255
2276

1/Exon 1

545451
n/a
Intron
CCAGAAGGCACTTGTA
eekddddddddddkke
42
93301
2277

1/Exon 1

545452
n/a
Intron
GGAAGATTTGCAGAAC
eekddddddddddkke
15
93340
2278

1/Exon 1

545453
n/a
Intron
CCTTGGTCATGGAAGA
eekddddddddddkke
35
93350
2279

1/Exon 1

545454
n/a
Intron
TGACCTTGGTCATGGA
eekddddddddddkke
55
93353
2280

1/Exon 1

545455
n/a
Intron
GAGGTGACCTTGGTCA
eekddddddddddkke
70
93357
2281

1/Exon 1

545456
n/a
Intron
ATCCAAAGAGGTGACC
eekddddddddddkke
41
93364
2282

1/Exon 1

545457
n/a
Intron
GCCAATCCAAAGAGGT
eekddddddddddkke
56
93368
2283

1/Exon 1

545458
n/a
Intron
GGTCTGCCAATCCAAA
eekddddddddddkke
79
93373
2284

1/Exon 1

545459
n/a
Intron
CCCTGGGTCTGCCAAT
eekddddddddddkke
68
93378
2285

1/Exon 1

545460
n/a
Intron
GAGATCTCAACAAGGG
eekddddddddddkke
52
93427
2286

1/Exon 1

545461
n/a
Intron
CGCCCATCACTCTTCC
eekddddddddddkke
68
93988
2287

1/Exon 1

545462
n/a
Intron
CACCTGTCGCCCATCA
eekddddddddddkke
67
93995
2288

1/Exon 1

545463
n/a
Intron
CATCACCTGTCGCCCA
eekddddddddddkke
78
93998
2289

1/Exon 1

545464
n/a
Intron
CACCATCACCTGTCGC
eekddddddddddkke
74
94001
2290

1/Exon 1

545465
n/a
Intron
AATAGTTGTCACCATC
eekddddddddddkke
76
94010
2291

1/Exon 1

545466
n/a
Intron
GCCACCTTTCATGAGA
eekddddddddddkke
58
94048
2292

1/Exon 1

545467
n/a
Intron
CTCTTGGAAGTAGGTA
eekddddddddddkke
89
198762
2293

2/Exon 2

545468
n/a
Intron
GTTCTCTTGGAAGTAG
eekddddddddddkke
80
198765
2294

2/Exon 2

545469
n/a
Intron
TAAACAGGTTGGTCTG
eekddddddddddkke
68
198854
2295

2/Exon 2

Example 8: Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by Deoxy, MOE and cEt Gapmers

