WO2015002971A2 - Modulators of growth hormone receptor - Google Patents

Modulators of growth hormone receptor Download PDF

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Publication number
WO2015002971A2
WO2015002971A2 PCT/US2014/045088 US2014045088W WO2015002971A2 WO 2015002971 A2 WO2015002971 A2 WO 2015002971A2 US 2014045088 W US2014045088 W US 2014045088W WO 2015002971 A2 WO2015002971 A2 WO 2015002971A2
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Prior art keywords
intron
compound
modified
seq
certain embodiments
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PCT/US2014/045088
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French (fr)
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WO2015002971A3 (en
Inventor
Sanjay Bhanot
Susan M. Freier
Huynh-Hoa Bui
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Isis Pharmaceuticals, Inc.
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Priority to MX2015017863A priority Critical patent/MX2015017863A/en
Priority to RU2016102833A priority patent/RU2700244C2/en
Application filed by Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Priority to CN201480038401.4A priority patent/CN105452461B/en
Priority to US14/902,446 priority patent/US20170073689A1/en
Priority to EP20159386.0A priority patent/EP3730614A3/en
Priority to NZ715151A priority patent/NZ715151B2/en
Priority to JP2016524320A priority patent/JP6487913B2/en
Priority to CA2917229A priority patent/CA2917229A1/en
Priority to ES14819490T priority patent/ES2787600T3/en
Priority to AU2014284398A priority patent/AU2014284398B2/en
Priority to BR112015033069A priority patent/BR112015033069A2/en
Priority to EP14819490.5A priority patent/EP3017044B1/en
Priority to KR1020167001790A priority patent/KR102236784B1/en
Publication of WO2015002971A2 publication Critical patent/WO2015002971A2/en
Publication of WO2015002971A3 publication Critical patent/WO2015002971A3/en
Priority to IL243093A priority patent/IL243093B/en
Priority to HK16111878.6A priority patent/HK1223652A1/en
Priority to US16/234,409 priority patent/US20190225970A1/en
Priority to AU2020200570A priority patent/AU2020200570A1/en
Priority to IL272828A priority patent/IL272828A/en
Priority to US16/884,782 priority patent/US20210108211A1/en
Priority to US17/748,961 priority patent/US20230147811A1/en

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Abstract

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

Description

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 BIOL0226WOSEQ_ST25.txt, created on June 30, 2014, 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'-0-methoxyethyl" (also 2'-MOE and 2'-0(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of a furanose ring. A 2'-0-methoxyethyl modified sugar is a modified sugar.
"2'-MOE nucleoside" (also 2'-0-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 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) a-L- Methyleneoxy (4'-CH2-0-2') LNA , (B) β-D-Methyleneoxy (4'-CH2-0-2') LNA , (C) Ethyleneoxy (4'- (CH2)2-0-2') LNA , (D) Aminooxy (4'-CH2-0-N(R)-2') LNA and (E) Oxyamino (4'-CH2-N(R)-0-2') LNA, as de icted below.
Figure imgf000005_0001
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(Ri)(R2)]n-, -C(Ri)=C(R2)-, -C(RL)=N- , -C(=0)-, -C(=S)-, -0-, -Si(Ri)2-, -S(=0)x- and -N(Ri)-; wherein: x is 0, 1 , or 2; n is
1, 2, 3, or 4; each RL and R2 is, independently, H, a protecting group, hydroxyl, Ci-Ci2 alkyl, substituted Q- alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-C12 alkynyl, substituted C2-Ci2 alkynyl, C5-C2o aryl, substituted C5-C20 aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJiJ2, SJi, N3, COOJi, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=0)-Ji); and each { and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C Ci2 aminoalkyl, substituted CrCi2 aminoalkyl 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(Ri)(R2)]n-, -[C(Ri)(R2)]„-0-, -C(RiR2)-N(R -O- or -C(RiR2)-O-N(R -. Furthermore, other bridging groups encompassed with the definition of LNA are 4'-CH2-2', 4'-(CH2)2-2', 4'- (CH2)3-2', 4'-CH2-0-2', 4'-(CH2)2-0-2', 4'-CH2-0-N(R1)-2' and 4'-CH2-N(R -O-2'- bridges, wherein each Ri and R is, independently, H, a protecting group or CrCi2 alkyl.
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'-CH2-0-2') 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'- CH2-0-2') LNA is used. Furthermore; in the case of the bicylic sugar moiety having an ethylene bridging group in this position, the term ethyleneoxy (4'-CH2CH2-0-2') LNA is used, a -L- methyleneoxy (4'-CH2- 0-2'), an isomer of methyleneoxy (4'-CH2-0-2') 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)-0-2'.
"Constrained ethyl nucleoside" (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' 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'-0- 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 occuring 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 monomelic 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(=0)-0- 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 occuring 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 OO 1242400.2 (incorporated herein as SEQ ID NO: 10), GENBANK Accession No. NM OO 1242401.3 (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 OO 1242462.1 (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 having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases 100% complementary to an equal length portion of nucleobases 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 the nucleobase sequence of the modified oligonucleotide is 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, 18734-21219037, 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, 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: 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 (GENBAN 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 (GENBAN 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'-0-methoxyethyl group. In certain aspects, at least one modified sugar is a bicyclic sugar, such as a 4'-CH(CH3)-0-2' group, a 4'-CH2- 0-2' group, or a 4'-(CH2)2-0-2'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'-0-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'-0-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'-0-methoxyethyl group. In certain aspects, the at least one modified sugar is a bicyclic sugar, such as a 4'-CH(CH3)-0-2' group, a 4'-CH2-0-2' group, or a 4'-(CH2)2-0- 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.
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, 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'-0-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 compoun 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'-0-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 lie:
Figure imgf000036_0001
lie wherein:
Ti is an optionally protected phosphorus moiety; T2 is an internucleoside linking group linking the compound of Formula lie to the oligomeric compound;
A has one of the formulas:
Figure imgf000036_0002
Qi and Q2 are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl
Q3 is 0, S, N(R5) or C(R6)(R7); each R3, R4 R5, R6 and R7 is, independently, H, Ci-C6 alkyl, substituted Ci-C6 alkyl or CrC6 alkoxy;
M3 is O, S, NR14, C(R15)(R16), C(R15)(R16)C(R17)(R18), C(R15)=C(R17), OC(R15)(Ri6) or
Figure imgf000036_0003
Ri4 is H, C1-C6 alkyl, substituted C1-C6 alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
Ri5, Ri6, Ri7 and R18 are each, independently, H, halogen, Ci-C6 alkyl, substituted C -C6 alkyl, Ci-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
Bxi is a heterocyclic base moiety; or if Bx2 is present then Bx2 is a heterocyclic base moiety and Bxi is H, halogen, Q-C6 alkyl, substituted Ci-C6 alkyl, Ci-C6 alkoxy, substituted Ci-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2- C6 alkynyl or substituted C2-C6 alkynyl;
J4, J5, J6 and J7 are each, independently, H, halogen, C C6 alkyl, substituted Ci-C6 alkyl, CrC6 alkoxy, substituted CrC6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; or J4 forms a bridge with one of J5 or J7 wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, R19, C(R20)(R2i), C(R20)=C(R21), C[=C(R20)(R2i)] and C(=0) and the other two of J5, J6 and J7 are each, independently, H, halogen, CrC6 alkyl, substituted CrC6 alkyl, CrC6 alkoxy, substituted CrC6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; each R19, R20 and R2i is, independently, H, CrC6 alkyl, substituted CrC6 alkyl, CrC6 alkoxy, substituted CrC6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
G is H, OH, halogen or 0-[C(R8)(R9)]n-[(C=0)m-X1]j-Z; each R8 and R9 is, independently, H, halogen, Ci-C6 alkyl or substituted Ci-C6 alkyl; Xi is O, S or N(E ;
Z is H, halogen, Ci-C6 alkyl, substituted CrC6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or (E2)(E3);
Ei, E2 and E3 are each, independently, H, Ci-C6 alkyl or substituted Ci-C6 alkyl; 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, OJb N(J (J2),
Figure imgf000037_0001
X2 is 0, S or NJ3; each Ji, J2 and J3 is, independently, H or Ci-C6 alkyl; 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, M3 is O, CH=CH, OCH2 or OC(H)(Bx2). In certain embodiments, M3 is O. In certain embodiments, J4, J5, J6 and J7 are each H. In certain embodiments, J4 forms a bridge with one of J5 or J7.
In certain embodiments, A has one of the formulas:
Figure imgf000038_0001
wherein: Qi and Q2 are each, independently, H, halogen, Ci-C6 alkyl, substituted Ci-C6 alkyl, Ci-C6 alkoxy or substituted Ci-Ce alkoxy. In certain embodiments, Qi and Q2 are each H. In certain embodiments, Qi and Q2 are each, independently, H or halogen. In certain embodiments, Qi and Q2 is H and the other of Qi and Q2 is F, CH3 or OCH3.
In certain embodiments, Ti has the formula:
Rc
wherein:
Ra and Rc are each, independently, protected hydroxyl, protected thiol, CrC6 alkyl, substituted Ci-C6 alkyl, CrC6 alkoxy, substituted CrC6 alkoxy, protected amino or substituted amino; and
Rb is O or S. In certain embodiments, Rb is O and Ra and Rc are each, independently, OCH3, OCH2CH3 or CH(CH3)2.
In certain embodiments, G is halogen, OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, 0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-SCH3, 0(CH2)2-OCF3, 0(CH2)3- N(R10)(Rii), O(CH2)2-ON(R10)(Rii), O(CH2)2-O(CH2)2-N(R10)(Rn), OCH2C(=O)-N(R10)(Rn), OCH2C(=0)- N(R12)-(CH2)2-N(R10)(Rn) or O(CH2)2-N(R12)-C(=NR13)[N(R10)(R11)] wherein R10, Ru, R12 and R13 are each, independently, H or C C6 alkyl. In certain embodiments, G is halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-0(CH2)2-N(CH3)2, OCH2C(=0)-N(H)CH3, OCH2C(=0)-N(H)- (CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2. In certain embodiments, G is F, OCH3 or 0(CH2)2-OCH3. In certain embodiments, G is 0(CH2)2-OCH3.
In certain embodiments, the 5'-terminal nucleoside has Formula He:
Figure imgf000039_0001
He
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 modificatios are 2'-F and 2'- OMe. Such regions may be contiguous or may be interupted 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 patern is (AB)xAy wheren 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 certan 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
AB ABBAABBABABAA; 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 lie or He.
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 modifed nucleosde;
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'- (QJ- (AB)xAy-(D)z wherein:
Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula lie or He;
A is a first type of modifed 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 O or l;
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 lie or He;
A is a first type of modifed 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 certiain 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 R A. 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 OO 1242400.2 (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 OO 1242460.1 (incorporated herein as SEQ ID NO: 17), GENBANK Accession NM OO 1242461.1 (incorporated herein as SEQ ID NO: 18), GENBANK Accession No. NM OO 1242462.1 (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 occuring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
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(Ri)(R2) (R, Ri and R2 are each independently H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on 8/21/08 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 June 16, 2005) or alternatively 5'-substitution of a BNA (see PCT International Application WO 2007/134181 Published on 11/22/07 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'- 0(CH2)2OCH3 substituent groups. The substituent at the 2' position can also be selected from allyl, amino, azido, thio, O-allyl, O-Ci-Cio alkyl, OCF3, OCH2F, 0(CH2)2SCH3, 0(CH2)2-0-N(Rm)(Rn), 0-CH2-C(=0)- N(Rm)(Rn), and 0-CH2-C(=0)-N(Ri)-(CH2)2-N(Rm)(Rn), where each ¾, Rm and Rn is, independently, H or substituted or unsubstituted Ci-Cio alkyl.
As used herein, "bicyclic nucleosides" refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4'-(CH2)-0-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-0-2' (ENA); 4'-CH(CH3)-0-2' (also referred to as constrained ethyl or cEt) and 4'-CH(CH2OCH3)- 0-2' (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH3)(CH3)-0-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4'- CH2-N(OCH3)-2' (and analogs thereof see published International Application WO/2008/150729, published December 11, 2008); 4'-CH2-0-N(CH3)-2' (see published U.S. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH2-N(R)-0-2', wherein R is H, C1-C12 alkyl, or a protecting group (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH2-C(H)(CH3)-2' (see Chattopadhyaya et al, J. Org. Chem., 2009, 74, 1 18-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof see published International Application WO 2008/154401, published on December 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 Oram et al, Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent 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 Serial 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 a-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 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(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=0)-, -C(=NRa)-, -C(=S)-, -0-, -Si(Ra)2-, -S(=0)x-, and -N(Ra)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted CrCi2 alkyl, C2-Ci2 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJiJ2, SJi, N3, COOJi, acyl (C(=0)- H), substituted acyl, CN, sulfonyl
Figure imgf000054_0001
or sulfoxyl and each J i and J2 is, independently, H, CrCi2 alkyl, substituted CrCi2 alkyl, C2-C12 alkenyl, substituted C2-Ci2 alkenyl, C2-C12 alkynyl, substituted C2-Ci2 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=0)- H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group. In certain embodiments, the bridge of a bicyclic sugar moiety is -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-0-
, -C(RaRb)-N(R)-0- or -C(RaRb)-0-N(R)-. In certain embodiments, the bridge is 4'-CH2-2', 4'-(CH2)2-2', 4'- (CH2)3-2', 4'-CH2-0-2', 4'-(CH2)2-0-2', 4'-CH2-0-N(R)-2' and 4'-CH2-N(R)-0-2'- wherein each R is, independently, H, a protecting group or CrCi2 alkyl.
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 a-L configuration or in the β- D configuration. Previously, a-L-methyleneoxy (4'-CH2-0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et ah, Nucleic Acids Research, 2003, 21, 6365- 6372).
In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L-methyleneoxy (4'-CH2-0-2') BNA , (B) β-D-methyleneoxy (4'-CH2-0-2') BNA , (C) ethyleneoxy (4'-(CH2)2-0-2') BNA , (D) aminooxy (4'-CH2-0-N(R)-2') BNA, (E) oxyamino (4'-CH2-N(R)-0-2') BNA, and (F) methyl(methyleneoxy) (4'-CH(CH3)-0-2') 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:
Figure imgf000055_0001
wherein Bx is the base moiety and R is independently H, a protecting group, C1-C12 alkyl or CrCi2 alkoxy.
In certain embodiments, bicyclic nucleosides are provided having Formula I:
Figure imgf000055_0002
wherein:
Bx is a heterocyclic base moiety;
-Qa-Qb-Qc- is -CH2-N(Rc)-CH , -C(=0)-N(Rc)-CH2-, -CH2-0-N(Rc)-, -CH2-N(Rc)-0- or -N(EQ-0-
CH2;
Ro is C1-C12 alkyl or an amino protecting group; and
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium. In certain embodiments, bicyclic nucleosides are provided having Formula II:
Figure imgf000056_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Za is CrC6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, 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, OJc, NJcJd, SJC, N3, OC(=X)Jc, and NJeC(=X)NJcJd, wherein each Jc, Jd and Je is, independently, H, CrC6 alkyl, or substituted CrC6 alkyl and X is O or NJC.
In certain embodiments, bicyclic nucleosides are provided having Formula III:
Figure imgf000056_0002
wherein:
Bx is a heterocyclic base moiety; Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Zb is Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(=0)-).
In certain embodiments, bicyclic nucleosides are provided having Formula IV:
Figure imgf000057_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Rd is Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; each qa, qb, qc and qd is, independently, H, halogen, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, Ci-C5 alkoxyl, substituted Cr C6 alkoxyl, acyl, substituted acyl, Ci-C6 aminoalkyl or substituted Ci-C6 aminoalkyl;
In certain embodiments, bicyclic nucleosides are provided having Formula V:
Figure imgf000057_0002
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; qa, qbj qe and qf are each, independently, hydrogen, halogen, C1-C12 alkyl, substituted CrCi2 alkyl, C2-
C12 alkenyl, substituted C2-Ci alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, C1-C12 alkoxy, substituted C1-C12 alkoxy, OJj; SJj, SOJj5 S02Jj, NJjJk, N3, CN, C(=0)OJj; C(=0)NJjJk, C(=0)Jjs 0-C(=0)NJjJk, N(H)C(= H)NJjJk, N(H)C(=0)NJjJk or N(H)C(=S)NJjJk; or qe and qf together are
Figure imgf000058_0001
qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted CrCi2 alkyl.