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

TABLE 66

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

541396
30
51
68
74
67
1.4

541262
55
87
90
94
97
0.2

541393
30
38
52
66
81
2.1

541375
41
45
54
64
79
1.6

541438
44
49
75
80
91
0.9

541428
35
32
56
78
88
1.8

541491
13
46
67
55
95
2.0

541435
21
46
55
72
94
1.9

541471
11
49
50
77
89
2.0

541430
24
44
56
57
79
2.2

541492
32
40
65
80
85
1.5

541431
22
46
73
84
92
1.5

TABLE 67

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

541487
36
46
66
85
92
1.3

541423
33
55
64
80
93
1.2

541452
37
60
79
87
94
0.9

541505
51
75
86
92
97
0.4

541522
54
76
81
90
95
0.3

541539
65
76
85
94
98
0.2

541503
54
65
80
93
97
0.5

541520
43
61
86
94
96
0.7

541515
57
72
85
92
94
0.3

541564
57
72
88
90
97
0.3

541554
43
65
81
89
93
0.7

541509
11
8
19
6
8
>10

541584
59
65
84
91
96
0.3

541585
70
80
93
92
98
0.1

TABLE 68

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

541598
26
43
75
80
76
1.5

541592
35
48
67
85
95
1.2

541641
22
63
70
91
93
1.2

541590
27
59
70
94
95
1.2

541615
40
65
84
88
94
0.7

541595
35
57
73
84
95
1.0

541575
49
60
79
84
95
0.6

541571
41
50
76
80
94
1.0

541582
0
10
25
50
82
4.4

541262
66
79
93
94
99
<0.6

541652
1
44
80
82
87
1.9

541670
29
40
63
79
89
1.6

541662
17
13
45
62
84
3.1

541724
37
47
72
85
95
1.2

TABLE 69

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

541748
86
94
96
98
98
<0.6

541767
83
91
95
96
98
<0.6

541797
78
89
93
97
99
<0.6

541766
59
82
92
97
99
<0.6

541742
65
87
93
95
99
<0.6

541750
80
86
96
96
99
<0.6

541262
79
88
93
97
97
<0.6

541749
71
84
93
95
98
<0.6

541793
71
88
94
97
98
<0.6

541785
56
79
89
93
98
<0.6

541746
34
61
85
94
97
0.9

541752
49
72
88
93
93
<0.6

541826
86
94
95
99
98
<0.6

541811
66
87
93
97
98
<0.6

TABLE 70

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

541822
83
88
95
96
96
<0.6

541870
77
87
95
97
98
<0.6

541262
85
93
96
97
98
<0.6

541873
32
77
93
94
97
0.7

541819
60
91
97
97
99
<0.6

541841
86
91
95
96
97
<0.6

541825
78
88
95
98
98
<0.6

541863
63
77
87
93
97
<0.6

541827
42
80
87
94
97
<0.6

541875
77
84
93
96
97
<0.6

541835
56
73
90
95
98
<0.6

541838
72
90
93
98
97
<0.6

541833
52
69
83
92
97
<0.6

541813
47
75
86
95
97
<0.6

TABLE 71

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

541853
74
79
88
93
91
<0.6

541842
69
85
91
97
99
<0.6

541877
79
91
93
98
97
<0.6

541848
58
90
96
98
98
0.7

541804
23
81
89
95
95
0.8

541881
87
94
98
98
99
<0.6

541936
91
96
98
99
98
<0.6

541909
56
80
89
95
97
<0.6

541907
75
91
95
97
98
<0.6

541952
68
81
93
97
98
<0.6

541953
68
80
94
97
98
<0.6

541914
60
78
94
97
97
<0.6

541880
56
74
89
94
95
<0.6

541903
37
74
87
96
98
0.6

TABLE 72

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

541895
47
72
85
93
94
<0.6

541882
60
67
89
93
97
<0.6

541889
63
80
87
94
97
<0.6

541904
26
78
23
89
93
1.4

545418
0
81
91
94
95
1.7

541930
58
71
82
88
92
<0.6

545439
67
87
93
96
98
<0.6

542024
15
58
78
87
90
1.4

541985
59
81
88
93
97
<0.6

541972
47
58
83
90
92
0.6

541991
57
64
88
92
83
<0.6

541980
33
50
76
72
93
1.2

TABLE 73

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

541264
26
44
64
79
89
1.6

541265
29
32
62
79
91
1.8

541263
25
40
62
78
93
1.7

541268
57
73
85
90
95
0.3

541266
15
33
46
66
90
2.5

542107
93
97
98
98
98
<0.6

542052
93
96
97
96
98
<0.6

542105
80
92
96
98
97
<0.6

542102
94
96
96
97
98
<0.6

542108
90
92
94
97
99
<0.6

542080
87
93
95
95
97
<0.6

TABLE 74

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

542101
90
97
97
97
95
<0.6

542051
89
96
95
98
97
<0.6

542106
83
93
96
96
98
<0.6

542071
84
91
94
97
97
<0.6

542094
85
92
94
97
98
<0.6

542069
89
94
97
95
98
<0.6

542086
83
94
96
97
98
<0.6

542085
85
92
96
97
97
<0.6

542053
64
83
94
98
97
<0.6

542087
69
84
99
95
98
<0.6

542109
87
94
96
98
98
<0.6

542126
96
98
99
98
98
<0.6

542127
94
96
97
98
97
<0.6

542128
90
96
98
98
97
<0.6

TABLE 75

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

542118
97
97
98
95
43
<0.6

542186
93
96
98
99
98
<0.6

542150
95
97
98
99
99
<0.6

542122
90
94
98
98
99
<0.6

542125
88
97
98
98
99
<0.6

542145
90
96
98
99
99
<0.6

542112
86
94
99
99
99
<0.6

542149
88
93
99
98
99
<0.6

542146
79
93
96
97
98
<0.6

542153
87
94
97
98
99
<0.6

542119
64
84
93
97
98
<0.6

542137
76
91
97
97
98
<0.6

542152
84
94
96
96
97
<0.6

542157
83
95
98
99
98
<0.6

TABLE 76

0.625
1.250
2.50
5.00
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

542185
82
93
96
96
94
<0.6

542143
81
91
96
98
98
<0.6

542144
77
93
95
96
99
<0.6

542139
87
93
98
98
98
<0.6

542134
83
90
90
95
96
<0.6

545333
68
85
91
96
98
<0.6

545373
57
73
86
92
97
<0.6

545438
84
96
98
97
99
<0.6

545431
77
91
93
97
98
<0.6

545447
70
85
96
96
97
<0.6

545417
62
82
90
93
95
<0.6

545467
77
88
91
94
95
<0.6

545441
63
82
92
94
96
<0.6

Example 9: Dose-Dependent Antisense Inhibition of Human GHR in Hep3B Cells by Deoxy, MOE and cEt Gapmers

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.04 μM, 0.11 μM, 0.33 μM, 1.00 μM, and 3.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

TABLE 77

0.04
0.11
0.33
1.00
3.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

539380
11
16
57
93
98
0.2

541724
0
27
71
66
83
0.3

541748
28
40
71
90
97
0.1

541767
19
38
54
87
98
0.2

541797
23
46
70
88
97
0.1

541766
15
26
49
82
96
0.3

541742
17
28
41
80
95
0.3

541750
33
27
60
89
98
0.2

541749
27
16
62
84
82
0.2

541793
0
14
44
77
96
0.4

541785
4
11
39
75
95
0.4

541752
14
6
45
70
94
0.4

541826
8
34
74
94
99
0.2

541811
6
4
45
79
97
0.4

541822
9
29
67
89
97
0.2

TABLE 78

0.04
0.11
0.33
1.00
3.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

539380
0
16
47
82
98
0.4

541819
3
12
50
76
94
0.3

541841
0
19
47
80
95
0.3

541825
0
6
40
74
96
0.4

541827
5
26
48
76
95
0.3

541835
7
11
33
74
93
0.4

541838
21
26
61
90
97
0.2

541833
0
9
41
63
89
0.5

541813
0
17
28
65
92
0.5

541842
5
15
30
72
90
0.4

541804
0
12
3
49
79
1.1

542024
0
0
26
54
76
1.0

542107
15
45
78
92
99
0.1

542105
2
14
55
88
98
0.3

542102
10
16
73
88
98
0.2

TABLE 79

0.04
0.11
0.33
1.00
3.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

539380
4
18
50
86
95
0.3

542108
15
13
65
86
97
0.2

542101
17
40
68
92
98
0.2

542106
4
23
56
88
98
0.3

542094
0
30
51
86
96
0.3

542086
13
38
50
84
97
0.2

542085
0
27
57
90
98
0.3

542087
7
3
49
80
92
0.4

542109
17
10
56
88
98
0.3

542126
40
63
91
96
99
<0.03

542127
27
47
69
93
97
0.1

542128
11
30
66
90
98
0.2

542118
14
42
77
95
98
0.1

542150
31
46
72
94
98
0.1

542122
13
14
59
90
97
0.3

TABLE 80

0.04
0.11
0.33
1.00
3.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

539380
0
2
50
86
97
0.4

542125
31
32
69
89
96
0.1

542145
15
29
64
91
97
0.2

542112
14
38
61
87
96
0.2

542149
9
37
63
90
97
0.2

542146
13
33
59
82
95
0.2

542153
22
26
63
86
96
0.2

542119
10
20
34
70
87
0.4

542137
3
19
47
77
95
0.3

542152
0
9
47
82
96
0.4

542157
0
26
56
84
96
0.3

542143
8
12
44
81
95
0.3

542144
0
21
42
75
95
0.4

542139
0
14
46
82
97
0.4

542134
3
23
43
72
92
0.4

TABLE 81

0.04
0.11
0.33
1.00
3.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

539380
0
9
64
85
97
0.3

541870
7
15
48
80
92
0.3

541262
0
29
63
90
98
0.2

541863
0
26
40
82
93
0.4

541875
6
30
71
84
91
0.2

541853
0
13
39
67
91
0.5

541877
0
26
41
79
94
0.4

541881
0
30
54
87
94
0.3

541936
20
41
73
93
98
0.1

541909
0
16
34
64
90
0.5

541907
6
31
59
84
96
0.2

541952
0
0
50
72
92
0.5

541953
0
22
50
80
92
0.4

541914
0
0
46
76
93
0.4

541880
0
13
48
79
89
0.4

TABLE 82

0.04
0.11
0.33
1.00
3.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

539380
0
5
53
78
94
0.4

541903
12
20
26
62
88
0.5

541895
3
12
29
66
92
0.5

541882
2
0
27
65
86
0.7

541889
12
12
47
68
87
0.4

541930
0
6
40
59
85
0.6

541985
0
16
41
66
93
0.4

542031
1
0
22
55
80
0.8

541972
0
1
23
46
83
0.9

541991
4
35
42
67
89
0.4

542052
5
28
70
92
98
0.2

542080
0
18
54
87
96
0.3

542051
0
18
52
86
97
0.3

542071
5
3
51
74
95
0.4

542069
0
7
56
85
94
0.3

TABLE 83

0.04
0.11
0.33
1.00
3.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

539380
11
20
54
89
92
0.3

542053
6
14
38
69
74
0.6

542186
14
43
70
90
98
0.2

542185
0
26
48
80
96
0.3

545333
0
4
27
65
90
0.6

545336
0
15
24
43
79
0.9

545373
0
2
9
42
86
1.0

545438
0
24
56
81
92
0.3

545431
0
18
50
73
91
0.4

545447
0
15
34
78
93
0.4

545417
0
11
39
66
87
0.5

545467
12
16
37
76
93
0.4

545441
21
15
20
60
87
0.6

545439
17
24
49
82
91
0.3

Example 10: Dose-Dependent Antisense Inhibition of Rhesus Monkey GHR in LLC-MK2 Cells

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested for their potency for rhesus GHR mRNA in LLC-MK2 cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

TABLE 84

0.12
0.37
1.11
3.33
10.00
IC₅₀

ISIS No
Chemistry
μM
μM
μM
μM
μM
(μM)

541262
Deoxy, MOE and
9
25
42
85
91
1.1

cEt

541742
Deoxy, MOE and
0
24
19
58
77
3.2

cEt

541767
Deoxy, MOE and
6
10
30
68
88
2.0

cEt

541875
Deoxy, MOE and
7
19
64
84
96
0.9

cEt

541881
Deoxy, MOE and
6
24
59
79
91
1.0

cEt

542101
Deoxy, MOE and
0
5
38
71
81
2.0

cEt

542112
Deoxy, MOE and
5
17
33
67
76
2.0

cEt

542118
Deoxy, MOE and
1
6
35
68
86
2.0

cEt

542125
Deoxy, MOE and
0
12
57
83
93
1.0

cEt

542127
Deoxy, MOE and
1
0
30
68
84
2.4

cEt

542128
Deoxy, MOE and
12
0
26
58
83
2.7

cEt

542153
Deoxy, MOE and
4
0
0
36
59
6.6

cEt

542185
Deoxy, MOE and
4
0
25
56
87
2.5

cEt

542186
Deoxy, MOE and
15
23
51
73
90
1.1

cEt

542051
Deoxy, MOE and
5
19
40
81
94
1.2

cEt

TABLE 85

0.12
0.37
1.11
3.33
10.00
IC₅₀

ISIS No
Chemistry
μM
μM
μM
μM
μM
(μM)