The synthesis and preparation of the methyleneoxy (4'-CH2-0-2') 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'-CH2-0-2') 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-affmity 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:
Figure imgf000058_0002
wherein: Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; each q;, q-, qk and qi is, independently, H, halogen, CrCi2 alkyl, substituted C1-C12 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-C12 alkynyl, CrCi2 alkoxyl, substituted Cr C12 alkoxyl, OJj; SJjs SOJj, S02Jj, NJjJk, N3, CN, C(=0)OJj5 C(=0)NJjJk, C(=0)Jj5 0-C(=0)NJjJk, N(H)C(=NH)NJjJk, N(H)C(=0)NJjJkorN(H)C(=S)NJjJk; and
¾ and q, or qi and qk together are
Figure imgf000059_0001
are each, independently, H, halogen, C1-C12 alkyl or substituted Ci-C12 alkyl. 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: 0[(CH2)nO]mCH3, 0(CH2)nNH2, 0(CH2)nCH3, 0(CH2)nF, 0(CH2)nONH2, OCH2C(=0)N(H)CH3; and 0(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-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, F, CF3, OCF3, SOCH3, S02CH3, ON02, N02, 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, modifed nucleosides comprise a 2'-MOE side chain (Baker et al, J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'- O- methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al, Chimia, 1996, 50, 168-176; Altmann et al, Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al, Nucleosides Nucleotides, 1997, 16, 917-926).
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:
Figure imgf000060_0001
In certain embodiments, sugar surrogates are selected having Formula VII:
Figure imgf000060_0002
VII wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII: Bx is a heterocyclic base moiety;
Ta and Tb are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of Ta and Tb is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of Ta and Tb is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group; qi, q2, q3, q4, qs, qe and q7 are each independently, H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and each of Ri and R2 is selected from hydrogen, hydroxyl, halogen, subsitituted or unsubstituted alkoxy, NJiJ2, SJi, N3, OC(=X)Ji, OC(=X)NJ!J2, NJ3C(=X)NJ!J2 and CN, wherein X is O, S or Ji and each J J2 and J3 is, independently, H or d-C6 alkyl.
In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein qb q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of qi, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of qi, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R2 is fluoro. In certain embodiments, Ri is fluoro and R2 is H; Ri is methoxy and R2 is H, and Ri is methoxyethoxy and R2 is 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. Patents 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:
Figure imgf000061_0001
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 "modifed morpholinos."
Combinations of modifications are also provided without limitation, such as 2'-F-5 '-methyl substituted nucleosides (see PCT International Application WO 2008/101 157 published on 8/21/08 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 June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181 , published on 1 1/22/07 wherein a 4'-CH2-0-2' 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 ei fl/., 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 April 10, 2010, Robeyns et al, J. Am. Chem. Soc, 2008, 130(6), 1979-1984; Horvath 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.
Figure imgf000062_0001
X wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula X: Bx is a heterocyclic base moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5'-or 3'-terminal group; and
Qi, ¾2, q3, q4, qs, ¾6, ¾7, qs and q9 are each, independently, H, Ci-C6 alkyl, substituted CrC5 alkyl, C - C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl 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-d-do alkyl, -OCF3, 0-(CH2)2-0-CH3, 2'-0(CH2)2SCH3, 0-(CH2)2-0- N(Rm)(Rn), or 0-CH2-C(=0)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Ci-Ci0 alkyl. 2'-modifed 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'-0-methyl" each refers to a nucleoside comprising a sugar comprising an -OCH3 group at the 2' position of the sugar ring.
As used herein, "MOE" or "2'-MOE" or "2'-OCH2CH2OCH3" or "2'-0-methoxyethyl" each refers to a nucleoside comprising a sugar comprising a -OCH2CH2OCH3 group 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.: 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 June 2, 2005 and published as WO 2005/121371 on December 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)-0-2') bridging group. In certain embodiments, the (4'- CH(CH3)-0-2') 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 0-6 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 January 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 l,2-di-0-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., US 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, CA). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, CA) 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, CA). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, CA) 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, CA) 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 often 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 b 5-10-5 MOE a mers tar etin exonic re ions of SE ID NO: 1 and 2
Figure imgf000069_0001
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
Exon 4-
523317 792 811 exon 5 TCATTTTCCATTTAGTTTCA 19 n/a n/a
junction 47
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
Exon 5-
523331 964 983 exon 6 TAGAAATCTTCTTCACATGT 4 n/a n/a
junction 53
Exon 5-
523332 971 990 exon 6 TGGAAAGTAGAAATCTTCTT 9 n/a n/a
junction 54
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 SEQ ID SEQ ID
SEQ
ISIS NO: NO: Target % NO: 2 NO: 2
Sequence ID NO 1 1 Region inhibition Start Stop
NO
Start Stop Site Site Site Site
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
SEQ SEQ SEQ ID ID ID ID
ISIS NO: Target % SEQ
NO: 1 Sequence NO: 2 NO: 2 NO 1 Region inhibition ID NO
Stop Start Stop
Start
Site Site Site
Site
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
26495 26514
533133 n/a n/a Intron 1 CAGGAATGGAAAACCAAATA 74 129
121986 122005
26496 26515
533134 n/a n/a Intron 1 TCAGGAATGGAAAACCAAAT 73 130
121987 122006
26498 26517
533135 n/a n/a Intron 1 ACTCAGGAATGGAAAACCAA 77 113032 113051 131
121989 122008
26499 26518
533136 n/a n/a Intron 1 AACTCAGGAATGGAAAACCA 79 113033 113052 132
121990 122009
26500 26519
533137 n/a n/a Intron 1 TAACTCAGGAATGGAAAACC 67 113034 113053 133
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
Figure imgf000073_0001
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
Figure imgf000075_0001
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
26497 26513
539311 n/a n/a Intron 1 AGGAATGGAAAACCAAA 18 113031 113047 246
121988 122004
26498 26514
539312 n/a n/a Intron 1 CAGGAATGGAAAACCAA 40 113032 113048 247
121989 122005
26499 26515
539313 n/a n/a Intron 1 TCAGGAATGGAAAACCA 49 113033 113049 248
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
SEQ SEQ ID ID SEQ
ISIS %
Sequence NO: 2 NO: 2 ID
NO inhibition
Start Stop NO
Site Site
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
26497 26516
523577 CTCAGGAATGGAAAACCAAA 71 113031 113050 311
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
intron l of SEQ ID O: 2
SEQ SEQ ID ID SEQ
ISIS %
Sequence NO: 2 NO: 2 ID NO inhibition
Start Stop NO Site Site
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
; intron l of SEQ ID NO: 2
SEQ SEQ ID ID SEQ
ISIS %
Sequence NO: 2 NO: 2 ID NO inhibition
Start Stop NO Site Site
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 SEQ
ISIS %
Sequence NO: 2 NO: 2 ID NO inhibition
Start Stop NO Site Site
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 ID ID SEQ SEQ
ISIS NO: NO: Target % ID NO: ID NO: SEQ ID
Sequence
NO 1 1 region inhibition 2 Start 2 Stop NO
Start Stop Site Site
Site Site
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 1 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
SEQ SEQ ID ID SEQ
ISIS Target %
Sequence NO: 2 NO: 2 ID NO Region inhibition
Start Stop NO Site Site
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 SEQ
ISIS %
Sequence NO: 2 NO: 2 ID NO inhibition
Start Stop NO Site Site
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
ISIS Target % SEQ ID
Sequence NO: 2 NO: 2
NO region inhibition NO
Start Stop
Site Site
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 SEQ
ISIS Target % NO: 2
Sequence NO: 2 ID NO region inhibition Stop
Start NO
Site Site
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
SEQ
ISIS Target % ID NO: ID NO:
Sequence ID NO region inhibition 2 Start 2 Stop
NO
Site Site
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
SEQ SEQ
ID ID
ISIS Target % SEQ ID
Sequence NO: 2 NO: 2
NO region inhibition NO
Start Stop
Site Site
532502 GAGTATTTCAGGCTGGAAAA Intron 3 43 214623 214642 1064
26501 26520
533404 GTAACTCAGGAATGGAAAAC Intron 1 56 113035 113054 1065
121992 122011
26502 26521
533405 AGTAACTCAGGAATGGAAAA Intron 1 41 113036 113055 1066
121993 122012
26503 26522
533406 AAGTAACTCAGGAATGGAAA Intron 1 43 113037 113056 1067
121994 122013
143207 143226
143235 143254
143263 143282
143291 143310
143319 143338
533407 GAGATTTCAAATAAATCTCA Intron 1 0 1068
143347 143366
143375 143394
143403 143422
143431 143450
143459 143478
143208 143227
143236 143255
143264 143283
143292 143311
143320 143339
533408 TGAGATTTCAAATAAATCTC Intron 1 11 1069
143348 143367
143376 143395
143404 143423
143432 143451
143460 143479
143209 143228
143237 143256
143265 143284
533409 GTGAGATTTCAAATAAATCT Intron 1 0 143293 143312 1070
143321 143340
143349 143368
143377 143396 143405 143424
143433 143452
143461 143480
143210 143229
143238 143257
143266 143285
143294 143313
143322 143341
533410 TGTGAGATTTCAAATAAATC Intron 1 0 1071
143350 143369
143378 143397
143406 143425
143434 143453
143462 143481
143183 143202
143211 143230
143239 143258
143267 143286
143295 143314
533411 TTGTGAGATTTCAAATAAAT Intron 1 10 143323 143342 1072
143351 143370
143379 143398
143407 143426
143435 143454
143463 143482
143184 143203
143212 143231
143240 143259
143296 143315
533412 TTTGTGAGATTTCAAATAAA Intron 1 0 1073
143324 143343
143352 143371
143380 143399
143464 143483
143185 143204
143213 143232
143241 143260
143297 143316
533413 CTTTGTGAGATTTCAAATAA Intron 1 20 1074
143325 143344
143353 143372
143381 143400
143465 143484
143186 143205
533414 ACTTTGTGAGATTTCAAATA Intron 1 57 143214 143233 1075
143242 143261 143298 143317
143326 143345
143354 143373
143382 143401
143466 143485
143187 143206
143215 143234
143243 143262
143299 143318
533415 CACTTTGTGAGATTTCAAAT Intron 1 69 1076
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
SEQ SEQ ID ID SEQ
ISIS %
Sequence NO: 2 NO: 2 ID NO inhibition
Start Stop NO Site Site
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
207510 207529
532495 GGCCTTGGGCACACTTTCTC 82 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
SEQ SEQ
SEQ
ISIS % ID NO: ID NO:
Sequence Target region ID NO inhibition 2 Start 2 Stop
NO
Site Site
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
Exon 5- intron 5
523844 ACCTGTGATTTACCTATTTC 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
Exon 6- intron 6
523849 TACCATTTTCCATTTAGTTT 7 279088 279107 1274 junction
1275
523850 ATTGGCATCTTTTTCAGTGG Intron 6 34 279902 279921
1276
523851 TCAAGCTCACGGTTGGAGAC Intron 6 36 280799 280818
1277
523852 AAATGAAATCAGTATGTTGA Intron 6 0 281622 281641
1278
523853 TGATTTATCACAAAGGTGCT Intron 6 29 282437 282456
1279
523854 AAAACAGTAGAAAAGATTAA Intron 6 14 284073 284092
1280
523855 CTACATCACAGCAGTCAGAA Intron 6 23 285187 285206
286349 286368
523856 AAAAGATGTAAGTGTGACAT Intron 6 28 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
Exon 7- intron 7
523860 CCTTCTTCACATGTAAATTG 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
SEQ SEQ ID ID SEQ
ISIS Target %
Sequence NO: 2 NO: 2 ID NO Region inhibition
Start Stop NO Site Site
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
47 277045 277064
532582 ATATAACAGAATCCAACCAT Intron 5 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 μΜ, 1.25 μΜ, 2.50 μΜ, 5.00 μΜ and 10.00 μΜ 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
μΜ μΜ μΜ μΜ μΜ (μΜ)
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
μΜ μΜ μΜ μΜ μΜ (μΜ)
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 ICso
ISIS No
μΜ μΜ μΜ μΜ μΜ (μΜ) 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
ISIS No ic50
μΜ μΜ μΜ μΜ μΜ (μΜ)
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
ISIS No ic50
μΜ μΜ μΜ μΜ μΜ (μΜ)
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
Figure imgf000109_0001
Table 26
Figure imgf000109_0002
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
Figure imgf000110_0001
Table 28
Figure imgf000110_0002
Table 29 0.625 1.250 2.50 5.00 10.00 ICso
ISIS No
μΜ μΜ μΜ μΜ μΜ (μΜ)
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
μΜ μΜ μΜ μΜ μΜ (μΜ)
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 ICso
ISIS No
μΜ μΜ μΜ μΜ μΜ (μΜ)
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
Figure imgf000112_0001
Table 33
0.625 1.250 2.50 5.00 10.00 IC50
ISIS No
μΜ μΜ μΜ μΜ μΜ (μΜ)
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 μΜ, 0.625 μΜ, 1.25 μΜ, 2.50 μΜ, 5.00 μΜ and 10.00 μΜ 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 (IC5o) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Table 34
Figure imgf000113_0001
Table 35
0.3125 0.625 1.250 2.50 5.00 10.00 IC50
ISIS No
μΜ μΜ μΜ μΜ μΜ μΜ (μΜ)
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
μΜ μΜ μΜ μΜ μΜ μΜ (μΜ)
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
μΜ μΜ μΜ μΜ μΜ μΜ (μΜ)
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 μΜ, 1.25 μΜ, 2.50 μΜ, 5.00 μΜ and 10.00 μΜ 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 (IC5o) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Table 38
Figure imgf000115_0001
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
Figure imgf000116_0001
Table 41
Figure imgf000116_0002
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
Figure imgf000117_0002
Table 43
Figure imgf000117_0001
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
Figure imgf000118_0001
Table 45
Figure imgf000118_0002
Table 46
Figure imgf000119_0001
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 μΜ, 0.625 μΜ, 1.25 μΜ, 2.50 μΜ, 5.00 μΜ and 10.00 μΜ 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 (IC5o) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Table 47
Figure imgf000120_0001
Table 48
Figure imgf000120_0002
Table 49
0.3125 0.625 1.250 2.50 5.00 10.00
ISIS No ic50 μΜ μΜ μΜ μΜ μΜ μΜ (μΜ)
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
ISIS No IC50
μΜ μΜ μΜ μΜ μΜ μΜ (μΜ)
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 intemucleoside 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 5 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
SEQ
SEQ ID ID SE
ISIS NO: %
Target Region Sequence Chemistry NO: 2 II NO 1 inhibition
Start Nl
Start
Site Site
541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 84 156891 13'
541263 164 Intron 1 CCGAGCTTCGCCTCTG eekddddddddddkke 89 3040 13'
541264 167 Intron 1 CCTCCGAGCTTCGCCT eekddddddddddkke 90 3043 13'
Junction
541265 170 spanning two GGACCTCCGAGCTTCG eekddddddddddkke 89 n/a 13' exons
Junction
541266 176 spanning two CCTGTAGGACCTCCGA eekddddddddddkke 83 n/a 13' exons
541268 214 Exon 2 CCAGTGCCAAGGTCAA eekddddddddddkke 87 144998 13'
541269 226 Exon 2 CACTTGATCCTGCCAG eekddddddddddkke 67 145010 13'
541270 244 Exon 2 CACTTCCAGAAAAAGC eekddddddddddkke 34 145028 13'
541278 365 Exon 4/Intron 3 GTCTCTCGCTCAGGTG eekddddddddddkke 77 268028 13'
541279 368 Exon 4/Intron 3 AAAGTCTCTCGCTCAG eekddddddddddkke 76 268031 13'
541280 373 Exon 4/Intron 3 ATGAAAAAGTCTCTCG eekddddddddddkke 66 268036 13! exon 2-exon 3
541283 445 TCCTTCTGGTATAGAA eekddddddddddkke 37 n/a 13! junction
541288 554 Exon 5 CAATAAGGTATCCAGA eekddddddddddkke 49 274114 13!