523723
5-10-5 MOE
23
14
31
43
71
3.5

532254
5-10-5 MOE
29
35
42
69
87
0.8

532401
5-10-5 MOE
27
28
46
73
88
1.2

533932
5-10-5 MOE
10
24
48
70
92
1.2

539376
3-10-4 MOE
21
8
8
35
81
4.3

539399
3-10-4 MOE
2
10
14
18
57
8.3

539404
3-10-4 MOE
39
12
25
27
57
7.7

539416
3-10-4 MOE
24
35
44
79
89
1.0

539432
3-10-4 MOE
9
29
42
73
89
1.2

541262
Deoxy, MOE
0
43
63
88
94
0.8

and cEt

541742
Deoxy, MOE
3
19
35
56
85
1.9

and cEt

541767
Deoxy, MOE
3
24
39
64
86
1.6

and cEt

545439
Deoxy, MOE
19
15
43
74
80
1.7

and cEt

545447
Deoxy, MOE
25
34
58
80
90
0.6

and cEt

Example 11: Dose-Dependent Antisense Inhibition of GHR in Cynomolgus Primary Hepatocytes

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested for their potency for GHR mRNA in cynomolgus monkey primary hepatocytes. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

TABLE 86

0.12
0.37
1.11
3.33
10.00
IC₅₀

ISIS No
Chemistry
μM
μM
μM
μM
μM
(μM)

541262
Deoxy, MOE
40
52
75
92
98
0.3

and cEt

541742
Deoxy, MOE
40
57
51
91
96
0.2

and cEt

541767
Deoxy, MOE
36
59
60
78
91
0.4

and cEt

541875
Deoxy, MOE
54
76
88
95
95
<0.1

and cEt

541881
Deoxy, MOE
53
75
85
98
98
<0.1

and cEt

542101
Deoxy, MOE
38
55
78
89
97
0.2

and cEt

542112
Deoxy, MOE
28
50
74
89
96
0.4

and cEt

542118
Deoxy, MOE
20
45
69
84
91
0.5

and cEt

542125
Deoxy, MOE
33
62
77
92
97
0.3

and cEt

542127
Deoxy, MOE
30
50
65
86
92
0.4

and cEt

542128
Deoxy, MOE
25
40
52
80
93
0.7

and cEt

542153
Deoxy, MOE
10
31
51
73
85
1.0

and cEt

542185
Deoxy, MOE
12
45
65
85
93
0.6

and cEt

542186
Deoxy, MOE
36
54
74
90
96
0.3

and cEt

542051
Deoxy, MOE
9
29
32
32
42
>10

and cEt

TABLE 87

0.12
0.37
1.11
3.33
10.00
IC₅₀

ISIS No
Chemistry
μM
μM
μM
μM
μM
(μM)

523435
5-10-5
35
47
61
74
85
0.5

MOE

523723
5-10-5
4
16
40
66
86
1.8

MOE

532254
5-10-5
14
15
24
16
9
>10

MOE

532401
5-10-5
37
54
73
88
94
0.3

MOE

533932
5-10-5
23
40
69
78
86
0.6

MOE

539376
3-10-4
3
0
44
65
91
2.0

MOE

539399
3-10-4
0
0
9
42
67
5.0

MOE

539404
3-10-4
0
0
26
52
71
3.5

MOE

539416
3-10-4
8
29
62
89
93
0.7

MOE

539432
3-10-4
0
24
55
85
93
0.9

MOE

541262
Deoxy,
23
52
73
92
96
0.4

MOE and

cEt

541742
Deoxy,
15
51
73
86
97
0.5

MOE and

cEt

541767
Deoxy,
19
20
39
68
81
1.8

MOE and

cEt

545439
Deoxy,
0
0
30
61
90
2.4

MOE and

cEt

545447
Deoxy,
0
17
17
19
27
>10

MOE and

cEt

Example 12: Dose-Dependent Antisense Inhibition of GHR in Hep3B Cells

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested for their potency for GHR mRNA at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

TABLE 88

0.12
0.37
1.11
3.33
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

541262
25
43
76
85
94
0.5

541742
32
55
76
88
97
0.3

541767
29
56
83
89
97
0.3

541875
38
68
84
93
94
0.1

541881
32
57
81
94
97
0.3

542051
34
66
83
95
98
0.2

542101
25
55
85
95
98
0.3

542112
18
56
83
95
98
0.4

542118
42
61
88
95
97
0.1

542125
30
63
87
95
98
0.2

542127
50
70
91
91
98
0.1

542128
38
63
88
96
98
0.2

542153
37
59
85
94
97
0.2

542185
44
51
76
89
96
0.2

542186
46
59
84
95
97
0.1

TABLE 89

0.12
0.37
1.11
3.33
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

523435
9
26
49
78
93
1.0

523723
7
16
39
72
90
1.4

532254
36
46
69
86
94
0.4

532401
25
54
71
86
91
0.4

533932
8
47
69
80
94
0.7

539376
26
31
54
73
86
0.8

539399
23
43
72
89
94
0.5

539404
30
60
88
95
98
0.2

539416
30
59
84
93
98
0.3

539432
35
62
88
95
98
0.2

541262
43
60
84
89
98
0.2

541742
23
53
73
84
97
0.4

541767
22
49
74
85
92
0.4

545439
41
69
88
95
96
0.1

545447
31
47
63
74
82
0.5

Example 13: Dose-Dependent Antisense Inhibition of GHR in Cynomolgus Primary Hepatocytes

Gapmers from studies described above exhibiting significant in vitro inhibition of GHR mRNA were selected and tested at various doses in cynomolgous monkey primary hepatocytes. Cells were plated at a density of 35,000 cells per well and transfected using electroporation with 0.04 μM, 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

TABLE 90

0.04
0.12
0.37
1.11
3.33
10.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
μM
(μM)

541767
8
17
29
48
59
58
0.4

541875
20
39
48
51
55
58
0.2

541881
23
36
49
60
56
58
0.1

542112
23
21
35
42
54
68
0.5

542118
19
14
26
38
54
59
0.8

542153
17
20
27
39
46
52
2.2

542185
20
23
27
46
39
56
2.0

532254
1
20
23
11
1
23
>10

532401
0
15
24
39
47
55
1.6

523723
0
0
7
24
49
54
2.0

Example 14: Comparative Analysis of Dose-Dependent Antisense Inhibition of GHR in Hep3B Cells

ISIS 532401 was compared with specific antisense oligonucleotides disclosed in US 2006/0178325 by testing at various doses in Hep3B cells. The oligonucleotides were selected based on the potency demonstrated in studies described in the application. 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, 1.11 μM, 3.00 μM, and 9.00 μM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and GHR mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3437_MGB was used to measure mRNA levels. GHR mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN©. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. The results indicate that ISIS 532401 was markedly more potent than the most potent oligonucleotides of US 2006/0178325.