541289 561 Exon 5 CTTGATACAATAAGGT eekddddddddddkke 66 274121 13!
541290 569 Exon 5 CTAGTTAGCTTGATAC eekddddddddddkke 61 274129 13! exon 3 -exon 4
541293 628 GATCTGGTTGCACTAT eekddddddddddkke 57 n/a 13! junction
541294 639 Exon 6 GGCAATGGGTGGATCT eekddddddddddkke 38 278933 13!
541295 648 Exon 6 CCAGTTGAGGGCAATG eekddddddddddkke 67 278942 13!
541296 654 Exon 6 TAAAGTCCAGTTGAGG eekddddddddddkke 43 278948 13! 541301 924 Exon 7 TACATAGAGCACCTCA eekddddddddddkke 86 290422 13!
541302 927 Exon 7 TGTTACATAGAGCACC eekddddddddddkke 78 290425 13'
541303 930 Exon 7 AAGTGTTACATAGAGC eekddddddddddkke 59 290428 13'
541304 958 Exon 7 CTTCACATGTAAATTG eekddddddddddkke 26 290456 13'
541305 981 Exon 8 GAGCCATGGAAAGTAG eekddddddddddkke 66 292535 13'
Exon 7-exon 8
541310 1127 CCTTCCTTGAGGAGAT eekddddddddddkke 26 n/a 13' junction
541320 1317 Exon 10 CTTCACCCCTAGGTTA eekddddddddddkke 38 297734 13'
541321 1322 Exon 10 CCATCCTTCACCCCTA eekddddddddddkke 81 297739 13!
541322 1326 Exon 10 GTCGCCATCCTTCACC eekddddddddddkke 79 297743 13'
541323 1331 Exon 10 CCAGAGTCGCCATCCT eekddddddddddkke 64 297748 13'
541325 1420 Exon 10 GTGGCTGAGCAACCTC eekddddddddddkke 79 297837 13'
541326 1434 Exon 10 CCCTTTTAACCTCTGT eekddddddddddkke 67 297851 141
541331 1492 Exon 10 CATCATGATAAGGTGA eekddddddddddkke 16 297909 141
541332 1526 Exon 10 TGGATAACACTGGGCT eekddddddddddkke 30 297943 141
541333 1532 Exon 10 TCTGCTTGGATAACAC eekddddddddddkke 63 297949 141
541335 1597 Exon 10 GAATATGGGCAGCTTG eekddddddddddkke 33 298014 141
541336 1601 Exon 10 AGCTGAATATGGGCAG eekddddddddddkke 34 298018 141
541337 1607 Exon 10 TTGCTTAGCTGAATAT eekddddddddddkke 39 298024 141
541338 1611 Exon 10 TGGATTGCTTAGCTGA eekddddddddddkke 79 298028 141
541339 1614 Exon 10 ACTTGGATTGCTTAGC eekddddddddddkke 73 298031 141
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
SEQ
SEQ ID ID
ISIS NO: Target % SEQ ID
Sequence Chemistry NO: 2
NO 1 Region inhibition NO
Start
Start
Site
Site
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
Figure imgf000125_0001
541426 4528 Exon 10 TTGGTGTTTGATAACA eekddddddddddkke 31 300945 146:
541427 4531 Exon 10 ATGTTGGTGTTTGATA eekddddddddddkke 32 300948 146:
541429 30 Exon 1 CCGCCACTGTAGCAGC eekddddddddddkke 77 2906 146:
541430 35 Exon 1 CGCCACCGCCACTGTA eekddddddddddkke 88 2911 146-
541431 63 Exon 1 GCCGCCCGGGCTCAGC eekddddddddddkke 86 2939 146;
541432 67 Exon 1 CGCCGCCGCCCGGGCT eekddddddddddkke 61 2943 146(
541433 144 Exon 1 GAGAGCGCGGGTTCGC eekddddddddddkke 57 3020 146'
541434 n/a Exon 1/Intron 1 CTACTGACCCCAGTTC eekddddddddddkke 80 3655 146S
541435 n/a Exon 1/Intron 1 TCACTCTACTGACCCC eekddddddddddkke 90 3660 146'
541436 n/a Exon 1/Intron 1 TCATGCGGACTGGTGG eekddddddddddkke 56 3679 147(
541437 n/a Exon 3/Intron 3 ATGTGAGCATGGACCC eekddddddddddkke 82 225438 147:
541438 n/a Exon 3/Intron 3 TCTTGATATGTGAGCA eekddddddddddkke 93 225445 147:
541439 n/a Exon 3/Intron 3 TTCAAGTTGGTGAGCT eekddddddddddkke 72 226788 147:
541440 n/a Exon 3/Intron 3 TGCTTCCTTCAAGTTG eekddddddddddkke 68 226795 147'
541441 n/a Exon 3/Intron 3 TGTAATTTCATTCATG eekddddddddddkke 62 226809 147;
541442 n/a Exon 3/Intron 3 CCTTTTGCCAAGAGCA eekddddddddddkke 85 226876 147(
541443 n/a Exon 3/Intron 3 GATCCTTTTGCCAAGA eekddddddddddkke 77 226879 147'
541444 n/a Exon 3/Intron 3 GCTAGTAATGTTACAT eekddddddddddkke 68 238331 1475
541445 n/a Exon 3/Intron 3 GCAACTTGCTAGTAAT eekddddddddddkke 65 238338 147<
541446 n/a Exon 3/Intron 3 TGTGCAACTTGCTAGT eekddddddddddkke 44 238341 148(
541447 n/a Exon 3/Intron 3 GGATTTCAGTTTGAAT eekddddddddddkke 0 238363 148:
541448 n/a Exon 3/Intron 3 CTCAGAGCCTTGGTAG eekddddddddddkke 65 238428 148:
541449 n/a Exon 1/Intron 1 CAAACGCGCAAAAGAC eekddddddddddkke 1 3608 148:
541450 n/a Exon 1/Intron 1 GCCCGCACAAACGCGC eekddddddddddkke 11 3615 148-
541451 n/a Exon 1/Intron 1 GGTTAAAGAAGTTGCT eekddddddddddkke 60 93190 148;
541452 n/a Exon 1/Intron 1 CCCAGTGAATTCAGCA eekddddddddddkke 85 93245 148(
541453 n/a Exon 1/Intron 1 GCGCCCAGTGAATTCA eekddddddddddkke 74 93248 148'
541454 n/a Exon 1/Intron 1 AAGATGCGCCCAGTGA eekddddddddddkke 71 93253 148S
541455 n/a Exon 1/Intron 1 TGTAAGATGCGCCCAG eekddddddddddkke 75 93256 148
541456 n/a Exon 1/Intron 1 AATTACTTGTAAGATG eekddddddddddkke 15 93263 149(
541457 n/a Exon 1/Intron 1 CCCAGAAGGCACTTGT eekddddddddddkke 61 93302 149:
541458 n/a Exon 1/Intron 1 TTGCAGAACAAATCTT eekddddddddddkke 3 93333 149:
541459 n/a Exon 1/Intron 1 CATGGAAGATTTGCAG eekddddddddddkke 17 93343 149:
541460 n/a Exon 1/Intron 1 GGTCATGGAAGATTTG eekddddddddddkke 57 93346 149-
541461 n/a Exon 1/Intron 1 GACCTTGGTCATGGAA eekddddddddddkke 51 93352 149;
541462 n/a Exon 1/Intron 1 TGCCAATCCAAAGAGG eekddddddddddkke 34 93369 149(
541463 n/a Exon 1/Intron 1 GGGTCTGCCAATCCAA eekddddddddddkke 67 93374 149'
541464 n/a Exon 1/Intron 1 TCCCTGGGTCTGCCAA eekddddddddddkke 82 93379 149S
541465 n/a Exon 1/Intron 1 AAGTGTGAATTTATCT eekddddddddddkke 16 93408 149!