TABLE 91

0.11
0.33
1.00
3.00
9.00
IC₅₀

ISIS No
μM
μM
μM
μM
μM
(μM)

227452
11
12
46
73
92
1.4

227488
26
25
39
76
88
1.2

272309
16
14
39
66
91
1.6

272322
13
20
44
70
86
1.4

272328
22
20
24
43
56
5.7

272338
22
24
52
71
85
1.1

532401
34
53
72
87
94
0.3

Example 15: Tolerability of 5-10-5 MOE Gapmers Targeting Human GHR in CD1 Mice

CD1 ® mice (Charles River, Mass.) are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of eight- to ten-week old male CD1 mice were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS oligonucleotides (100 mg/kg/week dose). One group of male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 92. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 92

Plasma chemistry markers in CD1 mice plasma at week 6

ALT
AST
Bilirubin
Creatinine
BUN

(IU/L)
(IU/L)
(mg/dL)
(mg/dL)
(mg/dL)

PBS
31
50
0.28
0.15
28

ISIS 523271
366
285
0.18
0.11
29

ISIS 523324
222
139
0.19
0.10
31

ISIS 523604
2106
1157
0.41
0.06
48

ISIS 532254
66
84
0.11
0.10
27

ISIS 533121
176
155
0.19
0.09
27

ISIS 533161
1094
904
0.23
0.07
29

ISIS 533178
78
83
0.18
0.08
28

ISIS 533234
164
147
0.21
0.09
26

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and platelets, and total hemoglobin content. The results are presented in Table 93. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 93

Hematology markers in CD1 mice plasma at week 6

HCT
Hemoglobin
RBC
WBC
Platelets

(%)
(g/dL)
(10⁶/μL)
(10³/μL)
(10³/μL)

PBS
45
13
8.2
4.1
689

ISIS 523271
42
12
7.9
4.5
1181

ISIS 523324
39
11
7.5
7.9
980

ISIS 523604
33
10
6.9
14.1
507

ISIS 532254
35
10
6.9
7.2
861

ISIS 533121
39
12
7.9
8.4
853

ISIS 533161
49
14
9.3
9.0
607

ISIS 533178
44
13
8.5
6.9
765

ISIS 533234
42
12
7.8
9.2
1045

Example 16: Tolerability of 5-10-5 MOE Gapmers Targeting Human GHR in CD1 Mice

CD1 @ mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of eight- to ten-week old male CD1 mice were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide (100 mg/kg/week dose). One group of male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 94. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 94

Plasma chemistry markers in CD1 mice plasma at week 6

ALT
AST
Bilirubin
Creatinine
BUN

(IU/L)
(IU/L)
(mg/dL)
(mg/dL)
(mg/dL)

PBS
30
59
0.26
0.14
20

ISIS 523715
636
505
0.24
0.14
22

ISIS 523723
57
80
0.20
0.16
23

ISIS 523726
165
167
0.18
0.15
23

ISIS 523736
140
177
0.20
0.15
23

ISIS 523747
96
108
0.17
0.14
23

ISIS 523789
45
74
0.20
0.15
22

ISIS 532395
64
ill
0.23
0.12
21

ISIS 532401
47
88
0.21
0.17
22

ISIS 532411
225
426
0.17
0.16
22

ISIS 532420
60
99
0.21
0.12
25

ISIS 532468
319
273
0.15
0.14
21

ISIS 533932
62
81
0.18
0.14
21

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WB), RBC, and platelets, and total hemoglobin content. The results are presented in Table 95. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 95

Hematology markers in CD1 mice plasma at week 6

HCT
Hemoglobin
RBC
WBC
Platelets

(%)
(g/dL)
(10⁶/μL)
(10³/μL)
(10³/μL)

PBS
43
13
8.1
3.3
1047

ISIS 523715
40
12
8.1
4.2
1153

ISIS 523723
35
11
6.8
2.9
1154

ISIS 523726
32
10
6.8
5.8
1056

ISIS 523736
35
11
7.1
3.6
1019

ISIS 523747
37
11
7.7
2.8
1146

ISIS 523789
37
11
7.3
2.5
1033

ISIS 532395
37
11
7.4
4.5
890

ISIS 532401
36
11
7.1
3.7
1175

ISIS 532411
27
8
5.3
3.2
641

ISIS 532420
35
11
7.0
3.3
1101

ISIS 532468
36
11
7.4
4.0
1043

ISIS 533932
36
11
7.2
3.8
981

Example 17: Tolerability of 3-10-4 MOE Gapmers Targeting Human GHR in CD1 Mice

CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 96. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 96

Plasma chemistry markers in CD1 mice plasma at week 6

ALT
AST
Bilirubin
Creatinine
BUN

(IU/L)
(IU/L)
(mg/dL)
(mg/dL)
(mg/dL)

PBS
48
63
0.20
0.13
28

ISIS 539302
204
192
0.15
0.15
24

ISIS 539321
726
455
0.17
0.12
27

ISIS 539360
3287
2495
0.58
0.13
22

ISIS 539361
310
226
0.17
0.11
21

ISIS 539376
77
75
0.14
0.12
27

ISIS 539379
134
136
0.16
0.13
24

ISIS 539380
180
188
0.14
0.12
23

ISIS 539383
80
81
0.15
0.12
25

ISIS 539399
119
127
0.13
0.12
24

ISIS 539401
1435
1172
0.24
0.11
24

ISIS 539403
1543
883
0.18
0.12
26

ISIS 539404
75
109
0.16
0.13
23

ISIS 539416
100
107
0.19
0.15
26

ISIS 539432
55
64
0.20
0.14
22

ISIS 539433
86
91
0.12
0.13
22

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and platelets, and total hemoglobin content. The results are presented in Table 97. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 97

Hematology markers in CD1 mice plasma at week 6

HCT
Hemoglobin
RBC
WBC
Platelets

(%)
(g/dL)
(10⁶/μL)
(10³/μL)
(10³/μL)

PBS
46
13
8.5
6
954

ISIS 539302
40
11
8.1
13
830

ISIS 539321
39
11
7.8
16
723

ISIS 539360
49
14
9.0
14
671

ISIS 539361
45
13
8.5
9
893

ISIS 539376
42
12
7.7
6
988

ISIS 539379
42
12
8.1
7
795

ISIS 539380
38
10
7.7
8
950

ISIS 539383
45
12
8.4
8
795

ISIS 539399
41
12
8.0
10
895

ISIS 539401
41
11
8.2
9
897

ISIS 539403
33
9
6.2
13
1104

ISIS 539404
42
12
8.4
7
641

ISIS 539416
41
11
7.5
5
686

ISIS 539432
44
12
8.0
6
920

ISIS 539433
40
11
7.4
6
987

Example 18: Tolerability of Deoxy, MOE and cEt Gapmers Targeting Human GHR in CD1 Mice

CD1@ mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of eight- to ten-week old male CD1 mice were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS oligonucleotide (50 mg/kg/week dose). One group of male CD1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 98. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 98

Plasma chemistry markers in CD1 mice plasma at week 6

ALT
AST
Bilirubin
Creatinine
BUN

(IU/L)
(IU/L)
(mg/dL)
(mg/dL)
(mg/dL)

PBS
36
71
0.22
0.18
22

ISIS 541262
115
133
0.21
0.18
21

ISIS 541724
543
531
0.34
0.17
21

ISIS 541742
44
71
0.18
0.16
21

ISIS 541748
269
582
0.16
0.15
22

ISIS 541749
626
491
0.20
0.20
22

ISIS 541750
1531
670
0.20
0.18
23

ISIS 541766
2107
1139
0.21
0.21
23

ISIS 541767
42
62
0.21
0.17
20

ISIS 541822
493
202
0.13
0.16
22

ISIS 541826
889
398
0.21
0.14
17

ISIS 541838
266
172
0.16
0.15
20

ISIS 541870
445
272
0.23
0.16
23

ISIS 541875
103
114
0.20
0.15
20

ISIS 541907
940
725
0.16
0.19
35

ISIS 541991
1690
1733
0.31
0.20
23

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and platelets, and total hemoglobin content. The results are presented in Table 99. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 99

Hematology markers in CD1 mice plasma at week 6

HCT
Hemoglobin
RBC
WBC
Platelets

(%)
(g/dL)
(10⁶/μL)
(10³/μL)
(10³/μL)

PBS
37
11
7
3
1083

ISIS 541262
38
11
7
6
1010

ISIS 541724
52
16
10
9
940

ISIS 541742
47
14
9
6
1134

ISIS 541748
41
12
8
7
941

ISIS 541749
41
12
8
5
1142

ISIS 541750
42
12
8
4
1409

ISIS 541766
39
11
7
7
989

ISIS 541767
46
14
9
2
994

ISIS 541822
42
12
8
3
1190

ISIS 541826
41
12
8
10
1069

ISIS 541838
44
13
8
6
1005

ISIS 541870
38
11
7
8
1020

ISIS 541875
44
13
8
6
1104

ISIS 541907
40
11
8
9
1271

ISIS 541991
34
10
6
6
1274

Example 19: Tolerability of Deoxy, MOE and cEt Gapmers Targeting Human GHR in CD1 Mice

CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers. The 3-10-4 MOE gapmer ISIS 539376 was also included in the study.

Treatment

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 100. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 100

Plasma chemistry markers in CD1 mice plasma at week 6

ALT
AST
Bilirubin
Creatinine
BUN

(IU/L)
(IU/L)
(mg/dL)
(mg/dL)
(mg/dL)

PBS
43
66
0.21
0.11
20

ISIS 541881
63
109
0.28
0.13
23

ISIS 541936
3260
2108
0.40
0.13
24

ISIS 542051
97
119
0.23
0.12
23

ISIS 542052
454
236
0.23
0.12
25

ISIS 542069
293
211
0.23
0.13
27

ISIS 542085
91
87
0.18
0.10
21

ISIS 542086
137
133
0.24
0.10
23

ISIS 542094
86
143
0.23
0.13
21

ISIS 542101
46
74
0.19
0.10
21

ISIS 542102
4920
2432
2.30
0.15
29

ISIS 542105
1255
575
0.35
0.13
21

ISIS 542106
3082
2295
3.42
0.17
23

ISIS 542107
4049
3092
0.50
0.14
20

ISIS 542108
1835
859
0.32
0.11
21

ISIS 539376
40
79
0.27
0.08
22

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and total hemoglobin content. The results are presented in Table 101. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 101

Hematology markers in CD1 mice plasma at week 6

HCT
Hemoglobin
RBC
WBC

(%)
(g/dL)
(10⁶/μL)
(10³/μL)

PBS
46
13
8
6

ISIS 541881
53
15
10
7

ISIS 541936
41
11
8
18

ISIS 542051
49
14
9
8

ISIS 542052
46
13
9
9

ISIS 542069
43
13
8
7

ISIS 542085
38
11
7
5

ISIS 542086
49
14
9
9

ISIS 542094
36
10
6
5

ISIS 542101
44
13
9
5

ISIS 542102
27
7
5
25

ISIS 542105
42
12
8
7

ISIS 542106
37
10
7
14

ISIS 542107
41
12
7
17

ISIS 542108
51
14
8
10

ISIS 539376
49
14
10
5

Example 20: Tolerability of Deoxy, MOE and cEt Gapmers Targeting Human GHR in CD1 Mice

CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 102. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 102

Plasma chemistry markers in CD1 mice plasma at week 6

ALT
AST
Bilirubin
Creatinine
BUN

(IU/L)
(IU/L)
(mg/dL)
(mg/dL)
(mg/dL)

PBS
51
63
0.3
0.14
27

ISIS 542109
3695
2391
0.8
0.19
24

ISIS 542112
119
104
0.3
0.16
28

ISIS 542118
66
86
0.3
0.15
26

ISIS 542122
1112
350
0.3
0.16
27

ISIS 542125
79
92
0.2
0.13
26

ISIS 542126
381
398
0.5
0.14
23

ISIS 542127
54
85
0.3
0.16
26

ISIS 542128
55
89
0.2
0.12
24

ISIS 542145
834
671
0.3
0.11
24

ISIS 542146
163
107
0.2
0.14
30

ISIS 542149
974
752
0.3
0.12
26

ISIS 542150
2840
2126
2.4
0.17
23

ISIS 542153
53
75
0.2
0.14
28

ISIS 542157
137
122
0.3
0.13
25

ISIS 542185
57
72
0.2
0.11
23

ISIS 542186
62
84
0.2
0.12
24

ISIS 545431
2622
1375
3.0
0.15
28

ISIS 545438
1710
1000
0.3
0.14
26

ISIS 545439
70
117
0.2
0.12
28

ISIS 545447
141
108
0.3
0.13
26

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and total hemoglobin content. The results are presented in Table 103. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 103

Hematology markers in CD1 mice plasma at week 6

HCT
Hemoglobin
RBC
WBC
Platelets

(%)
(g/dL)
(10⁶/μL)
(10³/μL)
(10³/μL)

PBS
40
12
7
6
1210

ISIS 542109
47
13
9
16
1244

ISIS 542112
50
13
8
7
1065

ISIS 542118
42
12
8
8
1120

ISIS 542122
37
11
7
7
1064

ISIS 542125
42
13
8
7
1063

ISIS 542126
34
10
7
9
1477

ISIS 542127
41
12
7
7
1144

ISIS 542128
40
12
7
6
1196

ISIS 542145
42
12
8
8
1305

ISIS 542146
45
13
8
7
1310

ISIS 542149
33
10
6
12
903

ISIS 542150
27
7
5
18
1202

ISIS 542153
46
13
8
5
1130

ISIS 542157
44
12
9
6
791

ISIS 542185
45
13
8
3
1031

ISIS 542186
44
12
8
6
985

ISIS 545431
28
7
6
13
2609

ISIS 545438
40
11
8
8
1302

ISIS 545439
48
13
9
4
857

ISIS 545447
45
13
9
9
964

Example 21: Tolerability of MOE Gapmers Targeting Human GHR in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide (100 mg/kg weekly dose). Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 104 expressed in IU/L. Plasma levels of bilirubin were also measured using the same clinical chemistry analyzer and the results are also presented in Table 104 expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 104

Liver function markers in Sprague-Dawley rats

ALT
AST
Bilirubin

(IU/L)
(IU/L)
(mg/dL)

PBS
69
90
0.15

ISIS 523723
79
123
0.12

ISIS 523789
71
105
0.15

ISIS 532254
67
97
0.14

ISIS 532401
61
77
0.12

ISIS 532420
102
127
0.17

ISIS 533178
157
219
0.34

ISIS 533234
71
90
0.11

ISIS 533932
58
81
0.12

ISIS 539376
75
101
0.14

ISIS 539380
86
128
0.16

ISIS 539383
64
94
0.14

ISIS 539399
52
95
0.14

ISIS 539404
88
118
0.13

ISIS 539416
63
104
0.14

ISIS 539432
63
90
0.13

ISIS 539433
69
92
0.13

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 105, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 105

Kidney function markers (mg/dL)

in Sprague-Dawley rats

BUN
Creatinine

PBS
24
0.32

ISIS 523723
20
0.39

ISIS 523789
19
0.37

ISIS 532254
21
0.43

ISIS 532401
17
0.36

ISIS 532420
20
0.31

ISIS 533178
20
0.43

ISIS 533234
22
0.41

ISIS 533932
19
0.43

ISIS 539376
19
0.36

ISIS 539380
18
0.35

ISIS 539383
19
0.35

ISIS 539399
18
0.39

ISIS 539404
23
0.39

ISIS 539416
17
0.39

ISIS 539432
20
0.39

ISIS 539433
20
0.34

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and total hemoglobin content. The results are presented in Table 106. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 106

Hematology markers in Sprague-Dawley rats

HCT
Hemoglobin
RBC
WBC
Platelets

(%)
(g/dL)
(10⁶/μL)
(10³/μL)
(10³/μL)

PBS
46
15
8
11
1078

ISIS 523723
38
12
7
19
626

ISIS 523789
38
12
8
12
702

ISIS 532254
36
12
7
11
547

ISIS 532401
42
14
8
12
858

ISIS 532420
37
12
7
17
542

ISIS 533178
37
12
7
15
1117

ISIS 533234
38
12
7
8
657

ISIS 533932
40
13
7
9
999

ISIS 539376
43
14
9
8
910

ISIS 539380
33
11
5
6
330

ISIS 539383
39
13
7
10
832

ISIS 539399
37
11
7
4
603

ISIS 539404
37
12
7
6
639

ISIS 539416
33
11
6
9
601

ISIS 539432
44
14
9
10
810

ISIS 539433
38
12
7
9
742

Organ Weights

Liver, heart, spleen and kidney weights were measured at the end of the study, and are presented in Table 107. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 107