541466 n/a Exon 1/Intron 1 GGAGATCTCAACAAGG eekddddddddddkke 38 93428 150(
541468 n/a Exon 1/Intron 1 TCGCCCATCACTCTTC eekddddddddddkke 43 93989 150:
541469 n/a Exon 1/Intron 1 CCTGTCGCCCATCACT eekddddddddddkke 61 93993 i5o:
541470 n/a Exon 1/Intron 1 TCACCTGTCGCCCATC eekddddddddddkke 70 93996 i5o: 541471 n/a Exon 1/Intron 1 CCATCACCTGTCGCCC eekddddddddddkke 89 93999 150'
541472 n/a Exon 1/Intron 1 TCACCATCACCTGTCG eekddddddddddkke 72 94002 150;
541473 n/a Exon 1/Intron 1 TAATAGTTGTCACCAT eekddddddddddkke 42 94011 150(
541474 n/a Exon 1/Intron 1 TTCAGATCTTATTAAT eekddddddddddkke 0 94023 150'
541475 n/a Exon 1/Intron 1 TTGCAAATTCAGTCTG eekddddddddddkke 32 94096 150S
541477 n/a Exon 2/Intron 2 CGTTCTCTTGGAAGTA eekddddddddddkke 78 198766 150!
541478 n/a Exon 2/Intron 2 TCTTGAATAAATTTCG eekddddddddddkke 25 198780 15K
541479 n/a Exon 2/Intron 2 AAGCTCACTCTTCAAT eekddddddddddkke 60 198810 151 :
541480 n/a Exon 2/Intron 2 TCCAAGCTCACTCTTC eekddddddddddkke 49 198813 151.
541481 n/a Exon 2/Intron 2 GCTCCTGCCACTCTGT eekddddddddddkke 75 198837 151:
541482 n/a Exon 2/Intron 2 ATGGGCAAAGGCATCT eekddddddddddkke 60 198874 151'
541483 n/a 5' UTR AGTCTTCCCGGCGAGG eekddddddddddkke 32 2571 151.
5' and overlappig
541484 n/a CCGCCGCTCCCTAGCC eekddddddddddkke 73 2867 15K with exon 1
541485 n/a Intron 1 GCCCGCAACTCCCTGC eekddddddddddkke 37 3341 151'
541486 n/a Intron 1 CGCCTCCCCAGGCGCA eekddddddddddkke 34 4024 1515
541487 n/a Intron 1 GAGTGTCTTCCCAGGC eekddddddddddkke 86 4446 151*
541488 n/a Intron 1 CTGAAGACTCCTTGAA eekddddddddddkke 39 4721 152(
541489 n/a Intron 1 GGCTAGCCAAGTTGGA eekddddddddddkke 54 5392 152:
541490 n/a Intron 1 TGACTCCAGTCTTACC eekddddddddddkke 76 5802 152:
541491 n/a Intron 1 ATTCATTGTGGTCAGC eekddddddddddkke 91 6128 152:
541492 n/a Intron 1 GAAGTGGGTTTTTCCC eekddddddddddkke 86 6543 152'
541493 n/a Intron 1 GCCTTGGTTCAGGTGA eekddddddddddkke 79 6786 152;
Table 54
Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting SEQ ID NO: 3 and 4
Figure imgf000127_0001
Table 55
Inhibition of GHR mRNA by deoxy, MOE and cEt gapmers targeting intronic regions of SEQ ID NO: 2
SEQ SEQ ID ID SEQ
ISIS Target %
NO: 1 NO: 2 Sequence Chemistry ID NO Region inhibition
Start Start NO Site Site
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
Figure imgf000129_0001
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
ISIS NO: 2 NO: 2 Target % SEQ
Sequence Chemistry
NO Start Stop Region inhibition ID NO
Site Site
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
ISIS ID NO: ID NO: Target % SEQ
Sequence Chemistry
NO 2 Start 2 Stop Region inhibition ID NO
Site Site
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
SEQ
ISIS ID NO: NO: 2 Target %
Sequence Chemistry ID NO 2 Start Stop Region inhibition
NO
Site Site
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
207517 207532
541863 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
210281 210296
541868 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
SEQ ID
ISIS ID NO: Target % SEQ ID NO: 2 Sequence Chemistry
NO 2 Stop Region inhibition NO Start Site
Site
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 2261 10 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
SEQ
ISIS ID NO: Target % NO: 2
Sequence Chemistry ID NO 1 Start Region inhibition Start
NO
Site Site
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
Exon 8-
542011 n/a intron 8 CCTACCTTTGCTGTTT eekddddddddddkke 12 292611 2040 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'IJTR 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
ISIS ID NO: Target % SEQ
Sequence Chemistry NO: 2
NO 1 Start Region inhibition ID NO
Start
Site
Site
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
Figure imgf000143_0001
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
ISIS % SEC NO: 1 Target Region Sequence Chemistry NO: 2 NO inhibition N Start Start Site Site
541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 93 156891 13 exon 1- intron 1
545316 168 ACCTCCGAGCTTCGCC eekddddddddddkke 80 3044 21 junction
exon-exon
545317 173 GTAGGACCTCCGAGCT eekddddddddddkke 74 n/a 21 junction
exon-exon
545318 177 ACCTGTAGGACCTCCG eekddddddddddkke 70 n/a 21 junction
545321 213 Exon 2 CAGTGCCAAGGTCAAC eekddddddddddkke 77 144997 21
545322 225 Exon 2 ACTTGATCCTGCCAGT eekddddddddddkke 36 145009 22
545332 361 Exon 4/ Intron 3 CTCGCTCAGGTGAACG eekddddddddddkke 57 268024 22
545333 366 Exon 4/ Intron 3 AGTCTCTCGCTCAGGT eekddddddddddkke 88 268029 22
Exon 4-intron 4
545337 444 CCTTCTGGTATAGAAC eekddddddddddkke 21 268107 22 junction
545340 570 Exon 5 GCTAGTTAGCTTGATA eekddddddddddkke 39 274130 22 exon 3- exon 4
545343 626 TCTGGTTGCACTATTT eekddddddddddkke 34 n/a 22 junction
exon 3- exon 4
545344 629 GGATCTGGTTGCACTA eekddddddddddkke 30 n/a 22 junction
545345 632 Exon 6 GGTGGATCTGGTTGCA eekddddddddddkke 18 278926 22
545346 638 Exon 6 GCAATGGGTGGATCTG eekddddddddddkke 50 278932 22
545347 647 Exon 6 CAGTTGAGGGCAATGG eekddddddddddkke 71 278941 22 545348 651 Exon 6 AGTCCAGTTGAGGGCA eekddddddddddkke 58 278945 22
545349 655 Exon 6 GTAAAGTCCAGTTGAG eekddddddddddkke 34 278949 22
545350 660 Exon 6 GTTCAGTAAAGTCCAG eekddddddddddkke 52 278954 22
545351 685 Exon 6 CTGCATGAATCCCAGT eekddddddddddkke 77 278979 22
545355 923 Exon 7 ACATAGAGCACCTCAC eekddddddddddkke 38 290421 22
545356 926 Exon 7 GTTACATAGAGCACCT eekddddddddddkke 79 290424 22
545357 929 Exon 7 AGTGTTACATAGAGCA eekddddddddddkke 70 290427 22
Exon 7- exon 8
545362 1124 TCCTTGAGGAGATCTG eekddddddddddkke 3 n/a 22 junction
545363 1170 Exon 10 GCTATCATGAATGGCT eekddddddddddkke 69 297587 22
545364 1180 Exon 10 CGGGTTTATAGCTATC eekddddddddddkke 58 297597 22
545369 1320 Exon 10 ATCCTTCACCCCTAGG eekddddddddddkke 46 297737 22
545370 1328 Exon 10 GAGTCGCCATCCTTCA eekddddddddddkke 60 297745 22
545371 1332 Exon 10 TCCAGAGTCGCCATCC eekddddddddddkke 51 297749 22
545373 1418 Exon 10 GGCTGAGCAACCTCTG eekddddddddddkke 80 297835 22
545374 1422 Exon 10 CTGTGGCTGAGCAACC eekddddddddddkke 63 297839 22
545380 1524 Exon 10 GATAACACTGGGCTGC eekddddddddddkke 60 297941 22
545381 1530 Exon 10 TGCTTGGATAACACTG eekddddddddddkke 76 297947 22
545382 1533 Exon 10 CTCTGCTTGGATAACA eekddddddddddkke 60 297950 22
545386 1600 Exon 10 GCTGAATATGGGCAGC eekddddddddddkke 29 298017 22
545387 1613 Exon 10 CTTGGATTGCTTAGCT eekddddddddddkke 59 298030 22