Organ weights (g)

Heart
Liver
Spleen
Kidney

PBS
0.35
3.6
0.2
0.8

ISIS 523723
0.31
4.9
0.7
0.8

ISIS 523789
0.34
4.8
0.6
0.8

ISIS 532254
0.32
5.0
0.6
1.0

ISIS 532401
0.32
3.8
0.4
0.8

ISIS 532420
0.29
4.6
0.7
1.0

ISIS 533178
0.34
5.2
0.7
0.9

ISIS 533234
0.30
4.4
0.6
1.0

ISIS 533932
0.31
3.9
0.5
0.9

ISIS 539376
0.29
4.4
0.4
0.8

ISIS 539380
0.31
6.3
1.6
1.2

ISIS 539383
0.31
4.5
0.6
1.0

ISIS 539399
0.31
4.5
0.8
1.0

ISIS 539404
0.34
4.9
0.6
1.0

ISIS 539416
0.32
4.7
0.7
0.9

ISIS 539432
0.30
3.8
0.4
0.8

ISIS 539433
0.28
4.1
0.7
1.0

Example 22: Tolerability of Deoxy, MOE, and cEt Gapmers Targeting Human GHR in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide (50 mg/kg weekly dose). Two groups of rats were injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT and AST were measured and the results are presented in Table 108 expressed in IU/L. Plasma levels of bilirubin were also measured using the same clinical chemistry analyzer and the results are also presented in Table 108 expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 108

Liver function markers in Sprague-Dawley rats

ALT
AST
Bilirubin

(IU/L)
(IU/L)
(mg/dL)

PBS group 1
34
56
0.08

PBS group 2
37
54
0.09

ISIS 541881
53
77
0.12

ISIS 542051
61
96
0.09

ISIS 542101
64
214
0.10

ISIS 542112
46
72
0.10

ISIS 542118
42
60
0.08

ISIS 542125
39
67
0.10

ISIS 542127
56
75
0.12

ISIS 542128
45
71
0.12

ISIS 542153
44
69
0.11

ISIS 542185
44
93
0.09

ISIS 542186
51
107
0.12

ISIS 545439
41
73
0.10

ISIS 545447
103
114
0.10

ISIS 541262
106
133
0.12

ISIS 541742
56
102
0.11

ISIS 541767
53
69
0.09

ISIS 541875
70
133
0.08

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 109, expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 109

Kidney function markers (mg/dL) in

Sprague-Dawley rats

BUN
Creatinine

PBS group 1
16
0.2

PBS group 2
15
0.2

ISIS 541881
22
0.3

ISIS 542051
18
0.2

ISIS 542101
22
0.3

ISIS 542112
18
0.2

ISIS 542118
18
0.3

ISIS 542125
18
0.3

ISIS 542127
19
0.3

ISIS 542128
18
0.3

ISIS 542153
17
0.3

ISIS 542185
19
0.3

ISIS 542186
19
0.3

ISIS 545439
16
0.2

ISIS 545447
16
0.2

ISIS 541262
21
0.4

ISIS 541742
19
0.2

ISIS 541767
15
0.2

ISIS 541875
16
0.2

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WBC, RBC, and total hemoglobin content. The results are presented in Table 110. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 110

Hematology markers in Sprague-Dawley rats

HCT
Hemoglobin
RBC
WBC
Platelets

(%)
(g/dL)
(10⁶/μL)
(10³/μL)
(10³/μL)

PBS group 1
43
14
7
7
775

PBS group 2
49
15
8
8
1065

ISIS 541881
41
13
8
6
553

ISIS 542051
39
13
7
9
564

ISIS 542101
37
12
7
15
603

ISIS 542112
45
14
8
10
587

ISIS 542118
47
15
8
7
817

ISIS 542125
41
13
7
7
909

ISIS 542127
44
14
8
10
872

ISIS 542128
44
14
8
7
679

ISIS 542153
48
15
8
7
519

ISIS 542185
44
14
8
9
453

ISIS 542186
44
14
8
12
433

ISIS 545439
40
12
7
11
733

ISIS 545447
43
13
8
9
843

ISIS 541262
46
14
8
17
881

ISIS 541742
47
15
8
10
813

ISIS 541767
53
16
9
9
860

ISIS 541875
42
13
7
9
840

Organ Weights

Liver, heart, spleen and kidney weights were measured at the end of the study, and are presented in Table 111. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 111

Organ weights (g)

Heart
Liver
Spleen
Kidney

PBS group 1
0.4
3.7
0.2
0.9

PBS group 2
0.3
3.2
0.2
0.7

ISIS 541881
0.4
3.4
0.4
0.9

ISIS 542051
0.4
3.8
0.4
1.0

ISIS 542101
0.3
4.2
0.6
1.1

ISIS 542112
0.3
3.7
0.4
0.8

ISIS 542118
0.4
3.6
0.2
0.8

ISIS 542125
0.4
3.7
0.3
1.1

ISIS 542127
0.3
4.2
0.3
0.8

ISIS 542128
0.3
3.5
0.3
0.8

ISIS 542153
0.3
3.5
0.3
0.8

ISIS 542185
0.4
3.8
0.4
0.9

ISIS 542186
0.3
3.8
0.6
0.9

ISIS 545439
0.4
4.1
0.3
0.9

ISIS 545447
0.4
3.4
0.3
1.1

ISIS 541262
0.3
3.4
0.3
2.0

ISIS 541742
0.3
3.8
0.3
0.8

ISIS 541767
0.3
3.4
0.2
0.8

ISIS 541875
0.3
5.2
0.4
1.0

Example 23: Effect of ISIS Antisense Oligonucleotides Targeting Human GHR in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described in the Examples above. Antisense oligonucleotide efficacy and tolerability, as well as their pharmacokinetic profile in the liver and kidney, were evaluated.

At the time this study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed. Instead, the sequences of the ISIS antisense oligonucleotides used in the cynomolgus monkeys was compared to a rhesus monkey sequence for homology. It is expected that ISIS oligonucleotides with homology to the rhesus monkey sequence are fully cross-reactive with the cynomolgus monkey sequence as well. The human antisense oligonucleotides tested are cross-reactive with the rhesus genomic sequence (GENBANK Accession No. NW_001120958.1 truncated from nucleotides 4410000 to 4720000, designated herein as SEQ ID NO: 2296). The greater the complementarity between the human oligonucleotide and the rhesus monkey sequence, the more likely the human oligonucleotide can cross-react with the rhesus monkey sequence. The start and stop sites of each oligonucleotide to SEQ ID NO: 2296 is presented in Table 112. “Start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey gene sequence.

TABLE 112

Antisense oligonucleotides complementary to the rhesus

GHR genomic sequence (SEQ ID NO: 2296)

Target
Target

Start
Stop

SEQ ID

ISIS No
Site
Site
Chemistry
NO

523723
149071
149090
5-10-5 MOE
918

532254
64701
64720
5-10-5 MOE
479

532401
147560
147579
5-10-5 MOE
703

541767
152700
152715
Deoxy, MOE
1800

and cEt

541875
210099
210114
Deoxy, MOE
1904

and cEt

542112
146650
146665
Deoxy, MOE
2122

and cEt

542118
149074
149089
Deoxy, MOE
2127

and cEt

542185
245782
245797
Deoxy, MOE
2194

and cEt

Study 1

Prior to the study, the monkeys were kept in quarantine during which the animals were observed daily for general health. The monkeys were 2-4 years old and weighed between 2 and 4 kg. Nine groups of 5 randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS using a stainless steel dosing needle and syringe of appropriate size into the intracapsular region and outer thigh of the monkeys. The monkeys were dosed three times (days 1, 4, and 7) for the first week, and then subsequently once a week for 12 weeks with 40 mg/kg of ISIS oligonucleotide. A control group of 5 cynomolgus monkeys was injected with PBS in a similar manner and served as the control group.