545388 1645 Exon 10 CCTGGGCATAAAAGTC eekddddddddddkke 47 298062 22
545392 1832 Exon 10 ACCTTGATGTGAGGAG eekddddddddddkke 44 298249 22
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
ISIS NO: Target %
Sequence Chemistry NO: 2 SEQ ID Nl NO 1 Region inhibition
Start
Start
Site
Site
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
Intron
545434 n/a TACTGACCCCAGTTCC eekddddddddddkke 68 3654 2260
1/Exon 1
Intron
545435 n/a ACTCTACTGACCCCAG eekddddddddddkke 70 3658 2261
1/Exon 1
Intron
545436 n/a GTCACTCTACTGACCC eekddddddddddkke 83 3661 2262
1/Exon 1
Intron
545437 n/a TTCATGCGGACTGGTG eekddddddddddkke 68 3680 2263
1/Exon 1
Intron
545438 n/a GTGAGCATGGACCCCA eekddddddddddkke 94 225436 2264
3/Exon 3
Intron
545439 n/a TGATATGTGAGCATGG eekddddddddddkke 88 225442 2265
3/Exon 3
Intron
545440 n/a AAGTTGGTGAGCTTCT eekddddddddddkke 85 226785 2266
3/Exon 3
Intron
545441 n/a CCTTCAAGTTGGTGAG eekddddddddddkke 88 226790 2267
3/Exon 3
Intron
545442 n/a GTAAGATCCTTTTGCC eekddddddddddkke 70 226883 2268
3/Exon 3
Intron
545443 n/a CAGCTGTGCAACTTGC eekddddddddddkke 50 238345 2269
3/Exon 3
Intron
545444 n/a GCCTTGGTAGGTAGGG eekddddddddddkke 68 238422 2270
3/Exon 3
Intron
545445 n/a AGAGCCTTGGTAGGTA eekddddddddddkke 85 238425 2271
3/Exon 3
Intron
545446 n/a CCCGCACAAACGCGCA eekddddddddddkke 10 3614 2272
1/Exon 1
Intron
545447 n/a GTCTTCAAGGTCAGTT eekddddddddddkke 92 93208 2273
1/Exon 1 Intron
545448 n/a GCCCAGTGAATTCAGC eekddddddddddkke 76 93246 2274
1/Exon 1
Intron
545449 n/a AGATGCGCCCAGTGAA eekddddddddddkke 60 93252 2275
1/Exon 1
Intron
545450 n/a GTAAGATGCGCCCAGT eekddddddddddkke 78 93255 2276
1/Exon 1
Intron
545451 n/a CCAGAAGGCACTTGTA eekddddddddddkke 42 93301 2277
1/Exon 1
Intron
545452 n/a GGAAGATTTGCAGAAC eekddddddddddkke 15 93340 2278
1/Exon 1
Intron
545453 n/a CCTTGGTCATGGAAGA eekddddddddddkke 35 93350 2279
1/Exon 1
Intron
545454 n/a TGACCTTGGTCATGGA eekddddddddddkke 55 93353 2280
1/Exon 1
Intron
545455 n/a GAGGTGACCTTGGTCA eekddddddddddkke 70 93357 2281
1/Exon 1
Intron
545456 n/a ATCCAAAGAGGTGACC eekddddddddddkke 41 93364 2282
1/Exon 1
Intron
545457 n/a GCCAATCCAAAGAGGT eekddddddddddkke 56 93368 2283
1/Exon 1
Intron
545458 n/a GGTCTGCCAATCCAAA eekddddddddddkke 79 93373 2284
1/Exon 1
Intron
545459 n/a CCCTGGGTCTGCCAAT eekddddddddddkke 68 93378 2285
1/Exon 1
Intron
545460 n/a GAGATCTCAACAAGGG eekddddddddddkke 52 93427 2286
1/Exon 1
Intron
545461 n/a CGCCCATCACTCTTCC eekddddddddddkke 68 93988 2287
1/Exon 1
Intron
545462 n/a CACCTGTCGCCCATCA eekddddddddddkke 67 93995 2288
1/Exon 1
Intron
545463 n/a CATCACCTGTCGCCCA eekddddddddddkke 78 93998 2289
1/Exon 1
Intron
545464 n/a CACCATCACCTGTCGC eekddddddddddkke 74 94001 2290
1/Exon 1
Intron
545465 n/a AATAGTTGTCACCATC eekddddddddddkke 76 94010 2291
1/Exon 1
Intron
545466 n/a GCCACCTTTCATGAGA eekddddddddddkke 58 94048 2292
1/Exon 1
Intron
545467 n/a CTCTTGGAAGTAGGTA eekddddddddddkke 89 198762 2293
2/Exon 2
Intron
545468 n/a GTTCTCTTGGAAGTAG eekddddddddddkke 80 198765 2294
2/Exon 2
Intron
545469 n/a 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 μΜ, 1.25 μΜ, 2.50 μΜ, 5.00 μΜ and 10.00 μΜ 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 (IC5o) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Table 66
Figure imgf000149_0001
Table 67
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 1 1 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 ICso l l 1 O
μΜ μΜ μΜ μΜ μΜ (μΜ)
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
ISIS No ic50
μΜ μΜ μΜ μΜ μΜ (μΜ)
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 T„ 0.625 1.250 2.50 5.00 10.00 l l ΓΝ Ο ic50
μΜ μΜ μΜ μΜ μΜ (μΜ)
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
T¾T¾ Nn 0.625 1.250 2.50 5.00 10.00 ICso
μΜ μΜ μΜ μΜ μΜ (μΜ)
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
Figure imgf000152_0001
Table 73
Figure imgf000152_0002
Table 74
0.625 1.250 2.50 5.00 10.00
ISIS No ICso μΜ μΜ μΜ μΜ μΜ (μΜ)
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
Figure imgf000153_0001
Table 76
0.625 1.250 2.50 5.00 10.00 ICso
ISIS No
μΜ μΜ μΜ μΜ μΜ (μΜ)
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 μΜ, 0.11 μΜ, 0.33 μΜ, 1.00 μΜ, and 3.00 μΜ 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 (IC5o) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Table 77
Figure imgf000154_0001
Table 78
Figure imgf000155_0003
Table 79
Figure imgf000155_0001
Table 80
Figure imgf000155_0002
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
Figure imgf000156_0001
Table 82
Figure imgf000156_0002
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
Figure imgf000157_0001
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 μΜ, 0.37 μΜ, 1.11 μΜ, 3.33 μΜ, and 10.00 μΜ 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. The half maximal inhibitory concentration (IC5o) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Table 84
Figure imgf000158_0001
Table 85
Figure imgf000158_0002
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
Deoxy, MOE
541262 and cEt 0 43 63 88 94 0.8
Deoxy, MOE
541742 3 19 35 56 85 1.9
and cEt
Deoxy, MOE
541767 3 24 39 64 86 1.6
and cEt
Deoxy, MOE
545439 19 15 43 74 80 1.7
and cEt
Deoxy, MOE
545447 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 μΜ, 0.37 μΜ, 1.11 μΜ, 3.33 μΜ, and 10.00 μΜ 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.
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 86
Figure imgf000159_0001
and cEt
Deoxy, MOE
542118 20 45 69 84 91 0.5 and cEt
Deoxy, MOE
542125 33 62 77 92 97 0.3 and cEt
Deoxy, MOE
542127 30 50 65 86 92 0.4 and cEt
Deoxy, MOE
542128 25 40 52 80 93 0.7 and cEt
Deoxy, MOE
542153 10 31 51 73 85 1.0 and cEt
Deoxy, MOE
542185 12 45 65 85 93 0.6 and cEt
Deoxy, MOE
542186 36 54 74 90 96 0.3 and cEt
Deoxy, MOE
542051 9 29 32 32 42 >10 and cEt
Table 87
Figure imgf000160_0001
Deoxy,
545439 MOE and 0 0 30 61 90 2.4 cEt
Deoxy,
545447 MOE and 0 17 17 19 27 >10 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 μΜ, 0.37 μΜ, 1.11 μΜ, 3.33 μΜ, and 10.00 μΜ 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 (IC5o) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Table 88
Figure imgf000161_0001
Table 89
Figure imgf000162_0001
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 μΜ, 0.12 μΜ, 0.37 μΜ, 1.11 μΜ, 3.33 μΜ, and 10.00 μΜ 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.