During the study period, the monkeys were observed twice daily for signs of illness or distress. Any animal experiencing more than momentary or slight pain or distress due to the treatment, injury or illness was treated by the veterinary staff with approved analgesics or agents to relieve the pain after consultation with the Study Director. Any animal in poor health or in a possible moribund condition was identified for further monitoring and possible euthanasia. Scheduled euthanasia of the animals was conducted on day 86 by exsanguination after ketamine/xylazine-induced anesthesia and administration of sodium pentobarbital. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).

Target Reduction
RNA Analysis

On day 86, RNA was extracted from liver, white adipose tissue (WAT) and kidney for real-time PCR analysis of measurement of mRNA expression of GHR. Results are presented as percent inhibition of mRNA, relative to PBS control, normalized with RIBOGREEN®. ‘n.d.’ indicates that the data for that particular oligonucleotide was not measured. As shown in Table 113, treatment with ISIS antisense oligonucleotides resulted in significant reduction of GHR mRNA in comparison to the PBS control. Specifically, treatment with ISIS 532401 resulted in significant reduction of mRNA expression in all tissues.

The expression of the growth hormone-responsive gene, ALS was also measured in liver, kidney and adipose tissue. Treatment with ISIS 532401 resulted in ALS RNA expression reduction in liver by 44±9%, correlating with GHR levels. There was no reduction observed in adipose tissue. The expression of IGF1 in the liver was also measured. Treatment with ISIS 532401 resulted in IGF1 RNA expression reduction in liver by 71±10%, correlating with GHR levels.

TABLE 113

Percent inhibition of GHR mRNA in the cynomolgus

monkey liver relative to the PBS control

ISIS No
Liver
Kidney
WAT

532401
60
47
59

532254
63
65
n.d.

523723
38
0
n.d.

542112
61
60
36

542118
0
22
27

542185
66
53
n.d.

541767
0
14
n.d.

541875
34
77
n.d.

Protein Analysis

Approximately 1 mL of blood was collected from all available animals at day 85 and placed in tubes containing the potassium salt of EDTA. The tubes were centrifuged (3000 rpm for 10 min at room temperature) to obtain plasma. Plasma levels of IGF-1 and GH were measured in the plasma. The results are presented in Table 114. The results indicate that treatment with ISIS oligonucleotides resulted in reduced IGF-1 protein levels.

Plasma levels of IGF1 after treatment with ISIS 532401 are also presented in Table 115 and demonstrate the effect of antisense inhibition of GHR in reducing IGF1 levels at day 7 and day 85.

TABLE 114

Plasma protein levels in the cynomolgus monkey

IGF-1
GH

(% baseline)
(ng/mL)

PBS
121
19

532401
57
39

532254
51
26

523723
77
16

542112
46
48

542118
97
6

542185
59
32

541767
101
22

541875
45
47

TABLE 115

Plasma IGF1 levels in the cynomolgus monkey

Day 7
Day 85

PBS
458
643

ISIS 532401
326
263

Tolerability Studies
Body and Organ Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health of the animals, body and organ weights were measured. Body weights were measured on day 84 and are presented in Table 115. Organ weights were measured on day 86 and the data is also presented in Table 115. The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides. Specifically, treatment with ISIS 532401 was well tolerated in terms of the body and organ weights of the monkeys.

TABLE 115

Final body and organ weights in cynomolgus monkey

Body
Spleen
Kidney
Liver

Wt (kg)
(g)
(g)
(g)

PBS
2.7
2.8
12.3
56.7

532401
2.6
4.0
11.5
58.5

532254
2.6
4.8
15.4
69.5

523723
2.8
3.1
14.8
69.4

542112
2.6
3.5
13.6
60.0

542118
2.7
2.7
11.9
58.6

542185
2.6
5.5
17.2
68.5

541767
2.8
5.1
11.7
65.1

541875
2.8
5.5
13.2
55.0

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, blood samples were collected from all the study groups. The blood samples were collected via femoral venipuncture, 48 hrs post-dosing. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes containing K₂-EDTA anticoagulant, which were centrifuged to obtain plasma. Levels of various liver function markers were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Plasma levels of ALT and AST and bilirubin were measured. The Tables below present the results for ALT and AST levels at various time points. The results indicate that antisense oligonucleotides had no effect on liver function outside the expected range for antisense oligonucleotides. Specifically, treatment with ISIS 532401 was well tolerated in terms of the liver function in monkeys.

TABLE 116

ALT levels (IU/L) in cynomolgus monkey

Day 16
Day 44
Day 86

PBS
46
37
40

ISIS 532401
63
59
88

ISIS 532254
62
46
56

ISIS 523723
50
77
86

ISIS 542112
53
54
60

ISIS 542118
38
41
52

ISIS 542185
58
59
91

ISIS 541767
56
45
46

ISIS 541875
70
54
71

TABLE 117

AST levels (IU/L) in cynomolgus monkey

Day 16
Day 44
Day 86

PBS
58
40
45

ISIS 532401
47
48
61

ISIS 532254
71
81
98

ISIS 523723
56
61
73

ISIS 542112
58
65
89

ISIS 542118
41
40
46

ISIS 542185
61
63
98

ISIS 541767
52
39
63

ISIS 541875
70
50
70

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, blood samples were collected from all the study groups. The blood samples were collected via femoral venipuncture, 48 hrs post-dosing. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes containing K₂-EDTA anticoagulant, which were centrifuged to obtain plasma. Levels of BUN and creatinine were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). The Tables below present the results for BUN and creatinine levels at various time points.

The plasma chemistry data indicate that most of the ISIS oligonucleotides did not have any effect on the kidney function outside the expected range for antisense oligonucleotides. Specifically, treatment with ISIS 532401 was well tolerated in terms of the kidney function of the monkeys.

TABLE 118

BUN levels (mg/dL) in cynomolgus monkey

Day 16
Day 44
Day 86

PBS
29
26
26

ISIS 532401
27
27
27

ISIS 532254
21
22
25

ISIS 523723
25
24
22

ISIS 542112
26
24
24

ISIS 542118
29
27
29

ISIS 542185
22
21
22

ISIS 541767
29
24
24

ISIS 541875
29
24
21

TABLE 119

Creatinine levels (mg/dL) in cynomolgus monkey

Day 16
Day 44
Day 86

PBS
0.9
0.8
0.9

ISIS 532401
1.1
1.0
1.1

ISIS 532254
1.0
1.0
1.0

ISIS 523723
1.0
1.0
1.0

ISIS 542112
1.0
0.9
1.0

ISIS 542118
0.9
0.9
0.9

ISIS 542185
1.0
0.9
0.9

ISIS 541767
1.1
0.9
0.9

ISIS 541875
1.2
1.1
1.1

Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys on hematologic parameters, blood samples of approximately 1.3 mL of blood was collected from each of the available study animals in tubes containing K₂-EDTA. Samples were analyzed for red blood cell (RBC) count, white blood cells (WBC) count, individual white blood cell counts, such as that of monocytes, neutrophils, lymphocytes, as well as for platelet count, hemoglobin content and hematocrit, using an ADVIA120 hematology analyzer (Bayer, USA). The Table below presents the results for platelet count at various time points. ‘n/a’ indicates that the data for that time point is not available.

The data indicate the oligonucleotides did not cause any changes in hematologic parameters outside the expected range for antisense oligonucleotides at this dose. Specifically, treatment with ISIS 532401 was well tolerated in terms of the hematologic parameters of the monkeys.