The half maximal inhibitory concentration (IC5o) of each oligonucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Table 90
Figure imgf000162_0002
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 μΜ, 0.33 μΜ, 1.00 μΜ, 1.11 μΜ, 3.00 μΜ, and 9.00 μΜ 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
Figure imgf000163_0001
Example 15: Tolerability of 5-10-5 MOE gapmers targeting human GHR in CD1 mice
CD 1® mice (Charles River, MA) 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, NY). 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
Figure imgf000164_0001
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
Figure imgf000165_0001
Example 16: Tolerability of 5-10-5 MOE gapmers targeting human GHR in CD1 mice
CD 1® 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, NY). 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
Figure imgf000166_0001
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/μ > (103/μί) (103/μ >
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
CD 1® 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, NY). 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
Figure imgf000167_0001
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
Figure imgf000168_0001
Example 18: Tolerability of deoxy, MOE and cEt gapmers targeting human GHR in CD1 mice
CD 1® 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, NY). 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
Figure imgf000169_0001
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
Figure imgf000170_0001
Example 19: Tolerability of deoxy, MOE and cEt gapmers targeting human GHR in CD1 mice
CD 1® 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
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, NY). 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
Figure imgf000171_0002
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
Figure imgf000171_0001
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 CDl mice
CDl® 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 CDl 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 CDl 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, NY). 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 CDl mice plasma at week 6
Figure imgf000172_0001
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/μί) (103 iL) (103/μί)
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, NY). 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
Figure imgf000174_0001
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, NY). 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
Figure imgf000175_0001
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
Figure imgf000176_0001
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
gan weights
Figure imgf000177_0001
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, NY).
Plasma levels of ALT and AST were measured and the results are presented in Table 108 expressed in IU/L.
Plasma levels of bilirubm 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
Figure imgf000178_0001
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, NY). 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
Figure imgf000179_0001
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/ViL) (103 L) (103/μ¾
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
gan weights
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)
Figure imgf000181_0001
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 IGFl in the liver was also measured. Treatment with ISIS 532401 resulted in IGFl 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
Figure imgf000183_0001
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 IGF 1 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
Figure imgf000183_0002
Table 115
Plasma IGF1 levels in the cynomolgus monkey
Figure imgf000184_0001
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
Figure imgf000184_0002
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 200F 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
Figure imgf000185_0001
Table 117
AST levels (IU/L) in cynomolgus monkey
Figure imgf000185_0002
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 2- 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
Figure imgf000186_0001
Table 119
Creatinine levels (mg/dL) in cynomolgus monkey
Figure imgf000186_0002
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 (x 103/μ¾ in cynomolgus monkey
Figure imgf000187_0001
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
in cynomolgus monkey
Figure imgf000187_0002
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
Figure imgf000188_0001
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'-0-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 IGFl in the liver, muscle and fat tissues was also measured. Treatment with ISIS 532401 resulted in IGFl 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
Figure imgf000189_0001
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/niL) in the cynomolgus monkey
Figure imgf000190_0002
Table 126
Plasma growth hormone levels (ng/mL) in the cynomolgus monkey
Figure imgf000190_0003
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 μί) the preheated 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 μί) 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 oli onucleotides targeting human GHR
Figure imgf000190_0001
Deoxy, MOE
542112 10
and cEt
Deoxy, MOE
542118 14
and cEt
Deoxy, MOE
542185 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, IGFl, and ALS were measured. The results are presented in Table 128. Antisense inhibition of GHR resulted in inhibition of GHBP, IGFl and ALS gene expression levels.
Table 128
mRNA expression (% Inhibition) in CD1 mice liver
Figure imgf000191_0001
Protein expression
Plasma levels of IGFl and growth hormone were measured. The results are presented in Table 129. Antisense inhibition of GHR resulted in decrease in IGFl levels, and had no effect on growth hormone levels.
Table 129 IGFl protein levels (ng/mL) in CDl mice liver
Figure imgf000192_0001

Claims

WHAT IS CLAIMED:
1. 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.
2. 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.
3. 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 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 the nucleobase sequence of the modified oligonucleotide is complementary to SEQ ID NO: 1.
4. 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.
5. 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.
6. The compound of claim 5, wherein 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.
7. 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.
8. A compound comprising a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 20-2295.
9. The compound of any one of claims 1-8, wherein the modified oligonucleotide comprises at least one modified sugar.
10. The compound of claim 9, wherein the at least one modified sugar comprises a 2'-0- methoxyethyl group.
11. The compound of claim 9, wherein the at least one modified sugar is a bicyclic sugar.
12. The compound of claim 11, wherein the bicyclic sugar comprises a 4'-CH(CH3)-0-2' group.
13. The compound of claim 11, wherein the bicyclic sugar comprises a 4'-CH2-0-2' or 4'- (CH2)2-0-2'group.
14. The compound of any one of claims 1-13, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
15. The compound of claim 14, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
16. The compound of any one of claims 1-15, wherein the modified oligonucleotide comprises at least one modified nucleobase.
17. The compound of claim 16, wherein the modified nucleobase is 5-methylcytosine.
18. The compound of any one of claims 1-17, wherein 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.
19. 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.
20. The compound of claim 19, wherein 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'-0-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine.
21. The compound of claim 19, wherein 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'-0-methoxyethyl sugar or a constrained ethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.
22. 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.
23. The compound of claim 22, wherein the modified oligonucleotide comprises at least one modified sugar.
24. The compound of claim 23, wherein the at least one modified sugar comprises a 2'-0- methoxyethyl group.
25. The compound of claim 23, wherein the at least one modified sugar is a bicyclic sugar.
26. The compound of claim 25, wherein the bicyclic sugar comprises a 4'-CH(CH3)-0-2' group.
27. The compound of claim 25, wherein the bicyclic sugar comprises a 4'-CH2-0-2' or 4'- (CH2)2-0-2'group.
28. The compound of any one of claims 22-27, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
29. The compound of claim 28, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
30. The compound of any one of claims 22-29, wherein the modified oligonucleotide comprises at least one modified nucleobase.
31. The compound of claim 30, wherein the modified nucleobase is 5-methylcytosine.
32. The compound of any one of claims 22-31 , wherein 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.
33. 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, 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'-0-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.
34. The compound of any one of claims 1-33, wherein the modified oligonucleotide is at least 90% complementary to a nucleic acid encoding growth hormone receptor.
35. The compound of any of claims 1-33, wherein the modified oligonucleotide is 100% complementary to a nucleic acid encoding growth hormone receptor.
36. The compound of any one of claims 1-35, wherein the nucleic acid encoding growth hormone receptor comprises the nucleotide sequence of any one of SEQ ID NOs: 1-19.
37. The compound of any one of claims 1-36, wherein the compound is single-stranded.
38. A composition comprising the compound of any of claims 1-37 or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent.
39. 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 one of claims 1-38, thereby treating the disease associated with excess growth hormone.
40. The method of claim 39, wherein the disease associated with excess growth hormone is acromegaly.
41. The method of claim 40, wherein the treatment reduces IGF-1 levels.
42. 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 one of claims 1-38, thereby preventing the disease associated with excess growth hormone.
43. The method of claim 42, wherein the disease associated with excess growth hormone is acromegaly.
44. The method of any of claims 39-43, comprising co-administering the compound or composition and a second agent.
45. The method of claim 44, wherein the compound or composition and the second agent are administered concomitantly.
46. 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 one of claims 1 -38, thereby reducing GHR levels in the human.
47. The method of claim 46, wherein the human has a disease associated with excess growth hormone
48. The method of claim 47, wherein the disease associated with excess growth hormone is acromegaly.
49. The method of any one of claims 46-48, comprising co-administering the compound or composition and a second agent.
50. The method of claim 49, wherein the compound or composition and the second agent are administered concomitantly.
51. A compound consisting of ISIS 532401.
52. A composition comprising ISIS 532401 or salt thereof and a pharmaceutically acceptable carrier or diluent.
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