TABLE 120

Platelet count (×10³/uL) in cynomolgus monkey

Day 30
Day 58
Day 86

PBS
538
464
403

ISIS 532401
493
465
395

ISIS 532254
334
328
306

ISIS 523723
352
304
268

ISIS 542112
454
430
368

ISIS 542118
418
379
377

ISIS 542185
370
303
296

ISIS 541767
435
326
325

ISIS 541875
437
359
n/a

C-Reactive Protein and Complement C3 Level Analysis

To evaluate any inflammatory effect of ISIS oligonucleotides in cynomolgus monkeys, blood samples were taken for analysis. The monkeys were fasted overnight prior to blood collection. Approximately 1.5 mL of blood was collected from each animal and put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 min and then centrifuged at 3,000 rpm for 10 min at room temperature to obtain serum. C-reactive protein (CRP), which is synthesized in the liver and which serves as a marker of inflammation, was measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). The Tables below present the results for CRP and C3 levels at various time points. The results indicate that treatment with ISIS 532401 did not cause inflammation in monkeys.

TABLE 121

CRP (mg/L) in cynomolgus monkey

Day 16
Day 44
Day 86

PBS
3.8
2.0
2.0

ISIS 532401
2.3
1.7
1.9

ISIS 532254
2.1
3.2
5.9

ISIS 523723
6.1
4.5
4.4

ISIS 542112
2.5
2.7
2.7

ISIS 542118
2.2
2.8
2.1

ISIS 542185
2.3
11.9
9.3

ISIS 541767
1.9
1.5
6.7

ISIS 541875
4.9
3.5
8.4

TABLE 122

C3 (mg/dL) on day 85 (24 hours after

dosing) in cynomolgus monkey

C3

PBS
114

ISIS 532401
96

ISIS 532254
100

ISIS 523723
87

ISIS 542112
100

ISIS 542118
110

ISIS 542185
98

ISIS 541767
99

ISIS 541875
81

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide in the liver and the kidney of the monkeys was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 2300) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. Half-lives were then calculated using WinNonlin software (PHARSIGHT).

The results are presented in Table 123, expressed as μg/g of tissue, as well as the ratio of concentration in kidney versus liver.

TABLE 123

Oligonucleotide concentration in the liver

and kidney of cynomolgus monkeys

K/L

Chemistry
Liver
Kidney
ratio

ISIS 532401
5-10-5 MOE
725
2154
3.0

ISIS 532254
5-10-5 MOE
911
4467
4.9

ISIS 523723
5-10-5 MOE
657
3093
4.7

ISIS 542112
3-10-3 cEt/MOE
491
2863
5.8

ISIS 542118
3-10-3 cEt/MOE
429
1222
2.8

ISIS 542185
3-10-3 cEt/MOE
432
3126
7.2

ISIS 541767
3-10-3 cEt/MOE
280
994
3.5

ISIS 541875
3-10-3 cEt/MOE
766
3892
5.1

Study 2

One group of 5 randomly assigned male cynomolgus monkeys was injected subcutaneously with ISIS 532401 or PBS using a stainless steel dosing needle and syringe of appropriate size into the intracapsular region and outer thigh of the monkeys. The monkeys were dosed a loading dose per week (days 1, 3, 5, and 7) for the first week, and then subsequently once a week (days 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91) with 40 mg/kg of ISIS 532401. A control group of 5 cynomolgus monkeys was injected with PBS in a similar manner and served as the control group.

Target Reduction
RNA Analysis

On day 93, RNA was extracted from liver, white adipose tissue (WAT) and muscle for real-time PCR analysis of measurement of mRNA expression of GHR. Treatment with ISIS 532401 resulted in significant reduction of GHR mRNA in liver and white adipose tissue.

The expression of the growth hormone-responsive gene, ALS was also measured in the liver. Treatment with ISIS 532401 resulted in ALS RNA expression reduction in liver by 38%, correlating with GHR levels. ‘n.d.’ indicates that the levels were not checked in that particular tissue. The expression of IGF1 in the liver, muscle and fat tissues was also measured. Treatment with ISIS 532401 resulted in IGF1 RNA expression reduction in liver and in the WAT, correlating with GHR levels.

TABLE 124

Effect of treatment with ISIS 532401 on mRNA levels

(% inhibition compared to the PBS control) in the

cynomolgus monkey

Liver
WAT
Muscle

GHR
64
75
21

ALS
38
n.d.
n.d.

IGF1
73
56
35

Protein Analysis

Plasma levels of IGF-1 and GH were measured in the plasma. The results are presented in the Table below. The results indicate that treatment with ISIS 532401 resulted in reduced IGF-1 protein levels. There was no increase in plasma growth hormone levels.

TABLE 125

Plasma IGF1 levels (ng/mL) in the cynomolgus monkey

Day 7
Day 49
Day 91

PBS
625
776
850

ISIS 532401
378
455
363

TABLE 126

Plasma growth hormone levels (ng/mL) in the cynomolgus monkey

Day 7
Day 49
Day 91

PBS
25
27
33

ISIS 532401
16
13
17

Example 24: Measurement of Viscosity of ISIS Antisense Oligonucleotides Targeting Human GHR

The viscosity of select antisense oligonucleotides from the study described in the Examples above was measured with the aim of screening out antisense oligonucleotides which have a viscosity more than 40 cP. Oligonucleotides having a viscosity greater than 40 cP would be too viscous to be administered to any subject.

ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μL of water was added and the antisense oligonucleotide was dissolved into solution by heating the vial at 50° C. Part of (75 μL) the pre-heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometer was set to 25° C. and the viscosity of the sample was measured. Another part (20 μL) of the pre-heated sample was pipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UV instrument). The results are presented in Table 127 and indicate that all the antisense oligonucleotides solutions are optimal in their viscosity under the criterion stated above.

TABLE 127

Viscosity of ISIS antisense oligonucleotides targeting human GHR

ISIS

Viscosity

No.
Chemistry
(cP)

523723
5-10-5 MOE
8

532254
5-10-5 MOE
22

532401
5-10-5 MOE
12

541767
Deoxy, MOE
13

and cEt

541875
Deoxy, MOE
33

and cEt

542112
Deoxy, MOE
10

and cEt

542118
Deoxy, MOE
14

and cEt

542185
Deoxy, MOE
17

and cEt

Example 25: Effect of Antisense Inhibition of GHR in Mice

In order to confirm the effect of antisense inhibition of GHR in the primate model, an ISIS oligonucleotide targeting murine GHR was employed to replicate the result in a mouse model.

ISIS 563223 (GAGACTTTTCCTTGTACACA, designated herein as SEQ ID NO: 2301) is a 5-10-5 MOE gapmer murine antisense oligonucleotide targeting murine GHR (GENBANK Accession No; NM_010284.2, designated herein as SEQ ID NO: 2302) at target start site 3230. A group of male and female CD1 mice were injected with a loading dose (on days 1, 3, 5, and 7) on the first week and subsequently with a once weekly dose (on days 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91) with 40 mg/kg of ISIS 563223. One group of CD1 mice was injected in a similar manner with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

mRNA Expression

Liver mRNA expression of GHR, GHBP, IGF1, and ALS were measured. The results are presented in Table 128. Antisense inhibition of GHR resulted in inhibition of GHBP, IGF1 and ALS gene expression levels.

TABLE 128

mRNA expression (% Inhibition) in CD 1 mice liver

% (in male mice)
% (in female mice)

GHR
98
96

GHBP
74
66

IGF1
60
48

ALS
84
74

Protein Expression

Plasma levels of IGF1 and growth hormone were measured. The results are presented in Table 129. Antisense inhibition of GHR resulted in decrease in IGF1 levels, and had no effect on growth hormone levels.

TABLE 129

IGF1 protein levels (ng/mL) in CD 1 mice liver

in male mice
in female mice

PBS
949
1002

ISIS 563223
439
740

TABLE 130

Growth hormone protein levels (ng/mL) in CD1 mice liver

in male mice
in female mice

PBS
3.3 ± 2.2
2.2 ± 1.3

ISIS 563223
5.6 ± 6.7
2.9 ± 1.7

	Number	Date	Country
Parent	16884782	May 2020	US
Child	17748961		US
Parent	16234409	Dec 2018	US
Child	16884782		US
Parent	14902446	Dec 2015	US
Child	16234409		US

MODULATORS OF GROWTH HORMONE RECEPTOR

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